Nanoparticles and Gut Microbiota in Colorectal Cancer

Recent years have witnessed an unprecedented growth in the research area of nanomedicine. There is an increasing optimism that nanotechnology applied to medicine will bring significant advances in the diagnosis and treatment of various diseases, including colorectal cancer (CRC), a type of neoplasm affecting cells in the colon or the rectum. Recent findings suggest that the role of microbiota is crucial in the development of CRC and its progression. Dysbiosis is a condition that disturbs the normal microbial environment in the gut and is often observed in CRC patients. In order to detect and treat precancerous lesions, new tools such as nanotechnology-based theranostics, provide a promising option for targeted marker detection or therapy for CRC. Because the presence of gut microbiota influences the route of biomarker detection and the route of the interaction of nanoparticle/drug complexes with target cells, the development of nanoparticles with appropriate sizes, morphologies, chemical compositions and concentrations might overcome this fundamental barrier. Metallic particles are good candidates for nanoparticle-induced intestinal dysbiosis, but this aspect has been poorly explored to date. Herein, we focus on reviewing and discussing nanotechnologies with potential applications in CRC through the involvement of gut microbiota and highlight the clinical areas that would benefit from these new medical technologies.


INTRODUCTION
Colorectal cancer (CRC) is the second most common cancer in females and third most common cancer in males worldwide. Over time, it has become a leading cause of morbidity and mortality. There is a broad geographical variation in the incidence of CRC globally, and there has been a rapid rise in its incidence in Asian countries for the past few years (Siegel et al., 2020). In Malaysia, for example, the National Cancer Patient Registry has reported that CRC is the second most common cancer in both males and females, with a total number of 4,501 cases diagnosed from 2008 to 2013 (Abu Hassan et al., 2016). In general, CRC incidence is higher in the developed countries as compared to the developing ones; however, the burden of this disease is rising globally, including that in the low-to-middle-income nations (Siegel et al., 2020).
Currently, ample research is being conducted in order to find the definite cause of CRC. Based on the actual findings, it is theorized that the bacteria present in the human colon are linked to the development of carcinogenesis (Saus et al., 2019). Large numbers of bacterial cells live in commensal relationships with the host. However, once the gastric ecosystem is altered, various bacterial species become prone to develop pathogenic phenotypes (Sekirov et al., 2010). In recent years, there has been a surge of interest in assessing the relationship between the gut microbiota and the gut modifications that eventually lead to CRC. From here on, modification in the composition of the gut microbe is suggested as the cause underlying the development of colorectal malignancies (Saus et al., 2019).
Nanomedicine can be broadly defined as comprehensive monitoring, control, construction, repair, defense and/or improvement of all human biological systems, working from the molecular level to more complex wholes, with the use of nanomaterials. Nanoparticles (NPs) have been increasingly applied in the disease diagnosis and treatment during the last few decades. This use of NPs for medical purposes has led to encouraging prospects of their use for the betterment of human health (Riaz Rajoka et al., 2021). Diagnostic and therapeutic research on nanomedicine formulations has brought about a number of effective platforms, including those for combined diagnosis, targeted drug delivery and therapy (Saini et al., 2010). Still, despite the immense prospect of nanomedicine for improving human health, the prognosis for advanced stages of CRC is still relatively poor. The current treatment method for CRC includes surgery and/or chemotherapy. Although CRC is curable, the survival rates are still low. With the current advancements in the world of nanomedicine, it is expected that utilization of NPs will be a vital future approach in CRC theragnostics (Werner and Heinemann, 2016). Correspondingly, this review primarily elaborates on the dysbiosis condition that leads to the development of CRC, and the clinical aspect of NPs and their limitations based on published research.

MICROBIOTA
Human body is inhabited by a vast number of bacteria, archaea, viruses and unicellular eukaryotes. The collection of microorganisms that live in peaceful coexistence with their hosts has been referred to as the microbiota, microflora or normal flora (Dieterich et al., 2018). By far the most heavily colonized organ is the gastrointestinal tract (GIT); the colon alone is estimated to host over 70% of all the microbes in the human body (Jandhyala et al., 2015). The concentration of microbiota increases steadily along the GIT. Hence, lesser concentrations of microbiota are found in our stomach and significantly higher in the colon (Anselmo and Mitragotri, 2019), as shown in Figure 1.
The GIT shelters trillions of microbes which mostly live in a harmonized relationship with the host. In spite of the fact that most of the microbiome shows a favorable symbiotic relationship with the host, when the microbiome composition and function get disturbed, preconditions for various diseases arise, including cancers, obesity, metabolic diseases, diabetes, allergies, depression and disorders of the immune system (Quigley, 2013). Here, a selective group of bacteria such as Enterococcus faecalis, Bacteroides fragilis, Escherichia coli and Fusobacterium nucleatum are seen as fundamental causes of the pathogenesis of CRC (Kong and Cai, 2019). These bacteria are listed in Table 1.

