Kai Guo,Nanyan Xiao,Yixuan Liu,Zhnmin Wan,Juit T′oth,J′anos Gynis,Vijay Kumar Thakur,Ayako Oyan,Quazi T.H.Shubhra

a Stomatology Hospital of Guangzhou Medical University,Guangzhou Medical University,Guangzhou,510140,China

b Department of Microbiology,The University of Chicago,Chicago,IL,60637,USA

c Department of Pathology,School of Basic Medical Sciences,Nanjing Medical University,Nanjing,21116,China

d State Key Laboratory of Oral Diseases,West China Hospital of Stomatology,Sichuan University,Chengdu,610041,China

e Research Centre for Natural Sciences,Institute of Materials and Environmental Chemistry,1117,Budapest,Magyar Tud′osok K¨orútja 2,Hungary

f University of Pannonia,Egyetem u.10,H-8200,Veszpr′em,Hungary

g Nanomaterials Research Institute,National Institute of Advanced Industrial Science and Technology(AIST),Tsukuba,305-8565,Japan

h School of Engineering,University of Petroleum & Energy Studies(UPES),Dehradun 248007,India

Keywords:Cell membrane(CM)Nanoparticles(NPs)Cancer Camouflage Biomimetic coating Drug delivery

ABSTRACT Nanotechnology has revolutionized cancer drug delivery,and recent research continues to focus on the development of“one-size-fits-all,”i.e.,“all-in-one”delivery nanovehicles.Although nanomedicines can address significant shortcomings of conventional therapy,biological barriers remain a challenge in their delivery and accumulation at diseased sites.To achieve long circulation time,immune evasion,and targeted accumulation,conventional nanocarriers need modifications,e.g.,PEGylation,peptide/aptamer attachment,etc.One such modification is a biomimetic coating using cell membrane(CM),which can offer long circulation or targeting,or both.This top-down CM coating process is facile and can provide some advantageous features over surface modification by synthetic polymers.Herein,an overview is provided on the engineering of CM camouflaged polymer nanoparticles.A short section on CM and the development of CM coating technology has been provided.Detailed description of the preparation and characterization of CM camouflaged polymer NPs and their applications in cancer treatment has been reported.A brief comparison between CM coating and PEGylation has been highlighted.Various targeting approaches to achieve tumor-specific delivery of CM coated NPs have been summarized here.Overall,this review will give the readers a nice picture of CM coated polymer NPs,along with their opportunities and challenges.

1.Introduction

Cancer,one of the most lethal diseases with the high expense of treatment,is threatening human health due to its diverse pathological progress[1].Some chronic diseases have the potential to be evolved into cancers[2].Nonetheless,the risk factors in daily life,e.g.,alcohol consumption,smoking,and air pollution,can also contribute to cancer development.With the advances in the understanding of nature and science,myriad cancer therapies such as chemo-[3],immuno-[4],radio-[5],gene-[6],photothermal[7],etc.therapies have been researched and achieved inspiring results in increasing the survival and improving the life quality of cancer patients[8].However,the different disadvantages of various therapies hinder the success of clinical treatment.For example,chemotherapy usually has strong side effects on the healthy organs,e.g.,harming the hair follicle and hemopoietic system;immunotherapy varies from patient to patient due to differences in their immune systems,gene therapy is effective against tumor growth,but its high expense is an obstacle[9].For addressing the shortcomings in those therapies,tumor-targeting strategies have been employed,and they have already shown great promise.Drugging tumor sites by using nanobiotechnology to construct the engineered nanoparticles(NPs)containing therapeutic agents is one of the most effective solutions to treat cancers[10].

Over the past two decades,nanotechnology has emerged as an amazing therapeutic strategy in biomedical applications,especially in cancer research.Due to their unique characteristics,nanomaterials have been developed as very important tools in drug delivery,tumor imaging,synthetic vaccine development,and gene therapy[11–15].In particular,NPs with different size ranges(from 10 to 1000 nm in diameter)have already been constructed for targeted drug delivery to the pathological foci with high efficiency and specificity besides minimizing the risk of damage to healthy tissues.NPs are prepared from various biomaterials,such as polymers,liposomes,inorganic materials,carbon nanotubes(CNTs),and even viruses[16–19].The main features NPs can offer as drug carriers are:high stability in vivo,enhanced bioavailability,high carrier capacity,and relative controllability,which dramatically reduce chemotherapeutic drug's dosage and adverse effects[20,21].

The properties of NPs are essential for their intracellular trafficking and can significantly influence the delivery and release of drugs.Biodegradable polymers are most extensively used as a carrier of drug to prepare various drug delivery systems(DDSs)due to their ease in preparation and in vivo degradation.One cancer therapy paradigm is poly(lactic-co-glycolic acid)(PLGA)-based NPs.As a biodegradable polymer,PLGA can be hydrolyzed to lactic acid(LA)and glycolic acid(GA)in vivo.These products are endogenous monomers to the human metabolism causing minimal systemic toxicity for PLGA-based DDSs,and PLGA was approved by the US Food and Drug Administration(FDA)to be used for clinical therapeutic applications[22,23].PLGA NPs are easily internalized into cells by endocytosis unless specifically targeted using suitable targeting agents[24].PLGA NPs can show rapid escape from the host endolysosomes to enter cytoplasm followed by slow release of the entrapped therapeutic drug during its degradation process[25,26].

Although NPs have shown great values and tremendous potential in cancer therapies,some issues still restrain their clinical translation and applications.The significant challenges are poor ability to overcome in vivo barriers and our immune defenses system against NPs.The synthetic NPs interfering with body homeostasis are identified as a dangerous signal and activate the body's immune system to destroy them.Systemically administered NPs are monitored and removed from the body within a few minutes[27–29].Moreover,another major side effect of NPs is inflammation,which significantly hinders the translation and clinical applications of many NPs[27–29].

These limitations promote the development of novel bioinspired NPs that integrate characteristics of the natural cell membrane(CM)to bypass host immune cells’uptake and systemic clearance[30–32].CM-nanotechnology has been developed by coating NPs with a layer of CM extracted from red blood cells(RBCs),cancer cells,or platelets,etc.[32,33].These CM coated NPs mimic the properties of their membrane source cells,survive for hours unharmed inside the body,and function as long-circulating and pathology targeting systems[33].Throughout this review,CM coated NPs have been termed as CM-NPs,and cancer CM(CCM)coated ones as CCM-NPs.CM-NPs and CCM-NPs can offer various features,e.g.,targeting,signal transduction,immune evasion,and other therapeutic advantages.This top-down process has emerged as a new era in nanomedicine,and over the years,the field is growing with more and more published articles,as can be seen from Fig.1.

CM-NPs find widespread application in treating cancers which have been thoroughly discussed in this review.However,CM-NPs have also shown promise in imaging and theranostic applications too.For example,Li et al.reported fibroblast CM coated phototheranostic system to target cancer-associated fibroblasts[34].Another research group reported that MCF-7 CM coated PLGA NPs loaded with indocyanine green could exhibit photothermal therapy and dual-modal imaging capability[35].Immune modulation is one of the most attractive approaches in treating complex diseases like cancers,viral infections,bacterial infections,autoimmune diseases,etc.CM-NPs have the potential to leverage the immune system since CMs contain components that are capable of instructing intercellular communication.Till now,RBC,white blood cell(WBC),platelet,cancer cell,bacteria,and hybrid cells have been exploited to prepare CM-NPs for immunomodulatory therapies[36].The application of CM-NPs in the treatment of bacterial infection is also mentionable.The growing resistance of bacteria to available conventional antibiotics and increased bacterial infection demand the need for novel antibiotics.Nanotechnology has found application in treating bacterial infections,and some NPs,e.g.,silver and zinc,show inherent antibacterial properties.Interesting work was reported by Hu et al.,who coated vancomycin loaded PLGA NPs by platelet membrane[33].CM coating could facilitate the binding to MRSA252 bacteria by 12-fold compared to uncoated NPs.Such efficient binding ultimately resulted in improved bactericidal efficacy.Most toxins(endo-and exotoxins)interact with specific cell surface molecules,and CM-NPs are anexcellent candidate for detoxification treatments[37].Hu et al.first reported a detoxification approach using CM-NPs in 2013[38].The aim was to absorb toxins using RBC coated PLGA NPs since RBC has an affinity for toxins,and such absorption prevents toxins from reaching their cellular targets.As a result,toxins fail to cause harmful effects in vivo.

Fig.1.Graphical representation of(a)Research areas,and(b)Percentage of works published from different countries using CM-NPs.Published articles in the last 10 years found with the search“cell membrane coated nanoparticles”in(c)Scopus and(d)Web of science.

Fig.2.(a)Simplified structure of the cell membrane.(b)Timeline showing major developments and some exciting works in the field of cell membrane coating technology.

This review systematically describes recent advances in CM coating nanotechnology to engineer polymeric DDSs.The evolution of this technology over the last decade has been briefly mentioned here.Indepth,a discussion was made on the preparation and characterization to illustrate them clearly.After that,a summary of the application of CM coated polymer NPs to treat various cancers can be found.We have also reported a brief comparison between CM coating and the most extensively used synthetic polymer(polyethylene glycol;PEG)coating.Finally,approaches to target CM-NPs have been reported,followed by a discussion on challenges and future perspectives before ending this review.

