# NANOTECHNOLOGY IN CARDIOVASCULAR REGENERATIVE MEDICINE

EDITED BY : Wuqiang Zhu, Wenguo Cui, Aijun Wang and Chao Zhao PUBLISHED IN : Frontiers in Bioengineering and Biotechnology

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ISSN 1664-8714 ISBN 978-2-88966-277-7 DOI 10.3389/978-2-88966-277-7

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# NANOTECHNOLOGY IN CARDIOVASCULAR REGENERATIVE MEDICINE

Topic Editors: Wuqiang Zhu, Mayo Clinic Arizona, United States Wenguo Cui, Shanghai Jiao Tong University, China Aijun Wang, University of California, Davis, United States Chao Zhao, University of Alabama, United States

Citation: Zhu, W., Cui, W., Wang, A., Zhao, C., eds. (2020). Nanotechnology in Cardiovascular Regenerative Medicine. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-277-7

# Table of Contents


Yudi Deng, Xudong Zhang, Haibin Shen, Qiangnan He, Zijian Wu, Wenzhen Liao and Miaomiao Yuan


Sijia Yi, Nicholas B. Karabin, Jennifer Zhu, Sharan Bobbala, Huijue Lyu, Sophia Li, Yugang Liu, Molly Frey, Michael Vincent and Evan A. Scott

*90 Nanoscale Technologies in Highly Sensitive Diagnosis of Cardiovascular Diseases*

Chaohong Shi, Haotian Xie, Yifan Ma, Zhaogang Yang and Jingjing Zhang

*108 Nanoparticle-Mediated Drug Delivery for Treatment of Ischemic Heart Disease*

Chengming Fan, Jyotsna Joshi, Fan Li, Bing Xu, Mahmood Khan, Jinfu Yang and Wuqiang Zhu

*121 Nitric Oxide-Producing Cardiovascular Stent Coatings for Prevention of Thrombosis and Restenosis*

Jingdong Rao, Ho Pan Bei, Yuhe Yang, Yu Liu, Haodong Lin and Xin Zhao

*131 Extracellular Matrix Mimicking Nanofibrous Scaffolds Modified With Mesenchymal Stem Cell-Derived Extracellular Vesicles for Improved Vascularization*

Dake Hao, Hila Shimshi Swindell, Lalithasri Ramasubramanian, Ruiwu Liu, Kit S. Lam, Diana L. Farmer and Aijun Wang

*144 An Aligned Patterned Biomimetic Elastic Membrane Has a Potential as Vascular Tissue Engineering Material*

Juanjuan Tan, Jing Bai and Zhiqiang Yan

#### *153 Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease*

Caroline Hu, Tatiana S. Zaitseva, Cynthia Alcazar, Peter Tabada, Steve Sawamura, Guang Yang, Mimi R. Borrelli, Derrick C. Wan, Dung H. Nguyen, Michael V. Paukshto and Ngan F. Huang

*163 Developing an Injectable Nanofibrous Extracellular Matrix Hydrogel With an Integrin* a*v*b*3 Ligand to Improve Endothelial Cell Survival, Engraftment and Vascularization*

Dake Hao, Ruiwu Liu, Kewa Gao, Chuanchao He, Siqi He, Cunyi Zhao, Gang Sun, Diana L. Farmer, Alyssa Panitch, Kit S. Lam and Aijun Wang

*175 Scalable Biomimetic Coaxial Aligned Nanofiber Cardiac Patch: A Potential Model for "Clinical Trials in a Dish"*

Naresh Kumar, Divya Sridharan, Arunkumar Palaniappan, Julie A. Dougherty, Andras Czirok, Dona Greta Isai, Muhamad Mergaye, Mark G. Angelos, Heather M. Powell and Mahmood Khan

# Editorial: Nanotechnology in Cardiovascular Regenerative Medicine

Wenguo Cui <sup>1</sup> \*, Aijun Wang<sup>2</sup> \*, Chao Zhao<sup>3</sup> \* and Wuqiang Zhu<sup>4</sup> \*

*<sup>1</sup> Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, <sup>2</sup> Department of Surgery and Biomedical Engineering, University of California, Davis, Davis, CA, United States, <sup>3</sup> Department of Chemical and Biological Engineering, University of Alabama at Tuscaloosa, Tuscaloosa, AL, United States, <sup>4</sup> Department of Cardiovascular Diseases, Physiology and Biomedical Engineering, Center of Regenerative Medicine, Mayo Clinic, Scottsdale, AZ, United States*

Keywords: nanotechnology, cardiovascular, regenerative medicine, nanoparticle, tissue engineering, heart failure

**Editorial on the Research Topic**

#### **Nanotechnology in Cardiovascular Regenerative Medicine**

### Edited and reviewed by:

*Gianni Ciofani, Italian Institute of Technology (IIT), Italy*

#### \*Correspondence:

*Wenguo Cui wgcui@sjtu.edu.cn Aijun Wang aawang@ucdavis.edu Chao Zhao czhao15@eng.ua.edu Wuqiang Zhu Zhu.Wuqiang@mayo.edu*

#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

Received: *21 September 2020* Accepted: *28 September 2020* Published: *23 October 2020*

#### Citation:

*Cui W, Wang A, Zhao C and Zhu W (2020) Editorial: Nanotechnology in Cardiovascular Regenerative Medicine. Front. Bioeng. Biotechnol. 8:608844. doi: 10.3389/fbioe.2020.608844* Due to their unique properties, nanomaterials can provide novel opportunities in cardiovascular tissue engineering. Thus far, nanomaterial and nanotechnologies have been extensively tested in various fields of cardiovascular tissue engineering, including drug delivery, engineered cardiac muscle, and vascular grafts. In this special issue, we have focused on various aspects of nanotechnologies (and nanomaterials) relevant to cardiovascular regenerative medicine.

#### NANOTECHNOLOGIES IN VASCULAR AND MICROVASCULAR REGENERATION AND REPAIR

Many types of vascular diseases lead to stenosis and reduced blood perfusion. The mainstay treatments include thrombolytic medications and surgical procedures to re-establish the blood flow, such as angioplasty and bypass surgery with autologous or artificial vessel grafts. Nanotechnologies possess unique value in treating vascular disease. Novel nanomaterials may deliver drugs to lesion sites after intravascular administration. In this special issue, several studies demonstrated that novel nanotechnologies promotes angiogenesis from vascular cells and accelerate wound healing.

The stromal vascular fraction (SVF) is a heterogeneous population of cells that are derived from subcutaneous fat. Hu et al. reported that aligned nanofibrillar scaffolds promote cell attachment and enhance the secretion of VEGF from attached SVF cells. Transplantation of SVF-seeded nanomaterials improves blood perfusion recovery in a mouse model of peripheral arterial disease. Endothelial cells are the major component of the vascular structure. Activation of integrin signaling via hydrogel scaffold-mediated delivery of integrin ligands promotes endothelial cell (EC) proliferation and angiogenesis. First, Hao, Liu et al. reported that conjugation of LXW7, an integrin αvβ3 ligand, to the collagen backbone increases EC specific integrin binding sites on the collagen hydrogel. LXW7-treated collagen surface significantly improves cell attachment, proliferation, and survival of ECs under hypoxia conditions. In a mouse subcutaneous implantation model, LXW7-modified collagen hydrogel improves the engraftment of transplanted ECs and enhances vascular formation. Extracellular vesicles (EVs) modified biomaterials represent a new functional biomaterial and hold promise for tissue engineering and regenerative medicine applications. Hao, Swindell et al. further demonstrated that EVs from human placenta-derived mesenchymal

**5**

stem cells (PMSCs) expressed integrin α4β1. Immobilizing the PMSC-EVs onto the electrospun extracellular matrix-mimicking scaffolds promotes EC migration and vascular sprouting in an ex vivo rat aortic ring assay. Zhou et al.reported that a copper sulfide nanoparticles-incorporated hyaluronic acid (CuS/HA) injectable hydrogel promotes wound healing in the rat skin wound model via promoting the expression of vascular endothelial growth factor (VEGF) and enhancing angiogenesis. This novel biomaterial holds promising potential for treating skin wounds.

Engineered vessel grafts represent a promising alternative to autografts and are frequently employed in the surgery clinics. Poly (styrene-block-butadieneblock-styrene) (SBS) is a kind of widely used thermoplastic elastomer with good mechanical properties and biocompatibility. Tan et al. demonstrated that synthesized anthracene-grafted SBS (SBS-An) promotes adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) on a biomimetic elastic membrane with a switchable Janus structure. This approach may have great implication in vascular tissue engineering.

#### NANOTECHNOLOGIES IN MYOCARDIAL REGENERATION AND REPAIR

Engineered heart tissue emerges as a promising approach for repairing the injured heart. However, the fabrication of a cardiac tissue with optimal biomechanical properties and high biocompatibility remains a challenge. Kumar et al. fabricated an aligned PCL-Gelatin coaxial nanofiber patch using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and electrospinning. Nanofibers improve the maturation of hiPSC-CMs and their response to cardioactive drugs, and promote the formation of functional syncytium of these cardiac tissues. These biomimetic cardiac patches are potential models for "clinical trials in a dish" and for heart repair.

Nanomaterials carrying drugs are promising in enhancing the cardioprotective potential of drugs in patients with cardiovascular diseases. Deng et al. provided a comprehensive review of different types of nanomaterial-drug delivery systems and their applications in cardiovascular imaging and the treatment of various cardiovascular diseases. Nanomaterials have been shown to provide sustained exposure precisely to the ischemic myocardium. Fan et al. systemically reviewed the recent advances and challenges of different types of nanoparticles loaded with agents for the treatment of ischemic heart disease. Liao et al. summarized the composition, advantages, and disadvantages of different injectable hydrogels in the diagnosis and treatment of cardiovascular diseases. Angioplasty with an intra-vessel stent is an effective method for treating coronary heart disease. However, thrombosis and restenosis are the two major issues that often lead to device failure. Rao et al. provided a comprehensive review of the nanotechnologies on generating NO-leasing stents and the NO-producing strategies in anti-thrombosis and restenosis treatments.

Molecular imaging (MOI) or biomarkers has been commonly used in the diagnosis of cardiovascular diseases. However, sensitivity, specificity, and accuracy of the assay are still challenging for the early stage of cardiovascular diseases. Shi et al. nicely summarized the cardiac biomarkers and imaging techniques that are currently used for CVD diagnosis, and discussed the applications of various nanotechnologies on improving the value of cardiac immunoassays and molecular imaging in the detecting early stage of cardiovascular diseases.

### NANOTECHNOLOGIES IN THE TREATMENT OF CARDIOVASCULAR INFLAMMATION

Inflammation contributes to the pathogenesis of vessel diseases such as arteriosclerosis and restenosis. It is not clear whether the morphology of vascular ECs has any impact on the activation of monocytes and other inflammatory cells. Liang et al. showed that elongated ECs cultured on poly-(dimethyl siloxane) membrane surface with microgrooves significantly suppressed the activation of the monocytes in co-culture, in comparison to the ECs with a cobblestone shape. Further investigation demonstrated that EC morphology can regulate the response of inflammatory cells through the secretion of specific miRNAs. These data provides a basis for the design and the optimization of biomaterials for vascular tissue engineering.

The objective of cardiovascular immunotherapy is to develop approaches that suppress excessive inflammatory responses. Yi et al. engineered an injectable filamentous hydrogel depot (FM-depot) for low dosage, sustained delivery of anti-inflammatory nanocarriers. Specifically, the bioactive form of vitamin D (aVD; 1, 25-Dihydroxyvitamin D3), which inhibits pro-inflammatory transcription factor NF-kB via the intracellular nuclear hormone receptor vitamin D receptor (VDR), was stably loaded into poly(ethylene glycol)-blockpoly(propylene sulfide) (PEG-b-PPS) filomicelles. Following crosslinking with multi-arm PEG for in situ gelation, aVDloaded FM-depots maintained high levels of Foxp3+ Tregs in both lymphoid organs and atherosclerotic lesions for weeks following a single subcutaneous injection into ApoE−/<sup>−</sup> mice. These data suggested that nanomaterial-based slow release of anti-inflammatory chemicals may be effective to enhance cardiovascular immunotherapy.

Deferoxamine (DFO) has long been used as an FDA-approved iron chelator. Conjugation of DFO with polymers improves their plasma half-life and reduces toxicity when administered intravenously. However, the blood interactions and antioxidation of the DFO-conjugates and the mechanisms underlying these outcomes remain to be elucidated. Sun et al. reported that alginate-DFO conjugates (ADs) inhibit the intrinsic pathways in the process of coagulation, and activate the complements C3a and C5a Ds in a dose-dependent manner through an alternative pathway. These data implicated that Ads induce the cross-talking among coagulation, complement and platelet.

The editors are grateful to the authors who contributed their original research articles and invited reviews to this Special Issue. These articles provided an excellent outline of the current status of nanotechnologies in cardiovascular regenerative medicine.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

We thank Dr. Jyotsna Joshi for comments on the Editorial.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Cui, Wang, Zhao and Zhu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Application of the Nano-Drug Delivery System in Treatment of Cardiovascular Diseases

Yudi Deng1,2†, Xudong Zhang2†, Haibin Shen<sup>2</sup> , Qiangnan He<sup>2</sup> , Zijian Wu<sup>2</sup> , Wenzhen Liao<sup>2</sup> \* and Miaomiao Yuan<sup>1</sup> \*

*<sup>1</sup> The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, China, <sup>2</sup> Guangdong Provincial Key Laboratory of Tropical Disease Research, Department of Nutrition and Food Hygiene, School of Public Health, Southern Medical University, Guangzhou, China*

Cardiovascular diseases (CVDs) have become a serious threat to human life and health. Though many drugs acting via different mechanism of action are available in the market as conventional formulations for the treatment of CVDs, they are still far from satisfactory due to poor water solubility, low biological efficacy, non-targeting, and drug resistance. Nano-drug delivery systems (NDDSs) provide a new drug delivery method for the treatment of CVDs with the development of nanotechnology, demonstrating great advantages in solving the above problems. Nevertheless, there are some problems about NDDSs need to be addressed, such as cytotoxicity. In this review, the types and targeting strategies of NDDSs were summarized, and the new research progress in the diagnosis and therapy of CVDs in recent years was reviewed. Future prospective for nano-carriers in drug delivery for CVDs includes gene therapy, in order to provide more ideas for the improvement of cardiovascular drugs. In addition, its safety was also discussed in the review.

Keywords: nano-drug delivery system, cardiovascular disease, targeting strategy, application progress, safety

### INTRODUCTION

Cardiovascular diseases (CVDs) have become a serious worldwide public health problem, and the morbidity and mortality rank first above other diseases in the world (Gaurav et al., 2015). Faced with such a severe situation, developing drugs for the treatment of CVDs has become a top priority. Owing to the rapid development of nanoscience and outstanding performance of nanomaterials, nanotechnology has become a new solution to overcome the bottleneck of cardiovascular disease treatment. Nano-drug delivery systems (NDDSs) are a class of nanomaterials that have abilities to increase the stability and water solubility of drugs, prolong the cycle time, increase the uptake rate of target cells or tissues, and reduce enzyme degradation, thereby improve the safety and effectiveness of drugs (Quan et al., 2015; Gupta et al., 2019). NDDSs can be administered by various routes including inhalation, oral administration, or intravenous injection, remaining better bioavailability. In recent years, more scholars have started to develop nano-drug carrier system for the diagnosis and therapy of CVDs.

Additionally, as the application of nanomaterials increases, the exposure hazard of nanomaterials in clinical application also raises, resulting in the consequence that nanomaterials will have more opportunities to interact with blood vessels, blood, and their components, which will have an important impact on the human health. Therefore, this article mainly introduced the different types of NDDSs, their targeting strategies and application in CVDs, and the safety of nanomaterials was discussed as well.

#### Edited by:

*Wenguo Cui, School of Medicine, Shanghai Jiao Tong University, China*

#### Reviewed by:

*Jian Zhong, Shanghai Ocean University, China Zhilu Yang, Southwest Jiaotong University, China*

#### \*Correspondence:

*Wenzhen Liao wenzhenliao@163.com Miaomiao Yuan yuanmm2019@163.com*

*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

Received: *21 November 2019* Accepted: *31 December 2019* Published: *31 January 2020*

#### Citation:

*Deng Y, Zhang X, Shen H, He Q, Wu Z, Liao W and Yuan M (2020) Application of the Nano-Drug Delivery System in Treatment of Cardiovascular Diseases. Front. Bioeng. Biotechnol. 7:489. doi: 10.3389/fbioe.2019.00489*

**8**

## TYPES OF THE NDDSs

NDDSs refer to material in which at least one dimension is in the range of nanometer scale (1–100 nm) or composed of them as basic units in three-dimensional space (Cooke and Atkins, 2016; Zhou et al., 2018). As an effective means to optimize the drug delivery, NDDSs have become a research hotspot in the field of pharmacy and modern biomedicine (Matoba et al., 2017). The investigation of NDDSs has been for more than 40 years, creating a mass of nano-drug carriers. According to the composition of the materials, the nanomaterials used in NDDSs can be divided into organic, inorganic and composite materials. The following is a description of several common NDDSs and their features (**Figure 1**, **Table 1**).

#### Liposomes

In general, liposomes are lipid vesicles formed by ordered phospholipid bilayer with cell-like structure (Landesman-Milo et al., 2013). As a type of drug carrier, liposomes show many advantages, such as non-toxic, non-immunogenicity, sustained-release drugs, prolonging drug action time, changing drug distribution in vivo, improving drug treatment index, reducing drug side effects, and so on (Yingchoncharoen et al., 2016). Liposomes can not only be easily developed for the entrapment of hydrophilic and ionic molecules, but compatible with hydrophobic drug (Chandrasekaran and King, 2014). Hydrophobic drugs can be surrounded by the bimolecular structure of phospholipids, and hydrophilic drugs, especially those containing genes, can be attached to the hydrophilic region of liposomes. The particle size, potential, and surface chemistry can be adjusted by modification of different lipid materials. Among various type of liposomes, cationic liposomes are positively charged, indicating that they may result in dosedependent cytotoxicity and inflammatory responses, and as a kind of complexes, they may interact non-specifically with negatively charged serum proteins. Neutral lipids (Chapoy-Villanueva et al., 2015) and pH sensitive liposomes (Fan et al., 2017) are two ways to solve the above problems.

#### Polymer Micellar Co-delivery System

Polymer nanoparticles, another carriers for the delivery of drug, can be classified into non-biodegradable materials and biodegradable materials (Shi et al., 2019a,b). The synthetic polymer materials mainly include poly(lactic-co-glycolic acid) (PLGA), polyvinyl imine (PEI), polycaprolactone (PCL), polyvinyl alcohol (PVA), and so on (Danhier et al., 2012; Wei et al., 2018). These polymers exhibit biocompatibility, non-toxicity and no teratogenicity. Its degradation products, including oligomerization and final products, have no toxic effect on cells, and can coexist stably with most drugs. Natural polymers are mainly categorized into polysaccharides, peptides (Li et al., 2012), Chol and cyclodextrin inclusion complexes. Polymer nanoparticles usually formed by self-assembly of Amphiphilic block copolymers, are stable in the core and can be used to intercept insoluble drugs (Afsharzadeh et al., 2018). The stable structure of polymer nanoparticles is beneficial to the uniformity of particle size and the controlled release of drugs (Wang et al., 2011), and can effectively overcome the influence of gastrointestinal environment during oral administration. Their nanoscale and large surface area are conducive to uptake of drugs in cells and better bioavailability. Unfortunately, some polymer nanoparticles, have some drawbacks. For example, Chitosan, a natural polymer, is incompatible with biologic fluids, which can lead to particle degradation and reduce the working efficiency. Structural changes can be taken to solve its deficiency. Combining chitosan with polyethylene glycol, the conjugate has a unique endocytosis and macrophage phagocytosis mechanism (Yang et al., 2017). In addition, the modification of chitosan with a polypeptide can improve its working efficiency (Ping et al., 2017).

#### Dendritic Macromolecules

Macromolecules are synthetic, various-shaped and usually branched. Macromolecules shaped as sphere can be arranged in monodisperse space and mostly used as nano-carriers to be used for the administration and dissolution of insoluble targeted drugs. Dendritic macromolecules with unique branch structure, are also monodispersion and their molecular weight can be controlled. Besides, a large number of ready-made surface functional groups and hydrophobic environment are exist in the packaging, which make them an excellent drug delivery material (Kesharwani et al., 2012). Because of their excellent biological properties, dendritic macromolecules are widely used in biomedical and pharmaceutical fields, but the existence of surface cationic charge also limits their clinical application.

#### Metal Nanomaterials

The most commonly used metal nanomaterials are gold and silver nanomaterials, shaped in different structures that can be divided into/like nanoparticles, nanorods, nanocapsules, nanocuboid, and nanowire (Baeza et al., 2017). In addition to being used as nano-contrast agent for CT and surfaceenhanced Raman spectroscopy, gold nanomaterials are also used in photothermal treatment of tumors and rheumatoid arthritis. As many studies shown, the application fields of silver nanomaterials mainly involved antibacterial, anti-infection and anti-tumor (Pietro et al., 2016). Moreover, some therapeutic drugs can be physically loaded into hollow gold or silver nanostructures (Liang et al., 2014), or chemically bonded to the surface of nanoparticles to achieve targeted delivery of the drugs. However, the removal of gold nanomaterials in human body is too slow, and the toxicity of silver ions in vivo limits the application of these metal nanomaterials in the treatment of chronic diseases.

#### Inorganic Non-metallic Nanomaterials

Inorganic non-metallic nanomaterials mainly include quantum dots, iron oxide, silicon, grapheme, and so on (Khafaji et al., 2019). Quantum dots (QDs), that is, semiconductor nanocrystals, are particularly focused on fluorescence imaging because of their unique luminous properties, while iron oxide nanoparticles are chiefly lay on the study of new MRI contrast agents (Jayagopal et al., 2009; Hauser et al., 2016; Su et al., 2017; Wei H. et al., 2017). Among them, mesoporous silicon nanomaterials have

TABLE 1 | Category and features of nano-drug carriers.


attracted more and more attention in the therapy of diseases in recent years due to its large surface area and porous structure (Wang W. et al., 2016). Those Inorganic nanomaterials can be used to improve the transport efficiency of drugs and genes in mammal cells through the integration of different functional groups. Meanwhile, they are suggested to be a kind of joint carrier with development potential. However, the bio-safety of inorganic non-metallic nanomaterials would be a considerable obstacle to their application in clinic (Perioli et al., 2019).

#### Composite Nanomaterials

In addition to the above nanomaterials, the preparation of composite nanomaterials with different properties is also under exploration in many studies. For example, metal or inorganic non-metallic nanomaterials are introduced into polymer or lipid nanomaterials to prepare multifunctional NDDSs containing both therapeutic drugs and contrast agents. Metal and inorganic nanomaterials are decorated or modified by organic materials to improve their physical and chemical properties, in vivo kinetic behavior and biocompatibility; and some NDDSs with special structure and diversified functions can be prepared by the combination of different metals and inorganic materials.

### TARGETING STRATEGY OF THE NDDSs

The targeted design of NDDSs focuses on the diagnosis and therapy of cancer in the early stages of development, but recent researches argued that lesion cells or tissues of CVDs can also be targeted, even easier to targeted than tumor tissues with multiple physiological barriers. Compared with conventional Deng et al. Application of the NDDSs

preparations, the metabolic time of nano-transporter drugs in the blood circulation may be prolonged. By regulating pH value (Gao et al., 2018; Yi et al., 2018), temperature (Wei L. et al., 2017), light (Ding et al., 2011), ultrasound or biological enzyme (Zhang et al., 2019), the rate of those targeted nano-transporter drugs can be controlled to function longer.

## Passive Target Transfer

#### Enhanced Vascular Permeability

Passive targeted transport mainly utilizes high permeability and high retention (EPR) effects (**Figure 2**) (Holback and Yeo, 2011). EPR refers to the fact that some molecules or particles tend to accumulate in tumor tissues (Dinarvand et al., 2011). The microvascular endothelial cell space in normal tissue is dense and intact, and NDDSs loaded with drug, generally in high molecular weight, are not easy to pass through the vascular wall. The tumor tissue is rich in blood vessels and poor in structural integrity (Torchilin, 2011). Those drug-loaded NDDSs in high molecular weight can selectively pass through the vascular wall and remain in the tumor tissue. A large number of studies have shown that nano-drug carriers with particle size <100 nm can be located and targeted to solid tumor tissues by EPR. Compared with the direct administration method, the nano-drug carrier can increase the accumulation of the drug in the tumor tissue by more than 10 times, greatly improving the bioavailability (Maeda et al., 2013). But it is discovered that EPR effect can also be used in various CVDs, not only for tumors. In some course of CVDs, for example, the occurrence and development of AS is a chronic inflammatory process, where vascular permeability is often increased, which is very similar to that of solid tumors. Vascular endothelial permeability provides an effective means for NDDSs to deliver from the lumen side to the interior of the plaque. The nano-drug carriers entering the circulation are also ingested by inflammatory cells (monocytes or macrophages), and these drug-carrying cells migrate to plaque inflammation, allowing drugs to be delivered in another way (Flogel et al., 2008).

Due to the size and surface characteristics of a portion of nanomaterials, they are rapidly cleared in the blood during intravenous injection, making nanomaterials unsuitable for drugs that require long cycle times. In this case, nano-coating technology can be applied to the nano-system for certain concealment, and the rate of administration of the coating agent can be also controlled and adjusted. This technology is particularly suitable for NDDSs in the treatment of CVDs. Developers on NDDSs have employed poly (ethylene glycol) (PEG) in particle design. In fact, PEG is a flexible hydrophilic polymer that can form a hydrated layer when grafted onto the surface, effectively reducing the adsorption of proteins on the surface (Jokerst et al., 2011). The tissue plasminogen activator is encapsulated in the nanoparticles, making the nanosystem concealed in some degree, thus protecting the tissue plasminogen activator from inactivation by plasma inhibitors and prolonging the half-life (Hemmati and Ghaemy, 2016).

#### Shear-Induced Targeting

Studies have shown that as the intima grew outward (toward the lumen) in CVDs, such as advanced AS or myocardial infarction, thrombosis or microthrombus occurs, stenosis of the blood vessels follows and blood flow rate through the plaque increases, and thus the fluid shear force increases. The mean blood fluid shear force in the normal vasculature is <70 dyne·cm−<sup>2</sup> , while the blood fluid shear force in the AS plaque stenosis is up to 1,000 dyne·cm−<sup>2</sup> (Korin et al., 2012). Therefore, the design of blood fluid shear-sensitive nanoparticles can achieve physicochemical targeting by utilizing the difference of blood fluid shear force between AS plaque and normal blood vessels. Holme et al. (2012) prepared a lenticular lipid nanoparticle vesicle with two sides convex. The drug-loaded nanometer can maintain structural stability in normal blood vessels, and the configuration change can be utilized to release the drug under the action of high blood fluid shear force through the blood circulation to the AS plaque. Inspired by the activation of platelets under the action of local high blood fluid shear forces in AS plaques and adhesion to plaque blood vessels, the researchers constructed a nanoparticle aggregate that can be assembled locally in plaques (Korin et al., 2012). First, the authors prepared PLGA nanoparticles with a particle size of about 180 nm and entrapped tissue plasminogen activator, and then obtained a PLGA nanoparticle aggregate with a particle size of 3.8 nm through spray drying. When the nanoparticles were exposed to the local high fluid shear stress of the AS plaque, they could be decomposed into 180 nm PLGA nanoparticles, and relied on the strong penetrability of the small particle size nanoparticles to enter the local thrombus of the plaque. The thrombolytic effect maximized the efficacy, significantly reduced the dose required for thrombolysis and the side effects of thrombolysis. In ischemic cardiomyopathy, the endothelial gap in ischemic myocardium widened, thus altering the shear of blood flow, and the concentration of polysaccharide from Ophiopogon japonicus in ischemic myocardium was twice as high as that in normal rats (Lin et al., 2010). Tan et al. found that both shear stress and blood flow shear rate of vascular wall could affect the aggregation of nanoparticles (Tan et al., 2011).

#### Magnetically Guided

Magnetically guided nanoparticle is an interesting "pseudopassive" targeting method. Theoretically, the application of an external magnetic field can direct magnetic nanoparticles to the disease site (Prijic and Sersa, 2011). Recent evidence suggests that this strategy is beneficial for CVDs (Chandramouli et al., 2015). Alam et al. (2015) compared the effects of several nano drug carriers on atherosclerotic plaque imaging. Those Nanoparticles include iron oxide particles, superparamagnetic iron oxide nanoparticles, ultra-small superparamagnetic iron oxide nano-carrier, and very small superparamagnetic iron oxide nanoparticles. The results showed that the ultra-small superparamagnetic iron oxide nanoparticles have better vascular wall penetration ability and plaque retention than other groups. Some researchers have pointed out that the external magnetic field helps to transport particles from the cell-free layer which lacks red blood cells to the vessel wall (Freund and Shapiro, 2012).

#### Active Targeted Transhipment

On the basis of passive targeting, using the special pathological features of CVDs to develop an active targeting strategy for

CVDs can improve the targeted delivery efficiency of drugs to the lesions of CVDs, which aroused researchers strong interest. Active targeting is primarily directed to functional modification of NDDSs with one or more targets to allow the drug to reach a particular site (**Figure 3**) (Matoba and Egashira, 2014). That is to say, introducing a functional group or active substance that specifically interacts with diseased tissues or cells into the surface of the nano-drug carrier will enhance carriers targeting (Lee et al., 2006; Gullotti and Yeo, 2009). Some active targets are discussed in detail below (**Table 2**).

#### Active Targeting of Vascular Endothelial Cells

At different stages of CVDs, vascular endothelial cells are in an inflammatory activation state. Compared with normal vascular endothelial cells, some small molecules including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), integrins, selectins, and so on, are often overexpressed, which provides the active target for NDDSs (Glass and Witztum, 2001). It is showed that the conjugation of lungtargeted single-stranded variable fragment/liposome together with platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody increases liposome transport to the pulmonary vascular system and strengthen its anti-inflammatory effects (Hood et al., 2018). In 2013, Yang et al. decorated the surface of silica nanoparticles with anti-VCAM-1 monoclonal antibody. The nanoparticles were able to bind to sites of inflammation before they were taken up by endothelial cells (Yang et al., 2013).

Based on the pathological features of high expression of ICAM-1 in early vascular endothelial cells of AS, Paulis et al. (2012) modified the antibody anti-ICAM-1 which actively targets ICAM-1 on the surface of liposomes and used it to load contrast agents (gadolinium). Studies have shown that the liposome could achieve the activated targeting of vascular endothelial cells and AS plaques through the specific action of anti-ICAM-1 and ICAM-1. However, competitive binding of circulating white blood cells to the ICAM-1 site and blood flow shearing could reduce the targeting function of liposomes to AS plaques. The authors optimized the binding degree of liposome to ICAM-1 by screening liposome particle size, antibody and lipid ratio, and obtained higher active targeting efficiency.

E-selectin is a surface glycoprotein of endothelial cells, which can promote the attachment of monocytes/macrophages and lymphocytes to induce inflammatory response, and eventually cause the occurrence and development of CVDs, such as AS (Ma et al., 2016). E-selectin can also be used as a target for nanotransport drugs. Functional liposomes carrying mouse H18/7 mAb (specific antibody to E-selectin) were used to act on interleukin (IL)-1β-activated human umbilical vein endothelial cells and non-interleukin (IL)-1β-activated human umbilical cord Vein endothelial cells. It was found that the ability of functional liposomes to target activated human umbilical vein endothelial cells is 275 times that of the non-activated type (Flaht-Zabost et al., 2014).

AT1 rises in myocardial when myocardial infarction or heart failure happened. Dvir et al. (2011) designed a polyethylene glycol liposomes (142 ± 8 nm), that could carry therapeutic payloads (such as growth factors, cytokines, etc.) and released them in a controlled manner. The ligand attached on these liposomes is a string of amino chain sequenced Gly-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (binding sequence of AT1 receptor), which could direct the nanoparticles to the infarction heart.

#### Active Targeting of Macrophages or Foam Cells

Macrophages or foam cells play a key role in the development of AS. In the early stage of AS, mononuclear/macrophages were recruited to activate vascular endothelial cells, and

overexpressed some inflammation-related receptor molecules in an inflammatory environment, such as CD44 and interleukin-4 (IL-4) receptors, etc. Imaging and drug delivery for macrophages or foam cells using NDDSs will facilitate monitoring of disease progression and drug treatment in AS.

For example, Lee et al. (2015) linked 5β-cholic acid and fluorescent dye Cy5.5 to the carboxyl group of the HA skeleton by chemical bonding and formed nanoparticles (HA-NPs) by selfassembly. Compared with nanoparticles (HGC-NPs) constructed with chitosan backbones that did not target CD44 receptors, HA-NP could significantly increase the uptake of activated macrophages, and the plaque site of ApoE−/<sup>−</sup> mouse (AS model) was more targeted. Fluorescence co-localization studies indicated HA -NP was mainly distributed in macrophages in plaques.

Park et al. (2008) used phage library screening technology to optimize the amphiphilicity of the target IL-4 receptor peptide (CRKRLDRNC) which was modified on amphiphilic chitosan (with ethylene glycol chitosan as the backbone and 5β-cholate bonded) by chemical bond. Then nanoparticles with the function of targeted macrophages in AS plaque are obtained in a selfassembled method.

#### Targeting Vascular Basement Membrane Collagen

It has been reported that the vascular basement membrane of damaged blood vessels and inflammation sites is rich in collagen IV (Col IV) (Duner et al., 2015). In 2013, Kamaly et al. (2013) ligated the 7 amino acid oligopeptide molecule KLWVLPK (PLEA-β-PEG-Col IV) targeting collagen IV at the PEG end of the PLGA-β-PEG block copolymer and used it to package Act-26 (With anti-inflammatory and inhibition of leukocyte extravasation), thus nanoparticles (Ac2-26 Col IV NPs) targeting damaged blood vessels and collagen sites of inflammation sites were prepared. The results showed that Act-26 Col IV NPs reduced the migration and adhesion of neutrophils to the inflammation site and inhibited the development of inflammation. Further, in 2016, some researchers prepared nanoparticles (Col-IV IL-10 NPs) containing anti-inflammatory factor IL-10 by self-assembly using PLGA-p-PEG-Col IV and



PDLA-PEG-OMe targeting collagen LV (Kamaly et al., 2016). After intravenous administration of Ldlr−/<sup>−</sup> mice, it was found that Col-IV IL-10 NP significantly increased the content of IL-10 in the plaque, and had better AS treatment effect than free IL-10.

In addition, multi-target nano-carriers with multiple inflammatory cell characteristics have been studied. PLNs incorporated these often ignored biophysical design criteria of platelet-mimetic discoid morphology and flexibility, then integrated these design parameters with the platelet-mimetic biochemical heteromultivalent interactive functions by dendritic presentation of multiple peptides that bind simultaneously to both activated natural platelets and injured endothelial sites (Anselmo et al., 2014).

Whether it is passive targeting or active targeting, the final targeting efficiency depends on the biological and physical properties of nanoparticles. The biological and physical properties includes particle size and distribution, targeting unit types, surface chemistry, morphology and density (Morachis et al., 2012). For the body, the development stage, type as well as location of CVDs and tumor, vascular wall shear rate, blood composition and its fluid type, together with other factors will greatly affect the targeting efficiency (Charoenphol et al., 2011). Although the application of active targeting NDDSs in clinical diagnosis and therapy is extremely attractive, its development is still facing great challenges. Those challenges are mainly reflected in two aspects: one is the limitation of the discovery of ideal target; the other is that there are still many bottleneck problems in the design and preparation of effective targeting nanosystem.

#### Multifunctional Responsiveness NDDSs

Multifunctional responsive NDDSs is a kind of drug carrier with better targeting ability, which is developed on the basis of the above two targeting modes of nano-drug carrier. In addition to having the previous targeting ability, this kind of carrier is generally composed of stimulating responsive materials, which can be released under the stimulation of the special environment of the focus site, thus reducing the release in the normal tissue and increasing the drug accumulation of the lesion tissue. At the same time, diagnostic molecules can be assembled or labeled on nano-carriers to compose an integrated diagnosis and therapy system.

### APPLICATION OF THE NDDSs IN THE DIAGNOSIS OF CVDs

Early, rapid and accurate detection is important for effective prevention and treatment of CVDs. The application of molecular imaging in the diagnosis of CVDs has been paid more and more attention in recent years. In addition to the constant innovation of various imaging technologies, new contrast agents are the key to real-time, fast, high sensitivity and high resolution diagnostics. Compared with conventional contrast agents, nanocontrast agents have the following advantages: (1) in vivo stabilization, regulable distribution, and prolonging the half-life of contrast agents or drugs; (2) controllable physical and chemical properties (such as chemical composition, size) and imaging performance; (3) specific identification of certain biomolecules; (4) ability of multimodal imaging realization; (5) values in individualized diagnosis and therapy are expected to be realized (Attia et al., 2016). By designing specific nano-probes with the unique chemical signal molecules of diseased tissues determined by pathological studies, the contrast agent can be directed to the lesion area in the early stage of the disease for magnetic resonance imaging (MRI), X-ray imaging, fluorescence imaging, and contrast-enhanced ultrasound (US) imaging (**Figure 4**).

#### Magnetic Resonance Imaging

In many imaging methods, magnetic resonance imaging is noninvasive, safe, and high resolution, and it is good for soft tissue imaging. However, the sensitivity of MRI is not high (10−<sup>3</sup> -10−<sup>9</sup> M). The complexes of gadolinium commonly used in clinical

practice are used as T1-weighted imaging contrast agents, and gadolinium has certain nephrotoxicity. Fe3O<sup>4</sup> nanoparticles are considered to be non-toxic T2-weighted imaging contrast agents (Corot et al., 2006). Compared with tinctures, they have high sensitivity, good tissue compatibility and superparamagnetism (Kim et al., 2007). Targeted contrast agents are used to accumulate MRI probes at a sufficiently high concentration (in micrograms to milligrams) in the target tissue to achieve a high signal to noise ratio.

on the targeting ligand or associated with the nanoparticle shell.

It is discovered that vascular imaging can be performed in the early stage of cardiovascular disease formation, and drugs can be administered for treatment after the magnetic nanoparticles are injected into the body. Yoo et al. (2016) loaded the hydrophilic lipid (amphiphilic) gadolinium chelating agent diethylenetriamine pentaacetic acid (DTPA) into a dendritic polymer and then wrapped it in the kernel of amphiphilic micelles and connected with fibrin binding agent. Thus, its targeting to atherosclerotic plaque was enhance, and can be used for early detection of thrombus. Winter et al. chose paramagnetic nanoparticles targeting integrin αvβ3 to inject intravenously into high fat fed New Zealand white rabbits to detect neovascularization in plaques in the early stage of AS (Winter et al., 2008).

### X-Ray Imaging

Imaging with radionuclides plays a crucial role in the field of nuclear medicine (Mottu et al., 1999, 2002). Radionuclides are not only sensitive but also quantifiable. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are the most common types (Alie et al., 2015). At present, radionuclide-labeled nanomaterials can be used to monitor the embolization process and the distribution of nanomedicine to achieve targeted imaging (Mottu et al., 2002; Okamura et al., 2002; Torchilin, 2002; James et al., 2006). For example, the researchers used <sup>186</sup>Re-BMEDA (Bao et al., 2003) and 99mTc-PEGylate-labeled (Bao et al., 2004) doxorubicin liposomes to perform SPECT, which can trace the distribution of drugs in the body, and also promote drug release. The nanoparticles can be used to detect the formation of atherosclerotic plaques by CT and to judge the prognosis as well. Galperin et al. injected iodine nanoparticles contrast agent (N1177) into mice via vein. It was found that the contrast agent gathered in macrophage rich tissue, and the signal of atheromatous plaques could be significantly enhanced, and the enhancement time could last for more than 30 min (Galperin et al., 2007). In 2016, Chhour et al. (2016), used 11 mercaptoundecanoic acid (11-MUDA) to encapsulate gold nanoparticles, found that gold nanoparticles could accumulate in foam cells of atherosclerotic plaques and increase the contrast of imaging.

#### Fluorescence Imaging

Optical imaging is a powerful imaging method with the advantages of no radiation, no invasion, high resolution and good controllability, but its penetration is poor. Fluorescence imaging is usually performed by using fluorescein to generate fluorescence signals. Near-infrared fluorescence (NIRF) probes are widely used because of their strong penetrating power and safety. They have been used in small animal living imaging systems and clinical tumor transformation. At present, a large number of nano-drug carriers, such as liposomes, metal, or non-metallic nanoparticles can enclose NIRF to achieve optical imaging of blood vessels (Weissleder and Ntziachristos, 2003; Setua et al., 2010; Sevick-Muraca, 2012). Its application in cardiovascular disease imaging has been paid more and more attention. McCarthy et al. bound the group of near infrared light activated therapeutic (NILAT) with macrophage-targeted magnetic nanoparticles(MNP) and prepared a kind of diagnostic and therapeutic nanoparticles (McCarthy et al., 2006). The experiment results shown that within 24 h of administration, the nanoparticles were reached in area. Wang Y. et al. (2016) injected profilin-1 magnetic iron oxide nanoparticles (PF1- Cy5.5-DMSA-Fe3O4-NPs) focusing on profilin-1 into the vein of atherosclerotic mice. It was found that the magnetic iron oxide nanoparticles were aggregated in carotid atherosclerotic plaques. There was a good correlation between the MRI signal of the animals injected with PC-NPs and the fluorescence intensity of NIRF imaging in vitro.

#### Ultrasound Imaging

Compared with fluorescence imaging, ultrasound imaging has natural advantages in medical imaging including safe, convenient, and real-time. Nano-ultrasound imaging materials that can be targeted to vascular-related markers have been developed. For example, vascular ultrasound nanoparticles that can be targeted to high expression of the vascular endothelial growth factor receptor 2 (VEGFR2) not only provide a more clear ultrasound imaging of tumor blood vessels, but also promote drug localization in blood vessels (Rojas et al., 2018). Marsh et al. had developed perfluorocarbon nanoparticles targeting blood fibrin, carrying the thrombus drug streptokinase for the diagnosis and therapy in thrombus (Marsh et al., 2007). The drug-loaded particles are synthesized by evaporation/dispersion technique with a diameter of about 250 nm and can be used for ultrasonic imaging.

#### Multi-Modal Bioimaging

At present, multi-modal imaging technology using a combination of different types of imaging methods can integrate different imaging methods to produce synergistic effects, providing more comprehensive and accurate image information for accurate diagnosis and precise treatment of CVDs. For instance, it was found that <sup>64</sup>Cu-labeled SPIO-loaded doxorubicin nanoparticles could be used for MRI and PET (Yang et al., 2011). It has been reported that Cy5, sputum, and folic acid can be embedded in gold nanoparticles to achieve trimodal optical imaging, MRI and CT imaging in mice (Chen et al., 2016). This multimodal imaging and integration of diagnosis and treatment will be a new direction for the development of cardiovascular nanomedicine in the future.

#### APPLICATION OF THE NDDSs IN THE TREATMENT OF CVDs

#### The NDDSs in AS

AS is the most common type of CVDs, often leading to a stroke or heart attack. The formation of AS begins with endothelial dysfunction. Plaque-induced coronary artery stenosis can cause ischemic cardiomyopathy, while plaque rupture can cause acute myocardial infarction (Nabel and Braunwald, 2012; Wall, 2013). Mechanisms of plaque instability include enhanced vascular permeability, Platelet endothelial cell adhesion molecules (PECAM) expression, macrophage aggregation, and expression of proteases, which can be targets for intervention. The drug can be delivered to atherosclerotic plaques by nanodrug carrier, to effectively prolong the half-life of drug plasma, increase the concentration of lesions and reduce side effects. The treatment strategies of these nano-drug carriers including regulating lipoprotein level, reducing the degree of inflammation, inhibiting of neovascularization, preventing coagulation, and so on (**Table 3**). These treatment strategies are used as interventions to development of AS, reduce plaque area or stabilize vulnerable plaques (Chetprayoon et al., 2015; Bejarano et al., 2018).

### The NDDSs in Hypertension

At present, many kinds of drugs are applied for the treatment of hypertension, including angiotensin converting enzyme inhibitors, vascular angiotensin antagonists, central sympathetic nerve drugs, adrenergic receptor blockers, diuretics and vasodilators (Sharma et al., 2016). However, all these antihypertensive therapeutic drugs have obvious defects, including short plasma half-life, low bioavailability, toxic and side effects (upper respiratory tract abstraction, angioedema, reflex tachycardia, extreme hypotensive effect, and so on) (Alam et al., 2017; Martin et al., 2017; Niaz et al., 2017). Conversely, nanodrug carriers can provide prominent advantages mentioned above (**Table 4**) (Kimura et al., 2009). Some researchers have made olmesartan into a nanoemulsion system. Compared with the conventional dose, the nanoemulsion group has better blood pressure lowering effect, longer maintenance time, and can produce nearly three times the dose reduction (Alam et al., 2017).

### The NDDSs in Pulmonary Hypertension

Pulmonary hypertension, a progressive highly dangerous disease, is characterized by increased pulmonary vascular resistance and elevated pulmonary artery pressure. Prostaglandin I, Endothelin receptor antagonist, type 5 phosphodiesterase inhibitor, etc. are common vasodilators for pulmonary hypertension. These vasodilators have shown some effects, but the overall therapeutic ability is limited. For solving this problem, nano-mediated drug delivery system has gradually become an important alternative strategy (**Table 5**). Bosentan is a selective and competitive Endothelin receptor antagonist, which is loaded into nanoparticles and has a solubility of seven times as much as that of unprocessed bosentan (Ghasemian et al., 2016).

#### The NDDSs in Myocardial Infarction

Reperfusion is mainly used in the early stage of myocardial infarction, but it can cause apoptosis, calcium overload and reactive oxygen species. These factors cause the opening of the mitochondrial membrane permeability transition pore (MPTP) and the increase of mitochondrial outer membrane permeability, thereby promoting cardiomyocyte apoptosis and necrosis (Hausenloy and Yellon, 2013). Clinically, the drug therapy for myocardial ischemia mainly depends on growth factors, cytokines and some small molecular compounds. These drugs have the same disadvantages of the above traditional drugs. The high permeability of blood vessels and enrichment of monocytes in ischemic myocardium can be harnessed to deliver drugs by targeting ability of nano-drug carriers (**Table 6**).

### The NDDSs in Other CVDs

As a new drug delivery platform, nano-drug delivery system also performs well in other CVDs. Coronary artery allogeneic

#### TABLE 3 | Application of the NDDSs in the AS.



angiopathy is an inflammatory proliferation process that undermines the long-term success of heart transplantation. Lipid nanoparticles coated with methotrexate or paclitaxel were injected intravenously into rabbits which fed cholesterol-rich diet and received an ectopic heart transplant, both of which reduced macrophage infiltration in the graft (Barbieri et al., 2017). Myocardial ischemia is mainly due to the decrease of aortic perfusion in the heart, resulting in insufficient oxygen supply and unstable myocardial energy metabolism, thus forming a pathological state that cannot support the normal work of the heart. Liposomes coated with phenytoin (PHT, a nonselective VGSC inhibitor) were prepared by thin film dispersion. The results showed that PHT-encapsulated liposomes partially inhibited I/R injury-induced CD43<sup>+</sup> inflammatory monocyte expansion and reduced infarct size and left ventricular fibrosis after intravenous injection of the rat myocardial I/R injury model (Zhou et al., 2013).

Vascular restenosis is the process of stenosis and obstruction after the interventional treatment of the blood vessels, such as angioplasty, arteriotomy, implantation of an endovascular stent, and so on (Wang et al., 2018). Some scientists (Banai et al., 2005; Kamath et al., 2006; Nakano et al., 2009; Schröder et al., 2018; Xi et al., 2018) have proposed that in the site of angioplasty, catheterintervention techniques are used to infuse the drug-loaded nanoparticles into the injury site, enabling angioplasty, and topical administration in one step. The nanoparticles can enter the arterial wall through the damaged endothelium, localize, reside in and between cells, and then slowly release the drug (Wu et al., 2019). Therefore, the lesion vessel can be maintained at a relatively high concentration for a long period of time, which is beneficial to fully exerting the drug effect, and finally effectively prevents and treats vascular restenosis.

### APPLICATION OF THE CO-LOADED NANO-SYSTEM IN THE CVDs

Drug combination therapy (including genes) is the treatment of two or more drugs to patients at the same time. In clinical practice, this therapy has been widely used for disease treatment. The purpose of this combination therapy is often due to the synergistic effect between drugs, or the therapeutic effect of multiple drugs is greater than that of a single drug. In recent years, many co-loaded nano-systems have been developed to carry common drugs and/or genes, especially siRNA to treat CVDs.

#### Application of RNAi in the Treatment of CVDs

RNA interference (RNAi) is a gene-specific silencing mechanism present in eukaryotic cells and an important measure for resisting foreign genes and infections during biological evolution. RNAi was first discovered in Caenorhabditis elegans (Braukmann et al., 2017), then in 2001, it was demonstrated to occur in mammalian cells (Lendeckel et al., 2001). RNA interference includes micro RNA (miRNA), small interfering RNA (siRNA),



TABLE 6 | Application of the NDDSs in myocardial infarction.


Piwi-interacting RNA, and long non-coding (lncRNA). RNAi technology, also known as gene silencing, introduces doublestranded RNA (dsRNA) consisting of sense and antisense RNAs corresponding to a certain mRNA sequence into cells, degrading mRNA homologously complementary thereto, and inhibiting the expression of cell-specific genes. The rapid development of RNAi research has driven it from experimental technology to therapeutic development tools (Katyayani et al., 2017), and RNAi has potential value in the treatment of CVDs (Kwekkeboom et al., 2014; Tadin-Strapps et al., 2015; Hoelscher et al., 2017). At the same time, RNA interference therapy also has challenges in the treatment of CVDs, including the toxicity, targeting, time-effect, and effective delivery system of RNA, which limits its widespread use in the clinic and is urgently needed to be solved and improved (Cotten et al., 1992; Sioud, 2015; Kasner et al., 2016; Navickas et al., 2016; Zhou et al., 2016). **Table 7** indicated the future direction of cardiovascular RNA interference.

#### Co-loaded Gene and Drug Nano-System

For overcoming the problems in the delivery process and realizing the broad potential of RNAi-based therapeutics, safe and efficient nano delivery systems are needed. The apolipoprotein B (ApoB) siRNA was encapsulated into the liposome vector. After 48 h, the ApoB mRNA of the macaque liver decreased, and the maximum silencing rate exceeded 90%. ApoB protein, serum cholesterol, and low-density lipoprotein levels began to decrease 24 h after treatment and continued until day 11 (Zimmermann et al., 2006). Some researchers have used chitosan nanoparticles to construct and package small interfering RNA (siRNA) against PDGF-B mRNA expression vector, and then transfected into vascular smooth muscle cells (vSMC) of rabbit arterial wall damaged by balloon catheter, using therapeutic ultrasound for gene delivery. The results showed that the nanoparticles significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs while the local intimal thickness and area were also reduced remarkably (Xia et al., 2013). Nox2-NADPH expression is significantly increased in the infarcted myocardium. Somasuntharam et al. (2013) demonstrated acid-degradable polyketal particles for Nox2 siRNA to the post-MI heart, which not only reduced siRNA degradation, but also inflammation.

Some pharmaceutical companies have developed new nanodosages that deliver siRNA to the right cells at the right time (Hayden, 2014). Healthy volunteers (serum LDL levels of 3 mmol/L or higher) were injected intravenously with ALN-PCS or



placebo developed by Alnylam Pharmaceuticals (Fitzgerald et al., 2014). ALN-PCS is a siRNA that inhibits the synthesis of PCSK9 and is assembled in lipid nanoparticles. PCSK9 protein in the human body cycle was reduced 70%, and LDL was reduced by 40% after intravenous injection of ALN-PCS.

The co-loaded gene and drug nano-system combined with nanotechnology and gene interference technology, the packaged substances have a synergistic effect, and the therapeutic effect is much better than the single treatment (**Figure 5**). Carvedilol, a kind of anti-hypertrophic drug that simultaneously blocks β-adrenergic receptors non-specifically in various organs, is widely used and effective. The non-specific genomewide downregulation of p53 expression by specific siRNA efficiently abrogates cardiac hypertrophy. However, it can cause extensive tumorigenesis affecting bystander organs. Rana et al. (2015) encapsulated these bioactive molecules with stearic acid modified carboxymethyl chitosan (CMC) nanopolymers conjugated to a homing peptide for delivery in vivo to hypertrophied cardiomyocytes, resulted in effective regression of cardiac hypertrophy.

#### SAFETY OF THE NDDSs

As the researches of nanomaterials go further, an increasing number of nanomaterials are prepared as NDDSs, but the unclear toxicity and the lack of systematically study of materials themselves restrict their further application. When the particle size enters the nanometer scale, it will show strong surface effect, small scale effect, quantum scale effect and macroscopic quantum tunneling effect (Gatoo et al., 2014).

Relatively few studies discuss about toxicity of NDDSs, particular in cardiovascular toxicity. But the tissue of cardiovascular system is considered to be the key site of NDDSs induced toxicity, which can produce great impact on the disease

prognosis. Studies have revealed that nanomaterials could enter the blood circulation through respiratory tract, digestive tract, skin and other mucous membranes, and inevitably interacted with the blood system, immune system and other organs or tissues including plasma proteins and immune proteins, blood cells and immune cells, and so on.

The safety evaluation of the NDDSs is mainly focused on the toxicological study of the health effect. At present, the cardiovascular toxicity of nanomaterials based on animal and cell level shown that the toxicity was closely related to a series of undesirable effects induced by nanomaterials, including oxidative stress, inflammation apoptosis, blood aggregation and cardiac signal transduction (Donnini et al., 2000; Savic et al., 2003; Chen and Von, 2005; Qinghua et al., 2005). Among them, inflammatory reaction and oxidative stress are recognized as the main mechanisms of cardiovascular toxicity of nanomaterials.

Inflammatory response can affect the occurrence and development of CVDs including hypertension, myocarditis, AS, acute myocardial infarction and heart failure. Some researchers have found that if nano-carriers have not been removed in time, they could reach all organs through blood, stimulate the body to produce a series of inflammatory cytokines, and eventually lead to cytotoxicity, which increases the risk of cardiovascular events (Suwa et al., 2002).

Nanomaterials have a large number of surface atoms and are highly reactive, which can generate free radicals and stimulate the formation of ROS, thereby interfering with antioxidant systems (Chen and Von, 2005). Oxidative stress can induce oxidative damage to macromolecular substances, such as DNA and proteins, which leads to cell growth inhibition, cell cycle abnormalities, and cell death.

The study on the toxic mechanism of cardiovascular system damage caused by nanomaterials in global is still in its infancy. There is very few relevant research evidence on the biological endpoints to determine the relationship between the physicochemical parameters (shape, size, size distribution, surface structure, electrochemical properties, etc.) of the nanoparticles and the toxic effects of the cardiovascular system. Therefore, scientists need to carry out more researches on the cardiovascular system toxic effects and mechanisms of typical nanomaterial exposure, which can make better use of the positive effects of nanomaterials to prevent, reduce or eliminate the possible adverse effects on health. Furthermore, it would provide theoretical and technical basis for the establishment of nanomaterial safety evaluation technology and standards.

#### REFERENCES


#### SUMMARY AND PERSPECTIVE

In conclusion, the nano-carrier, as an efficient, specific and controllable intracellular drug delivery method, has shown unique advantages in the diagnosis and therapy of CVDs. It can effectively solve the problems of targeting, local drug delivery, controlled release, sustained release, and reducing toxicity while it is developing toward the multifunctional and integrated direction of diagnosis and therapy. With the innovation of nanotechnology and the deepening studies on molecular pathological mechanism of CVDs, the application of NDDSs will be promoted, and new techniques and methods will be provided for clinical diagnosis and therapy. In addition, since the study on these nano-carriers is in its infancy, many problems still remain unclear. The main challenge is how to solve the biocompatibility of nano-drug-loaded particles themselves or their degradation products, which is need to be solved in the field of nano-biomedicine in the future.

#### AUTHOR CONTRIBUTIONS

WL proposed and developed the research outline, MY contributed to the concept and content framework, YD wrote the first draft and XZ prepared the drawings and contributed to improving the draft. HS and QH polished the article. ZW modified the format.

#### FUNDING

This review was supported by National Natural Science Foundation of China (Nos. 81972488, 81701836, 81973013), Guangdong Key R&D Program (No. 2019B020210002), Guangdong Natural Science Foundation (C1051164), The Eighth Affiliated Hospital of Sun Yat-sen University Outstanding Youth Reserve Talent Science Fund (FBJQ2019002).


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Deng, Zhang, Shen, He, Wu, Liao and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Interactions of Alginate-Deferoxamine Conjugates With Blood Components and Their Antioxidation in the Hemoglobin Oxidation Model

Tong Sun1,2, Xi Guo1,2, Rui Zhong<sup>3</sup> , Chengwei Wang1,4, Hao Liu1,2, Hao Li<sup>2</sup> , Lu Ma<sup>2</sup> , Junwen Guan<sup>2</sup> , Chao You1,2,5 and Meng Tian1,2,5 \*

#### Edited by:

Wenguo Cui, Shanghai Jiao Tong University, China

#### Reviewed by:

Jianxun Ding, Changchun Institute of Applied Chemistry (CAS), China Wei Tao, Harvard Medical School, United States

> \*Correspondence: Meng Tian tianmong007@gmail.com; 6744710@qq.com

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 16 December 2019 Accepted: 22 January 2020 Published: 11 February 2020

#### Citation:

Sun T, Guo X, Zhong R, Wang C, Liu H, Li H, Ma L, Guan J, You C and Tian M (2020) Interactions of Alginate-Deferoxamine Conjugates With Blood Components and Their Antioxidation in the Hemoglobin Oxidation Model. Front. Bioeng. Biotechnol. 8:53. doi: 10.3389/fbioe.2020.00053 <sup>1</sup> Neurosurgery Research Laboratory, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China, <sup>2</sup> Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu, China, 3 Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China, <sup>4</sup> Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, China, <sup>5</sup> West China Brain Research Centre, West China Hospital, Sichuan University, Chengdu, China

While deferoxamine (DFO) has long been used as an FDA-approved iron chelator, its proangiogenesis ability attracts increasing number of research interests. To address its drawbacks such as short plasma half-life and toxicity, polymeric conjugated strategy has been proposed and shown superiority. Owing to intravenous injection and application in blood-related conditions, however, the blood interactions and antioxidation of the DFO-conjugates and the mechanisms underlying these outcomes remain to be elucidated. In this regard, incubating with three different molecularweight (MW) alginate-DFO conjugates (ADs) red blood cells (RBCs), coagulation system, complement and platelet were investigated. To prove the antioxidant activity of ADs, we used hemoglobin oxidation model in vitro. ADs did not cause RBCs hemolysis while reversible aggregation and normal deformability ability were observed. However, the coagulation time, particularly APTT and TT, were significantly prolonged in a dosedependent manner, and fibrinogen was dramatically decreased, suggesting ADs could dominantly inhibit the intrinsic pathways in the process of coagulation. The dosedependent anticoagulation might be related with the functional groups along the alginate chains. The complements, C3a and C5a, were activated by ADs in a dose-dependent manner through alternative pathway. For platelet, ADs slightly suppressed the activation and aggregation at low concentration. Based on above results, the cross-talking among coagulation, complement and platelet induced by ADs was proposed. The antioxidation of ADs through iron chelation was proved and the antioxidant activity was shown in a MW-dependent manner.

Keywords: deferoxamine, blood components, antioxidation, alginate, conjugates

### INTRODUCTION

fbioe-08-00053 February 10, 2020 Time: 15:2 # 2

Deferoxamine (DFO) has long been used as an FDA-approved intravenously injected iron chelator in clinic for half a century in the treatment of iron overload diseases, while in the recent years, a great deal of attention was given to the important applications of DFO in the growing field of tissue regeneration as a result of its unique properties to inhibit inflammation and promote vascularization (Guo et al., 2019; Holden and Nair, 2019). In the early studies, DFO was proved to stimulate angiogenesis and neovascularization in the ischemia model of sheep and rabbit, and since then, increasing number of studies attempt to elucidate the related mechanisms (Chekanov et al., 2003). To date, it is widely accepted that DFO is contributed to the prevention of degradation of hypoxia-inducible factor-1 alpha (HIF-1α), an oxygen-sensitive molecule to upregulate the expression of vascular endothelial growth factor (VEGF) (Wu et al., 2010). In the meanwhile, some studies indicated that angiogenesis induced by DFO was closely associated with the property of antioxidation, as a secondary effect (Jiang et al., 2014). Despite the dispute, the ability of angiogenesis in addition to the antioxidation make it serve as a promising candidate for various biomedical use.

However, the application of DFO is still of much limitation owing to its drawbacks, like other small molecular drugs, including short plasma half-life and toxicity (Cassinerio et al., 2014). In this regard, polymer-drug conjugation strategy is appropriate for DFO to overcome these obstacles (Yan et al., 2016). Polymer-drug conjugates belonging to nano-sized drug delivery system attracts great research interest and are becoming established as a shining platform for drug delivery due to the reasons that the conjugation strategy deeply changes the behavior of the corresponding parent drugs and offers them many benefits, including high drug-loading, prolonged circulatory half-life, and reduced toxicity (Zhou et al., 2015; Ding et al., 2019a; Jesus et al., 2019; Macha et al., 2019; Qi et al., 2019). However, the therapeutic efficiency in clinic is not as expected, e.g., less than 10% of a systemically administered dose accumulates within the lesion, and in some cases there is no significant improvement for patient survival rate such as FDA approved doxorubicin HCl liposome (DOXIL) for anticancer (Zhang et al., 2013). Hence, the reasons why these conjugates with fine-tuned structures do not function as intended need to be elucidated in order to advance the therapy in clinic (Feng et al., 2019).

One of the most factors that contributes to this discrepancy is the interactions that exist between intravenously injected conjugates and the blood, since these interactions may change the target and transport capabilities of conjugates, thus determining the fate and the final therapeutic efficiency of conjugates in body (Lazarovits et al., 2015; Ding et al., 2019b). To develop solutions to these barriers, it is crucial to study the bloodconjugates interactions, e.g., RBCs, coagulation function, protein adsorption, complement system and platelets, which depended on the properties of conjugates such as structure, molecular weight (MW), and the functional groups along the chains (Wu et al., 2017). Besides, several pathways and mechanisms underlying these interactions suggest that some cross-talking among them has to be involved (Fornaguera et al., 2015). For instance, protein adsorption is deemed as the initial event in blood-conjugates interactions (Kenry et al., 2016), leading to the activation of coagulation cascade that can contribute significantly to both of complement system and platelet through certain coagulation enzymes (Liu et al., 2014). Nevertheless, the interplay among coagulation, platelet and complement is still of much limitation to be fully understood in the process of thrombus formation and inflammation response (Speth et al., 2015).

Actually, some studies had proven that conjugation of DFO into polymer carriers, such as hydroxyethyl starch, dextran, dialdehyde cellulose, nanoparticles, and polyethylene glycol copolymer, significantly prolonged the half-life comparing to the free of DFO (Tian et al., 2016a). To better of our knowledge, there is no report on systematic study of the DFO-based bloodconjugates interactions. Alginate, consisting of α-L-guluronate (G unit) and β-D-mannuronate (M unit), has long been used in delivery systems and in terms of the interactions with blood, alginate could induce aggregation of RBCs, allowing it be used as a viscosity modifier for blood substitutes. Contributing to the versatile functional groups, e.g., carboxyl and hydroxyl, along the molecular chains, alginate is also suggested to have effects on the protein adsorption and complement activation (Zhao et al., 2010; ¸Sen, 2011). In this light, the interactions of alginate-DFO conjugates is rather crucial to quest the potential mechanism. On the other hand, alginate has its intrinsic antioxidant activity due to the reductive ability of residues along the molecular chains, which may benefit to protect hemoglobin from oxidation in a different manner comparable to DFO. However, the polymer drug carrier also leads to steric hindrance for DFO cheating, resulting in iron binding occurring at a slower rate, depending on the density and location of conjugated DFO molecules.

In this work, alginate was chosen as a polymer carrier to prepare a series of alginate-DFO conjugates (ADs) with various MW and study their interactions with blood. We hypothesize that the molecular weight and functional groups along the alginate chains have specific interactions with blood. To address this hypothesis, a series of ADs with different MW were synthesized (**Figure 1A**). Incubating with ADs, RBCs, coagulation system, complement, and platelet, were investigated (**Figure 1B**). Specifically, the RBCs' properties including hemolysis, aggregation and deformability, were determined. Blood coagulation time including prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT), and the concentration of fibrinogen (Fib), were tested. The concentration of completement 3a (C3a) and completement 5a (C5a) were investigated. In terms of platelet, the activation and aggregation of platelet were determined. Based on above results, the interplay among coagulation system, complement and platelet incubating with ADs was proposed. Finally, the antioxidation of ADs was determined using the hemoglobin oxidation model, in which the effect of MW on iron chelation was discussed (**Figure 1C**).