Immunomodulation Through
Host-Microbiota Interactions immune responses are triggered by microbes via interactions with immune cells that express pattern recognition receptors (PRRs). Local dendritic cells (DCs) get activated by microbes or microbederived elements such as constituents, products, metabolites via interactions with PRRs. Activated DCs travel from the gastrointestinal tract to mesenteric lymph nodes (mLNs), where they present microbe-derived antigens and subsequently induce the differentiation of immature T cells into effector T cells, particularly regulatory T cells (Tregs) and T helper 17 (Th17) cells (Inamura, 2021). A subset of these effector T cells migrates back to the gastrointestinal tract and influences local immune responses. The remaining population enters the systemic circulation and influences systemic immunity. The conversion of the immune system from a pro-inflammatory to an antiinflammatory state occurs through the release of antiinflammatory cytokines such as IL-10, TGF-β or the engagement of DCs mediated by Tregs. Conversely, Th17 cells mediate the conversion of the immune system to a proinflammatory state by secreting immunostimulatory cytokines (e.g., IL-17) or by activating and recruiting neutrophils. This intriguing relationship strongly suggests the vital role of microbes in Th17 cell activation (Inamura 2021).

Impact of Nanoparticles and Gut Microbiota
NPs could be present in food in the form as additives, supplements and packaging and directly impact the composition and/or metabolic activities of the gut microbiota (Lamas et al., 2020). Furthermore, inorganic NPs, particularly, such as silver, titanium dioxide, silicon dioxide and zinc oxide have been shown to affect gut microbiota through their interaction with immune system. This alteration in gut microbiota-immune axis is associated with many chronic diseases such as inflammatory bowel disease (IBD), diabetes and even colorectal cancer (Ni et al., 2017;Meijnikman et al., 2018;Richard et al., 2018). Silver NPs are used in hundreds of commercial products due to their anti-microbial properties and intentional and accidental uptake of silver NPs, which might affect the gut microbiome, are underestimated. Dahiya et al. (2018) have reviewed evidence from both animal and human studies and concluded that silver NPs altered the gut microbiota, thus making the host susceptible to certain diseases. They also illustrated that the mucus present in the gut lining prevents absorption of silver NPs into intestinal cells, hence the interaction with gut microbiota. Meanwhile, titanium dioxide NPs, which are commonly available in daily products, also cause changes in both gut microbiota morphology and metabolism (Juan et al., 2018;Mao et al., 2019;Zhangjian et al., 2019). Specifically, a mix of commensal and pathogenic bacteria was modulated upon administration of titanium dioxide NPs and manifested as oxidative stress and inflammatory responses in the intestine (Zhangjian et al., 2019). However, another study indicates that titanium dioxide NPs have limited effect on gut microbiota compared to silver NPs that drastically changes the commensal density. In this study, titanium dioxide NPs formed large agglomerates that appeared to loosely interact with microbial cells, whereas Silver NPs were found both within and outside of microbial cells. This explains the significantly lower effect of titanium dioxide NPs on community growth (Agans et al., 2019). There is also optimistic opportunity of NPs in their interaction with gut microbiota. Selenium NPs are shown to be a promising tool in livestock industry due to their efficacy in intestinal pathogen control (Gangadoo et al., 2019). These NPs are observed to reduce emerging poultry pathogen, Enterococcus cecorum, without any significant disturbance to the gut total commensal community. Another type of NPs, fullerenol NPs with good biocompatibility properties, markedly increased the production of short-chain fatty acids (SCFAs)-producing bacteria in vivo (Li et al., 2018). Fullerenol NPs contain furan-and pyran-like structure which is similar to those of polysaccharides in dietary fiber such as inulin, thus the capacity to promote this kind of gut microbes. The increase of this gut main metabolites-producing bacteria is accompanied with anti-hyperlipidemic effect of fullerenol NPs when both triglycerides and total cholesterol levels in liver and blood were decreased. Interestingly, fabrication of fullerenol NPs to form carbonyls in low pH would induce a peroxidase-like activity and eradicate Helicobacter pylori both in vivo and in vitro (Zhang C. et al., 2020). In addition, approach to design NPs with natural sources and line up into a systematic drug delivery tool is demonstrated to be beneficial to gut microbiota. Curcumin-and ginger-derived NPs are shown to improve absorption by gut microbiota, so that they can produce their respective effects (Ohno et al., 2017;Teng et al., 2018). Curcumin NPs are shown to suppress development of mouse colitis through the increase of butyrate-producing bacteria and regulatory T cells (Tregs) while ginger NPs is design to contain microRNA that could ameliorate mouse colitis. Another natural sources such as milk is developed as extracellular vesicles and these NPs modulated intestinal SCFAs metabolites and increased intestinal immunity (Tong et al., 2020).

