Overcoming Biological Barriers With Block Copolymers-Based Self-Assembled Nanocarriers. Recent Advances in Delivery of Anticancer Therapeutics

Cancer is one of the most common life-threatening illness and it is the world’s second largest cause of death. Chemotherapeutic anticancer drugs have many disadvantages, which led to the need to develop novel strategies to overcome these shortcomings. Moreover, tumors are heterogenous in nature and there are various biological barriers that assist in treatment reisistance. In this sense, nanotechnology has provided new strategies for delivery of anticancer therapeutics. Recently, delivery platforms for overcoming biological barriers raised by tumor cells and tumor-bearing hosts have been reported. Among them, amphiphilic block copolymers (ABC)-based self-assembled nanocarriers have attracted researchers worldwide owing to their unique properties. In this work, we addressed different biological barriers for effective cancer treatment along with several strategies to overcome them by using ABC‐based self-assembled nanostructures, with special emphasis in those that have the ability to act as responsive nanocarriers to internal or external environmental clues to trigger release of the payload. These nanocarriers have shown promising properties to revolutionize cancer treatment and diagnosis, but there are still challenges for their successful translation to clinical applications.


INTRODUCTION
Cancer is the world's second largest cause of death, endangering tens of millions of people. Albeit the substantial development of significant drugs has advanced; their delivery is far from satisfactory (Zhou et al., 2020). Moreover, anticancer drugs have many disadvantages (undesirable biodistribution, low specificity, limited targeting, inefficient cellular uptake, produce side/toxic effects) (Yin et al., 2016;Avramović et al., 2020). These limitations have prompted the development of novel strategies to overcome these shortcomings. Advanced nanotechnology has provided new approaches for efficient delivery of anticancer therapeutics and supposed to be a promising tool against this illness (Chan, 2017). Nevertheless, considering the relatively few nanomedicine choices available in clinical trials, this promise has apparently fizzled (Huo et al., 2019). Some failures originate from safety issues respect to long term exposure to engineered materials despite the advancements made to understand their nanotoxicity (Yan et al., 2019). Also, tumor multifaceted nature clarifies why most nanomedicines have not prevailing in clinical trials (Kievit and Zhang, 2011;Ernsting et al., 2013). It has been proposed that tumors are heterogenous in nature, possessing various biological barriers that assist in treatment resistance. Recent observations into the tumor niche indicate that many obstacles still exist and insufficient attention is given to their biological consequences when novel nanomedicines are designed (Huo et al., 2019). As a result, in the last decade new delivery platforms to overcome biological barriers caused by tumor cells and tumor-bearing hosts have been evaluated.
Many delivery systems on nanoscale with significant achievements have been reported to date. Among them, amphiphilic block copolymers (ABC)-based self-assembled nanocarriers (SAN) for delivery of anticancer therapeutics have attracted researchers worldwide owing to their unique chemical and physical properties, improved biocompatibility, tunable compositions, extended blood circulation, and facile functionalization (Yin et al., 2016;García and Quiroz, 2018;García, 2019b). With the advancements in polymer chemistry, different ABC with preferred number and monomer type, each having different hydrophobic and hydrophilic properties have been synthetized (Yorulmaz Avsar et al., 2019). By tailoring monomer combinations, SAN with upgraded properties to overcome biological barriers can be obtained. ABC can self-assemble into distinctive nanostructural arrangements among which micelles and more recently polymer vesicles (also termed as polymersomes) are the most reported (Yin et al., 2016;García and Quiroz, 2018;. Inspired by their promising properties, nanomedicines could be intended to successfully mimic, improve or interact with these cross-talks for improving cancer therapy.
In this review, we addressed different biological barriers for numerous anticancer therapeutic moieties for effective cancer treatment along with several strategies to overcome them by using ABC-based SAN, with special emphasis in those that have the ability to act as responsive nanocarriers to internal or external environmental clues, producing on-demand triggered release of the payload.

BIOLOGICAL BARRIERS AND STRATEGIES TO OVERCOME BARRIERS FOR EFFECTIVE DELIVERY OF ANTICANCER THERAPEUTIC
Cancer nanomedicine efficacy is measured primarily by how much drug can reach to the tumor-site. The properties of the biological barriers are diverse and represent both a challenge as well as an opportunity to develop tailor-made drug delivery system to effectively reach the target site. Distinct biological barriers and their peculiarities ( Figure 1A), and strategies to overcome them are discussed in the following section.