### MATERIALS AND METHODS

#### Materials

Deferoxamine and sodium alginate were purchased from Sigma-Aldrich (St. Louis, MO, United States). NaBH3-CN and NaBH<sup>4</sup> were obtained from AAD-50din Co. (Shanghai, China). Normal saline (NS), phosphate buffer saline (PBS), sodium periodate, peroxide hydrogen, polyetherimide and ferric sulfate, were obtained from Kelong Co., Ltd. (Chengdu, China). Heparin sodium was purchased from Alading Co., Ltd. (Chengdu, China). Blood was collected from Chengdu Blood Center. Coagulation-related kits were purchased from Union Biotech Co., Ltd. (Chengdu, China). The ELISA Kit II was purchased from Becton-Dickinson Co., Ltd. (United States). Flow cytometry-related agents, anti-CD61-fluorecein isothiocyanate (FITC), anti-CD62p-phycoerythrin (PE) and IgG1 (mouse) – PE, were purchased from BD Pharmingen, BD Bioscience Co., Ltd. (San Jose', CA, United States). Platelet aggregation-inducers, adenosine-disphosphate and epinephrine, were obtained from Kelong Co., Ltd. (Chengdu, China).

#### Synthesis of the ADs

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The ADs were synthesized by Schiff-base reaction through the terminal amine groups in DFO and reactive aldehyde groups in oxidized alginate (OA) and followed by reduction as our previous report (Tian et al., 2016b). In brief, 10.0 g of sodium alginate was dissolved with 700 mL deionized water (DW) and 100 mL ethanol followed by different ratios of sodium periodate to uronate units (15, 30, 50 mol%) were used. The mixture was stirred in dark for 6 h and then mixed with 20 mL ethylene glycol before stirred for another 2 h. After that, to separate and purify each prepared ADA, the precipitant, presented by adding 10 g of NaCl and 2000 mL ethanol, was redissolved in 400 mL DW and 2000 mL ethanol again, and the progress was repeated three times. The products prepared from 15, 30, and 50 mol % of periodate/uronate units are corresponding to OA-15, OA-30, and OA-50 respectively. To obtain AD conjugates, 1 g of each OA was incubated with 2.5 g of DFO under stirring in room temperature. After 2 h, NaBH3CN (125 mg/mL) was slowly added to the solution under stirring for 4 h and then NaBH<sup>4</sup> was added to the mixture for another 24 h. It respectively takes 3 days to dialyze against 1 M NaCl and DW using a dialysis tube (MWCO, 3500), totally 6 days. The conjugates were synthesized by OA-15, OA-30, and OA-50 named AD-15, AD-30, and AD-50 respectively. The structure of the synthesized conjugates was characterized as our previous report and shown in **Table 1**. The degree of DFO (% DFO) incorporation was calculated and described as moles of DFO attached per uronate residue.

#### Blood Collection and Preparation of AD Solutions

The study was approved by Ethical Committee of Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College. We collected blood samples at Chengdu Blood Center from three healthy donors. The whole blood was obtained through venipuncture and then mixed with 3.8% sodium citrate at radio of 9:1(blood/sodium citrate) to obtain citrated whole blood (CWB). To prepare 10% hematocrit of RBCs suspension, CWB was used to mix with the same volume of NS and centrifuged at 5000 r/min for 4 min, and then removed the supernatant. Repeated wash three times by NS for a total of four washes. While the supernatant was clear, removed it


out and then added corresponding volume of NS to ensure the hematocrit of RBC suspension was 10%. CWB was centrifuged at 1200 g for 20 min to obtain platelet-rich plasma (PRP). Serum was prepared by centrifuging CWB he citrated at 1200 g for 30 min. The fresh frozen plasma (FFP) supplied by Chengdu blood center has anticoagulated with citrate-phosphate-adenine. ADs were dissolved in normal saline (NS) to obtain solutions with different concentration.

# Red Blood Cells

#### Hemolysis

The percent of RBC lysis was measured by the Free Hemoglobin Colorimetric Assay Kit using. Two hundred and seventy µl of RBC suspension (hematocrit: 10%) was respectively incubated with 30 µl of three ADs solutions with different concentrations for 1 h at 37◦C to obtain a final concentration of 1, 5, and 10 mg/ml. We investigated the concentration of free hemoglobin by the method of ortho-tolidine to determine the hemolysis of incubated RBCs according to previous study (Lin et al., 2019). The absorbance was obtained by spectrophotometer (EON, Bio-Tech CO., Ltd., United States) at 435 nm. The percent of hemolysis was calculated by the following eq. (1). The RBCs suspension incubated with distilled water (DW) was used as a positive control.

$$\text{Hemolysis (\%)} = \frac{\text{A(l)}}{\text{A(0)}} \times \frac{100 - \text{Hct (\%)}}{\text{Hb (g/l)} \times 1000} \times 100\% \quad \text{(1)}$$

A(1) : the absorbance of sample. A(0) : the absorbance of standard sample. Hct, hematocrit. Hb, 100 mg/L

#### Aggregation

Two hundred and seventy µl of CWB was respectively incubated with 30 µl of three ADs solutions with different concentrations for 1 h at 37◦C to obtain a final concentration of 1, 5, and 10 mg/ml followed by centrifuging at 1000 g for 3 min. Three µl of RBCs sediment and 40 µl of supernatant were mixed and examined by optical microscopy. We captured the imagines by a digital microscope camera. CWB incubated with NS and polyetherimide (PEI) were respectively used as a normal control and positive control.

#### Deformability

Two hundred and seventy µl of CWB was respectively incubated with 30 µl of three ADs solutions with different concentrations for 1 h at 37◦C to obtain a final concentration of 1, 5, and 10 mg/ml After incubation, the mixture (20 µl) was suspended in PBS (1 mL) containing 15% polyvinylpyrrolidone and then was tested by a Laser-diffraction Ektacytometer (LBYBX, Beijing Pencil Instrument CO., Ltd., China) according to the manufacture's manual. Four shear stress, 0.39, 0.77, 1.54, and 7.7 Pa, were used, corresponding to the shear rate 50, 100, 200, and 1000 respectively. CWB incubated with NS was used as a normal control.

#### Blood Coagulation

Sixty µl of three ADs solutions at 10 and 50 mg/ml were respectively incubated with 540 µl of FFP at 37◦C for 3 min. After incubation, we used Prothrombin Time Diagnostic Kit to determine PT in the presence of Ca2+, and Activated Partial Thromboplastin Time Diagnostic Kit to determine APTT by adding kaolin and encephalin. We tested TT using Thrombin Time Determination Kit and the concentration of Fib in the presence of thrombin using Fibrinogen Determination Reagent Kit respectively. The coagulation time of incubated FFP, including APTT, prothrombin time (PT), thrombin time (TT), and the concentration of fibrinogen (Fib) were determined by an automated coagulation analyzer (Instrumentation Laboratory ACL ELITE, United States). To be specific, FFP incubated with NS and heparin (HP, 2 IU/mL) were respectively used as normal control and negative control.

#### Complement Activation

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The concentration of C3a and C5a were tested to prove the effect of ADs on complement system. Thirty µl of three ADs solutions at 10 and 50 mg/ml were respectively incubated with 270 µl of serum at 37◦C for 3 min followed by analysis of complement activation using ELISA Kit II (Becton-Dickinson Co., Ltd., United States). The standards and samples were finally read by spectrophotometer (EON, Bio-Tech Co., Ltd., United States) at 450 nm and the concentration of C3a and C5a were calculated by utilizing standard curve. Serum incubated with zymosan (7 mg/ml) was used as a positive control.

### Platelet

#### Platelet Activation

Thirty µl of three ADs solutions at 10 and 50 mg/ml were respectively incubated with 270 µl of PRP at 37◦C for 1 h. Five µl of incubated PRP, 5 µl of CD61, 5 µl of CD62p and 40 µl of PBS buffer (10 mM) were mixed in dark at 15 min and then added to 400 µl PBS (10 mM) for flow cytometric analysis. The activation of platelet was defined as the percentage of marker CD62p detected in 10000 total events counted by flow cytometry (Becton-Dickinson) using anti-CD61-fluorecein isothiocyanate (FITC), anti-CD62p-phycoerythrin (PE) and IgG1 (mouse)-PE (BD Pharmingen, BD Bioscience, San Jose', CA, United States). The data was analyzed by BD FACSD via Software (Version 8.0.1.1). Human thrombin (10 IU/mL) and NS were respectively used as positive and normal control.

#### Platelet Aggregation

Thirty µl of three ADs solutions at 10 and 50 mg/ml were respectively incubated with 270 µl of PRP at 37◦C for 1 h. Incubated PRP (225 µl) were mixed with two aggregation-inducers, adinosinedisphosphate (0.1 mM, 12.5 µl) and epinephrine (0.15 mM, 12.5 µl), and then we used platelet aggregometer (MODEL700, Chrono-Log Co., Ltd., United States) to determine the platelet aggregation.

#### Antioxidation

To prove the effect of ADs on prevention of iron-mediated oxidation in the presence of hemoglobin (Hb), we observed the dynamic changes of oxyhemoglobin (oxyHb) while mixing ADs with ferric solutions containing Hb. Briefly, washed RBCs were frozen and thawed and then mixed with NS to obtain hemolysate (22 µM). To guarantee each sample, except for Hb control, contains 0.4 mM of DFO equivalent and 0.4 mM of ferric ion, ADs or DFO solutions (10 µl) and ferric sulfate solution (50 µl) were mixed with hemolysate solution (440 µl). The full wavelength scanning of each group was immediately performed by spectrophotometer (EON, Bio-Tech Co., Ltd., United States). In addition, the absorbance at 560, 576, 630, and 700 nm was respectively measured again every 30 s, in total of 5 min. The containing of oxyHbA was respectively calculated using the method of Winterbourn in different times, shown as the following eq. (2) (Winterbourn, 1985). The percent of oxyHbA was the ratio of oxyHbA to the primary containing of oxyHbA in blood samples.

$$\begin{aligned} \text{row} &HbA(\%) &= \\ \frac{1.013 \times (A1 - A4) - 0.3269 \times (A3 - A4) - 0.7353 \times (A2 - A4)}{10000} \times 100\% \end{aligned} \tag{2}$$

A1 means the absorbance at 560 nm; A2 means the absorbance at 570 nm; A3 means the absorbance at 630 nm; A4 means the absorbance at 700 nm.

Further assessing the antioxidation activity of three AD conjugates and DFO, the degree of ADs and DFO chelating to iron was investigated. Fifty µl ferric sulfate solution (0.11 mM) was mixed with 50 µl AD conjugates or DFO solutions (DFO equivalent, 0.2 mM), and then the absorbance at 430 nm was respectively measured every 5 s, in total of 1 min.

#### Statistical Analysis

We use analysis of variance and paired t-test to perform the statistical analysis. The probability (P) values less than 0.05 was considered to have significant difference and was calculated with the software assistance of Excel 2007 and SPSS 19.0. The results are presented as mean ± SD (n = 3).

## RESULTS AND DISCUSSION

#### Synthesis of ADs

ADs were synthesized through a two-step process in which alginate was oxidized to ADA firstly, and then the conjugates were prepared by Schiff-base reaction through the terminal amine groups in DFO and reactive aldehyde groups in ADA and followed by reduction with NaBH3CN and NaBH<sup>4</sup> (**Figure 1A**). To prepare a series of conjugates with different molecular weight, alginate was oxidized to different oxidation degree by adjusting the mol ratio of periodate/uronate units, since oxidation of alginate with sodium periodate results not only cleavage of the C2-C3 bond, but also main chain scission as a simultaneous reaction. The structure characteristics of the prepared conjugates are summarized in **Table 1**. As expected, the Mw of the conjugates are 245, 128, and 62.7 kDa, respectively. The DFO contents in the conjugates were increased with the decrease of the Mw due to the coupling of the oxidation and Mw, with 8.7, 14.7, and 20.4% by molar and corresponding to around one DFO per eleven, seven, and five uronate units for AD-15, AD-30, and AD-50, respectively.

### Red Blood Cells

Hemolysis is characterized as the rupture of RBCs and the release of the cytoplasm, and the evaluation of hemolysis is essential regarding the biosafety of exogenous materials. To investigate the device-associated hemolysis, static or semi-static testing in vitro was widely used (Haishima et al., 2014). Based on ISO10993-5, ISO10993-4 and ASTMF756-00, the percent less than 5% will be considered as a very low risk of hemolysis (Zhen et al., 2015). According to the results as shown in **Figure 2**, the percent of hemolysis in AD-15, AD-30, AD-50 and DFO at any concentration fluctuated around 0.2%, while the hemolysis of DW group is approximately 90%. Except for the DFO in 10 mg/mL, the others rarely showed any significant difference compared with normal saline (NS), indicating ADs had a quite low risk of hemolysis.

The property of RBCs to aggregate is one of the typical features that plays a vital role in the blood circulation (Claveria et al., 2016). The aggregation of RBCs has been proved to affect their viscosity ability owing to the non-Newtonian behavior of blood in the body and reversible aggregation may physiologically occur in the presence of macromolecules and calcium ion, serving as the bridge among RBCs (Mehri et al., 2014). However, irreversible RBCs aggregation, induced by some chemicals, such as phosphodiesterase and polyethyleneimine (PEI), could damage RBCs and even potentially disrupt capillaries (Muravyov and Tikhomirova, 2014). Herein, photomicrographs of RBCs incubated with PEI, used as positive control, and ADs at 1, 5, and 10 mg/mL were shown in **Figure 3**. Compared with PEI, neither ADs nor DFO showed any irreversible aggregation. The likely aggregation in ADs group as well as NS control was contributed to rouleau formation, as a typical result of reversible aggregation (Flormann et al., 2015). According to the previous study, it has long been known that alginate increased whole blood viscosity and induced aggregation of RBCs thus being used as a viscosity modifier for blood substitutes (Xu et al., 2017). In this work, ADs showed negative response to RBCs aggregation, which could be ascribed to the possibly low content of alginate in the reaction system (Zhao et al., 2010).

The property of allows them to change their shape according to the diameter of blood vessel as a result of natural shape of RBCs. The regulation of RBCs deformability depends on three aspects, cytoplasm viscosity, membrane mechanical properties and surface area and volume of RBCs (Uhl et al., 2018). Malfunction of RBCs deformability is contributed to the occurrence of several diseases, such as sickle cell anemia, malaria and hereditary spherocytosis (Zhao et al., 2010). **Figures 4A–D** showed the deformability of incubated RBCs at various shear stress. The results indicated there is no significant difference among three ADs and NS at any concentration or shear stress. The deformability of DW group was significantly lower than three ADs at the shear rate 100, 200, and 1000 while at 50, DW group was slightly lower than AD-15 at 5 mg/ml (P = 0.046) and AD-30 at 1 mg/ml (P = 0.038). Generally, the elongation index (EI) significantly increased while being exposed to a higher shear stress. For DW group, the EI stayed a low level as the shear

rate changed, suggesting that the deformability of RBCs could be significantly suppressed in the presence of hemolysis.

### Coagulation Function

To determine the effect of ADs on coagulation system, we tested the coagulation time, including PT, APTT, and TT, together along with the concentration of Fib. The results of blood coagulation were shown in **Figure 5**. Comparing with DFO or NS group, the PT of three ADs, on behalf of extrinsic coagulation pathway, were simultaneously lengthened to some extent. Unlike AD-15 and AD-30, the PT of AD-50 presented a dose-dependent effect. For APTT and TT, reflecting the intrinsic coagulation pathway and common coagulation pathway respectively, three ADs significantly prolonged APTT and TT compared with DFO or NS in a dose-dependent manner and at the concentration of 5 mg/mL, the values of APTT and TT were too high to be tested by analyzer (the detection range for APTT is 6–245 s, for TT is from 3–169 s). In terms of Fib, the concentration of Fib of three ADs was approximately 1.2 g/L while DFO or NS group was approximately 1.6 g/L. Additionally, ADs could decrease the concentration of Fib in a dose-dependent manner since the concentration of Fib ADs at 5 mg/mL was approximately 0.6 g/L with statistical difference.

To the best of our knowledge, this is the first study demonstrated that polymer-drug conjugate using alginate as drug carrier was capable of anticoagulant activity. ADs significantly prolonged the coagulation time, particularly APTT, indicating that the intrinsic pathway was dominantly blocked in the process of anticoagulation. To explain this phenomenon, two main mechanisms were proposed to involve in anticoagulation pathway. Firstly, interacting with the coagulation factors, such as factor I (Fib), II, V, and X, ADs can significantly inhibit their activity, thus preventing them from participating in coagulation cascade reaction (Zou et al., 2014; Xin et al., 2016). Secondly, the anticoagulative activity relates with the negative functional groups along the chains of the polysaccharides since the negative functional group would form a complex interaction with antithrombin in plasma (Li et al., 2017). Alginate is the only polysaccharide that naturally contains negative carboxyl groups along the chains, indicating that anticoagulant activity probably has to be involved. Besides carboxyl groups, there are many hydroxyl groups along the chains of alginate, both of which have been suggested to have significant effect on the coagulation function (Shiu et al., 2014; Guo et al., 2018). The anticoagulation induced by carboxyl groups and hydroxyl groups is supported by the work of Sperling et al. (2005), who studied the influence of different content of carboxyl groups or hydroxyl groups on coagulation function and indicated that pure-carboxyl groups showed a strong anticoagulative effect while the effect of purehydroxyl groups was milder.

Among three ADs, AD-50, with the lowest MW, showed a weaker anticoagulative activity than that of AD-15 and 30, indicating that ADs-induced anticoagulation was partially depended on the MW, which was consistent with previous

analyzer. HP, heparin.

studies. For instance, Xu et al. (2017) studied the blood compatibility of alginate with different MW (1170–50075 kDa) prepared by heterogeneous phase acid degradation and the results indicated that the blood clotting time was prolonged with increasing of MW of alginate. Similarly, the anticoagulation of sulfated polysaccharides depends not only on the substituted functional groups along main chains, but also the MW. Xin et al. (2016) investigated the anticoagulative properties of a series of low MW propylene glycol alginate sodium sulfate (2.99– 8.91 KDa) and they found it could prolong the APTT and clotting time and the anticoagulative activity declined with the decrease in MW. Besides, Fan et al. (2011) aimed at prove the correlation between MW and anticoagulative activity and indicated that the clotting time induced by sodium alginate sulfate (14900– 35300 KDa) initially increased as the MW decreased but fall with the further decrease in MW. Therefore, based on the previous study, it is suggested that within a certain MW range, the degree of anticoagulative activity through absorption coagulation factors through negative charged groups along the chains has a main dependence on MW, and with the reduction of MW, the anticoagulation activity is much weaker probably ascribing to the decrease of negative groups.

In addition, it should be noted that the anticoagulative effect induced by ADs were weaker than HP, a widely used anticoagulant in clinic. Besides, the mild anticoagulative activity of ADs would rather extend the application in the treatment of iron-overload diseases. For instance, in some iron-overload diseases like thalassanemia and sideroblastic anaemia, long-term repeated-transfusion is needed to improve the content of hemoglobin while transfusion-associated thrombosis is unacceptably common in clinic as well (Xenos et al., 2012). To simultaneously overcome iron-overload and hypocoagulability state, ADs show superiorities contributing to their anticoagulative effect. Additionally, hemorrhage is another concern in clinic following the application of anticoagulant and some research indicated that the concentration of Fib had a closely link with the incidence of hemorrhage and there is a much higher risk of bleeding as Fib concentration is less than 0.5 g/L (Lunde et al., 2014). In this light, our results showed that the concentration of Fib of three ADs at 1 or 5 mg/mL were higher than HP group, indicating that ADs had a quite lower risk of hemorrhage than HP.

#### Complement Activation

Complement can be activated by cascade reaction in three pathway, classic pathway, alternative pathway and mannose binding lectin pathway (Koscielska-Kasprzak et al., 2014). It has long been known that exogenous biomaterials mainly

activate alternative pathway, which could cause severe sideeffects, such as inflammation (Liu et al., 2013). In alternative pathway, C3, directly activated by biomaterials, cleaves to produce C3a and C3b. C3b can regulate the function of monocytes and macrophages. C3a is component of C3ADesArg, and make up C5 convertase, which causes change of C5 to C5a and C5b. C5a and C5b are component of C5ADesArg and membrane attack complex respectively. In a word, concentrations of C3a and C5a are canonical indexes to access the activation of complement pathway (Nesargikar et al., 2012). Serum incubated with 1 and 5mg/mL AD conjugates or DFO were adopted to measure the concentrations of C3a and C5a. The results of incubated complement were shown in **Figures 6A,B**. At the concentration of 1 mg/mL, except for the mild activation of C3 induced by AD-15, C3 and C5 were rarely activated by AD-30 or AD-50. At the concentration of 5 mg/mL, C3a and C5a of three ADs sharply increased almost two times than low concentration suggesting high-dose of ADs could activate complement system through alternative pathway. The activation of C3a and C5a induced by ADs, however, were significantly lower than that of positive control, zymosan. The dosedependent complement activation is closely dependent on the content of hydroxyl groups along the chains, one of negative groups that can trigger the complement cascade response (Zhong et al., 2013). Besides binding to complement directly, hydroxyl group also interact with complement through absorption numerous complement-related proteins (Wetterö et al., 2002).

#### Platelet

Platelets, derived from megakaryocytes in the bone marrow with plasmolemma, usually maintain the integrity of blood vessel and participate in the process of hemostasis following vascular injury (Sun et al., 2019). CD62P (platelet surface P-selectin) has long been used as markers to determine the activation of platelet since its considerable stability (Lu and Malinauskas, 2011). In this work, CD62P was used to determine the effect of ADs on platelet activation using flow cytometry. To further measure platelet function, the aggregation of incubated platelet was investigated using platelet aggregometer. The expression of CD62P and the percent of platelet aggregation were shown in **Figure 7**. At the concentration of 1 mg/mL, AD-15 and AD-30 slightly suppressed platelet activation compared with NS, which is accordant with the results of platelet aggregation. Compared with ADs at 1 mg/mL, mild activation of platelet can be observed at the concentration of 5 mg/mL. Based on the results, AD-50 rarely influenced the platelet function.

Platelet activation, in which fib has been reported one of requisite stimulating factors, involving in a complicated multistep process including adhesion, release and aggregation for hemostatic plug formation and thrombosis, has been reported that could be influenced by coagulation function and complement system in the blood (Khanbeigi et al., 2015). For instance, coagulants, like thrombin, can significantly activate platelet and inhibit the coagulation process while some complement inhibitors can similarly inhibit the platelet function (Speth et al., 2015). However, few studies fully revealed the interplay of coagulation and complement in platelet function in vitro. Therefore, according to our findings, it is proposed that AD conjugates can inhibit the platelet by the means of inhibition of Fib through functional groups along the chains and in the meantime, the inhibition can be antagonized by complement activation to some extent. However, few studies fully revealed the interplay of coagulation and complement in platelet function in vitro. According to the results of coagulation, complement, and platelet function, it is proposed that ADs can inhibit the platelet by the means of inhibition of Fib through functional groups along the chains, which could be antagonized by complement-associated activation to some extent. As shown in **Figure 8**, ADs, as mild anticoagulants, can absorb Fib to make them be absence of the process of platelet activation through the functional groups along the chains, like hydroxyl and carboxyl group. However, with the elimination of anticoagulative activity, the anticoagulation-associated platelet inhibition is negligible since AD-50 barely influenced the platelet activation. On the

other hand, it has been reported that platelets express many complement molecules, including some complement receptors and complement regulatory molecules that can generate C3a and C5a and as a return, the complement system and its activation products can also stimulate platelets by several complement factors, such as the anaphylatoxins C3a and C5a

#P < 0.05 versus NS and thrombin. Th, thrombin.

aggregation was determined by aggregometer. \*P < 0.05 for NS; \*\*5 mg/mL has significant differences with 1 mg/mL, P < 0.05; \*\*\*P < 0.05 for the others;

(Speth et al., 2015). Herein, at the concentration of 1 mg/mL, comparing to the platelet inhibition through absorbing Fib, complement-associated activation induced by ADs is milder as a result of slight reduction on platelet activation. However, at the concentration of 5 mg/mL, with the raised activity in both anticoagulation and complement, the complement-induced activation is significantly enhanced against the anticoagulationassociated inhibition, being balanced and resulting in equal level of platelet activation between ADs and NS.

#### Antioxidation

To prove the prevention of iron-mediated oxidation, hemoglobin (Hb) oxidation model, in which mimic the process underlying the pathology of hemoglobinopathies such as sickle cell anemia and the thalassemia, was widely used (Xu et al., 2014). Under normal condition, the Hb converts to oxyHb in ferrous state (Fe2+), able to bind and transport oxygen, and methemoglobin in the presence of ferric iron (Fe3+) (Pichert and Arnhold, 2015). As shown in **Figure 9A**, oxyHb has two obvious peaks between 500 and 600 nm while methemoglobin (oxidized Hb) has another new peak at 630 nm, a feature that can be utilized to distinguish these two proteins. Upon addition of DFO, the absorption spectrum that refers to content of oxyHb is similar to oxyHb rather than oxidized Hb control, suggesting that the conjugates can bind to ferric iron to protect Hb from being oxidized to methemoglobin in Fe3+-rich condition. To detail the prevention, the percent of oxyHb was calculated on the basis of absorbance at 560, 576, 630, and 700 nm. The results are shown in **Figure 9B**. The content of oxyHb in ADs and DFO are dramatically higher than oxidized Hb control (containing Hb and Fe3+) all the time, suggesting that ADs or DFO as a chelator for ferric iron prevents Hb from being oxidized. However, the binding to iron could be time-dependent since a sharply reduction is observed in the first 30 s, and then reaches a stable lever. In terms of three ADs, additionally, AD-50 seems to exhibit a slightly better capability on antioxidation comparing to AD-15 and AD-30. Therefore, the ability of the conjugates and the free DFO binding to Fe3<sup>+</sup> were further determined by spectrophotometer and the results are shown in **Figure 9C**. The degree of ADs or DFO chelating Fe3<sup>+</sup> rapidly reach to the peak in the first 5 s, which means the binding to Fe3<sup>+</sup> occurs as soon as they contact. For three ADs with similar DFO content, the level of chelates significantly augment as the MW reduction while the free of DFO is slightly higher than that of conjugates. As a result, it is indicated that the ADs are capable of antioxidant activity mainly through iron chelation in a MW dependence manner.

Although there are several reports attempt to reveal the links between MW and antioxidative property, that found biomaterials with a lower MW have a higher antioxidant activity, the mechanism remains controversial. Alginate has its intrinsic antioxidant activity due to the reductive ability of residues along the molecular chains and its antioxidant activity is increased with the decrease of the MW (Xu et al., 2017), which may benefit to protect Hb from oxidation in a different manner comparing to the free of DFO. However, the conjugates did not show superior antioxidant activity as the expected. The reasons for the

MW dependence of the iron chelation probably relate with the polymer carrier-induced steric hinderance. By determining the scavenging ability of 1, 1-diphenyl-2- picrylhydrazyl free radical (DPPH), alginate with a lower MW shows a better antioxidant activity probably ascribe to the increase in the a-L-guluronic acids (G) content of alginate and in the meanwhile, the nature of alginate chain was extended due to the diaxial linkage in G-blocks, which may hind the rotation around glycosidic acid as the MW increase (¸Sen, 2011). Nevertheless, the polymer drug carrier also leads to steric hinderance for DFO chelating, resulting in iron binding occurring at a slower rate, depending on the density and location of conjugated DFO molecules (Banerjee, 2009; Yu et al., 2019). As shown in **Figure 9D**, iron chelation results in a locally ordered molecular configuration which is a process of losses of entropy. The higher MW and longer molecular chains, the higher losses of entropy, which in turn makes it more difficult for iron chelation.

FIGURE 9 | Protection against iron-mediated oxidation in hemolysate. Each group, except for the hemolysate (Hb) and oxyHb (containing Hb and Fe3+) control, similarly contains 0.4 mM of DFO equivalent (AD or DFO) and Fe3+, dissolving in Hb solution (22 µM). (A) The full wavelength scanning. (B) The tendency of the percent of oxyHb in each group, scanning every 30 s in total of 5 min. (C) The lever of DFO-Fe3<sup>+</sup> chelates. The absorbance was obtained at 430 nm, which means the degree of ADs or DFO chelating to iron. (D) The diagrammatic sketch of different MW ADs to Fe3+. With the Mw of AD conjugates increasing, the ability to chelating to iron will be repressed ascribing to the steric hinderance effect.

### CONCLUSION

In conclusion, we successfully synthesized a series of ADs with various MW and the interactions with RBCs, coagulation, complement, and platelet had been studied for the conjugates as a function of their dose and MW. Interactions of ADs with RBCs did not reveal any hemolysis and showed reversible aggregation contributing to rouleau formation of RBCs and normal deformability ability. On the contrary, ADs significantly prolonged the coagulation time in a dose-dependent manner, particularly APTT and TT, suggesting ADs could dominantly inhibit the intrinsic and common pathways in the process of coagulation. The results of complement and platelet tests showed ADs could activate complements C3a and C5a, presenting dose-dependence while AD-15 and AD-30 slightly inhibit the platelet activation and aggregation in low concentration. Besides, the interplay among coagulation, complement, and platelet activation was proposed. Finally, the antioxidant activity of ADs was demonstrated in a MW-dependent manner.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material.

#### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by the Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College. The patients/participants provided their written informed consent to participate in this study.

#### AUTHOR CONTRIBUTIONS

fbioe-08-00053 February 10, 2020 Time: 15:2 # 13

TS performed the blood evaluation, wrote the manuscript, and discussed the results. XG performed the blood evaluation. RZ performed the other experiments. CW, HLiu, HLi, LM, JG, and CY were involved in the results discussion. MT was

#### REFERENCES


responsible for conceptualization, results discussion, and revising the manuscript.

#### FUNDING

This work was sponsored by the National Natural Science Foundation of China (Nos. 51403238 and 81401528) and Sichuan Province Science and Technology Key R&D Project (Nos. 2018SZ0029, 2018SZ0100, and 2019YFS0120).



**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Sun, Guo, Zhong, Wang, Liu, Li, Ma, Guan, You and Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Injectable Hydrogel-Based Nanocomposites for Cardiovascular Diseases

Xiaoshan Liao1,2† , Xushan Yang<sup>1</sup>† , Hong Deng<sup>1</sup> , Yuting Hao<sup>1</sup> , Lianzhi Mao<sup>1</sup> , Rongjun Zhang<sup>1</sup> , Wenzhen Liao<sup>1</sup> \* and Miaomiao Yuan<sup>2</sup> \*

<sup>1</sup> Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China, <sup>2</sup> The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, China

#### Edited by:

Chao Zhao, The University of Alabama, United States

#### Reviewed by:

Chiara Tonda-Turo, Politecnico di Torino, Italy Enza Torino, University of Naples Federico II, Italy

#### \*Correspondence:

Wenzhen Liao wenzhenliao@163.com Miaomiao Yuan yuanmm2019@163.com †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 22 November 2019 Accepted: 11 March 2020 Published: 31 March 2020

#### Citation:

Liao X, Yang X, Deng H, Hao Y, Mao L, Zhang R, Liao W and Yuan M (2020) Injectable Hydrogel-Based Nanocomposites for Cardiovascular Diseases. Front. Bioeng. Biotechnol. 8:251. doi: 10.3389/fbioe.2020.00251 Cardiovascular diseases (CVDs), including a series of pathological disorders, severely affect millions of people all over the world. To address this issue, several potential therapies have been developed for treating CVDs, including injectable hydrogels as a minimally invasive method. However, the utilization of injectable hydrogel is a bit restricted recently owing to some limitations, such as transporting the therapeutic agent more accurately to the target site and prolonging their retention locally. This review focuses on the advances in injectable hydrogels for CVD, detailing the types of injectable hydrogels (natural or synthetic), especially that complexed with stem cells, cytokines, nano-chemical particles, exosomes, genetic material including DNA or RNA, etc. Moreover, we summarized the mainly prominent mechanism, based on which injectable hydrogel present excellent treating effect of cardiovascular repair. All in all, it is hopefully that injectable hydrogel-based nanocomposites would be a potential candidate through cardiac repair in CVDs treatment.

Keywords: injectable hydrogel, nanocomposite, angiogenesis, stem cell homing, cardiovascular diseases

### INTRODUCTION

Cardiovascular diseases (CVDs), the group of pathological disorders, including atherosclerosis, myocardial infarction (AMI), stroke and heart failure (HF), remains the leading cause of death globally (Ujcic-Voortman et al., 2012; Nichols et al., 2014; Cainzos-Achirica et al., 2019). In the United States, there were 12.3 million deaths caused primarily by CVD from 2003 to 2017, among which, ischemic heart disease accounted for 48.2%, followed by cerebrovascular disease or stroke (16.7%), and heart failure or cardiomyopathy (10.6%) (Cross et al., 2019). CVDs affects the life of quality of patients, and causes enormous health and economic burdens (Gersh et al., 2010) all over the world, both in the developing countries (Lopez-Jaramillo, 2008; Gersh et al., 2010; Celermajer et al., 2012; McAloon et al., 2016) and in the rich ones (Gersh et al., 2010).

To date, current clinical regimens largely rely on the administration of drugs and other therapeutic agents such as the stem cell and the growth factors (Madonna and De Caterina, 2011; Bagno et al., 2018), basing on the hypothesis that a certain disease consists of dysfunctional cells and molecules within healthy organs and body. As well known, on the one hand, drugs or other therapeutic materials need to overcome physiological barriers to reach targets sites and during the process of transporting them, potential adverse effects may be produced; on the other hand, another

hamper is the retention time of agents in the injury site is not adequate for new vessel growth. Therefore, the use of drug delivery systems (DDS) is necessary for enhancing the efficacy and safety of therapeutic agents (Matoba et al., 2017).

In the past decades, DDS has been investigated for improving the transportation efficiency of drugs or other agents of interest (Miyake et al., 1998). Currently, great advance about DDS has been made, for example, electrospun polymeric nanofibers (Torres-Martinez et al., 2018), Lipid-based DDSs (Semalty et al., 2009) and Metallic nanoparticles (Mody et al., 2010), Electrospun polymeric nanofibers (Torres-Martinez et al., 2018), as one of promising DDSs, has the capacity to improve drug's bioavailability and release them in a controlled way via making the low solubility drugs loaded into the fibers. Besides, the high surface-to-volume ratio of the fibers can promote cell adhesion and proliferation, drug loading, and mass transfer processes. However, because of its high cost, the matter of manufacturing drug loaded electrospun mats has to be considered before wide utilization. As one of the lipid-based DDS, pharmacosomes (Semalty et al., 2009) were able to improve dissolution and absorption efficiency through the lipophilic membrane tissue owing to its amphiphilic property (Wang et al., 2011), so that the bioavailability of drugs was greatly improved. However, the targeting of the lipid-based DDS is still a challenge. Metallic nanoparticles (Mody et al., 2010) such as iron oxide nanoparticles have been widely used in targeted drug delivery since they were able to conjugate with antibodies and drugs of interest via modification of different chemical functional groups. However, the toxicity of these magnetic nanoparticles to certain kinds of neuronal cells remain unclear (Pisanic et al., 2007).

Recently, the utilization of injectable hydrogel-based DDSs has attracted considerable attention in many medicine fields, including chemotherapeutics (Norouzi et al., 2016), tissue engineering and regenerative medicine such as cartilage (Li J. et al., 2019) and spinal cord (Macaya and Spector, 2012). Injectable hydrogel has mechanical properties to closely match the targeting organ, and can also be loaded with cellular and a cellular therapeutics to modulate the wound environment and enhance regeneration (Frith et al., 2013; Seo et al., 2017; Cipriani et al., 2018; Mao et al., 2019). In the past years, hydrogels have been paid considerable attention as potential candidates for restoration of ischemia myocardial, in particular, those stem from natural extracellular matrix (ECM) components (e.g., collagen, fibronectin, as well as glycosaminoglycans) could favor greatly endothelial cells adhesion and their transformation to microvessels in vitro (Moon et al., 2010) attributing to their high water content and structural similarity to the natural ECM (Peppas et al., 2006; Seliktar, 2012). Additionally, when in an extremly swollen state, hydrogel-based materials such as chitosan hydrogels show good ability to deliver cells and bioactive agents (Liu et al., 2006). Besides, owing to its pH- and temperature-responsive properties, injectable hydrogel exhibits good capacities as a minimally invasive biomaterial scaffolding (Van Vlierberghe et al., 2011) applied for CVDs. Here, we review the wide application of various kinds of injectable hydrogel and the major strategies for the cardiovascular disease therapy.

### SINGLE USE OF INJECTABLE HYDROGELS

It is of significant potential for injectable hydrogels to be applied for cardiovascular diseases. The single use of injectable hydrogels characterized by minimally invasive has a suitable effect in cardiovascular disease treatment (Johnson and Christman, 2012). Injectable hydrogels are able to form a network structure at a certain temperature, to provide a morphological environment for supporting myocardial cells and retaining self-differentiated growth factors to promote myocardial repair (MacArthur et al., 2017). The current research and development focused on injectable hydrogels mainly divided into two categories: natural hydrogels and synthetic hydrogels.

#### Natural Hydrogel

Natural hydrogels are attracting attention because of their nontoxicity, immunogenicity, and excretion of metabolites (Li L. et al., 2019). Generally, natural hydrogels are composed of polysaccharides or proteins whose water-swelling properties making them easy to adsorb and contain nutrients and small molecules (Ahmed, 2015) and improving cell survival and exercise performance (Ahearne, 2014).

Among them, the application of ECM (Extracellular matrix) hydrogel is the representative of natural hydrogel (Francis et al., 2017). Once the nanofiber hydrogel is formed by thermal induction at physiological temperature, the decellularized myocardial matrix hydrogels are possible to quickly create a natural cellular microenvironment for heart tissue and promote myocardial cell repair (Stoppel et al., 2016). Currently, ECM hydrogels are transformed into clinically available injectable biomaterial therapy stages by clinical trials (Wang and Christman, 2016). However, ECM is currently encountered with the lack of effective extraction methods with the reason that the use of chemical reagents for decellularization to remove the nucleus and cytokines of tissue organs can cause damage and denaturation of ECM proteins. Some scholars have proposed the use of supercritical carbon dioxide to extract to reduce damage while with an inevitable challenge of higher cost (Seo et al., 2018).

Therefore, there are many scholars who have developed other natural hydrogels and studied their role in promoting cardiovascular disease repair to replace ECM. Currently developed hydrogels biomaterials include chitosan natural hydrogels (Li J. et al., 2013), hyaluronic acid hydrogels (Yoon et al., 2009), sodium alginate hydrogels (Rocca et al., 2016), and so on. As an immunological linear neutral polysaccharide, hyaluronic acid has multiple acid and hydroxyl groups in the molecule, which can be modified into different forms of hydrogels, including soft or hard hydrogels, as well as nanoparticles and electrospinning. HA-based biomaterial (Burdick and Prestwich, 2011; Larraneta et al., 2018). The presence of reduced left ventricular volume of the glue, increased ejection fraction and the increased wall thickness evaluated by nuclear magnetic resonance (MRI) combined with finite element (FE) models following the treatment of injectable hyaluronic acid hydrogels confirmed the cardiovascular properties of injectable

hyaluronic acid hydrogels, including mechanical properties and degradation properties which have been strongly verified before (Rodell et al., 2016).

Perivascular macrophages maintain the balance between endothelial cells and vascular permeability, but when exposed to foreign substances, they activate the inflammatory response and break the balance leading to vascular embolism (Lapenna et al., 2018). Fortunately, chitosan not only has a group that can be modified to change its properties (Vukajlovic et al., 2019), but also has good compatibility with macrophages (Aussel et al., 2019), suggesting that chitosan can treat cardiovascular diseases through vascular repair. Chitosan injectable hydrogels can also be used to remove free radicals due to their antioxidant properties and degradability, resulting in anti-inflammatory effects to promote heart and blood vessel repair (Dorsey et al., 2015). Similarly, due to the easy modification of chitosan, a suitable biocompatible conductive polypyrrole (PPy)-chitosan hydrogel was designed to effectively maintain myocardial function by connecting isolated cardiomyocytes to increase the electrical conductivity of cardiac tissue (Mihic et al., 2015).

In addition, the easily degradable, non-toxic sodium alginate hydrogel can be modified without modification and induced specific properties for a wide range of applications (Hadley and Silva, 2019). Once the degradable alginate hydrogel was designed to own a microstructure to sustain the release of angiopoietin, it can promote cardiac repair (Rocca et al., 2016) and that is why it widely used in cardiac engineering (Ruvinov and Cohen, 2016). According to the rapid development of alginate hydrogel, a multicenter prospective randomized controlled trial called AUGMENT-HF followed up for 1 year was conducted and found that patients with advanced heart failure (HF) using calcium alginate-injected hydrogel presented better cardiac function and clinical outcome rather than who accepting clinical standard medicine therapy (SMT) (Mann et al., 2016).

Furthermore, the sericin-injected hydrogel also performed an excellent biodegradability whose advantage is promoting the recovery of acute myocardial infarction (MI) by promoting inflammation and promoting cardiomyocytes and vascular repair, with limited application due to the high cost and weaker mechanical properties (Song et al., 2016). In order to improve mechanical properties, silk fibroin (SF) is used as a raw material for hydrogel to increase hydrogel toughness and obtain an appropriate degradation rate for better therapeutic effects (Kambe and Yamaoka, 2019). The following is a classification description of several common natural hydrogel materials (**Figure 1**).

Obviously, natural injectable hydrogels own good cardiovascular repair and biocompatibility, while the defects in uncontrolled function, rapid degradation rate, long gel formation time (Ahearne, 2014; Pena et al., 2018) and high production cost play (Song et al., 2016) a tough role of obstacle in the way to cardiovascular application. Therefore, the development of synthetic hydrogels had become researchers' hot spot.

#### Synthetic Hydrogels

Compared to natural hydrogels, synthetic hydrogels perform a strong mechanical properties and a possibility of being linked to

new functional groups by physical and chemical means to achieve the desired function (Highley et al., 2016; Pena et al., 2018). Besides, extensively alternative synthetic materials range and the low risk of immune rejection implanted in the body (Wang R. M. et al., 2017) also facilitates the development of synthetic hydrogels. However, synthetic hydrogels are encountered with low adhesion, due to the lack of cell attachment sites, and poor biocompatibility (Do et al., 2015).

The biochemical properties of hydrogels would be altered to be suitable to play a role in cardiovascular regeneration engineering due to the addition of chemical groups, the following is the introduction of several common synthetic hydrogels (**Figure 2**). The addition of 2-methylene-1,3-dioxepane (MDO) provided biodegradability, and the introduction of tetraaniline endowed copolymers with desirable electrical properties and antioxidant activities, were added to an in situ hydrogel composing of poly (NIPAM-based) copolymer that presented superior biocompatibility and conductivity (Cui et al., 2014). Furthermore, the biomimetic hydrogel visible-crosslinking with the GelMA provided biodegradability perform a good biocompatibility, while the biosafety of which has been questioned to some extent (Noshadi et al., 2017). It is worth noting that a functional polyion complex added by static cross-linking create a controlled release system of NO to inflammatory tissues to remove the ROS by redox reaction to promote angiogenesis and prolong the retention period for more than 10 days, which solved the problem of short retention time of natural hydrogel (Vong et al., 2018). Equally, the cross-linking with oxygen-suppressing microspheres to release oxygen to infarcted tissue increase myocardial cell survival rate (Fan et al., 2018). Clearly, the plasticity of synthetic hydrogels provides an effective way to treatment based on cardiovascular disease pathways. Moreover, the commercialization of synthetic hydrogels has developed rapidly owing to its designability. An

injectable bioabsorbable stent (IK-5001) was used in patients with clinical MI before a 6-month follow-up, the result evaluated by laboratory examinations showed that IK-5001 was well tolerated without damage to the myocardium (Süselbeck, 2014). Undoubtedly, the prominent superiority of synthetic hydrogels are low manufacturing cost, low immunogenicity and controllability, which provides a huge space for the design and development, while biocompatibility, degradability and biosafety of synthetic hydrogels are issues worthily to be discussed.

Since injectable hydrogels have proven to be a good treatment in clinical practice (Wang H. et al., 2018) while the method is also encountered with the lack of suitable injectable hydrogel materials owing to the respective characteristics of natural hydrogels or synthetic hydrogels. Therefore, the exploration of clinically appropriate injectable hydrogel materials is also one of the research priorities (**Table 1**).

#### INJECTABLE HYDROGEL-BASED NANOCOMPOSITES

It is urgent to develop an injectable hydrogel with a stronger intervention effect as the result of the suitable cardiovascular repair effect of a single natural or synthetic hydrogel usually dissatisfy the needs of clinical treatment. Generally, the hydrogels cross-linking with other substances present a better effect on the cardiovascular repair than which of hydrogel alone (Singelyn and Christman, 2011). It is significant that the nanofiber network structure of hydrogels provides a possibility of the combination with nanocomposites (Johnson et al., 2011). At the same time, in addition to the degradability of the injectable hydrogel (Tous et al., 2011), the particle size of the hydrogel (Yoon et al., 2014) is equally important to cardiovascular repair effects so that nanocomposite with hydrogel considered as a carrier plays a great potential role in the field of cardiovascular tissue engineering (Kurdi et al., 2010). We will review the common types of active nanomaterials complexed in injectable hydrogels for tissue repair as followed (**Figure 3**).

#### Nanoparticles and Nanotubes

Limitations of clinical application of natural hydrogels and the lack of cell sites of synthetic hydrogel was ameliorated by the introduction of nanoparticles and nanotubes. Biocompatibility of nanoparticle composite injectable hydrogel have been demonstrated that the addition of nanoformulations into the ECM maintained the functional behaviors and balance of electrical conductivity into cardiomyocytes (Zhang et al., 2019). The addition of nanotubes not only avoided the low conductivity of natural injectable hydrogels, but also retained the strong mechanical properties of synthetic injectable hydrogels. A pHEMA [poly(2-hydroxyethyl methacrylate)] hydrogel consisting of RNT (rosette nanotubes) and CNF (carbon nanofibers) was designed to increase the conductivity of the myocardium and the mechanical properties to promote the adhesion of cardiomyocytes to enhance cells survival rate (Meng et al., 2013). In addition, the Au-loaded Laponite nanoparticles/ECM injectable hydrogel with superior electrical conductivity to reduces the long-short structure of the hydrogel to create a good environment for the cells (Zhang et al., 2019). Nanotube-injectable hydrogel can increase cell adhesion sites and ameliorate the arrangement structure of hydrogels to ensure cell-to-cell integrity to increase the survival rate of cardiomyocytes. For instance, carbon nanotube-incorporated collagen hydrogels can improve arrangement to promote cell– cell integrity and accelerate the regeneration of functional tissues in 3-D hydrogels (Sun et al., 2017). Recently, in addition to superior biocompatibility and electrical conductivity as well as appropriate adhesion sites, the nanotube composite injectable hydrogels were designed to provide sites for bioactive substance adhesion. In terms of vascular tissue engineering applications, Pacelli et al. (2017) proposes a Nanodiamond-based injectable hydrogel for controlled release of angiogenic factors since the chemical functional group on the surface of the ND efficiently interact with the VEGF and facilitate sustained release from the Polymer staggered network structure. The design of the bioactive substance adhesion site not only provides convenience for retaining the active factors produced by the cardiomyocytes itself, but also provides the possibility for carrying foreign biologically active factors. Significantly, the combination of bionanomaterials and tissue engineering is a definite effective means for cardiac tissue engineering.

#### Drugs

Based on the insufficient therapeutic effect of oral medicine and cardiac stent treatment (Johnson and Christman, 2012), the drug-delivered injectable hydrogel treatment method, a minimally invasive surgical treatment, was proposed. Drugs or natural active substances can be introduced into the site of


TABLE 1 | Preclinical efficacy studies in the last 5 years using natural or synthetic injectable hydrogels for treating myocardial infarction.

MI, myocardial infarction; pcECM, porcine cardiac extracellular matrix cells; hpECM, human placenta-derived hydrogel; CSCL- Ro γ, chloride-Roγ hydrogel; TEMPO Gel, 2,2,6,6-tetramethylpiperidine-1-oxyl hydrogel; HA, hyaluronic acid; BZ, extension of the border zone; ES, end-systolic.

inflammation through using injectable hydrogels as carriers. Oxidative stress usually occurs with MI and lead to excessive generation of free radicals, which damages transplanted cell membrane lipid, proteins and DNA, seriously affecting the treatment of MI. Drug delivery with hydrogel can change the harsh environment of diseased tissue (Hasan et al., 2015). Hydrogels have a highly porous structure in which irregular pores are connected to each other throughout the structure (Trombino et al., 2019), and the drug or a biologically active substance like a liposome-encapsulated alpha-tocopherol (Qu et al., 2019) or Ferulic acid (FA) (Cheng et al., 2016) is uniformly distributed in the porous structure. The inlaid structure of the hydrogel creates a sustained release system to sustained-release to resists oxidative stress inflammatory response and improved cardiomyocyte survival rate. In addition to repairing blood vessels and promoting myocardial cell repair through the antioxidant action of biologically active substances, targeted therapy for drug delivery to damaged myocardium is also an effective means of treating adverse tissue remodeling. For instance, metalloproteinase inhibitor-containing injectable hydrogel was used to locally inhibit matrix metalloproteinases (MMPs), with the aim of reducing adverse tissue remodeling contributed by excess MMP activity (Purcell et al., 2014). At present, drug-encapsulated hydrogel treatment mainly focuses on finding suitable natural or chemical drugs that change the environment of tissue lesions, and designing suitable injectable hydrogel delivery systems. Moreover, the sustained-release effect of the DDS also affects the treatment of cardiovascular disease (Singh et al., 2019). It is worth noting that an injecting TIIA@PDA Nanoparticle-Cross-linked ROS-Sensitive Hydrogels as a nanoscale DDS roperly control of the drug release amount because TIIA@PDA NPs can be seized via the chemical bond between thiolate and quinone groups on PDA (Wang W. et al., 2019). There generally are a variety of sites of hydrogels that

can be modified by reactive groups, such that the drug or active material to forms a composite gel by a cross-linking reaction such as a click chemistry or a supramolecular assembly of a guesthost pair (Highley et al., 2016). This design provides ideas for the development of sustained-release injectable hydrogels and it is an inevitable challenge of controlled release of the drug to be solved by the injectable hydrogel nanoscale DDS.

### Stem Cells

Stem cell therapy, a treatment that has developed concurrently with drug-loaded injectable hydrogels therapy, is well known to play a very important role in cardiac engineering (Cheraghi et al., 2016). Hydrogels protect cells from host inflammation and enable functional integration with damaged myocardium by providing physical support for transplanted cells to maintain their location in the injured area (Sepantafar et al., 2016). Therefore, hydrogels for CVDs ought to be suitable for CMs owing to superior function in tissue repair (**Figure 4**). One of the aspects of current research on injectable hydrogels for transporting cells is to design a hydrogel that is more compatible with cells (Lovett et al., 2009). A polyethylene glycol (PEG) PEGylated fibrin proposed by Geuss et al. (2015) and an injectable hydrogel combained poly (propylene fumarate-co-sebacate-coethylene glycol) with PEGDA designed by Komeri and Muthu (2016) are also suitable for cardiomyocytes. In addition, hydrogel for CVDs should be electrically conductive to generate electrical signals to the myocardium (Sepantafar et al., 2016). An Injectable, flexible, antioxidant and electroconductive hydrogel with suitable biocompatibility, which is equivalent to CMs and provides a porous network structure suitable for embedding of CMs and sustained- generated electrical signal (Komeri and Muthu, 2017).

Myocardium contains approximately four basic cell types: 60–80% heart Fibroblast, 20–40% Cardiomyocyte (CM), smooth muscle cells (SMC) and endothelial cells (EC) (Dolnikov et al., 2006). It is necessary to recruit cardiac precursor cells to compensate for cell loss for high levels of cell slippage occurring during MI (Leri et al., 2005). Injectable hydrogel-based cell therapy techniques provide sufficient cell populations to support the ability to electromechanically couple to Cardiomyocytes (CMS) of host tissues, as well as provide appropriate vascular and connective tissue (Li and Weisel, 2014). The application and effects of various cell re-myocardia repair projects have been fully studied by researchers, among which embryonic stem cells (Lu et al., 2009) and CMs (Habib et al., 2011) are commonly used

materials for cardiac engineering. In addition, mesenchymal stem cells (MSCs) are able to differentiate into cardiomyocytes for acute myocardial repair so that some researchers tried to combine the injectable hydrogel with MSCs to explore more effective therapeutic effects owing to the extremely low differentiation rate of MSCs in the heart and the function of hydrogelinjected network that provide a suitable environment and induce MSC differentiation (Li Z. et al., 2012). A tunable bioactive semi-interpenetrating polymer network (sIPN) hydrogels have been developed with matrix metalloproteinase (MMP) to create an assistive microenvironment for delivery of bone marrowderived mesenchymal stem cells (BMSCs) into the Inflammatory myocardium. The cardiac function of the mice with the injection of hydrogel used as a carrier was improved which provided the basis for the long-term use of transplantation therapy for cardiac stem cells (Wall et al., 2010). Furthermore, an injectable hyaluronic acid (HA) shear-thinning hydrogel (STG) loaded endothelial progenitor cell (EPC) construct (STG-EPC) resulted in prolonged cell retention time and angiogenesis following injection into a myocardial infarction mouse model (Alarcin et al., 2018). In addition to the above-mentioned cells, researchers also used hydrogels to load human amniotic fluid stem cells (Yeh et al., 2010), cardiosphere-derived cells (Li Z. et al., 2011), brown adipose derived stem cells (Wang H. et al., 2014), autologous bone marrow cells (Chen et al., 2014) to promote cardiomyocyte differentiation and angiogenesis and so on (**Table 2**), and the achieved successful results among the cells above indicate that cell nanocomposites based on injectable hydrogels are a useful strategy for cardiac tissue engineering.

It is worth noting that the emerging 3D printing technology also provides a new idea for the design of injectable hydrogel cell nanocomposites (**Table 3**), since fine detail can be included on the micron level with high complexity which provide cells for a superior microenvironment with 3D printing (3DP) technology (Do et al., 2015; Kuo et al., 2015). There is no doubt that the cell composites based on the injectable hydrogel


HA, hyaluronic acid; FA, folic acid; iPS, induced pluripotent stem cells; MSCs, mesenchymal stem cells.

TABLE 3 | Studies of using injectable hydrogels to formulate 3D structure for treating cardiovascular diseases.


3D printable MEGEL/PEGDA3350/alginate hydrogel, extrusion 3D printable mixture of methacrylated gelatin/poly-ethylene glycol diacrylate/alginate (MEGEL/PEGDA3350/alginate); PEG, Polyethylene glycol; HADMSC, human adipose derived mesenchymal stem cells; HAVIC, human aortic valve interstitial cells; HASSMC, human aortic valve sinus smooth muscle cells; NSCs, neural stem cells; ECs: endothelial cells; CMs, cardiomyocytes.

of 3D printing technology will be one of the hotspots of cardiac tissue engineering in the future (Alonzo et al., 2019; Han H. W. et al., 2019).

#### Cell Active Factor

The common injectable hydrogels-based treatments for cardiovascular disease are drug-loaded therapy and stem cell therapy, but both have limitations. The drugs currently in use are usually angiotensin receptor blockers, betablockers, angiotensin-converting enzyme (ACE) inhibitors and aldosterone antagonists, which possibly cause severe adverse reactions in patients, including sleep disturbances, hypotension and difficulty breathing (Jin and Yu, 2018). In order to clinically reduce the incidence of adverse reactions of CVDs, cell active factor therapy, including small molecule protein and exosomes, is considered as a cell-free treatment alternative to drug therapy (Cohen et al., 2014).

Endogenous and exogenous low molecular proteins are usually used in clinically applied cell-free therapies with difficult control of delivery and local release. Obviously, the application of injectable hydrogel probably significantly improved the biological activity of small molecule protein. An injectable hydrogel with a light-sensitive bond and photoresolvability, including polyethylene glycol and heparin-based polymers, successfully wrapped fibroblast growth factor 2 (FGF-2), whose activity was comparable to that before embedding and significantly altered the release profile of FGF-2 (Kharkar et al., 2017). It is worth noting that some researchers attempted to embed horseradish peroxidase (HRP) with a bioactive peptide with a phenolic hydroxyl group into hydrogel to cause a coupling reaction to enhance the function of the active peptide (Wang L. S. et al., 2014). The combination of injectable hydrogel and small molecule protein, which can not only improve its biological activity but also significantly increase the retention time of active protein in the myocardium and achieve sustained release, promotes cardiovascular repair by promoting cell homing and regulating key proteins. MacArthur et al. (2013) successfully loaded the synthetic analog of stromal cell-derived factor 1-α (engineered stromal cellderived factor analog [ESA]) into an injectable hyaluronic acid hydrogel and successfully induced the persistence of endothelial progenitor cells Homing. Moreover, a delivery system

of MMP-2 specific inhibitor peptide CTTHWGFTLC (CTT), which enables CCT to be released continuously within 4 weeks, effectively preventing ECM degradation worsens the condition (Fan et al., 2017). The fusion protein (TAT-HSP27), consisting of the heat shock protein 27 (HSP27) and transcriptional activator (TAT), loaded into microsphere/hydrogel combination delivery devices for controlled release behavior for prolonged periods because the heat shock proteins is a favorable target for protecting cardiomyocytes under environmental stimulation (Lee et al., 2009). Similarly, researchers designed low molecular protein injectable hydrogel nanocomposites with sustained release function according to the mechanism of action of different low molecular proteins: an Poly(ethylene glycol) dimethacrylate(PEGDMA) hydrogel storing local increasing mechano growth factor (MGF), a member of the IGF-1 family with an anti-apoptotic E domain playing a role of a stem cell homing factor (Doroudian et al., 2014), a temperature-sensitive chitosan chloride-RoY (CSCl-RoY) hydrogel (Shu et al., 2015), a hydrogel loading Neuregulin-1β (NRG) which is a member of the epidermal growth factor family (Cohen et al., 2014), a hydrogel loading high-mobility group box 1 (HMGB1) (He et al., 2013), and so on. Song M's findings on association between stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) also demonstrate a better therapeutic effect in combination with bioactive substances (Song et al., 2014).

In addition to the aforementioned small molecule regulatory proteins, certain growth factors, including Thymosin β4 (Tβ4), especially vascular endothelial growth factor (VEGF), should be delivered to heart tissue to reduced poor heart remodeling and improving ventricular function because of the poor cardiac remodeling that occurs later in the myocardial infarction (Anselmi et al., 2000). Thymosin β4 (Tβ4), a 43-amino acid peptide which performs angiogenic and cardioprotective properties, combined with injectable hydrogel resulted in stimulation of Vascular regeneration and cardiomyocyte migration (Shaghiera et al., 2018). Transportation of vascular endothelial growth factor (VEGF) and other angiogenic factors to promote angiogenesis are both potential treatment for cardiovascular disease and a vital aspect of tissue regeneration (Cao et al., 2009). The myocardial thickness and the density blood vessels of the rat myocardial infarction model were larger than that of the group without treating, following the injection of a novel temperature-susceptible aliphatic polyester hydrogel (HG) crosslinked with VEGF (Wu et al., 2011). Retention of highly vascularized cardiomyocytes is a limiting factor in growth factor therapy although it presents superior performance in cardiovascular repair (Rufaihah et al., 2017). The Dex-PCL-HEMA/PNIPAAm hydrogelcon containing VEGF developed by Zhu et al. (2016) and the injectable hydrogel amalgamated polyethylene glycol with fibrinogen (PEG-fibrinogen) loaded with VEGF-A designed by Rufaihah et al. (2013) both are able to release and store VEGF in a controlled manner and achieve better cardiac repair than VEGF alone. Moreover, in order to present a superior repair effect, an polyethylene glycol-fibrinogen (PF) hydrogels was manufactured for sustained dual transportation of VEGF and angiopoietin-1 (ANG-1) to promote myocardial therapy (Rufaihah et al., 2017). Recently,

researchers' research hotspots have shifted from the development of nano-growth factor injectable hydrogels to exploring which nano-growth factor injectable hydrogel complexes present superior cardiac repair functions. Therefore, the growth factors, including hepatocyte growth factor (HGF) (Ruvinov et al., 2010), insulin-like growth factor 1 (IGF-1) (Koudstaal et al., 2014; Fang et al., 2015), etc. that have been explored in combination with injectable hydrogels and present good myocardial repair effects, are suitable in myocardial regeneration.

Though stem cell treatment is one of the effective strategies for the CVDs, the stem cell clinical transplantation is limited by the low cell implantation and survival rate (Li Z. et al., 2018). Exosomes have recently become recognized as new candidates for cell-free treatment (Emanueli et al., 2015; Davidson et al., 2017; Zhang et al., 2017). Exosomes, extracellular vesicles derived from endosomes and the vital mediators of intercellular communication (Ibrahim and Marbán, 2016), are released by major cardiac cells, including cardiomyocytes, fibroblasts and endothelial cells (Barile et al., 2017), to regulate cellular function (Poe and Knowlton, 2018). Direct use of paracrine factors is an attractive strategy that play a role in therapy via cytokine regulatory pathway, taking cell implantation or survival rate out of considered (Han C. S. et al., 2019). The development of injectable hydrogel nanocomposites for composite exosomes has raise researchers' attention since hydrogels are appropriate carrier materials. Significant improvement of exosome implantation on injured myocardium has been proven by that an injectable shear-thinning gel (STG) carrying EVs probably effectively improve myocardial function and increased the hemodynamics as well as the number of blood vessels (Chen et al., 2018). Furthermore, exosomes generated by human adipose-derived stem cells (hASCs), Gelatin and Laponite <sup>R</sup> were combined to formulate a shear-thinning, nanocomposite hydrogel (nSi Gel) which was considered as an injectable carrier of secretome (nSi Gel+), and the results indicate an increasing density of blood vessels around the myocardium, an improvement in myocardial function and a reduction in scar area (Waters et al., 2018). However, the residence time and stability of exosomes are the major challenges in the clinical application of exosomes in recent years. Therefore, good biocompatibility and retention time are the vital research directions of exosomes-delivered injectable hydrogels. The stability and cardiovascular application of chitosan-injectable hydrogel-encapsulated paracrine factors in vivo were demonstrated by the results which indicated that exosomes showed high retention rates and promote vascular repair and formation (Zhang K. et al., 2018). Since the principle of stem cell therapy is based on the release of paracrine factors around the myocardial injury tissue to interfere with the progression of myocardial infarction (Mirotsou et al., 2011), exosome nanocomplexes with injectable hydrogels plays a significant role as promising alternative therapies.

#### Genetic Material: RNA/DNA

Since stem cell and foreign active substance suppression is prone to collective immune rejection (Lu et al., 2010), embedding exogenous genetic material (DNA/RNA) into injectable hydrogels to produce autologous histocompatibility

stem cells to promote myocardial regeneration, is an appreciated method in CVDs therapy. According to the pathway of MMP2 related to the cardiac harmful remodeling process, an injectable hydrogel complexed with siRNA up-regulate the hydrolytic activity of MMP2 protein to inhibit the harmful remodeling process of the heart and promote heart repair (Wang L. L. et al., 2018). In addition, an injectable Hyaluronan-Based hydrogel modulate remodeling of the myocardial extracellular matrix (ECM) by injecting a hyaluronic acid-based reservoir delivering exogenous microRNA-29B (miR-29B) (Monaghan et al., 2018). Noteworthily, protocols for injection-based delivery of Cre-CPP by ultrasound-guided injection to cardiac muscle in mice is mature owing to widely used technique of Cre-mediated DNA recombination at loxP sites (Chien et al., 2017), which provides a feasible mean for genetic material composite hydrogel. The study results above strongly demonstrated that genetic material (DNA/RNA) would be considered as the potential candidate for myocardial regeneration.

### Composite Use of Nano-Bioactive Substances

Cell therapy is currently the most mature treatment in cardiac tissue engineering which encounters with the problems of immune rejection of foreign cells, low survival rate and short residence time (Lu et al., 2010) so that researchers have begun to combine biologically active substance with stem cells to increase stem cell functional activity. It is common to carry out the mixture of cell growth factor and stem cells: combined polyethylene glycol hydrogel (PEG), a hydrogel consisting of human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and erythropoietin (EPO) (Chow et al., 2017), a hydrogel consisting of insulin-like growth factor (IGF-1) and delivering mesenchymal stromal cell (MSC) (Wang et al., 2010), injectable linear engineering protein hydrogels encapsulating VEGF and human induced pluripotent stem cellderived endothelial cells (hiPSC-EC) (Mulyasasmita et al., 2014) and the like. What raise researchers' attention is the combined use of multiple nano-bioactive substance. An injectable matrix metalloproteinase (MMP)-responsive, bioactive hydrogel used as an in situ forming scaffold to deliver thymosin β4 (Tβ4), along with vascular cells derived from human embryonic stem cells (hESC), which useful in engineering sustained tissue preservation (Kraehenbuehl et al., 2011). Noteworthily, Karam et al. (2014) proposed to integrate human adipose-derived stem cells (ADSCs) and pharmacologically active microcarriers (PAMs), a threedimensional (3D) carrier of cells and growth factors, into an injectable hydrogel (HG), to obtain a system that stimulates the survival and/or differentiation of the grafted cells toward a cardiac phenotype. This study suggests that the use of 3D nanocomposites is one of the more effective means and a hot spot in cardiovascular repair development. From a gene therapy perspective, an injectable biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene is significant for myocardial therapy (Paul et al., 2014), which suggested the feasibility of gene therapy combined with cardiac tissue engineering treatment is illustrate.