NANOMEDICINES AND COLORECTAL CANCER
The unique optical, magnetic, and electrical properties of metallic NPs such as gold and silver have rendered these systems a great tool for the detection of several markers of cancer. For example, fluorescent gold NPs have been used to detect mechano-growth factor (MGF), a unique marker of colon cancer (Kasprzak and Szaflarski, 2020). Another study demonstrated that the incorporation of theragnostic gold nanorods as cores inside tumour-specific antibody shells as allowed to make the distinction between the different types of tumors and destroy the surrounding tumor cells (Lee et al., 2015). A non-invasive NPbased breath sensor was also shown to distinguish between the healthy and cancerous patients, including those with CRC (Peng et al., 2010). This nanomaterial-based sensor array would analyze volatile organic compounds associated with and differently regulated in different diseases using cross-reactive absorption sites on nanomaterials such as gold NPs, thus producing a unique breath fingerprint (Xu et al., 2013). Local staging of colon cancer is routinely done with computerized tomography (CT) scans and magnetic resonance imaging (MRI), which both benefit from the use of NPs, which can improve the sensitivity for the detection of tumor invasion (Nerad et al., 2017). In MRI, superparamagnetic iron oxide (γ-Fe 2 O 3 or Fe 3 O 4 ) NPs coated with a polymer are demonstrated as promising contrast agent for MRI, whereas conjugation with doxorubicin further displayed the multimodality of these NPs in both diagnostic and therapeutic assays. Conjugation of these NPs to chemotherapeutic drugs did not only effectively kill tumor cells via active targeting, but also inhibited tumor growth using near-infrared (NIR) irradiation due to the photothermal activity of the NPs (Fan et al., 2019). In another study, Fe 2 O 3 -gold NPs were observed to accumulate within the tumor mass instead of other organs in a colon cancer model, while also enhancing the tumor detection selectivity as MRI contrast agents in a pancreatic cancer model (Kumagai et al., 2010). Conventional chemotherapy has been reported to be associated with severe side effects, mainly due to the nonspecific biodistribution, whereby both the cancer cells and the healthy cells are affected by the therapy. In CRC, common side effects over the course of chemotherapy include fatigue, diarrhea, constipation and dyspnoea, as reported in routine healthcare settings (Pearce et al., 2017). However, over the past three decades, the concept of enhanced permeability Frontiers in Nanotechnology | www.frontiersin.org July 2021 | Volume 3 | Article 681760 retention (EPR) effect conditioned by the hypervasculature in solid tumors, as proposed by Matsumura and Maeda (1986), greatly improved the logistics of cancer treatments using NPs (Matsumura and Maeda, 1986). Nowadays, the application of NPs in cancer therapeutics enhances the efficacy of the conventional cancer management, particularly relative to that of chemotherapy. This enhanced efficacy is largely owing to the passive targeting of tumors by the NPs, which are able to pass through the tumor vascular pores and accumulate the nanomedicine in the tumor zone. Furthermore, the poor response to anticancer drugs due to the development of multidrug resistance (MDR) phenotypes has shown improvement following the incorporation of NPs in various chemo-photothermal therapies Jiang et al., 2020). Various preclinical and clinical studies have explored the potential of nanomedicines, including metallic NPs, in the CRC treatment. Drugs such as 5-fluorouracil (5-FU) and doxorubicin (DOX) have been incorporated into NP delivery systems to enhance the efficacy of the treatment of CRC. One example of a recent in vitro study using NPs was that of enhancing the antitumour efficacy in a CRC model, as reported by Jiang et al. (2020). In this study, DOX-resistant SW620/Ad300 cells treated with a nanocomposite based on mesoporous silica-coated gold nanorods (GNRs/mSiO2) loaded with DOX showed a higher toxicity compared to the free DOX treatment, the reason being the increased intracellular DOX accumulation achieved by the NP drug carrier. Apart from biocompatibility, the GNRs exhibit a highly efficient photothermal conversion, which enabled the chemophotothermal therapy on CRC cells through functionalization with anticancer drugs (Zhou et al., 2017). In another study, manganese-based (Mn) NPs stabilized by L-arginine were developed as a nanocarrier system to deliver 5-FU to colon cancer cell lines (Jain et al., 2020). In that case, cell viability was significantly reduced after the treatment with 5-FU-loaded Mn-based NPs as compared to the treatment with free 5-FU, being twice lower in the former specimen group than in the latter.