Opsonization/Sequestration by the Mononuclear Phagocytic System
After intravenous administration, nanocarriers are exposed to a several sequential hurdles that must be overcome in order to efficiently reach therapeutic levels of drugs at disease sites, avoiding nonspecific uptake in healthy organs (Blanco et al., 2015;Chevalier et al., 2017). As per the literature, when nanocarriers are intravenously administered, they can interact with thousands of proteins. Biological media consists of numerous active biomolecules such as lipids, nucleic acids, proteins, and blood plasma contains roughly 3,700 identified proteins (Lazarovits et al., 2015;Chen et al., 2017). Protein corona effect i.e. due to the presence of charge on nanocarriers can result into interaction with oppositely charged serum protein. Thus, nanocarriers or the targeting moiety decorated over nanocarriers get buried in protein corona resulting into prevention of nanocarriers's interaction with the targeted receptor in cancer cells (Tonigold et al., 2018). Opsonins are key proteins in the blood serum that promotes elimination of vulnerable nanocarriers by marking them off for an immune response. The major part of opsonized nanocarriers are cleared within seconds by a receptor-mediated mechanism because of the phagocytic cells in the liver, lymph nodes, and spleen of the mononuclear phagocyte system, contributing to nonspecific distribution of nanocarriers in healthy organs (Blanco et al., 2015;Chevalier et al., 2017).
One most established strategy to mitigate the effect of protein corona involves the nanocarrier functionalization with amphiphilic or hydrophilic polymers, such as poly (ethylene glycol) (PEG) (Bros et al., 2018). PEG forms strong adhered hydration layer, which sterically avoid the interaction/adsorption of serum protein (Huo et al., 2019). PEG-decorated nanocarrriers reduce their recognition by the components of the mononuclear phagocytic system, increasing their blood circulation time (Xiong et al., 2009;Naeye et al., 2010). Zwitterionic ligands (i.e., sulfobetaine and carboxybetaine) have also been explored as antifouling agents (Kane et al., 2003;Tsai et al., 2004;Shao and Jiang, 2014;Li et al., 2018). These strategies are different since stacking of PEG chains is driven by Van der Waals forces and hydrophobic interactions, resulting into hard to form an ideal self-assembled monolayer; whereas zwitterionic ligands exhibit strong electrostatic interactions within their molecules, resulting into dense packing and minimizing protein adsorption on their surface. Additionally, charges on zwitterionic polymers can prompt strong hydration which retards protein adsorption (Huo et al., 2019).

Vascular Flow
Blood vessels at tumor site develop gaps between endothelial cells during angiogenesis process through which nanocarriers can easily pass for reaching tumors by EPR effect. However, this effect is now in dispute in clinical trials since nanocarriers have shown moderate therapeutic efficacy. It could have happened due to heterogeneity of EPR effect in humans. For instance, mouse xenograft models develop tumor very fast and endothelial cells in blood vessels tend to present many fenestrations. Aversely, tumors in humans develop very slowly and not all tumor vessels develop inter-endothelial gaps (Li J.-X. et al., 2020). Recently, Matsumoto et al. demonstrated that tumor blood vessels form transient opening and closing as they dynamically generate vascular bursts, resulting into eruption and vigorous fluid outflow into tumor interstitial space which ultimately lead to nanocarrier extravasation (Matsumoto et al., 2016). In contrast, Sindhwani et al. proposed that extravasation takes place via interconnected vesiculovacuolar organelles (trans-endothelial pathway), revealing that 97% of nanocarrier extravasation occurred through this pathway (Sindhwani et al., 2020).
Different strategies to overcome tumor vascular barrier and improve accumulation of nanocarriers at tumor site can be use. 1) Enhancing tumor vascular permeability: a) physical stimulation FIGURE 1 | (A) Main and sequential biological barriers (after intravenous administration) faced to therapeutic delivery nanocarriers for effective cancer therapy: a) they may undergo opsonization and uptake by macrophages of the mononuclear phagocytic system; b) nonspecific distribution that lead accumulation of nanocarriers in other healthy organs such as spleen, livers and lungs, and extravasation and renal clearance faced by nanocarriers smaller than 5-6 nm; c) flow in blood vessels and endothelial surfaces; d) cancer microenviroment, including interstitial fluid pressure (IFP); e) cellular internalization and endosomal escape; and f) upon entry into tumor cells, multidrug resistant (MDR) system, including drug efflux pumps that remove anticancer therapeutics from the tumor cell. (B) Schematic representation of tumor environment and summary of internal and external stimuli for triggered delivery of anticancer therapeutics from self-assembled nanocarriers.