### THE MAJOR MECHANISM USING BY INJECTABLE HYDROGEL IN CVDS

Although injectable hydrogel as a desirable candidate for CVDs with numerous outstanding properties has been widely used in clinical treatment, its mechanism of promotion restoration of CVDs remains unclear. Herein several possible paths are illustrated in the following parts.

### The Promotion Effect of Recovery in CVDs via Angiogenesis

Recently, therapeutic angiogenesis, or the delivery of angiogenic agents such as growth factors (GFs) (Madonna and De Caterina, 2011), NO (Vong et al., 2018), and some drugs (Qi et al., 2018) to promote revascularization of ischemic tissue, holds great promise in the fields of treating CVDs. As shown in the **Figure 5**, a variety of GFs are indispensable for the different phrase of neovascularization. Nevertheless, this approach has been confronted with several obstacle when hydrogel used as

a delivery device, among which, the difficulties of keeping angiogenic GFs retained locally at the injury site and released gradually to allow adequate time for growth of new blood vessels must be overcome before successful clinical implementation. Basing on the status, recently a growing body of evidences have shown evidence of injectable hydrogel's promising effects on cardiac recovery through addressing the problems mentioned above in the process of revascularization.

GFs therapy shows great promises in treating ischemia, but the retention of GFs in the highly vascularized myocardium is mainly obstacle of its widely application. Some researchers (Feng et al., 2017) designed an injectable hydrogel scaffold composed of Konjac glucomannan (KGM, a naturally derived polysaccharide with capability to activate macrophages/monocytes to secrete pro-angiogenic/-mitogenic GFs) and heparin (Hep, one of the glycosaminoglycan molecule that binds numerous proangiogenic GFs and sequester them). Therefore, the injectable hydrogel was capable of promote revascularization via first stimulating the secretion of endogenous pro-angiogenic growth factors (GFs) and next sequestering these GFs inside the scaffold. Furthermore, controlling the degradation kinetics of injectable hydrogel would be an effective strategy to prolong the retention of GFs. Gel-CDH/HA-mCHO (Hozumi et al., 2018) hydrogels, a new injectable hydrogel synthetized by carbohydrazide -modified Gel (Gel-CDH) and mono-aldehyde modified-HA (HA-mCHO), was degraded much more slowly because of stable Schiff's base formation between aldehyde and carbohydrazide groups. Additionally, the limited function of one single GF in the delivery system was one of the restrictions. Therefore, polyethylene glycol-fibrinogen (PF) hydrogels (Rufaihah et al., 2017) was employed and incorporating with vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to achieve the effect of dual delivery of GFs in a sustained release way. Besides, other materials also play the crucial roles on cardiovascular diseases. It is generally known the significance of nitric oxide (NO) but its therapeutic application is hampered because of its highly short half-life and rapidly consumed by excessive producing of ROS. Thereby, a new injectable hydrogel, namely NO-RIG (Vong et al., 2018), was prepared which consisted of PArg-PEG-PArg (NO releasing polymer) and PMNT-PEG-PMNT (ROS scavenging polymer), in a complex with polyanion PAAc, so that NO's effect on promoting angiogenesis were improved.

In addition to the adequate retention time of GFs at the targeted district, transporting the GFs to the injury site accurately is also important for inducing angiogenesis. Given the acidic microenvironment (Khabbaz et al., 2001; Kumbhani et al., 2004; Ding et al., 2011; Zhao et al., 2012; Wei et al., 2017) of ischemic myocardium, a pH- and temperature-responsive, injectable hydrogel has been synthesized (Garbern et al., 2011) with several pH- and temperature-responsive random copolymer, including N-isopropylacrylamide (NIPAAm), propylacrylic acid (PAA), and butyl acrylate (BA) by reversible addition fragmentation chain transfer polymerization. This polymer existed as a liquid at room temperature and pH 7.4 but becomes a gel at 37◦C and pH 6.8. Thereby, the hydrogel successfully provided sustained release of basic fibroblast growth factor (bFGF) at the injury site locally and the angiogenesis effect of bFGF were improved. Similar, another new (Wu et al., 2011), temperature-sensitive, aliphatic polyester hydrogel (HG) conjugated with (VEGF) was designed and also shown good therapeutic effect on attenuating adverse cardiac remodeling and improved ventricular function when injected after an MI.

In a word, therapeutic angiogenesis showed remarkably therapeutic potential in cardiovascular disorders by changing the status of one single material delivering, prolonging the retention of pro-angiogenic factor and transmitting them accurately to the targeted site.

#### The Therapeutic Effect in CVDs Through Promoting Stem Cell Homing

Stem cell homing, the capability of stem cells to find their destination in a targeted organ through the bloodstream (Zhao and Zhang, 2016), was another promising therapeutic strategy in CVDs, especially in Myocardial infarction (MI). Here, an example of mesenchymal stromal cells (MSC) in **Figure 6** (Marquez-Curtis and Janowska-Wieczorek, 2013) was used to illustrate the mechanisms of stem cell transendothelial migration toward injured tissue. As we can see in the **Figure 6**, the effect of MSC homing was achieved by production of a series of some critical factors such as homing receptors including CXCR4. Although the mechanism of stem cell homing has been understood recently, the clinical utilization of stem cells was mainly hindered by their poor homing efficiency. In the recent years, a growing body of clinical evidence suggests that injectable hydrogel is a promising biomaterial that were capable of enhancing stem cell homing efficiency in treatment of numerous filed of regeneration medicine, such as in periodontal regeneration (He et al., 2019), cartilage regeneration (Lu et al., 2018), as well as corneal epithelium regeneration (Tang et al., 2017).

"Homing" directs stem cells migration through different signaling pathways, mediated by released chemokines or growth factor receptors on the surface of stem cells. Over the past decade, the most thoroughly studied stem cell homing factor is the chemokine SDF-1α/CXCL12 (Ghadge et al., 2011), based on which, a number of researchers committed themselves to develop some new delivery devices loaded with these promoting homing factors in order to improve the myocardium repair. Recently, a combined strategy (Naderi-Meshkin et al., 2016) was implemented via mixing human adipose tissue-derived MSCs (hASCs) into chitosan-glycerophosphate-hydroxyethyl cellulose (CH-GP-HEC) injectable hydrogel and as a result, site-directed homing efficacy and retention of ASCs increase by harnessing SDF1/CXCR4 axis. Similar, The E domain of mechano growth factor (MGF) (Doroudian et al., 2014) peptide is anti-apoptotic and a stem cell homing factor. As shown in a study, a microrod delivery device of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogel could absorb cells and decrease apoptosis of myocytes via incorporating MGF.

On the other hand, a comfortable microenvironment for stem cell survival is also of great significance. For example, as shown in a current report, ROS (Song et al., 2010) in MI microenvironment negatively regulated graft cell death and stem cell adhesion,

finally caused anoikis of transplanted cells. Hence, changing the unfavorable MI microenvironment for stem cell homing and proliferation would have better therapeutic efficiency in cellular cardiomyoplasty. Chitosan hydrogel (Liu et al., 2012) were able to improve the MI microenvironment, enhance stem cell engraftment and survival through ROS scavenging. Furthermore, adequate blood vessel would be another crucial supportive condition for cell survival and proliferation. Thus, some scientists (Song et al., 2014) designed a biomimetic hydrogel incorporated with both stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) in treating chronic myocardial infarction (CMI) and consequently, regeneration of cardiac function model were significantly promoted. By and large, the stem cell homingbased injectable hydrogel emerged as a promising therapy in treatment ischemic infarction.

All in all, as for treating CVDs, revascularization and stem cell homing are the two major effective strategies through injectable hydrogel as a delivery system in the recent years. Besides, there existing other approach that would hold great therapeutic potential in the field of CVDs treatment, for instance, taking advantage of an injectable hyaluronic acid (Zhang Y. et al., 2018) (HA) hydrogel to deliver miRNA in order to induce proliferation in cardiomyocytes through its inhibition of Hippo signaling via a direct binding site on the 3<sup>0</sup> UTR, such as miR-302 (Wang L. L. et al., 2017) and miR-1825 (Pandey et al., 2017), developing a new hydrogel (Qi et al., 2018) from supramolecular assembling of a synthetic glycol peptide which endows the hydrogel with the capacity of endothelial cell adhesion and proliferation due to its high density of glucose moieties, as well as using Ferulic Acid (Kanki and Klionsky, 2009; Wu et al., 2012; Wiley et al., 2013) (a natural antioxidant that is most abundant in vegetables, especially in eggplants and maize bran) to form a new injectable hydrogel (Cheng et al., 2016) to effectively promote the recovery of Cisd2 deficiency induced damage.

## SUMMARY AND PERSPECTIVE

Injectable hydrogels have shown promise in promoting cardiovascular disease repair for years from single hydrogels (natural or synthetic hydrogels) to hydrogel- based nanocomposite. To the begin, natural hydrogels were attracting attention because of their non-toxicity, immunogenicity, and excretion of metabolites (Li L. et al., 2019), such as. However, due to the lack of effective extraction methods (Francis et al., 2017), ECM were gradually replaced by other natural hydrogels, such as hyaluronic acid hydrogels (Yoon et al., 2009) (an immunological linear neutral polysaccharide with multiple acid and hydroxyl groups, which can be modified into different forms of hydrogels, including soft or hard hydrogels, as well as nanoparticles and electrospinning), chitosan natural hydrogels (which had good compatibility with macrophages and antioxidant properties and degradability) (Aussel et al., 2019), sodium alginate hydrogels (Hadley and Silva, 2019), and so on. On the other hand, synthetic hydrogels have been attached much importance since their strong mechanical properties and various and controllable function by physical and chemical means (Pena et al., 2018). Synthetic hydrogels are low manufacturing cost and could provide a huge space for the design and development, while their biocompatibility, degradability, biosafety and low adhesion for cell (Do et al., 2015) are issues worthily to be discussed.

Recently, since the porosity of hydrogel of hydrogels provides a possibility to combine with nanocomposites (Johnson et al., 2011), and the hydrogels cross-linking with other substances

show better cardiovascular repair effect than which of hydrogel alone (Singelyn and Christman, 2011), several types of active nanomaterials complexed in injectable hydrogels for tissue repair have been explored. For instance, injectable hydrogel-based composite carrying drug and/or other bioactive materials have been explored and the effective have been achieved.

According to different treatment mechanisms and different aspects of concern, the invention of different nano-composite injectable hydrogels was designed. For example, drugs-delivered injectable hydrogels mainly improve the environment of myocardial tissue with excessive oxidative stress, and small molecule proteins-delivered and exosomes-delivered injectable hydrogels are mainly involved in the mechanism of hormone regulation in the process of self-repair of myocardium. Celldelivered injectable hydrogels therapy is mainly to provide a large number of favorable healthy cells to promote the process of myocardial repair, while pure hydrogel therapy is mainly to provide the stent of myocardial cells. Recently, because of foreign material is prone to collective immune rejection, embedding foreign genetic material (DNA/RNA) into injectable hydrogels might be an appreciated method in CVDs therapy.

Although much progress has been made due to injectable hydrogel's wide application in the CVDs, some limitations remain challenges that need to be overcome before successful clinical implementation, for instance, the exploration of appropriate approach for injection (Chen et al., 2017), the method for controlling and tailoring release profiles of targeting agents confronting the complicated biological processes (Kharkar et al., 2013; Annabi et al., 2014; Yesilyurt et al., 2016), the substantial requirement for hydrogel's rheological and mechanical properties (Unterman et al., 2017), their capacities to be scaled up to a

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#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

This review was supported by National Natural Science Foundation of China (Nos. 81972488, 81701836, 81973013), The Basic Research Start-up Project (QD2018N005), Guangdong Key R&D Program (No. 2019B020210002), Guangdong Natural Science Foundation (C1051164), High-level Talent Introduction Project (C1034220), and The Eighth Affiliated Hospital of Sun Yat-sen University Outstanding Youth Reserve Talent Science Fund (FBJQ2019002).

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Liao, Yang, Deng, Hao, Mao, Zhang, Liao and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fbioe-08-00251 March 28, 2020 Time: 18:58 # 18

# Copper Sulfide Nanoparticles-Incorporated Hyaluronic Acid Injectable Hydrogel With Enhanced Angiogenesis to Promote Wound Healing

#### Wencheng Zhou1,2† , Liu Zi1,3† , Ying Cen<sup>2</sup> , Chao You1,4,5 and Meng Tian1,2,4,5 \*

#### Edited by:

Wenguo Cui, School of Medicine, Shanghai Jiao Tong University, China

fbioe-08-00417 May 6, 2020 Time: 19:47 # 1

#### Reviewed by:

Jianxun Ding, Changchun Institute of Applied Chemistry (CAS), China Chaenyung Cha, Ulsan National Institute of Science and Technology, South Korea Wei Tao, Harvard Medical School, United States

#### \*Correspondence:

Meng Tian tianmong007@gmail.com; 6744710@qq.com

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology Received: 19 March 2020 Accepted: 14 April 2020 Published: 08 May 2020

#### Citation:

Zhou W, Zi L, Cen Y, You C and Tian M (2020) Copper Sulfide Nanoparticles-Incorporated Hyaluronic Acid Injectable Hydrogel With Enhanced Angiogenesis to Promote Wound Healing. Front. Bioeng. Biotechnol. 8:417. doi: 10.3389/fbioe.2020.00417 <sup>1</sup> Neurosurgery Research Laboratory, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China, <sup>2</sup> Department of Burns and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, China, <sup>3</sup> Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China, <sup>4</sup> Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu, China, <sup>5</sup> West China Brain Research Centre, West China Hospital, Sichuan University, Chengdu, China

Skin wound caused by trauma, inflammation, surgery, or burns remains a great challenge worldwide since there is no effective therapy available to improve its clinical outcomes. Herein, we report a copper sulfide nanoparticles-incorporated hyaluronic acid (CuS/HA) injectable hydrogel with enhanced angiogenesis to promote wound healing. The prepared hydrogel could not only be injected to the wound site but also exhibited good photothermal effect, with temperature increasing to 50◦C from room temperature after 10 min of near-infrared light irradiation. The cell culture experiments also showed that the hydrogel has no cytotoxicity. In the rat skin wound model, the hydrogel treated wounds exhibited better healing performances. Masson's trichrome staining suggested that collagen deposition in wounds treated with the hydrogel was significantly higher than other groups. The immunohistochemical staining showed that the hydrogel can effectively upregulate the expression of vascular endothelial growth factor (VEGF) in the wound area at the incipient stage of healing, and the CD 31 immunofluorescence staining confirmed the enhanced angiogenesis of the hydrogel. Taken together, the prepared CuS/HA hydrogel can effectively increase the collagen deposition, upregulate the expression of VEGF, and enhance the angiogenesis, which may contribute to promote wound healing, making it a promising for application in treating skin wound.

Keywords: copper sulfide, nanoparticle, hydrogel, angiogenesis, wound healing

## INTRODUCTION

Skin wound resulting from trauma, inflammation, surgery, or burns is a common issue that needs to be treated immediately (Browne and Pandit, 2015). However, up to now, an effective wound healing remains a significant challenge due to its complex biological process where various intracellular and intercellular pathways should be activated and coordinated to accelerate and enhance the healing

(Das and Baker, 2016). Among these biological processes, angiogenesis plays many important roles during the healing since the newly formed vessels are of great significance to nutrients and growth factors transport and granular tissue growth, and thus encouraging angiogenesis is regarded as one of the most essential steps for promotion of wound healing. In this regard, earlier studies mainly focused on the introduction of angiogenic growth factors such as vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). Nevertheless, the use of growth factors was limited by their high cost and side effects.

Recently, some metal ions have attracted more and more research interest due to their vascularization effect (Yu et al., 2019). For example, copper ions have been reported that could stimulate angiogenesis by secretion of VEGF and thus promote wound healing. Moreover, nano-formed copper such as copper sulfide nanoparticles (CuS NPs) is capable of photothermal therapy induced by near-infrared (NIR) light irradiation which would be effective in killing bacteria as a non-resistant and minimally invasive process (Zhou et al., 2015; Feng et al., 2019). As a result, CuS NPs may offer both angiogenesis and antibacterial ability, both of which are beneficial to accelerate wound healing (Li Z. et al., 2018; Shanmugapriya and Kang, 2019). However, the use of copper ions or CuS NPs alone is not suitable for wound healing since direct contact may produce inflammation in skin tissues and the administration will easily detach from the wound as well. Considering these shortages, it is potential to incorporate copper ions or CuS NPs into carriers for wound healing.

Hydrogel composed of cross-linked hydrophilic polymer chains has its innate merits as wound dressing or drug carrier for wound healing, resulting not only from its good water-reserving ability that is suitable for absorption of wound exudates and skin cell survival and metabolism but also from its interconnected pores that provide three-dimensional networks with large volume and surface area for the incorporation of a drug (Li S. et al., 2018). The hydrogel can be prepared by natural polymers or synthesized ones. Compared to synthesized polymers, natural ones are more biocompatible, and more importantly, they are usually derived from the extracellular matrix (ECM). For instance, hyaluronic acid (HA) is one of the major components of the ECM consisting of disaccharide units of D-glucuronic acid-N-acetylglucosamine which contains many carboxyl and hydroxyl groups that is beneficial to water conservation for the skin. Furthermore, it was found that HA also plays an important role in the process of angiogenesis, suggesting that HA is a promising candidate to prepared hydrogel for wound healing.

In this work, we hypothesize to prepare a CuS NPsincorporated HA injectable hydrogel, which can not only be injected to the wound site but can also be capable of photothermal therapy induced by NIR light irradiation (as shown in **Figure 1**). Moreover, both the use of CuS NPs and HA may benefit to enhance angiogenesis and thus promote wound healing. To address this hypothesis, CuS NPs and thiolated HA were first synthesized, and then CuS/HA hydrogel was prepared and characterized in terms of gelation time, morphology, photothermal effect, and cytotoxicity, before the

in vivo angiogenesis and wound healing ability was evaluated in a rat skin wound model.

### MATERIALS AND METHODS

#### Materials

Copper chloride (CuCl2), sodium sulfide (Na2S 9H2O), and sodium citrate were purchased from Kelong Co., Ltd. (Chengdu, China). Methoxy-PEG-thiol (SH-PEG, molecular weight 5000 Da) was purchased from ToYongBio Co., Ltd. (Shanghai, China). HA sodium salt from Streptococcus Equi was supplied by Aladdin Co., Ltd. (Shanghai, China) 0.5, 5<sup>0</sup> -Dithiobis (2-nitrobenzoic acid; DTNB), N-(3-Dimethylaminopropyl)- N'-ethyl carbodiimide hydrochloride (EDAC), cysteamine, and dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO, United States). N-hydroxysuccinimide (NHS) was obtained from Pierce. Isoflurane was obtained from RWD Life Science Co., Ltd. (Shenzhen, China). Cell counting kit-8 (CCK-8) was obtained from Dongren Chemical Technology Co., Ltd. (Shanghai, China). Hematoxylin and eosin (H&E) stains and Masson's Trichrome Stain Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The antibody of endothelial growth factor anti-VEGF was bought from Proteintech (Chicago, United States). The anti-CD31 antibody was from Affinity Biosciences, Inc. (Cincinnati, OH, United States). Deionized water (18 M) was obtained from a Milli-Qsynthesis system (Millipore, Billerica, MA, United States). All the above reagents are used directly without further purification.

### Synthesis and Characterization of CuS Nanoparticles (NPs)

The synthesis of CuS NPs was carried out as a facile hydrothermal route as follows (Zhou et al., 2010). Briefly, 10 mL of sodium citrate (1.0 mg mL−<sup>1</sup> ), and 10 mL of CuCl2·2H2O (0.85 mg mL−<sup>1</sup> ) aqueous solutions were added into 30 mL of ultrapure water in sequence. The whole solution was protected by argon and stirred for 30 min at room

temperature. After that, dropwise added 50 µL of Na2S·9H2O (78 mg mL−<sup>1</sup> ) aqueous solution into the reaction and kept on stirring another 5 min. In this process, the color of the reaction solution gradually changed from light blue to light yellow, orange, and finally to dark brown. Next, the reaction was transferred to a 90◦C oil bath to react for 15 min to form green-colored CuS-citrate NPs. The whole mixture was moved to ice-cold water. To obtain PEG-coated NPs, 1 mg of SH-PEG was added to the 1 mL CuS-citrate NPs solution (200 µgmL−<sup>1</sup> ) to introduce the PEG coating. The reaction was performed overnight at room temperature to obtain PEGcoated CuS NPs.

The morphology of the CuS NPs was observed by transmission electron microscopy (TEM) performed on a Hitachi HT7700 (Japan). Specimens were prepared by adding 50 µL micellar solutions onto a copper grid followed by staining with phosphotungstic acid (1 wt%) for 1 min and then dried with filter paper. The size, zeta potential, and polymer dispersity index (PDI) of the CuS NPs were determined by dynamic light scattering (DLS) on a NanoBrook Series Particle/Protein Size and Zeta Potential Analyzer.

### Synthesis and Characterization of Thiolated HA

The synthesis of thiolated HA is illustrated in **Supplementary Figure S1A**. The thiolated HA was synthesized by the coupling of cysteamine onto the HA molecular chains through EDC chemistry. Briefly, 1 g of HA sodium salt was dissolved in 200 ml distilled water, and then cysteamine and EDAC were added as solids to the reaction with a molar ratio of -COOH/cysteamine/EDAC 1:2:2. The pH of the reaction solution was maintained at 4.75 by the addition of 1 M HCl. After 4 h the reaction was stopped by neutralizing the solution by addition of 4 M NaOH. The solution was dialyzed (3500 cutoffs) against distilled water for 3 days at room temperature. 5 g DTT was then added to the resulting solution and the pH of the solution adjusted to 8.5. After stirring for 8 h under N2, the pH of the solution was adjusted to 4.0 by the addition of 1 M HCl. The resulting solution was first dialyzed (3500 cutoffs) against HCl solution (pH 4.0) containing 100 mM NaCl under N2, followed by dialysis (8000 cutoffs) against HCl solution (pH 4.0) under N2. The solution was clarified by centrifugation, and the supernatant was sterilized with a 0.2 µm Millipore filter and then lyophilized. The product was stored at −20◦C and protected under N2. The degree of substitution (DS) of free thiols was determined using the Ellman method (Shu et al., 2002). The structure of thiolated hyaluronan was characterized by 1H NMR spectrum in D2O.

## Preparation of the Injectable CuS/HA Hydrogel

The CuS/HA hydrogel was prepared by simply mixing with thiolated HA solution and CuS NPs solution to obtain the precursor gelation solution, and then the gelation was initiated with the hydrogen peroxide solution. Gelation time was determined by a test tube inverting method. The final concentration of thiolated HA, CuS NPs, and hydrogen peroxide was 2% w/v, 200 µg/ml, and 0.03% v/v, respectively. This injectable CuS/HA hydrogel was named G II. The blank hydrogel without CuS NPs was used as a control (named G I). Scanning electron microscopy (SEM) was used to observe the morphology of the freeze-dried hydrogel. After coating with gold, the cross-sectional morphology was viewed with a ZEISS EVO 10 microscope. The storage and loss modulus were measured with a plate-to-plate rheometer (MCR 302, Anton Paar, Ashland, VA, United States) using a 25 mm plate under a constant strain of 1% and frequency of 10 rad/s.

### In vitro Photothermal Effect

The photothermal effect of CuS NPs was carried out in two parts, nanoparticles solution, and hydrogels. For the solution, different concentrations (200, 100, 50, 20, and 10 µg/mL) were exposed to 808 nm laser at 1 W/cm<sup>2</sup> for 10 min and the temperatures were recorded every other min interval by an infrared imaging camera (FLIR ONE PRO, United States). To further test the stability of the photothermal effect, 200 µg/mL CuS NPs were determined to heat by the laser in the same condition and then cool down naturally for several cycles. On the other hand, the photothermal effect of CuS/HA hydrogels was evaluated in the same approach as above.

### Cell Experiments

Mouse embryonic fibroblast (NIH/3T3) cell lines were chosen to perform the cell experiments. The cells were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, United States), 100 mg/ml penicillin, and 100 mg/ml streptomycin. The cells were grown at 37◦C in an atmosphere of 95% humidified air containing 5% CO2. Experiments were carried out when cells were in the logarithmic phase of growth. Cytotoxicity is a classic problem for biomaterials. According to ISO10993 part 5 guidelines 3, CCK-8 was used to examine the cytotoxic effects of the hydrogel, and solution of CuS NPs. For the test of CuS NPs solution, samples of three concentrations were used for testing (200, 100, and 50 µg/ml). For the injectable hydrogel test, both GI and GII were included. The extracts of these samples were prepared by the incubation of each sample with 1 ml of DMEM supplemented with 10% FBS for 48 h at 37◦C.3T3 cells were cultured with common conditions [DMEM supplemented with penicillin (100 mg/ml), streptomycin (100 mg/ml), and 10% FBS, 37◦C, 5% CO2] until they reached approximately 80% confluency before cytotoxicity assay. After that, the cells were seeded into 96-well plates at a density of 3000 cells/well. After another 24 h incubation, the cells cultured in medium without test samples were used as a control group and the culture media of other wells were discarded and replaced with the medium containing the three concentration solutions of samples or two extracts (12.5%, v/v). After further cultured for designed time (0, 24, 48, and 72 h), the cells were subjected to cell toxicity assays by CCK-8. Briefly, the cells washed by phosphate buffer saline (PBS) and were incubated in a fresh culture medium containing 10% CCK-8 to incubate for 3 haway from light. Finally, the supernatant was transferred

and the absorbance of the solution was determined at a test wavelength of 450 nm. The ratio of the optical density (OD) of the test samples to that of the control group presented the cell proliferation rate.

#### Cutaneous Wound Healing Experiments

The wound healing experiment in vivo for samples was conducted on Sprague–Dawley male rats (SD rats). All the animal experiments and procedures were approved by the Animal Ethical Committee of the West China Hospital of Sichuan University in compliance with Chinese national guidelines for the care and use of laboratory animals. Eight weeks old SD male rats (200 ∼ 220 g) were purchased from Da-Shuo Laboratory Animal Co., Ltd. (Chengdu, China). The rats were kept under controlled temperature and humidity with food and water ad libitum. After standard anesthetization with anesthetic ventilator using isoflurane (4%for induction in an induction chamber, and 2% for maintenance), the back of each animal was shaved to expose the injection site. A skin biopsy apparatus (6 mm) was used to make the round section of the full-thickness skin injury on the back of the rat skin. All procedures were performed under aseptic conditions during the surgery process. There were six round wounds (6 mm in diameter) created on the back of each rat and 7 rats were included, so there were 30 wounds served as experimental subjects, and 12 wounds as back-ups in case of experimental animal death. After the removal of wound skin, 100 µL of different injectable hydrogels were synthesized in situ (methods as mentioned before) 0.30 wounds were randomly divided into 6 groups (I-VI). Group I was the blank control group with nothing dressed after a skin injury, group II wounds received NIR radiation after the operation, group III wounds were dressed with the hydrogel G II, wounds in group IV were dressed with the hydrogel G II and treated with NIR, group V wounds were dressed with the hydrogel G I, and group VI wounds were dressed with the hydrogel G I and treated with NIR. For NIR treated groups, the wound area was irradiated with an 808 nm NIR laser (1 W/cm<sup>2</sup> ) for 10 min after the operation 10 min, 2 days, and 3 days and infrared photos were taken at the same time. The gross image of the wounds was taken after the operation on days 0, 3, 7, 10, and 14. To monitor the wound healing process, ImageJ software was employed to process the images to obtain the wound area. The healing rate (HR) was calculated, according to the formula as below:

$$HR = \frac{(\text{S}\_0 - \text{S}\_{\text{x}})}{\text{S}\_0} \times 100\%$$

where S<sup>0</sup> was the initial area on day 0 and S<sup>x</sup> represented the wound areas on days 3, 7, 10, and 14, respectively.

#### Hematoxylin-Eosin and Masson's Trichrome Staining

Rats were sacrificed on the 3rd, 7th, and 14th days after the operation, respectively, and the skin tissue (1 × 1 cm<sup>2</sup> ) containing the wound was taken for dermatological examination. The samples were fixed in 4% paraformaldehyde, dehydrated in graded alcohol, and embedded in paraffin. The tissue blocks were sectioned into 5 µm thick slices. The sections were stained via H&E and Masson's trichrome to carry on histological analysis and collagen formation assessment. 8 All photos were captured by a microscope (BX43, Olympus, Japan). Histopathological scores of HE slices were employed to a semiquantitative evaluation of wound healing according to the criterion of Eldad et al. (1998). Collagen volume fraction (CVF) was utilized to assess collagen formation (Singh et al., 2019). Specifically, 6 regions of interest (ROIs) per HPF were recorded under a microscope. Then, the CVF of each ROI was assessed using the ImageJ software to semiquantitative analysis collagen content.

#### Immunohistochemical and Immunofluorescence Staining

Angiogenic vessel markers CD31 and VEGF were chosen in our experiment because they play an important role in angiogenesis (Nowak-Sliwinska et al., 2018). Immunofluorescence and immunohistochemical staining of CD31 and VEGF were carried out as previous literature. The rehydrated skin tissue sections were boiled in sodium citrate buffer for about 20 min before anti-CD31 and anti-VEGF (vascular endothelial growth factor) antibody incubated the sections overnight at 4◦C, respectively. For CD31, DAPI stained the nuclei. The distribution of CD31 was observed by a fluorescence microscope and vascular density (VD) was expressed as the percentage of positive CD31 staining per ROI which were calculated using ImageJ software (Du et al., 2017). For VEGF, hematoxylin counterstained the nuclei and the integral optical density (IOD) of each view was assessed using the ImageJ software to determine its content (Zhou et al., 2020).

#### Statistical Analysis

The experimental data are expressed as mean ± standard deviation. Multiple comparisons among groups were determined using two-way ANOVA followed by Tukey's multiple comparisons test with adjusted P value. Significance was presented as: ∗∗∗∗for P < 0.0001, ∗∗∗for P < 0.001, ∗∗for P < 0.01, and <sup>∗</sup> for P < 0.05.

### RESULTS AND DISCUSSION

#### Synthesis and Characterization of CuS NPs

The CuS NPs were synthesized by a simple one-pot approach where CuS-citrate NPs were first prepared by reacting CuCl<sup>2</sup> and Na2S in the presence of sodium citrate, and then PEG coating was introduced by incubation of CuS-citrate NPs with thiolated-PEG to disperse the NPs in the solution and incorporate into the hydrogel uniformly. The morphology of the synthesized CuS NPs was confirmed by TEM showing in **Figure 2A** where the NPs were in good dispersion and uniform size, with an average diameter of 12 nm. This diameter is of great significance for drug delivery through the blood vessels (Ding et al., 2019). The hydrodynamic average diameter of the CuS NPs

was 35 nm as shown in **Figure 2B** determined by DLS. The Zeta-potential and PDI of the NPs was -6.07 mV, and 0.219, respectively. The negative charge of the NPs was probably due to the PEG layer, which is consistent with a previous report (Lin et al., 2019).

### Synthesis and Characterization of Thiolated HA

Thiolated HA was synthesized using an amide condensation reaction between HA and cysteamine in the presence of EDC and followed by reduced using DTT. As shown in Supporting Information **Supplementary Figure S1**, the chemical structure of the final product was confirmed by 1H NMR spectrum in D2O where two new peaks have appeared, one is at 2.8 ppm corresponding to the hydrogen of methylene close to thiol, and the other is at 2.6 ppm that assigned to the hydrogen of methylene adjacent to amide, suggesting that the Thiolated HA was successfully synthesized. The content of thiol was 0.49 mmol/g corresponding to 40% of the substitution degree as determined by the Ellman method.

## Preparation of the Injectable CuS/HA Hydrogel

Both the CuS/HA hydrogel and blank hydrogel were formed by crosslinking with the disulfide bonds that initiate by oxidization

of the thiolated groups along the HA chains with hydrogen peroxide. The gelation time of the CuS/HA and blank hydrogel was similar, approximately 6 min. Within this gelation time, the hydrogel could be injected into the skin wounds. **Figures 3A,B** show the injection operation of the CuS/HA hydrogel before gelation and the gelated CuS/HA hydrogel, respectively. The SEM images of a cross-section of CuS/HA hydrogel and blank hydrogel were shown in **Figures 3C,D**, both of which exhibited interconnected pores that provide three-dimensional networks with large volume and surface area. The storage (G') and loss modulus (G") were measured with a plate-to-plate rheometer. As shown in **Supplementary Figure S2**, the G' for HA hydrogel was 1820 Pa, while the one for CuS/HA hydrogel was 1930 Pa, and indicating that the hydrogel containing CuS NPs exhibited a higher modulus.

#### In vitro Photothermal Effect

The in vitro photothermal effect of the CuS NPs and CuS/HA hydrogel was studied to confirm their photothermal conversion. As shown in **Figures 4A,B**, the CuS NPs with different concentration (10, 20, 50, 100, and 200 µg/ml) was irradiated with NIR light at 808 nm and the results showed that the CuS NPs exhibited a significant increase in temperature upon 808 nm irradiation at all concentrations. In particular, the increase in temperature was dependent on the concentration of the CuS NPs, with the final temperature of the five concentrations reaching 34.4, 38.2, 46.7, 50.9, and 53.1◦C after 10 min exposure, respectively, indicating that the synthesized CuS NPs has a good photothermal effect. The photothermal stability of the synthesized CuS NPs was also studied. As shown in **Figure 4C**, the heating curve of the CuS NPs of each cycle was identical upon repeated irradiation, with temperature from 27 to 53◦C, and there was no significant change after repeated laser exposure. These results suggested that the synthesized CuS NPs displayed good photothermal stability. The photothermal

images of CuS/HA and blank hydrogels upon 808 nm laser exposure were shown in **Figure 4D**. Like NPs, the CuS/HA hydrogel also exhibited good photothermal effect, with increasing temperature of 50◦C after 10 min exposure. In contrast, there was no increase in temperature for blank hydrogel due to without CuS NPs.

### Cytotoxicity

The evaluation of biocompatibility such as cytotoxicity is the primary problem in the application of biomaterials before further experiments that can only be carried out when the material is proved to be non-toxic (Savoji et al., 2018). Herein, we first tested the cytotoxicity of the CuS NPs and CuS/HA hydrogel. As shown in **Figure 5A**, the CuS NPs with different concentrations show not only hardly any toxicity to the cells, but also promoting the proliferation of cells with the culture time prolonged. Cell viability shows significantly CuS NPs concentration dependency, e.g., the higher the NPs concentration, the better the cell growth. In particular, the NPs with a concentration of 200 µg/ml can significantly promote cell proliferation during the whole test. The mechanism underlying the promotion of the proliferation of the cells has been suggested to relate with them directly or indirectly stimulation effects of the copper ions (Li M. et al., 2018). For example, the copper ions stimulated the cells to secrete growth factors such as bFGF, which is known to promote the proliferation of the cells (Gopal et al., 2014). **Figure 5B** demonstrates that both hydrogels do not have toxicity to cells too. The above results indicated that the CuS NPs could be biosafe for the following experiment and application.

#### Animal Experiments

To evaluate the effects of CuS/HA injectable hydrogel on the wound healing process, a full-thickness skin wound model was used. As shown in **Figures 6A,B**, in general, the wound area of all groups decreased with the prolongation of the healing time. On day 0, immediately after injection the hydrogel onto the wounds, the hydrogel can effectively stop bleeding. On day 3, compared with the blank control group I, the wounds were going to form a scar in all the other groups, and while

the edema and exudation in group I were observed. By the 7th day, the difference in the healing area in each group was gradually obvious. On the 10th day, to observe and measure the area of wound healing, the hair around the rat's wound was shaved off and we could perceive that the scars in the groups without NIR irradiated were smaller than those in the corresponding control groups. At the end of the experiment (Day 14), it is easy to notice that the groups with NIR treated present better recovery outcomes than their counterparts, not only from the smaller areas of the scars but also the lighter degree of pigmentation. Consistently, the HR for each group was shown in **Figure 6C**. To evaluate the safety of the photothermal effect in vivo, the temperature change on the wound site was also recorded. As shown in **Supplementary Figure S3** the temperature of CuS/HA injectable hydrogel in the wound site changed from 32.4 to 50.3◦C. On the other hand, the temperature of HA injectable hydrogel changed from 34.2 to 37.7◦C. The time-temperature relationship determines the burn injury. It has been reported that burn injury occurs when the temperature reaches 50◦C and the time need at least 60 min. The treated time of the highest temperature is shorter than 10 min in our experiment. Thus, the treatment is safe for animals (Martin and Falder, 2017).

#### Pathomorphological Evaluation

The process of wound healing was further evaluated on the pathological level through HE staining. **Figure 7A** demonstrated pathological changes of skin in each group on day 14 postoperation. The skin structure was formed without a residual wound in all groups. For groups I, III, and V without NIR treated, the thickness of epidermal was thin and there were few layers of epidermal cells. They all shared these commonalities: edematous bundles of collagen, no obvious proliferation, more lasting inflammatory cells infiltration in the wound, slow formation of granulation tissue, and less neovascularization. On the contrary, the groupII, IV, and Vexposed to NIR exhibited better healing performances, e.g., cell proliferation was obvious; epidermis and dermis were thickened to a certain extent; clear epidermis cell structure was seen; inflammatory cells were reduced; necrotic tissue fell off, and cells were found to crawl. Especially in group VI, most of the wound epithelium was completely covered; there was more neovascularization; granulation tissue was thicker and collagen was more and arranged in order. To semiquantitative evaluation of wound healing, histopathological scores of HE slices were calculated based on the research of Eldad et al. As shown in **Figure 7B**, the scores of all groups were calculated on days 3,7, and 14, respectively. The results were following the wound healing tendency that all groups were going to heal with different degrees. It is intriguing to notice that there are differences between groups V and VI, but there is no statistical significance discrepancy between groups III and IV. Therefore, it indicates that NIR promotes wound healing on account of CuS/HA hydrogel. Furthermore, it is uncalled for to discuss the groups without NIR treated in the following tests.

Collagen deposition played an important role in the course of repair (Yen et al., 2018). It can be a temporary scaffold and a bed enabling migration of epithelium, keratinocytes, and microvasculature. Thus, collagen deposition was chosen as an indirect indicator to reflect the wound healing. More collagen deposition would promote wound repair in a better way and CVF would be on behalf of the collagen deposition. To determine the formation of collagen, Masson's trichrome staining was used in this study. As depicted in **Figure 8A**, collagen was dyed blue in all groups. CVF was calculated using ImageJ to the semiquantitative analysis of collagen content. As shown in **Figure 8B**, collagen deposition in group VI had been significantly higher than other groups, even though all groups were growing. This trend consistent with HE results further clued that CuS/HA hydrogel might accelerate wound healing through increasing collagen deposition.

#### Angiogenic Evaluation

Vascular endothelial growth factor, a specific growth factor, can bind to specific receptors on the surface of endothelial cells, which effectively stimulates the mitosis of cells and make them proliferate. Additionally, VEGF acts a pivotal part in stimulating the formation of granulation tissue and epithelium,

keratinocytes, and fibroblasts, and promotes the healing of the wound. Therefore, VEGF was employed to unveil the truth of wound healing at a molecular level. As shown in **Figure 9A**, the skin samples of the groups treated NIR were dyed by immunohistochemical staining at predesigned day (3, 7, and 14). Further statistical analysis results in **Figure 9B** revealed that the tendencies among the three groups were different. Specifically, the expression of VEGF in group VI reached the peak value at the beginning (the 3rd day), then it weakened gradually over time. However, for groups II and IV, they

both gradually increased from less to the maximum and then decreased. In particular, the expression of VEGF in group II and IV had never outstripped group VI from beginning to end, even though all of them hitting bottom on the 14th day. These observations indicate that CuS/HA hydrogel can effectively upregulate the expression of VEGF in the wound area at the incipient stage of wound healing to promote the formation of new blood vessels.

Wound healing requires enough nutrients and metabolism for repair and reconstruction, and neovascularization is the premise and basis for the transportation of these ingredients (Burgoyne and Morgan, 2003). As a biomarker of endothelial cells, the expression of CD31 was used to evaluate the neovascularization of the wounds. As shown in **Figure 10A**, immunofluorescence staining of CD31 confirmed the number of newly formed blood vessels in the wounds of different groups. The expression of CD31 in group VI was always higher than the other groups and reached a peak on the 7th day (**Figure 10B**). However, for groups II and IV, CD31 gradually increased and finally hit the maximum on the 14th day. These observations indicated that the CuS/HA hydrogel enhanced the angiogenesis of the wound at the early stage of wound healing.

To figure out whether the VEGF expression was induced by PTT or not, we compared the VEGF expression in groups I,

II, V, and VI. As shown in **Supplementary Figure S4**, there was a relatively low expression of VEGF in groups I and II. In contrast, both the PTT and copper ions released from CuS NPs can upregulate the VEGF expression, and the VEGF expression induced by PTT was significantly higher than that of copper ions release alone.

### DISCUSSION

Wound healing refers to the process of healing after disconnection or defect of skin and other tissues caused by external forces on the body, including regeneration of various tissues, hyperplasia of granulation tissues and complex combination of scar formation, and showing the synergistic effect of various processes. Among them, granulation tissue has an important function of anti-infection to protect the wound and filling the gap of tissue defects (Suarato et al., 2018). Especially, angiogenesis is the critical process to form granulation tissue, because newly formed blood vessels, like a farm irrigation system, and can provide nutrients and oxygen to cells in the wound site. Therefore, promoting angiogenesis can be used as a strategy to promote wound healing.

In our study, we have found that the CuS/HA hydrogel can promote the secretion of VEGF at the beginning of the healing process. This time is pivotal for the angiogenesis, while VEGF in the other groups does not increase rapidly and sensitively. As time went on, the VEGF in the other groups then increased gradually, but they might miss the golden window phase. In the initial stage of healing, the more angiogenesis appears, the faster wound healing will be. However, the increased expression of angiogenesis in the middle and later stages is not beneficial to epidermal remodeling for the healing. The wound healing process generally consists of several intersecting and fused parts, including thrombosis, inflammation, hyperplasia, and remodeling (Wang et al., 2019). Each part is not independent, but a complex whole of intersecting and interacting with each other. The cellular proliferation status, granulation tissue formation, collagen matrix deposition, and remodeling processes are all making great contributions to efficient wound healing. Our CuS/HA hydrogels accelerate the wound healing not only by angiogenesis but also through boosting collagen matrix deposition which was proved by the observation of Masson's trichrome staining. Why could our CuS/HA hydrogels have this talent to kill two birds with one stone? These good characteristics should attribute the success to the biological

effect of copper. Copper ion is involved in the activity of many transcription factors and combines with the release complex of the cell membrane to promote the release of growth factors and cytokines. Moreover, copper can stimulate angiogenesis and collagen deposition at the same time, which has been proved by Gérard et al. (2010).

As a wound dressing, the long-term stability of CuS NPs in vivo should be considered. When ingested by the organism, what is the fate of NPs? All forms [zero-valent state (Cu0), ionic copper (Cu1 + orCu2 +), copper NPs] of copper may cause varying degrees of biotoxicity at high exposure concentrations (Ameh and Sayes, 2019). Some studies have shown that copper NPs have cytotoxic and genotoxic effects on human skin epidermal cells (HaCaT), which is mediated by mitochondrial pathways triggered by reactive oxygen species (ROS; Alarifi et al., 2016). Because the size of copper NPs is very small, a single copper nanoparticle can be transferred through intercellular or permeated through the cell membrane. Eventually, the particles enter the bloodstream. Most copper nanoparticles were mainly found in feces. This suggests that the colon removes most of the unabsorbed particles (Lee et al., 2016).

To make sure the needs of copper can meet while minimizing adverse effects associated with defects or overages, organisms have developed a series of strategies to maintain homeostasis. As

for mammalians, they can make the Cu change not significantly through intestinal absorption, biliary excretion, and intrahepatic storage, when the organisms expose in mild to moderate concentration of Cu (Gaetke et al., 2014). These mechanisms have significant meanings in exploring the appropriate concentration of copper nanoparticles in the process of fabricating Cu NPs contained biomaterials. In our study, there was no obvious toxicity appearance, indicating that the concentration of Cu NPs used might be safe in our condition.

#### CONCLUSION

In conclusion, we successfully prepared CuS/HA hydrogel for wound healing. Within the gelation time, the hydrogel could be injected into the skin wounds. The hydrogel exhibited good photothermal effect, with increasing temperature of 50◦C after 10 min irradiation with NIR light at 808 nm, as well as no toxicity to the cells in vitro. In the rat skin wound model, the CuS/HA hydrogel can effectively increase the collagen deposition, upregulate the expression of VEGF, and enhance angiogenesis, which might contribute to promote wound healing.

#### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

#### REFERENCES


#### ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Ethical Committee of the West China Hospital of Sichuan University.

#### AUTHOR CONTRIBUTIONS

WZ performed the experiments, writing the manuscript, and the discussion of the results. LZ performed the experiments. YC and CY were involved in the discussion of the results. MT was responsible for conceptualizing, performed the experiments, the discussion of the results, and revising the manuscript.

### FUNDING

This work was in part sponsored by the Founds of West China Hospital (HX2019nCoV041).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2020.00417/full#supplementary-material



**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Zhou, Zi, Cen, You and Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Endothelial Cell Morphology Regulates Inflammatory Cells Through MicroRNA Transferred by Extracellular Vesicles

Jiaqi Liang<sup>1</sup> , Shuangying Gu<sup>1</sup> , Xiuli Mao<sup>1</sup> , Yiling Tan<sup>1</sup> , Huanli Wang<sup>1</sup> , Song Li<sup>2</sup> and Yue Zhou<sup>1</sup> \*

<sup>1</sup> Shanghai Jiao Tong University Affiliated Sixth People's Hospital, School of Biomedical Engineering, Med-X Research Institution, Shanghai Jiao Tong University, Shanghai, China, <sup>2</sup> Department of Bioengineering, Department of Medicine, University of California, Los Angeles, Los Angeles, CA, United States

Vascular inflammation plays an important role in the pathogenesis and the development of cardiovascular diseases such as arteriosclerosis and restenosis, and the dysfunction of endothelial cells (ECs) may result in the activation of monocytes and other inflammatory cells. ECs exhibit an elongated morphology in the straight part of arteries but a cobblestone shape near the pro-atherogenic region such as branch bifurcation. Although the effects of hemodynamic forces on ECs have been widely studied, it is not clear whether the EC morphology affects its own function and thus the inflammatory response of monocytes. Here we showed that elongated ECs cultured on poly-(dimethyl siloxane) membrane surface with microgrooves significantly suppressed the activation of the monocytes in co-culture, in comparison to ECs with a cobblestone shape. The transfer of EC-conditioned medium to monocytes had the same effect, suggesting that soluble factors were involved in EC–monocyte communication. Further investigation demonstrated that elongated ECs upregulated the expression of anti-inflammatory microRNAs, especially miR-10a. Moreover, miR-10a was found in the extracellular vesicles (EVs) released by ECs and transferred to monocytes, and the inhibition of EV secretion from ECs repressed the upregulation of miR-10a. Consistently, the inhibition of miR-10a expression in ECs reduced their anti-inflammatory effect on monocytes. These results reveal that the EC morphology can regulate inflammatory response through EVs, which provides a basis for the design and the optimization of biomaterials for vascular tissue engineering.

Keywords: extracellular vesicles, microtopology, vascular inflammation, monocytes, miR-10a

### INTRODUCTION

Atherosclerosis is the major cause of cardiovascular diseases (Momiyama et al., 2005). Although atherosclerosis develops through multiple discrete stages (Puri et al., 2011), vascular inflammation is involved in the whole process of disease progression (Ross, 1999; Davignon and Ganz, 2004). Healthy endothelial cells (ECs) control vascular tone, limit vascular smooth muscle cell (VSMC) proliferation, inhibit leukocyte adherence, and block thrombosis (Landmesser et al., 2004).

#### Edited by:

Chao Zhao, University of Alabama, United States

#### Reviewed by:

Debanjan Sarkar, University at Buffalo, United States Wuqiang Zhu, Mayo Clinic Arizona, United States

> \*Correspondence: Yue Zhou yzhou2009@sjtu.edu.cn

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 27 January 2020 Accepted: 02 April 2020 Published: 19 May 2020

#### Citation:

Liang J, Gu S, Mao X, Tan Y, Wang H, Li S and Zhou Y (2020) Endothelial Cell Morphology Regulates Inflammatory Cells Through MicroRNA Transferred by Extracellular Vesicles. Front. Bioeng. Biotechnol. 8:369. doi: 10.3389/fbioe.2020.00369

However, a variety of vascular injuries destroy the functions of the endothelium from protecting the vessel wall (Vanhoutte et al., 2009). Endothelial injury represents a major initiating step in the pathogenesis of vascular disease and atherosclerosis (Boos et al., 2007), followed by the activation and the recruitment of circulating monocytes to the region of vascular injury or infection (Ley et al., 2007). After their entry into the vessel wall, the monocytes differentiate into macrophages, which contributes to inflammatory response to neutralize invading pathogens, repair tissue damage, or activate other immune cells (Shi and Pamer, 2011). In addition to EC injury, there is evidence that the flow pattern also regulates EC morphology and activation. ECs in the straight part of an artery have an elongated morphology and align in the direction of laminar flow. In contrast, in the branched area and the curved part of artery, the formation of vortex and disturbed flow activated ECs and promoted inflammatory signaling (Li et al., 2005), and the ECs in these areas exhibit a cobblestone shape. However, whether the EC morphology per se has direct effects on inflammatory cells is not well-understood.

Monocyte activation contributes to the pathogenesis of various inflammatory conditions and atherosclerosis (Woollard and Geissmann, 2010). Such inflammatory response is regulated by various signals in the microenvironment, such as microbial products, cytokines, and microRNAs. It has been reported that circulating microRNAs exert great influence in modulating monocyte/macrophage phenotype and function in the process of vascular inflammation (O'Connell et al., 2007; Tili et al., 2007; Ono et al., 2011). Vascular inflammation is an important early event in atherogenesis, where many microRNAs are involved, including miR-10 (Fang et al., 2010), miR-17, miR-31 (Suarez et al., 2010), miR-92 (Wu et al., 2011), miR-155 (Zhu et al., 2011), miR-221, and miR-222 (Liu et al., 2012; Chen et al., 2019). Circulating miRNAs are not only biomarkers for disease but also serve as cell-to-cell messengers (Hergenreider et al., 2012; Yamakuchi, 2012).

Since naked RNAs are easy to be degraded by ribonuclease, microRNAs in circulation can exist either in protein binding form or enclosed in extracellular vesicles (EVs). The transfer of microRNAs in EVs mediating through interactions between the wide varieties of cell types in the cardiovascular system (Das and Halushka, 2015) has now been reported in cardiovascular systems and disease. ECs can modulate myeloid inflammatory responses through the secretion of EVs containing anti-inflammatory miRNAs (Njock et al., 2015).

In addition to biochemical signals, biophysical factors in the microenvironment may cause significant changes in the gene expression and the cellular behavior to execute regulatory function in diverse vascular events. For example, nano/microtopographic cues can significantly affect the gene expression of human vascular ECs, which can also affect the progress of cardiovascular diseases (Biela et al., 2009; Gasiorowski et al., 2010). In addition, biophysical cues, in the form of parallel microgroove on the surface of cell-adhesive poly-(dimethyl siloxane) (PDMS) substrates, can replace the effects of smallmolecule epigenetic modifiers and significantly improve cell reprogramming efficiency (Downing et al., 2013). However, it is not clear whether micro/nano-topography regulates cell–cell communications through EVs.

Here we investigated whether and how a specific pattern on culture substrates could induce pronounced changes in EC morphology and functions through microRNA-enclosing EVs to modulate inflammatory cells.

## MATERIALS AND METHODS

### Fabrication and Characterization of the Culture Substrate

Microfabrication technique was used to fabricate PDMS membranes with desired surface topography (10 µm in width, 3 µm in depth, and 10 µm spacing between each microgroove). PDMS was prepared according to the manufacturer's instruction (Dow Corning, United States), spin-coated onto the patterned silicon wafers to achieve the desired thickness, degassed under vacuum, and cured at 75◦C for 1.5 h. The micropatterned membranes were removed from the template, cut to appropriate dimensions and thoroughly cleaned by sonication, treated with Plasma Prep III (11050Q-AX) to enhance the surface hydrophilicity, and coated with 2% gelatin for 1.5 h to promote cell attachment. The images of surface topography of the micropatterned PDMS membranes were collected by using scanning electron microscopy (SEM; JEOL JSM-5600, Japan).

#### Cell Culture

Human umbilical vein endothelial cells (Sciencell, United States) were cultured in ECM medium (Sciencell, United States) with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% endothelial cell growth supplement at 37◦C in a humidified 5% CO<sup>2</sup> incubator. The cells were seeded on the PDMS membrane and cultured to reach confluence before proceeding

TABLE 1 | Primer sequences for mRNA quantitation in qRT-PCR.


TABLE 2 | Primer sequences for microRNA quantitation in qRT-PCR.


to the experiments. Monocyte cell line THP-1 (human acute monocytic leukemia cell line) was obtained from the American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum, L-glutamine, and 0.05 mM β-mercaptoethanol. The cells were grown to a density of 1 × 10<sup>6</sup> cells/ml and treated with 50 ng/ml lipopolysaccharide (LPS) for 2 h for activation.

#### Co-culture

Monocytes and ECs co-culture was carried out in a TranswellTM system purchased from Thermo (Cat# Nunc, 140660). The PDMS membrane was attached to the outer surface at the bottom of the Transwell insert, with the ECs facing down to the monocyte culture in the lower well, leaving an ˜1 mm space in between. Before co-culturing with ECs, the monocytes were stimulated with LPS at a concentration of 50 ng/ml for 2 h and washed thoroughly to remove the additional LPS. The total volume of the medium in the coculture system was 2 ml, comprising of 1 ml of EC medium and 1 ml of RPMI medium (for monocyte). To make sure that the EVs that we extracted were from ECs and not from the FBS, exosome-depleted FBS (System Biosciences,

LLC, United States) was used in the co-culture and transmedium system.

#### Immunofluorescence Staining

The cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, United States) and blocked with 10% BSA-PBST (Merck, United States). For actin–cytoskeleton staining, the samples were incubated with fluorescein-isothiocyanateconjugated phalloidin (FITC; AAT Bioquest, United States) for 1 h. For immunostaining, primary antibodies were incubated overnight at 4◦C, followed by 1 h of incubation with proper secondary antibodies in the dark. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, China). The microscopic images were acquired with confocal laser scanning microscope TCS SP5II (Leica, Germany).

#### Nanoparticle Tracking Analysis

After culturing for 24 h, the EVs were isolated from the EC culture medium by ultracentrifugation. Briefly, cell culture medium was collected and centrifuged at 300 g for 10 min and at 2,000 g for 10 min to remove cell debris. Then, the supernatant was centrifuged at 100,000 g for 70 min (Beckman XE-90, United States) to separate the EVs and the EV-depleted medium. Finally, the EVs were resuspended in 1 ml Dulbecco's phosphate-buffered saline. The size of the EVs was analyzed by the manufacturer's default software of Zeta View 8.04.02 SP2 (Bachurski et al., 2019).

## EV Uptaking

The EVs extracted from the conditioned medium of ECs by ultracentrifugation were labeled with I-135 according to a previously published method (Zhang et al., 2016) and added into the monocyte medium. After 2 h, the intensity of the radiation from the monocytes was measured with a scintillation counter and expressed as counts. Normal culture medium was used as blank for background reading.

### RNA Isolation and qRT-PCR Analysis

The total RNA and microRNAs in the cells were isolated using TRIzol <sup>R</sup> plus RNA Purification (Invitrogen, United States) and miRcute miRNA isolation kit (Tiangen, China), respectively, following the manufacturer's instruction. For qRT-PCR, cDNA of mRNA synthesis was performed using the FastQuant RT kit (with gDNase) (Tiangen, China), and the mRNA level was measured using SuperReal PreMix Plus (SYBR Green) (Tiangen, China). Here the GAPDH was used as a normalization control. The sequences of the primers used for real-time PCR are listed in **Table 1**.

Total Exosome RNA and Protein Isolation Kit (Invitrogen, United States) was used for microRNA isolation from EVs. The EVs were dissolved in a 37◦C pre-warmed denaturing solution. The microRNAs were extracted by a mixture of phenol/chloroform solution and precipitated from the aqueous phase by ethanol, followed by a series of wash and elution steps. The purified microRNAs were finally dissolved in the

elution solution provided in the kit before the subsequent cDNA first-strand synthesis experiments. The cDNAs of the microRNAs were synthesized with miRcute miRNA First-Strand cDNA Synthesis Kit, and the quantitative real-time PCR was performed with miRcute miRNA qPCR Detection Kit (SYBR Green) (Tiangen, China) according to the manufacturer's instruction. RNU6-2 was used as a normalization control in all microRNA measurements. The relevant primers are listed in **Table 2**. The qRT-PCR was run on Applied Biosystems 7900HT Fast Real-Time PCR System (ABI, United States). The qRT-PCR data were analyzed using the comparative 2 <sup>−</sup>11CT method.

#### Enzyme-Linked Immunosorbent Assay

The total of IL-6 protein in monocyte culture media was quantified by enzyme-linked immunosorbent assay (ELISA) (R&D, United States). The operating procedure follows the manufacturer's recommendations.

#### EV Secretion Inhibition

N-SMase Spiroepoxide inhibitor is a potent and selective irreversible inhibitor of neutral sphingomyelinase (N-SMase), and it has been used to inhibit exosome release (Devhare et al., 2017). To inhibit EV production, N-SMase Spiroepoxide inhibitor (C20H30N2O5, Santa Cruz, United States) was used at a concentration of 2.5 µm to treat the ECs for 24 h. Dimethyl sulfoxide (Invitrogen, United States) was used as the solvent control.

### MicroRNA Inhibition

ECs were seeded on the flat or microgrooved PDMS membranes for 24 h, and then miR-10a inhibitor or negative control was added into the medium at a concentration of 100 nM, following the manufacturer's protocol. The trans-medium system was applied to culture monocytes for 24 h. The micrOFFTM hsa-miR-10a-5p inhibitor, the negative control of micrOFFTM inhibitor, and the FECTTM CP Transfection Kit (Ribobio, China) were used in this experiment.

#### Western Blotting

For Western blotting analysis of the exosome marker, the exosomes were isolated using Total Exosome Isolation Kit (Invitrogen, United States), according to the manufacturer's instruction. Exosome samples were directly denatured with the loading buffer, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride membranes. The membrane was blocked in 5% non-fat milk and incubated with primary antibody against CD63 (Thermo Fisher Scientific, United States) at a concentration of 2 µg/ml and HRP conjugated goat anti-mouse IgG (Jackson immunoResearch Laboratories, Inc., United States) as secondary antibody. Then, blots were incubated using Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, United States) and visualized and recorded on Tanon 5200 chemiluminescence imaging analysis system (Tanon Science & Technology Co Ltd., China).

FIGURE 3 | The miR-10a expression in monocytes was affected by morphology-modified endothelial cells. In both co-culture (panels in the upper row) and trans-medium (panels in the lower row) systems, the expression level of miR-10a in monocytes (A,C) was analyzed. Data were normalized to U6 expression. (B,D) The mRNA expression level of MAP3K7 in monocytes was analyzed by qRT-PCR. Data were normalized to GAPDH expression. All the experiments were repeated three times, \*p < 0.05.

### Statistical Analysis

Unless otherwise indicated, data represent the mean of at least three independent experiments and error bars represent the standard error of the mean. Pairwise comparisons were made using Student's t-test. A comparison of three or more groups was performed using ANOVA. Differences between groups were then determined using Tukey's post-hoc test. For all cases, p < 0.05 were considered as statistically significant. In all figures, the asterisks represent P < 0.05. GraphPad Prism 6.0 software was used for all the statistical evaluations.

## RESULTS

#### Elongated ECs Suppressed Inflammatory Response in Co-cultured Monocytes

ECs were seeded on either flat PDMS membrane (**Figure 1A**) or micropatterned PDMS with 10-µm parallel microgrooves (**Figure 1B**), as shown in the SEM image. The morphology of the ECs was demonstrated by both FITC-phalloidin (green) and DAPI (blue) (**Figures 1C,D**) staining. Phalloidin staining of the stress fibers indicated that the microgrooved substrates had a pronounced effect on EC cytoskeleton and morphology. In general, cells grown on microgrooved PDMS membranes exhibited a more elongated morphology, following the guide of parallel microgrooves, compared to those cultured on flat PDMS

FIGURE 4 | Morphology-modified endothelial cells (ECs) altered the miR-10a expression in monocytes through extracellular vesicles (EVs). ECs were cultured on flat or microgrooved poly-(dimethyl siloxane) membranes, and the conditioned medium of ECs was collected to extract EVs. (A) Nanoparticle tracking analysis was applied to analyze the size of EVs. (B) EVs extracted from the conditioned medium of ECs were uptaken by monocytes. (C) The expression level of miR-10a and miR-126 in EVs was determined by qRT-PCR. (D) N-SMase Spiroepoxide inhibitor was added to the cell medium to treat the ECs for 24 h. The expression level of miR-10a in EVs from the conditioned medium of ECs was determined. (E) The conditioned medium of ECs was transferred to culture monocytes for 24 h. The expression level of miR-10a in monocytes was determined by qRT-PCR. Data were normalized to U6 expression. Dimethyl sulfoxide was used as control. Inhibitor refers to N-SMase Spiroepoxide inhibitor. All the experiments were repeated three times, \*p < 0.05.

membrane, and the stress fibers were also parallel to the axis of the microgrooves. LPS was used to treat the monocytes, which induced a robust expression of IL-6, IL-1β, and TNF-α. The EC co-culture (**Supplementary Figure S1**) significantly decreased the expression of these inflammatory genes. This is consistent with the precious findings that ECs suppress inflammatory responses. In addition, ECs on microgrooves showed more suppressive effects. Therefore, in the rest of the studies, we focused on the effects of EC morphology on monocytes following LPS stimulation.

To determine whether ECs cultured on microgrooved PDMS membranes could modulate the inflammatory cells, THP-1 cell line was used as a pro-inflammatory monocyte model in this study. The monocytes were treated with LPS for 2 h and were co-cultured with ECs in the system illustrated in **Figure 1E**. After 24 h, the expression level of the inflammatory cytokines IL-6, IL-1β, and TNF-α was assessed (**Figures 1F,H**). Compared with ECs on the flat PDMS membrane, the mRNA expression level of IL-6, IL-1β, and TNF-α was significantly decreased. In addition, the amount of the soluble IL-6 protein in the culture medium was further verified by ELISA (**Figure 1G**), showing the same trend as its mRNA expression level. The decrease in the inflammatory cytokine level suggested that the inflammatory response of the monocytes was significantly suppressed by ECs grown on the microgrooved PDMS membrane.

#### EC Morphology Regulated Monocytes Through Soluble Factors

In order to identify the possible regulator in the EC-conditioned medium, which altered the monocyte inflammatory response, we used a trans-medium system (as shown in **Figure 2A**). The purpose was to verify whether ECs could exert a unidirectional regulatory effect on monocyte inflammation. The EC-conditioned medium was transferred to LPS-activated monocytes. The monocytes were treated for 24 h and the mRNA expression level of the inflammatory cytokines IL-6, IL-1β, TNFα, and IL-10 was assessed (**Figures 2B,D**). Similar to the results demonstrated in **Figure 1**, compared with ECs on the flat PDMS membrane, the level of IL-6, IL-1β, and TNF-α was significantly

treated with miR-10a inhibitor for 24 h. The conditioned medium of ECs was transferred to culture the LPS-treated monocytes for 24 h. (C) The expressions level of miR-10a in monocytes was determined. (D) The expression level of IL-6 was determined. NC refers to negative control. All the experiments were repeated three times, \*p < 0.05 (ns, non-significant).

decreased, except for IL-10. Therefore, we focused on IL-6 expression in the studies thereafter. The level of soluble IL-6 protein in the culture medium determined by ELISA (**Figure 2C**) also showed a significant decrease, which was consistent with its mRNA expression level. The decrease in the inflammatory cytokine level indicated that the inflammatory response of the monocytes was significantly suppressed by the factors secreted from the ECs grown on the microgrooved PDMS membrane.