2.Cell membrane(CM)and their attractive features for biomimetic coating(coating technology)

Cells are basic units of life,and they are found in all living organisms.All cells have CM,which acts as a physical barrier between the outside environment and the interior of cells and ultimately protects cells from their surroundings.CM is semipermeable and allows specific molecules to cross the barrier to enter cells.CM is a thin layer(thickness 5–10 nm)composed of three major classes of biomolecules:lipids,proteins,and carbohydrates[39](Fig.2a).Various cells contain various lipids(phospholipids,glycolipids,sphingolipids,cholesterol,free fatty acids,etc.)and proteins on their CM,which perform bunches of crucial functions.Lipids are primarily responsible for maintaining bilayer membrane structure,flexibility,membrane fluidity,and they also take part in regulating cell signaling.Proteins(e.g.,transmembrane and membrane-anchored proteins)play major roles in regulating cell signaling.Some proteins,which act like pumps and channels,take part in transporting molecules across the cell.Membrane proteins also participate in various cell physiological activities,such as adhesion,passive transport,and help cells respond to environmental cues.Approximately 30%of total proteins are membrane proteins in humans[40].Carbohydrates are found on CMs either bound with proteins(forming glycoproteins)or lipids(forming glycolipids),contributing to cellular recognition.

Biomimetic coating of NP surface by transferring the outermost cell layer can attribute proteins,lipids,and carbohydrates of CM to the surface of coated NPs.Consequently,coated NPs can exhibit various properties of the source cell.Specific cell's membrane can impart specific features to the coated NPs,e.g.,cancer cell membrane(CCM)coating facilitates homotypic tumor targeting(more given in section 8).Cells adhere to neighbor cells,forming a junction with the help of cell adhesion proteins(e.g.,cadherins)[35,41].CM coating can decorate the surface of NPs with such proteins that facilitate the attachment of NPs with similar types of cells[42].Moreover,CM coating can provide NPs with crucial biomarkers and can impart various attractive features to the decorated NPs,as shown in Table 1.For example,CD47 is a biomarker found on various types of cells,e.g.,cancer cells and RBC.The presence of CD47 on CM-NP's surface facilitates immune evasion,resulting in extended blood circulation time[43].

Table 1Various adhesion molecules/biomarkers found on the surface of different cells.

CM coating technology is relatively new compared to some other conventional coating technology of nanomedicines,e.g.,PEGylation.This technology was first reported in 2011(by Hu et al.)[32].Even cell-based nanovesicle platforms for drug delivery were reported earlier;for example,a macrophage-mediated NP platform to deliver drug was first reported in 2006[56].However,membrane ghost preparation was reported as early as the 1970s by Steer et al.,who prepared erythrocyte ghosts utilizing hypotonic lysis and resealing[57,58].In 2011,Hu et al.applied a top-down biomimetic approach to coat polymeric PLGA NPs by directly transferring RBC's outermost layer onto the PLGA surface[32].The complexity of the RBC membrane containing various carbohydrates,lipids,and proteins could be successfully preserved,and coated NPs exhibited many properties of the source cell.Later on,in 2014,the same group also first reported CCM coating and revealed that CCM coated NPs have homotypic targeting capability to treat cancer[59].

Since 2011,CM coating technology has grown(Fig.1),and plenty of research works have been published over the past 10 years in different research fields.In 2013,leukocyte CM coated nanoporous silicon NPs were developed and coated NPs showed enhanced circulation time with improved tumor accumulation[60].Different types of CMs have been employed so far for coating purposes,including stem cells[61],platelets[62],neutrophils[63],natural killer cells[64],etc.Recent years have witnessed utilization of hybrid membrane-coating(e.g.,platelet–leukocyte[65],cancer stem cell-platelet[66],etc.),which can impart properties of both cells to the functionalized NPs.CM-NPs have already shown remarkable success in treating various cancers,including glioblastoma[42].We have recently reported successful breast cancer treatment overcoming drug resistance using CCM coated PLGA NPs[67].Some landmarks and exciting works on CM camouflaging nanomedicines have been illustrated in Fig.2b.

3.Most commonly used cells in biomimetic coating

This section has a brief overview of the most widely used cells that find applications in biomimetic coating.Fig.3 schematically shows major cells and their membrane coated NPs with key features.

3.1.Erythrocyte

Erythrocytes or RBCs are the major component of blood in mammals,derived from bone marrow differentiated by hematopoietic stem cells,undertake the mission of oxygen-carbon dioxide exchange during the circulation[68].As a natural vehicle of oxygen and carbon dioxide,RBCs are inherently suitable for intravascular delivery due to their intrinsical biocompatibility,biodegradability,and non-immunogenicity[69].Interestingly,natural compartments can be formed in RBCs to protect encapsulated materials which offer them a long circulation time in the bloodstream[70].The transportation capacity of RBCs inspired scientists to explore the possibility of using RBCs as a carrier in drug delivery.

Advances in molecular biology have provided an unprecedented understanding of the connections between the physicochemical characteristics of RBCs and their biological functions,enabling RBCs as an ideal vehicle[71].After the first application of RBC by Hu et al.,in 2011,the serviceability of RBCs as a DDS has been well-proven by the various applications in the last decade[71].Small-molecule drug,nucleic acid,and antibody can be successfully carried and delivered to the site of action by natural and mimical RBC vehicles.They could achieve excellent results in treating various diseases including immunodeficiency induced disease and different kinds of cancers in vitro and in vivo[72–74].Meanwhile,the RBC membrane is one of the most commonly used vehicles for drug delivery.

The RBC membrane is composed of 19.5%(w/w)of water,39.5%of proteins,35.1%of lipids,and 5.8%of carbohydrates,which is similar to most animal membranes.A large number of biomarkers expressed in the membrane surface as adhesion molecules,like CD44,CD47,and LW/ICAM-4,play key roles in cell recognition and participate in the communication between the RBCs and different kinds of cells and tissues[75–77].

3.1.1.CD44

The CD44 proteins form a ubiquitously expressed family of cell surface adhesion molecules involved in cell–cell,cell–matrix interactions and have additional functions in innate and adaptive immunity[78].The main property ascribed to CD44 is its ability to bind hyaluronic acid,which was the first function of CD44 that was believed to be important for the metastatic process[79].In addition,diverse functions attributed by CD44 involve not only cellular adhesion and migration but also include homing,activation,and proliferation of lymphocyte,monocyte,and cytocidal activity of natural killer cells[80].

Fig.3.Most common cells used to prepare CM coated polymer NPs and the key features of those cells.

3.1.2.CD47

CD47,also known as integrin associated protein(IAP),is a transmembrane glycoprotein with a highly glycosylated extracellular IgV domain at its N-terminus[81].The ability of CD47 to inhibit RBCs phagocytosis by hindering the expression of immunoreceptor SIRPα has been reported.Due to the“Do not eat me”signal constituted by the interaction of CD47 and SIRPα,the RBC's membrane is widely used as an efficient vehicle for therapeutic agents'delivery[82].

3.1.3.LW/ICAM-4/CD242

LW is known as intercellular adhesion molecule-4(ICAM-4)or CD242[83].This glycoprotein is specifically expressed on the RBC's membrane and plays an important role in cell adhesion as well as in cellular interaction events,including hemostasis and thrombosis.The ability of ICAM-4 to interact with several types of integrins expressed on blood and endothelial cells is already proven.Moreover,the integrin CD11a,b,c/CD18 in monocyte and macrophage is reported as receptors for ICAM-4.RBC's function is affected by selective binding of ICAM-4 to different integral proteins.Moreover,the specific binding of ICAM-4 is associated with the pathology of thrombotic events in sickle cell disease[84].

3.2.Macrophage

Macrophages,which were originally identified by Metchnikoff on account of their phagocytic nature,are ancient cells in metazoan phylogeny[85].In mammals,macrophages could be found in all tissues where they display great anatomical and functional diversity.Macrophages play important roles and participate in almost every aspect of an organism's biology,such as development,homeostasis,tissue repair,immune responses to pathogens,and so on[86].

Macrophages are rich in diversity,and the most well-known phenotypes are M1 macrophages(M1Φ)and M2 macrophages(M2Φ).These two phenotypes are defined by responses to the cytokine interferon-γ(IFN-γ)and activation of Toll-like receptors(TLRs)(M1Φ phenotype),and to interleukin-4(IL-4)and IL-13(M2Φ phenotype),respectively[87].When tissues are damaged after pathogens infection or injury,macrophages migrate into the affected tissues and respond to different occasions accordingly[88].The pro-inflammatory phenotype M1Φ often appears in the early stages of a wound-healing response.M1Φ can activate anti-microbial defense mechanisms by secreting a variety of inflammatory mediators such as tumor necrosis factor-α(TNF-α),IL-1,and nitric oxide.Similarly,the M1Φ is able to be activated by IFN-γ and/or after signaling transduction of TLR pathway,leading to the activation of the NF-κB and STAT1 signaling pathways,called classically activated macrophage[89].The M2Φ,which is induced by IL-4 and IL-13,represents an‘a(chǎn)lternative’state of macrophage activation that participates in wound healing,fibrosis,insulin sensitivity,and immunoregulatory functions by expressing platelet derived growth factor(PDGF),transforming growth factor beta-1(TGF-β1),vascular endothelial growth factor(VEGF),ligands of Wnt/β-catenin,and various matrix metalloproteinases.M2Φ is also capable to secret plenty of immunoregulatory proteins,e.g.,arginase 1(ARG1),RELMα,PDL2,and IL-10,etc.that regulate the magnitude and duration of immune responses[86,90].

Tumors are abundantly populated by macrophages.However,the macrophages,which are involved in chronic diseases and persistent infections,seem to be a critical factor in the imitation and formation of tumors[91].In established tumors,the roles of tumor-associated macrophages(TAMs)in stimulating tumor cell migration,invasion,and intravasation,as well as the angiogenic response required for tumor growth,have been described[91–94].

3.2.1.CD11b

Integrin CD11b is mainly expressed on the surface of immune cells and myeloid cells.It is an important adhesion and signaling molecule with both pro-inflammatory and immunomodulatory effects by responding to TLR and FcγR[95,96].Integrin CD11b is inactive conformation in circulating leukocytes and it gets rapidly activated upon TLR stimulation[95,97].