Tumor Microenvironment
After crossing the vascular barrier, nanocarriers come across tumor microenvironment barrier composed by dense extracellular matrix (ECM), high interstitial fluid pressure (IFP) and stromal cells (Ding et al., 2019). ECM is a complex molecular structure mainly consisting of collagen network, proteoglycan, glycosaminoglycan, microfiber elastin and other polysaccharides with distinct biochemical and physical properties (Koláčná et al., 2007;Lu et al., 2012). Nanocarriers cannot cross this barrier because of the following reasons: 1) densely packed gel-like structure of tumor ECM increased lysyloxidase levels and the presence of integrin receptors with high viscosity (Barua and Mitragotri, 2014); 2) ECM has pores with 40 nm in size; thus, nanocarriers larger than 60 nm exhibit difficulties to cross this barrier (Zhang et al., 2018); 3) cancer cell density compresses the ECM and increases the IFP (solid tumors 5-40 mmHg, pancreatic tumor 75-130 mmHg whereas normal IFP 0-3 mmHg) (Miao et al., 2015). Moreover, stromal cells that consist of cancer-associated fibroblasts (CAF) are mostly found in tumor microenvironment. CAF produce dense ECM, facilitate the tumor growth, and angiogenesis as they secrete cytokines viz. interleukin (IL)-6, hepatocyte growth factor and vascular endothelial growth factor, which hinder delivery of nanocarriers to target site (Kalluri, 2016). Also, CAF may express receptors similar to tumor cells to which nanocarriers can be targeted, leading to off target distribution (Sherman et al., 2014;Miao et al., 2016).
For overcoming this barrier, physical methods, such as ultrasound, phototherapy and hyperthermia, can be used since they disrupt ECM. However, these methods can affect normal healthy nearby tissues (Eggen et al., 2014). Enzymes viz. hyaluronidase (Zhou et al., 2016) and collagenase (Kuhn et al., 2006) constitute another strategy. They can effectively degrade the hyaluronan in tumor ECM and reduce IFP, enhancing nanocarrier penetration. microenvironment, such as stimuliresponsive (García, 2019b) or size-switching (Cabral et al., 2011) delivery platforms, are other strategies for enhancing cancer therapy. Size-switching nanocarriers are those with large particle size in blood circulation, which when become in contact with tumor tissue switch their size to small to effectively extravasate, leading to deep penetration (Cabral et al., 2011). Because of the advancements in the development of stimuli-responsive nanocarriers, in Stimuli-Responsive Self-Assembled Nanocarriers Nanocarriers they are described.
Furthermore, it has to be addressed that an additional important feature of tumor microenvironment is the overall immunosuppressive microenvironment because of the presence of numerous immune cells viz. dendritic cells (DC), tumor associated macrophages (TAM, including M1immunostimulatory and M2-immunosuppressive phenotypes), natural killer (NK) cells, B and T lymphocytes, which have complex and diverse roles (Zhou et al., 2020). M2 phenotype is primarily triggered in tumors in response to hypoxic condition and facilitate metastasis, angiogenesis and decrease in immune response of T cells. Targeting TAM and switching from M2 to M1 phenotype, which reverse the immune suppression, can be an efficient anticancer strategy for preventing tumor progression (Zhang B. et al., 2017). Myeloid-derived suppressor cells and regulatory T-cells are also responsible for immune suppression (Munn and Bronte, 2016). They generate large amount of IL-10, arginine-1 and NO; and IL-10 and transforming growth factor (TGF)-β, respectively, which directly suppresses T cell function and release granzyme and perforin to destroy effector T cells, resulting into immunosuppression (Talmadge and Gabrilovich, 2013). Additionally, prostaglandin E-2, programmed death-1 molecule, TGF-β and IL-10 could suppress the antigen cross presenting ability of DC. Tumor cells also downregulate NK cells to prevent exposure of tumor necrosis factor-related apoptosis inducing ligand (Waldhauer and Steinle, 2008). Due to decrease trafficking in tumor sites (including downregulation of cluster of differentiation (CD) 62L on CD8 + T cells, the adhesion molecules VCAM-1 and ICAM-1, and IL-12) and lack of activation caused by the aforementioned immunosuppressive tumor microenvironment, the CD8 + T cells are not able to exhibit an inhibitory response against tumors (Buoncervello et al., 2019). Thus, targeting immune cells or immune related specific molecules could reshape the immune microenvironment and increase the treatment efficacy.