#### miR-10a Expression in Monocytes Was Affected by Morphology-Modified ECs

Among the soluble factors in the conditioned medium, microRNAs have been reported to have a great regulatory effect on cellular inflammatory response, and miR-10a has an inhibitory effect on IL-6 expression (Njock et al., 2015). Therefore, we explored further to determine whether miR-10a was the upstream regulator of the inflammation-suppressed status of the monocytes. The expression level of miR-10a in monocytes was analyzed in both co-culture and trans-medium systems. In the co-culture system, the miR-10a expression level in monocytes was elevated while they were co-cultured with ECs on the microgrooved PDMS membrane (**Figure 3A**). Consistently, the mRNA expression of MAP3K7, known as miR-10a direct target (Fang et al., 2010), was suppressed in monocytes (**Figure 3B**). Similarly, while only the ECconditioned medium was used to treat the monocytes, the expression level of miR-10a in monocytes was elevated by the conditioned medium of ECs on the microgrooved PDMS membrane in comparison to that on the flat PDMS membrane (**Figure 3C**), and the mRNA expression of MAP3K7 in monocytes was suppressed accordingly (**Figure 3D**). Both the co-culture and the trans-medium experiments showed that miR-10a expression in monocytes was affected by morphologymodified ECs.

#### miR-10a Expression in Monocytes Was Affected by EC-Secreted EVs

Since miR-10a expression in the monocytes was affected by soluble factors in the conditioned medium, we further explored whether miRNA could be transferred by EVs. Thus, EVs from the culture medium of the ECs were isolated and analyzed. Nanoparticle tracking analysis demonstrated that the isolated EVs were 40–200 nm in diameter (**Figure 4A**), which is consistent with the size of small to medium EVs. Since EVs are heterogeneous and exosome is one of the major subpopulations in EVs, we performed exosome isolation and verified the presence of exosomes in isolated EVs by transmission electron microscopy and Western blotting analysis of CD63 expression (**Supplementary Figure S2**).

In order to confirm whether the EVs were uptaken by monocytes, the EVs extracted from the EC-conditioned medium were labeled with I-135 and added into the culture medium of the monocytes. Normal culture medium was used as blank control. The intensity of the radiation from the monocytes incubated with I-135-labeled EVs within 2 h was measured with a scintillation counter and expressed as counts. The result indicated that the EVs were uptaken by monocytes immediately and increased gradually (**Figure 4B**). Then, we analyzed the expression level of microRNAs in EVs. The miR-10a showed a robust increase in EVs (**Figure 4C**). In contrast, there was no significant change of another inflammatory-modulator miR-126.

If the anti-inflammatory effect of the morphology-modified ECs was indeed passed by EVs enclosing a specific microRNA, the inhibition of EV secretion from the ECs should block the change of miR-10a in monocytes. N-SMase Spiroepoxide inhibitor was used to block the formation and the release of the EVs from the ECs. To avoid the possible side effects of the inhibitor on the monocytes, a trans-medium system was used to culture the cells separately. The ECs were treated with the inhibitor for 24 h. Then, the conditioned medium was transferred to the monocytes for an additional 24 h. Upon the inhibition of EV secretion, the level of miR-10a was significantly decreased in EVs (**Figure 4D**) and monocytes (**Figure 4E**). In order to confirm that the monocyte response was affected by miR-10a in EVs, we used EVs only

and EV-excluded medium (both from flat and microgrooved surfaces) to culture LPS-stimulated monocytes (**Supplementary Figure S3**). We found that EVs from ECs cultured on the microgrooved surface significantly inhibited the inflammatory response of the monocytes, while EV-excluded medium showed no significant change. These results confirmed that monocyte response was affected by miR-10a in EVs, not random miR-10a in trans-medium.

#### The Inflammatory Response of the Monocytes Was Inhibited by miR-10a Suppression in ECs

To determine whether the change of the EV-microRNA expression was sourced from ECs, we directly examined the level of miRNAs in ECs. The results in **Figure 5A** showed that the change of miRNA levels in ECs was consistent with those in EVs, i.e., miR-10a and miR-126 in ECs were also significantly induced. This result was confirmed by checking the mRNA level of MAP3K7, a miR-10a target gene, which had a significant decrease in ECs cultured on a microgrooved PDMS membrane (**Figure 5B**).

To investigate the effects of EC miRNA on miRNA and cytokine expression in monocytes, miR-10a inhibitor was applied to ECs cultured on a flat or a microgrooved surface. After 24 h, the EC-conditioned medium was transferred to culture the LPS-treated monocytes for additional 24 h. qRT-PCR analysis demonstrated that the expression level of miR-10a in monocytes decreased significantly and there was no more significant difference between ECs cultured on a flat surface and those cultured on a microgrooved membrane (**Figure 5C**). At the same time, the expression level of IL-6 in the monocytes was upregulated after inhibiting miR-10a, and also there was no more significant difference between ECs on a flat surface and those on a microgrooved surface (**Figure 5D**).

#### DISCUSSION AND CONCLUSION

The pathogenesis and the progression of a lot of cardiovascular diseases such as atherosclerosis and thrombosis are accompanied by an inflammation response (Gistera and Hansson, 2017). In the blood vessel, the athero-prone regions are usually near the bifurcation of the arteries, where the hemodynamic forces are different from those in the straight part of the artery due to the blood flow (Wang et al., 2016). In this athero-prone lesion area, ECs show a cobblestone morphology in contrast to the elongated ECs in the straight part of the artery. Our study provides a direct evidence that this EC morphology change in the atheroprone area may upregulate microRNAs that can be transferred to monocytes via EVs and thus induce inflammatory signals.

MicroRNAs have long been implicated as key regulators of inflammatory responses in monocytes or macrophages (Fish and Cybulsky, 2012; Sun et al., 2013). In addition, the fluctuations in shear stress contribute to microRNA-mediated epigenetic regulation on the function of ECs, which are essential for the maintenance of vascular homeostasis (Ando and Yamamoto, 2011). A recent study identified miR-10a as a flow-responsive microRNA in ECs in vitro (Davignon and Ganz, 2004), and miR-10a could be induced by high shear stress as an atheroprotective mechanism (Neth et al., 2013). Another study revealed that the expression of endothelial miR-10a was lower in the athero-susceptible regions of the inner aortic arch and the aorta– renal branches than that in other regions (Fang et al., 2010). Interestingly, the physical effect of the microgroove surface on EC morphology is similar to laminar shear stress, and consistently, elongated ECs have a higher level of miR-10a, suggesting that EC morphology change is sufficient to induce miR-10a expression.

Previous studies have demonstrated that miR-10a acts as a post-transcriptional modulator of the IκB/NF-κB signaling pathway by inhibiting MAP3K7 and βTRC (Fang et al., 2010; Njock et al., 2015). The increased proteolysis of IκBα and nuclear p65 in atherosclerotic susceptible areas resulted in a significantly up-regulated expression of the inflammatory biomarkers including IL-6, IL-8, MCP-1, VCAM-1, etc. (Fang et al., 2010). This provides a possible mechanism of how miR-10a regulates inflammatory cytokines in monocytes.

Based on our investigation, miR-10a was not the only microRNA with a significant change in the expression level. However, the level of miR-10a in both ECs and EVs was increased significantly, while miR-126 was increased significantly only in ECs on microgrooved PDMS membranes. A previous report (Asgeirsdottir et al., 2012) shows that miR-126 targets VCAM-1 in ECs to regulate the adhesion of ECs to monocytes. Therefore, miR-126 mainly functions in ECs but not in extracellular vesicles.

Another significant finding is that miR-10a made by ECs could be transferred to monocytes through EVs to modulate the expression of IL-6 in monocytes, which was demonstrated by the inhibition of EV secretion and miR-10a inhibition in ECs. This contact-independent EV transfer mechanism suggests a novel EC–monocyte signaling in the vascular microenvironment. These findings provide another explanation of how elongated ECs in the straight part of the arteries are anti-inflammatory and athero-protective.

This study not only unravels a new mechanism of EC– monocyte communication through EVs but also provides an insight into the biophysical regulation of cell–cell signaling in a vascular microenvironment. Our findings provide a rational basis for the design and the fabrication of micro/nano-materials that can be used to regulate EC and monocyte functions for vascular tissue engineering.

#### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

#### AUTHOR CONTRIBUTIONS

SL and YZ: conception or design of the work. JL, SG, XM, YT, and HW: acquisition of the data. JL, SG, YZ, and SL: manuscript writing. All authors have made significant contributions to the work, including analysis and interpretation of data, have

approved the submitted version, and have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work.

#### FUNDING

This work was supported by the National Key Research and Development Program of China (2016YFC1100202), the

#### REFERENCES


Multidisciplinary Research Foundation of Shanghai Jiao Tong University (YG2016MS67), and the Key Laboratory Open Project of Shanghai Municipality (2016SGK-001).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2020.00369/full#supplementary-material



inflammation and migration. Atherosclerosis 215, 286–293. doi: 10.1016/j. atherosclerosis.2010.12.024

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Liang, Gu, Mao, Tan, Wang, Li and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# An Injectable Hydrogel Platform for Sustained Delivery of Anti-inflammatory Nanocarriers and Induction of Regulatory T Cells in Atherosclerosis

Sijia Yi, Nicholas B. Karabin, Jennifer Zhu, Sharan Bobbala, Huijue Lyu, Sophia Li, Yugang Liu, Molly Frey, Michael Vincent and Evan A. Scott\*

*Department of Biomedical Engineering, Northwestern University, Evanston, IL, United States*

#### Edited by:

*Aijun Wang, University of California, Davis, United States*

#### Reviewed by:

*Francesca Taraballi, Houston Methodist Research Institute, United States Piergiorgio Gentile, Newcastle University, United Kingdom*

> \*Correspondence: *Evan A. Scott evan.scott@northwestern.edu*

#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

> Received: *21 March 2020* Accepted: *05 May 2020* Published: *05 June 2020*

#### Citation:

*Yi S, Karabin NB, Zhu J, Bobbala S, Lyu H, Li S, Liu Y, Frey M, Vincent M and Scott EA (2020) An Injectable Hydrogel Platform for Sustained Delivery of Anti-inflammatory Nanocarriers and Induction of Regulatory T Cells in Atherosclerosis. Front. Bioeng. Biotechnol. 8:542. doi: 10.3389/fbioe.2020.00542* Chronic unresolved vascular inflammation is a critical factor in the development of atherosclerosis. Cardiovascular immunotherapy has therefore become a recent focus for treatment, with the objective to develop approaches that can suppress excessive inflammatory responses by modulating specific immune cell populations. A benefit of such immunomodulatory strategies is that low dosage stimulation of key immune cell populations, like antigen presenting cells, can subsequently propagate strong proliferation and therapeutic responses from effector cells. We have previously demonstrated that intravenous injections of anti-inflammatory nanocarriers provided atheroprotection that was mediated by regulatory T cells (Tregs) upregulated in lymphoid organs and atherosclerotic lesions. Here, we demonstrate an injectable filamentous hydrogel depot (FM-depot) engineered for low dosage, sustained delivery of anti-inflammatory nanocarriers. The bioactive form of vitamin D (aVD; 1, 25-Dihydroxyvitamin D3), which inhibits pro-inflammatory transcription factor NF-κB via the intracellular nuclear hormone receptor vitamin D receptor (VDR), was stably loaded into poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-*b*-PPS) filomicelles. These aVD-loaded filaments underwent morphological transitions to release monodisperse drug-loaded micelles upon oxidation. This cylinder-to-micelle transition was characterized *in vitro* by cryogenic transmission electron microscopy (CryoTEM) and small angle X-ray scattering (SAXS). Following crosslinking with multi-arm PEG for *in situ* gelation, aVD-loaded FM-depots maintained high levels of Foxp3<sup>+</sup> Tregs in both lymphoid organs and atherosclerotic lesions for weeks following a single subcutaneous injection into ApoE−/<sup>−</sup> mice. FM-depots therefore present a customizable delivery platform to both develop and test nanomedicine-based approaches for anti-inflammatory cardiovascular immunotherapy.

Keywords: hydrogel, atherosclerosis, sustained delivery, regulatory T cells, immunotherapy, nanoparticle, filament

## INTRODUCTION

Atherosclerosis is a chronic inflammatory disorder and a major source of cardiovascular disease (CVD) that involves the accumulation of fatty deposits and inflammatory cells within the intimal walls of arterial vessels (Virani et al., 2020). Lipidlowering drugs, such as statins, have been widely used in the treatment of CVD. Statins inhibit HMG-CoA reductase and reduce cholesterol levels risk of coronary heart disease and stroke. However, numerous undesired side effects discourage patients from continuing statin treatment, such as stain-associated muscle symptoms, diabetes mellitus, and central nervous system complaints (Thompson et al., 2016). Many patients with reduced low-density lipoprotein (LDL) cholesterol levels still have a high risk of heart attack and stroke (Hu et al., 2003; Sampson et al., 2012; Fernández-Friera et al., 2017). Furthermore, systemic and plaque inflammation are not resolved by statin treatment (Sansbury and Spite, 2016). Recently, attention has shifted from focusing on lipids toward addressing the immune cell-mediated inflammation contributing to CVD. In particular, an imbalance between effector T cells and regulatory T cells (Tregs) can trigger a cascade of inflammatory responses leading to atherosclerosis progression and plaque vulnerability (Ou et al., 2018). Effector type 1 helper T (Th1) cells have been demonstrated to promote the migration of monocytes and T cells into plaques and activate antigen-presenting cells (APCs) by secreting interferon-γ (IFNγ) and IL-6 (Dietel et al., 2013; Tabas and Lichtman, 2017). However, as an essential immunoregulatory cell population, Tregs induce and maintain systemic immune homeostasis and tolerance by suppressing diverse immune cells, including effector T cells (Th1 and Th17 cells), monocytes, dendritic cells, and natural killer cells, as well as by secreting anti-inflammatory cytokines (IL-10, TGF-β, and IL-35) (George et al., 2012; Chistiakov et al., 2013; Dietel et al., 2013). In the clinic, decreased numbers and dysfunction of Tregs are suggested to be involved in atherosclerosis pathogenesis as evidenced by low levels of circulating and lesional Tregs in patients with vulnerable plaques and acute coronary syndrome (George et al., 2012; Dietel et al., 2013; Jia et al., 2013). Strategies to controllably elicit Tregs in vivo are therefore needed to better investigate, develop, and harness their atheroprotective mechanisms.

As essential mediators of immunity and tolerance, APCs play a pivotal role in the induction of Tregs. A variety of approaches to modulate APCs and increase Tregs have yielded promising results in the treatment of atherosclerosis, such as oral administration of anti-inflammatory immunomodulators (Chistiakov et al., 2013), anti-inflammatory cytokine treatment (Ji et al., 2017), and adoptive transfer of tolerogenic dendritic cells (DCs) (Hermansson et al., 2011). Despite these major advances, the clinical use of immunotherapies faces several challenges in both efficacy and safety due to off-target effects. Biomaterials and nanotechnology have been shown to improve the efficacy and safety of immunomodulatory molecules through controlling the colocalization, biodistribution, and release kinetics of drugs (Kim et al., 2011; Shao et al., 2015; Allen et al., 2016). We have previously developed multiple strategies to inhibit inflammation and the progression of atherosclerosis by nanocarrier-enhanced immunomodulation of APCs (Allen et al., 2019; Yi et al., 2019). The nanocarriers were composed of poly(ethylene glycol) block-poly(propylene sulfide) (PEG-b-PPS) and are both noninflammatory and non-toxic in non-human primates (Allen et al., 2018), humanized mice (Dowling et al., 2017), or mouse models of atherosclerosis (Yi et al., 2016). By adjusting the hydrophilic PEG fraction (FPEG) relative to the hydrophobic PPS blocks, PEG-b-PPS can self-assemble into diverse nanostructure morphologies that are highly stable in vivo due to their lyotropic mesophases and low critical micelle concentration of ∼10−<sup>7</sup> M (Napoli et al., 2002, 2004). Using vesicular polymersomes, we selectively targeted DCs in atherosclerotic mice for intracellular delivery of 1, 25-Dihydroxyvitamin D3 (aVD) (Yi et al., 2019). 1, 25-Dihydroxyvitamin D3 is the active metabolite of vitamin D and has been shown to induce a tolerogenic DC phenotype via interaction with the vitamin D nuclear receptor (VDR) (Mathieu and Adorini, 2002). With a logP of 7.6, aVD stably partitioned into the hydrophobic domains of PEG-b-PPS assemblies. Indeed, weekly intravenous administration of these DC-targeted anti-inflammatory polymersomes induced tolerogenic DCs, promoted the proliferation of Tregs and significantly inhibited atherosclerosis in ApoE−/<sup>−</sup> mice (Yi et al., 2019). Although effective in mice, weekly intravenous administration is not a clinically practical option for human patients, which would instead be better served by a long-term delivery platform to sustain Treg levels after a single injection.

Recently, we reported an injectable filamentous (FM) PEG-b-PPS hydrogel, which can be employed for the sustainable delivery of drug-loaded nanocarriers in response to physiological levels of oxidation (Karabin et al., 2018). We found that subcutaneously injected FM hydrogel depots (FM-depots) can sustainably deliver monodisperse micelles (MC) to APC and lymphoid organs such as spleen and lymph nodes in vivo for months. We therefore hypothesized that FM-depots might serve as an excellent platform to maintain therapeutic immunomodulation of chronic inflammatory diseases. Although thoroughly characterized for in vivo sustained release of diagnostic micelles, FM-depots have never before been employed for the delivery of micelles transporting a bioactive or therapeutic molecule. Here, we demonstrate a controllable anti-inflammatory FM-depot, which can sustainably release aVD-loaded PEG-b-PPS MC for months following subcutaneous (s.c.) injection. In this proof of concept work, we aimed to verify that delivery of the immunomodulator aVD in a controlled spatiotemporal manner using FM-depots exhibits the superior capacity to induce Foxp3<sup>+</sup> Tregs compared to free aVD after 2 months of treatment. This nanocarrier delivery system may therefore serve as an excellent tool to investigate and optimize strategies employing Tregs for CVD immunotherapy and presents new opportunities for long-term, low dosage anti-inflammatory treatment regiments.

#### MATERIALS AND METHODS

#### Materials

All chemical solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless indicated. All antibodies and

reagents used for flow cytometry were purchased from BioLegend (San Diego, CA, USA).

#### PEG-b-PPS Polymer Synthesis

Poly(ethylene glycol)-block-poly(propylene sulfide) copolymers PEG45-b-PPS<sup>44</sup> and vinyl sulfone functionalized PEG45-b-PPS<sup>44</sup> (VS-PEG45-b-PPS44) were synthesized as described previously (Karabin et al., 2018). Briefly, benzyl mercaptan initiated the living anionic ring-opening polymerization of propylene sulfide. The thiolate was then end-capped with either monomethoxy poly (ethylene glycol)-mesylate to form PEG45-b-PPS<sup>44</sup> or α-tosyl-ωhydroxyl PEG to form OH-PEG45-b-PPS44. The vinyl sulfone functionalized PEG45-b-PPS<sup>44</sup> was obtained from converting the hydroxyl group to a vinyl sulfone group. The obtained polymers (PEG45-b-PPS<sup>44</sup> and VS-PEG45-b-PPS44) were purified by precipitation in cold diethyl ether, dried under vacuum and characterized by <sup>1</sup>H NMR (CDCl3) and gel permeation chromatography (GPC) (ThermoFisher Scientific) using Waters Styragel THF columns with refractive index and UV-Vis detectors in a tetrahydrofuran (THF) mobile phase.

#### Preparation of Filomicelle (FM) Depots

Filomicelles were self-assembled from PEG45-b-PPS<sup>44</sup> and VS-PEG45-b-PPS<sup>44</sup> polymers via thin-film rehydration method in PBS as described previously (Karabin et al., 2018). Briefly, the mixture of PEG45-b-PPS<sup>44</sup> (40 mg) and VS- PEG45-b-PPS<sup>44</sup> (10 mg) polymers were dissolved in 1 ml dichloromethane within 1.8 ml glass vials (ThermoFisher Scientific) and placed under vacuum to remove the solvent. The resulting thin films were hydrated with 493 µl of phosphate-buffered saline (PBS) and gently mixed using a Stuart SB3 rotator for at least 36 h. To prepare FM-depots, eight-arm PEG-thiol (10% w/v in PBS solution, Creative PEGWorks) was added to FM solution corresponding to a 1.1:1 molar ratio of thiol:vinyl sulfone. The obtained mixture was vortexed and a Teflon mold (6 mm) was filled with 55 µl of the mixture. The FM-depots were formed after incubation at 37◦C in a humidified environment for 30 min.

#### Material Characterization

The morphology of nanostructures was determined using cryogenic transmission electron microscopy (CryoTEM). Prior to plunge-freezing, 200 mesh Cu grids with a lacey carbon membrane (EMS Cat# LC200-CU-100) were glow-discharged in a Pelco easiGlow glow discharger (Ted Pella Inc., Redding, CA, USA) using an atmosphere plasma generated at 15 mA for 15 s with a pressure of 0.24 mbar. This treatment created a negative charge on the carbon membrane, allowing liquid samples to spread evenly over the grid. 4 µL of FM samples (10 mg/ml in PBS) was pipetted onto the grid and blotted for 5 s with a blot offset of +0.5 mm, followed by immediate plunging into liquid ethane within a FEI Vitrobot Mark III plunge freezing instrument (Thermo Fisher Scientific, Waltham, MA, USA). Grids were then transferred to liquid nitrogen for storage. The plunge-frozen grids were kept vitreous at−180◦C in a Gatan Cryo Transfer Holder model 626.6 (Gatan Inc., Pleasanton, CA, USA) while viewing in a JEOL JEM1230 LaB6 emission TEM (JEOL USA, Inc., Peabody, MA,) at 100 keV. Image data were collected by

a Gatan Orius SC1000 CCD camera Model 831 (Gatan Inc., Pleasanton, CA, USA). The images were processed and analyzed using ImageJ.

Small angle X-ray scattering (SAXS) experiments were completed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) beamline at Argonne National Laboratory's Advanced Photon Source (Argonne, IL, USA) with 10 keV (wavelength λ = 1.24 Å) collimated X-rays. The qrange calibration was performed using a diffraction standard, silver behenate. All the sample measurements were in the qrange 0.001 to 0.5 Å−<sup>1</sup> . The data reduction procedure including subtraction of solvent buffer scattering to obtain a final scattering curve was made using PRIMUS 2.8.2 software. The filomicelle and micelle samples were confirmed using flexible cylinder and polymer micelle model fits, respectively, using SasView.

#### In vitro Release Study

To determine the release kinetics of payload from FMdepots, lipophilic dye DiIC18(3) (DiI) (ThermoFisher Scientific) was loaded into the polymer mixture at a final fluorophore concentration of 0.067% w/w. The DiI-loaded polymers were mixed with eight-arm PEG-thiol (10% w/v in PBS solution, Creative PEGWorks) to form a DiI-loaded scaffold in Teflon molds. The DiI-loaded FM-depots were incubated in 1 ml DI water with different concentrations of hydrogen peroxide (0, 1, 100 and 500 mM). At different time points (from 1 h to 30 days), 0.5 ml of supernatant was collected and replaced with 0.5 ml fresh DI water. The amount of payload that had been released was then determined using a fluorescence plate reader (SpectraMax M3, Molecular Devices) at an excitation of 549 nm and an emission of 565 nm.

Zetasizer Nano (Malvern Instruments) equipped with a 4mW He-Ne 633 laser was performed to characterize the size distribution of released nanostructures in the supernatant under different oxidation conditions at different time points. Given that the size of micelles in CryoTEM aligned very well with the number average in DLS (Karabin et al., 2018), the number average was used for the representation of the released nanostructure population. The polydispersity index (PDI) was calculated using a two-parameter fit to the DLS correlation data.

#### Animals

The apolipoprotein E-deficient (ApoE−/−) female mice with C57BL/6 background were purchased from The Jackson Laboratory at 4–6 weeks old. The mice were fed a highfat diet (HFD, Harlan Teklad TD.88137, 42% kcal from fat) starting at 7 weeks old for 18 weeks until sacrifice. All mice were housed and maintained in the Center for Comparative Medicine at Northwestern University. All experimental animal procedures were performed according to protocols approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC). For each experiment, mice were allocated randomly to each group.

#### Treatment

Seven weeks old female ApoE−/<sup>−</sup> were fed a high-fat diet (HFD, Harlan Teklad TD.88137, 42% kcal from fat) for 3 months before

treatment. To prepare the 1,25-Dihydroxyvitamin D3 (aVD) loaded FM scaffold, 1,25-Dihydroxyvitamin D3 (0.0067% w/w) (Sigma) was loaded into the polymers (PEG45-b-PPS<sup>44</sup> and 20% VS-PEG45-b-PPS44) to form aVD-loaded FMs. The aVD-loaded FMs in PBS were then quickly vortexed with eight-arm PEGthiol (10% w/v in PBS solution) before use. After 4 months on a high fat diet, 50 µl of various treatment groups were injected s.c. into the mid-scapular region of ApoE−/<sup>−</sup> mice every month for 2 months: 1, PBS control; 2, free aVD; 3, aVD-FM-depots. The same amount of aVD (8 µg/kg/month) was used in groups 2 and 3. Mice were kept maintaining on a high-fat diet, and their activities were monitored during treatment.

#### Flow Cytometry Analysis

After 2 months of treatment, spleen and lymph nodes (two brachial and two axillary from both sides of the mouse) were collected from all groups. Single-cell suspensions from spleen and LNs were prepared as described previously. RBC lysis buffer was used to eliminate red blood cells in spleen samples. Anti-mouse CD16/CD32 was used to block FcRs and Zombie Aqua fixable viability dye was used to determine live/dead cells. For flow cytometric analysis, cells were stained using cocktails of fluorophore-conjugated anti-mouse antibodies: BUV396 anti-CD45, FTIC anti-CD3, PerCP/Cy5.5 anti-CD4, PE anti-CD25, and Alex Fluor 647 anti-Foxp3. After washes, cells were suspended in cell staining buffer and then fixed by IC cell fixation buffer. Intracellular staining of Foxp3 was performed using Foxp3 Fix/Perm Buffer Set following the instruction (Biolegend). At least 200,000 events were recorded per tube on a BD LSRFortessa 6-Laser flow cytometer (BD Biosciences) and data were analyzed with FlowJo software.

#### Immunohistochemistry

For immunohistochemical analysis, mice were anesthetized and aortas were carefully harvested after perfusion with PBS under a microscope. The heart with aorta was fixed with 4% paraformaldehyde (PFA)/5% sucrose in PBS solution 12 h at 4 ◦C. The tissue samples were immersed in 15% sucrose solution for 12 h and then 30% sucrose solution for 24 h. The resulting specimens were embedded in Tissue-Tek OCT and frozen at −80◦C and then sectioned with a cryostat as described previously (Yi et al., 2019). Briefly, serial sections (10 µm thick) of the aortic roots were collected (10–12 sections per mouse) starting at the appearance of aortic valves. The distance between each section was 150µm, and totally of 100–120 serial cross-sections were obtained. To determine the immune cell populations in aortic lesions, the slides with multiple frozen aortic root sections were fixed in acetone and washed twice with PBS. Antibodies were performed on consecutive cross-sections for Treg cells (anti-Foxp3, 1:500, Abcam). Slides were stained using the Tyramide Signal Amplification kits in MHPL core facility of Northwestern University. All slides containing the cross-sections were digitally imaged with Leica DM6B widefield fluorescent microscope. An in-house software written in Python was used for automated and quantitative image analysis (Yi et al., 2019).

### Statistical Analysis

The sample sizes were determined based on the results of pilot experiments so that relevant statistical tests would reveal significant differences. For animal studies, 5–8 mice per group were selected in each experiment. GraphPad Prism software (version 8) was used for data analysis. Data are presented as means ± SD. The two-tailed unpaired t-test was performed to determine statistical significance.

### RESULTS AND DISCUSSION

### Preparation and Characterization of aVD-FM Hydrogel

To achieve sustainable delivery of micellar nanocarriers in vivo, an injectable filamentous hydrogel drug depot has been applied. FM-depots allow the sustained delivery of nanocarriers to APC via the cylinder-to-sphere transition at the injection site, wherein the synthetic FM that comprise the depot reassemble into nanocarrier vehicles. In contrast to alternative sustained delivery platforms that employ more stable porous scaffolds to retain nanocarriers for diffusion-based release, FM-depots efficiently degrade into monodisperse nanocarriers to both minimize the amount of administered polymer and avoid chronic inflammation (Yi et al., 2016; Karabin et al., 2018). Cylindrical filomicelles composed of PEG45-b-PPS<sup>44</sup> (FPEG ∼0.38) and 20% (w/w%) vinyl sulfone (VS)-functionalized PEG45-b-PPS<sup>44</sup> were assembled and loaded with aVD using the thin-film hydration method (**Figure 1A**), as previously described (Yi et al., 2016; Karabin et al., 2018). The crosslinking density can significantly affect the hydrogel stability, degradation, and the rate of nanostructure release. We have previously investigated the physicochemical properties of the crosslinked hydrogels composed of 10, 20, and 30% (w/w%) VS-PEG45-b-PPS44. Mixed filomicelles containing 20% (w/w%) VS-PEG45 b-PPS<sup>44</sup> were found to achieve optimal hydrogel stability and degradation rate while releasing monodisperse nanocarriers following crosslinking with 8-arm PEG-thiol. We, therefore, used 20% VS-PEG45-b-PPS<sup>44</sup> to form the FM-depots in this study. Given the filamentous structure is critical to the hydrogel formation, the structure of aVD-loaded filomicelles (aVD-FM) was verified using both cryogenic transmission electron microscopy (CryoTEM) and small angle x-ray scattering (SAXS). Furthermore, CryoTEM confirmed that loading of hydrophobic immunomodulator aVD into filomicelles does not change the filamentous structure (**Figures 2A,B**). aVD-FM showed micron-scale length, which was comparable with the unloaded filomicelles. SAXS scattering profiles of both unloaded and aVD-FM were fitted using a flexible cylinder model with a cylinder length of 2.1µm and a core radius of 16 nm (**Figures 2D,F**). Suspensions of filomicelles were simply mixed with the 8-arm PEG thiol, which can be spontaneously crosslinked with VSfunctionalized filomicelles. The aVD-FM-depot stably formed within minutes. Due to the oxidation sensitivity of PPS blocks, photo- or physiological oxidation converts the hydrophobic poly(propylene sulfide) into more hydrophilic poly(propylene sulfoxide) and ultimately poly(propylene sulfone) derivatives (**Figure 1B**). Changing the hydrophilic/hydrophobic ratio can trigger a cylinder-to-sphere transition in filomicelles, which we have previously demonstrated via thermodynamic modeling and interfacial measurements to be driven by interfacial tension (Karabin et al., 2018). This morphological transition at the end of the PEG-b-PPS filaments into spherical MC, highlighted by white arrows, was clearly observed for aVD-FM using CryoTEM (**Figure 2C**). Moreover, the SAXS analysis showed that the scattering of the oxidized aVD-FM in 500 mM H2O<sup>2</sup> was fitted well to a MC model with a diameter of 18.4 nm, which is consistent with the oxidized filomicelles without aVD (**Figures 2E,G**). These data suggested that the aVD-FM demonstrated a similar morphological transition with blank FMs upon oxidation.

### Sustained Release of Drug-Loaded Micelles From the FM-Depots Upon Oxidation

Excessive ROS is widely acknowledged in atherosclerosis, and high levels of ROS induce systemic oxidative stress, leading to cell apoptosis and redox-dependent signaling disruption (Goncharov et al., 2015). Given the continuous and dynamic production of ROS in the progress of atherosclerosis, it is difficult to accurately quantify the dynamic ROS concentrations in mice. We have previously demonstrated that PEG-b-PPS nanostructure morphology is sensitive to physiologic levels of ROS (Du et al., 2017; Karabin et al., 2018), and here we further verified the oxidative responses of FM-depots under various degrees of oxidation in vitro. To investigate the release of drug-loaded MC from FM-depots, we chose lipophilic indocarbocyanine dye DiI as a model for hydrophobic drugs. The DiI-loaded FMdepots were immersed in PBS solution with a variety of H2O<sup>2</sup> concentrations (0, 1, 100, and 500 mM). The poor water solubility of DiI induces its aggregation and precipitation in aqueous environments, allowing fluorescence in the supernatant to be attributed solely to DiI-loaded MC released from FM-depots in response to oxidation. The degradation rate of the hydrogels was found to be dependent on the degree of oxidation (**Figure 3A**), as increasing the H2O<sup>2</sup> concentration accelerated the disassembly of FM-depots. The supernatants were collected and the release of DiI-loaded MC was then quantified for up to 30 days using a fluorescence plate reader. The in vitro release kinetics suggested that the release rate of DiI-loaded MC was correlated with the concentration of H2O<sup>2</sup> (**Figure 3B**), with a rapid release of >90% after only 1 day in the highest concentration solution of

500 mM H2O2. However, the release rate significantly decreased with decreasing H2O<sup>2</sup> levels, and excellent stability was observed for the 0% H2O<sup>2</sup> sample over the course of 30 days. These data are consistent with oxidation induced reassembly of the filomicelle network within the hydrogel into DiI-loaded MC. The DLS data confirmed the morphology transition from FM to MC with an average diameter ranging from 16 to 30 nm (**Figure 3C**). It is noticeable that smaller MC diameters were observed in higher concentrations of H2O<sup>2</sup> (500 mM) compared to a low H2O<sup>2</sup> (1 mM) or the PBS control (**Figure 3C**). These data further indicate that the lower interfacial tension of the oxidized PPS drives the disassembly of FM and reassembly into more thermodynamically stable MCs. In addition, more oxidation may lead to a higher hydrophilic/hydrophobic balance and the formation of smaller MC, as the increased steric repulsion within the thicker hydrophilic corona would induce higher MC curvature. It's noteworthy that the released MC are highly uniform with a polydispersity index (PDI) < 0.1 (**Figure 3D**). These results verify that FM-depots can serve as controllable delivery systems for the sustainable release of drug-loaded MC under a wide range of oxidative conditions.

#### aVD-Loaded FM-Depots Elicit Treg Responses in Atherosclerotic Mice

Although typically associated with modulating bone metabolism, aVD also has potent effects on the regulation of immune responses. Of note, many patients with chronic inflammation like CVD have low levels of the precursor to aVD, 25(OH)VD3, suggesting a potential role for vitamin D in inflammation (Kassi et al., 2013; Yin and Agrawal, 2014). However, clinical

studies showed that oral supplementation of vitamin D has very limited effects on systemic inflammation, likely due to the low bioavailability and the broad distribution of the vitamin D receptor (VDR) (Anderson et al., 2010; Carvalho and Sposito, 2015). Intravenous administration of vitamin D in the form of calcitriol is associated with extensive side effects (Goodman et al., 1994), most notably hypercalcemia (Andress, 2001). We have previously demonstrated that encapsulation of aVD in PEG-b-PPS nanostructures significantly enhances the efficacy of aVD without side effects and induces tolerogenic DCs, Foxp3<sup>+</sup> Tregs, and anti-inflammatory effects in atherosclerotic mice (Yi et al., 2019). Building upon this work, an injectable hydrogel delivery system may present a more practical option for the administration of aVD-loaded nanocarriers and provide a means for sustained, low-dosage delivery (Li and Mooney, 2016). It is noteworthy that the monodisperse MC released from FM-depots were within the optimal size range for efficient lymphatic drug delivery following subcutaneous injection (Reddy et al., 2006, 2007). As a proof of concept, we loaded aVD at half the dosage employed in our previous work into FM-depots, sustained the delivery over the course of 2 months and assessed the increased levels of Tregs in the lymph nodes and spleen of ApoE−/<sup>−</sup> mice.

We evaluated the in vivo generation of Tregs by aVD loaded FM-depots in 8–10 weeks old female ApoE−/<sup>−</sup> mice. After receiving a high-fat diet for 3 months, mice were injected s.c. once per month with a PBS control, free aVD (8 µg/kg/month), or an aVD-loaded FM-depot (8 µg aVD/kg/month) (**Figure 4A**). After 2 months of administration, the mice were euthanized and no inflammation (swelling or irritation) was observed at the injection site. Activation of antigen-presenting cells (APCs) has been demonstrated to occur within atherosclerotic lesions and in peripheral lymphoid organs, especially draining lymph

nodes and spleen, where T cells migrate back to lesions to manipulate local immune responses (Weber et al., 2008; Zhu et al., 2016). The released MC from s.c. FM-depots have been demonstrated to strongly associate with APCs (macrophages and DCs) in both spleen and lymph nodes (Karabin et al., 2018). We hypothesized that our aVD-FM-depots would deliver aVDloaded MC to APCs and elicit Treg generation in spleen and migration to vascular lesions. We, therefore, characterized Tregs in lymphoid organs of ApoE−/<sup>−</sup> mice after a 2-month treatment using flow cytometry. The number of Foxp3+CD25<sup>+</sup> Treg cells was significantly increased in CD4<sup>+</sup> T cell populations in both lymph nodes (p < 0.001) (**Figure 4B**) and spleen (p < 0.05) (**Figure 4C**) for the aVD-FM-depot treatments compared to PBS control. However, no statistical difference was observed in the free aVD group compared to the PBS control group. The T cell responses were further evaluated using immunohistochemistry analysis in serial cross-sections of the aortic root. The levels of Foxp3<sup>+</sup> Tregs were significantly higher in mice receiving aVD-FM-depots compared to both the free aVD (p < 0.01) and PBS controls (p < 0.01) (**Figure 5**). Our results indicated that our FM-depots could sustainably deliver aVD-loaded MC and induce accumulation of Tregs responses for at least 2 months in atherosclerotic mice.

### CONCLUSIONS

An injectable s.c. hydrogel delivery system for sustained release of aVD-loaded nanocarriers was characterized and validated in vivo to elicit Treg responses in a mouse model of atherosclerosis. As a proof of concept, we used a simple system that delivered solely non-targeted, aVD-loaded MC monthly at low dosages. We verified that aVD could be stably loaded into PEG-b-PPS filomicelles without modulating the filamentous structure or inhibiting the crosslinking of filomicelles into hydrogels. Both CryoTEM and SAXS showed these aVD-loaded filaments to undergo cylinder-to-sphere transitions as expected under oxidative conditions to release monodisperse MC. In ApoE−/<sup>−</sup> mice fed a high-fat diet, significantly increased levels of Tregs were found in the lymph nodes, spleen and atheroma following monthly s.c. injections of aVD-loaded FM-depots. Thus, our work indicated that even at low doses and with less frequent administration, the sustained delivery of aVD via FM-depots could significantly induce the proliferation, expansion and homing of Foxp3<sup>+</sup> Tregs. This capability may prove to be a promising strategy for clinical translation by offering a practical monthly drug administration regimen and decreased systemic side effects. Although we previously demonstrated that PEG-b-PPS filamentous hydrogels could deliver a model fluorescent dye as a payload (Karabin et al., 2018), this is the first demonstration of the sustained delivery of a therapeutic molecule using the cylinder-to-sphere transition to modulate cell function. In future work, we will investigate the therapeutic efficacy of these sustained released FM-depots for inhibition of atherosclerotic plaque development by employing our previously developed nanomedicine-based anti-inflammatory strategies, which include cell-specific targeting (Yi et al., 2019) and alternative atheroprotective immunomodulators (Allen et al., 2019). In summary, this work demonstrates that FM-depots can serve as an effective sustainable delivery platform for the development of combinatorial and sustained delivery approaches to CVD immunotherapy.

### REFERENCES


#### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

#### ETHICS STATEMENT

The animal study was reviewed and approved by Northwestern University Institutional Animal Care and Use Committee.

### AUTHOR CONTRIBUTIONS

SY designed experiments, synthesized and characterized the materials and performed the mouse experiments, wrote the manuscript, and discussed the results. NK, SB, SL, and MF synthesized and characterized the materials and discussed the results. JZ, HL, MV, and YL assisted with the animal experiments. ES was responsible for conceptualization, designing experiments, results discussion, and revising the manuscript.

#### FUNDING

This research was supported by the National Science Foundation (CBET-1806007 and CAREER Award No. 1453576) and the National Institutes of Health Director's New Innovator Award (NHLBI 1DP2HL132390-01).

### ACKNOWLEDGMENTS

The authors would like to thank Dr. Eric W. Roth for CryoTEM assistance. The CryoTEM made use of the BioCryo facility of Northwestern University's NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); and the State of Illinois, through the IIN. It also made use of the CryoCluster equipment, which has received support from the MRI program (NSF DMR-1229693).

status, and incident events in a general healthcare population. Am. J. Cardiol. 106, 963–968. doi: 10.1016/j.amjcard.2010.05.027


correlate with infiltrated mature dendritic cells. Atherosclerosis 230, 92–99. doi: 10.1016/j.atherosclerosis.2013.06.014


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Yi, Karabin, Zhu, Bobbala, Lyu, Li, Liu, Frey, Vincent and Scott. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nanoscale Technologies in Highly Sensitive Diagnosis of Cardiovascular Diseases

Chaohong Shi<sup>1</sup> , Haotian Xie<sup>2</sup> , Yifan Ma<sup>3</sup> \*, Zhaogang Yang<sup>4</sup> \* and Jingjing Zhang<sup>3</sup> \*

<sup>1</sup> Department of Rehabilitation Medicine, The First People's Hospital of Wenling, Wenzhou Medical University, Wenling, China, <sup>2</sup> Department of Mathematics, The Ohio State University, Columbus, OH, United States, <sup>3</sup> Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, United States, <sup>4</sup> Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, United States

#### Edited by:

Wuqiang Zhu, Mayo Clinic in Arizona, United States

fbioe-08-00531 June 4, 2020 Time: 15:51 # 1

#### Reviewed by:

Mahmood Khan, College of Medicine, The Ohio State University, United States Yun Chang, Purdue University, United States Yi Hong, University of Texas at Arlington, United States

#### \*Correspondence:

Yifan Ma ma.1711@buckeyemail.osu.edu Zhaogang Yang Zhaogang.Yang@UTSouthwestern.edu Jingjing Zhang zhang.8211@osu.edu

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 21 March 2020 Accepted: 04 May 2020 Published: 05 June 2020

#### Citation:

Shi C, Xie H, Ma Y, Yang Z and Zhang J (2020) Nanoscale Technologies in Highly Sensitive Diagnosis of Cardiovascular Diseases. Front. Bioeng. Biotechnol. 8:531. doi: 10.3389/fbioe.2020.00531 Cardiovascular diseases (CVD) are the leading cause of death and morbidity in the world and are a major contributor to healthcare costs. Although enormous progress has been made in diagnosing CVD, there is an urgent need for more efficient early detection and the development of novel diagnostic tools. Currently, CVD diagnosis relies primarily on clinical symptoms based on molecular imaging (MOI) or biomarkers associated with CVDs. However, sensitivity, specificity, and accuracy of the assay are still challenging for early-stage CVDs. Nanomaterial platform has been identified as a promising candidate for improving the practical usage of diagnostic tools because of their unique physicochemical properties. In this review article, we introduced cardiac biomarkers and imaging techniques that are currently used for CVD diagnosis. We presented the applications of various nanotechnologies on diagnosis within cardiac immunoassays (CIAs) and molecular imaging. We also summarized and compared different cardiac immunoassays based on their sensitivities and working ranges of biomarkers.

Keywords: cardiovascular disease (CVD), biomarker, molecular imaging, diagnostic, nanotechnology

### INTRODUCTION

Cardiovascular diseases (CVDs) are the most common causes of death in the world (Ho, 2018). CVDs can be medically defined as a group of disorders involving heart, brain, and blood vessels, including but not limited to coronary heart diseases, peripheral arterial diseases, rheumatic heart diseases, deep vein thrombosis, and cerebrovascular diseases – all of which result in ischemia and tissue death (Yang et al., 2009, 2012; Laflamme et al., 2012; Chakrabarti et al., 2013; Yu et al., 2015; Fan et al., 2020a,b). General CVDs can be characterized into five categories: atherosclerosis, acute myocardial infarction (AMI), heart failure (HF), stroke, and hypertension (Lichtenstein and Matthan, 2007; Govindappa et al., 2020; Joshi et al., 2020). Individuals who demonstrate tobacco smoking, high levels of low-density lipoproteins (LDL)-associated cholesterol, glucose, and diabetes as well as overweight and obesity, are especially susceptible to CVD morbidity and mortality (D'Agostino et al., 2008). Effectively diagnosing individuals who are most susceptible to CVDs opens the door to optimal treatment, thereby lowering the death rate. Given that early-stage CVDs demonstrate a high survival rate, predicting CVDs early on is essential.

Current common clinical CVD diagnosis methods include electrocardiography (ECG), plain X-ray, computed tomography (CT), and magnetic resonance imaging (MRI), and other MOI

techniques (Anderson et al., 2013). ECG measures variations in the conduction system of the heart and monitors chest pain in AMI patients (Fesmire et al., 1998). CT scans X-ray images around the body and generates slices images of bones, blood vessels and tissues, which is appropriate for CVD diagnosis on grounds of its high signal contrast and accuracy (Kirkpatrick et al., 2003). MRI has been widely used in atherosclerosis and stroke detection given it scans three-dimensional images of bodies in a non-invasive manner (Pykett et al., 1983). However, these traditional methods were limited to low sensitivity and specificity.

To overcome these aforementioned difficulties, various new platforms such as cardiac immunoassays (CIAs) and advanced molecular imaging (MOI) were introduced, which significantly improved the efficiency of CVD diagnosis over the past decades (Qureshi et al., 2012; Osborn and Jaffer, 2013). Cardiac biomarkers are substances in the blood when the heart and brain are damaged or act abnormally. For example, cardiac troponin I (cTnI) has been demonstrated as a promising biomarker for AMI (Apple et al., 1997). MOI is capable of identifying cellular and molecular biology process, however, each technique has advantages and limitations. Therefore, advanced MOI combined different MOI techniques have been invented (e.g., dual-module, triple-module-CT) to obtain more detailed imaging information, which has increased the accuracy of diagnostic results (Hur et al., 2011, 2012).

Despite the great merits of previous methods, early-stage diagnosis is still challenging due to its complex pathophysiology, vague symptoms, and low expression levels of cardiac biomarkers. These difficulties increase the aggravation and mortality of CVDs. For instance, atherosclerosis shows no signs or symptoms and extremely low-level of related biomarkers in some patients even after a heart attack (Libby, 2002). Moreover, quick and convenient measurements are inadequate in addressing the expanded needs of CVD patients. Hence, rapid, accurate, and highly sensitive and specific platforms are needed for early-stage CVDs.

Nanotechnology involves nanoscale dimension systems (Johnson, 2012; Zhou et al., 2014), has specific physicochemical properties that make them appealing for improving current diagnosis (Kuriyama et al., 2011; Sun et al., 2016, 2019; Chen Z. et al., 2017; Liu et al., 2017; Yang et al., 2020a). Nanomaterials have been extensively applied to CIAs, including electrochemiluminescence (ECL), Electrochemical (EC), and photoelectrochemistry (PEC) due to their unique optical property, electrical property, and excellent biocompatibility (**Figure 1**) (Abdorahim et al., 2016). For example, Gold nanoparticles (AuNPs) can be incorporated with biotinylated antibodies to reduce non-specific binding, or conjugated with biomolecules with specific physical properties [e.g., hybridization chain reaction (HCR)] for signal amplification. Liu G. et al. (2016) detected cTnI via antigen-antibody affinity with a low limit of detection using AuNPs and graphene oxide. AuNPs and other metal nanoparticles can modify substrates (e.g., boron nitride nanosheets, titanium) in EC assays as well because of their attractive electrical properties (Golberg et al., 2010). Other nanomaterials, like silica/Pt NPs, are common signal enhancers in PEC and surface plasmon resonance (SPR) thanks to their efficient photocurrent quenching ability and unique plasmonic properties (Homola et al., 1999). Besides, upconversion nanoparticles (UCNPs) have been used in fluorescence assay because of their excellent photon conversion ability (Haase and Schäfer, 2011). Also, knotted and hollow nanomaterials (e.g., nanosheets, nanotubes, nanowires, and nanoclusters) with the large surface area that increases loading efficiency of biomolecules for signal enhancement (Kong et al., 2000). Nanomaterials also play important roles in MOI. Nanomaterials coupled with photoacoustic, fluorescent, radioactive, paramagnetic substances can work as contrast agents in MOI settings to enhance their detection signal (Van Schooneveld et al., 2010).

In this review, we will introduce the recent progress of nanotechnology for diagnosing CVDs. The nanotechnology facilitated CIA and MOI applications will be covered. Firstly, we summarized common clinical cardiac biomarkers and MOI settings. Then the applications on nanotechnology to identify cardiac biomarkers within different platforms based on unique properties of various nanomaterials will be introduced, including ECL, fluorescence, PEC, EC Surface-enhanced Raman scattering (SERS), SPR, Field effect transistor (FET), Enzyme-Linked Immunosorbent Assay (ELISA), and lateral flow assay (LFA). We compared different CIAs based on their sensitivities and working ranges of cardiac biomarkers as well. Besides, we concluded current diagnosis achievements on MOI with the help of functionalized nanoparticle.

## CVD DIAGNOSTIC SETTINGS

### Cardiac Immunoassay (CIA)

Cardiac biomarkers present in human bodyfluids are reliable and reproducible indicators of the risk and progression of CVDs. They can be detected and quantified through various CIAs based on antigen-antibody immunoaffinity. Measuring expression levels of cardiac biomarkers within CIAs shows advantages, including high sensitivity, rapid, cheap, and non-invasive for the prediction and diagnosis of disease. Cardiac biomarkers can be classified as circulating biomarkers and exosomal biomarkers. Circulating biomarkers present in bodyfluids freely, and exosomal biomarkers are bound into or on the surface of extracellular vesicles (EVs) that are mobile and secreted from cells.

#### Cardiac Circulating Biomarkers

Circulating biomarkers include miRNA, mRNA, long-noncoding RNA, proteins, and other substances that present in human blood, milk, saliva, urine, and cerebral spinal biofluids (Durrani-Kolarik et al., 2017; Wang et al., 2017a,b; Wang X. et al., 2018; Yang et al., 2020b). Currently, several cardiac biomarkers, such as cardiac troponin I (cTnI), troponin I (TnI), myoglobin (MB), C-reactive protein (CRP), and creatine kinase-MB (CK-MB), have attracted interests as potential biomarkers for AMI (Christenson and Christenson, 2013). Particularly, cTnI has high specificity and sensitivity toward AMI (Jo et al., 2015), and MB is a good candidate for early diagnosis of AMI (Korff et al., 2006).

Myeloperoxidase (MPO), glycogen phosphorylase isoenzyme BB (GPBB), B-type natriuretic peptide (BNP), N-terminal pro-Btype natriuretic peptide (NT-proBNP), C-type natriuretic peptide (c-TNP), Matrix metalloproteinase-8 (MMP-8), MMP-9 and tissue inhibitor of MMP-8 (TIMP-1), and leukotriene B4 are promising biomarkers as well (Anderon, 2005).

Recently, researchers discovered that some miRNAs circulating in serum or plasma were closely correlated with CVDs. For example, miR-208 was undetectable in healthy donors but was detected successfully in 90.9% of AMI patients (Ji et al., 2009). There are almost 50 circulating miRNAs proven to be relevant to CVDs. Specifically, miR-208a, miR-208b, and

miR133 are up-regulated AMI biomarkers, while miR150, let-7b, and miR-126 are down-regulation AMI biomarkers (Corsten et al., 2010; Wang et al., 2010; Gidlöf et al., 2011; Long et al., 2012a,b; Devaux et al., 2013; Friese et al., 2013; Li et al., 2013). Furthermore, miR-423, miR-18b, miR-499, miR-142, miR-320a, miR-22, miR-20b, and miR-26b are positively associated with HF, but miR-103 is shown to be low in HF patients (Corsten et al., 2010; Tijsen et al., 2010; Goren et al., 2012; Ellis et al., 2013; Marfella et al., 2013). Additionally, let-7e, Hcmv-miR-UL112, miR-605, miR623, miR-516b, miR-132 are found to be relatively high in plasma in hypertension cases. In contrast, miR-296, miR-133b, miR-625, miR-1236 are down-regulation biomarkers (Li et al., 2011). Moreover, miR-145, miR21 are up-regulating biomarkers for strokes, and miR-221, miR-210, miR-30a, and miR-126 are shown to be low in human blood in stroke cases (Zeng et al., 2011; Gan et al., 2012; Long et al., 2012b; Tsai et al., 2013). In summary, circulating miRNAs are relatively stable and serve as sufficiently sensitive biomarkers for CVD diagnosis.

#### Cardiac Exosomal Biomarkers

EVs are submicron-sized vesicles ranging from 30 to 1000 nm secreted by cells. EVs play important roles in transferring proteins, mRNA, miRNA, and other molecules among cells (Yang et al., 2016; Ma et al., 2019; Walters et al., 2019; Liu et al., 2019). Increasing evidence showed that exosomal miRNAs are involved in the pathogenesis of CVDs (Zamani et al., 2019). It has been observed that exosomal miRNAs mediate intercellular communication in cardiovascular systems and play an indispensable role in the control of cellular functions (Bang et al., 2014). As such, exosomal miRNAs can be used as biomarkers for diagnosing subjects with CVDs (Rehman et al., 2017).

Recently, researchers found miR-1, miR-133a, miR-21, and miR-499 are AMI-related biomarkers (Cheng et al., 2012; Oerlemans et al., 2012). Also, miR-192, miR-194, miR-34a, miR-423-5p, miR320a, miR-22, and miR-92b are highly associated with HF (Goren et al., 2012; Matsumoto et al., 2013). Besides, miR-223 isolated from serum EVs is a potential biomarker for stroke (Chen Y. et al., 2017). Additionally, exosomal membrane proteins or internal proteins, such as TNF-α and fibronectin are also potential CVD biomarkers (de Jong et al., 2012; Yu et al., 2012). **Table 1** summarized the biomarkers mentioned above.

#### Molecular Imaging

Molecular imaging (MOI) is a common diagnostic tool for CVD in clinical practice. When paired with other approaches, MOI can reveal individual biology, including blood vessels, the brain, and the heart (Jaffer et al., 2007). Common MOI approaches include photoacoustic tomography (PAT), MRI, CT, positron emission tomography (PET), and single photon emission computed tomography (SPECT). PAT utilizes the photoacoustic effect that converts optical adsorption into acoustic energy and generates high-resolution imaged under optically ballistic and diffusive modules (Xia et al., 2014). Various materials such as dyes, nanoparticles, and probes can work as functional contrasts in PAT for vascular imaging (Li and Wang, 2009). CT takes X-ray images from different angles around the human body and produces cross-sectional images of the body to reveal blood vessels, tissues and organs (Article, 2018). MRI generates anatomical images based on the changes of protons in the body with a strong magnetic field (Hashemi et al., 2012). MRI sensors first turn on the radiofrequency current that spins the protons out of equilibrium and then detects the time and amount of released energy of realigning randomized protons within the magnetic field after turning off the radiofrequency current (McRobbie et al., 2017). Detected parameters are translated into images for diagnosis. PET uses injected radioactive tracers to review and evaluate tissues. The radiotracers used in PET produce positrons after decaying. Positrons react with electrons and produce photons that aggregate in specific, disease-related areas of the human body (Shan et al., 2013). The combination of PET with CT or MRI can create detailed, specific images. Similar to PET, SPECT provides tomographic images by recording and translating the activities of radioactive tracers that have gamma ray emissions (Hutton, 2014).

#### NANOTECHNOLOGY-BASED CARDIAC IMMUNOASSAY

Various advanced techniques like ECL, PEC, SERS, SPR, and ELISA have been utilized to detect cardiac biomarkers with precision. Despite satisfactory results from previous methods, pursuing high sensitivity and accuracy in testing results is still an ongoing endeavor. Combining nanotechnologies with cardiac immunoassays may serve as a solution for early-stage CVD diagnosis. Nanotechnologies may reduce non-specific binding sites, provide high binding efficiency of cardiac targets, offer excellent signal amplification, and possess multiple functions. **Table 2** summarized and compared performances of recent cardiac immunoassays based on various nanotechnology platforms.

### Electrochemiluminescence (ECL) Immunoassay

Electrochemiluminescence (ECL) involves electron-transfer reactions that generate excited states and light emission, thus works as a common diagnostic assay to quantify expression levels of biomarkers because detected emission intensity in ECL is proportional to the concentration of the biomarkers (Richter, 2004). Luminol is one of the most important signal enhancers in ECL but has a weak signal and poor solubility. Fortunately, luminol-functionalized nanomaterials can overcome the limitations of traditional luminol and greatly enhance the intensity and sensitivity due to their large specific surface area (e.g., carbon nanotubes, nanosheets), the ability to functionalize signal amplification materials (e.g., nucleic acid isothermal amplification, HCR signal amplification), conductivity, and surface charge. All of these advantages have contributed to intense attention of ECL assay in biomolecule analysis (Hao et al., 2014; Feng et al., 2016).

Dong et al. (2019) detected NT-proBNP using ECL immunoassay based on ECL resonance energy transfer (RET). The ECL-RET transfer was between semicarbazide-modified

#### TABLE 1 | Summary of cardiac biomarkers.

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gold nanoparticles (AgNC-sem@AuNPs) covered nanocubes (donor) and a Ti (IV)-based metal-organic framework of type MIL-125 (receptor). The partial overlap between the ECL emission of the AgNC-sem@AuNPs and the visible adsorption spectrum of MIL-135 created RET. The schematic was illustrated in **Figure 2A**. Firstly, 8 µL of AgNC-sem@AuNP solution was dropped on the pre-cleaned glassy carbon electrode (GCE) surface, then the primary antibody<sup>1</sup> (Ab1) was immobilized on it via Au-NH<sup>2</sup> bond. Then, NT-proBNP analytes were added to the assay and captured by Ab1. Finally, the MIL-125 labeled secondary antibody<sup>2</sup> (Ab2) was incubated onto the electrode to form the sandwich format and quench the ECL strength of luminophore. The assay was able to recover 96.8%∼100.2% of NT-proBNP and detect it as low as 0.11 pg/mL.

Zhu et al. (2019) detected cTnI using Au Nanocluster and HCR signal amplification. A sandwich immunocomplex composed of cTnI, Ab1, and Ab2-AuNP-T<sup>1</sup> was applied. Ab2- AuNP-T<sup>1</sup> is a smart probe in which the DNA initiator strands (T1) and Ab<sup>2</sup> are conjugated onto the AuNPs. The schematic was illustrated in **Figure 2B**. Once the cTnI was caught, the initiator strands T<sup>1</sup> of Ab2-AuNP-T<sup>1</sup> opened the hairpin DNA structures (H1 and H2) that were dual-labeled on the Au nanoclusters (Au NCs). This process triggered hybridization events, thus modified a large number of Au NCs on the surface of the electrode. Finally, a strong ECL signal was emitted owing to the reaction of the modified Au NCs and the coreactant K2S2O2. This ECL-HCR sensor was able to detect 1.01 fg/mL cTnI with high specificity stability and reproducibility.

Recently, various research groups have made great progress in CVD diagnosis using ECL assay. Wang et al. (2019) combined Co2+-based metal organic frameworks (MOF), zeolitic imidazolate frameworks (ZIF-67), and luminol-capped Ag nanoparticles (luminol-AgNPs) for fast and ultrasensitive detection of cTnI, with a 0.58 fg/mL detection limit. Zou et al. (2019) developed an ECL immunosensor to detect MB in human serum. They fabricated a basal electrode using AuNPs and platinum nanowires that were deposited onto indium tin oxide-coated glass linked with 3-aminopropyl-trimethoxysilane. Besides, Adhikari et al. (2019) presented an ultrasensitive labelfree ECL immunosensor for CK-MB detection using a novel nanocomposite-modified printed electrode. To fabricate this sensor, carbon nano-onions (CNOs)/Fe3O4/AuNP/chitosan (CS) nanocomposite was dropped in single-layered rolled-up carbon nanotubes (SWCNTs). Then Ab<sup>1</sup> against CK-MB was spiked onto the electrode and blocked by bovine serum albumin (BSA). Once CK-MB was captured on the electrode, the ECL signal was determined by Tris(2,2<sup>0</sup> -bipyridyl)-ruthenium(II) chloride ([Ru(bpy)3] <sup>2</sup>+Cl) and tri-n-propylamine (TPrA), in which Ru(bpy)3] <sup>2</sup>+Cl was used as luminophore and TPrA was the co-reactant. Moreover, Lakshmanakumar et al. (2019) functionalized graphene quantum dots with acetic acid (fGQDs) on the Au electrode to detect cTnI. The cTnI was recognized via carbodiimide conjugation between the N-H group of cTnI and the COOH group on fGQDs instead of antibody-antigen interaction. The interaction of cTnI and fGQDs was examined by cyclic voltammetry (CV) and amperometry (Lakshmanakumar et al., 2019). They detected cTnI over a linear range of 0.17– 3 ng/mL and offered a detection limit of 0.02 ng/mL with good stability and sensitivity. Based on the aforementioned discussion, ECL immunoassay is appealing for diagnosis since its high stability as well as sensitivity and specificity. Despite the considerable advantages for biomedical analysis, ECL often requires specialized and expensive equipment for generating excited states with light-emitting for detection, which to some extent impair its extensive application.

#### Fluorescence Immunoassay

Fluorescence immunoassay is by far the dominant analytical approach in biomedical engineering on account of its outstanding versatility and signal enhancement ability (Strianese et al., 2012).



Nanomaterials have promoted the efficiency of fluorescence assay because nanomaterials have good solubility, low toxicity, and high binding affinity of biomolecules, which can couple with various intensive fluorescence materials for amplification (e.g., Horseradish Peroxidase) (Yeh et al., 2010; Lu et al., 2017).

Miao et al. (2019) used a robust nanoceria-linked immunosorbent assay to detect cTnI based on colorimetric and ratiometric fluorescence. Ratiometric fluorescence overcomes some dependence defects, such as external environment luminophore, consequently, it can be used for trace detection. The schematic of the sensor was illustrated in **Figure 2C**. After the bonding of cTnI and Ab1, o-phenylenediamine (OPD) as an organic substrate was oxidized and converted into 2,3-diaminophenazine (oxOPD) by the robust nanozyme of nanoceria with peroxidase-like properties and the addition of H2O2. OxOPD was immobilized on the surface of g-C3N<sup>4</sup> QDs through hydrogen bonding and π–π stacking interactions. The g-C3N<sup>4</sup> QDs led to a ratiometric fluorescence response as a result of photoinduced electron transfer (PET). Besides, a visible color change was detected through the conversion of colorless OPD to an orange oxOPD, which acted as a colorimetric fluorescence. As a result, cTnI was quantified by combining the merits of the ratiometric assay and colorimetric assay. Similarly, Tan et al. (2019) oxidized the OPD to oxOPD through the Pd-Ir nanocubes catalysis, which possessed excellent peroxidase-like activity. The detection range of cTnI was between 1 pg/mL to 1 ng/mL, and the detection limit was 0.31 pg/mL.

Moreover, Guo et al. (2019) completed multiplexed detection of cTnI, human heart-type fatty acid binding protein (FABP), and MB using Zinc Oxide Nanowires to enhance fluorescence signal.

FIGURE 2 | (A) (a) The schematic of fabricating ECL immunoassay and its possible self-enhanced luminescence mechanism. A sandwich detection format was utilized to detect NT-proBNP. Ab<sup>1</sup> was immobilized via Au-NH<sup>2</sup> bond and dried on AgNC-sem@AuNP modified GCE surface, once the NT-proBNP was captured, the MIL-125 labeled Ab<sup>2</sup> was added to quench the ECL luminophore. The resonance energy transfer was due to the partial overlap between the ECL emission of the AgNC-sem@AuNPs (wavelength 470–900 nm) and the visible adsorption spectrum of MIL-135 (wavelength 406–900 nm). (b) The preparation of self-enhanced luminophore AgNC-Sem@AuNPs. AuNPs combined with Sem-AgNCs with Au-NH<sup>2</sup> bond. (Dong et al., 2019) Copyright 2020 Springer Nature Switzerland AG. (B) Schematic Illustration of ECL-HCR Immunosensor. AuNPs modified GCE immobilized Ab<sup>1</sup> via Au-N and Au-S bonds, and then AuNPs/GCE was prepared by electrodepositing in HAuCl<sup>4</sup> solution. The Ab2-AuNP-T<sup>1</sup> opened hairpin DNA structures (H1 and H2 after cTnI was caught), which triggered hybridization that modified Au NCs on the electrode. Au NCs reacted with K2S2O<sup>2</sup> thus emitted the ECL signal (Zhu et al., 2019). Copyright 2020 American Chemical Society. (C). The mechanism of nanozyme-linked immunosorbent assay for dual colorimetric and ratiometric fluorescent detection. OPD was oxidized and converted into oxOPD by the robust nanozyme of nanoceria with peroxidase-like properties, resulting an emission maximum of oxOPD at 578 nm. OxOPD was immobilized on the surface of g-C3N<sup>4</sup> QDs, which led to a ratiometric fluorescence response as a result of photoinduced electron transfer (PET). The combination of ratiometric fluorescent assay with colorimetric assay could quantify cTnI (Miao et al., 2019). Copyright 2020 from Elsevier B.V. (D) Schematic of UCNPs based immunoassay and the synthesis, surface modification of core-shell UCNPs. UCNPs-Ab<sup>1</sup> and UCNPs-rabbit IgG were added on the conjugate pad, the second anti-(MB Ab1) and anti- (rabbit IgG) antibodies were separately stripped onto the NC membrane. Samples will be captured on the sample pad. UCNPs on the test and control line was excited by a continuous wave laser diode at 908 nm for reading. The Poly (acrylic acid) PAA was added to the core-shell NaYF4: Yb, Er@NaLuF<sup>4</sup> nanoparticles, and yield UCNPs@PAA particles. Then UCNPs@PAA were conjugated with Ab<sup>1</sup> using 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC-HCl) and N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) as cross-linking agents (Ji et al., 2019). Copyright 2020 from Elsevier B.V.