3.2.2.CSF1R

The macrophage colony-stimulating factor-1 receptor(CSF1R)belongs to the type III receptor tyrosine kinase family.The overexpression of CSF1R and its ligand CSF1 is widely found in various solid tumors,and its important roles in tumor malignant proliferation,metastasis,and microenvironmental regulation have been proved[98,99].The CSF1R highly expressed TAM positively correlates with post-operative survival,revealing the potential and promise of CSF1R as a therapeutic target for cancer[100,101].

3.2.3.F4/80

F4/80,a 160-kDa glycoprotein,is a member of EGF-TM7 protein family and is widely used as a macrophage surface marker in the fluorescence activated cell sorting(FACS)[102,103].Based on this modular structure,F4/80 is supposed to be involved in macrophage adhesion,cell migration,or as a component of G protein-coupled receptor(GPCR)family in macrophages[104].

3.3.Dendritic cell

Classical dendritic cells(cDCs)are specialized antigen processing and presenting cells that possess high phagocytic activity as immature cells and high cytokine production capacity as mature cells[105].Although present in human circulation,cDCs are rare in mouse blood[106].cDC is a highly mobile cell that can use afferent lymphatic vessels and high endothelial venules to move tissues to the T cell and B cell areas of surrounding lymphoid organs.cDCs are able to regulate T cell responses not only in the steady-state but also during infection[107].However,plasmacytoid cell-like DCs(pDCs)are different from cDCs in that pDCshave a relatively long lifespan,and some of pDCs have the characteristics of immunoglobulin rearrangements[108].The massive production of type I interferons(IFNs)will be synthesized by pDCs for anti-virus response;moreover,pDCs also can act as antigen-presenting cells and control T cell responses[106].

DC precursor is a subset of proliferating cells in the bone marrow.It has the same phenotypic characteristics as the myeloid precursor cell,and it produces many macrophages and DC subsets[109].The common DC progenitors are proliferating cells that differentiate into pDCs and the precursors for cDCs in the bone marrow,whereas they have lost the potential to give rise to monocytes at the same time.At a steady-state,pre-cDCs are found in the bone marrow,blood,and spleen[106].They enter lymph nodes from the blood through high endothelial venules and acquire a mature cDC surface phenotype and morphology when they integrate into the DCs network.

3.3.1.HLA-DR

HLA-DR is a major histocompatibility complex class II antigen presentation molecule,critical for the activation of lymphocytes and the orchestration of adaptive immune responses[110].HLA-DR is normally expressed on monocytes,macrophages,DCs and B cells,but it can be expressed on epithelial cells and tumor cells due to cell response to inflammatory conditions[111].

3.3.2.CD11c(cDC)

CD11c is expressed prominently on monocytes,tissue macrophages,NK cells,and on most DCs.For achieving its capacity of modulation,CD11c binds to complement fragment(iC3b),provisional matrix molecules(fibrinogen),and the Ig superfamily cell adhesion molecule(ICAM-1)[112,113].It has been proposed that the binding of CD11c and ligands involves in phagocytosis,cell migration,and cytokine production by monocytes/macrophages as well as induction of T cell proliferation by Langerhans cells[114,115].

3.4.Platelet

Platelets are small anucleate blood cells with a short lifespan,circulating in the blood for 5–10 days in humans(for a shorter time in mice),and are destined to be eliminated by spleen and liver[116].Circulating platelets are subjected to balanced states of activating and inactivating biomolecules and conditions.When vascular activation or injury is absent,the spontaneous adhesion and activation of platelet are prohibited[117].

Platelet inhibition is initiated by nitric oxide and prostaglandin I2(PGI2)IP receptors,acting via guanylate cyclase(GC)and adenylyl cyclase(AC),with activation of protein kinase G(PKG)and protein kinase A(PKA),respectively[118,119].Platelet activation is initiated by the interaction of adhesion receptors such as integrins α6β1,α2β1,αIIbβ3,with their ligands,such as collagen and von Willebrand factor(vWF)[52].

3.4.1.CLEC2

C-type lectin-like receptor 2(CLEC-2),exclusively expressed in platelets and megakaryocytes,has been involved in signaling transduction and regulates the downstream pathways[120,121].The engagement of mucin-type glycoprotein podoplanin,which is the endogenous ligand of CLEC-2,leads to the activation of platelet[122].

3.4.2.α6β1,α2β1,αIIbβ3

In the basement membranes and extracellular matrix,α6β1 integrin can mediate platelet adhesion to laminin by interacting with cobalt,manganese,and magnesium[123].Morphologic changes in the platelets could be induced by the signaling transduction of PI3K pathway which initiates from α6β1[124].

α2β1 receptor,or GPIa-IIa,is an important platelet receptor that plays a critical role in the interaction with collagen due to its α2 subunits,which has I domain for Mg2+[125,126].α2β1 receptors promote platelet adhesion to collagen,stabilize thrombus growth,and promote pro-coagulant activity.

αIIbβ3,also known as GPIIb-IIIa complex,is the most abundant platelet adhesion receptor,specifically expressed on platelets[127].The ligands of αIIbβ3 are extracellular matrixes such as hemofibrinogen,fibronectin,bilirubin,and platelet-reactive protein,and αIIbβ3 is involved in platelet agglutination and activation,and mutations in its gene can lead to hereditary bleeding disorders.

3.5.Mesenchymal stem cell

Mesenchymal stem cells(MSCs)are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues[128].MSCs have the potential for multiple differentiation and can differentiate into adipocytes,osteocytes,and chondrocytes,as well as cells of other embryonic phenotypes[129].MSCs can lead to the modulation of several effector functions by communicating with cells of both the innate and adaptive immune systems.MSCs can migrate to injured tissues and inhibit the release of pro-inflammatory cytokines[130].

It is widely believed that human MSCs do not express the hematopoietic markers CD14,CD45,and CD34 or the co-stimulatory molecules CD40,CD80,and CD86.But human MSCs do express different levels of CD105,CD73,CD44,CD90,CD71,the ganglioside GD2,and CD271[131–133].

3.5.1.CD90

The CD90(Thy-1)protein is composed of 110 amino acids with molecular weight varying from 25 to 37 kDa,and it is a glycophosphatidylinositol(GPI)anchored protein that is located at the plasma membrane[134].The existed reports have demonstrated its critical role in MSC differentiation.Recently,it has been proved that CD90 is involved in osteogenic differentiation of MSCs for facilitating bone formation and restricting adipose tissue accumulation at the same time[135].However,CD90 is also able to transmit intracellular signals that lead to the activation of tumor cell migration/invasion program in liver and lung cancers,in glioblastoma(GBM),and in melanoma[134,136,137].

3.5.2.CD105

CD105(Endoglin)is a CM surface glycoprotein,a member of the transforming growth factor beta(TGF-β)superfamily,and a putative indicator of human endothelial cell proliferation[138].Based on its capacity of angiogenesis and tumorigenesis,using CD105 as a biomarker for tumor diagnosis,prognosis,and therapeutic target has been demonstrated[139,140].

3.5.3.CD146

CD146,also known as melanoma cell adhesion molecule,is a 11.3 kDa glycoprotein and is a member of the immunoglobulin superfamily[141].In 2007,a study showed that higher levels of CD146 were found to be expressed on human umbilical cord perivascular cells(HUCPVCs),which has a higher capacity of differentiation and proliferation compared with bone marrow stem cells(BMSCs),suggesting CD146 is a MSC marker[142].Interestingly,CD146 is not only related to the development,proliferation,differentiation,and immune response in MSC but also participates in the formation of various cancers[143,144].

3.6.Cancer cell

Cancer is a malignant disease related to aging,arises from genemutated biological cells.The diseased cells proliferate uncontrollably and destroy the order of tissue[145].Abnormal protein structures change the ability of cancer cells to contractor stretch by influencing their mechanics of deformation.Therefore,the shapes of cancer cells can be irregular and different from normal cells.Enhanced mobility of cancer cells will cause them to migrate through the tissue to different sites in the human body and induce tumor metastasis[146].There are six hallmarks of cancers:sustaining proliferative signaling,evading growth suppressors,resisting cell death,enabling replicative immortality,inducing angiogenesis,and activating invasion and metastasis[147].

In conclusion,the discovery of thousands of distinct adhesion molecules and metabolites has already led to significant changes in our understanding of the mechanism of tumor formation that operates in good health and disease[148,149].According to the reports from literature,programmed death-ligand 1(PD-L1),and epithelial cell adhesion molecule(EpCAM)are well-known biomarkers for cancer cells,and the corresponding small molecule drugs and antibodies for the targets mentioned above have been listed in the market[150–154].

3.6.1.PD-L1

PD-1,also referred to as CD279,was first discovered in interleukin-3(IL-3)-deprived LyD9 and 2B4-11 cell lines in 1992[155].PD-1 can regulate adaptive and innate immune responses,and is expressed on activated T,natural killer(NK)and B lymphocytes,macrophages,DCs,and monocytes[156–158].Of note,PD-1 is highly expressed on tumor-specific T cells[159].PD-1 plays two opposing roles in immunoregulation:it plays a key role in reducing the regulation of ineffective or harmful immune responses and maintaining immune tolerance and PD-1 causes the dilation of malignant cells by interfering with the protective immune response[160].

PD-L1,referred to as CD279 and B7–H1,is a 33-kDa type 1 transmembrane glycoprotein that contains 290 amino acids with Ig-and IgC domains in its extracellular region[161,162].PD-L1 is usually expressed by macrophages,some activated T cells and B cells,DCs,and some epithelial cells,particularly under inflammatory conditions[163].In addition,PD-L1 is expressed by tumor cells as an“adaptive immune mechanism”to escape anti-tumor responses[164].PD-L1 is associated with an immune environment rich in CD8 T cells,production of Th1 cytokines and chemical factors,as well as interferons and specific gene expression characteristics[163,165].