Previously, CRLX101, a 30-40 nm NP consisting of camptothecin (CPT), a topoisomerase 1 (topo-1) and hypoxiainducible factor 1-alpha (HIF-1α) inhibitor, conjugated to cyclodextrin and polyethylene glycol (PEG) was designed to enhance the CPT efficacy against CRC (Weiss et al., 2013). Promising findings of CRLX101 in a human-phase trial with advanced solid tumors has led to further studies in other in vitro and in vivo systems to support or complement the ongoing clinical trials. Nonetheless, the local recurrence of tumors has been a major concern in these tumor treatments. Thus, CRLX101 with 5-FU incorporation was investigated in a subcutaneous mouse xenograft model of CRC following radiotherapy (Tian et al., 2017). It was reported that the nano-formulation had the highest therapeutic effect by eradicating tumor repopulation after chemoradiotherapy. Meanwhile, a comparative study involving the use of gold-based NPs in a radio-iodide cancer treatment reported that iodine-131 alone reduced the tumor growth by 15%, while the incorporation of iodine-131 inside the NPs reduced the tumors by 50%, suggesting that functionalized NPs may open up a new venue for radiopharmaceutical therapies in cancer management (Le Goas et al., 2019).
The branch of nanomedicine is emerging into the biomimetic NPs, particularly cell membrane-based NPs to avoid the immune system clearance and, thereby, increase their therapeutic effects. This biomimetic approach mimics the native cells to mediate the interactions at the nano-bio interface as the fate of NPs in vivo is governed by their physicochemical surface (Sushnitha et al., 2020). The presence of natural cell membrane coatings to camouflage NPs offers the ability to remain the NPs in circulation and eventually to achieve targeted delivery. Red blood cell (RBC) membrane-coated NPs have been one of the most studied cell membrane-based nanocarriers in drug targeting delivery to enhance tumor cell death. Fabricating NPs with RBC membrane suited in vivo circulation due to the expression of CD47 protein on their surface to ensure self-recognition by the RES to allow longer circulation (Liu et al., 2019). In addition, RBCs have limited nuclei and are easily isolated from the blood donor . Poly(lactic-co-glycolic acid) (PLGA) NPs loaded with gambogic acid (GA) possessed antitumour potential when covered with RBC membrane obtained by BALB/c mice. They found that RBC membrane-GA/PLGA NPs showed significant inhibition of tumor growth in CRC in vivo model via necrosis. Comparing GA toxicity with RBC membrane-coated NPs to the free GA without NPs, they observed the higher median survival rate recorded at 30 days compared to the free-GA group that lasted for 4 days only. It should be noted that, RBC membrane-coated NPs also improved the poor aqueous solubility of GA, just as with the poor water solubility of the available chemotherapy drug in the market such as paclitaxel.
Still, to date, limited numbers of clinical trials have been conducted on CRC treatments using nanomedicine. The most prominent NPs used in these studies have been polymer-based. For instance, a phase I clinical study using PEGylated liposomal mitomycin C (NCT01705002) and PEG conjugate of SN38 (NCT00931840) showed that both nano-formulations were well-tolerated at higher doses compared to the free antitumour drugs (Norris et al., 2014;Golan et al., 2015). Nevertheless, further studies are warranted to evaluate therapeutic efficacies of nanomedicine formulations in CRC management. However, to our knowledge, no clinical trials using metallic NPs in CRC treatments have been reported yet. Still, considering the number of promising preclinical findings, the metallic NPs are expected to be utilized in human-phase trials with CRC patients in due time.