Han and Kim (2019) designed a AuNP-Antibody-HRP conjugate for cTnI detection. They bound aldehydeactivated(ald)HRP and the primary amine group based on adsorption or covalent coupling, to enhance the sensitivity of the AuNPbased conjugates.

Upconversion nanoparticles (UCNPs) are another type of fluorescent nanomaterials that have been used in luminescent and fluorescent detection (Tan et al., 2016). Ji et al. (2019) used core-shell upconversion nanoparticles (UCNP) to capture the MB in blood samples. MB was fixed between UCNPs-Ab<sup>1</sup> and second anti-(MB Ab1) antibody to form a sandwich format. UCNPs were excited by a continuous wave laser diode at 908 nm for fluorescent reading by external equipment. The detailed procedure was illustrated in **Figure 2D**. Applied UCNPs increased the sensitivity of the assay, which reached a limit of detection as low as 0.21 ng/mL with a 90.6–110.5% MB recovery rate (Ji et al., 2019). Ali et al. (2020) also designed a biosensor assisted with UCNPs for ultrasensitive detection of Vaspin.

#### Photoelectrochemical (PEC)

The photoelectrochemical (PEC) process refers to the electricity conversion of photons resulting from photoactive materials

absorbing photons upon illumination and forming electronhole pairs. This, in turn, causes the oxidization-reduction reaction of the molecules and generates charge separation and subsequent charge transfer. PEC is a promising diagnostic tool since the detected photocurrent change is caused by the biological interactions between biomarkers and corresponding recognitions (Zhao et al., 2014). Nanomaterials have improved the sensitivity of PEC assay because they present a low background signal. For instance, AuNPs, QDs, TiO<sup>2</sup> nanotubes (NTs), Pt NPs, and Silica NPs are suitable candidates for PEC biosensors thanks to their efficient photocurrent quenching ability and unique plasmonic properties (Zhao et al., 2012).

Xue et al. (2019) reported a split-type liposomal PEC immunoassay composed of immunoaffinity, cadmium sulfide (CdS) quantum dots-loaded liposomes (QDLL), and TiO<sup>2</sup> nanotubes (NTs) that capture QDs for cTnI detection. The thioglycolic acid (TGA)-capped QDs and QDLL were linked with the Ab<sup>2</sup> as signaling probes. The schema was explained in **Figure 3A**. cTnI was captured by Ab<sup>1</sup> in the 96 well plates, and then QDLL-Ab<sup>2</sup> was introduced after the immunorecognition. Triton X-100 (10%) was introduced to damage the QDLL and release the QDs that were later captured by the TiO2-NTs electrode. TGA-capped CdS QDs reacted with the Titania surface through the complexing between the carboxylic acid functionality on the CdS QDs and the hydroxyl groups of the TiO<sup>2</sup> NTs – via either chelating or bidentate binding modes (or both). Owing to the sensitization effect, the photocurrents were acquired for PEC immunoassay.

Similarly, Dong et al. (2020) modified the indium tin oxidepolyethylene terephthalate (ITO-PET) electrode with Bi2Se<sup>3</sup> and the flower-like ZnIn2S<sup>4</sup> nanospheres (ZIS). The latter was synthesized by a solvothermal method, which accelerated the electronic transition and improved the photocurrent conversion efficiency. Additionally, the cadmium selenide (CdSe) QDs, which modified Ab2, were able to increase the photocurrent blocked by immunoaffinity binding. This resulted in a detection limit of 0.026 ng/mL of cTnI (Dong et al., 2020).

Gao et al. (2019) fabricated cellulose paper-based, singlecrystalline, three-dimensional aloe like TiO<sup>2</sup> arrays (PSATs) as the electron transporting material. It was subsequently coupled with CdS to form PSATs/CdS to extend the solar spectrum response for cTnI detection (**Figure 3B**) (Gao et al., 2019). Single stranded DNAs (ssDNAs) that bound to cTnI specifically were coupled with positive-charged mesoporous silica nanoparticles (PMSNs). The complexes were prepared as the nanocarrier to entrap the Cu2+, which was regarded as a signal quencher because of its reaction with CdS to form CuxS. After the formation of cTnIssDNAs complexes, Cu2<sup>+</sup> formed CuxS, which decreased the photocurrent signal. This process was used to quantify the concentration of cTnI.

Despite the accomplishments of PEC assay in CVD diagnosis, PEC assay has some disadvantages, especially the inherent drawback of photoanodes. For example, the CdS photoanodes of PEC might have low charge separation and transfer efficiency. Notably, they are unstable in water upon illumination as well.

#### Electrical and Reduction

Electrochemical (EC) immunoassay has attracted considerable attention and is one of the most promising techniques for diagnosis owing to its high sensitivity and fast response time. Besides that, nanomaterials are highly appreciated for improving EC sensitivity and intensity because of their catalytic property, conductivity, binding affinity, and large surface area. Like, AuNPs can bind biomolecules and facilitate electron transfer for catalyzing electrochemical reactions (Zhang et al., 2014). Besides, nanosheets and other hollow-structured nanomaterials can increase the loading efficiency of agents on grounds of their large surface area that enhances signal (Zhu et al., 2017).

Lopa et al. (2019) developed a AuNP modified titanium (Ti) metal substrate to detect cTnI based on DNA aptamer. AuNPs were deposited on Ti sheets by the potential-step deposition method. They immobilized the DNA aptamer using a selfassembled monolayer mechanism. The sensor obtained high sensitivity and specificity with the assistance of the AuNP-Ti layer and detected cTnI with the minimum detection limit of ca. 0.18 pM (Lopa et al., 2019).

Moreover, Adeel et al. (2019) modified the boron nitride nanosheets (BNNS) with AuNPs to detect MB in a low-cost, label-free and simple way (**Figure 4A**). BNNSs were synthesized via the hydrothermal method and were deposited on the fluorine-doped tin oxide (FTO) electrode. Subsequently, AuNPs were chemically deposited on the BNNS/FTO electrode. The AuNPs/BNNSs/FTO electrode was then used as a transducer to fix a thiol-functionalized DNA aptamer (Apt) via the Au-S covalent. When the MB bound to the sensor, [Fe(CN)6]3-/4 was used as a redox probe to monitor the oxidation current variation. The Apt/AuNPs/BNNSs/FTO sensor showed a high signal response for MB, with a detection limit of 34.6 ng/mL.

Phonklam et al. (2020) presented a molecularly imprinted polymer-based (MIP) EC sensor using a screen-printed carbon electrode (SPCE) to detect cTnT. The MIP sensor possessed an electrodeposited polymethylene blue (PMB) redox probe on SPCE, which was functionalized with multi-walled carbon nanotubes (MWCNTs). Also, the electropolymerized polyaniline was around the cTnT immobilized platform. The sensor response was stimulated by pulse voltammetry, wherein the concentration of cTnT was negatively associated with the PMB current. The linearity range of the sensor was between 0.10–8.0 pg/mL, with a detection limit of 0.040 pg/mL. Similarly, Vasantham et al. (2020) integrated MWCNT with Ab<sup>2</sup> to detect cTnI via paper-based multi-frequency impedimetric transducers. The limit of detection was 0.05 ng/mL, and the response time was ∼1 min.

Singh et al. (2019) developed a microfluidic biosensorintegrated mesoporous nickel vanadate hollow-nanosphere modified chitosan (Ch-Ni3V2O8) to detect cTnI in patient samples. They synthesized chitosan-based (Ch) Ni3V2O<sup>8</sup> hollownanospheres that could load abundant antibodies made possible by their hydroxy and amino clusters in the nanocomposite, their good adhesion capability, and their larger surface. Moreover, they amplified the electrochemical readouts because

cTnI detection using PMSN/Cu2+/ssDNAs. Positive-charged PMSNs were added into Cu2<sup>+</sup> solutions. Cu2<sup>+</sup> entered the pores of PMSNs via diffusion. Later, ssDNAs bound cTnI specifically was added and attached on the surface of PMSNs. Once the cTnI was recognized by ssDNAs, the complexes reacted with CdS that was functionalized onto the PSATs, to form CuxS and diseased the photocurrent signal. The changes of photocurrent were correlated to the concentration of cTnI (Gao et al., 2019). Copyright 2020 from Elsevier B.V.

of tunable oxidation states of the Ch-Ni3V2O<sup>8</sup> matrix. The microfluidic biosensor was fabricated using a three-electrode system, including a patterned gold (Au), silver (Ag/AgCl), and Ch-Ni3V2O<sup>8</sup> electrodes on a glass substrate. A bare Au electrode represented as the counter electrode (CE). The Ch-Ni3V2O<sup>8</sup> composite was added on the Au-coated substrate to capture

FIGURE 4 | (A) Schematic illustration for aptasensor fabrication and MB detection process. BNNSs were obtained from filtered BN powder dissolved solution via hydrothermal method. BNNSs were spinning deposited onto FTO electrode. AuNPs were then functionalized onto BNNS/FTO electrode to form AuNPs/BNNSs/FTO electrode acted a transducer to fix a thiol-functionalized DNA aptamer (Apt) via the Au-S covalent interaction. The [Fe(CN)6]3-/4- was used as a redox probe to monitor the oxidation current variation after MB binding to the Apt (Adeel et al., 2019). Copyright (© 2020 from Elsevier B.V. (B). The illustration of electrochemical immunosensor. DIL was noncovalently non-covalently bonded with HCNTs to form the DIL-HCNTs composite, which provided sufficient binding sites for Ab1. After antigen was capture by Ab1, the sandwich immunocomplexes formed on the electrode hindered electron transfer thus decreased the peak current of DPV. The signals were corresponded to concentrations of cTnI (Shen et al., 2019). Copyright 2020 American Chemical Society. (C) The preparation procedure of the sandwich-type electrochemical immunosensor. Dispersed Fe3O4-NH<sup>2</sup> and glutaraldehyde was stirred to form Fe3O4-Ab1, BSA was added to block remaining active sites on the surface of Fe3O4. CoPc NPs were first dispersed in the mesoporous of Fe3O4-Ab1, then APSM was used to cap on the mesoporous of Fe3O4-Ab<sup>1</sup> by electrostatic interaction and formed APSM-capped CoPc NPs- Fe3O4-Ab1. Once the target cTnI was captured, CoPC NPs were released after APSM was separated. The CoPC NPs oxidized the cobalt element from Co (I) to Co (II) with H2O2. The reduction current was corresponded to concentrations of cTnI (Ma et al., 2019). Copyright 2020 from Elsevier B.V. (D) The schematic diagrams of preparing Au@AgNC/N, S-rGO-Ab2. Au solation was mixed with AA and AgNO3 in turn, then the Au@AgNC were collected using centrifugation. Later, Au@AgNC and S-rGO were reacted for 8 h for Au@AgNC and S-rGO composite. Ab<sup>2</sup> solution was added into Au@AgNC/N and S-rGO, and oscillated to form Au@AgNC/N, S-rGO-Ab2. AuNC/GO immobilized Ab<sup>1</sup> via amino-Au affinity. Once cTnI was captured, the catalyzed-oxidation of o-phenylenediamine (o-PD) with H2O<sup>2</sup> was accelerated. The oxidation generated 2,3-diaminophenazine that gained in electrons and hydrogen and generated a larger current signal of DPV at 0.34 V (Lv et al., 2019). Copyright 2020 Springer Nature Switzerland AG.

cTnI antibodies and connect the microchannels. Ag/AgCl was deposited on the substrate using e-beam evaporation for the reference electrode (RE). This device offered 5 pg/mL limit detection of cTnI with high stability, good selectivity, and high reproducibility, and detected BNP, MB, cardiac troponin C (cTnC), and cTnT with specific antigens (Singh et al., 2019).

Shen et al. (2019) presented a label-free electrochemical immunosensor using a helical carbon nanotube (HCNTs) supported aldehyde-functionalized ionic liquid (DIL). The DIL-HCNTs provided binding sites for Ab1, which simplified the sensor construction processes (**Figure 4B**) (Shen et al., 2019). The sandwich immunocomplexes influenced electron transfer on the electrode, thereby decreasing the peak current of differential pulse voltammetry (DPV). The signals corresponded to concentrations of cTnI.

Additionally, Sandil et al. (2019) immobilized cTnI (Ab1) on tungsten trioxide nanorods (WO<sup>3</sup> NRs), which were deposited on indium tin oxide (ITO) using the electrophoretic deposition technique that worked as the electrode. The authors detected cTnI in a linear detection range of 0.01–10 ng/mL

with high reproducibility (Sandil et al., 2019). Zhang et al. (2019) synthesized β-cyclodextrins-functionalized (CDs) 3D porous graphene-supported Pd@Au nanocubes (NCs) for cTnI detection. CDs were able to increase the dispersibility of the 3D-porous graphene and improve the capability of Ab<sup>2</sup> capture. Besides, the electrochemical signal was improving given the Pd@Au NCs. In addition, the amino-functionalized microporous carbon sphere was functionalized using AuNPs and Th (AuNPs-FMCS-Th) to immobilize Ab<sup>1</sup> effectively and accelerate the electron transfer process. The transfer was based on the reduction of H2O<sup>2</sup> on the Th-modified electrode surface, which further amplified the signal response. The authors detected cTnI with a low detection limit of 33.3 fg/mL through the EC signal amplification labeling in the sandwich format (Zhang et al., 2019).

Similarly, Ma et al. (2019) utilized mesoporous Fe3O4- NH<sup>2</sup> for loading cobalt phthalocyanine NPs (CoPc NPs) and captured Ab<sup>2</sup> to form Fe3O4-Ab<sup>1</sup> to detect cTnI. The aminated polystyrene microsphere (APSM) was used to cap on the mesoporous of Fe3O4-Ab<sup>1</sup> by electrostatic interaction and presented as a molecular gate. Once the target cTnI was captured, CoPC NPs were released after APSM was separated. The CoPC NPs showed a superb catalytic performance that oxidized the cobalt element from Co (I) to Co (II) with the addition of H2O2. The reduction of current corresponded to concentrations of cTnI. This novel controlled release system-based EC immunoassay presented a broad linear range from 1.0 pg/mL to 100 ng/mL with a low detection limit of 0.39 pg/mL (**Figure 4C**) (Ma et al., 2019). Lv et al. (2019) functionalized the gold nanocube with graphene oxide (AuNC/GO) as the substrate to immobilize Ab<sup>1</sup> via amino-Au affinity. To detect cTnI, the authors utilized Au@Ag core–shell nanocubes and nitrogen/sulfur doped GO as signal amplification labels to conjugate with Ab<sup>2</sup> via Ag-N bonds. Once cTnI was captured, the catalyzed-oxidation of o-phenylenediamine (o-PD) with H2O<sup>2</sup> was accelerated. The oxidation generated 2,3-diaminophenazine that gained in electrons and hydrogen and released an exaggerated current signal of DPV, which was used to electrochemical detect cTnI (**Figure 4D**) (Lv et al., 2019).

EC assay has shown advances in the sensitivity, but disadvantages remain. More specifically, the facility requires periodical calibration and care maintenance to guarantee accuracy, which is expensive and bothersome.

### Surface-Enhanced Raman Scattering (SERS)

Surface-enhanced Raman scattering (SERS) immunoassay shows strong potential for CVD clinical diagnosis in view of their excellent multiplexing ability, high sensitivity, and large dynamic range (Huang et al., 2019). Typical SERS-based immunoassay uses substrates modified with Ab<sup>1</sup> to capture targets, and the concentration of the targets is quantified by the SERS immunoprobe (Wang et al., 2017c). Nanomaterials have simplified the preparation and attachment of Raman labels thus have improved the sensitivity. For example, Fu et al. (2019) developed a capture probe/target/SERS nanotags platform to detect cTnI. They functionalized graphene oxide (GO) with AuNPs, which acted as both SERS nanotags and signal amplification carriers. The GO/AuNP complexes provided strong SERS enhancement ability and detected cTnI with a 5 pg/mL detection limit (Fu et al., 2019). Similarly, Cheng et al. (2019) loaded targets and polyclonal-antibody-conjugated Au@Ag core–shell nanoparticles in a gold-patterned chip that ran as a SERS active template for the ultrasensitive detection of cTnI and CK-MB. The limits of detection were 8.9 pg/mL and 9.7 pg/mL for cTnI and CK-MB, respectively.

While large surface area and outstanding physical properties of nanomaterials have facilitated sensitive SERS immunoassays, additional work is required on stabilizing the tag functionalization in SERS assays.

#### Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) biosensor measures the changes of the refractive index at the sensor surface. Its principle is based on charge-density oscillation that exists at the interface of two media, normally involving a metal (i.e., gold) and a dielectric. SPR has been used to detect proteins, DNA, and drugs (Mullett et al., 2000). SPR sensors are mainly classified as sensors with angular wavelength and intensity modulation (Dudak and Boyaci, 2009). Nanomaterials with plasmonic and optical properties, good distributing ability, strong photostability have amplified signals, hence, improved SPR sensitivity (Liu et al., 2013).

Recently, Chen et al. (2019) modified Au film with AuNPs and polydopamine (PDA), which ran as platforms for immobilizing Ab<sup>1</sup> and SPR sensing. The films attracted the detection antibody on the PDA-coated Fe3O<sup>4</sup> as the immune probe to detect cTnI (with a detection limit of 3.75 ng/mL) (Chen et al., 2019). The authors also introduced secondary antibody conjugated with multi-walled carbon nanotube (MWCNTs)-PDA-AgNPs to interact with cTnI exposed on the surface of probes, thus further amplifying the SPR response signal (**Figure 5A**).

#### Field Effect Transistor (FET)

Field effect transistor-based (FET) sensors have been applied in the biomedical analysis on grounds of their small size, high versatility, and low costs (Syedmoradi et al., 2019). FET-based sensors use an electric field to control the flow of current on platforms such as silicon nanowire and graphene (Kaisti, 2017).

Silicon nanoribbon fabricated biosensors are highly sensitive and uniform. However, they are expensive and complicated, requiring oxidation, photolithography, and wet etching. In contrast, semiconducting metal-oxide-based field-effect transistors (FET) biosensors show advantages, as simplicity and reliability. Photolithography-free shadow mask fabrication methods are also beneficial compared to silicon-based sensors. To give an example, Liu et al. developed a highly uniform, sensitive, and reusable In2O<sup>3</sup> nanoribbon biosensor array using a lithography-free, scalable, and facile fabrication with high time efficiency (**Figure 5B**) (Liu Q. et al., 2016). The nanoribbons were formed through a first-layer shadow mask that was attached to a silicon substrate using In2O<sup>3</sup> sputter-coating.

FIGURE 5 | (A) The schematic diagram of experimental procedure. A bare Au film with soaked into DA solution in Tris-buffer to form PDA-Au film. Afterward, AuNPs were deposited onto PDA-Au film with the help of HAuCl<sup>4</sup> to improve the sensitivity. After 30 min, Ab<sup>1</sup> was immobilized on the PDA-Au for further cTnI capture. As for detection probe, Fe3O4@PDA-detection antibody immune probe was collected with external magnet after Fe3O4@PDA and detection antibody were mixed and shake for 24 h. Then samples with different concentrations of cTnI were incubated with Fe3O4@PDA-detection antibody, and the nanoconjugates were collected by a magnet. Once the resonant wavelength of probes was stable, MWCNTS-PDA-AgNPs/secondary antibody was added to enhance SPR response signals (Chen et al., 2019). Copyright 2020 from Elsevier B.V. (B) Schematic of In2O<sup>3</sup> nanoribbon biosensor and electronic ELISA for cardiac biomarker detection. The first shadow mask was attached onto the SiO2/Si wafer, and then In2O<sup>3</sup> ribbons were deposited using radio frequency (RF). Nanoribbons were obtained after removing first-layer shadow mask. A second shadow mask was attached to define the Ti/Au disposition, which used e-beam evaporation. Finally, a FET-based sensor was completed after removing shadow mask. Captured antibodies were fixed on the In2O<sup>3</sup> surface and captured the biomarkers. Biomarkers were fixed between the capture antibody and a biotinylated secondary antibody that is specific to biomarkers. The biotin tales of the secondary antibody was to capture streptavidin, which bound to a biotinylated urease that led to deprotonation the hydroxyl groups on the nanoribbon and increased the negative surface charges. The change of surface charge was detected by FET sensor because negative charges decreased the conduction of nanoribbons (Liu Q. et al., 2016). Copyright 2020 American Chemical Society. (C) Schematic representation of the preparation of antibody-modified nanoprobes involved in the lateral flow immunoassay. Carboxylated Nanospheres (CNs) were incubated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crystalline (EDC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), the mixtures were collected centrifugation. TP110 (detection antibody) was incubated with CNs at 5.8, 6.8, or 7.8 pH. Florescent molecules were conjugated with CNs-TP110 for later detection on LFA. Briefly, sample flowed through sample pad, and conjugated with fluorescent labeled CNs-TP110 on the conjugate pad. The complexes went through NC membrane that immobilized Ab1 (3H9) on the test line and Goat anti-mouse on the control line for further antigen capture. The strip was read by a fluorescent analyzer after 15 min (Lou et al., 2019b). Copyright 2020 American Chemical Society. (D) Scheme of fabrication of the microfluidic paper-based device (µPAD) for multiplex detection of cardiac markers. Capture antibodies were immobilized onto NC membrane at three positions (G for GPBB, M for CK-MB, and T for troponin T). Serum sample was added at central sample zone. After antibody-antigen capture, AuNPs conjugated anti-cTnT (red), silver-nanoparticles (AgNPs) conjugated anti-GPBB (yellow) and Gold urchin nanoparticles conjugated anti-CKMB (purple) were added for color detection (Lim et al., 2019). Copyright 2020 from Elsevier B.V.

The nanoribbons went through a metal electrode deposition process that was defined by a second-layer shadow mask. Two shadow masks were simply removed instead of being lifted off, as in the case in photolithography. Captured antibodies were fixed on the In2O<sup>3</sup> surface using N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry. A biotinylated secondary antibody was introduced to attach biomarkers, and its biotin tales were bounded to the streptavidin that attracted biotinylated urease later. Urea increased the pH of the solution, hence, causing the deprotonation of hydroxyl groups on the nanoribbon and lowering surface potential. Consequently, the conduction of the n-type nanoribbon FETs was decreased due to increased negative surface charges. They detected cTnI, CK-MB, and BNP down to 1 pg/mL, 0.1 ng/mL, and 10 pg/mL concentration range, respectively.

#### Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-Linked Immunosorbent Assay (ELISA) is a commercialized method that identifies the concentration of targets through the color change of antigen-antibody reactions using an enzyme-linked conjugate and enzyme substrate. Even though ELISA shows advantages in reading results, it still requires more effort to improve sensitivity and accuracy (Aydin, 2015). Unique physical properties and biocompatibility of nanomaterials have greatly improved the performance of ELISA and below lateral flow assay (LFA). Nanofibers have the potential to reduce non-specific binding and improve binding efficiency due to their large surface area as an illustration. In addition, AuNPs conjugated with functional materials, such as HRP could amplify the detection signal in the assay (Wang W. et al., 2018).

Recently, Jiao et al. (2019) completed a multimodal ELISA diagnosis based on photothermal effect and the peroxidasemimicking property in Au@Pt nanodendrites. The cTnI target was quantified using photothermal, colorimetric, and ratiometric fluorescent signals simultaneously.

#### Paper Based Lateral Flow Assay (LFA)

Lateral flow assay detects targets in a fast, simple, and cheap manner that has attracted much interests in recent years (Koczula and Gallotta, 2016). Lou et al. (2019a) functionalized the LFA with a multi-layer structure, including the BSA layer to increase biotinylation sites, a streptavidin layer loaded with a fluorescent dye, and an outermost layer on which the biotinylated antibody is bounded to the streptavidin. Due to the high loading efficiency of fluorescent molecules, the authors detected the cTnI within the range of 0.049–50 ng/mL (Lou et al., 2019a). Recently, Lou et al. (2019b) conjugated Ab<sup>2</sup> onto polystyrene nanospheres at pH 5.8 for fast and sensitive immunodetection. At pH 5.8, the tail-on orientated antibodies (TP110) were conjugated on the carboxylated nanospheres (CNs) because of the charge distribution and the hydrophobic area of antibodies. As a result, the loading capacity of TP110 was increased, thus the authors detected cTnI with high sensitivity. The detailed procedure was illustrated in **Figure 5C**. Lim et al. (2019) utilized AuNPs conjugated anti-cTnT, silvernanoparticles (AgNPs) conjugated anti-GPBB, and Gold urchin nanoparticles conjugated anti-CKMB as probes to detect multiplexed biomarkers simultaneously. The serum sample was loaded on the central sample pad and flowed through the NC membrane where anti-GPBB, anti-CK-MB, and anti-cTnT were immobilized on at three positions. Nanoparticles-conjugated detection antibodies were added on the NC membrane for color detection in the end. The detailed illustration was in **Figure 5D** (Lim et al., 2019).

Despite the convenience of ELISA and LFA in analyzing biomolecules, they have some drawbacks. ELISA suffers from complicated procedures, long assay duration (at least 2–3 h), and large sample consumptions. LFA requires labor-intensive preparations and is highly susceptible to false positives/negatives due to improper operations.

### NANOTECHNOLOGY-BASED MOI

Nanomaterials with good bioavailability and versatility have increased the accuracy and specificity of clinical MOI applications thanks to improved resolution, signal amplification, and simple manipulation. Among other nanomaterials, nanoparticles are suitable for MOI since their mobilities in both internal and external vascular systems, high surface area to volume ratio, and imaging functionality. These advantages allow them to circulate through human bodies with low restrictions and produce functional imaging vehicles as contrast agents when they are applied in MOI settings, which leads to significantly improved diagnosis efficiency.

Nanoparticles can be used for RNA detection in intravascular systems because of their mobility. Injected or swallowed nanoparticles that are functionalized with MOI detectable molecules can circulate through the human body and target specific RNA for diagnosis. However, the size, shape, morphology, and density of functionalized nanoparticles should be explored carefully since the RNA detection requires nanoparticles entering and interacting with cells.

In addition, nanoparticles can work as nanoscale contrast agents by incorporating materials such as photoacoustic, fluorescent, radioactive, paramagnetic, superparamagnetic, electron-dense, light-scattering particles, and multimodal functional groups that are detectable by MOI.

The fluorescent material is a popular choice to conjugate with nanoparticles. For example, fluorescence labeled quantum dots (QDs) exhibit good performance in atherosclerotic plagues imaging (Jaffer et al., 2007). Popular radionuclides, including <sup>18</sup>F, <sup>64</sup>Cu, <sup>68</sup>Ga, <sup>124</sup>I, <sup>86</sup>Y, have been conjugated with QDs, UCNPs, AuNPs, and NCs and have demonstrated good diagnostic functions (Saraste et al., 2009). For instance, radiolabeled nanoparticles play significant roles for atherosclerotic plagues under PET (Dobrucki and Sinusas, 2010). Radionuclides can be labeled either on the surface or encapsulated inside nanoparticles.

Additionally, superparamagnetic nanoparticles, like iron oxide (IO) nanoparticles and predominately magnetite (Fe2O3/Fe3O4), can improve the sensitivity of MRI by providing dark contrast to enhance the signal. For instance, MRI can detect thrombus (Chen and Wu, 2011), and the composition of plaques, including the fibrous cap and necrotic core, macrophage content, plaque neovascularization, intraplaque hemorrhage, and mural thrombus using superparamagnetic nanoparticles as contrasts (Avelar et al., 2017).

Also, AuNPs with good optical properties are good signal enhancers for photoacoustic tomography (PAT) (Taruttis et al., 2010). For example, Pan et al. (2014) successfully detected thrombus using gold nanobeacons under PAT. AuNPs can enhance the signal of coherence tomography (OCT) based on light-scattering as well.

Moreover, AuNPs were also widely used in CT as contrast agents. Hyafil et al. (2007) identified macrophages in atherosclerotic plagues using iodinated AuNPs facilitated CT. Kim et al. (2012) used polyethylene glycol (PEG)-coated AuNPs to improve the performance of CT scanning. They utilized PEG-coated GNPs as a contrast agent, in which the PEG-coating

prevented antibiofouling and extended lifetime of AuNPs in the bloodstream. The PEG-coated GNPs obtained a better X-ray absorption coefficient and thus provided high-resolution images.

#### CONCLUSION AND FUTURE PERSPECTIVE

Current clinical challenges of CVDs include generating straightforward, accurate diagnostic clinical decisions, and monitoring drug responses frequently. To overcome these difficulties, various platforms have been proposed. Among these platforms, CIAs have gained attraction since they can measure and evaluate the expression of biomarkers for the prediction and diagnosis of disease in a sensitive, rapid, cheap, and noninvasive manner. Additionally, MOI settings have emerged as promising techniques for CVD diagnosis thanks to their excellent imaging capabilities that facilitate physicians to make decisions. Even though significant advances in CIAs and MOI have been made regarding CVD diagnosis, the early-stage diagnosis is still challenging since symptoms of early-stage CVDs are vague, and the expression level of cardiac biomarkers at early-stage is relatively low for detection.

Therefore, novel platforms with improved sensitivity and specificity are required. As summarized in this review, nanotechnology has greatly contributed to the developments of MOI and CIA on account of its specific physicochemical properties. By virtue of their excellent biocompatibility, nanomaterials can conjugate with various biomolecules because their excellent biocompatibility. These functional biomolecules were able to increase the sensitivity and specificity of diagnostic platforms. For example, AuNP-conjugated capture antibody can reduce non-specific bindings, thereby increasing the capture specificity and eliminating background noises in various CIAs. Besides, fluorescence lumiphore conjugated nanomaterials can work as enhancers to amplify the detective signals in ELISA, or as contrast agents in MOI. In addition, except for functionalized with other biomolecules, nanomaterial itself can serve as a signal enhancer. This is made possible by their unique optical, electrical and plasmonic properties, silica NPs can quench photocurrent effectively in PEC biosensors as an example. Moreover, nanomaterials with a large surface area (e.g., nanocubes) can improve the loading efficiency of biomolecules to increase the sensitivity of CIAs. To better elucidate CIAs in CVD diagnosis, the lasted outcomes of nanotechnology assisted CIAs (e.g., ECL) was introduced, with comparison to the sensitivity and detection range of cardiac targets among these

#### REFERENCES


assays. We also reviewed the clinical applications of nanoparticles in MOI settings. In this regard, we believe strongly bridging clinical platforms and nanotechnology is necessary for directing future research plans.

In this review, we have highlighted the advantages of CIAs. They are non-invasive, cheap, sensitivity, and convenient. Its repeat-sampling ability also makes it ideal for long-term diagnosis and prognosis. However, the CIA testing still faces challenges. An important concern is the sensitivity and specificity of the biomarker. Previous researchers have shown that a single marker may be insufficient, lack sensitivity and specificity for accurate CVD diagnosis. It is unrealistic to provide clear diagnosis results using only one biomarker owing to the complexity, heterogeneity and diversity of pathogenesis in different populations. Additionally, biomarkers may be regulated differently during the development of pathologies. Consequently, even though we can diagnosis some clinical subjects who have high risks for certain diseases using biomarkers (e.g., cTnI for AMI), we have insufficient information on the progression and states of the diseases (Madhurantakam et al., 2018). To solve this problem, multiplexed biomarker panels are critical. The multiplexed analysis of various biomarkers can establish correlations (or scores) with high specificities and predictive values, as a result, can improve the accuracy of diagnostic decisions. Hence, the fabrication of CIAs should focus on multiplexed analysis in the future. In addition, current findings on biomarkers are based on relatively small samples, which may possess large variations (Sayed et al., 2014). Most of the studies only focused on the correlation between biomarker expression level and single disease. The pathological significance behind the biomarker expression level changing is not addressed yet. As such, large-scale and systematic clinical studies are needed to recognize and better understand the mechanisms of biomarkers. Most importantly, combination with advanced analytic tools (e.g., machine learning) that is capable of developing objective and automatic algorithms to analyze large-scale and highdimensional-multiplexed data should be highlighted in future studies, which are supposed to greatly improve the efficiency and accuracy of diagnosis.

#### AUTHOR CONTRIBUTIONS

ZY and JZ coordinated this project. CS and HX wrote this manuscript. CS and HX collected and summarized the literatures. YM edited the figures in this manuscript and revised this manuscript.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer MK declared a shared affiliation, with no collaboration, with one of the authors, YM, to the handling Editor at the time of review.

Copyright © 2020 Shi, Xie, Ma, Yang and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nanoparticle-Mediated Drug Delivery for Treatment of Ischemic Heart Disease

Chengming Fan<sup>1</sup> \* † , Jyotsna Joshi<sup>2</sup>† , Fan Li<sup>2</sup> , Bing Xu<sup>2</sup> , Mahmood Khan<sup>3</sup> , Jinfu Yang<sup>1</sup> and Wuqiang Zhu2,4 \*

<sup>1</sup> Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, China, <sup>2</sup> Department of Cardiovascular Diseases, Mayo Clinic, Scottsdale, AZ, United States, <sup>3</sup> Department of Emergency Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, United States, <sup>4</sup> Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, United States

#### Edited by:

Qingxin Mu, University of Washington, United States

#### Reviewed by:

Nicolas Christoforou, Pfizer, United States Deqiang Li, University of Maryland, Baltimore, United States

#### \*Correspondence:

Chengming Fan fchmfchm@163.com Wuqiang Zhu Zhu.Wuqiang@mayo.edu †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

> Received: 04 March 2020 Accepted: 02 June 2020 Published: 24 June 2020

#### Citation:

Fan C, Joshi J, Li F, Xu B, Khan M, Yang J and Zhu W (2020) Nanoparticle-Mediated Drug Delivery for Treatment of Ischemic Heart Disease. Front. Bioeng. Biotechnol. 8:687. doi: 10.3389/fbioe.2020.00687 The regenerative capacity of an adult cardiac tissue is insufficient to repair the massive loss of heart tissue, particularly cardiomyocytes (CMs), following ischemia or other catastrophic myocardial injuries. The delivery methods of therapeutics agents, such as small molecules, growth factors, exosomes, cells, and engineered tissues have significantly advanced in medical science. Furthermore, with the controlled release characteristics, nanoparticle (NP) systems carrying drugs are promising in enhancing the cardioprotective potential of drugs in patients with cardiac ischemic events. NPs can provide sustained exposure precisely to the infarcted heart via direct intramyocardial injection or intravenous injection with active targets. In this review, we present the recent advances and challenges of different types of NPs loaded with agents for the repair of myocardial infarcted heart tissue.

Keywords: nanoparticles, controlled release, myocardial infarction, cardiac repair, drug delivery systems

### INTRODUCTION

Ischemic heart diseases, caused by coronary artery obstruction, account for almost 80% of deaths from cardiovascular diseases (Lloyd-Jones et al., 2010). Traditional clinical approaches for myocardial infarction rely on surgical revascularization procedures, such as coronary stenting or coronary artery bypass grafts (CABG). Although the novel therapeutics using cells (especially stem cells) (Gao et al., 2013; Madigan and Atoui, 2018; Terashvili and Bosnjak, 2019), genes (Oggu et al., 2017), exosomes (Davidson and Yellon, 2018), and growth factors (Crafts et al., 2015; Reboucas et al., 2016) are emerging and have shown significant research outcomes, numerous challenges still exist in translating those technologies into clinical practice (Egwim et al., 2017).

Nanoparticles have a long history. Faraday (1857) reported the synthesis of a colloidal Au NP solution for the first time. Similarly, Richard Feynman gave a talk in 1959 describing molecular machines built with atomic precision (Feynman, 1960). These were considered the very first reports on nanotechnology. Metal nanoparticles play a major role in the field of nanoparticle research (Jeevanandam et al., 2018). The 1950s and the 1960s saw the world turning its focus toward the use of nanoparticles in the field of drug delivery. Biological approaches for molecular nanotechnology were the first scientific conference held on the topic in the year 1996 (San Diego, CA, United States). Biological systems are organized at nanoscale dimensions and synthetic nanomaterials correlated in size with biological structures such as proteins, glycolipids, and DNA (Singh et al., 2011). Nanoparticles (NPs) are a type of nano-sized vesicles and can act as a sustained

release delivery system of therapeutic agents and provide enhanced myocardial recovery in ischemic heart diseases (Binsalamah et al., 2011; Oduk et al., 2018). Nanoparticles can be classified either as organic (**Figures 1A,B**), inorganic (**Figures 1C–G**), or hybrid. Organic NPs usually show good biocompatibility, whereas inorganic NPs provide advantages in tailoring varied functions and properties (Vinhas et al., 2017). Organic nanoparticles are fabricated from proteins, carbohydrates, lipids, and other organic compounds to a characteristic dimension, such as a radius around 100 nm (Pan and Zhong, 2016), and are widely used NPs in cardiac therapy (**Table 1**). Inorganic NPs include carbon-based NPs, such as carbon nanotubes, buckyballs, and graphene, with remarkable features, strength and unique electrical properties (conducting, semiconducting, or insulating) (Vinhas et al., 2017). Besides, these inorganic NPs also include metal NPs, made of gold, silver, and iron oxide (Vinhas et al., 2017). **Table 2** lists recent studies that used inorganic NPs for cardiac therapy. In the organicinorganic hybrid nanoparticles, the organic functional groups combine the unique properties of the inorganic counterparts to confer efficient utility for various in vivo biomedical and clinical applications (Haque and Chowdhury, 2018). The use of hybrid nanoparticles for the slow release of drugs has been gaining great interest, particularly, to improve the selectivity and efficacy of the drugs by combining features of both organic and inorganic components in one nanoparticle system (**Table 3**).

Here, we review the studies done over the last 10 years that investigated the applications of different NP types for repairing cardiac tissue after myocardial infarction and also summarize treatment efficacies of different NP types. Furthermore, some of the advances, challenges, and future strategies in this field are also provided.

### ORGANIC NANOPARTICLES

#### Lipid-Based NPs

Typical lipid-based NP formulations (**Figure 1A**) include solidlipid nanoparticles, nanostructured lipid carriers, lipid-drug conjugates, and nanoemulsions; all are primarily comprised of physiological lipid analogs with surfactants as stabilizers (Qi J. et al., 2017). According to the size of lipid-based nanoparticles, they are named as micelles (∼15 nm), liposomes (∼100 nm) or polymeric NPs (Paulis et al., 2012; Vinhas et al., 2017). Micelles consist of lipids and other amphiphilic artificial molecules that self-assemble in aqueous solution and form a monolayer with the hydrophobic phase inside that incorporates hydrophobic therapeutic agents (Katsuki et al., 2017). The enclosed space in the micelle is more confined than that in liposomes (Katsuki et al., 2017). Liposomes are heavily investigated in nanomedicine and are the first to get FDA approval for nanomedicine (Katsuki et al., 2017; Vinhas et al., 2017). Liposomes mainly consist of phospholipids that form bilayers with the aqueous phase inside, conferring superior biocompatibility to the liposomes (Katsuki et al., 2017). Polymeric nanoparticles, such as polylactic acid (PLA), polyglycolic acid (PGA), and poly lactic-co-glycolic acid (PLGA) are FDA-approved polymers. PLGA is a copolymer of PLA and PGA and is being tested for drug delivery systems for intractable diseases, including cardiovascular diseases (Pascual-Gil et al., 2017; Oduk et al., 2018).

Lipid NPs are broadly considered as promising candidates for the delivery of therapeutics in the infarcted heart. They possess morphology similar to that of cell membranes and can incorporate both lipophilic and hydrophilic substances (Saludas et al., 2018). They have successfully demonstrated the ability to deliver several biomaterials in the target tissue, such as low molecular weight drugs, imaging agents, peptides, proteins, and nucleic acids (Cheraghi et al., 2017). Paulis et al. (2012) reported that micelles are promising vehicles for the delivery of cardioprotective drugs, needed for the acute stage of MI, and also for the delivery of drugs that regulate infarct healing during the chronic stage of MI. On the other hand, liposomes are more suited for the delivery of pro-angiogenic drugs to the infarct microvasculature (Paulis et al., 2012).

#### Dendrimers

Dendrimers (**Figure 1B**) are the smallest of all the nanocarriers and they have their multiple end groups that are appropriate for a high degree of link targeting or the active agents (Morgan et al., 2005). Dendrimers are dendritically expanded macromolecules with monodisperse structure consisting of a central core, branching interior and exterior functional groups (Morgan et al., 2005). Dendrimers possess the advantage of enhancing the binding capacity upon modification of their exterior surface with some ligands or antibodies for active targeting (Thomas et al., 2013); also, they can carry drugs with poor solubility (Singh et al., 2016). Xue et al. (2018) reported that cardiomyocyte apoptosis and infraction size were significantly reduced following single intravenous administration of dendrimer (15 µg), loaded with microRNA-1 inhibitor, in the acute mice MI model.

Selective studies using organic nanoparticles for the delivery of therapeutics to repair infarcted myocardial tissue were listed as **Table 1**. Paulis et al. (2012) reported that micelles are promising vehicles for the delivery of cardioprotective drugs, needed for the acute stage of MI, and also for the delivery of drugs that regulate infarct healing during the chronic stage of MI. On the other hand, liposomes are more suited for the delivery of pro-angiogenic drugs to the infarct microvasculature. However, to achieve cardiac protection after myocardial infarction, some therapeutic cargoes were required. So far, a large number of agents were loaded into nanoparticles targeted for different purposes. Recent studies have demonstrated that encapsulating ROS, Puerarin or Baicalin into micelles or lipids to reduce infarct size of the animals' ischemic heart (Zhang et al., 2016; Dong et al., 2017; Vong et al., 2018). For cardiomyocyte apoptosis prevention, IGF1, liraglutide, Nitroxyl radical, Cyclosporine A, Pitavastatin or 2,2,6,6-tetramethyl piperidine-1-oxyl was embarked on lipidbased NPs and sent to the animals' ischemic heart (Chang et al., 2013; Yin et al., 2014; Asanuma et al., 2017; Mao et al., 2017; Qi Q. et al., 2017). Intravenous injection of collapsin response mediator protein-2 (CRMP2) lipid with the size of 50 nm was shown fibrosis reducing in the mice chronic MI heart (Zhou et al., 2015). For vasculogenesis enhancement, VEGF, FGF1, Ang-1, stromal cell-derived factor-1 (SDF-1) or CCR2 was loaded in

NPs and delivered to the ischemic myocardial tissue to stimulate angiogenesis (Paul et al., 2011; Lu et al., 2015; Oduk et al., 2018; Ding et al., 2020; Fan et al., 2020). Interestingly, Wang et al. (2018) recently reported that no statistically significant improvements in cardiac function and infarct size were detected in mice acute MI heart with the intravenous administration of CCR2 targeting-nanoparticles (micelles) vs. non-targeted micelles. Recently, nanoparticle delivery through intravenous injection with targeting peptides has merged has a promising strategy. Xue et al. (2018) reported an early targeting therapy for myocardial infarcted mouse through the tail vein with anti-miR-1 antisense oligonucleotide (AMO-1) loaded and myocardiumtargeting dendrimer: PEGylated dendrigraft poly-L-lysine with angiotensin II type 1 receptor (AT1-PEG-DGL AMO-1). They found that AT1-PEG-DGL quickly accumulated in the MI heart during the desired early period, significantly outperforming the group without AT1 targeting. Apoptotic cell death in the infarct border zone was significantly decreased and the myocardial infarct size was reduced by 64.1% with a single IV injection as compared with that in MI group (Xue et al., 2018).

## INORGANIC NANOPARTICLES

#### Carbon-Based Nanoparticles Carbon Nanotubes

Carbon nanotubes (CNT) (**Figure 1C**) are a subfamily of fullerenes and are composed of graphite sheets that are rolled up into tubular forms (Katsuki et al., 2017). As nano-carriers, they incorporate drugs in their inner space and present chemically modified external surfaces with biological molecules, such as nucleotides and proteins, to provide selective targeting (Katsuki et al., 2017). Based on their number of layers, carbon nanotubes are categorized as either single-walled or multi-walled (Sajid et al., 2016). The poor solubility of drugs, faster deactivation, and limited bioavailability can be addressed by using these carbon nanotubes which are preferentially used as drug carriers (Raphey et al., 2019). However, one of the major disadvantages of the CNT is the chance for their dissociation in biological fluids (Raphey et al., 2019). Nevertheless, carbon nanotube is a wellsuited drug carrier for enhanced penetration in the cells and also for offering privileged drug actions (Zhang et al., 2011). Their unique optical, electrical, and mechanical properties make them a suitable candidate for potential therapeutic applications (Gorain et al., 2018). Moreover, a couple of studies have validated the promising potentials of CNT in cardiac tissue engineering, such as in the support of cardiomyocyte function and growth (Ahadian et al., 2017; Sun et al., 2017a) and acceleration of the gap junction formation (Martinelli et al., 2013; Shin et al., 2013). Aside these studies, other investigations have suggested that scaffold consisting of col-hydrogel and CNT could be promising injectable biomaterial to deliver drugs and cells for cardiac tissue regeneration in the infarcted myocardial tissues (Sun et al., 2017b; Gorain et al., 2018).

#### Graphene

The nanotechnology field is in constant research of novel materials that can be engineered for the precise, sensitive, and selective detection of biomarkers (Tang et al., 2020). Recently, the graphene-based family of materials has shown huge potential

as their proposed biosensing applications have shown great diversity (Stankovich et al., 2006; Geim and Novoselov, 2007; Bitounis et al., 2013). The isolated two dimensional (2D) crystal structures composed of single atomic layers of graphite are called "graphene" (**Figure 1D**; Bitounis et al., 2013). In Novoselov et al. (2004) isolated and characterized a single sheet of graphene. Since then, research on graphene has been highly increasing and has attracted a deep interest in scientific fields (Bitounis et al., 2013; Paul et al., 2014). Feng et al. (2011) pioneered the successful use of graphene as a non-toxic nano-vehicle for efficient gene transfection. With all atoms exposed on its surface, graphene has an ultra-high surface area available for efficient loading of aromatic drug molecules via π-π stacking, providing a plethora of applications in drug delivery via stable complex formation and avoiding chemical conjugation (Sun et al., 2008; Zhang et al., 2010). Paul et al. (2014) reported that methacrylated gelatin hydrogel (GelMA) impregnated with functionalized graphene oxide (fGO) nanosheets, where the latter were complexed with pro-angiogenic human vascular endothelial growth factor plasmid DNA (pDNAVEGF), formed nanocomposite hydrogels (fGOVEGF/GelMA) that efficiently transfect the myocardial tissues and induce favorable therapeutic effects without invoking adverse cytotoxic effects. Nevertheless, adverse reactions induced by graphene-based materials on exposure will depend on multiple factors that need to be scrutinized (Bitounis et al., 2013). Therefore, clinical translation of graphene-based materials is still in its infancy, yet the field holds tremendous potential for the treatment of multiple diseases (Bitounis et al., 2013).

### Metal Nanoparticles

Nanogold, also called gold nanoparticles (GNPs) or colloidal gold (**Figure 1E**), has been actively investigated in a wide variety of biomedical applications (Zhou Y. et al., 2018). The unique physical and chemical properties, such as ease of bioconjugation, excellent stability, superior security, and strong biocompatibility of many GNPs make them promising candidates in nanomedicine (Sperling et al., 2008).

Silver nanoparticles (AgNPs) (**Figure 1F**) have been developed as potent anti-microbial agents and have a multitude of applications, such as in toothpastes, bedding, water purification, and nursing bottles (Priyadarsini et al., 2018). After oral exposure, it is shown that about 18% of silver could be absorbed in humans (Bostan et al., 2016). Animal studies showed that AgNPs exposure will cause enhanced superoxide anion production and cause deleterious effects in cardiac tissues (Ebabe Elle et al., 2013; Lin et al., 2017; Xu et al., 2018). Thus, toxicity concerns of AgNPs have limited their effective translation for the cardiac tissue repair.

Cerium oxide (CeO2) nanoparticles (**Figure 1G**) have wide applications, such as in oxygen sensors and automotive catalytic converters (Niu et al., 2011). These nanoparticles are considered potent remedial options for the treatment of smoking-related diseases (El Shaer et al., 2017) since intravenous injection of these nanoparticles have shown a marked reduction in the myocardial oxidative stress and have also shown a significant reduction of the left ventricular dysfunction in the murine models of heart failure (Niu et al., 2011). The well-known mechanism underlying the action of these nanoparticles is attributed to their dual oxidation state, where the loss of oxygen and the reduction of Ce4<sup>+</sup> to Ce3<sup>+</sup> are accompanied by the creation of an oxygen vacancy (Niu et al., 2007).

Selective studies using inorganic nanoparticles for the delivery of therapeutics to repair infarcted myocardial tissue were listed as **Table 2**. Unlike the organic nanoparticles, the inorganic nanomaterials alone (without therapeutic agents loaded) could provide mechanical support even enhance cell electrical signaling in some conducting nanomaterials (Zhou J. et al., 2018). Zhou et al., created a conductive hydrogel by introducing graphene oxide (GO) nanoparticles into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels and delivered to the Sprague Dawley rats' acute MI heart by peri-infarct intramyocardial injection. They found that injected OPF/GO hydrogels can not only provide mechanical support but also electric connection between normal cardiomyocytes and the myocardium in the scar via activating the canonical Wnt signaling pathway, thus upregulating the generation of Cx43 and gap junction-associated proteins (Zhou J. et al., 2018). However, inorganic nanoparticles loaded with potential therapeutic agents have been widely studied. Similar to the studies of organic nanoparticles, scholars mainly aim at oxidative stress-reducing, inflammation attenuation, cardiomyocyte apoptosis prevention, fibrosis reducing and vasculogenesis enhancement (Han et al., 2018; Sharma et al., 2018). Copper has shown the anti-inflammatory, anti-oxidant potential and cardioprotective effect. Sharma et al. treated treat the I/R rat with low dose copper nanoparticles (CuNP) (1 mg/kg/day, p.o., 4 weeks) and myocardial protection was detected like the reduction of oxidative stress, inflammatory cytokines and apoptosis through phosphorylate GSK-3β kinase pathways (Sharma et al., 2018). Gold nanoparticles (AuNPs) delivered intravenously (400 µg/kg/day, 14 consecutive days) may also improve myocardial injury after myocardial infarction in rats with the decrease of eNOs immunoreaction, Bcl-2 and collagen fibers (Ahmed et al., 2017). However, in another mouse acute MI model, AuNPs intravenous administration (100 µl/day, 7 days) accumulated in infarcted hearts, decreased infarction size, inhibited cardiac fibrosis but has no effect on apoptosis and hypertrophy (Tian et al., 2018). Inflammation attenuation was shown in mouse MI models intramyocardial injection of 50 µl interleukin-4 plasmid DNA-functionalized macrophagetargeting graphene oxide complex (MGC/IL-4 pDNA) via a reduction in intracellular ROS and developing M2 macrophage phenotypes in macrophages (Han et al., 2018). Similar to the organic nanoparticles, intramyocardial injection of a nanocomplex of graphene oxide loaded with vascular endothelial growth factor-165 (VEGF) gene in the rat acute MI model shows significant infarct size reduction and capillary density enhancement (Paul et al., 2014). Further studies are needed to elucidate the long-term biocompatibility and safety of these inorganic nanoparticles.

### ORGANIC-INORGANIC HYBRID NANOPARTICLES

Recently, interests in the applications of various organicinorganic hybrid nanoparticles (NPs) have risen tremendously. TABLE 1| List of selective studies using organic nanoparticles for the delivery of therapeutics to repair infarcted myocardial tissue.


Nanoparticle and Myocardial Regeneration

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TABLE 2 |Listof selective studies using inorganic nanoparticles for the delivery of therapeutics to repair infarcted myocardial tissue.


TABLE 3 | List of selective studies using organic-inorganic nanoparticles for the delivery of therapeutics for the repair of myocardial infracted heart tissue.


Nanoparticle and Myocardial Regeneration

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Hybrid NPs combine features of organic and inorganic building blocks and generate NPs with improved physicochemical properties, such as particle size and surface charge (Haque and Chowdhury, 2018). Hybrid organic-inorganic NPs hold great promise in overcoming the pitfalls being faced by existing inorganic materials in the delivery of therapeutics and contrast agents (Somasuntharam et al., 2016), such as unwanted interactions with serum proteins (particularly opsonins) and consequential removal from the circulation by macrophages of mononuclear phagocytic system, rapid renal clearance, prolonged body accumulation, and lack of targetability (Zhou and Zhang, 2019).

Magnetoliposomes (MLs) are composed of liposomes and magnetic NPs and are the first efficient hybrid liposome/NP systems produced for the drug delivery (Namdari et al., 2017). In this line, several experimental strategies have been investigated the potential scope of magnetic NPs to leverage the delivery of growth factors, cytokines, and biomolecules to the degenerating cardiac cells and tissues and enhance their regeneration (Allen and Cullis, 2013; Paul et al., 2014; Ottersbach et al., 2018).

Selective studies using organic-inorganic nanoparticles for the delivery of therapeutics to repair infarcted myocardial tissue were listed as **Table 3**. Somasuntharam et al. (2016) created deoxyribozyme (DNAzyme) functionalized AuNPs to catalytically silence tumor necrosis factor-α (TNF-α) as a potential therapeutic for acute myocardial infarction. After the intramyocardial injection, with the silencing of TNF-α, significant anti-inflammatory benefits, and cardiac function improvement were detected in the rat heart. With the same model, (Paul et al., 2011) design a new gene delivery method utilizing a self-assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine-rich Tat peptide/DNA nanoparticles (NP) together with Angiopoietin-1 (Ang-1) gene carried by. 3 weeks post intramyocardially delivery, cardiac function improvement, capillary density enhancement, and infarct sizes reduction were detected in Bac-NP(Ang1) compared to Bac(Ang1), NP(Ang1) and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions. Furthermore, (Zhu et al., 2016) designed and synthesized molecularly organicinorganic hybrid hollow mesoporous organosilica nanoparticles (HMONs) for gene transfection (hepatocyte growth factor, HGF) in BMMSCs and subsequent in vivo cardiac repair. The fabricated organic-inorganic hybrid HMONs with large pore size represent a generalizable strategy to promote the ischemic myocardium therapeutic potential of HGF transfected BMMSCs including reduction of apoptotic cardiomyocytes, infarct scar size, and interstitial fibrosis while increasing angiogenesis (Zhu et al., 2016).

#### ARTIFICIAL DNA NANOSTRUCTURES

The success of DNA nanotechnology lies in the artificially constructed special nanostructure design systems for DNA computing (Lee et al., 2016). DNA nanostructures, owing to their precise control over chemistry, size, and shape, provide vast opportunity to unfold the convoluted mass of information relating to nanoparticle-biological interactions (Lee et al., 2016). Drug delivery and therapeutics is considered as one of the most promising applications of the structural DNA nanotechnology (Ke et al., 2018). In this line, artificial nucleic acid nano-devices could be utilized to provide targeted drug delivery in the tissues upon sensing their environment (Singh et al., 2016). Moreover, several studies have proposed various DNA nanostructures and strategies to load, deliver, and release biomolecular drugs for cardiac therapy (Paul et al., 2011).

#### COMPARISON OF THE NANOPARTICLES AS FOR ISCHEMIC MYOCARDIUM REPAIR

Nanoparticles of different types (for example, inorganic, organic and hybrid) designed to target ischemic cardiac cells are promising candidates for the treatment of myocardial infarction. Organic nanoparticles are offering numerous advantages which embrace the simplicity of their preparation from well-understood biodegradable, biocompatible polymers, and their high stability in biological fluids during storage (Virlan et al., 2016). Since the emergence of nanotechnology in the past decades, polymeric materials such as poly (d-lactic acid), polyethylene glycol (PEG) and poly lacticco-glycolic acid (PLGA) have emerged as a major class of biodegradable and controlled release systems for delivering biomolecules/proteins to the plaque site (Fredman et al., 2015; Kamaly et al., 2016).

The use of inorganic nanoparticles for applications in drug delivery presents a wide array of advantages, which include: (1) Ease of functionality with a range of surface and conjugation chemistries; (2) High payload loadings; (3) Tunable degradation rates; and (4) Enhanced penetration into tissue (Pandey and Dahiya, 2016). Magnetic nanoparticles were shown to accelerate the expression of critical gap junction proteins (for example, connexin 43) in cardiomyoblasts. These new cells demonstrated higher levels of both engraftment capacity and desirable paracrine factors compared with conventional therapeutic cells, thus significantly enhanced heart function and reduced scar size when delivered into the peri-infarcted area in rats (Han et al., 2015). Superparamagnetic iron oxide nanoparticles, with biocompatibility and capacity for simultaneous imaging and targeting, have emerged as the major particles for enhancing the engraftment of therapeutic cells in heart tissue. However, it was recently revealed that these nanoparticles increase tumorassociated macrophage activation (Zanganeh et al., 2016).

Intensive studies have thoroughly probed the toxicities of a wide range of nanoparticles (organic, inorganic, and polymeric) in different types of cells and organs. However, the cardiotoxicity of nanoparticles has been poorly investigated, and data are still limited to a few types of nanoparticle including metal oxides, silver, and carbon (Bostan et al., 2016). The main limiting issue for the design of safe and efficient nanoparticles for the treatment of ischemic heart disease is the lack of a deep understanding of the biological identity of nanoparticles. To accelerate the clinical translation of nanoparticles for use in cardiac nanotechnology,

TABLE 4 | Advantages and disadvantages of different nanoparticles for the delivery of therapeutics to repair infarcted myocardial tissue.


their biological identities must be precisely assessed and reported (Mahmoudi et al., 2017). The advantage and disadvantage of each NP category were summarized in **Table 4**.

#### CLINICAL APPLICATION

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A large number of patents, pertinent to the invention of cardiovascular biomaterials, have been filed in the past decade. Importantly, an invention of the UV-crosslinkable gelatin methacrylate-based cardiac patch, impregnated with gold nano-rods, was recently patented (US20170143871 A1). The patent describes about patch that exhibits high surface area and electrical conductivity. Another recent invention describes a combination of gold nano-wires and engineered scaffolds for controlling the cellular function through electronic circuits (US20170072109 A1). Furthermore, a new strategy of nanoparticle-stem cell electrostatically conjugates for postinfarction treatment was patented in Japan (JP5495215 B2). A preparation method of nanomagnetic particles for the detection and treatment of coronary heart diseases was patented in China (CN102085380A). This method can be used for preparing magnetic nanoparticles for mid-late-stage treatment of coronary heart diseases; and good repairing and treatment effects on coronary heart diseases can be achieved. In another patent, a solid lipid nanoparticle of Gelan Xinning Ruanjiaonang (Chinese traditional medicine) for treating coronary heart disease was created and beneficial for clinical application (CN103027981B). The treatment effect of Gelan Xinning Ruanjiaonang for coronary heart disease is significantly improved with the solid lipid nanoparticle included. Despite several patented technologies for cardiovascular therapeutics, only a few have entered into the clinical trials, due to the stringent regulatory requirements (Lakshmanan and Maulik, 2018). Fortunately, some of the targeted drug nanocarriers for cardiac therapies successfully passed clinical trials (Galagudza et al., 2010) and are already commercially available (Chong et al., 2014). For instance, one of the clinical strategies that has been practiced since long for inducing angiogenesis in the ischemic tissues includes intramuscular transplantation of the micro-bubbles and causing ultrasound-mediated microbubble destruction for the delivery of entrapped bone marrow-derived mononuclear cells (BM-MNCs) to provide tissue regeneration (Tateishi-Yuyama et al., 2002).

#### CURRENT STATE-OF-THE-ART NANOTECHNOLOGY USED IN CARDIAC THERAPY AND FUTURE PERSPECTIVES

Despite initial encouraging results from nanotechnology-based cardiac protection, poor retention time, efficacy, side effects or off-target effects of the delivered NPs remain major obstacles for efficient myocardial regeneration (Chang et al., 2013; Somasuntharam et al., 2013; Yin et al., 2014; Zhou et al., 2015). Several delivery strategies like intracoronary, intramyocardial, or intravenous have been applied for cardiac repair. Traditionally, NPs were injected via intracoronary or intramyocardial route and they rely on open heart surgery (Chang et al., 2013; Somasuntharam et al., 2013). Most adverse effects were observed they were delivered either intravenously or orally (Yin et al., 2014; Zhou et al., 2015). So far, no strategy has been proven to replace the transmural scar tissue in the chronic infarcted heart tissues. However, the current state-of-the-art nanoparticle technologies have emerged as one of the most promising strategies for myocardial repair (Awada et al., 2016). With the application of heart targeted agents, efficacy could be highly improved, while lowering the adverse effects by delivering NPs by intravenous route (Nguyen J. et al., 2015; Ferreira et al., 2016). The active targeting agents include MMP-2 and MMP-9 targeting peptides (Nguyen M. M. et al., 2015), which may results in long-term retention at the site of infarction. The atrial natriuretic peptide (ANP) is a circulating cardiac hormone produced physiologically, which belongs to the natriuretic peptide family and has been shown to have cardioprotective properties through cGMP-dependent signaling involving guanylyl cyclase A (GC-A) receptors (Potter et al., 2009). Peptide CSTSMLKAC and CRSWNKADNRSC are cyclic structures and have shown alone selective targeting to the ischemic heart (Kanki et al., 2011; Ferreira et al., 2016). Furthermore, heart homing agents make oral administration or inhalation administration an alternative and promising approach (Miragoli et al., 2018; Sharma et al., 2018).

The majority of the in vivo studies have shown the great potential of the nanoparticle systems in improving the function and tissue regeneration of the infarcted myocardium. However, further improvement in the homing and delivery of these nanoparticles and their therapeutic effects, respectively, to the target tissues can be achieved via decoration of these nanoparticle systems using heart-targeting active molecules (**Figure 2A**) or using non-invasive physical cues (**Figure 2B**). Furthermore, future clinical strategy may involve the application of cardiac patch that not only delivers the therapeutic agents, but its scaffolding effect provides optimal mechanical support to the failing heart and also replaces lost cells and tissues with induced pluripotent stem cell-derived cardiovascular cells, such as cardiomyocytes, endothelial cells and smooth muscle cells (**Figure 2C**). Thus, it is expected that advances in drug therapy, nanomedicine, cell-therapy, and material science will provide robust functional improvement and tissue restoration in patients with myocardial infarction in the near future.

## AUTHOR CONTRIBUTIONS

CF, JJ, and WZ wrote the manuscript. FL, BX, MK, JY, and WZ, revised the manuscript. All authors approved the submission and publication of the manuscript.

#### FUNDING

This work was supported by the National Institutes of Health (National Heart, Lung, and Blood Institute R01 grant HL142627 to WZ and R01HL136232 to MK), and the American Heart Association Scientist Development Grant (16SDG30410018 to WZ). CF was supported by the Fundamental Research Funds from the Central South University (2017zzts234).

## REFERENCES

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## ACKNOWLEDGMENTS

The authors would like to thank Drs. Jianyi Zhang and Gangjian Qin in The University of Alabama at Birmingham for their continued support.




**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Fan, Joshi, Li, Xu, Khan, Yang and Zhu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nitric Oxide-Producing Cardiovascular Stent Coatings for Prevention of Thrombosis and Restenosis

Jingdong Rao1,2, Ho Pan Bei <sup>1</sup> , Yuhe Yang<sup>1</sup> , Yu Liu<sup>1</sup> , Haodong Lin<sup>3</sup> \* and Xin Zhao<sup>1</sup> \*

*<sup>1</sup> Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong, China, <sup>2</sup> State Key Laboratory of Molecular Engineering of Polymers, Department of Orthopedic Surgery, Fudan University, Shanghai, China, <sup>3</sup> General Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China*

Cardiovascular stenting is an effective method for treating cardiovascular diseases (CVDs), yet thrombosis and restenosis are the two major clinical complications that often lead to device failure. Nitric oxide (NO) has been proposed as a promising small molecule in improving the clinical performance of cardiovascular stents thanks to its anti-thrombosis and anti-restenosis ability, but its short half-life limits the full use of NO. To produce NO at lesion site with sufficient amount, NO-producing coatings (including NO-releasing and NO-generating coatings) are fashioned. Its releasing strategy is achieved by introducing exogenous NO storage materials like NO donors, while the generating strategy utilizes the *in vivo* substances such as *S*-nitrosothiols (RSNOs) to generate NO flux. NO-producing stents are particularly promising in future clinical use due to their ability to store NO resources or to generate large NO flux in a controlled and efficient manner. In this review, we first introduce NO-releasing and -generating coatings for prevention of thrombosis and restenosis. We then discuss the advantages and drawbacks on releasing and generating aspects, where possible further developments are suggested.