3.6.2.EpCAM

The structure of the extracellular domain of EpCAM is conserved to a great extent from a variety of species,reflecting the functional importance of the EpCAM protein[146,166].Due to the feature that the expression of EpCAM is limited to normal and malignant epithelial,EpCAM has been applied successfully as a diagnostic marker for carcinoma cells detected in mesenchymal organs such as blood,bone marrow,or lymph nodes[167,168].

4.Preparation of CM coated NPs

In general,preparation of CM-NPs requires three main steps:preparation of NPs to be used as the inner core,extraction of CM,and the attachment of the extracted CM onto the surface of NPs.Briefly,the steps are summarized in this section.

4.1.Preparation of inner core

Polymer NPs can usually be prepared from the polymer of interest or by polymerizing monomers applying polyreactions or classical polymerization[169].A scheme showing various commonly used methods to prepare polymer NPs are shown in Fig.4a.Encapsulation of drug inside polymer matrix can be achieved by micelle formation,nanoprecipitation,micro/nanoemulsion,solvent extraction/evaporation,emulsion polymerization,etc.[170].Discussion on the preparation of each type of polymer NP is out of the scope of this review and can be found in some excellent comprehensive reviews[169,171,172].PLGA is one of the most widely used polymeric drug carriers[173],and until now,it has been most extensively used in the preparation of CM camouflaged NPs[174,175].The most commonly used methods to prepare PLGA NPs are:emulsification–solvent evaporation(single or double or multiple emulsion-based),emulsification–solvent diffusion,coacervation,emulsification–reverse salting-out,spray drying,nanoprecipitation,etc.[176].Besides synthetic polyester PLGA,natural carbohydrate polymer chitosan(CS)has been investigated to prepare CM coated cancer drug carriers[177].Chitosan NPs can be prepared by using the emulsion solvent diffusion method,ionotropic gelation,coprecipitation,complex coacervation,reverse micellar and spray drying method.Lipids are also widely used as the inner core to prepare CM coated cancer drug carriers.We exclude lipids in this review since lipids are generally referred to as hydrocarbons without distinct repeating subunits like polymers[178].

4.2.CM isolation method

Only a few approaches have been investigated so far to extract CMs,and the outputs from the employed methods are also low[43].The commonly used methods of CM isolation are:extrusion[179,180],sonication[42,177],hypotonic lysis of cells[59,67],repeated freezing and thawing[43,57],and homogenizing using Dounce homogenizer[181,182].The isolation method is selected based on the type of cell.

CM isolation from nucleus-free cells like platelets and erythrocytes is relatively simple.From the whole blood sample,cells are first isolated bycentrifugation.Isolated cells are then subjected to a hypotonic medium for lysis,or their membranes are mechanically disrupted by repeated freeze-thaw cycles.Finally,purification by removal of soluble proteins/debris is done by centrifugation.

Fig.4.(a)Schematic diagram showing commonly used methods to prepare polymer NPs.(b)Illustration showing sonication,coextrusion,and electroporation methods for the preparation of CM coated NPs.

On the other hand,membrane isolation from eukaryotic cells like stem cells,cancer cells,etc.,is complex compared to that from nucleusfree cells.The cell of interest is first isolated from the blood or tissue and cultured to grow.CMs are isolated by combining hypotonic lysis,sonication or homogenization,and high-speed differential centrifugation.Centrifugation is essential to separate CMs from organelles and nuclear components.

Extrusion is a very common method to prepare smaller nano-sized materials by pushing large cross-sectional areas through a die with a nano-sized cross-section.Extrusion is used along with lysis or homogenization to isolate CMs[183].The utilization of an ultrasonic probe/bath can lead to an increase in solution temperature[184].Ice bath should be used to minimize the rise in solution temperature.The solutions can be kept at 4°C,and prior to subjecting to sonication,the cold solutions can be used to maintain solution temperature during sonication.Otherwise,CMs might denature during the sonication process.The use of lysis buffer is very effective in disrupting the CM[185],and hypotonic lysis buffer(pH 7.5;10 mm Tris-HCl)can lyse cells in 15 min.Lysed cells need to be homogenized and centrifuged to obtain CMs for coating.The freezing and thawing process can disrupt cells if the cycles are repeated 2–3 times.However,this method can cause some damage to biomolecules besides affecting the activity of sensitive enzymes[186].During this process,swelling and contraction of cells take place,causing their disruption.CM can be extracted by using Dounce homogenizer.But cells need to be lysed using lysis buffer before using a Dounce homogenizer[187].

4.3.Attachment of the extracted CM onto the surface of NPs(fusion process)

The final step in the preparation of CM-NPs is the attachment or fusion or merging of CM onto the surface of NPs.There are three most commonly used methods for fusing CM with NPs:extrusion[35,188,189],sonication[190,191],and microfluidic electroporation.Sometimes,the combination of sonication and extrusion is also performed to fuse CM and NPs.Less commonly used methods include:freeze-thaw/sonication,biomimetic mineralization,extrusion/sonication and stirring,Ostwald ripening,extrusion/electroporation,etc.[186,192].Fig.4b schematically depicts the preparation of CM-NPs using the three commonly used methods.

Extrusion:In nanotechnology,extrusion is commonly used to prepare monodisperse nanovesicles,especially from liposomes.In the extrusion technique,a suspension is forced through nanopore channels of a membrane having a well-defined pore size.As a result,nano-sized products(e.g.,CM-NPs,liposomes,etc.)of diameter near the membrane's pore size can be obtained[193].Among the commercially available extruder,mini extruder(e.g.,from Avanti)is the most convenient and commonly employed to prepare CM-NPs since it offers easy low-scale production.Membranes used in most extruders utilized in nanotechnology are composed of polycarbonate.

Initial works on CM-NPs relied exclusively on extrusion,which is till now most extensively used.The first work on CM coated PLGA NPs by Hu et al.reported the use of extrusion to fuse CM and NPs.PLGA NPs(1 mg)were fused with CM extracted from RBC employing extrusion using a polycarbonate porous membrane(pore size 100 nm)[32].Extrusion generates strong force,and the structural integrity of CM is disrupted,followed by reformation onto the core NPs[174].The mean diameter and polydispersity of CM-NPs strongly depend on the parameters of the extrusion technique.The number of cycles,pressure applied,and pore size of the membrane determine the size and size distribution of the final product[194].

Extrusion results in a homogeneous type of coatings and favors the formation of NPs with uniform size.This method is very effective for small-scale production,and the use of this method for large-scale production is a challenge.Moreover,it is a time-consuming method compared to sonication[195].Unlike sonication,extrusion requires a relatively mild environment which facilitates better preservation of biomolecules.But compared to the freeze/thaw method,extrusion can offer a better“right-side-out”orientation(a ratio of over 80%)and less denaturation of biomolecules(e.g.,proteins).However,nanopore filtration can cause a higher loss of biomolecules which can result in a lower yield[196].Besides,material deposition on the filter can restrict pore area;thus,the filter needs to be carefully monitored and washed.

Sonication:Sonication is a process which generates sound waves of high-frequency and converts electrical signals into physical vibrations.Sonication is widely used in nanotechnology by employing ultrasound(frequency>20 kHz;above human hearing frequency)from either ultrasonic probe or bath.In general,higher localized intensity is resulted from a probe-type sonicator compared to a bath type.The major applications of sonication in nanobiotechnology are:preparation of emulsion to fabricate NPs,agitation of NPs in liquid medium facilitating dispersion by reducing aggregation,and disruption of CMs.

Sonication is comparatively easier to perform and control.In this method,ultrasonic energy of specific frequencies is used to the dispersion of core NPs co-incubated with membrane components.Sonication can also provide disruptive forces facilitating the spontaneous coating of the core-shell structure.Fang's group reported coating of PLGA NPs by RBC membrane using bath sonicator(100 W;frequency of 42 kHz for 2 min)[191].Our group recently reported the fabrication of CCM coated PLGA-CS NPs employing sonication[67].This method is also very useful to prepare hybrid CM coated NPs.Gong et al.decorated the surface of PLGA NPs using hybrid(RAW264.7 and 4T1)membranes,and only 2 min sonication was enough for successful coating[190].Hence,sonication is a fast and easy method compared to extrusion.Material loss is less in the sonication-based method compared to extrusion.Sonicators are also easy to clean and handle compared to mini extruders.

Sound waves are generally accepted as safe and non-toxic,which is a major advantage of ultrasonication.Ultrasonication is also used in the food industry besides the pharmaceutical industry.Although sonication is a fast and easy process,improper control of this process can result in denaturation of membrane proteins and/or drug leakage.For best results,duration,frequency,and power of sonication must be optimized.Moreover,this method cannot always ensure the uniform diameter of resultant CM coated NPs[197].

Microfluidic electroporation:Electroporation or electropermeabilization is a technique that employs an external electric field to the cells to increase their membrane permeability.Application of such electric field can breakdown the dielectric layer of CM forming transient pores through which foreign molecules,drugs,DNAs,etc.,can be introduced to the cells[198].The history of electroporation can be dated back to the middle of the 18th century,whereas their medical applications started with Neumann and coworkers'report in 1982[199].

Microfluidics is the technology of very small volume(e.g.,micro-or nanoliters)fluid manipulation.When microfluidic electroporation is employed to a mixture of CM and NPs,entry of NPs into CM takes place due to the formation of transient pores on the CM,resulting in the formation of CM-NPs.Microfluidic electroporation method to coat NPs by CM is comparatively new and was reported in 2017[200].The device required for the coating consists of a Y-shaped channel(for merging),an S-shaped channel(for mixing),an electroporation zone,and an outlet.By appropriate tuning of the pulse duration and voltage along with the flow velocity,NPs are successfully coated by CM.Since this method is new,the device needs to be developed by researchers and is not commercially available.Hence,the scalability of this method needs to be explored in the future.However,this method can offer accurate control of the size and function of the prepared CM-NPs[200].