ADVANTAGES AND CHALLENGES IN NANOMEDICINE
Nanomedicine aims to provide more efficient tools for prevention and treatment of various diseases as it possess potential advantages (Desai, 2012), and might someday provide answers to long lasting problems in medical research, such as poor drug solubility and lack of target specificity for therapeutic compounds (Bawa, 2011). This field give new perspectives for the biomedical Frontiers in Nanotechnology | www.frontiersin.org July 2021 | Volume 3 | Article 681760 area as well as clinical for prevention, diagnosis, and treatment of severe diseases such as cancer (Chow and Ho, 2013). Making remarkable enhancements in drug delivery systems (Butcher, Mortimer et al., 2016), medical imaging and diagnosis platforms have been accounted as nanotechnology effects in health and medicine (Bayford et al., 2017).

Biological Barrier
NPs impart a range of properties to drugs that they carry into a living organism. Due to specific characteristics such as efficient transport through capillary blood vessels, longer circulation duration, higher binding capacity to biomolecules, higher accumulation in target tissues, and reduced inflammatory and oxidative stress in tissue, various nanomedicines have been developed and commercially applied in clinical and nonclinical areas as their characteristics are differ from those of conventional medicine (Liu et al., 2011). This range of improved properties often translates to a greater therapeutic efficacy of nanomedicines than that of conventional medicines. These features also endow nanomedicines with lower toxicity, improved bioavailability, and enhanced pharmacokinetics compared to the conventional drug therapies. Thus, nanomedicines overcome obstacles that limit conventional drugs, and have the potential to meet future market demands for disease treatments (Wang et al., 2018).

Specific Surface Area
Namomedicines often exhibit greater therapeutic efficacy than conventional small molecular drugs due to their size, large specific surface area, and flexibility of surface functionalization. These features endow nanomedicines with low toxicity, improved bioavailability, and enhanced pharmacokinetics and therapeutics effect. Thus, nanomedicines overcome obstacles that limit conventional drugs, and have the potential to meet future market demands for disease treatments (Wang et al., 2018). Nanomedicine is used in versatile aspects of the disease management process, including diagnosis, treatment and monitoring (Sajja et al., 2009). In view of the ability of NP carriers to slow down the release of drugs and prolong the pharmacokinetic half-life for many of them, nanomedicine has a key role in the controlled delivery of drugs or other biological products to improve the human health.

Solubility and Permeability
NPs are also capable of ensuring the solubility and permeability of insoluble drugs across biological barriers. Drugs with low bioavailability can thus be delivered directly to the target site (Galvin et al., 2012). Moreover, the large surface area and a higher reactivity of nanomedicines may allow for the dose reduction of a drug, which can improve toxicity profiles and patient compliance (Bawa, 2011;Galvin et al., 2012). The large surface area of nanomedicines can also increase the dissolution rate, the saturation solubility, and the intracellular uptake of the drugs, thereby improving in vivo performance (Bawa, 2011). In addition, combining encapsulation, release modalities, and surface modifications to improve targeting or bioavailability could improve the therapeutic efficacy (Bharali and Mousa, 2010).
Currently, 65% of nanomedicines undergoing clinical trials focus on cancer applications, as nanomedicine can offer great contributions for a better treatment and early diagnosis of this disease (Germain et al., 2020). In addition to the cancer treatments, nanomedicine has also been used to combat infectious, ophthalmic, neurological and other disease, as well as to facilitate gene editing, immunotherapies, and so on.

Challenges
The development of nanomedicines is growing rapidly; however, each of the nanomedicines has its own challenges such as safety, biological challenges, scale-up, cost, and regulation (Grenha, 2011). In addition, the development of nanomedicines requires massive preclinical research, carefully conducted clinical trials, and appropriate clinical indications (Etheridge et al., 2013). The drug development process takes years, and any mistakes or negative results may delay these process (Etheridge et al., 2013). Thus, to understand the current situation of nanomedicine, including common challenges and future demands for nanomedicines development (Moghimi et al., 2005) is important.