Keywords: cardiovascular stents, surface coating, nitric oxide, restenosis, thrombosis

## INTRODUCTION

Cardiovascular diseases (CVDs) are a common cause of morbidity worldwide, accounting for 18 million deaths per year, and serve as a third of all global deaths (Frieden and Jaffe, 2018; Yusuf et al., 2020). To treat CVDs, surgical interventions including heart valve replacements (Saito et al., 2003), angioplasty (Stone et al., 2002), and intravascular stents (Zhu et al., 2014) are employed. Among these surgical procedures, cardiovascular stent is most commonly used in coronary heart disease, myocardial infarction, and stenocardia due to its effectiveness at dilating blood vessels and maintaining the circulation of blood (Kubo et al., 2008; Geng et al., 2013; Wei et al., 2013). In the United States alone, ∼2 million patients undergo stent implantation each year (Cicha et al., 2016). However, stenting has a high tendency to cause thrombosis and restenosis. Intravascular injuries during surgical implantation stimulate coagulation pathways to facilitate platelet adhesion (Furie and Furie, 2008), and the production of thrombin further contributes to platelet activation and converses fibrinogen into fibrin-formed thrombus (Zhao et al., 2014). The accumulation of platelets and leucocytes causes acute inflammatory response and results in endothelial malfunction,

#### Edited by:

*Chao Zhao, University of Alabama, United States*

#### Reviewed by:

*Francesca Taraballi, Houston Methodist Research Institute, United States Federico Vozzi, Institute of Clinical Physiology (CNR), Italy*

#### \*Correspondence:

*Haodong Lin haodonglin@hotmail.com Xin Zhao xin.zhao@polyu.edu.hk*

#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

> Received: *26 March 2020* Accepted: *12 May 2020* Published: *24 June 2020*

#### Citation:

*Rao J, Pan Bei H, Yang Y, Liu Y, Lin H and Zhao X (2020) Nitric Oxide-Producing Cardiovascular Stent Coatings for Prevention of Thrombosis and Restenosis. Front. Bioeng. Biotechnol. 8:578. doi: 10.3389/fbioe.2020.00578* which can lead to excessive proliferation of smooth muscle cells (SMCs) (Naghavi et al., 2013). The extracellular matrix (ECM) produced by SMCs then induces thickening of the vessel wall, contributing to neointimal hyperplasia and restenosis (Scott and Panitch, 2014). As a result, thrombosis and restenosis hamper the long-term effectiveness of cardiovascular devices and induce other related symptoms.

To ease the risk of thrombosis and restenosis, cardiovascular stents are modified with different polymers, biomolecules, or coatings. Compared to the traditional bare metal stents (BMSs) with unacceptable levels of thrombosis and restenosis (Nakamura et al., 2016), drug-eluting stents (DESs) can reduce neointimal hyperplasia and preserve vessel patency by releasing drugs from surface polymers (Joner et al., 2006). However, DESs may induce late thrombosis, which can be attributed to the depletion of drug reservoir, off-target effect of drugs, and the inflammatory response (Mcfadden et al., 2004; Ma et al., 2010). The most recent DES technology fabricates dual-therapy stents (DTSs) that combine two therapeutic regents like aspirin and adenosine diphosphate-receptor blockade for anti-platelet therapy (Ohkubo et al., 2011). Meanwhile, bioresorbable vascular scaffolds (BVSs) can decrease the propensity for thrombosis, since BVSs allow implants to degrade over time and leave an intact vessel (Stone, 2016). Furthermore, bio-engineered stents (BESs) adopt biocompatible materials, cell capture technology, or autologous venous tissue for better therapeutic effects (Stone et al., 2002; Hara et al., 2006; Colombo et al., 2019). In addition to material innovation, novel stent designs also employ coatings to improve the surface properties and clinical behaviors (Yang et al., 2020a). The coating materials possess the feasibility to directly attach or deposit onto the stents' surface and the ability to maintain a high local therapeutic concentration at specific site (Yang et al., 2017). Drugs and biomolecules such as paclitaxel (Palmerini et al., 2015), adhesive peptides (Wei et al., 2013), anti-CD 34 antibodies (Yoon et al., 2002), and nitric oxide (NO)-producing moieties (Gunasekera et al., 2018) are employed to improve the clinical behaviors of stents (see **Table 1** for details). Although these molecules achieved good therapeutic effects, they still present some limitations. For example, high toxicity and non-selective function of paclitaxel raises safety concerns about its side effects while high cost and potential complications of antibodies and adhesive peptides may hinder treatment efficacy (Pacelli et al., 2017). Among all therapeutic molecules, while NO still possesses some drawbacks such as low diffusion distance in blood, it remains highly effective because of its anti-thrombosis and anti-restenosis ability. As an endogenous substance, its pro-proliferative activity to endothelium and antiadhesion/aggregation effect on platelets additionally allow NO to serve as a guardian of cardiovascular implants (Winther et al., 2018). As reported by the ClinicalTrials.gov website, there are 678 NO-related clinical studies in CVD treatment by April 2020, which reveals the highly promising research and clinical application prospects of NO.

In a healthy vasculature, endothelial cells (ECs) produce NO to achieve thrombotic homeostasis by preventing platelet activation (Seabra et al., 2015). The mechanism of NO inhibition of platelet activity is multifaceted. The main way is to activate the soluble guanylate cyclase (sGC), thereby increasing


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concentrations of cyclic guanosine monophosphate (cGMP) and stimulating the downstream platelet inhibition molecule— Protein Kinase G (PKG) (Francis et al., 2010; Kraehling and Sessa, 2017). The other anti-platelet activity of NO is to inhibit the thromboxane receptor on platelet membranes (Fuentes and Palomo, 2016). Meanwhile, NO is also closely related to cardiovascular homeostasis. In the cardiovascular system, NO can relax the surrounding smooth muscle, lead to vasodilation, and increase blood flow (Jon O Lundberg et al., 2015). Moreover, NO stimulates EC migration and suppresses SMC proliferation (Mel et al., 2011). It also influences angiogenesis and vascular remodeling as well as kills various pathogens (Devine et al., 2019).

Under physical condition, NO can only diffuse around 100µm and degrades within seconds in blood, so NO must be administered or produced at the diseased site (Thomas, 2015). Hence, incorporating NO production moieties with stent coatings will work as a functional platform to achieve the antithrombosis and anti-restenosis purpose. In this review, we will discuss NO-producing coatings, with the emphasis on the NOreleasing and -generating strategies (**Figure 1** and **Table 2**). NOreleasing materials such as NO donors or peptides work as a finite reservoir, while NO-generating strategies utilize catalytic substances or genes to achieve endogenous NO supply. These coatings overcome the challenges of NO administration, and serve as great delivery platforms since they can maintain the physiologically relevant concentrations of NO at a specific site.

### NO-RELEASING COATINGS

In NO-releasing strategies, the potential NO can be stored in small molecules or biomolecules, and through connecting, dispersing, or encapsulating those functional molecules as a part of delivery system, the platform will act as a reservoir for conditional and spontaneous release of NO.

#### NO Donors

NO donors [e.g., N-diazeniumdiolates (NONOates), S-nitroso-N-acetylpenicillamine (SNAP)] are pharmacologically active substances that carry NO for localized release (Major et al., 2010; Midgley et al., 2019). NONOates can be synthesized by reacting gaseous NO with secondary amines under high pressure (5 atm) and spontaneously decomposed to release two moles of NO per mole of donor at physiological condition (Midgley et al., 2019), while SNAP, a kind of synthetic S-nitrosothiols (RSNOs), requires reactions between nitrosating agents and thiols, and NO can be exhausted from SNAP by heat, light (340 and 590 nm), or copper ions (Naghavi et al., 2013).



For example, Joung et al. fabricated NONOates containing liposome stent coatings via layer-by-layer (LBL) method to control the release of NO (Elnaggar et al., 2016). The NO release profiles showed that the release rate sustained up to 5 days with good thrombo-resistant effect. As thrombus involves the formation of fibrin and activation of platelets, Yang et al. proposed a novel concept to integrate anticoagulant agent bivalirudin (BVLD) and NO as a bulk synergetic modification on surface coating (Yang et al., 2020b). BVLD was covalently connected with the primary amine groups on plasma polymerized allylamine (PPAm)-coated surface, and then the coating was immersed in basic solution under high-pressure NO gas to form a NONOate-like bulk depot. For circumventing NO burst release, hydrophobic hexafluoroethane was introduced into this system, which prolonged the release time of NO to more than 8 h. Compared to 316L SS, the occlusion rate and thrombus weight in BVLD/NO-PPAmF were reduced from 58.7 ± 3.9% to 3.3 ± 1.2% and 64.1 ± 3.9 mg to 1.7 ± 1.2 mg after 2 h of extracorporeal circulation (ECC), respectively. Since the complexity of thrombogenic process requires multitherapy procedures, the combination of anti-platelet and anticoagulant dual functions is a promising strategy to integrate into cardiovascular coatings.

However, the NO release period in these studies was limited. To extend the NO release time, Hopkins et al. designed a highly stable NO-releasing coating by covalently attaching SNAP with poly(dimethylsiloxane) (PDMS) (Hopkins et al., 2018). Previous studies demonstrated that SNAP-based polymers have exhibited significant leaching of SNAP, which will result in non-localized NO release (Brisbois et al., 2016). Through tethering to PDMS, SNAP leaching into surrounding environment was prevented and allowed for lengthened potency. The NO flux formation of SNAP-PDMS reached up to 125 days in vitro and maintained at 0.1 × 10−<sup>10</sup> mol × cm−<sup>2</sup> × min−<sup>1</sup> till the end of the testing period. The SNAP-PDMS showed long-duration anti-bacterial efficacy, and it exhibited 78% thrombus reduction (compared with blank control) after testing by ECC over 4 h. Although this strategy achieved long-term NO release, the low NO levels in the later release stages limited the long-lasting anti-thrombotic effect.

Despite these efforts on NO donor applications, the long-term therapeutic effect after implantation was unsatisfying. Integrating NO-donor loaded liposomes with stents faces the risk of low loading capacity, carrier detachment from the stents and delayed NO release. Chemical covalent bond offers bulk NO donor storage, but it also brings extra reaction regents and shows relative low NO flux. Besides, the instability of the NO donors is a big obstacle that cannot be solved by simple connection or encapsulation. Hence, many other coatings have been developed for better application.

#### NO Prodrugs

Since NO donors are unstable under thermal, acidic, or physiological conditions, researchers use enzyme prodrug therapy (EPT) to maintain the stability of donors and control the release (Chandrawati et al., 2017). By fabricating NO donors (e.g., NONOates) with enzyme-sensitive linkers, the formed prodrugs remain stable and inactive in non-enzymatic environment and only release NO upon enzyme-triggering.

In a study of NO EPT, Wang et al. designed a galactosidase (Gal) immobilized surface coating and prepared glycosylated NONOate (Gal-NONOate) as NO prodrug (Wang et al., 2015). After the in vivo implantation of enzyme-functional platform, Gal-NONOate was administrated by tail vein injection, it then circulated until contact with the coating and the enzyme would catalyze the decomposition of the prodrug to release NO locally. The in situ release of NO was verified using a fluorescent probe to trace the NO flux. The immobilized enzyme retained the catalytic property up to 1 month in vivo and the results revealed effective inhibition of thrombosis and enhancement of vascular tissue regeneration and remodeling in EPT group. To engineer EPT for diverse medicinal implants, Zelikin et al. optimized EPT coating by adopting LBL method to fabricate multilayered polyelectrolyte coating with immobilized β-Gal enzyme (Winther et al., 2018). This method was all-aqueous and solution-based, which could accommodate modification of any substrate with no restriction on surface topography and geometry.

In addition to maintaining NO donors' stability, through using different concentrations of prodrugs or regulating administration time to adjust physiological effect, EPT can achieve personalized, fine-tuned therapeutic delivery of NO (Pan et al., 2015). Nevertheless, EPT requires systemic delivery of NO prodrugs, which only has effect at the enzyme-modified lesion site. This means that even with a bulky administration, only a small portion of drugs would work, which causes drug waste and side effects due to the high cytotoxicity of the drug. Scientists thus have developed other biomolecules to achieve NO release.

#### NO-Releasing Peptide Amphiphiles

Peptide amphiphiles (PAs) consist of hydrophobic tails coupled to hydrophilic functional peptide sequences that are attractive in biomimetic scaffolds, since enzyme-mediated degradable sites and cell adhesion ligands can be incorporated into PAs to mimic some biochemical properties (Jun et al., 2005; Cheng et al., 2015).

In a report by Matson et al., a NO-releasing PA coating was designed (Kushwaha et al., 2010). The functional peptide sequences involved a matrix metalloprotease-2 (MMP2) mediated cleavage site Gly-Thr-Ala-Gly-Leu-Ile-Gly-Gln (GTAGLIGQ), coupled to an EC-adhesive ligand Tyr-Ile-Gly-Ser-Arg (YIGSR) or a polylysine (KKKKK) group to form NO donating residue peptide. Burst release of NO occurred within 48 h, followed by sustained release for 30 days. This functional peptide increased initial adhesion of ECs from 51 ± 3% to 67 ± 2%, while the proliferation of SMCs were inhibited from 35 ± 2% to 16 ± 3% after 48 h of incubation. Additionally, compared with the positive control collagen group, the peptide group showed 150-fold decrease in platelet attachment, suggesting the potential of such coating for modification of various cardiovascular implants. Similarly, Alexander et al. used the same PAs to form a nanomatrix coating (Alexander et al., 2016), and they proved the vasodilatory effects ex vivo and the anti-inflammatory ability in vitro. This coating could address the shortcomings of the implanted stents and had the potential to be developed in animal models with cardiovascular stents. However, it is difficult to popularize peptide clinical application due to the high cost of peptides and the lack of control over their activity.

NO-releasing strategies have showed appropriate therapeutic effects on thrombosis and restenosis, but they still possess some shortcomings. In addition to the problems of different NOreleasing materials discussed above, all these strategies have to face the biggest obstacle—the finite reservoir of NO. With the NO release reaction proceeding, the elements will be exhausted within a short time frame. Therefore, reliable long-term and sufficient NO supplies need to be achieved.

### NO-GENERATING COATINGS

Radical NO species are short-lived. To overcome this drawback, NO-generating materials were developed, which utilize genes and catalytic substances such as selenium (Se) or copper ions to stimulate NO production. These strategies can achieve sufficient and long-term NO release depending on the endogenous regulation and continuous supply of NO resources.

#### NOS Gene Therapy

In the cardiovascular system, nitric oxide synthase (NOS) especially endothelial NOS (eNOS) and inducible NOS (iNOS) play a very important role. eNOS is highly related to the function of the endothelium, which can catalyze the production of NO via enzymatic effect (Zhao et al., 2013). iNOS is normally absent in the vasculature under physiological conditions, but it expresses in blood vessels under pathological situations (Sessa, 2004), and it can generate large amounts of NO over long periods of time (Kleinert et al., 2005). Since NOS has emerged as an active player in NO generation, a lot of therapeutic strategies have been developed around it. Gene therapy in CVDs can improve the selectivity of exerted effects toward certain cells and has the potential for combination therapy. Many researchers have thus investigated the therapeutic effect of NOS genes (Forstermann et al., 2017). In a study, DiMuzio et al. developed a natural vascular tissue mimetic stent by seeding it with autologous adipose-derived stem cells (ASCs) differentiated endotheliallike cells, and they used the eNOS gene to transfect these ASCs (McIlhenny et al., 2013). The transfection initiated eNOS production, yielding eNOS to generate functional NO gas. They found that the transfected ASCs produced NO (247 ± 10 nM) at a similar level to EC controls (288 ± 29 nM) in vitro, and exhibited a non-thrombogenic surface compared to unseeded controls in vivo.

In addition to in vitro gene transfection therapy, gene delivery is another approach to achieve targeted protein expression. To deliver NOS gene, Levy et al. described a gene delivery platform that provided local arterial gene transfer via iNOSencoding adeno-associated virus serotype 2 (AAV2) vectors (Fishbein et al., 2017). Through affinity effect, the iNOS-cDNA sequence loaded AAV2 vectors could be immobilized onto stent surfaces. Compared to the non-gene group, AAV2iNOS showed a 16-fold higher NO production in vitro. AAV2iNOS demonstrated escalating expression of encoded transgene for 12 weeks and showed the anti-restenosis efficacy with 95% inhibition rate in vivo.

Although there are many attempts in using NOS gene strategies, the instability, deliverability, release kinetics, off-target transduction, and high cost of gene limit the clinical application. Researchers are hence working hard on more effective NOgenerating pathways.

#### Endogenous NO Catalytic Approaches

RSNOs, a kind of endogenous NO donors in the blood, has highly promising opportunities to achieve localized synthesis of NO for continuous supply (Li et al., 2018). It was found that glutathione peroxidase (GPx) and Se or copper ions with GPx-like catalytic

source: Tu et al. (2020).

activity can catalyze the decomposition of RSNOs into NO in vivo (Weng et al., 2011; McCarthy et al., 2016).

Recently, our group developed one-pot approach to incorporate selenocystamine (SeCA) in the framework of dopamine (DA) via co-immobilization (Yang et al., 2018). The NO release rates could be controlled (from 0.5 to 2.2 × 10−<sup>10</sup> mol × cm−<sup>2</sup> × min−<sup>1</sup> ) by regulating the SeCA–DA molar ratio. The study showed that the SeCA/DA coating could release NO for more than 60 days, which achieved long-lasting prevention of thrombosis and restenosis. Additionally, the simple operation avoided the tedious process and toxic chemicals. This coating is believed to be universally formed on diverse types of materials with great clinical potential. We further improved the NO production ability by employing CuII instead of SeCA, since CuII possesses superior NO catalytic efficacy (Zhang et al., 2019). The NO release rates could reach to natural endothelium rates (0.5 to 4 × 10−<sup>10</sup> mol × cm−<sup>2</sup> × min−<sup>1</sup> ) by adjusting the dose of CuII with NO flux ranging from 0.4 to 6.5 × 10−<sup>10</sup> mol × cm−<sup>2</sup>

× min−<sup>1</sup> . After implantation for 3 months, the DA-CuII-coated stents not only inhibited thrombosis, but also promoted reendothelialization and reduced neointimal formation. We then additionally endowed our stents with anti-bacterial properties (Tu et al., 2019). In this system, cystamine (CySA) was chosen to fabricate the metal-phenolic-amine-based coatings with gallic acid (GA) and CuII. GA was added as chelating agent with anti-bacterial properties, which worked synergistically with CuII in bacterial inhabitation. The integrated coating showed 99% anti-bacterial rate in vitro and 3.4 ± 0.2% occlusion rate in vivo after implantation for 30 days. In our most recent study, we further immobilized vascular endothelial growth factor (VEGF) with DA-CuII coating (**Figure 2A**) (Tu et al., 2020). The introduction of VEGF was found to accelerate the early-stage EC adhesion, migration, and growth, forming a new and complete endothelium on the stents. The NO flux generated by the DA-CuII coatings successfully suppressed thrombosis (**Figure 2B**). Scanning electron microscope images showed that after 1 and 3 months of implantation, VEGF/CuII-DA coating demonstrated greater re-endothelialization compared to 316L SS (**Figure 2C**). Altogether, the multiple programmed therapeutic strategies for spatiotemporal dual delivery of NO and VEGF significantly prevented thrombosis and restenosis.

Nevertheless, these RSNO catalytic stents face difficulty in smart control of NO production. Additionally, different stages of disease and therapeutic purposes require different amounts of NO, a flexible, extended, and controllable NO supply is therefore highly sought after.

#### NO-RELEASING AND -GENERATING INTEGRATING COATINGS

There are substantial achievements of the anti-thrombosis and anti-restenosis therapeutic effects of NO-releasing and generating strategies, but limitations still exist. Researchers thus make effort to combine those two strategies to supplement NO delivery. Maintaining NO release within the physiological region (0.5 to 4 × 10−<sup>10</sup> mol × cm−<sup>2</sup> × min−<sup>1</sup> , and preferably at the up end) can be more efficient for biomedical applications, but the NO donors such as SNAP have been shown to release NO near the low end of the physiological levels. Based on this, Handa et al. incorporated SNAP in a medical grade polymer coated with copper nanoparticles to achieve better regulation of NO release (Pant et al., 2017). This strategy not only ensured the supply of exogenous NO by SNAP, but also provided the enzymatic NO release via the reaction of copper ions on endogenous RSNOs in the blood. The Cu-SNAP had increased NO flux values to around 4.48 or 4.84 × 10−<sup>10</sup> mol × cm−<sup>2</sup> × min−<sup>1</sup> when using different dosage of Cu. Additionally, significant reduction in bacterial growth and effective prevention of platelet adhesion has been achieved. Similarly, Brisbois et al. developed a multi-layered SNAP-doped polymer with a blended Se interface, and they focused on the different needs during the therapy process (Mondal et al., 2019). The first few hours after device implantation are crucial in preventing infection, since biofilm formation can occur rapidly after insertion (<24 h). The initial NO flux of generating materials may, however, be inadequate in preventing platelet activation or infection at early onslaught. Hence, in this strategy, SNAP offered initial NO release and Se interface could continuously generate NO in the presence of RSNOs. The results showed that there was a burst NO release on day 1, then the coating maintained a high and continuous NO release in the subsequent days. As a result, the enhanced initial NO flux would provide a potent anti-microbial activity acutely at the time of surgical placement, while the continuous NO generation contributed to inhibiting blood clot formation and protecting chronic or late device infections.

In addition to the above strategies to regulate the NO flux by using different coating compositions, Chandrawati et al. incorporated zinc oxide (ZnO) particles with EPT (Yang et al., 2020c). ZnO possess innate glutathione peroxidase and glycosidase activities, which allowed ZnO to catalytically decompose both endogenous (RSNOs) and exogenous (β-Gal-NONOate) donors to produce NO at physiological conditions. This strategy could solve problems such as finite pool of NO donors, short shelf life and low stability of natural enzymes in EPT. ZnO preserved its catalytic property for at least 6 months and the activity in producing NO was demonstrated. By tuning ZnO and NO prodrugs, physiologically NO levels were achieved. This method will be beneficial in long-term NO production and extra NO supplement can be achieved by on-demand NO prodrug administration, which has promising development in diverse blood-contacting devices.

In a word, the combination of NO-releasing and NOgenerating chemistries within a single platform allows for a release profile that achieves the best of both worlds. However, further studies should effectively control the amount of NO flux to avoid excessive NO production and related potential toxicity.

#### CONCLUSION AND FUTURE WORK

The progress of the NO-producing stent coatings are highly inspiring in the prevention of thrombosis and restenosis, and researchers have also attempted to improve the fabrication process and committed to developing simple materials for future clinical application. However, the immune reaction, inflammation, anaphylaxis, and biocompatibility of the coatings need to be further investigated. Although these problems cannot be solved temporarily, in the long term, what can be done is to promote the development of NO production strategies from those achievable directions: (1) Combination strategies of NO and other pathway regulation need to be carefully considered about the dosage ratio between the drugs to avoid the antagonism effect and ensure synergistic effect. (2) The toxicity and metabolism of the NO reaction, by-products [like N-Acetyl-D-penicillamine (NAP) and disulfide] should be taken into account. (3) Scientists should develop precise methods of NO quantitative detection in vivo to meet different needs. (4) Smart NO production system can be further developed to respond to the microenvironment changes and to produce NO flux on-demand during different pathologic process. (5) Most researchers use healthy animals to evaluate the effect of NO; however, differences between the healthy and the CVD individuals should be assessed. Researchers should also combine the effects of NO-producing coatings and the implanted materials as a whole to investigate the therapeutic effect in their corresponding disease models, which will have much more clinical significance.

In summary, NO-releasing and NO-generating strategies still have significant room for improvement, and a thorough understanding of NO production and solutions for the corresponding pathological reaction will ensure the safe clinical practices for the next generation of technological devices. We hope that this review can be helpful for the further development in this research field.

#### AUTHOR CONTRIBUTIONS

XZ and HL supervised the whole review. JR and HP wrote the manuscript. YY and YL performed literature search and revised the manuscript. All the authors approved the review for publication.

#### ACKNOWLEDGMENTS

The authors acknowledge the financial support from the seed projects of the Hong Kong Innovation and Technology

#### REFERENCES


Support Programme (ITS/065/19), the Program of Outstanding Medical Talent of Shanghai Municipal Health Bureau (grant number 2017BR034), and the State Key Laboratory of Molecular Engineering of Polymers of Fudan University (K2019-20).


in 155 722 individuals from 21 high-income, middle-income, and lowincome countries (PURE): a prospective cohort study. Lancet 395, 795–808. doi: 10.1016/S0140-6736(19)32008-2


coated on cardia stents. RSC Adv. 4, 34405–34411. doi: 10.1039/C4RA0 4771K

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Rao, Pan Bei, Yang, Liu, Lin and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Extracellular Matrix Mimicking Nanofibrous Scaffolds Modified With Mesenchymal Stem Cell-Derived Extracellular Vesicles for Improved Vascularization

Dake Hao1,2, Hila Shimshi Swindell 1,2, Lalithasri Ramasubramanian1,2, Ruiwu Liu<sup>3</sup> , Kit S. Lam<sup>3</sup> , Diana L. Farmer 1,2 and Aijun Wang1,2,4 \*

#### <sup>1</sup> Department of Surgery, School of Medicine, University of California, Davis, Sacramento, CA, United States, <sup>2</sup> Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA, United States, <sup>3</sup> Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, Sacramento, CA, United States, <sup>4</sup> Department of Biomedical Engineering, University of California, Davis, Davis, CA, United States

Edited by:

Weien Yuan, Shanghai Jiao Tong University, China

#### Reviewed by:

Hae-Won Kim, Institute of Tissue Regeneration Engineering (ITREN), South Korea Olivier Huck, Université de Strasbourg, France

> \*Correspondence: Aijun Wang aawang@ucdavis.edu

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

> Received: 22 March 2020 Accepted: 22 May 2020 Published: 25 June 2020

#### Citation:

Hao D, Swindell HS, Ramasubramanian L, Liu R, Lam KS, Farmer DL and Wang A (2020) Extracellular Matrix Mimicking Nanofibrous Scaffolds Modified With Mesenchymal Stem Cell-Derived Extracellular Vesicles for Improved Vascularization. Front. Bioeng. Biotechnol. 8:633. doi: 10.3389/fbioe.2020.00633 The network structure and biological components of natural extracellular matrix (ECM) are indispensable for promoting tissue regeneration. Electrospun nanofibrous scaffolds have been widely used in regenerative medicine to provide structural support for cell growth and tissue regeneration due to their natural ECM mimicking architecture, however, they lack biological functions. Extracellular vesicles (EVs) are potent vehicles of intercellular communication due to their ability to transfer RNAs, proteins, and lipids, thereby mediating significant biological functions in different biological systems. Matrix-bound nanovesicles (MBVs) are identified as an integral and functional component of ECM bioscaffolds mediating significant regenerative functions. Therefore, to engineer EVs modified electrospun scaffolds, mimicking the structure of the natural EV-ECM complex and the physiological interactions between the ECM and EVs, will be attractive and promising in tissue regeneration. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LLP2A, an integrin α4β1 ligand, which had a strong binding to human placenta-derived mesenchymal stem cells (PMSCs). In this study, we isolated PMSCs derived EVs (PMSC-EVs) and demonstrated they expressed integrin α4β1 and could improve endothelial cell (EC) migration and vascular sprouting in an ex vivo rat aortic ring assay. LLP2A treated culture surface significantly improved PMSC-EV attachment, and the PMSC-EV treated culture surface significantly enhanced the expression of angiogenic genes and suppressed apoptotic activity. We then developed an approach to enable "Click chemistry" to immobilize LLP2A onto the surface of electrospun scaffolds as a linker to immobilize PMSC-EVs onto the scaffold. The PMSC-EV modified electrospun scaffolds significantly promoted EC survival and angiogenic gene expression, such as KDR and TIE2, and suppressed the expression of apoptotic markers, such as caspase 9 and caspase 3. Thus, PMSC-EVs hold promising potential to functionalize biomaterial constructs and improve the vascularization and regenerative potential. The EVs modified biomaterial scaffolds can be widely used for different tissue engineering applications.

Keywords: electrospun nanofibrous scaffold, mesenchymal stem cell, extracellular vesicle, integrin-based ligand, vascularization, tissue regeneration

#### INTRODUCTION

Natural extracellular space is a dynamic and responsive environment consisting of non-cellular components such as soluble factors, non-soluble extracellular matrix (ECM), and extracellular vesicles (EVs) (Mathivanan, 2017). The ECM regulates many important processes including cellular proliferation, adhesion, migration, differentiation, tissue homeostasis and remodeling. These functions do not only depend on ECM's three-dimensional network, but also on its biological components (Huleihel et al., 2016). Electrospinning is a powerful technology to manufacture nano/microfibrous scaffolds that imitate the natural ECM architecture for allowing the integration of the scaffolds with surrounding cells and promoting tissue regeneration (Sarkar et al., 2006; Gao et al., 2018). We have successfully constructed electrospun scaffolds for various tissue regeneration applications, such as vascular tissue regeneration (Yu et al., 2012; Hao et al., 2020a), wound healing (Lee et al., 2012), peripheral nerve regeneration (Wang et al., 2011; Zhu et al., 2011), spinal cord regeneration (Zhu et al., 2010; Saadai et al., 2011; Downing et al., 2012) and drug delivery (Qi et al., 2006). However, the electrospun scaffolds lack biological motifs that are also included in the natural ECM and can mediate biological signaling and intercellular communication (Lu et al., 2011; Demircan et al., 2014). Surface modification plays a key role in regulating the biological interactions between cell/tissue and biomaterials (Wang et al., 2003). The functionalities of biomaterials, such as biocompatibility, adhesion and biological signaling, can be further improved by surface modification with bioactive motifs (Jiao and Cui, 2007; de Mel et al., 2012). Our previous studies have successfully improved the biological functions of electrospun scaffolds for different types of stem cells by surface modification (Hao et al., 2017, 2020b). Thus, surface modification is essential for the new functional biomaterial development and innovative medical devices design, which are contributive for advancing biomaterials in tissue regeneration and clinical applications (Wu et al., 2012; Bose et al., 2018). Vascularization is crucial for tissue development, maintenance and regeneration by to supplying nutrients and oxygen for cells and tissue (Santos and Reis, 2010; Muangsanit et al., 2018). Thus, many different approaches have been used to functionalize the electrospun scaffolds for enhancing vascularization, such as growth factors (Zhao et al., 2016; Janse van Rensburg et al., 2017), functional molecules (Lee et al., 2015; Garcia and Garcia, 2016), DNA (Scharnweber et al., 2018) and so on. In our previous studies, we demonstrated that an integrin-based ligand modified electrospun scaffold improved EC functions in vitro and vascularization in vivo (Hao et al., 2017, 2020a). However, these approaches only improve the cell and tissue functions by promoting cell/tissue-biomaterial interaction, but do not promote the biological information and substance transfer simulating the dynamic native ECM (Teodori et al., 2014; Sood et al., 2019). Therefore, to construct biofunctional scaffolds with biological information exchange and transmission will further promote the applications of biological materials in tissue regeneration.

EVs are produced in the endosomal compartment of most eukaryotic cells (Yanez-Mo et al., 2015; van Niel et al., 2018), enriched with various molecular constituents of their original cell, including lipids, proteins and RNAs, and are capable of transferring cell-to-cell signaling (van der Pol et al., 2012; Dhondt et al., 2016). Therefore, EVs have been widely used in tissue engineering area due to their multiple functions, such as pro-angiogenesis, cancer dormancy, anti-inflammation, mineralization (Azoidis et al., 2018; Casson et al., 2018; Baruah and Wary, 2019; Zhang H. et al., 2019). It is also known that physiologically native EVs actively interact with the ECM (Buzas et al., 2018) and the EV-ECM complexes are mediating significant biological functions of both ECMs and EVs (Sung et al., 2015). Recently, matrix-bound vesicles (MBVs) are identified to play a significant role in mediating the regenerative functions of ECM scaffolds (Huleihel et al., 2016, 2017; van der Merwe et al., 2017; Rilla et al., 2019), which highlights the biological functions of the EV-ECM structural complexes. Therefore, designing and constructing biomaterial scaffolds to mimic the structure of the EV-ECM complex and the physiological interactions between the ECM and EVs, represents an attractive and promising novel approach for tissue engineering applications. Mesenchymal stem cells (MSCs) isolated from various tissues are multipotent stem cells (Fridenshtein et al., 1968; Jo et al., 2007; Oh et al., 2008; Zannettino et al., 2008; Xue et al., 2018), represent a promising regenerative treatment for a variety of diseases (Bouffi et al., 2010; Lankford et al., 2015, 2017; Wang et al., 2015; Brown et al., 2016; Hofer and Tuan, 2016; Kabagambe et al., 2017; Galganski et al., 2019; Vanover et al., 2019; Zhang Z. et al., 2019), especially vascular diseases (Pankajakshan and Agrawal, 2014; Premer et al., 2019), due to the biofunctional paracrine secretion, including EVs. However, in many of the cases where therapeutic effects were observed using MSCs, the transplanted stem cells did not persist following injection and thus did not contribute to tissue regeneration by integration. Therefore, MSC derived EVs represent a promising alternative with sustained paracrine functions of live MSCs and have great potential for cell-free therapy. Although the functions of the EVs are not as

comprehensive as the MSCs, EVs derived from different types of MSCs also have been demonstrated for improving tissue regeneration (Zhang et al., 2015; Liang et al., 2016; Merino-Gonzalez et al., 2016). Among other molecules, integrins on the surface of EVs are of critical functional significance as they regulate the interactions between EVs and the surrounding micro milieu, especially ECM molecules (Clayton et al., 2004; de Jong et al., 2016; Buzas et al., 2018). Therefore, developing integrinbased conjugation approaches to immobilize MSC-derived EVs onto biomaterial scaffolds will mimic the EV-ECM complexes and hold promise for tissue engineering applications.

One-bead one-compound (OBOC) combinatorial technology is an ultra-high throughput chemical library synthesis and screening method, which is suitable for integrin-based ligand discovery (Lam et al., 1991). Previously, we have identified various potent ligands, such as LXY30, LXW7, and LLP2A targeting integrins α3β1, αvβ3, and α4β1, respectively, by employing the OBOC combinatorial technology (Peng et al., 2006; Xiao et al., 2010, 2016). We also have identified LLP2A had strong binding to human placental chorionic villus MSCs (PMSCs) (Hao et al., 2019) via integrin α4β1 and established an approach to immobilize LLP2A onto electrospun scaffold (Hao et al., 2020b). It has been shown that EVs derived from MSCs, especially from PMSCs, could stimulate angiogenesis (Komaki et al., 2017). Thus, in this study, we propose to immobilize PMSC-derived EVs (PMSC-EVs) onto native ECM mimicking electrospun nanofibrous scaffold by using LLP2A as the conjugation linker to construct the functional biomaterial scaffold to mimic the natural EV-ECM structural complexes and promote the vascularization and regeneration potential for tissue regeneration applications.

### MATERIALS AND METHODS

#### Cell Culture

We used PMSCs isolated from early gestation placental chorionic villus tissue as described in our previous studies (Lankford et al., 2017; Hao et al., 2019; Kumar et al., 2019). PMSCs were expanded in D5 medium containing high-glucose DMEM (HyClone), 5% fetal bovine serum (FBS, HyClone), 20 ng/mL recombinant human basic fibroblast growth factor (bFGF, R&D systems), 20 ng/mL recombinant human epidermal growth factor (EGF, R&D systems), 100 UI/mL of penicillin and 100µg/mL of streptomycin and incubated at 37◦C, 5% CO2. PMSCs were used between P3 and P5 for all experiments. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and expanded in EGM-2 media (Lonza). HUVECs were used between P3 and P6 for all experiments.

#### EV Isolation and Characterization

EV isolation and characterization were performed using an established protocol described in our previous study (Kumar et al., 2019). Briefly, PMSCs were seeded at a density of 20,000 cells/cm<sup>2</sup> in tissue culture–treated T175 flasks in 20 mL of EVdepleted FBS containing D5 medium for 48 h at 37◦C, 5% CO2. Conditioned medium was collected and sequentially centrifuged at 300 g for 10 min, 2,000 g for 20 min, and passed through a 0.2 mm filter. Then, the medium was concentrated using Amicon Ultra 15 Centrifugal Filter Units with a 100 kDa MW cutoff (MilliporeSigma), transferred to thickwall polypropylene tubes (Beckman Coulter), and centrifuged at 8,836 g using the SW28 rotor and L7 Ultracentrifuge (Beckman Coulter). The supernatant was transferred to fresh tubes, centrifuged at 112,700 g for 90 min, and the pellet was resuspended in PBS (HyClone) and spun again at the same speed and time. The final pellet was resuspended in 10 mL of PBS per T175 flask and stored in aliquots at −80◦C. The number and size distribution of isolated EVs were characterized by nanoparticle tracking analysis (NTA) using the NanoSight LM10 (Malvern Panalytical, Malvern, United Kingdom) equipped with a 404 nm laser and sCMOScamera. The morphology of isolated EVs was characterized by Transmission electron microscopy (TEM) using a CM120 transmission electron microscope (Philips/FEI BioTwin, Amsterdam, Netherlands) at 80 kV. For Western blot analysis, 10 mL of EVs were treated with either NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) containing reducing agent DTT, for detecting ALIX, tumor susceptibility gene 101 (TSG101), calnexin, integrin α4 and integrin β1 proteins, or without DTT, for detecting CD9 and CD63 proteins), and heated to 90◦C. The samples were run, transferred, probed with primary antibodies ALIX, TSG101, CD9 (MilliporeSigma), calnexin (Cell Signaling Technology), CD63 (Thermo Fisher Scientific), Integrin α4 (MilliporeSigma) and Integrin β1 (MilliporeSigma) at 4◦C overnight. Subsequently, membranes were incubated for 1 h with conjugated secondary antibodies (Cell Signaling Technology) at room temperature and blots were imaged using a ChemiDoc MP<sup>+</sup> imaging system (Bio-Rad).

#### Cell Migration Assay

HUVECs were seeded in Culture-Insert 2 Well in µ-Dish (ibidi) and cultured in EBM-2 with 1% BSA (Thermo Fisher Scientific) with EVs or without EVs and incubated at 37◦C, 5% CO<sup>2</sup> for 12 h. Images were taken using a Carl Zeiss Axio Observer D1 inverted microscope. The cell migration area was quantified using ImageJ software (NIH).

#### Rat Aortic Ring Assay

All procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Ten-week-old male Sprague Dawley rats were purchased from the Charles River animal facility. Rat abdominal aortas were dissected and sliced into 1 mm sections. Aortic rings were embedded in Matrigel <sup>R</sup> Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning) and cultured in EBM-2 medium with 1% BSA with or without EVs and incubated at 37◦C, 5% CO2. Angiogenic sprouts were counted after 7 days of culture. The number of sprouts was quantified using Wimasis Image Analysis.

#### Exo-Glow Labeling of EVs

EVs were labeled using Exo-Glow (Green) (System Biosciences) according to manufacturer's instructions. Briefly, Exo-Glow (Green) was diluted 1:500 in PBS and added to EVs. The sample was mixed gently by flicking the tube and incubated at Hao et al. Scaffolds Modified With Extracellular Vesicles

37◦C for 10 min. Twenty microliters of ExoQuick-TC (System Biosciences) were added to stop the labeling reaction and the sample was incubated on ice for 30 min. Excess dye was removed by centrifuging at 16,000 g for 3 min at 4◦C. The EV pellet was resuspended in 500 µL of cold PBS, placed on ice for 5 min, and centrifuged again at 16,000 g for 3 min at 4◦C. Washes were repeated for a total of 3 times. After the final wash, labeled EVs were resuspended in 500 µL of PBS. As a control, the same process was repeated using with same volume of PBS that the EVs were re-suspended in.

#### EV Attachment on LLP2A Treated Surface

To modify the culture surface with ligands, cell culture wells in a 48-well plates were coated with 150 µL of 20µg/mL Avidin (Thermo Fisher Scientific) and incubated for 1 h at 37◦C. Avidin coated wells were rinsed three times with PBS and were treated with 150 µL molar equivalents (2µM) of D-biotin (Thermo Fisher Scientific) or LLP2A-bio. After 1 h, the wells were washed three times with PBS and blocked with 1% BSA for 1 h. After the wells were rinsed three times with PBS, for the EV attachment assay, 5 × 10<sup>6</sup> EVs suspended in PBS was added into each well and incubated for 10 min at 37◦C and 5% CO2. Then the wells were washed three times with PBS, and the adhered EVs were imaged using a Carl Zeiss Axio Observer D1 inverted microscope. Quantification of images was performed using the ImageJ software.

#### Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Assay and Caspase 3 Assay

For the treated culture surface assay, three different groups, untreated, LLP2A treated and EV treated were set up in 24 well plates. As described above, the untreated wells were coated with D-bio, the LLP2A treated wells were coated with LLP2A and the EV treated wells were seeded with EVs on the LLP2A treated surface. HUVECs were seeded in wells with different treatments and cultured in EBM-2 media with 1% BSA at 37◦C and 5% CO<sup>2</sup> for 48 h. RNA extraction from cells was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, and cDNA was synthesized using Superscript II Reverse transcriptase (ThermoFisher Scientific). PCR was performed using the Biorad CFX96 Real-Time PCR Detection System (BioRad Laboratories) machinewith the SsoAdvanced SYBR Green Supermix (Bio-Rad). Amplification conditions after an initial denaturation step for 90 s at 95◦C were 40 cycles of 95◦C, 10 s, for denaturation, 55◦C, 10 s, for annealing and 72◦C, 30 s, for elongation. GAPDH was used as the reference gene for calculations. Data were analyzed by the 2DDCT threshold cycle method. Primer sequences are listed in **Table 1**. For caspase 3 assay, to mimic the ischemic environment when ECs often experience after implantation in vivo into the wounded or defect area, EC survival was characterized in a hypoxic chamber as described previously (Hao et al., 2019). HUVECs were seeded on different treated surfaces and cultured in hypoxia chamber in EBM-2 media at 37◦C, 1% O<sup>2</sup> and 5% CO<sup>2</sup> for 6 h, and then lysed and analyzed by using a caspase TABLE 1 | Primers used for qRT-PCR.


3 Assay Kit (Cell Signaling Technology) according to the manufacturer's instruction. Fluorescence (ex 380 nm/em 450 nm) was measured using a SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices).

#### Preparation of EV-Modified Electrospun Scaffolds

Preparation of EV-modified electrospun scaffolds included three steps as shown in **Figure 5**: (1) construction of electrospun scaffolds using the established electrospinning technology, (2) LLP2A immobilization on the electrospun scaffolds via "Click chemistry," and (3) EV immobilization on the LLP2A modified electrospun scaffolds via integrin-based conjugation. Construction of electrospun scaffolds was performed as previously reported (Hao et al., 2020b). Briefly, The polymer blends (e.g., 19% PLLA and 5% PCL; w/v) were completely dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Aladdin). Electrospun scaffolds were prepared by electrospinning polymer fibers onto the rotating drum collector. A negative voltage of 4.5 kV was applied to the mandrel, and a positive voltage of 4 kV was applied to the spinneret, by using a high voltage generator (Gamma High Voltage). LLP2A was grafted onto the PLLA/PCL membrane surface as our previously reported in three steps (Hao et al., 2020b). First, scaffolds were incubated in 0.01 M sodium hydroxide for 10 min to expose the carboxyl groups on the surface. Second, the scaffolds were further incubated in a solution of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC, Thermo Fisher Scientific) and N-hydroxysulfosuccinimide (sulfo-NHS, Thermo Fisher Scientific) in 0.5 M morpholino ethanesulfonic acid (MES) buffer (Thermo Fisher Scientific) for 30 min. After washing with PBS, the scaffolds were incubated in a solution of azido-PEG11-amine (N3-PEG11-NH2, BroadPharm) in alkalescent PBS (pH = 7.8) for 2 h on a shaker. Third, 20 mM LLP2A-DBCO was conjugated to azido-decorated scaffolds via "Click chemistry" in water for 16 h.

To evaluate the EV immobilization on the LLP2A modified electrospun scaffolds, EVs were seeded on electrospun scaffolds modified with different density of LLP2A (0, 50% or 100%). Briefly, we used another peptide ligand LXW7, specifically binds to αvβ3 integrin, but not integrin α4β1, described in our previous studies (Hao et al., 2017, 2020a) as the competitor ligand to LLP2A to modify the electrospun scaffolds. We used three conditions with different molar ratios of LLP2A-DBCO/LXW7- DBCO to modify the scaffolds: (1) PBS, (2) solution with equal molar ratio of LLP2A-DBCO/LXW7-DBCO (0.5:0.5), and (3) solution with LLP2A only (LLP2A-DBCO/LXW7-DBCO=1:0) to represent 0, 50% density of LLP2A, and 100% density of LLP2A. The scaffolds were washed with PBS for three times. The EV immobilization was characterized by using Scanning Electron Microscope (SEM, Hitachi TM-1000). Quantification of images was performed using the ImageJ software.

#### Evaluation of Angiogenic Gene Expression, Apoptosis and Survival of HUVECs

For angiogenic gene expression, HUVECs were seeded on electrospun scaffolds modified with or without EVs and cultured in EGM-2 medium at 37◦C, 20% O<sup>2</sup> and 5% CO<sup>2</sup> for 48 h, and the gene expression was evaluated by qRT-PCR as described above. For apoptosis and survival assay, the ischemic environment was set up as described above. HUVECs were seeded on electrospun scaffolds modified with or without EVs and cultured in hypoxia chamber in EBM-2 medium at 37◦C, 1% O<sup>2</sup> and 5% CO2. For Annexin V staining, the cells were cultured for 6 h, the staining was performed by using the Annexin V-FITC antibody (Abcam) according to the manufacturer's instructions. The images were collected using a Carl Zeiss Axio Observer D1 inverted microscope. Quantification of images was performed using the ImageJ software. For caspase 9 assay and caspase 3 assay, the cells were cultured for 6 h, and then lysed and analyzed by using a caspase-9 Activity Assay Kit (Abcam) or the caspase 3 Assay Kit, respectively, according to the manufacturer's instructions. Fluorescence (ex 380 nm/em 450 nm) was measured using a SpectraMax i3x Multi-Mode Detection Platform. For cell survival assay, the cells were cultured for 4 days and then determined using the MTS assay according to the manufacturer's instruction. The amount of soluble formazan product produced by the reduction of MTS by metabolically active cells was measured at the 490 nm absorbance using the SpectraMax i3x Multi-Mode Detection Platform.

### Statistical Analysis

For two-sample comparison, a student's t-test was used. For multiple-sample comparison, analysis of variance (ANOVA) was performed to detect whether a significant difference existed between groups with different treatments. A p-value of 0.05 or less indicates a significant difference between samples in comparison.

#### RESULTS AND DISCUSSION

#### Characterization of PMSC-EVs

TEM analysis showed the characteristic cup shape and size of PMSC-EVs (**Figure 1A**). NTA showed that PMSC-EVs have a size range of 137.4 ± 3.6 nm, which is within the expected size range of EVs (**Figure 1C**). Western blot analysis confirmed the presence of characteristic EV markers CD9, CD63, ALIX and TSG101, and the absence of endoplasmic reticulum marker calnexin (**Figure 1B**). The mechanism of EV binding to natural ECM is important to explore the applications of EVs in tissue regeneration, especially to achieve the conjugation of EVs to biomaterials for modification (Huang et al., 2016). EVs adhere to ECM constituents in the integrin dependent manner (Clayton et al., 2004). Thus, the integrin expression on PMSC-EVs was also determined. Our previous study showed that PMSCs highly expressed integrin α4 and integrin β1 (Hao et al., 2020b). Also, EVs carry markers of their original cells (van der Pol et al., 2012). Thus, in addition to the characteristic EV markers, the Western blot results also confirmed that PMSC-EVs expressed integrin α4 and integrin β1 that could be used as the junction to conjugate PMSC-EVs to biomaterial for simulating the natural ECM. The integrin α4 and integrin β1 expression results were also consistent with the report related to protein identification of EVs in our previous study (Kumar et al., 2019).

### PMSC-EVs Promoted EC Migration and Sprouting

Angiogenesis is the crucial need for tissue regeneration (Bi et al., 2012). MSC derived EVs have already developed for the

FIGURE 2 | Proangiogenic capacities of PMSC-EVs. (A) HUVEC migration treated with PMSC-EVs (b, d) or without PMSC-EVs (a, c). Scale bar = 100µm. (B) Quantification of HUVEC migration area. Data were expressed as mean ± standard deviation: \*p < 0.05 (n = 3). (C) Rat aortic ring assay treated with PMSC-EVs (b) or without PMSC-EVs (a). Scale bar = 500µm. (D) Quantification of the number of sprouting per ring. Data were expressed as mean ± standard deviation: \*p < 0.05 (n = 3).

surface, LLP2A treated surface or PMSC-EV treated surface. (B) Caspase 3 activity of HUVECs cultured on untreated surface, LLP2A treated surface or PMSC-EV treated surface. Data are expressed as mean ± standard deviation: \*p < 0.05, \*\*p < 0.01 (n = 4).

new impetus for improving angiogenesis in tissue regeneration, and EVs derived from different types of MSCs have been demonstrated promoted angiogenesis (Shi et al., 2019). The EC migration results showed that PMSC-EVs significantly enhanced the migration of HUVECs compared to the control group (**Figures 2A,B**). The rat aortic ring assay is an ex vivo model of angiogenesis that studies the effects of mediators on normal vessel sprouting, which showed that PMSC-EVs significantly promoted vessel sprouting compared to the control group (**Figures 2C,D**). These results indicate that PMSC-EVs prossess strong proangiogenic capacitiy that will extend the PMSC-EV applications in tissue regeneration.

### LLP2A Treated Surface Improved the Attachment of PMSC-EVs

The addition of tool molecules, such as biotin, to ligands can be advantageous when used in combination with other components and when used to expand the bioengineering applications of the ligands (Hao et al., 2020b). Our previous work has shown that biotinylation of the ligand did not decrease its binding affinity and showed nearly identical binding strength to the targeted integrin (Hao et al., 2017). Therefore, we conjugated LLP2A to biotin (LLP2A-bio), as described in our previous study (Peng et al., 2006). We used LLP2A-bio or D-biotin (untreated, as control) to treat the culture surfaces and investigated the attachment of PMSC-EVs on different culture surfaces. Before the PMSC-EV seeding, the PMSC-EVs were labeled with Exo-Glow (green) to facilitate the imaging. The results showed that only a few PMSC-EVs attached on the untreated surface (**Figure 3A**, a) and a number of PMSC-EVs attached on the LLP2A untreated surface (**Figure 3A**, b). The numbers of attached PMSC-EVs on different treated surfaces were quantified, which showed the LLP2A-treated surface significantly improved PMSC-EV attachment compared to the untreated surface (**Figure 3B**).

#### PMSC-EV Treated Surface Improved Angiogenic Activity and Suppressed Apoptotic Activity of ECs

Angiogenic gene expression, such as KDR and TIE2, is the result of new vessel formation and the improvement of the process of tissue regeneration (Malecki et al., 2005). Upon ligand

binding, integrins activate signal transduction pathways that mediate cellular signals (Giancotti and Ruoslahti, 1999). Our previous study has demonstrated that LLP2A was an integrin α4β1 ligand (Hao et al., 2020b). To avoid the effect of LLP2A on the gene and protein expression of cells, we set up LLP2A treated surface as another control group. The results showed that PMSC-EV treated surface significantly increased the expression of the angiogenic genes, KDR and TIE2, compared to untreated surface and LLP2A untreated surface, and no significant difference was shown between untreated surface and LLP2A untreated surface (**Figure 4A**). Caspase-3 has been found to be necessary in apoptosis due to it is responsible for chromatin condensation and DNA fragmentation (Porter and Janicke, 1999). The results showed that PMSC-EV treated surface significantly decreased the expression of caspase 3 compared to untreated surface and LLP2A untreated surface, and no significant difference was shown between untreated surface and LLP2A untreated surface (**Figure 4B**). These results indicate that PMSC-EV treated surface is able to improve angiogenic activity and suppressed apoptotic activity of ECs, but LLP2A treated surface does not have impact on angiogenic and apoptotic activity of ECs.

## Preparation and Characterization of PMSC-EV Modified Electrospun Scaffold

ECM is a three-dimensional (3D) network of extracellular macromolecules and the crucial need to provide structural and biochemical support to surrounding cells (Bonnans et al., 2014; Rabelink et al., 2017). To mimic the natural ECM structure, we employed electrospinning technology to construct network electrospun scaffolds as the description in our previous study (Hao et al., 2020b). To increase the integrin binding sites on the electrospun scaffolds, we developed a protocol to immobilize LLP2A, an integrin α4β1 ligand, onto the electrospun scaffolds via "Click chemistry" as the description in our previous study (Hao et al., 2020b). To mimic the biological EV-ECM complexes of natural ECM, we conjugated the integrin α4β1 expressing PMSC-EVs onto the electrospun scaffolds via integrin-based binding approach (**Figure 5**). The PMSC-EV modified electrospun scaffold was evaluated using SEM. The results showed the 50% LLP2A modified electrospun scaffolds showed significantly increased immobilization of PMSC-EVs compared to the control untreated electrospun scaffolds, and the 100% LLP2A modified electrospun scaffolds further significantly increased the PMSC-EV immoblization compared to the 50% LLP2A modified electrospun scaffolds (**Figure 6A**), and the quantification of the numbers of immobilized PMSC-EVs on the electrospun scaffolds showed linear correlation with the densities of LLP2A used indicates that the PMSC-EV immobilization system we developed in this study is mechanistically mediated by the densities of LLP2A molecules (**Figure 6B**) and the PMSC-EVs have been successfully immobilized on the electrospun scaffold via LLP2A binding approach. The SEM results showed that some

FIGURE 7 | Effects of PMSC-EV modified electrospun scaffolds on angiogenic gene expression and apoptotic rate and protein expression of HUVECs. (A) Quantification of KDR and TIE2 expression of HUVECs cultured on untreated electrospun scaffolds and PMSC-EV modified electrospun scaffolds. (B) Quantification of HUVEC survival on untreated electrospun scaffolds and PMSC-EV modified electrospun scaffolds. (C) Annexin V staining of HUVECs cultured on untreated electrospun scaffolds (a) and PMSC-EV modified electrospun scaffolds (b). (D) Quantification of apoptotic rate of HUVECs cultured on untreated electrospun scaffolds and PMSC-EV modified electrospun scaffolds. (E) Quantification of caspase 9 expression of HUVECs cultured on untreated electrospun scaffolds and PMSC-EV modified electrospun scaffolds. (F) Quantification of caspase 3 expression of HUVECs cultured on untreated electrospun scaffolds and PMSC-EV modified electrospun scaffolds. Data were expressed as mean standard deviation: \*p < 0.05, \*\*p < 0.01 (n = 4).

EVs seemed aggregated on the ligand modified scaffolds. It is known that EV aggregation could occur during isolation, storage or upon freeze and thaw (Bosch et al., 2016) and the EVs we used in this study were frozen stored before the immobilization process. Therefore, we believe that the aggregation of EVs seen on the scaffolds happened due to the storage of the EVs. To minimize EV aggregation, avoiding the freeze-thaw cycles of EVs and adding Trehalose into the frozen stock solution could prevent aggregation and cryodamage of EVs according to the previous study (Bosch et al., 2016). We do not anticipate that the EV immobilization strategy we developed in this study will facilitate EV aggregation, because in our design, we conjugated EVs onto electrospun scaffolds by using LLP2A as a linker, and LLP2A was first conjugated onto the electrospun scaffold, then the EVs were immobilized onto the LLP2A modified electrospun scaffold. EVs were never in direct contact with free LLP2A molecules, therefore EV aggregation, if any, should not be caused due to their interactions with free LLP2A molecules. In addition, LLP2A is a small peptide molecule that can bind to integrin molecules on the surface of EVs, and one LLP2A molecule that is immobilized to the scaffold surface can only bind to one integrin molecule. Therefore, we do not anticipate the immobilized LLP2A molecules would affect the aggregation of EVs directly.

#### PMSC-EV Modified Electrospun Scaffolds Improved the Angiogenic Activity and Survival of ECs

Angiogenic gene expression is essential for the regulation of new vessel formation that is fundamental to the development and maintenance of regenerative tissues (Holden and Nair, 2019). The results showed that compared to the untreated electrospun scaffolds, PMSC-EV modified electrospun scaffolds significantly improved the expression of angiogenic genes, KDR and TIE2, of HUVECs (**Figure 7A**). EC transplantation is an effective approach to improve vascularization and integration of the transplanted biomaterial-based scaffolds, however, EC survival is a significant challenge for the success of cell-based functional biomaterial implants for tissue regeneration (Foster et al., 2018). Apoptosis is a form of programmed cell death that is regulated by the caspase family of proteins, such as caspase 9 and caspase 3, activation of which is the result of intrinsic apoptosis (Hassan and Amer, 2011; Brentnall et al., 2013). Many previous studies have demonstrated that MSC derived EVs prevent cell apoptosis by regulating the activation of caspase 9 and caspase 3 (Li et al., 2019; Liu et al., 2019). In addition, Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis expressed on the outer leaflet of the plasma membrane. The results showed PMSC-EV modified electrospun scaffolds significantly improved HUVEC survival (**Figure 7B**), decreased the apoptotic rate (**Figures 7C,D**) and the expression of caspase 9 and caspase 3 of HUVECs (**Figures 7E,F**). These results indicate PMSC-EV modified electrospun scaffolds will be worth expecting in vascular tissue regeneration. The main innovation of this study is that it established a novel approach to immobilize the PMSC-EVs onto the electrospun scaffolds by using an integrin-based method that mimics the MBVs in native ECM. This EV delivery system was to modify the polymeric scaffold with EVs to confer the biological functions of EVs to the scaffold. The EVs possess multiple functions, therefore, the EV-modified scaffolds could be widely applied in different areas. Further detailed evaluation of the functions of EV-modified scaffolds in different in vivo models is warranted in future studies. The release of the EVs from the scaffold is crucial for the regenerative capacity of this EVs modified scaffold. We anticipate that when used in vivo, the PMSC-EVs modified scaffold could transfer the biological information inside the EVs to the cells that are in direct contact with the scaffolds via EV-cell fusion without the need of releasing the EVs to the body system. Meanwhile, immobilized PMSC-EVs on the scaffold could also be released from the scaffold to the body system. The EV delivery system we designed in this study is based on chemical modification of the scaffolds with the N3-PEG11-NH<sup>2</sup> linker and DBCO-LLP2A via "Click chemistry" and then the molecular interaction mediated by LLP2A and integrin α4β1 on the surface of EVs. Since the LLP2A-integrin binding is an "on-and-off " non-covalent interaction, therefore, we anticipate PMSC-EVs will be released first off from the scaffold by the dissociation of the LLP2A from the binding pocket of integrin α4β1. In the long-term, PMSC-EVs will be released from the scaffold because of the break of the covalent bonds between LLP2A and electrospun scaffold as well as the biodegradation of the electrospun scaffold.

## CONCLUSION

In this study, we isolated EVs from PMSCs and demonstrated the PMSC-EVs possessed pro-angiogenic capacity and antiapoptotic capacity. We successfully established an integrinbased binding technology to immobilize the PMSC-EVs onto the electrospun ECM-mimicking scaffolds to mimic the EV-ECM complexes. The PMSC-EV modified electrospun scaffolds promoted EC angiogenesis and prevented EC apoptosis in ischemic environment. This study demonstrates that EV modified biomaterials represent a new functional biomaterial and hold promise for tissue engineering and regenerative medicine applications.

## DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article material.

## ETHICS STATEMENT

The animal study was reviewed and approved by institutional animal care and use committee at the University of California, Davis.

## AUTHOR CONTRIBUTIONS

DH performed the evaluation of PMSC-EVs, performed the construction and evaluation of the PMSC-EVs modified electrospun ECM-mimicking scaffolds, wrote the manuscript, and discussed the results. DH and HS performed the isolation and characterization of the PMSC-EVs. LR performed the labeling of PMSC-EVs. KL and RL performed the chemical synthesis and discussed the results. DF discussed the results. AW was responsible for conceptualization, results discussion revising the manuscript. All authors contributed to the article and approved the submitted version.

#### FUNDING

This work was in part supported by the Shriners Hospitals for Children Postdoctoral Fellowship (84705-NCA-19 to DH) and the UC Davis School of Medicine Dean's Fellowship (to AW) awards, NIH grants (5R01NS100761- 02, R03HD091601-01), Shriners Hospitals for Children research grants (87200-NCA-19, 85108-NCA-19), and the March of Dimes Foundation Basil O'Connor Starter Scholar Research Award (5FY1682).

#### REFERENCES


#### ACKNOWLEDGMENTS

The authors would also like to thank the Combinatorial Chemistry and Chemical Biology Shared Resource at University of California Davis for design and synthesis of LLP2A-Bio and LLP2A-DBCO. Utilization of this Shared Resource was supported by the UC Davis Comprehensive Cancer Center Support Grant awarded by the National Cancer Institute (P30CA093373). We acknowledge Jordan Elizabeth Jackson and Mounika LS Bhaskara for their help with the rat aortic ring collection. We acknowledge Alexandra Maria Iavorovschi and Olivia K Vukcevich for their help with manuscript editing and submission.


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Hao, Swindell, Ramasubramanian, Liu, Lam, Farmer and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# An Aligned Patterned Biomimetic Elastic Membrane Has a Potential as Vascular Tissue Engineering Material

Juanjuan Tan1,2† , Jing Bai<sup>1</sup>† and Zhiqiang Yan<sup>3</sup> \*

<sup>1</sup> School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composite Materials and Shanghai Key Lab of Electrical Insulation and Thermal Ageing, Shanghai Jiao Tong University, Shanghai, China, <sup>2</sup> Joint Research Center for Precision Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital South Campus, Shanghai, China, <sup>3</sup> Central Laboratory, Southern Medical University affiliated Fengxian Hospital, Shanghai, China

#### Edited by:

Wuqiang Zhu, Mayo Clinic Arizona, United States

#### Reviewed by:

Francesca Taraballi, Houston Methodist Research Institute, United States Chao Zhao, University of Alabama, United States Aijun Qiao, University of Alabama at Birmingham, United States

> \*Correspondence: Zhiqiang Yan zqyan@sjtu.edu.cn

†These authors share first authorship

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 20 March 2020 Accepted: 04 June 2020 Published: 30 June 2020

#### Citation:

Tan J, Bai J and Yan Z (2020) An Aligned Patterned Biomimetic Elastic Membrane Has a Potential as Vascular Tissue Engineering Material. Front. Bioeng. Biotechnol. 8:704. doi: 10.3389/fbioe.2020.00704 Cardiovascular disease is the leading cause of death worldwide, with an annual mortality incidence predicted to rise to 23.3 million worldwide by 2030. Synthetic vascular grafts as an alternative to autologous vessels have shown satisfactory long-term results for replacement of large- and medium-diameter arteries, but have poor patency rates when applied to small-diameter vessels. Nanoparticles with low toxicity, contrasting agent properties, tailorable characteristics, targeted/stimuli- response delivery potential, and precise control over behavior (via external stimuli such as magnetic fields) have made possible their use for improving engineered tissues. Poly (styrene-block-butadieneblock-styrene) (SBS) is a kind of widely used thermoplastic elastomer with good mechanical properties and biocompatibility. Here, we synthesized anthracene-grafted SBS (SBS-An) by the method for the fabrication of a biomimetic elastic membrane with a switchable Janus structure, and formed the patterns on the surface of SBS-An under ultraviolet (UV) light irradiation. By irradiating the SBS-An film at different times (0, 10, 20, 30, 60, and 120 s), we obtained six well-ordered surface-patterned biomimetic elastic film with SBS-An at different heights in the thickness direction and the same distances of intervals (named sample-0, 10, 20, 30, 60, and 120 s). The structural effects of the SBS-An films on the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) were studied, and the possible mechanism was explored. When the HUVECs were cultured on the SBS-An films at different heights in the thickness direction, the sample-30 s with approximately 4 µm height significantly promoted adhesion of the HUVECs at the early stage and proliferation during the culture period compared with the samples of the lower (0, 10, and 20 s) and higher (60 and 120 s) heights. Consistent with this, the sample 30 s showed a higher stimulatory effect on the proliferation- and angiogenesis-related genes. These results suggest that SBS-An with appropriate height could efficiently control bioactivities of the biomimetic elastic membrane and might have great potential in vascular tissue engineering application.

Keywords: vascular tissue engineering, nanoparticles, anthracene-grafted SBS, HUVECs, biocompatibility

### INTRODUCTION

fbioe-08-00704 June 27, 2020 Time: 19:53 # 2

Cardiovascular diseases are a serious threat to human health. The number of deaths caused by cardiovascular disease is as high as 15 million worldwide every year, ranking first among various causes of death. Another statistic enumerates approximately 850,000 vascular reconstruction operations in the world each year, most of which use autologous blood vessels, though postoperative data show that the surgical effect is not ideal given the scarcity of autologous blood vessel sources (McBane et al., 2012; Schwann et al., 2012). Therefore, the research and development of artificial blood vessels have great application prospects and social significance.