Successful and efficient coating of NPs by CM depends on several aspects,e.g.,the surface charge of the NPs,membrane/NP ratio and diameter of the NP.Study shows that to completely shield PLGA NP surface of 100 nm diameter by RBC membrane,85 μl of mouse blood isrequired[201].Biological membranes possess inherent charge asymmetry,and hydrophobic NPs with negatively charged surfaces can result in better membrane coating over positively charged NPs.Negatively charged NPs repel the CM's homo-charged outer leaflet and ensure correct topological orientation[202].If the core NP is positively charged,strong interaction between CM and NP surface will result in the cross-linked network,which might lead to aggregation[57,201].

Finally,the membrane extraction and coating steps should be performed as gently as possible.To maintain CM's function for the longterm,it is highly recommended that CM-NPs should be stored in an endotoxin-free solution with protease inhibitors at-80°C[203].

5.Validation and characterization of successful coating

Attachment of CM onto the surface of core materials can be validated by(1)physico-chemical,and(2)biological analyses.

5.1.Physico-chemical analyses

Size and surface charge observation:The fast and easiest way to estimate the attachment of a coating layer on a polymer NP is the observation of its size and zeta potential(surface charge)[204].The most common method to analyze the size of NPs along with their size distribution is the dynamic light scattering(DLS)method,and this method is non-invasive.Surface charge is usually observed by zeta potential analysis.CMs are negatively charged.Successful attachment of CM on polymer NPs increases the size and alters the surface charge.We have reported that CCM attachment on chitosan-coated PLGA NPs increases their particle size from 163.3±3.9 nm to 178.5±4.1 nm and decreases their zeta potential value from 21.2±1.5 mV to-26.5±2.1 mV(Fig.5a).Moreover,the time course of the size distribution profiles by the DLS method can help to understand the stability of CM coated NPs.Han et al.observed the stability and revealed that galactose-modified RBC coated PLGA NPs(named as NPs@RBC-Gala)were stable in PBS over 7 days[205].The NPs were more stable in pure fetal bovine serum(FBS)compared to uncoated ones over a period of 24 h since RBC coating could prevent serum protein adsorption on the NP surface(Fig.5b–d).

Morphological analysis:Morphology study of nanomaterials includes investigation of form,size,shape,and structure.Electron microscopy(Scanning Electron Microscopy(SEM)and Transmission Electron Microscopy(TEM))is considered as the gold standard for characterization of NPs.SEM provides 3D images of the NP surface to reveal the shape,size,pore size,and distribution of NPs.TEM provides 2D images of the sample and displays useful information about the inner structure,e.g.,morphology,crystal structure,and stress state.SEM usually applies relatively low accelerated voltages(up to 30 kV),whereas higher accelerated voltage is used for TEM(up to 300 kV).Sun et al.prepared macrophage CM-NPs using PLGA for treating breast cancer[206].The authors performed SEM and TEM analyses and obtained useful information on the attachment of CM onto the surface of PLGA(named as SCMNPs),as can be seen from Fig.5 e-f.Both PLGA NPs and SCMNPs were spherical in shape.Core-shell structure with smooth surface and 198 nm average particle size could be observed from the SEM and TEM results[206].TEM is mainly studied over SEM to characterize CM-NPs since CMs are composed of proteins and lipids and usually have different electron densities than the inner core material.

FTIR analysis:Fourier-transform infrared(FTIR)is a very effective tool to investigate the surface modification of NPs using biomolecules[209].FTIR analysis of CM coated NPs is reported in several nice articles[189,207].Li et al.analyzed CM-NPs using FTIR[207](Fig.5g).CM-NPs exhibited a typical peak at 1035 cm-1due to P––O stretching vibration resulting from the phospholipid of CM.Moreover,a stronger adsorption peak was visible at 1634 cm-1due to the amide group existing in the protein of CM.These peaks confirm the successful coating of CM onto the studied NP.

UV–vis analysis:Some CM coatings have been verified using UV–vis analysis.Characteristic UV–vis absorption peak of RBC membranederived vesicles is observed around 400 nm.NPs coated with RBCmembrane show absorption peak around that wavelength,declaring the successful RBC-membrane coating[210].Wang et al.prepared mouse RBC membrane coated magnetic PLGA NPs and observed their UV–vis absorption.The authors found that both RBC membrane and RBC coated NPs show absorption peaks around 400 nm,as shown in Fig.5h[208].CM coating imparts proteins on NPs surface which can be indicated by UV absorption at 280 nm unless the core material or entrapped drug contains amino groups.

Some other less commonly used analytical techniques to verify CM coatings are thermogravimetric analysis(TGA)[42],X-ray photoelectron spectroscopy(XPS)[42],and Raman spectroscopy[189].

5.2.Biological analyses

Surface protein analysis:In general,physico-chemical analyses are performed to confirm the attachment of CM,whereas biological analyses are performed to confirm the activity of biomolecules of the attached CM.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot are the most commonly used methods to detect and characterize CM coated NPs.A straightforward way to confirm CM attachment is to perform SDS-PAGE analysis of cell lysate,extracted CM,uncoated NPs,and CM coated NPs.For example,we have prepared CCM coated magnetic PLGA NPs,and coated NPs showed similar profiles to the cell lysate and CCM,confirming successful attachment of CCM onto PLGA(Fig.5i).The existence of specific makers is usually checked by Western-blot analysis.Upon successful coating,CM-NPs must exhibit the same specific marker that is shown by the source membrane.For example,we observed the expression of Histone H3(nucleus marker)and cytochrome C oxidase(COX IV,mitochondria marker)for CCM coated NPs.Histone H3 was distributed by cell lysate indicating that cell lysate contains nuclear components.However,CCM and CCM-NPs did not express Histone H3,which confirmed the high purity of extracted CCM(Fig.5j).It should be noted that presence of Histone H3 on CM-NPs indicates existance of nuclear component and indicates impurity.High expression of COX IV confirmed that our CCM-NPs could retain membrane antigens after the coating process(Fig.5j)[67].If NPs are coated by RBC membrane,western-blot analysis can confirm successful coating from the presence of CD47,which is highly expressed by RBC[211].

Moreover,by tagging the protein of CM by antibody,their presence on coated NPs can be determined using a flow cytometer.For example,Ben-Akiva stained RBC coated PLGA NPs by fluorescent CD47 antibody and analyzed by flow cytometry.RBC coated NPs showed a six-fold increase in fluorescent signal compared to the uncoated ones confirming the successful RBC coating[212].Asymmetric CM's correct topological orientation on the surface of NPs determines their functions.For characterizing the orientation of CM on the NP surface,colloidal gold conjugated antibodies or antibody decorated Dynabeads? can be used[202].

In vitro fluorescence colocalization study:In vitro fluorescence colocalization study can give two important information:(1)CM coating was successful or not,and(2)after cellular uptake,the coating was intact or damaged.For colocalization study,core NP and CM are both stained using different agents.For example,NPs can be labeled with red DiD dye.The CM can be stained by using different dyes,for example,DiO and fluorescein isothiocyanate(FITC).A recent colocalization study reported staining of macrophage membrane with green DiO dye and PLGA NPs with red DiD[213].Fang et al.reported the stability of CCM coated PLGA NPs by a colocalization study[59].PLGA core was stained with DiO and CCM with FITC.Confocal images of cells after uptake of NPs showed overlapping of fluorescent signals,revealing that the coating of CCM-NPs remained intact even after cellular uptake(Fig.5k).

Fig.5.Various physico-chemical and biological characterizations of CM coated NPs.(a)Size and zeta potential values of uncoated NPs(named as PIO-Dox-siRNA-P NPs)and CM-NPs(named as PIO-Dox-siRNA-PCSCM NPs)(reprinted with permission from Ref.[67],Copyright 2022,Elsevier).Stability observation of CM-NPs(named as NPs@RBC-Gala)(b)in PBS over 7 days by observing zeta potential,(c)in PBS over 7 days by observing size,and(d)in FBS over 24 h(b,c and d are reprinted with permission from Ref.[205],Copyright 2019 Royal Society of Chemistry).(e)SEM,and(f)TEM images of uncoated PLGA NPs and CM-NPs(named as SCMNPs);scale bar=200 nm.(e and f are reprinted with permission from Ref.[206],Copyright 2020,Elsevier).(g)FTIR spectra of various uncoated NPs(named as SSAP and SSAP-Ce6)and CM-NPs(named as SSAP-Ce6@CCM)(reprinted with permission from Ref.[207],Copyright 2018 Royal Society of Chemistry).(h)UV–Vis spectra of various NPs and CM-NPs(named as DOX@IRP@RBC NPs)(reprinted with permission from Ref.[208],Copyright 2020 Royal Society of Chemistry).(i)SDS-PAGE protein analysis of lysate,CCM and CCM-NPs.(j)Expression of markers of nucleus,mitochondria,and housekeeping gene detected by Western blot for lysate,CCM and CCM-NPs.(i and j are reprinted with permission from Ref.[67],Copyright 2022,Elsevier).(k)Colocalization of CCM coated PLGA NPs inside MDA-MB-435 cells(scale bar=10 μm)(reprinted with permission from Ref.[59],Copyright(2014),American Chemical Society).

6.Surface engineering:cell membrane vs PEG

Without a comparison between the natural CM coating and synthetic polymer coating,a complete picture of the coating of drug carriers cannot be depicted.Without such comparison,readers might find only the advantageous features of CM coating and might not compare them with the advantageous features of the synthetic polymer coating.