Cellular Membrane
This membrane not only supplies nutrients to cells but also acts as one of the barrier for cellular internalization of nanocarriers (Choudhury et al., 2019), which is mostly based on their physicochemical properties and the protein corona that coated them. Therapeutic moieties with low molecular weight and lipophilic nature cross the membrane lipid bilayer by passive diffusion while molecules with higher molecular weight require active transportation (Blanco et al., 2015). Furthermore, numerous factors of nanocarriers (particle size, shape, surface charge, and hydrophobicity) affect cellular internalization. The particle size determines the type of endocytosis through cell membrane. Shape structure other than sphere, viz. anisotropy and original orientation are critical for nanocarrier uptake through cellular membrane. Also, this parameter can have dramatic effects on targeting, circulation, internalization, immune cell association, and adhesion (Champion et al., 2007;Sharma et al., 2010;Truong et al., 2015). Mechanical properties can also affect cell adhesion/penetration. Enhanced permeability and retention (EPR) effect is favored if nanocarriers exhibit good flexibility. Their elasticity increases their chances of penetration between endothelial cancer cells and also, in targeted drug release, Frontiers in Pharmacology | www.frontiersin.org November 2020 | Volume 11 | Article 593197 nanocarriers' flexibility is a key factor in the interaction with cellular receptor-mediated endocytosis (Anselmo et al., 2015).

Multidrug Resistant System
MDR is a state of resistance toward structurally and/or functionally different therapeutic moieties and grouped into five classes: 1) increase efflux of drugs, primarily via adenosine triphosphate-driven extrusion pumps of adenosine triphosphatebinding cassette superfamily, such as breast cancer resistance proteins, MDR-associated protein 1, and P-glycoprotein, which form a unique defense system against anticancer drugs and exoand endotoxins, significantly reducing intracellular concentration of drugs or endogenous toxins; 2) decrease influx of drugs; 3) DNA repair activation; 4) inactivation of apoptosis pathways with parallel activation of antiapoptotic cellular defensive compartments; 5) metabolic modification and detoxification. MDR hinders to reach tumor-site by drug in the concentration and at the time required. Hence, only a fraction of dose remains into the cancer cells, while the remaining is removed by MDR (Choudhury et al., 2019;Huo et al., 2019).

AMPHIPHILIC BLOCK COPOLYMERS AND SELF-ASSEMBLED NANOCARRIERS
Self-assembly process refers to the spontaneous association of ordered and well-organized systems, which occurs under kinetic and thermodynamic conditions, allowing local and specific molecular interactions to form stable and perfectly structured aggregates. Electrostatic or hydrophobic interactions, hydrogen bonding, π-π aromatic-stacking, and van der Waals forces contribute to keep the molecules organized, achieving the minimal energy in the nanocarriers (Yadav et al., 2020).
SAN are easy to prepare, flexible and low-cost materials that have gained popularity for biomedical application in cancer (Berbezier and De Crescenzi, 2015;García and Quiroz, 2018). Moreover, their preparation involves generally large-scale techniques. Top-down and bottom-up approaches can be employed. While the former involves the transformation of a block of matter into a structured and organized nanostructure, the later requires the assembly of basic units/monomers for arriving at the nanocarrier structures through non-covalent interactions. Regardless of being weaks, altogether these interactions become the main force of the assembly process. Even though it is important to consider the balance between attractive driving, repulsive and directional forces (Yadav et al., 2020), simple but effective bottom-up approach is the most employed method. By tailoring experimental parameters different types of nanocarriers with distinctive morphologies and high performance for therapeutic purposes can be readily and selectively produced (Fratoddi et al., 2016;García and Quiroz, 2018). SAN represent a promising tool for targeted drug delivery, especially in personalized cancer treatments. They allow a site-directed delivery of anticancer therapeutics, increasing treatment efficacy and safety by modifying their physicochemical characteristics (i.e., size, shape, solubility, permeability) (García, 2019b;Yadav et al., 2020).

STIMULI-RESPONSIVE SELF-ASSEMBLED NANOCARRIERS
Even though passive and active targeting strategies contribute to intracellular accumulation of anticancer therapeutics, nanocarrier delivery performance can be improved by incorporating appropriate trigger responsiveness. Cutting-edge and emerging research focuses comprise the development of SAN since they can recognize some environmental clue, internal and/ or external stimuli ( Figure 1B), inducing physical/chemical changes and triggering cargo release in dose-, spatial-, and temporal-controlled manners (García, 2019b). Table 1 describes several examples of stimuli-responsive SAN and their application in cancer therapy.

External Stimuli-Responsive Nanocarriers
As stated, different strategies to overcome biological barriers for delivery of anticancer therapeutics, such as hyperthermia , ultrasound (Ho et al., 2016) and radiation (Potchen et al., 1972) are used. Interestingly, they can be also explored in the design of SAN that can be remotely controlled for triggering cargo delivery.