An artificial blood vessel is a substitute that can be used to replace or repair a diseased blood vessel, and its source does not belong to the tissue or organ contributed by the host's own or a foreign body. Thus, the design of the artificial blood vessel should meet certain performance requirements, such as a suitable microporous structure on the surface and the longterm compliance of the blood vessel in the body (Chen et al., 2001; Nerem, 2004). Therefore, materials play a vital role in the successful application of tissue engineering, and choosing the right materials will have a profound impact on the regeneration of new tissues in the human body. The main challenge for tissue engineering is to develop materials that can promote the required cells and identify tissue behavior (O'Brien, 2011). The development of artificial blood vessels began in the early 20th century. Scholars from various countries first used tubular materials made of metal, glass, polyethylene, silicone rubber, and other materials for a large number of animal experiments (Nerem and Seliktar, 2001). However, they were not widely used in clinical practice because of their susceptibility to intraluminal thrombosis in the short term. In 1952, Voorhees et al. (1952) first studied the use of vinylon as a vascular prosthesis, which changed the impermeability of the previous vascular wall. In the next few years, Voorhees et al. did a large number of clinical trials to develop a meshed artificial blood vessel, which is a milestone in the history of the development of vascular substitutes. Subsequently, experts tested many materials such as PVC (polyvinyl chloride), polyacrylonitrile (acrylic), silk, nylon, and viscose (Guidoin, 1992). Artificial blood vessels made of polyacrylonitrile (acrylic) and nylon will degrade in the body; thus, these two materials were quickly eliminated. In addition, polyurethane (PU) has been used in tissue engineering as a vascular implant for 30 years due to its excellent blood compatibility, mechanical strength, and ideal long-term patency. It is considered by many researchers as an ideal artificial blood vessel material. However, some studies have pointed out that after long-term implantation, aging degradation and calcification can occur, which results in material cracking (Kannan et al., 2005). Although the clinical demand for bioengineered blood vessels continues to rise, currently there are still limited choices for blood vessels. Therefore, finding new materials remains the focus of research.

Natural blood vessels are mainly composed of three layers: intima, media, and adventitia (Li et al., 2014). The innermost layer is the endothelial cell layer. Intact endothelial cells adhere to the basement membrane containing collagen and laminin to prevent infection and thrombosis. Vascular endothelial cells are not only a physical barrier on the surface of blood vessels, but more importantly, they play an indispensable function and role in the regulation of vasoconstriction and the maintenance of the balance of the coagulation-fibrinolytic system. Constructing an endothelial cell layer with normal morphology and physiological functions is an important means to solve the thrombosis after the in vivo implantation of tissue-engineered blood vessels. The endothelialization of tissue-engineered blood vessels can effectively resist thrombosis and inhibit intimal hyperplasia (Ballyk et al., 1997; Chlupác et al., 2009). Therefore, a hot spot in the study of vascular stent biocompatibility is the rapid endothelialization of the stent surface and the maintenance of normal endothelial function after endothelialization.

In recent years, micrographics technology has received increasingly extensive attention, particularly in the fields of microelectronics, optics, tissue engineering, and biochip manufacturing due to its important research value. The topological surface structure of biological materials, such as their surface roughness, pore size, pore distribution, shape, size, and micro-pattern orientation greatly influence cell adhesion, proliferation, and differentiation (Lim and Donahue, 2007; Nel et al., 2009). Experiments have shown that a stripe structure with a large pitch (100 µm) can promote endothelial cell orientation, but does not form a capillary lumen structure. Among them, smaller-spaced stripe structures (10–50 µm) are more oriented to induce the growth of endothelial cells, and can promote the formation of a tubular structure of the lumen and a vascular network (Lei et al., 2013). Whited et al. studied the effect of PCL/gelatin electrospun materials with different fiber diameters and orientations on the angiogenic capacity of surface-grown ECs (Whited and Rylander, 2014). Their immunofluorescence staining image results showed that EC on the single-layer growth on the material surface, and the state of cell alignment and stretching increased with the degree of fiber orientation. Along the arrangement direction of the fibers, thicker actin bundles (F-actin bundles) were aligned, and strong expression of cadherin (VE-cadherin) was also observed at the junction between cells. The results indicated that the electrospun fiber materials with a directional arrangement structure helped enhance the adhesion ability of the ECs (Whited and Rylander, 2014). The aforementioned studies have demonstrated that the micro-pattern structure on the surface of the material effectively influenced and further regulated the cell-growth behavior on the material.

Interestingly, a series of studies have demonstrated that poly SBS is a widely used thermoplastic elastomer with good mechanical properties and biocompatibility, and more importantly, lower biotoxicity (Cho and Paul, 2001; Fu and Qutubuddin, 2001; Kim et al., 2001; Novak and Florián, 2004). However, in our previous study, a kind of biomimetic Janus membrane was fabricated with anthracene-grafted SBS and carbon nanotubes (CNTs), and the gradient Janus structure was obtained via the combination of UV light-induced dimerization of anthracene and control of the UV light penetration depth by the CNTs (Bai and Shi, 2017). These results demonstrated that

Tan et al. Surface Patterned Biomimetic Elastic Membranes

this Janus membrane could be used as a shape actuator due to the Janus structure-induced impetus in its thickness direction. By designing different geometrical shapes and UV light irradiation conditions (time and location) or stimulated regions, the Janus membrane achieved more complex and diversified shape shifting or morphing triggered by the solvent effect or shape memory mechanism. In addition, we developed a new approach for making two-/three-dimensional (2D/3D) latent photopatterned morphologies on the modified SBS films. Therefore, we intend to investigate the potential of the SBS-An films as tissue-engineered vascular material. In this study, the branching ratio of anthracene was fixed at 10% of the double bonds on SBS chains, which can be crosslinked under UV light irradiation to easily form the pattern on the surface. Finally, the structure effects of the SBS-An films on the adhesion and proliferation of HUVECs were studied, and the possible mechanism was explored.

## MATERIALS AND METHODS

#### Materials

Toluene, 4-methylbenzenesulfonic acid, 4 dimethylaminopyridine, pyridine, and dichloromethane were purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). SBS (Mw∼153 000–185 000, 70 wt % PB block), 2-Mercaptoethanol, and succinic anhydride were purchased from Sigma-Aldrich. 9-anthracenemethanol was purchased from Alfa Aesar. All the reagents were used as received.

#### Preparation of the Anthracene-Grafted SBS

The UV light sensitive polymer (SBS-An) was prepared as our previous work (Bai and Shi, 2017). First, the hydroxylbranched SBS was synthesized via the thiol-ene click reaction. The anthracene group was then grafted to the polymer chains via esterification. In the manuscript, the branching ratio of anthracene was fixed at 10% of the double bonds on the SBS chains. Due to the dimerization of anthracene, this kind of polymer can be crosslinked under UV light irradiation, while was utilized to form the pattern on the surface.

### Preparation of the Surface Patterns

According to our previous study (Bai et al., 2019), the patterns can be written on the surface as shown in **Figure 1**. In detail, The SBS-An was dissolved in toluene, and the solution was cast on the glass plate. The films were then heated at approximately 80◦C for 12 h to evaporate solvent. To write the patterns, the films were exposed to 365 nm UV light covered with a photomask at different times (0, 10, 20, 30, 60, and 120 s; marked as sample-0, 10, 20, 30, 60, and 120 s) to initiate the photo-dimerization of An to obtain a well-ordered surface pattern that transferred the pattern on the mask with different height in the thickness direction without cumbersome processing steps. The pattern on the surface of films can be obtained almost the same as the designed masks.

FIGURE 1 | (A) The chemical structure of the anthracene modified SBS. Laser scanning confocal microscopy (LSCM) images showing patterns written with different geometric configuration controlled with masks. (B) Positive and negative hybrid concentric rings (size width/space: 50/50 µm). (C) Positive and negative hybrid concentric triangles (size width/space: 100/100 µm). (D) Positive and negative hybrid concentric quadrangles (size width/space: 50/50 µm). The thickness of the polymer blend film was ≈200 µm. The intensity and exposure time of 365 nm UV light were ≈50 mW/cm<sup>2</sup> and 30 s, respectively.

## Cell Culture

According to a previous description with modification (Tan et al., 2017), the HUVECs were isolated from the human umbilical cord vein with 0.1% type I collagenase. The HUVECs were cultured in the endothelial cell medium (ECM) containing 5% FBS and 1% endothelial cell growth supplement kit (Sciencell, Carlsbad, CA, United States) at 37◦C with 5% CO2. The purity of the ECs was confirmed using the von Willebrand factor (vWF) antibody (Abcam, Cambridge, United Kingdom). Cells from passages 3–5 were employed for all experiments.

## HUVEC Adhesion Assay

To evaluate the influence of the aligned patterned biomimetic elastic membrane on cell adhesion, samples were first sectioned into 10 × 10 mm<sup>2</sup> squares, and then soaked in ethanol for 0.5 h. The samples were then soaked in 75 vol% medical alcohol solution for another 2 h for sterilization and washed out with a large amount of sterile water prior to seeding cells on the samples.

The cell adhesion assay was performed as previously described (Winterbone et al., 2009). Briefly, the HUVECs were plated into the surfaces of those samples at a density of 2 × 10<sup>4</sup> cells per well in a 24-well culture plate. Following incubation for 6 h at 37◦C in 5% CO2, the wells were washed three times with 0.2 mL PBS, stained with 0.1% crystals, and the number of adherent cells in four high power fields of view were observed using a Leica BX-61 fluorescence microscope at ×100 magnification (Leica, Germany).

### HUVEC Proliferation Assay

To investigate the influence of the aligned patterned biomimetic elastic membrane on cell proliferation, the samples were treated

as described above. HUVECs were trypsinized and plated onto the surfaces of the samples at a density of 2 × 10<sup>4</sup> cells per well in a 24-well culture plate, and incubated in an atmosphere consisting of 5% CO<sup>2</sup> in air at 37◦C for 72 h. The absorbance of HUVECs was determined with a CCK8 assay (Dojindo Molecular Technologies, Japan) according to the instructions of the manufacturer, and measured at 450 nm using an enzyme-linked immunosorbent assay plate reader (Bio-TEK, United States). Cells cultured in normal condition were regarded as controls.

#### Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

To investigate the effect of aligned pattern of biomimetic elastic membrane on the proliferation and differentiation of HUVECs, the proliferation-related genes (p21, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 2**(**CDK2), and Cyclin A2) and the angiogenesis-related genes [vascular endothelial growth factor receptor 2 (KDR), endothelial nitric oxide synthase (eNOS), and vascular endothelial cadherin (VE-cad)] from the cells cultured on different samples for 72 h were detected by qRT-PCR. TRIzol Reagent (Invitrogen, United States) was used to extract total RNAs according to the instructions of manufacturer. The concentration and purity of RNA were measured by a nanodrop 1000 reader (Thermo Scientific, United States). qRT-PCR was performed with the 2 × SYBR green master mix (Takara, Japan) with a 7500 Real-Time PCR System (Applied Biosystems, United States). After an initial incubation step of denaturation for 1 min at 95◦C, 40 cycles (95◦C for 15s, 60◦C for 30s, 72◦C for 20 s) of PCR were performed. Reactions were performed in triplicate. The PCR primer sequences of the above genes and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are summarized in **Table 1**. All fold changes were calculated by the method of 2−11Ct. GAPDH was used as a housekeeping gene. Data were normalized to GAPDH mRNA expression of each condition and were quantified relative to the corresponding gene expression from the control samples (cells cultured on the surface of sample-0 s or in normal condition).

#### Statistical Analysis

Experimental data were expressed as a means ± standard deviation (SD). Three independent experiments were performed, and at least three samples per each test were taken for statistical analysis. The statistical significance was calculated by a Student's t-test or one-way ANOVA using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, United States). Differences were considered significant when p < 0.05 (<sup>∗</sup> ), p < 0.01 (∗∗).

### RESULTS

### The Preparation of the Patterns on the Polymer Films

As shown in **Figure 1**, **Figure 1A** shows the chemical structure of the anthracene modified SBS. SBS-An films can be patterned with different geometric configuration controlled with masks, such as rings (**Figure 1B**), triangles (**Figure 1C**), and hexagons (**Figure 1D**). Due to the different irradiating time, the patterns exhibited different height which increased with the irradiation time as reported in our previous work. **Figure 2** shows the 3D/2D images and the height of SBS-An films exposed to 365 nm UV light covered with a quadrangular photomask for 0, 10, 20, 30, 60, and 120 s. With the same distance of interval(50 µm), the height of samples 0-, 10-, 20-, 30-, 60-, and 120-s were 0, 1.36 ± 0.12, 2.56 ± 0.09, 4.47 ± 0.08, 7.22 ± 0.05, and 22.86 ± 0.28 µm, respectively.

#### Effect of the Aligned Patterned Biomimetic Elastic Membrane on Cell Adhesion

The effect of the aligned patterned biomimetic elastic membranes on the adhesion of HUVECs were evaluated, as shown in **Figure 3**. On the whole, the adhesion of HUVECs cultured on the SBS-An films were lower than those in the normal condition. The number of adhesive HUVECs cultured on the sample-20 and 30 s biomimetic elastic membranes were higher than those on the sample-0 and 10 s biomimetic elastic membrane. Specifically, the number of adhesive HUVECs on sample-30 s biomimetic elastic membrane was a 2.28-fold increase compared with those on the sample-0 s. By contrast, the adhesion of HUVECs cultured on the sample-10 and 120 s biomimetic elastic membranes were no significant difference compared with those on the sample-0 s.

#### Effect of the Aligned Patterned Biomimetic Elastic Membrane on Cell Proliferation

To determine the HUVEC proliferation on the biomimetic elastic membrane, the viabilities of HUVECs were determined by CCK8 assay. **Figure 4** summarized the cell viability after incubation for 72 h. Overall, the proliferation of HUVECs cultured on the SBS-An films were lower than those in normal condition. However, after 72 h, the number of HUVECs cultured on sample-20, 30, and 60 s were significantly higher than those cultured on the sample-0 and 120 s, which indicates that those samples have stimulatory effects on HUVEC proliferation, while the number of HUVECs cultured on sample-10 and 120 s had no significantly difference compared with sample-0 s. Consistent with this, the proliferation-related genes showed the same trend. As shown in **Figures 4C–E**, the markers of proliferation (PCNA, CDK2, and cyclin A2) were lower expressed in SBS-An films than in normal condition. While p21, acting as an inhibitor of cell cycle progression, was significantly increased in SBS-An films compared with in normal condition(**Figure 4B**). Together, these results indicate that SBS-An films with different height in the thickness direction have effect on the proliferation of HUVECs.

#### Effect of the Aligned Patterned Biomimetic Elastic Membrane on Angiogenesis Related Gene Expression

To investigate the effect of the aligned patterned biomimetic elastic membrane on angiogenesis related genes, the expression

#### TABLE 1 | Primer sequences used in qRT-PCR.

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of KDR, eNOS, and VE-Cadherin from HUVECs cultured on biomimetic elastic membrane with different aligned patterns has been investigated. **Figure 5** showed the mRNA expression of angiogenesis-related genes(KDR, eNOS and VE-Cad). The mRNA expression of KDR and VE-Cad were siginificantly higher in sample-30 s than in other samples and normal condition (**Figures 5A,C**). While, the mRNA expression of eNOS in sample-20, 30, and 60 s were significant differences compared with in sample-0, 10,120 s and normal condition. In a way, these results indicate that SBS-An films with different height in the thickness direction have effect on the differentiation of HUVECs.

#### DISCUSSION

With the development of micro/nano processing technology, many new methods such as soft lithography, laser particle beam engraving, electron beam etching, and hot stamping have been used to prepare morphologies on the surface of various substrates. There are three types of micro-nano pattern structures currently studied: micro-nano stripe/groove structure, micronano bump array, and micro-nano groove array. Among them, the micro-nano level stripe/groove structure has attracted much attention because it can make cells "contact guide" effect (Weiss and Hiscoe, 1948). Lei et al. used photolithography to prepare striped structures with different pitch sizes in polymer templates, and studied the effects of surfaces with striped structures on the adhesion, orientation, and morphological changes of endothelial cells (Lei et al., 2012). However, it is worth mentioning that the aforementioned micro-pattern preparation methods have their own limitations and disadvantages. For example, the requirements for the equipment are relatively high (electron beam etching), and they are limited by the limiting wavelength of the light source (lithography). It is difficult to control the elastic

deformation of the mold during operation to ensure the accuracy and repeatability of the pattern replication (soft lithography). In particular, due to the limited number of materials suitable for these technologies, it is not possible to directly achieve the controllable preparation of the micro-patterned structure on the surface of the required biodegradable material. In this work, the patterns were achieved via the UV light irradiation. This method is easy and convenient to operate, and can produce a variety of patterns. Meanwhile, the patterns can be design via the masks as needed.

Studies show that the choice of vascular endothelial cell carrier material and its traits have important effects on cell adhesion, growth, and expression of physiological functions (Jankowski and Wagner, 1999). In order to improve the adhesion and growth of endothelial cells on the surface of artificial blood vessels, people have tried to attach various cell adhesion factors such as collagen,

fibronectin, and laminin to the surface of vascular materials, and achieved relatively satisfactory results (Bellis, 2011). Later, it was discovered that the RGD (Arg-Gly-Asp) tripeptide is the smallest sequence for intercellular recognition shared by many cell membranes and multiple adhesion proteins in ECM, which plays a very important role in mediating cell adhesion and spreading (Pierschbacher and Ruoslahti, 1984; Ruoslahti, 1996; Arnaout et al., 2005). Currently, RGD has been widely used in the surface modification of biological materials to promote the adhesion and function of cells on the surface of biological materials (Barber et al., 2007). In addition to attaching various cell activation factors to the surface of the material, it has been found that the necessary 3D structure on the surface of the material is also one of the indispensable factors to promote cell adhesion and growth, especially to maintain cell functional differentiation. Numerous studies have well demonstrated that the appropriate surface topography of scaffolds could promote various cellular processes, including adhesion, proliferation, and migration (Dalby et al., 2007; Kirmizidis and Birch, 2009; Qi et al., 2010; Yang et al., 2010). Tunstall et al. (1994) compared the expression of tissue factor (TF) and prostacyclin (PG) during the growth of vascular endothelial cells on the surface of Dacron materials with different surface structures. During growth, the expression level of TF was significantly lower than that of endothelial cells grown on smooth surface Dacron membranes, while the expression level of PG was not significantly different between the two groups. Sun et al. have already demonstrated that the cell attachment would be greatly influenced by the diameter of electrospun fibers (Sun et al., 2007). Xu et al. (2015) investigated the effect of the fibrous patterns on cells, and found that the well-designed scaffolds with anisotropically and heterogeneously aligned patterns could significantly promote EC adhesion at the early stage and proliferation during the culture period.

Consistent with these studies, our results showed that biomimetic elastic membrane with different height in the thickness direction had an important influence on the adhesion and proliferation of HUVECs, which can significantly improve cell adhesion just inoculated cells, and cell proliferation during the cultivation process. However, the adhesion and proliferation of HUVECs cultured in SBS-An films were lower than these in normal condition. Therefore, future studies will try to attach some cell adhesion factors or the appropriate surface topography on the SBS-An films to improve the adhesion and growth of HUVECs on the biomimetic elastic membrane.

Moreover, we found that the biomimetic elastic membrane had a significant impact on the expression of the proliferationrelated genes (such as p21, PCNA, CDK2, and cyclin A2) and angiogenic genes (such as eNOS, KDR, and VE-Cadherin). The tumor suppressor protein p21 Waf1/Cip1 acted as an inhibitor for cell cycle progression, and functioned in stoichiometric relationships to form heterotrimeric complexes with cyclins and cyclin-dependent kinases. In association with CDK2 complexes, it served to inhibit kinase activity and block progression through G1/S (Pestell et al., 1999). Proliferating cell nuclear antigen (PCNA) is a member of the DNA sliding clamp family of proteins that assist in DNA replication (Kelman and O'Donnell, 1995).

PCNA expression is a well-accepted marker of proliferation). A number of studies have described the ability of over-expressed cyclin A to accelerate the G1 to S transition causing DNA replication, and cyclin A antisense DNA can prevent DNA replication (D'Urso et al., 1990; Zindy et al., 1992; Resnitzky et al., 1995). eNOS is a key enzyme for endothelial cells to produce NO. The expression level of eNOS can reflect the ability of endothelial cells to secrete NO and is an indicator of endothelial cell integrity and vitality (Sessa, 2004). Upregulation of KDR can promote the survival, proliferation, and differentiation of endothelial cells, and is a better indicator of endothelial cell proliferation and differentiation (Santos et al., 2007). VE-cadherin is expressed at the cell adhesion junction, and its expression can specifically reflect the differentiation of endothelial cells (Schäfer et al., 2003). Examination of the abundance of the above gene expression can prove that biomimetic elastic membrane has an effect on the proliferation and differentiation of HUVECs. Different expression of these genes in SBS-An films indicated that the surface height of the biomimetic elastic membrane may have a significant influence on it (**Figures 4**, **5**). According to the above results, the sample-20,30, and 60 s(height 2.56 ± 0.09, 4.47 ± 0.08, 7.22 ± 0.05 µm, respectively) had a stronger effect on the adhesion and proliferation of HUVECs, in contrast, sample-0,10, and 120 s (0, 1.36 ± 0.12, and 22.86 ± 0.28 µm, respectively.) had no siginificant difference. Therefore, this may be due to that too low or too high of the surface was not good for cell adhesion. However, the exact mechanism needs further study.

In conclusion, we easily achieved well-ordered surface patterned biomimetic elastic membrane with different height in the thickness direction through UV light-induced dimerization of anthracene grafted on SBS chains. All six membranes were found to be non-toxic against HUVECs. Among of them, sample-30 s had siginificantly effect on adhesion and proliferation of the HUVECs, and expression of the proliferation-related and angiogenic genes as compared with other samples, which makes it the best candidate for further improvement. Our results suggest that well-ordered surface-patterned biomimetic elastic membrane might have a potential in vascular tissueengineering application.

The repair and reconstruction of tissues and organs make tissue engineering a hot research topic. However, the internal tissues and organs of organisms are complex structures with specific morphology and function composed of different cells. Previous methods of experiments are difficult to reconstruct the specific topological conformation of cells in tissues and organs and simulate the microenvironment in which cells are located.

#### REFERENCES


After the continuous development of chemical technology, it has gradually become an experimental tool for studying and controlling cell behavior, making it an asset in the fields of cell biology, tissue engineering, cell sensing, drug screening, and wound treatment. Although some defects are still present, such as its non-biodegradability, SBS-An films have many advantages, such as good mechanical properties, low cost, abundance, good biocompatibility, precise and controllable surface patterns, diversified patterns, fast and easy preparation method, and good flexibility, making them a great potential tissue engineering vascular material.

#### DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

#### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by the Ethics Committees of Shanghai Jiao Tong University. The patients/participants provided their written informed consent to participate in this study.

### AUTHOR CONTRIBUTIONS

JT and JB carried out the experiments, analyzed the results, and wrote the manuscript, and were co-first authors. All authors contributed to conception and design of the study, and contributed to manuscript revision, read and approved the submitted version.

### FUNDING

National Nature Science Foundation of China (31570949).

### ACKNOWLEDGMENTS

We thank Central Laboratory and Southern Medical University affiliated Fengxian Hospital (Shanghai, China) for providing a research platform.


of peptide-modified p(AAM-co-EG/AAC) interpenetrating polymer networkcoated titanium implants. J. Biomed. Mater. Res. A 80, 306–320. doi: 10.1002/ jbm.a.30927


regulation of proliferation and differentiation. Endocr. Rev. 20, 501–534. doi: 10.1210/edrv.20.4.0373


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Tan, Bai and Yan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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# Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease

Caroline Hu<sup>1</sup>† , Tatiana S. Zaitseva<sup>2</sup>† , Cynthia Alcazar<sup>1</sup>† , Peter Tabada<sup>2</sup> , Steve Sawamura<sup>2</sup> , Guang Yang3,4, Mimi R. Borrelli<sup>5</sup> , Derrick C. Wan<sup>5</sup> , Dung H. Nguyen<sup>5</sup> , Michael V. Paukshto<sup>2</sup> and Ngan F. Huang1,3,4 \* ‡

#### Edited by:

Wuqiang Zhu, Mayo Clinic Arizona, United States

fbioe-08-00689 July 16, 2020 Time: 19:30 # 1

#### Reviewed by:

Arnaud Scherberich, University Hospital of Basel, Switzerland Nicolas Christoforou, Pfizer (United States), United States Junjie Yang, The University of Alabama at Birmingham, United States

\*Correspondence:

Ngan F. Huang ngantina@stanford.edu

†These authors have contributed equally to this work

‡ORCID:

Ngan F. Huang orcid.org/0000-0003-2298-6790

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

> Received: 15 April 2020 Accepted: 02 June 2020 Published: 17 July 2020

#### Citation:

Hu C, Zaitseva TS, Alcazar C, Tabada P, Sawamura S, Yang G, Borrelli MR, Wan DC, Nguyen DH, Paukshto MV and Huang NF (2020) Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease. Front. Bioeng. Biotechnol. 8:689. doi: 10.3389/fbioe.2020.00689 <sup>1</sup> Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, United States, <sup>2</sup> Fibralign Corporation, Inc., Union City, CA, United States, <sup>3</sup> The Stanford Cardiovascular Institute, Stanford University, Palo Alto, CA, United States, <sup>4</sup> Department of Cardiothoracic Surgery, Stanford University, Palo Alto, CA, United States, <sup>5</sup> Division of Plastic and Reconstructive Surgery, Stanford University, Palo Alto, CA, United States

Cell therapy for treatment of peripheral arterial disease (PAD) is a promising approach but is limited by poor cell survival when cells are delivered using saline. The objective of this study was to examine the feasibility of aligned nanofibrillar scaffolds as a vehicle for the delivery of human stromal vascular fraction (SVF), and then to assess the efficacy of the cell-seeded scaffolds in a murine model of PAD. Flow cytometric analysis was performed to characterize the phenotype of SVF cells from freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the SVF consisted of 33.1 ± 9.6% CD45<sup>+</sup> cells, a small fraction of CD45−/CD31<sup>+</sup> (4.5 ± 3.1%) and 45.4 ± 20.0% of CD45−/CD31−/CD34<sup>+</sup> cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF.

Keywords: angiogenesis, peripheral arterial disease, stem cell therapy, aligned scaffold, anisotropy, hindlimb ischemia

## INTRODUCTION

Peripheral arterial disease (PAD) affects over 10 million people in the United States (Benjamin et al., 2018). It is associated with reduced blood flow to the arms and legs, leading to pain and even limb amputation. Major risk factors include smoking, diabetes mellitus, and aging. Severe cases of PAD lead to critical limb ischemia (CLI) that is characterized by rest pain, gangrene formation,

and possible amputation of the limb (Davies, 2012). Current clinical treatments of PAD involve surgical interventions including bypass grafting and angioplasty to restore blood flow to the affected limb. However, a large portion of patients with severe disease lack suitable vessels for vascular intervention (Gerhard et al., 1995). Therefore, there is an urgent need for alternative approaches to stimulate angiogenesis.

Cell-based therapies hold promise for regenerating neovessels that support blood flow in the ischemic limb. Stem cells including bone marrow-derived mesenchymal stem cells (MSCs) are a potential therapy as they are easy to expand, resilient in hypoxic conditions, and secrete paracrine factors that have angiogenic effects (Kinnaird et al., 2004). Although some clinical studies report that the delivery of MSCs to the site of limb ischemia significantly improved rest pain and increased the ankle brachial index as a measurement of vascular function, limb amputation rates did not significantly change with treatment (Lu et al., 2011; Gupta et al., 2013). Similarly, clinical trials using mononuclear cells from bone marrow or peripheral blood have also showed mixed results (Rigato et al., 2017; Qadura et al., 2018). Consequently, alternative therapeutic cell types are desired for treatment of PAD.

The stromal vascular fraction (SVF) is a heterogeneous population of cells that is derived from subcutaneous fat (Levi et al., 2011; Blackshear et al., 2017). SVF contains various types of cells including adipose stromal and hematopoietic stem and progenitor cells, endothelial cells, erythrocytes, fibroblasts, lymphocytes, monocyte/macrophages, and pericytes (Zuk et al., 2001, 2002; Bourin et al., 2013). Cells from the SVF release a mix of cytokines and therapeutic growth factors such as vascular endothelial growth factor (VEGF) that promote angiogenesis (Thangarajah et al., 2009; Kapur and Katz, 2013; Comella et al., 2016). Compared to other cell types that require in vitro expansion, SVF can be derived autologously, extracted in a minimally invasive manner in a clinical setting (Levi et al., 2011), and transplanted back within hours. Consequently, SVF may have greater translational relevance than other stem cells types for treatment of limb ischemia. We have previously shown that collagen scaffolds seeded with human SVF and subcellular populations thereof significantly improved revascularization to dermal wounds (Brett et al., 2017b), which supports the safety of SVF-seeded collagen scaffolds.

Regardless of the kind of stem cell used, a major limitation to stem cell therapy is poor survival of the cells when transplanted in saline. As an alternative to saline as a cell delivery vehicle, biological scaffolds can localize cell delivery to the site of the scaffold, while also providing important extracellular matrix cues that modulate the survival and angiogenic capacity of the transplanted cells. In particular, cues derived from nanoscale anisotropic patterns of fibrillar collagen can modulate cellular organization, growth factor secretion, and upregulation of integrin gene expression (Huang et al., 2013a,b; Nakayama et al., 2015, 2019). We have previously demonstrated that parallel-aligned nanofibrillar scaffolds promote the survival and angiogenic capacity of transplanted primary human endothelial cells or human induced pluripotent stem cell-derived endothelial cells in a mouse model of PAD (Huang et al., 2013b; Nakayama et al., 2015). These studies suggest that nanoscale spatial patterning cues can directly modulate biological functions of therapeutic cells upon transplantation into the ischemic limb. Toward clinical translation, these nanofibrillar scaffolds have been demonstrated to improve angiogenesis (Huang et al., 2013b), arteriogenesis (Nakayama et al., 2015), and lymphangiogenesis (Hadamitzky et al., 2016) in vivo. Recently, these aligned collagen scaffolds have been demonstrated to be safe in a clinical study, without complication at a 1 year follow up (Rochlin et al., 2020). Accordingly, the delivery of SVF using aligned nanofibrillar scaffolds may synergistically enhance revascularization for treatment of PAD.

Therefore, the objective of this study was to characterize the adherent cells from human SVF on aligned nanofibrillar scaffolds, and to assess the therapeutic potential of aligned nanofibrillar scaffolds seeded with SVF in a murine model of PAD. We show that the subpopulation of SVF cells that adhered to the nanofibrillar scaffold had significantly higher release of VEGF, but without a significant change in the CD45−/CD31−/ CD34<sup>+</sup> cell fraction, compared to bulk SVF cells in suspension. When implanted into mice with induced hindlimb ischemia as an experimental model of PAD, the SVF-seeded scaffolds significantly increased blood perfusion, compared to cell delivery in saline. These findings suggest that aligned nanofibrillar scaffolds promoted the adhesion of SVF cells that induce angiogenesis and blood perfusion recovery, which has important therapeutic implications for the treatment of PAD.

### MATERIALS AND METHODS

#### Fabrication of Aligned Nanofibrillar Scaffolds

The aligned nanofibrillar collagen scaffolds were fabricated using shear-based fibrillogenesis technique as described previously (Huang et al., 2013b). In brief, purified monomeric type I collagen solution was concentrated to reach a liquid crystal state (Bobrov et al., 2002; Paukshto et al., 2008) and then sheared onto a rigid surface, creating thin film formed by parallel-aligned nanofibrils with 200–300 nm diameter (Muthusubramaniam et al., 2012). To make three-dimensional thread-like scaffolds, the membranes were dissociated from the rigid surface into a free-standing film that self-assembled by liquid–air surface tension into the thread-like scaffold (McMurtry et al., 2008). The scaffolds were then crosslinked by 1-ethyl-3- (3-dimethylaminopropyl)-1-carbodiimide (EDC) hydrochloride chemistry at 1 mg/ml and sterilized by e-beam per standard protocols (Fibralign Corporation). Surface topography of the scaffolds was assessed by routine scanning electron microscopy (SEM) (Huang et al., 2013b).

#### Isolation of SVF

Lipoaspirate was obtained from healthy female patients (n = 6) undergoing elective procedures in accordance with the Stanford University Institutional Review Board and kept at 4◦C until processing. All samples were processed within 24 h from the time of collection. SVF cells were isolated based on established

methods (Tevlin et al., 2016). Lipoaspirate was rinsed twice with equal volume of phosphate buffered saline (PBS) to separate fat from blood. Fresh collagenase digestion buffer was prepared using M199 medium containing 2.2 mg/ml type II collagenase (Sigma–Aldrich), 1000 U/ml DNAse, 0.5 µM calcium chloride, 0.1% bovine serum albumin, 1% polaxamer-188 (Sigma– Aldrich), and 2% hydroxyethyl piperazine ethanesulfonic acid (Life Technologies), and filtered using a 0.22-µm filter system. Aliquots of the rinsed fat (12.5 ml) were transferred into 50 ml Falcon tubes, and an equal volume of collagenase digestion buffer was added to the fat. The tube caps were sealed with Parafilm (Bemis NA). The fat/collagenase mixture was incubated at 37◦C in a water bath for 10 min to activate the collagenase. The tubes with fat/collagenase mixture were placed into the orbital shaker set at 220 r/min for 45 min. Collagenase activity was then neutralized by addition of an equal volume of cold buffer consisted of PBS containing 2% fetal bovine serum, 1% poloxamer-188, and 1% penicillin/streptomycin (FACS buffer). The solution was then centrifuged at 1250 r/min at 4◦C for 10 min. Supernatant was aspirated and the SVF pellets were resuspended again in FACS buffer again and filtered through a 100-µm cell strainer, before centrifuging again at 1250 r/min at room temperature for 10 min. The resulting cell pellet was resuspended in 15 ml FACS buffer and carefully layered over 15 ml of room-temperature equilibrated Histopaque (Sigma– Aldrich) in a new 50-ml Falcon tube. The cell suspension with Histopaque was centrifuged at 1500 r/min for 30 min at room temperature with acceleration set to low and deceleration settings inactivated. The cloudy interface was transferred to a new 50 ml Falcon tube, and 40 ml FACS buffer was added. The cell suspension was centrifuged at 1500 r/min at 4◦C for 5 min. The pellet was resuspended for cell culture or for flow cytometry analysis.

### Flow Cytometry

At indicated time points, cells were incubated for 30 min on ice in FACS buffer containing antibodies to CD34, CD31, and CD45 (BD Biosciences). Fluorescence-activated cell sorting was performed on a BD FACSAria II (BD Biosciences, San Jose, CA, United States), using a 100-µm nozzle. Propidium iodide was used as the live/dead discriminator. Compensation was performed using CD31-PB, CD45-PECy7, and CD43-FITC with antibody capture beads (Thermo Fisher). Acquisition was performed by BD FACSDiva (BD Biosciences). Analysis was performed using FlowJo (version 10). Gating for CD34-FITC positive utilized a full stain minus one control.

### Cell Culture

Aligned nanofibrillar scaffolds (1-cm long) were placed in 24 well ultra-low attachment plate (Corning), and pre-incubated with 1 ml DMEM/10% FBS with 1% penicillin-streptomycin in CO<sup>2</sup> incubator for 45 min. At the end of the pre-incubation, media was removed, and 1 ml SVF suspension in DMEM/10% FBS with 1% penicillin-streptomycin was added to the wells containing scaffold samples. The scaffolds were incubated with SVF suspension 5% CO<sup>2</sup> and 37◦C for 1 h, then the cell sample in each well was resuspended, and incubated again for 1 h. After the 2-h incubation, cell-seeded scaffold samples were transferred into new wells containing fresh media and incubated in CO<sup>2</sup> incubator at 37◦C overnight. As a control, 1 ml SVF suspension in DMEM/10% FBS with 1% penicillin-streptomycin was incubated at 10<sup>6</sup> cells/ml/well in 24-well ultra-low attachment plate in CO<sup>2</sup> incubator at 37◦C overnight. After overnight incubation, SVF cells cultured on scaffolds were dissociated using a 1:1 mixture of TrypLE (Fisher Scientific) and collagenase digestion buffer, pelleted, and resuspended in FACS buffer. SVF cells cultured in suspension were pelleted and resuspended in FACS buffer. Both scaffold- and suspension cultured SVF samples were analyzed by flow cytometry as described above.

## VEGF Enzyme Linked Immunosorbent Assay (ELISA)

Following the overnight incubation of control cell suspension and cell-seeded scaffolds, media samples were collected and kept frozen at −80◦C. Corresponding cell numbers were determined after counting by hemocytometer for cell suspension samples, and by DNA quantification for cell-seeded scaffold samples. VEGF concentration in media samples was measured by VEGF enzyme linked immunosorbent assay (ELISA) kit (R&D Systems) following manufacturer's instructions.

#### Preparation of Cell-Seeded Scaffolds and Control Cell Suspension for in vivo Implantation

Following the overnight incubation, cell-seeded scaffold samples were removed from the media, rinsed in PBS, placed in 1.5-ml Eppendorf tube filled with PBS, and transferred to the surgery room. Control samples were prepared from the SVF samples incubated in 24-well ultra-low attachment plate overnight. Cells were resuspended, centrifuged at 1250 r/min for 5 min at room temperature, and SVF suspension sample was prepared at 10<sup>4</sup> cells in 50 µl PBS per injection/animal. This cell number was based on pilot studies showed that 1-cm BioBridge scaffold could hold up to 10<sup>4</sup> cells. Quantification of scaffold-attached cells was performed by measuring DNA content in cell lysates using PicoGreen assay (Fisher Scientific), and calculation based on calibration curves obtained from series of cell samples with known cell numbers counted by hemocytometer.

### Immunofluorescence Staining of SVF-Seeded Scaffolds

Cell-seeded scaffolds were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X-100, then blocked with 1% bovine serum albumin. Scaffolds were incubated with up to two of the following agents to determine the cellular makeup of the SVF. A 1:100 dilution of anti-human CD31 antibody (DAKO, M082301-2), 1:100 dilution anti-CD105 (endoglin) antibody (Santa Cruz Biotechnology, sc-18838), 1:200 dilution anti-human CD34 antibody (Novus Biologicals, NBP2-44568), and 1:100 dilution Alexa Fluor 488 Phalloidin (ThermoFisher, A12379) for F-actin visualization. Samples were washed in PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (ThermoFisher). Scaffolds were washed in PBS

and counterstained with Hoechst 33342 nuclear dye (Invitrogen, H3570). Scaffolds were then imaged with a confocal microscope (LSM710, Zeiss).

### Hindlimb Ischemia and Blood Perfusion Assessment

Hindlimb ischemia was induced in 8–10 week old male NOD SCID mice (Jackson Laboratory) by unilaterally excising a portion of the femoral artery (Niiyama et al., 2009). The animals then received one of the following treatments: 10<sup>4</sup> SVF cells preseeded onto a 1-cm-long scaffold and delivered to the site of the ligated artery, or 1 × 10<sup>4</sup> SVF cell delivered to the adductor muscle of the ischemic leg by local intramuscular injection. Acellular scaffold and a PBS (50 µl) injection were included as reference control groups. Limb perfusion was measured up to 14 days post-surgery by using laser Doppler spectroscopy (PIM3, Perimed). Each animal was placed on a warming pad until they reached a core temperature of 37.5◦C before imaging (Niiyama et al., 2009). Results were assessed by taking perfusion value of the ischemic foot, relative to that of the non-ischemic foot to obtain a mean perfusion ratio (Huang et al., 2010; Rufaihah et al., 2011; Nakayama et al., 2015; Hou et al., 2018). All animal studies were approved by the Stanford University Administrative Panel on Laboratory Animal Care.

#### Immunofluorescence Assessment of SVF Cell Retention

After 14 days, the gastrocnemius tissues were harvested, snap frozen in OCT, and cryosectioned in 10-µm thick sections. To visualize the persistence of human SVF cells, tissue sections were immunofluorescently stained with antibodies directed against human-specific nuclear matrix antigen (Millipore), humanspecific CD34 (Novus Biologicals), and human-specific CD31 (Dako). After overnight incubation, the cells were washed in PBS and then incubated with secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488. The immunofluorescently stained slides were imaged using an epifluorescence microscope (Observer Z1, Zeiss).

#### Statistics

Data are shown as mean ± standard deviation. Statistical analysis of SVF in suspension, in comparison to on aligned nanofibrillar scaffolds, was performed using the Wilcoxon non-parametric paired t-test. For in vivo studies, mean blood perfusion recovery among treatment groups was statistically analyzed using a oneway analysis of variance (ANOVA) with Bonferroni post-test. Statistical significance was accepted at P < 0.05.

### RESULTS

### Characterization of Freshly Isolated Bulk SVF by Flow Cytometry

Human SVF from six independent donors were analyzed by flow cytometry to characterize the phenotype of bulk SVF immediately after isolation (**Figure 1**). Using antibodies targeting phenotypic markers of vascular (CD31 and CD34) and hematopoietic (CD45) lineages, the SVF was found to be heterogeneous and having subpopulations of CD45+, CD45−/CD31+, and CD45−/CD31−/CD34+cells (**Figure 1A**). The mean distribution of these subpopulations among six donors was 33.1 ± 9.6% for CD45+, 4.5 ± 3.1% for CD45−/CD31+, and 45.4 ± 20.0% for CD45−/CD31−/CD34<sup>+</sup> (**Figures 1B,C**), the latter subpopulation representing the largest segment of SVF. Among CD45−/CD31<sup>+</sup> subset, 85.4 ± 33.7% of the cells co-expressed CD34+.

### Characterization of SVF Cellular Attachment on Aligned Nanofibrillar Scaffolds

Aligned nanofibrillar scaffolds were fabricated using shearmediated extrusion technology that preferentially induced fibrillogenesis along one principle axis, creating 0.2-mm-wide thread-like scaffolds with longitudinally oriented bundles of nanofibrils. Each individual nanofibril was approximately 200 nm in diameter (**Figure 2A**), based on SEM imaging. After seeding of the freshly isolated bulk SVF onto the aligned nanofibrillar scaffolds for 1 day, the cell-seeded samples were fixed in 4% paraformaldehyde for immunofluorescence staining of phenotypic markers. As shown by the confocal microscopy images, overnight culture of freshly harvested SVF revealed diverse cellular morphologies, including elongated and adherent cells, loosely adherent cells with rounded morphology, as well as small cellular aggregates (**Figures 2B–D**). Some of the SVF expressed CD31 and CD34, in agreement with the flow cytometry data (**Figures 2B,C**). Additionally, CD105, which is expressed by both mesenchymal and endothelial lineages, was also found to be expressed by some cells attached to the aligned nanofibrillar scaffolds (**Figure 2D**). To quantitatively assess the phenotype of SVF 1 day after attachment to the nanofibrillar scaffold, in comparison to SVF in suspension culture, we performed flow cytometric analysis using the same phenotypic markers. Our results show that the percent population of vascular (CD31 and CD34) and hematopoietic (CD45) phenotypic markers did not significantly change over the course of 1 day of attachment to the scaffold (**Figure 3A**). Propidium iodide analysis confirmed > 80% viable cells in both treatment groups for all donor cells, which suggested high cell viability.

Although the cells remained phenotypically unchanged after attachment to aligned nanofibrillar scaffolds, the cells became functionally more angiogenic. Among individual SVF donor lines, the average VEGF level from the conditioned media derived from suspension cells was 4.3 ± 1.4 pg per 10<sup>4</sup> cells (**Figure 3B**). In stark contrast, the average VEGF secretion from cells seeded on the aligned collagen scaffold was 26.0 ± 23.5 pg per 10<sup>4</sup> cells, which represented a significant increase by more than a sixfold (p < 0.05). For all donor cell lines, there was a consistent increase in VEGF release on scaffold, compared to the bulk SVF suspension population, despite some degree of heterogeneity in the magnitude of VEGF increase among donor cells. These data suggested that aligned nanofibrillar scaffolds could promote angiogenic capacity of SVF.

FIGURE 1 | Flow cytometric analysis of human stromal vascular fraction (SVF) cells. (A) Representative flow cytometric plot depicts subpopulations of cells based on the expression of CD31 and CD45. (B) Flow cytometric analysis of the CD31−/CD45<sup>−</sup> subpopulation showing that the majority of the cells are CD34+. (C) Mean cell fraction data among six independent donor SVF extractions.

#### Therapeutic Efficacy of Aligned Nanofibrillar Scaffolds Seeded With SVF in a Mouse Model of PAD

To determine if the enhanced production of VEGF by SVF on aligned scaffolds could impart a therapeutic benefit, we implanted SVF-seeded scaffolds into immune compromised NOD SCID mice with hindlimb ischemia as an experimental model of PAD. Therapeutic improvement was assessed by non-invasive measurement of blood perfusion recovery by laser Doppler spectroscopy over the course of 14 days after implantation. Quantitative analysis of mean perfusion recovery demonstrated a significant improvement in animals treated with the SVFseeded scaffolds after 14 days (0.71 ± 0.21), in comparison to animals treated with PBS (0.34 ± 0.18) (**Figure 4**). In contrast, animals treated with injections of SVF or implantation of the acellular scaffold did not show statistically significant improvement in perfusion recovery, in comparison to animals treated with PBS (**Figure 4B**). Additionally, SVF could be visualized in some histological tissue sections using human specific nuclear matrix antigen near the site of implantation, suggesting the persistence of these cells (**Figure 5A**). The SVF cells appeared to retain the expression of CD34 and lacked the expression for CD31, based on staining with human-specific antibodies (**Figures 5B,C**). The persistence of CD34 and absence of CD31 is consistent with their cellular phenotype prior to transplantation, in which the majority of cells were CD34 with a low incidence of CD31 cells (**Figure 3**). Together, these results indicated that only SVF-seeded scaffolds promoted significant improvement in blood perfusion recovery, which concurs with the finding of SVF-seeded scaffolds releasing significantly more VEGF (**Figure 3B**).

## DISCUSSION

The salient results of this study are that SVF-seeded aligned nanofibrillar scaffold significantly improved blood perfusion recovery, compared to a control PBS injection (**Figure 4**), and that SVF cells attached to aligned nanofibrillar scaffolds produced significantly more VEGF than SVF cells in suspension (**Figure 3**). This is the first preclinical study that demonstrates that SVF-seeded aligned nanofibrillar scaffold significantly improves blood perfusion, compared to the control PBS injection

Scale bars: 2 µm (A); 50 µm (B–D).

group. In contrast, treatment with acellular scaffold alone had no therapeutic effect on the level of blood perfusion in the mouse ischemic limb. Likewise, treatment with SVF cellular injection alone did not have a significant benefit over PBS. Therefore, the combination of the SVF cells and aligned nanofibrillar scaffolds could be a promising therapeutic strategy to treat PAD.

Notably, cells attached to the scaffold in vitro produced significantly higher levels of VEGF, suggesting that cell attachment to the aligned collagen scaffold may be necessary for inducing VEGF production. This concurs with published literature showing that the gene expression of various proangiogenic factors such as VEGF could be regulated using hydrogels of various stiffnesses (Cai et al., 2016). Additionally, in our previous studies, we demonstrated that cellular attachment to aligned nanofibrillar scaffolds increased the gene expression of integrin α<sup>1</sup> subunit, which has been shown to be upregulated during angiogenesis (Nakayama et al., 2015). These studies highlight a potentially important role of nano-scale extracellular matrix interactions in mediating changes in cellular function. In a recent study, collagen biomaterial has been shown to enhance pro-angiogenic activity of CD34+ cells (McNeill et al., 2019). Together, the published literature supports our finding that mechanical and biophysical properties of biomaterials can modulate cellular secretion of angiogenic growth factors, which can contribute to proangiogenic capacity. However, further studies are warranted to elucidate the signaling mechanisms that govern how aligned nanofibrillar collagen scaffolds modulate cellular secretion of growth factors.

scaffolds, compared to bulk SVF cells in suspension by flow cytometry (n = 4). (B) Quantification of VEGF protein release in the bulk SVF population, in comparison to the adherent cells seeded on aligned nanofibrillar scaffolds, for six independent donors. \* denotes statistically significant relationship, compared to bulk SVF (p < 0.05).

Another mechanism that may explain the increased blood perfusion with SVF-seeded scaffold treatment is cell survivability and persistence. In our previous studies, we observed that nanofibrillar scaffolds had increased the survival duration of endothelial cells, and this potentially also applies to other cell types such as SVF cells (Huang et al., 2013b; Nakayama et al., 2015). It is plausible that the nanofibrillar scaffold prolonged the survival of SVF cells, resulting in a longer duration of therapeutic effect (Hoareau et al., 2018).Besides VEGF, other interesting cellular signaling mechanisms may also be at play. Additionally, the small sample size and variability of blood perfusion recovery in this study may have precluded the possibility of observing significant improvements in the other treatment groups.

Previous studies have been conducted with the use of mononuclear cells as a therapy for CLI. Many of such preclinical and clinical studies used a heterogeneous population of mononuclear cells which showed mixed results in resolving limb pain, ulcers, and amputation rates in particular (Matoba

FIGURE 5 | Immunofluorescence staining of retained human SVF adjacent to the site of cell-seeded scaffold transplantation. (A) Human SVF are visualized using human specific nuclear matrix antigen (NuMA). Immunofluorescence imaging of human-specific antibodies targeting CD34 (B) and CD31 (C). Scale bar: 50 µm (A), 100 µm (B,C).

et al., 2008; Teraa et al., 2015). Autologous stem cell therapy for PAD using mononuclear cells or MSC derived from bone marrow or peripheral blood improved the ulcer healing rate and reduced amputation rate, but no significant improvement in major limb salvage was reported (Gao et al., 2018). At least one clinical trial with the use of adipose-derived stem cells (ASC) to treat CLI is under way (Clinicaltrials.gov Identifier NCT03968198). ASC are multipotent cells that can differentiate in multiple cell types including endothelial cells (Cao et al., 2005). In addition to the multipotency of ASC, their benefits for vascular regeneration include paracrine secretion of cytokines and growth factors that may stimulate angiogenesis (Park et al., 2008). Pro-angiogenic potential of MSC is generally believed to be mediated by the secretion of multiple paracrine factors, and this property is largely similar between ASC (Park et al., 2008) and bone marrow derived MSC (Kwon et al., 2014).

The advantages of ASC over other stem cells include relative abundance of subcutaneous adipose tissue in many patients, ease of access without significant donor site morbidity (Tobita et al., 2011; Mizuno, 2013), and two orders of magnitude higher yield of cells per g tissue, compared to bone marrow (Mizuno, 2013; Ong and Sugii, 2013). However, MSC from both adipose tissue and bone marrow showed donor variability (Mohamed-Ahmed et al., 2018). Accordingly, the use of autologous ASCs showed a high variation in clinical outcome, due to differences in donor age, gender, and weight, and the anatomic harvest location and depth (Aksu et al., 2008; Baglioni et al., 2012). Although this current study is not powered to evaluate donorspecific outcomes, it is plausible that inherent differences in the quality of donor SVF may lead to different in vivo angiogenic outcomes.

Freshly isolated SVF includes an abundant population of ASC, which can be identified as CD34+/CD31−/CD45<sup>−</sup> cells (Tevlin et al., 2016; Brett et al., 2017a), and are mostly of pericytic or mesenchymal phenotype (Traktuev et al., 2008). Non-hematopoietic SVF cells also have a small numbers of CD31+/CD34<sup>+</sup> and CD31+/CD34<sup>−</sup> cells, which are generally identified as endothelial progenitor and mature cells, respectively (Zimmerlin et al., 2013; Polancec et al., 2019), although the latter were also referred as co-expressing CD34<sup>+</sup> (Tallone et al., 2011).

Preclinical data showed that CD34<sup>+</sup> cells represent a main subset of stem/progenitor cells in peripheral blood mononuclear cell transplants that potentiate neovascularization in the ischemic area (Schatteman et al., 2000), and improve blood perfusion (Li et al., 2010) and wound healing (Tanaka et al., 2018). In the present study, CD45−/CD31−/CD34<sup>+</sup> comprised the majority of the SVF cells. To determine the contribution of CD34<sup>+</sup> SVF cells on revascularization in the setting of limb ischemia model, mechanistic studies in which CD34<sup>+</sup> cells are depleted can be performed in future studies. Nevertheless, clinical studies of mononuclear stem cell-based therapeutic angiogenesis employed to treat no-option CLI demonstrated that the number of transplanted CD34<sup>+</sup> cells was an independent predictor of positive outcome (Pan et al., 2019). In addition, in a study of donor variability of MSC proangiogenic efficacy, Kim et al. (2019) found that a subset of paracrine factors including VEGF serve as efficient biomarkers for predicting vascular regenerative efficacy of stromal/stem cells. Together, these studies together suggest a promising therapeutic benefit of CD34<sup>+</sup> cell therapy.

The SVF population in this study contained a relatively high fraction of CD34<sup>+</sup> cells, which are pro-angiogenic cells that are known to produce VEGF (Bautz et al., 2000). We also observed that the cells that attached to our nanofibrillar scaffold had a high amount of CD34<sup>+</sup> cells compared to cells containing other markers CD31 and CD45. Therefore, it is likely that the CD34<sup>+</sup> cells within the SVF contribute to the observed pro-angiogenic effects in vitro and in vivo.

#### CONCLUSION

This is the first study to evaluate an SVF-seeded aligned nanofibrillar scaffold as a potential therapy for PAD. In vitro the SVF seeded onto aligned scaffolds produced significantly higher levels of VEGF, compared to cells in suspension. Furthermore,

### REFERENCES

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in vivo the SVF-seeded scaffolds significantly increased blood perfusion after 14 days in a murine hindlimb ischemia model. SVF is a promising candidate for regenerative medicine due to its availability and as a source of pro-angiogenic cells. Our results suggest that aligned nanofibrillar scaffolds play an important role in enhancing the angiogenic potential of therapeutic cells and have important implications in the design of cell-based therapies for treatment of PAD in patients.

### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by the Stanford University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Stanford University Administrative Panel on Laboratory Animal Care.

### AUTHOR CONTRIBUTIONS

CH, TZ, CA, DN, DW, MP, and NH contributed to the conceptual design of the study. CH, TZ, CA, PT, SS, GY, and MB collected and analyzed the data. CH, TZ, CA, DN, DW, MP, and NH interpreted the data. CH, TZ, CA, MP, and NH wrote the manuscript, with input from all authors.

### FUNDING

This work was supported in part by grants to NH from the US National Institutes of Health (R01 HL127113 and R01 HL142718), and the US Department of Veterans Affairs (1I01BX002310 and 1I01BX004259). This work was also supported in part by funding from Terumo Corporation to MP.

### ACKNOWLEDGMENTS

We acknowledge the technical assistance Tanaka Tetsuo, Kiminami Hideaki, Nakagawa Yuuji, and Ishii Naoki from Terumo Corporation.

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**Conflict of Interest:** MP is the Chief Scientific Officer of Fibralign Corporation. SS and TZ are the employees of Fibralign Corporation.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Hu, Zaitseva, Alcazar, Tabada, Sawamura, Yang, Borrelli, Wan, Nguyen, Paukshto and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fbioe-08-00689 July 16, 2020 Time: 19:30 # 10

# Developing an Injectable Nanofibrous Extracellular Matrix Hydrogel With an Integrin αvβ3 Ligand to Improve Endothelial Cell Survival, Engraftment and Vascularization

Dake Hao1,2, Ruiwu Liu<sup>3</sup> , Kewa Gao1,2, Chuanchao He<sup>1</sup> , Siqi He1,2, Cunyi Zhao<sup>4</sup> , Gang Sun<sup>4</sup> , Diana L. Farmer1,2, Alyssa Panitch1,5, Kit S. Lam<sup>3</sup> and Aijun Wang1,2,5 \*

<sup>1</sup> Department of Surgery, School of Medicine, University of California, Davis, Sacramento, CA, United States, <sup>2</sup> Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA, United States, <sup>3</sup> Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, Sacramento, CA, United States, <sup>4</sup> Department of Biological and Agricultural Engineering, University of California, Davis, Davis, CA, United States, <sup>5</sup> Department of Biomedical Engineering, University of California, Davis, Davis, CA, United States

#### Edited by:

Qingxin Mu, University of Washington, United States

#### Reviewed by:

Anna Laurenzana, University of Florence, Italy Martin F. Desimone, University of Buenos Aires, Argentina

> \*Correspondence: Aijun Wang aawang@ucdavis.edu

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

> Received: 22 March 2020 Accepted: 10 July 2020 Published: 29 July 2020

#### Citation:

Hao D, Liu R, Gao K, He C, He S, Zhao C, Sun G, Farmer DL, Panitch A, Lam KS and Wang A (2020) Developing an Injectable Nanofibrous Extracellular Matrix Hydrogel With an Integrin αvβ3 Ligand to Improve Endothelial Cell Survival, Engraftment and Vascularization. Front. Bioeng. Biotechnol. 8:890. doi: 10.3389/fbioe.2020.00890 Endothelial cell (EC) transplantation via injectable collagen hydrogel has received much attention as a potential treatment for various vascular diseases. However, the therapeutic effect of transplanted ECs is limited by their poor viability, which partially occurs as a result of cellular apoptosis triggered by the insufficient cell-extracellular matrix (ECM) engagement. Integrin binding to the ECM is crucial for cell anchorage to the surrounding matrix, cell spreading and migration, and further activation of intracellular signaling pathways. Although collagen contains several different types of integrin binding sites, it still lacks sufficient specific binding sites for ECs. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LXW7, an integrin αvβ3 ligand, which possessed a strong binding affinity to and enhanced functionality of ECs. In this study, to improve the EC-matrix interaction, we developed an approach to molecularly conjugate LXW7 to the collagen backbone, via a collagen binding peptide SILY, in order to increase EC specific integrin binding sites on the collagen hydrogel. Results showed that in the in vitro 2-dimensional (2D) culture model, the LXW7-treated collagen surface significantly improved EC attachment and survival and decreased caspase 3 activity in an ischemic-mimicking environment. In the in vitro 3 dimensional (3D) culture model, LXW7-modified collagen hydrogel significantly improved EC spreading, proliferation, and survival. In a mouse subcutaneous implantation model, LXW7-modified collagen hydrogel improved the engraftment of transplanted ECs and supported ECs to form vascular network structures. Therefore, LXW7-functionalized collagen hydrogel has shown promising potential to improve vascularization in tissue regeneration and may be used as a novel tool for EC delivery and the treatment of vascular diseases.

Keywords: collagen hydrogel, endothelial cell, integrin-based ligand, cell engraftment, tissue regeneration

### INTRODUCTION

fbioe-08-00890 July 27, 2020 Time: 18:11 # 2

Endothelial cell (EC) transplantation has been widely used for the treatment of various types of vascular diseases, such as myocardial ischemia, cerebrovascular disease, and peripheral vascular disease (Fadini et al., 2005; Singhal et al., 2010; Blum et al., 2012). However, exploration and tracking of EC behavior and fate after transplantation has indicated that ECs exhibit poor survival and engraftment rates, which proves to be the crucial limitation of EC transplantation (Hall and Jevnikar, 2003). To overcome this limitation, different approaches, such as cell pre-conditioning, genetic modification and co-transplantation (Penn and Mangi, 2008; Yu et al., 2013; Sun et al., 2016; Shafiee et al., 2017; Hao et al., 2019), have been used to improve EC engraftment after transplantation. However, these approaches have raised safety concerns and have complicated regulatory pathways. Thus, there is still an urgent need to develop safe and easy-for-translation approaches for EC transplantation that allow for improved cell engraftment rates.

Biomaterial carriers are attractive tools for improving cell delivery and survival (Qi et al., 2015). Hydrogel systems are currently widely studied in the field of stem cell transplantation, due to their 3-dimensional (3D) cross-linked networks, cytocompatibility, injectability, and biocompatibility (Mulyasasmita et al., 2014; Robinson et al., 2016; Hao et al., 2020b). Collagen is the main structural protein in natural extracellular matrix (ECM), which can mimic the physical characteristics of ECM (Di Lullo et al., 2002). Particularly, type I collagen has great potential as a cell delivery medium, because it is ubiquitous and can self-assemble under physiological conditions (Glowacki and Mizuno, 2008; Copes et al., 2019). Integrins are heterodimeric transmembrane receptors present on the cell surface (Giancotti and Ruoslahti, 1999). Upon ligand binding, integrins facilitate cell-cell and cell-ECM adhesion, activate signal transduction pathways, and regulate cell functions (Caiado and Dias, 2012; Malinin et al., 2012). Integrins have been shown to play critical roles in cartilage (Liang et al., 2015), bone (Kundu et al., 2009), neuronal (Chen et al., 2018), pancreatic (Krishnamurthy and Wang, 2009) and vascular (Li et al., 2017) regeneration. Consequently, increasing specific integrin binding sites will be significant in advancing the development of an engineered hydrogel cell delivery system for use in tissue regeneration applications. Although collagen has several integrin binding sites (Xu et al., 2000; Znoyko et al., 2006; Zeltz and Gullberg, 2016), such as α1β1, α2β1, α10β1, and α11β1, it lacks sufficient EC specific integrin binding sites. According to previous studies, αvβ3 integrin expressed on ECs has been shown to be crucial for EC adhesion and for advancing angiogenic development (Tzima et al., 2001; Sheppard, 2002; Avraamides et al., 2008). Therefore, identifying an integrin αvβ3 ligand specifically bound to ECs and using it to engineer the collagen hydrogel will be valuable for EC transplantation in tissue regeneration applications.

One-bead one-compound (OBOC) combinatorial technology is an ultra-high throughput chemical library synthesis and screening method, which is suitable for integrin-based ligand discovery (Lam et al., 1991). Previously, we have identified various potent ligands, such as LXY30, LXW7, and LLP2A targeting integrins α3β1, αvβ3, and α4β1, respectively, by employing the OBOC combinatorial technology (Peng et al., 2006; Yao et al., 2009; Xiao et al., 2010). We have also found that LXW7 had specific binding to ECs via αvβ3 integrin and biomaterial scaffolds decorated with LXW7 was found to improve EC adhesion and functions in vitro and promoted vascularization in a rat carotid artery bypass vascular graft model (Hao et al., 2017, 2020a). Thus, LXW7 will be an ideal choice to modify collagen hydrogel for increasing the number of EC specific integrin binding sites. SILY peptide, RRANAALKAGELYKSILY, is a high-affinity collagen binding ligand derived from platelet membrane receptors that bind to α1 chains in collagen (Paderi and Panitch, 2008; Goldbloom-Helzner et al., 2019). SILY has also been conjugated to other functional molecules and used in medical applications related to collagen (Paderi et al., 2011; Scott et al., 2013). Therefore, in this study, we propose to molecularly immobilize LXW7 onto collagen backbone by using SILY as the junction to increase the EC specific integrin binding sites of collagen hydrogel to improve EC binding, survival and ultimately engraftment after transplantation.

### MATERIALS AND METHODS

#### Cell Culture

We used endothelial colony forming cell (ECFC) banks as described in our previous study (Hao et al., 2017, 2020a). ECFCs were expanded and cultured in Endothelial Cell Growth Medium-2 BulletKit medium (EGM-2, Lonza). ECFCs between P3 and P6 were used for all experiments.

#### ECFC Attachment on LXW7-Treated Collagen Surface

To facilitate the LXW7 modification on 2-dimensional (2D) collagen culture surface and 3-dimensional (3D) collagen hydrogel, we synthesized (SILY)2-LXW7 and SILY-(LXW7)<sup>2</sup> (**Figure 1**) through three steps: 1) standard solid phase peptide synthesis (SPPS) of LXW7-2N<sup>3</sup> or SILY-2N3, 2) SPPS synthesis of SILY-DBCO or LXW7-DBCO, 3) DBCO-azido copper-free Click conjugation by mixing LXW7-2N<sup>3</sup> with 2 eq. of SILY-DBCO or SILY-2N<sup>3</sup> with 2 eq. of LXW7-DBCO, respectively. Detailed synthesis was described in **Supplementary Figure S1**. To modify the collagen culture surface with LXW7, target culture wells in a 24-well plate were coated with 500 µL of 100 ng/mL collagen type I (PureCol) and incubated for 1 h at 37 ◦C. Collagen coated wells were rinsed three times with PBS (HyClone) and were treated with 500 µL 20 µM of (SILY)2-LXW7 or SILY- (LXW7)2. After 1 h, the wells were washed three times with PBS and blocked with 1% BSA (Thermo Fisher Scientific) for 1 h. After, the wells were rinsed three times with PBS. For the cell attachment assay, 5 × 10<sup>3</sup> ECFCs were added to the wells and incubated for 5 min at 37◦C and 5% CO2. The wells were washed three times with PBS, and the adhered cells were fixed in 10% formalin (Azer Scientific) for 20 min. The wells were washed, and then nuclei were stained with 4<sup>0</sup> , 6-diamidino-2 phenylindole (DAPI, Sigma). After three washings with PBS,

the ECFCs were imaged using a Carl Zeiss Axio Observer D1 inverted microscope. Image quantification was performed using the ImageJ software (NIH).

#### ECFC Apoptosis and Survival on LXW7-Treated Collagen Surface Under Ischemic-Mimicking Hypoxic Environment

The ischemic-mimicking hypoxic environment was set up as cells cultured in EGM-2 with 1% fetal bovine serum (FBS) and low concentration growth factors (1/10 of the original bulk in the EGM-2 bullet kit) at 1% O2, 37◦C and 5% CO2. ECFCs were seeded in 96-well plates treated with collagen, collagen and (SILY)2-LXW7, or collagen and SILY-(LXW7)2. For caspase 3 assay, the cells were cultured in ischemicmimicking hypoxic environment for 6 h, then lysed and analyzed by using a Caspase 3 Assay Kit (Cell Signaling Technology) according to the manufacturer's instruction. Fluorescence (ex 380 nm/em 450 nm) was measured using a SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices). For the cell survival assay, the ECFCs were cultured for 5 d and determined using a CellTiter 96 <sup>R</sup> AQueous One Solution Cell Proliferation Assay (MTS, Promega) according to the manufacturer's instruction. The amount of soluble formazan product produced by the reduction of MTS by metabolically active cells was measured at the 490 nm absorbance using the SpectraMax i3x Multi-Mode Detection Platform.