Specific and Precise Targeting
One of the highest priorities for the next generation of nanomedicines is to create precise and highly efficient drugs that accumulate in targeted pathological tissues and not effect the healthy tissue. There are many nanomedicines that cannot avoid damage to healthy tissues because they do not specifically target the pathological tissues. Recently, smart drug delivery systems that possess active targeting and stimuli responsiveness are actively being developed. These smart drug delivery systems represent a promising strategy to eliminate pathological tissues while protecting normal tissues (Zhang J. et al., 2020).

Safety and Quality
It is imperative to understand the toxicology of the drugs, the physicochemical properties of NPs, and the properties of nanocarriers since safety is the crucial issues for nanomedicines, even after a nanomedicine is approved for clinical use, it can be withdrawn from the market due to safety issues. In human body, nanomedicines get exposed to biological environments such as the blood, extracellular matrix, cytoplasm, and cellular organelles, which may alter their biological performance, but also elicit undesired toxicities. Currently, there is a lack of standardized methods for evaluation of the safety of nanomedicines. It should be emphasized that the methods used for traditional drugs cannot accurately evaluate the safety of nanomedicines (Wolfram et al., 2015). For example, drugs such as paclitaxel or doxorubicin, which have been extensively studied, still facing the complex toxicity issues when used with novel nanomedicines and nanocarriers (Hua et al., 2018). To increase the safety and quality, many countries and organizations have enacted and implemented good laboratory and manufacturing practices. It is important to formulate good laboratory practices for nanomedicines to improve the success rate and to promote the development of safe and effective nanomedicines (Zhang J. et al., 2020).

Biodistribution of Drugs
Other challenges involve the drug biodistribution and biological barrier penetration which aim to enhance the accumulation of the drug at the target site and reduced the accumulation in healthy tissues (Lu et al., 2021). Biodistribution is affected by interactions with external and internal biological barriers, for which reason the interaction between nanomedicines and biological barriers must be well understood (Elsabahy and Wooley, 2012). Furthermore, nanomedicines often do not directly interact with living cells, but instead become coated with a protein corona, which alters the biological effects of the NPs and influences the cell uptake, biodistribution, clearance, toxicity, and immune response. Therefore, it is important to also focus on the protein coronae formed around NPs and the resulting biological responses to nanomedicines (Mahmoudi et al., 2016).

Scale up, Cost and Regulations
Additionally, nanomedicines may fail to enter the market because unmeet the requirements of scale-up synthesis and reproducibility. In both laboratory or preclinical investigations and in clinical studies, nanomedicines have been mainly synthesized in small batches (Anselmo and Mitragotri, 2019). Not only the manufacturing cost of nanomedicines scale-up high, but the cost of preclinical and clinical development is also increasing. Acquiring regulatory approvals is difficult for new nanomedicines, especially when existing products on the market have the same target indication (Ventola, 2017). The lack of regulation and standards for nanomedicines in manufacturing practices, quality control, safety, and efficacy evaluation are all barriers for the development of nanomedicines. There are currently no global regulatory standards specific to clinical translation of nanotherapeutics. Only initial guidance documents for nanotechnology products have been issued by regulatory authorities such as the FDA to provide guidance. There are also significant differences between different geographical regions in addressing the application of nanomedicines, so that nanomedicines approved in one country may not be approved in other countries. Strengthening of regulatory standards is a major challenge but must be overcome to achieve efficient nanomedicine development (Mühlebach, 2018).

CONCLUSION
This review has highlighted the existing use of diverse nanomedicines for the diagnosis and treatment of CRC. Despite the fact that the field is young and growing, a number of studies have reported on how combining metallic NPs and multiple targeting strategies establishes conditions for the improved treatment and diagnosis of CRC. Although considerable challenges and issues remain to be addressed, the potential impact of nanomedicine on the diagnosis and treatment of CRC is extensively recognized. As discussed through several examples in this review, careful modification and characterization of metallic NPs is needed in order to exploit the potential impact of nanomedicine on the clinical management of CRC and advance its use in the CRC treatment and early diagnosis.

AUTHOR CONTRIBUTIONS
KP, SA, MM-Z, and WNWH wrote this paper. ZI, J-LS, KF, VU, JJ, and RM supervised this work and revised the manuscript. All authors contributed to the paper and approved the submitted version.