Temperature-Responsive Nanocarriers
Temperature is the most widespread stimulus to trigger the specific responsiveness of SAN for applications in cancer nanomedicine (Mu et al., 2019). Local temperature is slightly higher in solid tumors than in normal tissues; hence, nanocarriers may accumulate into the tumor by adjusting the thermoresponsiveness of ABC (phase transition temperature: upper or lower critical solution temperatures, UCST and LCST, respectively) (Ward and Georgiou, 2011) to be between body and tumor temperature (Onaca et al., 2009;Thambi et al., 2016;García, 2019b). Poly-N-isopropylacrylamide (PNIPAM) as block, with LCST 32°C (Che and van Hest, 2016), has been widely evaluated in ABC-based SAN (Qin et al., 2006;Onaca et al., 2009;Xu et al., 2009;Moughton and O'Reilly, 2010;Li G. et al., 2011;Li W. et al., 2011;Chen et al., 2013;Luo et al., 2014;Che and van Hest, 2016;Panja et al., 2016;Hu et al., 2017;García, 2019b). Magnetic nanocarriers that incorporated PNIPAM have also been reported, exhibiting dual responsiveness as well as usefulness for hyperthermia therapy (Bixner et al., 2016). Despite plenty PNIPAM-based nanocarriers have been described so far, their translation to clinical applications is still controversial, since preclinical studies indicated systemic toxicity (Johnson and Preman, 2018). Alternatively, poly(N-vinylcaprolactam)-containing ABC have been evaluated (Liu F. et al., 2015;Kozlovskaya et al., 2019). Moreover, the wide variety of polymers and possible conjugations for synthetizing thermo-sensitive ABC allow broad horizons in the development of temperature-responsive SAN for cancer therapy.

Ultrasound-Responsive Nanocarriers
Ultrasound is a promising stimulus due to its easy administration, low-cost, and deep tissue penetration by tuning the frequency, duty cycles and time of exposure. It has been used as adjuvant in cancer treatment, behaving as a sensitizer to improve chemotherapy and overcome drug resistance (Thambi et al., 2016;Hu et al., 2017;García, 2019b). Ultrasound induces bubbles and air-containing assemblies can trigger drug release and ultrasound-targeted cancer imaging (Zhou et al., 2006).

Electric Field-Responsive Nanocarriers
Electric fieldor voltage-responsive SAN have shown interesting properties as well. This stimulus may produce changes in charge or polarity of ABC, affecting their chemical composition or structure, and evoking disassembly with the subsequent cargo release (Yan et al., 2010;Jang et al., 2014;García, 2019b;. Further studies are still needed to better understand their usefulness for cancer therapy.

Internal Stimuli-Responsive Nanocarriers
Endogenous stimuli-responsive SAN exploit the characteristics of tumor microenvironment, which are completely different from normal tissue physiology; thus, allowing cargo release in a programmed manner to specific intracellular stimuli viz. low pH, redox state, reactive oxygen species (ROS), and enzymes (García, 2019b).

CONCLUDING REMARKS AND FUTURE TRENDS
Cancer treatment mainly relies on the use of anticancer drugs; however, their disadvantages negatively impact in therapeutic success. Besides, to reach the tumor-site they need overcome different biological barriers. However, these barriers can be overcome by developing well-designed nanocarriers. Several efforts have been made to comprehensively understand the ability of ABC to self-assemble into nanocarriers and disassemble depending on different environmental stimuli. Representative examples of ABC-based stimuli-responsive SAN were reviewed and their applications for delivery of anticancer therapeutics were highlighted. As stated, the incorporation of stimuli-triggered responsiveness allows them recognizing changes in external/internal environment, inducing on-demand release behavior in spatial-, temporal-, and dosecontrolled fashions. Even though these nanocarriers have shown promising properties to revolutionize cancer therapy and diagnosis, there are still challenges for their successful translation to clinical applications. Their biocompatibility, long-term toxicity and immunogenicity need be more studied for establish their safety profile, and advanced in vivo studies are also required to better understand nanocarrier-organism interaction. Considering scalable/reproducible manufacturing process, significant efforts in design, synthesis, and optimization of ABC and their self-assemblies are needed.

AUTHOR CONTRIBUTIONS
MG conceived and proposed the idea. MG compiled the manuscript with support from JT, ND, ML.

ACKNOWLEDGMENTS
ML and MG are members of CONICET scientific career. The authors wish to acknowledge the assistance of the CONICET and the Universidad Nacional de Cordoba (UNC), both of which provided facilities for this work. JT is also thankful to UNC. ND thanks to Institute of Pharmacy, Nirma University for providing the necessary facilities to carry out this work.