## Preparation and Characterization of LXW7-Modified Collagen Hydrogel

For optimization of collagen hydrogel concentration, the original 10 mg/mL collage type I was diluted to 1, 2, 4, and 8 mg/mL with PBS, respectively. The same number of ECFCs was loaded into the collagen hydrogel with different concentration and cultured at 37◦C, 5% CO<sup>2</sup> for 5 days, then the number of cells was determined using MTS as described above. For the preparation and evaluation of LXW7-modified collagen hydrogel, the diluted 2 mg/mL collagen was mixed with SILY-(LXW7)<sup>2</sup> at the different concentration of SILY-(LXW7)2/collagen (nmol/mg), such as 0, 0.005, 0.05, 0.25, 0.5, 2.5, 5, 12.5, or 25 nmol/mg. The LXW7 modified collagen (0.1 mL) was then put in 48-well plates and incubated at 37◦C for 1 h, then 100 µL PBS was added into the wells. After 24 h, the PBS was collected for High-performance liquid chromatography (HPLC) analysis to quantify the amount of unbound free SILY-(LXW7)<sup>2</sup> that was eluted to the solution. Briefly, HPLC analysis was performed on the Waters 2996 HPLC system equipped with a Waters XTerra <sup>R</sup> MS column (5 µm, C18, 150 × 4.6 mm). A linear gradient was run from 100% solution A (water/0.1% trifluoroacetic acid) to 100% solution B (acetonitrile/0.1% trifluoroacetic acid) within 20 min with a flow rate at 1.0 mL/min. The UV detection wavelength was 214 nm. The compressive modulus of the untreated collagen hydrogel and the LXW7-modified collagen hydrogel were determined using the Instron 5566 Universal Testing Machine.

## Lentiviral Vector Transduction

All lentiviral constructs were generated at the UC Davis Institute for Regenerative Cures (IRC) Vector Core. To track the cell fate and behavior in vitro and in vivo, ECFCs were transduced with the pCCLc-MNDU3-LUC-PGK-EGFP-WPRE vector as previously described (Kumar et al., 2018; Gao et al., 2019, 2020; Rose et al., 2020). Transduction was performed in transduction medium consisting of DMEM high glucose (HyClone), 10% FBS (HyClone), and 8 µg/mL protamine sulfate (MP Biomedicals) for 6 h. The vectors were transduced at a multiplicity of infection (MOI) of 10. After that, ECFCs were cultured in EGM-2 medium for 72 h. After 72 h, ECFCs were screened for neomycin resistance for 7 days cultured in medium containing 2 µg/mL of G418 (EMD, Millipore). Successfully transduced ECFCs were then cultured and expanded in EGM-2 medium.

## ECFC Behavior in LXW7-Modified Collagen Hydrogel

For cell sprouting assay, ECFCs were loaded in the untreated collagen hydrogel, or LXW7-modified collagen hydrogel, respectively, and cultured in EGM-2 for 3 d. Images were taken at day 1 and day 3 by using the Carl Zeiss Axio Observer D1 inverted microscope. Image quantification was performed using the ImageJ software. For capillary network formation, three groups were set up: (1) ECFCs cultured in untreated collagen hydrogel, (2) ECFCs cultured in LXW7-modified collagen hydrogel, (3) ECFCs incubated with a monoclonal anti-αvβ3 integrin blocking antibody (MAB1876, Millipore) first to block the integrin αvβ3 expressed on the ECFCs, then cultured in the LXW7-modified collagen hydrogel. Images were taken at day 5 by using the Carl Zeiss Axio Observer D1 inverted

microscope. Image quantification was performed using the ImageJ software. For cell proliferation, ECFCs were loaded in the untreated collagen hydrogel or LXW7-modified collagen hydrogel, respectively, and cultured in EGM-2 at 37◦C, 20% O<sup>2</sup> and 5% CO<sup>2</sup> for 5 days. Cell metabolic activity and proliferation was quantified every 24 h using MTS as described above. For cell survival, ECFCs were loaded in the untreated collagen hydrogel or LXW7-modified collagen hydrogel, respectively, and cultured under ischemic-mimicking hypoxic environment for 5 days. Cell metabolic activity was quantified every 24 h using MTS, as described above.

#### In vivo Cell Transplantation

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Davis. All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). NSG (NOD/SCID/ IL2Rγ <sup>−</sup>/−) immunodeficient mice were purchased from The Jackson Laboratory. 5 × 10<sup>5</sup> transduced ECFCs were loaded in 500 µL untreated collagen hydrogel or LXW7-modified collagen hydrogel, respectively, and injected subcutaneously to the groin at both sides of each mouse.

### Bioluminescence Imaging

Cells transplanted in NSG mice were monitored via the In Vivo Imaging Spectrum (IVIS) system (PerkinElmer) as previously described (Kumar et al., 2018; Gao et al., 2019; Rose et al., 2020). Briefly, animals were injected intraperitoneally with luciferase substrate D-luciferin (Gold Biotechnology) at 100 mg/kg body weight and maintained under anesthesia with 2% inhaled isoflurane for 5 min before imaging. The transplanted NSG mice were imaged at the day of transplantation (week 0) and weekly thereafter for up to 10 weeks after transplantation. Images were analyzed by using Living Image <sup>R</sup> 2.50 (Perkin Elmer). Total intensity was measured within a defined area of the signal. Baseline intensity was determined by using the same defined area where there is no positive signal in the same animal.

### Immunohistochemistry

Samples were collected, fixed with 4% paraformaldehyde for 24 h, protected by 30% sucrose dehydration for 48 h, and embedded in the O.C.T compound (Sakura Finetek USA, Inc). Serial sections were made at the thickness of 15 µm using a Cryostat (Leica CM3050S) and collected onto microscope slides (Matsunami Glass). Tissue sections were extensively washed with PBS, blocked with 5% BSA in PBS at room temperature for 1 h, and stained with primary antibody at 4◦C overnight. The dilution of primary antibody was goat anti-GFP (Novus Biologicals) at 1:100. Sections were incubated with secondary antibodies diluted at 1:500 for 1 h at room temperature. The secondary antibody was donkey anti-goat (Thermo Fisher Scientific) conjugated with Alexa488. The slides were counterstained with 1:5000 dilution of DAPI for 5 min, mounted with Prolong Diamond Antifade Mountant (Invitrogen), and imaged with the Zeiss Observer Z1 microscope.

## Statistical Analysis

For two-sample comparison, a student's t-test was used. For multiple-sample comparison, analysis of variance (ANOVA) was performed to detect whether a significant difference existed between groups with different treatments. A p-value of 0.05 or less indicates a significant difference between samples in comparison.

### RESULTS AND DISCUSSION

### LXW7-Treated Collagen Surface Improved ECFC Attachment

Natural ECM displays numerous copies of several cell binding sites to support cell-matrix adhesion that is crucial for cell functions (Werb, 1997; Yue, 2014). Cell-matrix adhesion is formed through the utilization of cell adhesion molecules that bind to the cell surface (Zhong and Rescorla, 2012). Integrins are

a very important class of cell adhesion heterodimer molecules, comprised of two subunits that facilitate attachments between the cell surface and the ECM, and can greatly influence cell behavior (Gumbiner, 1996). Although collagen is the main component of natural ECM, it still lacks specific EC adhesion sites. Our previous study demonstrated an integrin αvβ3 ligand LXW7 possessed specific binding to ECs (Hao et al., 2017). Thus, to increase ECFC binding sites that facilitate ECFC attachment on the collagen surface and evaluate density effects of binding sites on ECFC functions, we synthesized two different types of SILY-LXW7 derived compounds, (SILY)2-LXW7 and SILY-(LXW7)2, that can be used conveniently to conjugate LXW7 onto the collagen backbone. The results showed that cell culture surface treated with both (SILY)2-LXW7 and SILY-(LXW7)<sup>2</sup> significantly improved ECFC attachment compared to the untreated collagen surface (**Figure 2**). The SILY-(LXW7)<sup>2</sup> treated collagen surface supported more ECFC attachment, compared to the (SILY)2- LXW7 treated collagen surface indicating that a higher density of LXW7 was more beneficial for ECFC attachment (**Figure 2**).

### LXW7-Treated Collagen Surface Suppressed ECFC Apoptosis and Improved ECFC Survival Under Ischemic-Mimicking Hypoxic Environment

Cell-matrix interaction regulates cellular homeostasis in multiple ways (Almeida et al., 2000; Niit et al., 2015). Disruption of this connection has deleterious effects on cell binding and survival, which leads to a specific type of apoptosis known as anoikis in most cell types (Reddig and Juliano, 2005). Caspase-3 is

FIGURE 7 | ECFC proliferation and survival in LXW7-modified collagen hydrogel. (A) Quantification of cell metabolic activity and proliferation of ECFCs cultured in collagen hydrogel with or without LXW7 modification for 5 days. (B) Quantification of survival of ECFCs cultured in collagen hydrogel with or without LXW7 modification under ischemic-mimicking hypoxic environment for 5 days. Data were expressed as mean ± standard deviation: <sup>∗</sup>p < 0.05, ∗∗p < 0.01 (n = 5).

responsible for chromatin condensation and DNA fragmentation that is necessary in apoptosis (Porter and Janicke, 1999). The results showed both (SILY)2-LXW7 and SILY-(LXW7)<sup>2</sup> treated collagen surfaces suppressed expression of caspase 3 in ECFCs, compared to the collagen surface under the ischemic-mimicking hypoxic environment, and only SILY-(LXW7)<sup>2</sup> treated collagen surface significantly decreased the expression of caspase 3 in ECFCs, compared to the collagen surface (**Figure 3A**). These

(B) High magnification imaging of the boxed area of (A).

results may be caused by the different binding sites provided by (SILY)2-LXW7 or SILY-(LXW7)2. Stoichiometrically, the LXW7 binding sites provided by SILY-(LXW7)<sup>2</sup> was four times greater compared to the LXW7 binding sites provided by (SILY)2-LXW7. Subsequently, the results showed that the SILY-(LXW7)<sup>2</sup> treated collagen surface significantly improved ECFC survival under the ischemic-mimicking hypoxic environment (**Figure 3B**), indicating that the LXW7-treated collagen surface was beneficial for ECFC survival by increasing the number of ECFC specific integrin binding sites.

#### Evaluation of LXW7 Conjugation on Collagen Hydrogel

The effect of ECM stiffness on cell biology, signaling and response has been well-established, which has significant implications for tissue regeneration (Antoine et al., 2014). Particularly, ECM stiffness has also been linked with endothelial integrity and consequently regulation of angiogenesis (Sieminski et al., 2004). The concentrations of hydrogel could significantly influence cell behavior and growth, due to their physical properties such as stiffness (Banerjee et al., 2009; Ahearne, 2014). Before LXW7 modification, we identified the optimal concentration of collagen hydrogel for ECFC growth. The results showed that both 1 mg/mL and 2 mg/mL collagen hydrogel significantly improved ECFC growth, compared to 4 mg/mL and 8 mg/mL collagen hydrogel. There was no significant difference between the 1 mg/mL and 2 mg/mL collagen hydrogel (**Supplementary Figure S2**). In addition, to consider the difference in solidification time, we chose 2 mg/mL as the optimal concentration of collagen hydrogel for the following tests. For the LXW7 modification on collagen hydrogel, different concentration of SILY-(LXW7)<sup>2</sup> were used to modify the collagen hydrogel. HPLC has been widely used

to separate and analyze the sample mixture in a discrete small volume (Gerber et al., 2004). The HPLC results did not show any peak of SILY-(LXW7)<sup>2</sup> until the amount of SILY-(LXW7)<sup>2</sup> used to modify collagen hydrogel was up to 5 nmol/mg, and the peaks increased as the amount of SILY-(LXW7)<sup>2</sup> increased (**Figure 4**). These results demonstrated that LXW7 had been successfully conjugated onto the collagen hydrogel via the "SILYcollagen" binding approach, and the saturation concentration of modification was between 2.5 and 5 nmol/mg. Compressive modulus of the collagen hydrogel before and after LXW7 modification showed no significant difference (**Supplementary Figure S3**) indicating the SILY-(LXW7)<sup>2</sup> modification approach did not significantly change the stiffness of the collagen hydrogel. This was probably because only one end of the SILY-(LXW7)<sup>2</sup> molecule carried the –SILY group that could bind to collagen, and the –(LXW7)<sup>2</sup> end could not bind to collagen, therefore the SILY-(LXW7)<sup>2</sup> modification approach did not induce molecular cross-linking of collagen.

#### LXW7-Modified Collagen Hydrogel Improved ECFC Sprouting and Promoted ECFC Vascular Network Formation by Increasing Integrin αvβ3 Binding Sites

Endothelial cell sprouting is important for the EC migration and vascular network formation, which are essential for angiogenesis and vascularization (Blanco and Gerhardt, 2013). Improving EC binding in the hydrogel, by increasing integrin-based binding sites, is vital to promote EC sprouting and branching (Li et al., 2017). The results of ECFC sprouting showed that most of the ECFCs cultured in LXW7-modified collagen hydrogel exhibited long spindle morphology after 1 day culture (**Figure 5A,b**), but the ECFCs cultured in untreated collagen hydrogel still displayed a round morphology (**Figure 5A,a**). After 3-day culture, the ECFCs cultured in LXW7-modified collagen hydrogel exhibited obvious sprouting (**Figure 5A,d**), but only few ECFCs cultured in untreated collagen hydrogel exhibited sprouting (**Figure 5A,c**). Quantification of cell area (**Figure 5B**) and number of sprouts (**Figure 5C**) showed that LXW7 modification significantly improved ECFC spreading and sprouting in collagen hydrogel, indicating that the LXW7-modified collagen hydrogel could promote EC migration and new vascular network formation in vascular tissue regeneration. Capillary morphogenesis is a reliable in vitro analog of in vivo angiogenesis. Also, it is know that cell-matrix interaction could generate cellular force that can impact capillary network formation. The results of network formation showed the ECFCs cultured in LXW7 modified collagen hydrogel for 5 days formed uniform and integrated network (**Figure 6A,c**), but the ECFCs cultured in untreated collagen hydrogel (**Figure 6A,a**) and the anti-αvβ3 integrin antibody blocked ECFCs cultured in LXW7-modified collagen hydrogel (**Figure 6A,b**) for 5 days only formed few intermittent network and some branches. Quantification of the number of vessel network (**Figure 6B**) and total vessel network length (**Figure 6C**) showed the LXW7-modified collagen hydrogel significantly improved the network formation of ECFCs compared to the untreated collagen hydrogel, and the network formation of the ECFCs cultured in LXW7-modified collagen hydrogel was significantly decreased by blocking the anti-αvβ3 integrins using a monoclonal anti-αvβ3 integrin antibody, which indicated that LXW7-modified collagen hydrogel promotes the vascular network formation of ECFCs compared to the untreated collagen hydrogel by increasing integrin αvβ3 binding sites.

### LXW7-Modified Collagen Hydrogel Improved ECFC Proliferation and Survival

Cell proliferation and survival play key roles in stem cell therapy and tissue regeneration (Lambrou and Remboutsika, 2014; Soteriou and Fuchs, 2018). Integrin-mediated cell binding to the ECM strictly regulates cell cycle progression in mammalian cells, which is crucial for cell proliferation and survival (Schwartz and Assoian, 2001; Vachon, 2011). For ECFC metabolic activity and proliferation evaluation, MTS results showed that the number of ECFCs cultured in LXW7-modified collagen hydrogel was higher, compared to the number of ECFCs cultured in untreated collagen hydrogel, and showed the significant difference after day 4 (**Figure 7A**). For ECFC survival evaluation, results showed that the number of ECFCs cultured in LXW7-modified collagen hydrogel under the ischemic-mimicking hypoxic environment was significantly higher, compared to the number of ECFCs cultured in untreated collagen hydrogel at day 5 (**Figure 7B**). Thus, LXW7-modified collagen hydrogel can promote ECFC proliferation and survival and therefore will be beneficial to vascular tissue regeneration.

#### LXW7-Modified Collagen Hydrogel Improved ECFC Survival and Engraftment in a Mouse Subcutaneous Implantation Model

Different types of hydrogel have been widely studied for cell transplantation (Drury and Mooney, 2003), however, cell survival and engraftment after transplantation remains as one of the key limiting factors for translational applications (Cao et al., 2001). Integrin-ECM interaction is important to regulate cell survival and engraftment (Kumaran et al., 2005). IVIS results showed that LXW7-modified collagen hydrogel prevented the decline in the number of implanted ECFCs at week 1 and improved the engraftment of implanted ECFCs in the next few weeks, compared to the untreated collagen hydrogel (**Figure 8A**), and further showed the significant difference from week 4, according to the luciferase intensity quantification results (**Figure 8B**). Because the implanted ECFCs have also been transduced with GFP before implantation, the engraftment of implanted ECFCs was further evaluated using immunohistological staining with anti-GFP antibody. The immunohistological staining results showed that LXW7-modified collagen hydrogel promoted the ECFC survival and engraftment, compared to the untreated collagen hydrogel, and some ECFCs formed vascular network structures in the LXW7-modified collagen hydrogel construct (**Figure 9**). Thus, LXW7-modified collagen hydrogel represents a good candidate for promoting ECFC

engraftment and holds promise for improving vascularization and tissue regeneration. It is known that neovascularization plays the key role in many different kinds of tissue regeneration, therefore, the LXW7-functionalized collagen hydrogel developed in this study could be widely used as a novel injectable EC delivery biomaterial tool for a variety of tissue regeneration applications, including hindlimb ischemia, myocardial ischemia and so on. Therefore, in this proof-of-concept study, we chose to use a more generic but widely applicable subcutaneous implantation model to evaluate the function of the novel LXW7-modified collagen hydrogel on neovascularization. Data obtained from this model have broad implications for a wide range of tissue regeneration applications. Further detailed evaluation of the functions of LXW7-modified collagen hydrogel in specific disease models is warranted in future studies.

#### CONCLUSION

This study sought to engineer collagen hydrogel, a widely used ECM biomaterial hydrogel, to improve its function for EC transplantation and tissue regeneration. To improve the ECmatrix interaction, we engineered the collagen hydrogel by increasing EC specific integrin binding sites. We successfully developed a technology to molecularly conjugate an integrin αvβ3 ligand LXW7 onto the collagen backbone via "SILYcollagen" binding approach. The LXW7-modified collagen hydrogel exhibited the capacities for promoting EC survival in an ischemic-mimicking environment in vitro and improved the engraftment of transplanted ECs and supported ECs to form functional vascular network structures in vivo. The LXW7 functionalized collagen hydrogel holds the promise to be used as a novel EC delivery tool and injectable biomaterial for tissue engineering and regenerative medicine.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

#### ETHICS STATEMENT

The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the University of California, Davis.

#### REFERENCES


#### AUTHOR CONTRIBUTIONS

DH performed the construction and the in vitro and in vivo evaluation of LXW7-modified collagen hydrogel, wrote the manuscript, and discussed the results. RL performed the synthesis of (SILY)2-LXW7 and SILY-(LXW7)2, performed the HPLC analysis, and discussed the results. KG assisted the in vivo evaluation. CH, SH, and CZ assisted the in vitro evaluation. GS, DF, AP, and KL were involved in the results discussion. AW was responsible for conceptualization, results, discussion, and revision of the manuscript. All authors contributed to the article and approved the submitted version.

#### FUNDING

This work was in part supported by the Shriners Hospitals for Children Postdoctoral Fellowship (84705-NCA-19 to DH) and the UC Davis School of Medicine Dean's Fellowship (to AW) awards, NIH grants (5R01NS100761-02 and R03HD091601-01), Shriners Hospitals for Children research grants (87200-NCA-19 and 85108-NCA-19), and the March of Dimes Foundation Basil O'Connor Starter Scholar Research Award (5FY1682). Utilization of this Shared Resource was supported by the UC Davis Comprehensive Cancer Center Support Grant awarded by the National Cancer Institute (P30CA093373).

#### ACKNOWLEDGMENTS

The authors would like to thank the Combinatorial Chemistry and Chemical Biology Shared Resource at University of California, Davis, for design and synthesis of (SILY)2-LXW7 and SILY-(LXW7)2. We acknowledge Alexandra Maria Iavorovschi and Olivia K. Vukcevich for their help with manuscript editing and submission.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2020.00890/full#supplementary-material

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Hao, Liu, Gao, He, He, Zhao, Sun, Farmer, Panitch, Lam and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Scalable Biomimetic Coaxial Aligned Nanofiber Cardiac Patch: A Potential Model for "Clinical Trials in a Dish"

Naresh Kumar1,2† , Divya Sridharan1,2† , Arunkumar Palaniappan1,2,3, Julie A. Dougherty1,2 , Andras Czirok<sup>4</sup> , Dona Greta Isai<sup>5</sup> , Muhamad Mergaye1,2, Mark G. Angelos1,2 , Heather M. Powell5,6,7 and Mahmood Khan1,2,8 \*

<sup>1</sup> Department of Emergency Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, United States, <sup>2</sup> Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, United States, <sup>3</sup> Centre for Biomaterials, Cellular and Molecular Theranostics, Vellore Institute of Technology, Vellore, India, <sup>4</sup> Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, United States, <sup>5</sup> Department of Biomedical Engineering, The Ohio State University, Columbus, OH, United States, <sup>6</sup> Department of Materials Science and Engineering, The Ohio State University, Columbus, OH, United States, <sup>7</sup> Research Department, Shriners Hospitals for Children, Cincinnati, OH, United States, <sup>8</sup> Department of Physiology and Cell Biology, The Ohio State University Wexner Medical Center, Columbus, OH, United States

Recent advances in cardiac tissue engineering have shown that human inducedpluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a threedimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and

#### Edited by:

Chao Zhao, The University of Alabama, United States

#### Reviewed by:

Yun Chang, Purdue University, United States Arghya Paul, University of Western Ontario, Canada

#### \*Correspondence:

Mahmood Khan mahmood.khan@osumc.edu

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 30 May 2020 Accepted: 13 August 2020 Published: 16 September 2020

#### Citation:

Kumar N, Sridharan D, Palaniappan A, Dougherty JA, Czirok A, Isai DG, Mergaye M, Angelos MG, Powell HM and Khan M (2020) Scalable Biomimetic Coaxial Aligned Nanofiber Cardiac Patch: A Potential Model for "Clinical Trials in a Dish". Front. Bioeng. Biotechnol. 8:567842. doi: 10.3389/fbioe.2020.567842

synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D in vitro model for "clinical trials in a dish" and for in vivo cardiac repair applications for treating myocardial infarction.

Keywords: nanofibers, cardiac patch, myocardial infarction, cardiovascular disease, multielectrode array (MEA), 3D model, induced pluripotent stem cell-derived cardiomyocytes

#### INTRODUCTION

Cardiovascular diseases (CVDs) are the number one cause of morbidity in North America. However, the development of therapeutics for CVDs has been limited by the paucity of efficient model systems for drug screening and toxicology studies (Schroer et al., 2019). Most pharmacological studies make use of primary cell lines or model organisms like rodents, rabbits, pigs, and non-human primates for assessing the effect of putative drug molecules (Savoji et al., 2019). Small animal models like rodents, which have been extensively used for pre-clinical cardiovascular drug testing, are not ideal models, since their cardiomyocytes differ significantly from humans in their structure and function (Milani-Nejad and Janssen, 2014). On the other hand, large animal models (pigs and non-human primates) are good systems as their cardiovascular system and the associated hemodynamics are similar to humans. However, the costs associated with their housing, maintenance, and ethical concerns make them less favorable for pre-clinical drug testing applications (Milani-Nejad and Janssen, 2014). While primary cultures of adult cardiomyocytes are a good, cost-effective system to study drug effects in vitro, their use is restrained by their limited availability and lack of efficient culture protocols (Ribeiro et al., 2019).

In this context, human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become increasingly popular for use as an in vitro model system (Karakikes et al., 2015). These cells have shown great potential in developing strategies for cardiac repair (Park and Yoon, 2018). Additionally, the advances in techniques for hiPSC generation, ease of scalability of hiPSC-CMs, and development of next-generation genetic manipulation techniques make hiPSC-CMs an attractive model for the development of patient-specific personalized precision medicine (Park and Yoon, 2018; Gintant et al., 2019; Ribeiro et al., 2019). Hence, hiPSC-CMs have become increasingly popular as an in vitro model for cardioprotective and cardiotoxic drug screening (Lynch et al., 2019; Ribeiro et al., 2019). However, in most of these studies, hiPSC-CMs used were cultured in twodimensional (2D) culture dishes (Antoni et al., 2015; Savoji et al., 2019). The 2D culture system has multiple drawbacks: (a) immature phenotype of hiPSC-CMs, (b) heterogeneity of the cells in culture, (c) lack of alignment of hiPSC-CMs, and (d) inconsistent response to drug treatment (Karakikes et al., 2015; da Rocha et al., 2017). However, recent studies have shown improved function and maturation of hiPSC-CMs in 2D cultures with increased culture time (Kumar et al., 2019) or electrical and mechanical stimulation (Machiraju and Greenway, 2019).

Overall, three-dimensional (3D) culture systems have been shown to improve the maturation as well as functionality of hiPSC-CMs (Savoji et al., 2019). Cells cultured in a 3D environment are shown to have better physiological characteristics and more closely resemble native tissue than the same cells grown in classical 2D culture flasks (Savoji et al., 2019). Hence, 3D cultures provide a better and more relevant model for cardiotoxicity and drug screening studies (Zuppinger, 2016, 2019; Archer et al., 2018; Savoji et al., 2019). The 3D models currently explored as CVD models include: (a) hydrogelbased engineered heart tissue (Weinberger et al., 2017), (b) selfassembling spheroids formed via hanging drop method (Figtree et al., 2017), (c) cardiac cell-sheets (Shimizu et al., 2003), and (d) bioengineered scaffolds (Khan et al., 2015, 2018; Kc et al., 2019). Of these, the scaffold-based models have been extensively studied for the development of engineered heart tissues (Gao et al., 2018; Dattola et al., 2019; Jabbour et al., 2019).

Three-dimensional scaffolds have been fabricated using different bioengineering techniques, like microfluidics (Wang et al., 2010), 3D bioprinting (Zhu et al., 2016), gas foaming (Costantini and Barbetta, 2018), and electrospinning (Jun et al., 2018). Of these, electrospinning provides for reduced batch-to-batch variation, better uniformity within scaffolds, nano-dimensional architecture similar to cardiac tissue, and controlled alignment of nanofibers (Kai et al., 2011; Khan et al., 2015; Kitsara et al., 2017). Further, nanofiber-based scaffolds have been fabricated using a wide variety of natural and synthetic biocompatible materials. It has been reported that scaffolds made using natural polymers like gelatin and collagen (Aldana and Abraham, 2017) exhibit efficient cell adhesion but poor mechanical properties. Alternatively, the scaffolds made using synthetic polymers like poly(lactic-coglycolic) acid (PLGA; Khan et al., 2015), polylactic acid (PLA; Wang et al., 2017), and polycaprolactone (PCL; Chen et al., 2015) have poor biomimetic and cell adhesion properties but improved mechanical support (Bhattarai et al., 2018). In our study, we fabricated an aligned coaxial nanofibrous scaffold, with nanofibers having a PCL core with a gelatin shell. The PCL imparts mechanical strength while gelatin provides the required biomimetic properties, thereby improving cell attachment. The hiPSC-CMs were seeded and cultured on the surface of a 3D aligned coaxial nanofibrous scaffold to obtain a functional 3D 'cardiac patch,' which was then used for drug screening and toxicity studies.

### MATERIALS AND METHODS

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#### Fabrication of PCL-Gelatin Aligned Coaxial Nanofibrous Patch

Gelatin (12% w/v) [gelatin from bovine skin, Sigma-Aldrich, St. Louis, MO] and PCL (8% w/v) [Sigma-Aldrich, St. Louis, MO; Mn = 42,500] solutions were prepared in 1,1,1,3,3,3-hexafluoro-2-propanol. The gelatin and PCL solutions were fed to the outer (at a flow rate of 4 ml/h) and inner tube (at a flow rate of 1 ml/h), respectively, of the coaxial spinneret as shown in **Figure 1A**. The distance between the spinneret tip and the grounded rotating collector was maintained at 20 cm and a 20 kV voltage was applied at the spinneret tip. The aligned coaxial nanofibers collected were dried inside a chemical fume hood overnight, to remove remnant solvent.

The morphology of the nanofibrous patch was assessed by scanning electron microscopy (SEM). To confirm the coaxial morphology of the nanofibrous patch, gelatin and PCL solutions were mixed with 1% w/v fluorescein (Sigma-Aldrich, St. Louis, MO) and rhodamine (Sigma-Aldrich, St. Louis, MO), respectively, and nanofibrous patches were fabricated as mentioned above and imaged using a confocal microscope (Olympus FV3000 Confocal microscope).

Patches were first cross-linked, sterilized, and hydrated using the protocol described previously (Drexler and Powell, 2011). Briefly, patches were cut into the desired diameter using biopsy punches (8 mm) and treated with 7 mM 1-ethyl-3-(3Dimethylamino propyl)carbodiimide hydrochloride (EDC) solution in ethanol for 24 h, followed by incubation in 70% ethanol for hydration and sterilization. The patches were then washed in phospahate-buffered saline (PBS), two times for 24 h each. Patches were then used for confocal imaging, mechanical testing, and cell seeding.

#### Fourier-Transform Infrared Spectroscopy (FTIR) Studies

Surface chemical analysis of the gelatin-only, PCL-only, and coaxial patches was performed using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) between the range of 400–4000 cm−<sup>1</sup> (Thermo Nicolet Nexus 670 FTIR spectrometer, MN). Approximately 25–40 scans were performed on the three types of patches.

#### Mechanical Testing of Nanofiber Scaffolds

The mechanical properties of acellular aligned PCL, gelatin, and PCL-gelatin coaxial nanofibrous patches were tested using a TestResources 100R (Shakopee, MN) as per the American Society for Testing and Materials (ASTM) D638 Type V standard, using the protocol described previously (Johnson et al., 2007; Blackstone et al., 2018). For aligned gelatin and coaxial patches, tests were performed after crosslinking in EDC solution and further hydration in PBS (described above). For aligned PCL patches, incubation was carried out in ethanol (to mimic the crosslinking process) before hydration in PBS. The thickness of each sample was measured using digital calipers. Samples were strained (along the axis of alignment) at a grip speed of 2 mm/s until failure. Samples were made using a dog-bone shaped punch, 3 mm gauge width, and 10 mm gauge length. The thickness of each sample was quantified using digital calipers. Stress-strain curves were generated for each sample and the ultimate tensile strength and Young's modulus were determined and reported as mean ± SD. Ultimate tensile strength was determined at the point of greatest stress, before failure. Young's modulus was calculated from the stress-strain curve using linear regression analysis for the first linear region of the curve (where R <sup>2</sup> ∼0.98) past the toe-in region, if present.

### Degradation Studies

The scaffold was cut into small 8 mm diameter circular patches and incubated in medium at 37◦C for 2 weeks. After 1 and 2 weeks, the patches were rinsed in water, air-dried and weighed to determine their weight loss by degradation. The weights of four independent samples were measured for each time point. Additionally, the patches were imaged using SEM to assess any morphological changes in the fibers during the 2-week culture duration.

#### Culturing and Maintenance of hiPSC-CMs on Aligned Coaxial Nanofibrous Patches

The hiPSC-CMs were obtained from Fujifilm Cellular Dynamics International (CDI, Madison, WI, United States, Cat# R 1007). The cells were plated in sterile six-well plates according to the manufacturer's protocol and after 48 h, cells were cultured in CDI hiPSC-CMs maintenance medium in a humidified atmosphere at 37◦C at 5% CO2, as previously described (Citro et al., 2014; Khan et al., 2015).

Once the hiPSC-CMs showed contractions, the cells were seeded onto aligned coaxial nanofibrous patches. For this, sterile cross-linked 8 mm aligned coaxial patches were transferred onto N-terface (Winfield Labs, Richardson, TX, United States) and coated with 30 µl of fibronectin (50 µg/ml) for 1 h in a humidified atmosphere at 37◦C. The patches on N-terface were then transferred onto sterile sponges soaked in the hiPSC-CMs' culture medium placed in a 100 mm culture dish. The hiPSC-CMs were harvested using 0.25% trypsin-EDTA and seeded onto the coaxial patches at a final density of 1 × 10<sup>6</sup> cells/cm<sup>2</sup> (50 µl of cell suspension/patch) and incubated at 5% CO<sup>2</sup> at 37◦C for 1 h. After 4–6 h, the aligned coaxial cardiac patches (aligned coaxial nanofibrous patches seeded with hiPSC-CMs) were transferred to six-well plates (one patch/well) containing 2 ml CDI hiPSC-CMs maintenance medium. The medium was replaced every alternate day for 2 weeks.

### Scanning Electron Microscopy (SEM)

Scanning electron microscopy was used to observe the morphology of the aligned PCL, gelatin, and PCL-gelatin

coaxial nanofibrous patches. SEM was also performed on aligned coaxial cardiac patches to determine the distribution and alignment of the seeded hiPSC-CMs. For these studies, the samples were prepared as described previously (Huang et al., 2013; McBane et al., 2013; Khan et al., 2015). In brief, the cardiac patches were fixed in 4% paraformaldehyde (PFA, MilliporeSigma, Milwaukee, WI, United States) for 1 h. Patches were then washed with de-ionized water and further dehydrated by gradually increasing ethanol concentration (50, 70, 80, 95, and 100%). The patches were then dried chemically using an increasing gradient of hexamethyldisilazane. Finally, patches were sputter-coated with gold-palladium (Pelco Model 3) and imaged on FEI NOVA nanoSEM.

#### Cellular Viability Staining of hiPSC-CMs

LIVE/DEADTM Cell Imaging Kit (Cat # R37601) was procured from Invitrogen (Life Technologies Corporation, Carlsbad, CA, United States) and used to stain the live and dead cells in culture per the manufacturer's instructions. After staining, the hiPSC-CMs were washed with maintenance medium three times and imaging was performed on an EVOSTM FL Auto 2 Fluorescent microscope (Thermo Fisher Scientific, Waltham, MA, United States).

#### Lactate Dehydrogenase (LDH) Assay

To measure the cytotoxicity of the aligned coaxial patch on hiPSC-CMs, LDH assay was performed using the in vitro toxicology assay kit, lactic dehydrogenase based (Cat# TOX7-1KT, MilliporeSigma, Milwaukee, WI, United States). The culture medium was collected from hiPSC-CMs cultured in tissue culture plates or on aligned coaxial patches after 48 h of culture and LDH release assay was performed according to the manufacturer's protocols. Background and primary absorbance of the plate were measured on a spectrophotometer (2030 Multilabel Reader, VictorTM

×3, PerkinElmer, Inc., Waltham, MA, United States) at 690 and 490 nm, respectively. The assay was performed in quadruplicate (n = 4) and data obtained was analyzed on WorkOut 2.5 (build 0428, PerkinElmer, Inc., Waltham, MA, United States), by subtracting background absorbance from primary absorbance.

#### Immunostaining for Cardiac Markers

As described previously (Khan et al., 2015), immunofluorescence staining was performed to analyze the expression of cardiac markers in aligned coaxial cardiac patches. Briefly, aligned coaxial cardiac patches, after 2 weeks in culture, were washed twice with PBS and fixed with 4% PFA for 10 min at room temperature. The patches were then washed twice with PBS and incubated in blocking buffer (PBS, 5% normal goat serum, and 0.3% Triton X) for 1 h to block non-specific antibody binding. Following this, the patches were incubated with anti-α-sarcomeric actinin (A7811, MilliporeSigma, Milwaukee, WI, United States), anti-GATA4 (PA1-102, Thermo Fisher Scientific, Waltham, MA, United States), anti-Troponin-T (HPA017888, MilliporeSigma, Milwaukee, WI, United States), and anti-Connexin-43 (MAB 3067, MilliporeSigma, Milwaukee, WI, United States) antibodies, overnight at 4◦C and after which the patches were washed thrice in PBS, 5 min. each. Cells were then incubated with the corresponding secondary antibodies conjugated either with Texas Red or FITC against rabbit (1:5000, 8889S, Cell Signaling Technology, Danvers, MA, United States) or mouse (1:5000, 4408S, Cell Signaling Technology, Danvers, MA, United States) for 1 h at room temperature in the dark and washed thrice in PBS. Nuclei were counterstained with NucBlue (R37605, Invitrogen, Carlsbad, CA, United States). Finally, the patches were washed thrice with PBS, transferred onto slides and mounted with ProLongTM Glass Antifade mounting medium (Cat# P36984, Invitrogen, Carlsbad, CA, United States). Imaging was performed on a confocal microscope (Olympus FV 1000 spectral, Olympus Corporation, Center Valley, PA, United States) and images were processed using the Olympus FLUOVIEW Ver. 4.2a Viewer.

#### Assessment of Calcium Cycling in hiPSC-CMs

Calcium transients were imaged in hiPSC-CMs cultured on fibronectin-coated glass coverslips and aligned coaxial cardiac patches using the calcium-binding dye Fluo-3, AM (F1242, Invitrogen, Carlsbad, CA, United States). For staining, the hiPSC-CMs cultured on coverslips or patches were washed three times with Dulbecco's Modified Eagle's Medium (DMEM) and incubated in 5 µM Fluo 3-AM in DMEM for 1 h in dark at 37◦C, 5% CO<sup>2</sup> in a humidified atmosphere. The cells were then washed three times in DMEM and incubated for an additional 30 min in serum-containing medium at 37◦C, 5% CO<sup>2</sup> in a humidified atmosphere. The cell culture plate was then placed on the microscope (Leica Microsystems, Wetzlar, Germany) stage to record a movie using Leica Application Suite X 3.0.6.17580 software.

### Particle Image Velocimetry (PIV) Cross-Correlation ("PIV") Analysis of Images

Cell contractility kinetics were assessed using the optical flow/PIV method described previously (Zamir et al., 2005; Aleksandrova et al., 2012). Briefly, a motion pattern (velocity field) captured on a pair of images was calculated by dividing the first image into overlapping tiles, each 64 pixels wide. The second image was then scanned pixel-by-pixel, by shifting an equally sized (64 pixels × 64 pixels) window. The most similar (by Euclidean distance) tile on the second image was then assumed to be the location where the pattern in the first image moved. The resulting displacement vectors characterizing each image tile were then interpolated and denoised by a thin-plate spline fit, yielding our coarse displacement field. The coarse estimate was used to construct a second, higher resolution displacement field. In this second step, the cross-correlation search for pattern similarity was repeated with tiles that were only 32 pixels wide but in a much smaller search area allowing only for fourpixel displacements around the location predicted by the coarse displacement field.

#### Beat Patterns

To determine the beat patterns, a suitable reference image taken at time t ∗ , which is a frame between two contraction cycles, where movement is minimal: V(t) ≥ V(t ∗ ), in a motionfree state, was first identified. This reference image was then compared to all other images of the recordings with crosscorrelation ("PIV") analysis. The result is a series of displacement vector fields d(t,x), which estimate for each time point t and location x the total movement (magnitude and directionality) relative to a resting state. For each time point t the beat pattern D(t) is the spatial average of the magnitude of d(t,x) as D(t) ≤ |d(t,x)|>x, where <. . .><sup>x</sup> denotes spatial averaging over all possible locations x.

#### Frequency Analysis

Fourier spectra were calculated from D(t) beat patterns using the discrete Fourier transform algorithm as described previously (Rajasingh et al., 2018). Power densities were calculated as the magnitudes of the squared Fourier spectra, and indicate periodicity within the signal in the form of peaks at the corresponding frequencies. When the analyzed signal is not a pure sine wave, harmonics are expected to appear at integer multiples of the fundamental frequency f (2f, 3f, etc.). The magnitude of a peak in the power spectrum indicates the amplitude of the signal oscillating with the corresponding frequency.

#### Convergence Analysis

Cell layers often move passively without actively contracting. The optical flow-based method does not distinguish between active and passive (elastic response of the adjacent cell layer) contractility. To identify contractile centers, we estimated the convergence maps of the displacement field as its negative divergence from optical flow data d(t,x) as previously described (Czirok et al., 2017).

### Functional Characterization Using a Multi-Electrode Array (MEA) System

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The field potentials of hiPSC-CMs cultured in 2D or on aligned coaxial patches (3D) were measured using an MEA system. For this, hiPSC-CMs were either cultured directly on 24-well MEA plates (M384-tMEA-24W, Axion Biosystems, Atlanta, GA, United States) having 16 PEDOT microelectrodes per well (as described previously; Kumar et al., 2019) or on 8 mm aligned coaxial patches; both were cultured for 2 weeks. The aligned coaxial cardiac patches were transferred into a sterile six-well MEA plate (M384-tMEA-6W, Axion Biosystems, Atlanta, GA, United States) having 64 PEDOT microelectrodes per well. The plate was equilibrated in the MEA system (Maestro Edge, Axion Biosystems, Atlanta, GA, United States) for 30 min in 5% CO<sup>2</sup> with a humidified atmosphere at 37◦C. For the patches, the excess culture medium was removed to facilitate better contact with the electrodes. The baseline was recorded for each well for 5 min. After which the hiPSC-CMs in 2D, as well as the cardiac patches, were treated with different cardiac drugs: (a) Isoproterenol (ISO, 10 and 100 nM), (b) Verapamil (VER, 0.1 and 0.3 µM), and (c) E4031 hydrochloride (E4031, 50 and 100 nM). The stock solutions of all drugs were prepared in dimethylsulphoxide (DMSO). The plates were equilibrated for 5 min after the addition of drugs and the field potentials were recorded for 5 min for each drug treatment. AxIS Navigator versionTM 1.4.1.9 was used for data recording while CiPATM analysis tool version 2.1.10 (Axion Biosystems, Atlanta, GA, United States) was used for data analysis. The beat period, field potential duration (FPD), spike amplitude, and the incidences of arrhythmias were calculated. Further, the Fredericia's correction was applied to the FPD, to interpret the effect of drugs on the QT interval. Data are expressed as mean ± SD (n = 3).

#### Statistical Analysis

Data acquired is expressed as mean ± SD. Statistical significance was determined using one-way ANOVA. All pairwise multiple comparison procedures were performed by the Holm–Sidak method. p-value < 0.05 was considered statistically significant.

## RESULTS

#### Fabrication of Aligned Nanofibrous Coaxial, PCL, and Gelatin Patches

Aligned nanofibrous PCL-gelatin coaxial patches were successfully fabricated via electrospinning (**Figure 1A**). Coaxial nanofibers were highly aligned with a mean diameter of 578 ± 184 nm (**Figure 1B**). The overall mean thickness of the aligned coaxial nanofibrous patches was 115 ± 11 µm. SEM imaging of a single nanofiber showed a core-shell structure indicating successful coaxial morphology (**Figure 1C**). Further, confocal microscope images of the coaxial patches after mixing of rhodamine and fluorescein with PCL and gelatin, respectively, validated the coaxial morphology. These images clearly showed the presence of PCL (red) in the core and gelatin (green) in the shell (**Figure 1D**). Following hydration, confocal image analysis showed that the nanofibers had a diameter of 2.21 ± 0.50 µm. Additionally, a comparison of the SEM images of pure PCL and pure gelatin nanofibrous patches versus coaxial patches clearly showed that the coaxial nanofibers were more uniform and cylindrical in morphology (**Figure 2A**). AFTIR analysis of the PCL and gelatin patches showed peaks corresponding to C = O ester of PCL at 1722 cm−<sup>1</sup> (green) and peaks corresponding to the amide groups of gelatin at 1544 and 1657 cm−<sup>1</sup> (brown; **Figure 2B**). Coaxial patches exhibited all three peaks (**Figure 2B**), further reiterating the presence of both polymers in the nanofibers.

#### Mechanical Testing of Aligned Coaxial Patches

The mechanical strength of PCL, gelatin, and the PCLgelatin coaxial patches was determined by tensile testing. The stress-strain curves obtained showed that the aligned PCL-only patch had the highest values for both tensile strength (1.490 ± 0.290 MPa) as well as Young's modulus (0.093 ± 0.017 MPa; **Figures 2C,D**). This was followed by the aligned coaxial patch, which had a tensile strength and Young's modulus of 0.780 ± 0.098 and 0.039 ± 0.007 MPa, respectively (**Figures 2C,D**). The aligned gelatin patches had the least mechanical strength with tensile strength and Young's modulus of 0.308 ± 0.032 and 0.009 ± 0.001 MPa (**Figures 2C,D**), respectively. However, in terms of percent elongation, gelatin showed the highest elongation, while no significant difference was observed between the PCL and coaxial patches (**Figure 2E**). Therefore, the aligned coaxial patches had strength and stiffness intermediate to PCL and gelatin, a percent elongation at failure similar to PCL alone, and their elongation at failure matched the PCL patches.

### Degradation of Aligned Coaxial Nanofiber Scaffolds

The degradation of the coaxial scaffolds at 1 and 2 weeks was determined by measuring their weight loss and studying their morphology. SEM images of the scaffolds after 7 and 14 days (**Figures 3A,B**) in culture did not show any striking differences when compared to the crosslinked patches at the beginning of the experiment (**Figure 1B**). Furthermore, no significant difference was observed in the weight of the scaffold after 7 and 14 days in the culture (**Figure 3C**). Taken together, this data suggests that the scaffolds did not undergo rapid degradation under the culture conditions used in our study.

#### Morphology and Viability of hiPSC-CMs on Aligned Coaxial Nanofibrous Patches

The hiPSC-CMs cultured on aligned coaxial patches and their morphology was assessed at 2 weeks by SEM. The SEM images showed a uniform distribution and attachment of the hiPSC-CMs on the coaxial patches (**Figure 3D**). The hiPSC-CMs also showed a parallel alignment with the nanofibers (**Figure 3E**). The viability of the hiPSC-CMs cultured on the coaxial patches was assessed by staining the cardiomyocytes with Calcein-AM (for live cells), BOBO-3 iodide (for dead

cells), and LDH assay (**Figure 3F**). Fluorescence images showed that a majority of cardiomyocytes stained positive for calcein-AM, indicating that they are viable and metabolically active (**Figures 3G,I**). Additionally, some cardiomyocytes stained positive for BOBO-3 iodide (**Figures 3H,I**), indicating that the patch was biocompatible. Furthermore, no significant differences

coaxial patch. (A,B) SEM images of the aligned coaxial patches after 1 and 2 weeks in culture. (C) Weight of the coaxial scaffolds after 1 and 2 weeks in culture. NS, not significant. (D,E) SEM images of hiPSC-CMs cultured on aligned coaxial (CoA) PCL/Gel nanofibrous patches. (F) Fold change in LDH released by hiPSC-CMs cultured in tissue culture plates (2D) and CoA patches (3D) as compared control. Data expressed as mean ± SD, n = 3. NS, not significant. (G–I) Live-dead cell staining of hiPSC-CMs cultured on coaxial aligned scaffolds showing live cells stained with Calcein-AM (green) and dead cells stained with BOBO-3 iodide (red) after 2 weeks in culture.

were observed in LDH released from hiPSC-CMs cultured on the aligned coaxial nanofibrous patches (3D) versus on a flatplate (2D) (**Figure 3F**). Taken together, these results showed that aligned coaxial PCL/Gel patches are biocompatible and support the 3D culture of hiPSC-CMs.

#### Confocal Imaging to Assess the Expression of Cardiac Markers in hiPSC-CMs Cultured on an Aligned Coaxial Patch

The expression of cardiac makers in hiPSC-CMs was assessed by confocal imaging to understand the intracellular sarcomere arrangement in the cells following culture on the aligned coaxial patches. At 2 weeks, the hiPSC-CMs cultured on aligned coaxial patches stained positive for the cardiac lineage markers; GATA 4, Nkx2.5, α-sarcomeric actinin (α-SA), cardiac Troponin T (TnT), and connexin-43 (Cx-43) (**Figure 4**). Expression of the cardiac transcription factors GATA4 (**Figures 4A–F**) and Nkx2.5 (**Figures 4J,K**) was detected in the nucleus of the hiPSC-CMs. Additionally, these hiPSC-CMs showed parallelly arranged sarcomeres, as observed by α-SA (**Figures 4A–F,I**) and TnT staining (**Figures 4G,H,J,K**). Also, the hiPSC-CMs showed the expression of Cx-43, indicating good intercellular contact between neighboring cardiomyocytes (**Figures 4G,H**). The distribution of the hiPSC-CMs was also assessed across the depth of the patch. The cross-sections and Z-stack confocal images of aligned coaxial cardiac patches showed migration of the hiPSC-CMs up to 40–50 microns below the surface of the scaffold (**Figures 4C,D,F,J**), evidenced by the expression of α-SA, TnT, GATA4, and Nkx2.5. Additionally, increased magnification of a single hiPSC-CM imaged on the patch showed

a multi-nucleated rod-shaped morphology with well-organized sarcomeres (**Figure 4I**). These observations indicated that the aligned coaxial nanofibrous patch can be used as a model for understanding the structural maturation of hiPSC-CMs on 3D scaffolds.

#### Assessment of Calcium Transients in hiPSC-CMs Seeded on an Aligned Coaxial Cardiac Patch

The calcium transients in hiPSC-CMs cultured on tissue culture plates (2D) and aligned coaxial patches (3D) were assessed after 2 weeks in culture. The hiPSC-CMs cultured in 2D and 3D showed synchronous calcium transients (**Figure 5A**). This data showed that the hiPSC-CMs cultured on aligned coaxial patches formed a functional syncytium as indicated by synchronous calcium waves.

#### Assessment of Cell Contractility in hiPSC-CMs Cultured on Aligned Coaxial Patches

Particle image velocimetry analysis was performed to evaluate the contractility of hiPSC-CMs cultured on aligned coaxial patches. For this, the response of the patches, at 2 weeks of culture, to 100 nM ISO, a non-specific β-adrenoreceptor agonist, was analyzed by an optical measure of contractility (**Figures 5B–G**). For both treated and control cultures, six videomicroscopic recordings were obtained from two parallel cultures. Beat patterns (**Figures 5B,E**) indicated an ISO-induced increase in spontaneous beat frequency and amplitude. Also, Fourier power spectra of the beat patterns indicated an increase in frequency by shifting the dominant peak toward higher frequencies (right) after ISO treatment (**Figures 5C,F**). A significant increase (p < 0.0004) in the average frequency of spontaneous beating from 0.136 ± 0.0069 to 1.497 ± 0.0394 SEM Hz was observed following ISO treatment (**Figures 5C,F**). Additionally, ISO treatment also promoted better-defined beating activity that is indicated by the tall narrow peaks. The contractility maps showed a stronger, spatially extended beating activity in ISO-treated patches (**Figures 5D,G**). These results demonstrate the ability of the aligned coaxial patches to respond to cardiac drugs in a reproducible manner.

#### Electrophysiological Assessment of hiPSC-CMs Cultured on Aligned Coaxial Patches

The hiPSC-CMs cultured in 2D and 3D (aligned coaxial patch) was assessed on the MEA system for field potentials, which result from spontaneous cardiac action potentials propagating across cells on neighboring electrodes. The hiPSC-CMs were treated with different concentrations of ISO, VER, and E4031, and the changes in their field potential was measured. An increase in the beating frequency (beats per minute) was observed in both the 2D as well as 3D cultures following treatment with ISO (**Figures 6A,B**) and VER (**Figures 6A,C**), while a decrease in beating frequency was observed following E4031 treatment (**Figures 7A,B**). These changes were as expected for these drugs' mechanisms of action.

A significant, dose-dependent decrease in the beat period was observed in hiPSC-CMs in both 2D and 3D cultures after treatment with ISO. After treatment with 10 nM ISO, the beat period in the 2D and 3D cultures decreased from 1.04 ± 0.04 and 1.22 ± 0.16 s to 0.67 ± 0.04 and 0.8 ± 0.15 s, respectively (**Figures 6D,E**). Similarly, after treatment with 100 nM ISO, the beat period of the hiPSC-CMs in 2D and 3D cultures was 0.66 ± 0.03 and 0.66 ± 0.1 s, respectively (**Figures 6D,E**), indicating that the dose-dependent response was improved in case of 3D cultures. Furthermore, as expected, ISO treatment resulted in a dose-dependent shortening of the QT interval, as evidenced by the decrease in Friedrica's corrected field potential duration (FPDc) in both the 2D and 3D cultures (**Figures 6D–F**). Also, between the 2D and 3D cultures, a significant difference in the FPDc was observed after treatment with 10 and 100 nM ISO, indicating that the 3D cultures responded more robustly to the treatment (**Figure 6F**). A similar decrease in the beat period and FPDc was observed in the hiPSC-CMs cultured in 2D and 3D cultures after treatment with the L-type Ca channel blocker VER (**Figures 6C,G–I**). A significant fold decrease in the beat period was observed in both the 2D as well as 3D cultures after treatment with 0.1 and 0.3 µM VER, as compared to their respective baseline controls (**Figure 6I**). However, a dose-dependent decrease in the FPDc was observed only in the case of the 3D culture indicating that the 3D culture system was a more responsive model for drug testing (**Figure 6I**).

We also assessed the effect of the hERG-type K<sup>+</sup> channel blocker E4031, which is an anti-arrhythmic drug. However, this drug is pro-arrhythmic in vitro especially at higher doses (Goto et al., 2018). As expected, following treatment with the E4031, a significant increase in the beat period and FPDc was observed in the 2D cultures following treatment with 50 and 100 nM of E4031, while a significant increase was observed in 3D cultures only after treatment with 100 nM E4031 (**Figures 7C–E**). Furthermore, we also observed higher variability in the FPDc in 3D cultures as compared to the 2D cultures (**Figure 7C**). This was reiterated by a Comprehensive in vitro Pro-arrhythmia Assay (CiPA), which showed that 16.67% (4/24) and 33% (8/24) of the samples in 2D cultures showed beat period irregularity after treatment with 50 and 100 nM E4031, respectively, while patches showed beat period irregularity of 60% following treatment with E4031(6/10). This data indicated the increased occurrence of arrhythmias in these cardiac patches (**Figure 7G**) making them a more powerful system for arrhythmia detection. Additionally, a significant decrease in the spike amplitude was observed in both the 2D and 3D cultures, following treatment with 100 nM E4031 (**Figure 7F**). Taken together, these results showed that while hiPSC-CMs cultured in 2D and 3D cultures responded to drug treatments, the 3D cultured hiPSC-CMs had more efficacious responses and would be a better model for in vitro drug testing.

FIGURE 4 | Confocal imaging to assess the expression of cardiac markers in human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured on the aligned coaxial nanofiber patch. (A–F) Confocal images showing the expression of α-SA and GATA4 in hiPSC-CMs cultured on aligned coaxial patches. (G,H) Confocal images showing the expression of TnT and Cx-43 in hiPSC-CMs cultured on aligned coaxial patches. (J,K) Confocal images showing the expression of TnT and Nkx2.5 in hiPSC-CMs cultured on aligned coaxial patches. (C,F) Z-stack images showing the distribution of the hiPSC-CMs through the depth of the coaxial patches. (D,J) Cross-section images of the coaxial patches showing the distribution of hiPSC-CMs in the patch. (I) Confocal image of a single hiPSC-CM cultured on an aligned coaxial patch.

FIGURE 5 | Calcium cycling and optical contractility analysis of the aligned coaxial cardiac patch. (A) Representative images of calcium transients in 2D and 3D cultures. (B–D) Representative data of untreated control aligned coaxial (CoA) cardiac patches (B–D), and (E–G) aligned CoA cardiac patches treated with 100 nM Isoproterenol (ISO). (B,E) Spontaneous beat patterns expressed as displacement relative to a resting reference state vs. time. (C,F) Fourier power spectra of beat patterns show the dominant beat frequency as a peak and spatially resolved contractility analysis. (D,G) Representation of particle image velocimetry (PIV) with warmer colors corresponding to higher divergence, a numerical estimate of contractile strength.

## DISCUSSION

The main focus of the present study was to develop a potent and efficient 3D culture system for cardiac drug testing. Our findings demonstrate that aligned PCL-Gel coaxial nanofibrous scaffolds successfully culture hiPSC-CMs in a 3D microenvironment. The comparative assessment of hiPSC-CMs cultured in 2D and 3D culture systems carried out in the present study showed improved functional characteristics and increased responsiveness to cardiac drugs in the latter.

Coaxial PCL-gelatin scaffolds demonstrated excellent biological properties including cell attachment, organization, and expression of cardiac lineage markers along with high mechanical strength and Young's modulus allowing for the scaffold to be handled easily. Our observations are consistent with previous reports, which have shown coaxial electrospinning is an efficient strategy for surface modification of nanofibrous scaffolds made from synthetic polymers like PCL, PLGA, and polyvinyl alcohol (PVA) (Kim and Cho, 2016). While coaxial electrospinning has been more commonly used for controlled drug/biomolecule release (Zhang et al., 2006; Ji et al., 2010; Sun et al., 2019), it has also been used to coat

FIGURE 7 | Functional assessment of human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in 2D and 3D cultures after treatment with E4031. (A) Heat maps showing the beat rate in representative wells of 2D and 3D cultures before treatment (baseline) and after treatment with E4031. (B) Representative images showing changes in beat detection before (baseline) and after treatment with E4031. (C) Quantitative assessment of fold change in corrected field potential duration (FPDc) following E4031 treatment in 2D and 3D cultures. Representative images showing changes in beat period in (D) 2D and (E) 3D cultures after treatment with E4031. Quantitative assessment of change in (F) spike amplitude and (G) arrhythmicity after E4031 treatment in 2D and 3D cultures. Arrows in (D,E) show field potential duration (FPD). Scale in A represents the beats per minute. Data shown in (C,F,G) is mean ± SD, n = 8, from three independent cultures. \*p < 0.05, \*\*\*p < 0.001 vs. corresponding baseline; #p < 0.01 vs. corresponding 2D cultures.

synthetic polymer-based nanofibers with a natural polymer (like gelatin, alginate, and collagen) to make them more biomimetic (Zhang et al., 2007; Hu et al., 2020). The use of coaxial nanofibers with a PCL inner core and gelatin outer shell has previously been used for wound healing (Blackstone et al., 2014) and vascular (Coimbra et al., 2017) and bone (Alissa Alam et al., 2020) tissue engineering applications. This coaxial structure has been shown to improve the biocompatibility of

the scaffolds as well as provide structural support to the cells for in vivo applications.

Another striking observation made in our study was the alignment of hiPSC-CMs with the nanofibers in the scaffold. As a result, the hiPSC-CMs cultured on the aligned scaffolds showed an elongated rod-shaped morphology. Additionally, the sarcomeres in these hiPSC-CMs showed parallel organization inside the cell, with some cells being binucleated. These observations indicate the maturation of the cells cultured on the coaxial scaffolds. Our observations are consistent with previous reports demonstrating a similar alignment of hiPCMs when cultured on aligned scaffolds (Khan et al., 2015; Han et al., 2016; Wanjare et al., 2017). These studies have clearly shown enhanced maturation of hiPSC-CMs cultured on aligned 3D scaffolds based on increased expression of cardiac genes, re-organization of sarcomeres, and an adult cardiomyocytelike rod-shaped morphology of the cells. Additionally, it has been observed that scaffolds with fibrous, aligned structures mimic the structure of heart tissue, thereby providing a 3D microenvironment for anisotropic arrangement of hiPSC-CMs similar to cardiomyocytes in the heart (Kitsara et al., 2017). Of relevance is another observation made in a previous study by our group (Kumar et al., 2019), wherein hiPSC-CMs cultured in 2D showed similar morphological maturation, but only after 4 weeks in culture. This further reiterates that 3D cultures would be more relevant for structural and functional maturation of hiPSC-CMs, when compared to 2D cultures.

Another important aspect of an in vitro model system for cardiac tissue, is the development of a functional syncytium of beating cardiomyocytes. It is a well-established fact that the electrical interconnectivity of cardiomyocytes is an essential pre-requisite for developing in vitro cardiac tissues for drug testing applications. This is especially important to determine the effect of a drug molecule on the heart function (e.g., heart rate, arrhythmia-inducing potential; Stoppel et al., 2016; Ugolini et al., 2017). Hence, cell-cell interaction between cardiomyocytes is extremely critical. Previous studies have shown the formation of a functional syncytium in 2D cultures mainly due to hypertrophic growth of hiPSC-CMs (Kumar et al., 2019). On the contrary, functional coupling between cardiomyocytes cultured on 3D constructs has been reported only after mechanical or electrical stimulation (Valls-Margarit et al., 2019). Interestingly, in our study, we observed the formation of a functional syncytium of hiPSC-CMs cultured on the coaxial patches, evidenced by the expression of the gap junction protein, Cx-43, and synchronous calcium transients across the patch. These observations are similar to the behavior of hiPSC-CMs cultured in 2D (Kumar et al., 2019), further indicating that the aligned coaxial scaffolds can be used as an in vitro culture system.

It has been shown that 3D culture systems making use of hiPSC-CMs are better suited for cardiac drug testing and toxicity studies because they mimic the in vivo response (Kussauer et al., 2019). However, the use of 3D cultures has been restrained due to lack of (a) extensive literature, (b) reproducible culture protocols, and (c) precise analysis tools (Zuppinger, 2019). In recent years, several different techniques have been developed and optimized for analyzing the effect of different cardiac drugs on hiPSC-CMs in 3D cultures, such as calcium imaging using fluorescent dyes, PIV, and measurement of field potentials by MEA (Zuppinger, 2019). In our study, these analysis tools were used to monitor the effectiveness of a few commonly used cardiac drugs on hiPSC-CMs cultured in 2D vis-à-vis 3D cultures. As expected, an increase in the beat rate along with a decrease in beat period and FPDc was observed in both the 2D as well as the 3D cultures after ISO treatment. Our observations are consistent with previous studies, where a dose-dependent increase in contractility was observed in 2D cultured hiPSC-CMs following treatment with ISO (Mehta et al., 2011; Blazeski et al., 2012; Kitaguchi et al., 2017), as a result of β1 and β2 receptor stimulation. Additionally, although VER has been known to inhibit voltage-dependent calcium channels (Harris et al., 2013), an increase in contractility was observed in both 2D and 3D cultures. However, consistent with our observation. a recent study identified that this discrepancy in the effect of VER in vitro and in vivo, resulted from the low potassium concentration in cell culture medium (Zeng et al., 2019). On the other hand, E4031, an hERG channel blocker, resulted in prolonged QT interval (FPDc) in addition to decreased spike amplitude in a dose-dependent manner for 3D cultured cells. The incidence of arrhythmicity increased following the addition of E4031 in both the 2D as well as 3D cultures. However, 3D cultures showed a four-times greater response to drugs than 2D cultures. Our observations are in agreement with earlier reports (Harris et al., 2013) using hiPSC-CMs. Overall, these results strongly indicated an elevated sensitivity of hiPSC-CMs in 3D cultures to various drugs used in this study. Similar observations were reported in the case of 3D culture of primary hepatocytes (Bell et al., 2018; Zhou et al., 2019) and cancer cells (Chaicharoenaudomrung et al., 2019). Likewise, a recent study demonstrated that 3D cardiac spheroids mimics an in vivo environment for drug testing and cardiotoxicity studies (Polonchuk et al., 2017).

#### CONCLUSION

Overall, our study demonstrated the fabrication and testing of an aligned coaxial PCL-gelatin nanofiber patch with mechanical and biomimetic properties desired for cardiac applications. hiPSC-CMs cultured on these patches showed a rod-shaped morphology and were aligned in parallel with the nanofibers. These cells on the cardiac patch showed synchronous contractions and exhibited quick response to cardiac drugs. Finally, these cardiac patches can be scaled and used as an in vitro drug screening platform for cardiotoxicity studies, as well as to developed into future strategies of cardiac patch transplantation for ischemic heart disease.

#### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

#### AUTHOR CONTRIBUTIONS

fbioe-08-567842 September 14, 2020 Time: 15:54 # 15

MK, NK, DS, and AP conceived and designed the experiments. MK, NK, DS, AP, JD, DI, AC, HP, and MM performed the experiments and data analysis. NK, DS, AP, JD, and MM analyzed the data. MK, HP, MA, and AC contributed reagents, materials, and analysis tools. DS, NK, AP, JD, HP, DS, and MK wrote and reviewed the manuscript. The input from all the authors was incorporated in finalizing the manuscript draft.

#### REFERENCES


#### FUNDING

Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R01HL136232 (MK) and GM102801 (AC), and OSU start-up funds to MK. We would like to thank the Ohio State Campus Microscopy Imaging Facility for helping with SEM and confocal microscopy studies. These facilities were supported in part by grant P30 CA016058, National Cancer Institute, Bethesda, MD, United States.




**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Kumar, Sridharan, Palaniappan, Dougherty, Czirok, Isai, Mergaye, Angelos, Powell and Khan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.