This section provides a brief comparison between the biomimetic CM coating and the synthetic polymer coating on polymer NPs.Among the synthetic polymers used for coating drug delivery systems(both inorganic and organic),water-soluble and biocompatible PEG is most extensively used[214,215].It is the only coating polymer that has received FDA approval to be used in therapeutics,foods,and cosmetics[216].Hence,to shorten this section,the comparison has been made with PEG coating only.The attachment of PEG to a molecule or surface of NP is known as“PEGylation”.The idea of PEGylation was first proposed by Davis et al.,who successfully PEGylated proteins in 1977[217,218].Before the first scientific article,a patent was filed(in January 1976)and got issued(in January 1977)on PEGylation of enzymes[217].Hence,PEGylation is a much older and well-documented method compared to the CM coating.The toxicity and immunogenicity of PEG are well documented since it has already gotten FDA approval.In 1990,Adagen?became the first FDA-approved PEGylated drug,whereas in 1995,liposomes containing Doxorubicin(Dox)became the first FDA-approved PEGylated NP[219].On the contrary,polymeric CM-NPs are yet to get FDA approval.Many PEGylated drugs reached clinical trials[220],whereas CM-NPs are yet to reach the clinical trial stage[174].Main PEGylation strategies to coat polymer NPs include:physisorption(non--covalent),grafting(covalent)and inclusion & affinity complexes(Fig.6a).PEGylated polymer NPs can also be prepared by directly using PEGylated polymer(e.g.,PEG-PLGA).Various strategies to coat NPs by PEG and their conformations are shown in Fig.6b.The two major conformations of PEG on the surface of NPs are“brush”and“mushroom”[221].The CMs can have“inside-out”and“outside-out(also known as right-side-out)”arrangements onto the surface of NPs[222].CM can be attached in two different ways on positively charged and negatively charged NP surfaces,as can be seen from Fig.6c.The CM is negatively charged and can significantly affect the surface charge of coated NPs.For example,positively charged chitosan NPs exhibit a negative zeta potential value after CM coating[177].On the contrary,PEG coating may slightly affect the surface charge of NPs[223].To shorten this section,some features have been listed in Table 2 to compare CM and PEG coatings on polymer NPs.

Table 2Comparison between CM and PEG coating of NPs.

7.Treatment of major cancers using CM coated NPs

In 2020,estimated new cancer cases were 19.3 million(Fig.7a),and death from cancers was 10 million,with lung cancer causing the leading number of deaths(Fig.7a)[1].This section will discuss the application of CM-NPs to treat the top few cancers causing leading deaths or top new cases.Some mouse tumor models are difficult and rarely studied or not studied to investigate CM coated NPs.Hence,our discussion was limited to the more studied cancer types out of the top cancer types shown in Fig.7a.In Fig.7b,we have depicted some common cancers and their common receptors,which are overexpressed and can be targeted by various DDSs,including CM-NPs.

Lung cancer shows the highest cancer mortality worldwide and contributes to 30% and 25% of cancer deaths in China and USA,respectively[234].Lung cancers have mainly two subtypes:small-cell lung carcinoma(SCLC;accounts for 15% of lung cancers)and non-small-cell lung carcinoma(NSCLC;accounts for 85% of lung cancers),and NSCLC can further be divided into three types(adenocarcinoma,squamous-cell carcinoma,and large-cell carcinoma)[235].Depending on the stage,lung cancer patients are treated by surgery,radiation,and chemotherapy.For stage IV NSCLC,the first-line therapy is the cytotoxic combination chemotherapy[235].Based on the literature survey,it was found that very few studies were reported on lung cancer treatment using CM coated NPs.Wu et al.treated chemo-resistant NSCLC by Dox and icotinib[236].Drugs were entrapped inside PLGA NPs prepared by the double emulsion solvent evaporation method,and the prepared NPs were coated with H1975 CM(named as MDI NPs).Extrusion(mini-extruder from Avanti,LF-1,Canada)was used to extract the H1975 CM.The size of uncoated PLGA NPs and MDI NPs were 124.2±1.10 and 157.5±2.45 nm,respectively.Over the 25 days treatment period,free Dox could reduce tumor volume by only 21.45%.On the other hand,MDI NPs showed 87.56% tumor inhibition,and tumor weight was 8.75-fold less for that group than the control group(PBS).Such a result clearly indicates that CM coated polymer NPs can be a very promising tool for targeted lung cancer therapy.Using platelet CM coating onto docetaxel(DTX)loaded PLGA NPs(DTX dosage of 5.0 mg(kg BW)-1),lung cancer volume could be reduced by 81.3%in the mouse model,whereas free DTX could reduce tumor volume by only 44.1%[237].Drug loading efficiency was quite high(92.4%),and CM coated NPs had a size less than 100 nm(98.2 nm),which was quite impressive,as was reported by Chi et al.[237].pH-sensitive drug delivery to liver cancer using CM-NPs showed good therapeutic results in vitro and in vivo.A research group prepared pH-sensitive poly(L-γ-gluta mylcarbocistein)-paclitaxel(named as PGSC-PTX)NPs and coated the NPs by RBC membrane[238](Fig.8a).It was hypothesized that the RBC coating would prolong circulation of CM-NPs(named as PGSC-PTX@RBCm)in the blood,and the formulated NPs will degrade in the tumor's acidic environment.CM and NPs were fused applying extrusion.The resulting CM-NPs(PGSC-PTX@RBCm)were 130 nm in diameter,and showed good dispersibility(PDI=0.108).The uptake of PGSC-PTX@RBCm NPs by NCI–H460 cancer cells was significantly reduced compared to the uncoated ones due to RBC membrane coating(mean fluorescence intensities of uncoated and RBC coated NPs were 1602.87 and 929.6,respectively)(Fig.8b).Treatment of NCI–H460 tumor-bearing mice using PGSC-PTX@RBCm NPs showed the best therapeutic result compared to all other groups tested,which the authors attributed to the long circulation time of the CM-NPs and pH-responsive release of PTX at the tumor site.Such results indicate that CM-NPs can be designed to prepare smart DDSs for efficient tumor treatment.

Liver cancer was the second deadliest cancer in 2020.Primary liver cancer has four subtypes:hepatocellular carcinoma(HCC),hepatoblastoma,intrahepatic cholangiocarcinoma(ICC),and combined HCC and ICC(cHCC-ICC)[240].HCC is the most common type of primary liver cancer,accounting for almost 90%of liver cancers.Main risk factors causing liver cancers include infection with hepatitis virus(B or C type),nonalcoholic fatty liver disease,alcoholic liver disease,and nonalcoholic steatohepatitis[241].Chemo-photothermal combination therapy was used very recently to treat HCC using platelet membrane coated polypyrrole(PPy)NPs.PPy acted as photothermal agents and was entrapped inside CM along with Dox(named as PLT-PPy–DOX).Upon NIR laser(808 nm)irradiation,PLT-PPy–DOX NPs heated the tumor area to 50°C or higher as can be seen from Fig.8c and d.Chemo-photothermal combination therapy could reduce tumor volume to less than 100 mm3,whereas for the control group,the volume was more than 800 mm3at the end of the treatment period[239].Using the chemotherapeutic drug,HCC has been treated by Liu et al.,who prepared HepG2 CM coated PLGA NPs for that purpose[242].The authors prepared fluorescently labeled(FITC)PLGA NPs entrapping Dox.2 mg of drug-loaded PLGA NPs were mixed with 1 ml of CM(0.5 mg/ml)and mixed by sonication(15 min;40 kW)to prepare CM coated NPs.Over the treatment period of 11 days,the fluorescence intensity increased with time in the tumor region,and Dox signal was strongest on the 11th day(Fig.9a).All organs observed in that study showed minimum tissue damage(Fig.9b).CM-NPs could reduce tumor volume by 90%and showed decent biocompatibility in vivo[242].A similar DDS was also tested by Xu et al.[243].The authors prepared Dox-loaded PLGA NPs using the double emulsion method and coated the NPs by HepG2 CM(name as Dox-HepM-PLGA).CM was collected from the source cell using extrusion.Free Dox,PLGA/Dox,and CM-NPs(CC/PLGA/Dox)showed 20.1,52.0,and 80.8%tumor volume reduction at the end of 17 days treatment period.Platelet membrane coating was also investigated to treat liver cancer in a mouse model using PLGA NPs to deliver the drug bufalin[244].

Fig.6.(a)Main strategies for PEGylation of the surface of a nanoparticle(reprinted with permission from Ref.[224],Copyright 2011,Wiley-VCH).(b)Various PEG coatings via the adjustment of PEG chain length,conformation,and density on a nanoparticle surface(reprinted with permission from Ref.[225],Copyright 2021,Wiley-VCH).(c)Schematic illustration showing electrostatic interactions between polymer NPs(-ve and+ve surface charge)and negatively charged CM(reprinted with permission from Ref.[226],Copyright 2019,Elsevier).

Currently,breast cancer is the biggest challenge to health regarding its incidence in new cancer cases(2.26 million cases in 2020),and it accounts for the highest death of female cancer patients worldwide.In general,breast cancers are of two types:invasive and noninvasive[245].Besides surgery and radiation therapy,chemotherapy is used to treat breast cancer patients.Commonly used chemotherapeutic drugs for breast cancers are Doxil?,DaunoXome?,Myocet?,Abraxane?,etc.Many researchers have studied breast cancer treatment using CM-NPs,which might be due to the simple mouse model.Nice work has been reported by Chen et al.,who prepared MCF-7 CM coated PLGA NPs that had homologous targeting and dual-modal imaging properties[35].The authors entrapped indocyanine green(ICG)which is a good imaging and photothermal agent.MCF-7 CM coated PLGA NPs were prepared using extrusion.Due to homologous targeting capability,CM-NPs showed significantly higher uptake by MCF-7 cells than uncoated ones which is evident from Fig.9c.Photothermal therapy(PTT)in combination with CM-NPs could kill cancer cells to a greater extent as was observed by dead cell staining(Fig.9d).Upon NIR laser irradiation after cellular uptake of ICG containing CM coated NPs,most of the cells died due to the photothermal effect.The NPs accumulated in the tumor,increased the tumor temperature up to 55.3°C(upon laser irradiation for 5 min),and completely inhibited the tumor growth within 6 days of treatment.

Treatment failure in breast cancer due to the development of drug resistance is a major problem.To overcome this problem,our group developed CCM coated PLGA NPs.Mcl-1-siRNAs were employed to MCF-7/ADR(drug-resistant breast cancer)both in in vitro and in vivo.Photothermal iron oxide(IO)NPs were prepared by covalently attaching ICG to the prepared IO NPs,which were entrapped inside PLGA NPs along with Dox and Mcl-1-siRNAs.The prepared CCM-NPs(named as PIO-DoxsiRNA-PCSCM NPs)had a hydrodynamic diameter of 178.5±4.1 nm and zeta potential of-26.5±2.1 mV.These NPs could be successfully targeted to the tumor site by an external magnet.Upon NIR laser irradiation(808 nm),cell viability significantly decreased in vitro.In the MCF-7/ADR tumor model,this group could inhibit almost 80% tumor growth upon laser irradiation,showing the excellent therapeutic outcome.Mcl-1-siRNAs could reduce Dox efflux in vitro and increased cell death remarkably.There are dozens of exciting works on breast cancer treatments using CM-NPs,and some of them are listed in Table 3.

In recent years,several research groups have tested less common cancer models using CM-NPs.A very recent work reported the treatment of multi-drug resistant(MDR)esophageal cancer using TE10 CM coated PLGA NPs[246].The authors prepared Dox and curcumin loaded PLGA NPs using the solvent volatilization method.CM coating was performed using extrusion and sonication to prepare PMPNs(PEG-TE10@PLGA@-DOX-Cur NPs),which could avoid the rapid clearance by reticuloendothelial system(RES)to a satisfactory extent and prolonged the circulation time.PMPNs exhibited a good therapeutic effect on the MDR esophageal carcinoma tested,because they could be targeted to tumor sites,reduced the side effects,and increased the residence time of chemotherapeutic drugs in the tumor.Such results indicate that CM coated polymer NPs can be very attractive and effective tools for cancer treatments.

To concise the section,Table 3 has been presented to show some additional results.

Table 3Summary of various CM coated polymer NPs that are used for the treatment of major cancers.

8.Drug targeting to tumors:strategies

Nanocarriers have revolutionized cancer treatments due to improved delivery of drugs at tumor sites and less accumulation of drugs in healthy organs compared to free drugs.Potency,plasma half-life time,and therapeutic index of free drugs can be increased by 100s folds using carriers[264,265].For example,Depo/methotrexate(a lipid-based DDS of anti-cancer drug Methotrexate)could increase plasma half-life time of methotrexate from 0.53 to 100 h,and Depo/methotrexate could increase the potency of a single Methotrexate dose by 334-fold[266].

A significant problem of cancer treatment using free drugs is their accumulation in healthy organs which can cause severe side effects.The endothelial junctions among the cells of normal vasculature usually range from 5 nm to 10 nm[267].Most commonly used anti-cancer drug molecules possess a size well below that limit and can easily accumulate in healthy tissues.Moreover,small drug molecules also show poor accumulation in tumors and high renal clearance[268].Hence,drug targeting to the tumor site is a prerequisite to achieve a satisfactory therapeutic outcome.Although targeted drug delivery to the tumor using free drugs[269]or by using NPs made of drug molecules[270]have been tested to some extent,the majority of research works report drug targeting using carriers.This is because the large surface area of nano-DDSs offers plenty of options to tune their surface by targeting molecules.In general,it is more expensive to modify drug molecules compared to modifying a nano-DDS entrapping the same drug[17].

It is well known that drug targeting to tumor sites relies on two different mechanisms:(1)passive targeting and(2)active targeting.

Passive targeting:Passive targeting directly depends on the blood circulation parameters(e.g.,blood flow,pressure,resistance,etc.).The passive targeting of DDSs to tumor sites is achieved by utilizing their size taking advantage of the abnormal and defective structure of tumor vasculature that can be easily extravasated by small DDSs resulting in“enhanced permeability”.In tumors,the lymphatic vessels are either absent or ineffective,which is responsible for inefficient drainage from the tumor tissues resulting in“enhanced retention”.Inefficient drainage,in turn,helps in the accumulation of drugs to a higher concentration.These two phenomena are collectively known as the enhanced permeability and retention(EPR)effect[271].Due to the insufficient supply of oxygen and nutrients,tumors show extensive angiogenesis.Hence blood vessel formation in tumors is chaotic,causing large gaps in the junctions of endothelium cells.As mentioned earlier,the endothelial junction among the cells of normal vasculature ranges 5–10 nm,whereas these gaps are comparatively large in tumor tissues(100–780 nm)[267].This gap is well suitable for various DDSs to extravasate and thereafter enter tumor cells to exhibit therapeutic effects.Until now,no active targetingtumor nanocarriers have received clinical approval,and all the clinically approved ones are passive targeting nanocarriers,although less than 1%of nanocarriers accumulate at a tumor site by passive targeting[272].

Fig.7.(a)Percentage of various cancers in new cases(upper one)and deaths(lower one).(b)Common cancers and receptors overexpressed by their cancer cells.

Active targeting:Active targeting strategy exploits the surface functionalization of the DDSs by using ligands having strong specificity and affinity for overexpressed receptors and molecules on diseased tissues and cell surfaces(e.g.,folate receptor,HER2,CD44,etc.).Before binding with the receptors of cancer cells,DDSs must reach the tumor site utilizing the mechanism of passive targeting[273].The main reason for the poor clinical translational results of active targeting cancer DDSs compared to passive targeting ones is that active targeting is investigated in xenografted mice models with dense vasculature,and such model does not recapitulate most solid tumors in humans[272].Moreover,studied tumors are a few centimeters in size,and nano-DDSs bypass a tumor of that size in sub-seconds(our blood flow rate is>5 L/min and approximately 1.5–33 cm/s in capillaries and venules)[274].For achieving active targeting,the surface of DDSs is usually modified by peptides,small proteins,antibodies,oligosaccharides,and aptamers.There aremany nice reviews on active drug targeting to the tumors,which readers can go through,e.g.,Refs.[272,275,276].

Fig.8.(a)Schematic illustration showing the preparation of PTX loaded RBC coated CM-NPs(named as PGSC-PTX@RBCm),and(b)Cellular uptake patterns of uncoated NPs and CM-NPs(named as PGSC-PTX@RBCm)by NCI–H460 cells.(a and b are reprinted with permission from Ref.[238],Copyright(2017)American Chemical Society).(c)Infrared thermal images of tumor-bearing(Huh7 orthotopic tumor)mice that went under NIR irradiation after various treatments with uncoated and CM-NPs(named as PLT-PPy–DOX),and(d)Temperature curves(based on infrared thermal images)of various mice groups shown in(c).(c and d are reprinted with permission from Ref.[239],Copyright 2020 Royal Society of Chemistry).

Fig.9.(a)Ex vitro fluorescence images showing organs and tumor tissues of HepG2 tumor-bearing mice after the treatment with CM-NPs(name as Dox-HepM-PLGA)for various time periods(1,3,7,and 11 days),and(b)Stained(H&E)histological sections of the respective tissues and organs shown in(a)(Scale bar:50 μm).(Adapted with permission from Liu et al.(2019).This is an open access article distributed under the terms of the Creative Commons Attribution(CC BY-NC)license (https://creativecommons.org/licenses/by-nc/4.0/).Copyright ?Ivyspring International Publisher”).(c)Confocal images showing MCF-7 cells after the treatment with free ICG,ICG-containing PLGA NPs(INPs),and CM coated INPs(ICNPs),and(d)Fluorescence images of MCF-7 cells after the treatment with free ICG,INPs,and ICNPs along with NIR laser irradiation(dead cells were stained red using PI and viable cells were stained green)(c and d are reprinted with permission from Ref.[35],Copyright(2016)American Chemical Society).

8.1.Targeting CM-NPs to the tumor site

Till now,CM-NPs have been targeted to tumors by applying three major strategies:(1)homotypic recognition(2)peptide-based targeting(3)magnetic targeting,or a combination of them.

Homotypic targeting utilizes the concept of“self-recognition”,and CCM shows high affinity towards the same type of cancer cells[277,278].The detailed mechanism is yet to be fully understood.Since cancer cells develop strong contacts plus adhesive interactions,components of CM proteins,receptors,and cognate ligands help in homotypic recognition facilitating homotypic targeting[42,67,278].Serval studies discussed in the previous section reported homotypic targeting to various cancers and are not repeated here.A study shows that MDA-MB-435 CCM coated PLGA NPs can show significantly higher uptake by MDA-MB-435 cancer cells compared to uncoated PLGA NPs[59].Among two different CM coated NPs(RBC and MDA-MB-435),uptake by MDA-MB-435 cells was higher for MDA-MB-435 coated NPs over RBC coated NPs.The result confirms and supports the homotypic targeting capability of CCM coated NPs.MDA-MB-435 coated NPs showed 40-and 20-fold higher cellularuptake over RBC coated and uncoated PLGA NPs,respectively(Fig.10a).Utilizing a homotypic targeting strategy,NCI–H1299 tumors were treated by using CM-NPs which could successfully deliver siPlk1 for gene therapy[248].Many other studies have reported higher uptake of CCM-NPs when NPs are coated by the same type of cancer cells[254,255,279,280].

More than 150 peptide drugs are already in active clinical development,whereas many of them have already got approval making peptidebased targeting up-and-coming tools[281].Researchers widely use peptides,especially cell penetrating peptides(CPP),in targeting various DDSs since their attachment onto the surface of NPs is not very difficult.Excellent work has been reported by Chai et al.on peptide-based brain targeting of CM coated Dox loaded PLGA NPs[259](Fig.10b i)).The authors used CDX peptide,which can be derived from candoxin and this peptide exhibits good binding affinity with nAChRs(nicotinic acetylcholine receptors)expressed on the brain's endothelial cells.The authors did not attempt homotypic targeting and did not coat NPs with the same type of cells.Instead,RBC coating was applied onto PLGA NPs,and CDX peptide was used to target U87 cells.By using a coupling reaction,CDX was attached to biotin-PEG3500 to get biotin-PEG3500-DCDX(Fig.10b ii)).Dox loaded PLGA NPs were prepared by the emulsion solvent evaporation method,and extrusion was applied to prepare RBC coated PLGA NPs.Finally,they prepared CDX attached RBC coated PLGA(DCDX-RBCNPs)NPs by a two-step process.First,CMs of RBC were incubated with streptavidin-PEG3400-DSPE,and then they were used to coat PLGA NPs to prepare streptavidin-inserted RBC coated NPs

(RBCNPs).Second, streptavidin-inserted RBCNPs and biotin-PEG3500-DCDX were incubated to ensure binding between streptavidin and biotin,which ultimately resulted inDCDX-RBCNPs.Prepared NPs were spherical with core-shell structure and had~75 nm size as was observed by TEM(Fig.10b iii).By analyzing the cell's mean fluorescence intensity,it was found that the cells incubated withDCDX-RBCNPs show 2-fold higher intensity compared to the cells incubated with the RBCNPs group(Fig.10b iv).The novel system could cross the blood brain barrier(BBB)and significantly improved glioma-bearing mice's survival due to efficient drug delivery by targeting NPs with CDX peptides.In another study,Su et al.applied a similar approach and prepared iRGD peptide attached RBC membrane coated PTX-loaded polymer NPs[253].Due to efficient tumor targeting,the therapeutic outcome was impressive in vivo.Developed NPs could reduce breast cancer to a significant extent due to iRGD peptide-based targeting.RGD modified polymer NPs were also used to deliver PTX to treat breast cancer in another recent study[252].

Fig.10.(a)Investigation of homotypic targeting capability of PLGA NPs,RBC coated PLGA NPs,and MDA-MB-435 coated PLGA NPs by observing their uptake by MDA-MB-435 cells i)Fluorescent imaging,ii)Flow cytometric analysis,and iii)Quantification of fluorescence intensities detected in“ii)”.(reprinted with permission from Ref.[59],Copyright(2014)American Chemical Society.(b)i)Preparation of DCDX-RBCNPs for brain tumor targeting,ii)chemical reaction for the preparation of biotin-PEG3500-DCDX,iii)TEM images of various NPs ((i) RBCNPs, (ii)DCDX-RBCNPs,(iii)RBCNPs incubated with Au NPs-linked nAChRs,and(iv)MeO-RBCNPs incubated with Au NPs-linked nAChRs;Scale bar=50 nm),iv)in vivo biodistribution of RBCNPs and DCDX-RBCNPs in the brain.The left side image shows the average radiant efficiency of brains in healthy nude mice,whereas the right side one is for glioma tumor-bearing mice(reprinted with permission from Ref.[259],Copyright 2017,Elsevier).

Another promising targeting approach is magnetic drug targeting with the help of an external magnetic field.This approach can offer additional advantages,including diagnosis and observation of biodistribution by magnetic resonance imaging(MRI).By using CM coated Dox loaded magnetic nanoparticles(MNPs),UM–SCC–7 tumor was treated by Zhu et al.,who applied magnetic targeting for improved drug delivery[277].Our group applied both magnetic and homotypic targeting of Dox to treat drug-resistant breast cancer[67].We preparedPLGA NPs entrapping photothermal MNPs(PIO),Dox,and siRNA using the double emulsion solvent evaporation method.PLGA NPs were first coated with chitosan(CS)to prepare PLGA-CS NPs,and then MCF-7 CM was used to coat the NPs(named as PIO-Dox-siRNA-PCSCM NPs)(Fig.11a).In vitro and in vivo targeting by an external magnet and homotypic recognition showed the best therapeutic effects among the groups tested.More than 95 and 75% viability could be reduced for MCF-7 and MCF-7/ADR cells,respectively,using PIO-Dox-siRNA-PCSCM NPs upon exposure to the external magnetic field and NIR laser.The tumor inhibition rate was 79.1% for the same group,whereas the saline-treated control group exhibited a 12.3-fold increase in tumor volume(Fig.11 b-d).Recently,U-251 CM coated magnetic NPs have been tested as a theranostic system for glioblastoma[282].

Fig.11.(a)Schematic illustration showing the preparation of CCM coated PLGA-CS NPs(named as PIO-Dox-siRNA-PCSCM NPs).(b)Photographs of excised tumors,(c)Change in tumor volume,and(d)Tumor inhibition rate in the breast cancer mouse model after the treatment with PBS(control),free Dox,and the PIO-Dox-siRNAPCSCM NPs with and without exposure to the external magnetic field and NIR laser.(reprinted with permission from Ref.[67],Copyright 2022,Elsevier).

We expect more studies to be reported in the near future on various targeting of CM-NPs since current studies mostly utilize homotypic targeting.

9.Challenges and future perspective

CM coating technology is a top-down approach and quite a new area of research.Hence,it is quite expected that there remain some significant challenges in this new area of research.Clinical translation of CM-NPs is very challenging.The two most used techniques to prepare CM-NPs are extrusion and ultrasonication,which can result in batch-to-batch variation or hinder a large-scale production required for translation.The general procedure to collect CM includes cell lysis and/or sonication and centrifugation.Loss can result in each step,and each step must ensure the intactness of membrane components.This issue can be well addressed if an automated system is developed.Moreover,it must be carefully observed that during the camouflaging process,the protein sequence of CM proteins should not change due to the use of the hypotonic solution or lysis buffer in vitro[36].

Cell phenotype maintenance on a large-scale is not easy.Cell phenotype might get altered during expansion[203].Monitoring of changes in phenotype is not available in most of the existing ex vivo culture systems.Moreover,monitoring of changes in phenotype will increase the workload.In large-scale production,controlling the quality and purity of the large cell population from which CM will be collected,will be very challenging.

Some cancer-associated proteins existing on the CCM are not easy to remove,and their presence on CM-NPs might trigger immune responses and undesired side effects[36].Moreover,preventing CM and CM-NPs from biological(e.g.,virus and bacteria)and chemical contaminants remains another challenge.If the CM collection step(s)gets contamination and virus or bacteria can enter the step(s),CM-NPs will ultimately trigger the undesired immune response and result in side effects.

Clinical translation will require thorough investigations of the potential adverse effects.Till now,the scientific literature on the adverse effects of CM-NPs is not enough for a clear conclusion on their short-and long-term effects after in vivo administration.Uncertainties in the biodistribution and pharmacokinetics will hinder the clinical translation.Long-term accumulation,decomposition,metabolism,excretion,and safety/toxicity of CM-NPs in vivo must be thoroughly investigated to get clinical approval.Moreover,the use of unstandardized protocol and immature manufacturing processes(e.g.,membrane protein's improper orientation)will result in poor and unstable therapeutic outcomes and side effects[36].

Functionalization of CM-NPs,e.g.,to prepare smart DDS,will be challenging.For example,to achieve triggered release,CM-NPs might need to be further modified.Further modification of CM-NPs might damage or alter the membrane properties and might trigger undesirable side effects.Hence,surface modification of CM-NPs will be challenging,unlike surface modification of PEGylated NPs.

Despite some remaining challenges,it is undeniable that this technology has a bright future if the challenges can be overcome.Future research should focus on the challenges,to overcome them,and possibly to develop new methods which can be used in large scale production with precise/automated control.In brief,the challenges,associated problems,and possible solutions to overcome them are listed in Table 4.The unique properties of biomimetic CM-NPs include extended blood circulation time,immune evasion,homologous targeting,etc.CM-NPs have shown success in various drug delivery,especially for cancer therapies(chemo-,immuno-,photothermal,photodynamic,etc.),inflammation therapy[283],detoxification,etc.The core materials investigated for CM coating include various polymers,liposomes,and inorganic materials(especially silica and iron oxide NPs).Some cells have been investigated,and many more are yet to be investigated,which might surprise the scientific community with some remarkable features.Continuous efforts and dedication of nanotechnologists,pharmacists,and cell biologists are expected and required to boost this field.We believe and hope that in thenear future,CM-NPs will offer invaluable contributions to human health and might change the landscape of cancer nanomedicine.

Table 4Challenges for CM coating technology,associated problems,and possible solution to overcome them.

10.Conclusion

CM coated cancer nanomedicines integrate both versatile properties of NPs and intrinsic properties of cells to deliver drugs to tumors.CM-NPs have already exhibited their capabilities to target diseased sites,immune evasion,extended blood circulation,and the reduction of interaction with protein corona.In this review,we have depicted the CM coating technology,the methods to prepare CM coated NPs,the techniques to characterize them,their exciting applications in cancer treatments,possible ways to target them to tumor sites,and a comparison with synthetic PEG coating.This area of research is comparatively new but blooming,and there has been a very sharp increase in the number of published works over the last five years.Although some types of CMs have been tested so far,many more are expected to be tested in the future.However,future success lies in precise reproduction with minimum batch-to-batch variation,precise preparation methods,and exploring the adverse effects of this technology-based DDSs.We expect future research to exploit the convergence of nanotechnology,material science,bioengineering,pharmaceutical science,and medicine to address these challenges and to develop such DDSs considering cost and translation.Despite some issues to be solved,this emerging field has the enormous potentiality to change the landscape of future cancer nanomedicines.

Declaration of competing interest

There are no conflicts to declare.

Acknowledgement

We thank Ayesha Akhter(School of Science and Technology,Georgia Gwinnett College,USA)for assisting in language editing.