Advancing cell surface modification in mammalian cells with synthetic molecules

Biological cells, being the fundamental entities of life, are widely acknowledged as intricate living machines. The manipulation of cell surfaces has emerged as a progressively significant domain of investigation and advancement in recent times. Particularly, the alteration of cell surfaces using meticulously crafted and thoroughly characterized synthesized molecules has proven to be an efficacious means of introducing innovative functionalities or manipulating cells. Within this realm, a diverse array of elegant and robust strategies have been recently devised, including the bioorthogonal strategy, which enables selective modification. This review offers a comprehensive survey of recent advancements in the modification of mammalian cell surfaces through the use of synthetic molecules. It explores a range of strategies, encompassing chemical covalent modifications, physical alterations, and bioorthogonal approaches. The review concludes by addressing the present challenges and potential future opportunities in this rapidly expanding field.


Introduction
][3][4] Over the past few decades, the manipulation of cells has provided a powerful tool to enhance our understanding of the underlying mechanisms governing various biological behaviors in basic research and has also promoted the development in biomedical applications, such as medical diagnosis and cellbased therapy. 57][8] Immune cells, including T cells and natural killer (NK) cells, have emerged as highly prominent candidates for tumor immunotherapy owing to their specic cytotoxicity against tumor cells while sparing normal cells. 9,10espite these exciting achievements, the functions of natural cells themselves are limited.The cell surface, also known as the cell membrane, is a highly heterogeneous and dynamic milieu comprising lipids, proteins, carbohydrates, and their complexes, which governs numerous intracellular and He Yang   He Yang received his BS degree from the Soochow University in 2019, and currently is pursuing his PhD degree under the supervision of Prof. Hong Chen and Prof. Gaojian Chen at the College of Chemistry, Chemical Engineering and Materials Science, Soochow University.His research interests are in cell surface modication and polymer biomimetic materials.extracellular processes. 11Simultaneously, the complex cell surface provides plenty of opportunities for further modication aimed at achieving particular functionalities, a process referred to as cell surface modication.This process serves as a powerful means to facilitate the biomedical application of natural cells. 124][15] Chemical manipulation of cell behavior and function through modication of cell surfaces using precisely synthesized and well-characterized synthetic molecules, such as polymers, is a captivating area of research. 16arious strategies have been devised for this purpose.][18] Hence, this paper provides a comprehensive review of the latest advancements in strategies related to the modication of cell surfaces accompanied by a discussion on their biomedical applications, with a particular emphasis on developments from 2017 onwards.These strategies encompass a range of approaches, including chemical covalent approaches, physical techniques, and bioorthogonal methods for synthetic molecules (Fig. 1).The synthetic molecules discussed include specic functional groups, synthetic functional small molecules, synthetic polymers, synthetic nanoparticles, synthetic cell coatings, and synthetic DNA, among others.Lastly, the paper discusses the existing challenges and potential future prospects in this rapidly expanding eld.

Chemical covalent modification
Chemical covalent modication is a strategic approach that entails the utilization of the chemically reactive functionalities found on the surface of the cell membrane.The cell membrane, a multifaceted chemical structure composed of lipids, proteins, carbohydrates and other components, offers a diverse array of functional groups that can be employed for chemical covalent binding. 19Previous researches have predominantly focused on employing amine, thiol, and vicinal diol groups present on amino acid residues within proteins or sugar residues as the most frequently utilized groups (Fig. 2). 20The stable attachment of synthetic molecules and the absence of cell pretreatment are the primary benets of this approach, making it a simple yet effective method for modifying cell membranes.However, it is widely recognized that directly modifying cell membranes with reactive functional groups through covalent bonds can potentially impair the functionality of membrane proteins and subsequently impair cellular functions.Consequently, when employing this strategy, careful attention must be paid to both cell viability and effector functions.
widely used for chemical modication of cell membrane surfaces due to their ease of chemical covalent modication and mild reaction conditions.The amine-mediated covalent binding strategy can be achieved through two primary pathways: acylation or alkylation.Generally, these reactions exhibit rapidity and selectivity, resulting in the formation of stable bonds (such as amide or secondary amine bonds) and high yields.
Among various kinds of reagents, N-hydroxysuccinimide (NHS) ester is the most frequently used to covalently bind to -NH 2 on cell membranes.Recently, Cai et al. modied human umbilical vein endothelial cells (HUVECs) and human skin broblasts (HSFs) with succinimide ester-methoxy polyethylene glycol (NHS-mPEG), resulting in a signicant enhancement of cell migration ability and motility through reduction of the focal adhesion area. 21In a separate study shown in Fig. 3A, Wang et al. employed acrylic acid NHS ester (NHS-AA) to immobilize vinyl onto cell membranes, followed by free radical polymerization to covalently attach polymers to the membrane. 22Aer subsequent ion exchange and electroless deposition (ELD), the polymer-functionalized cells could be converted into metallic biocomposites, which can be applied in the elds of biosensors, electronics, and energy.Sulfo-NHS ester is a more suitable reagent for covalent reactions with -NH 2 groups on the cell membrane due to its enhanced water solubility and negative charge, which reduces the transmembrane permeability of the sulfo-NHS ester.For instance, Jasiewicz et al. employed sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo SMCC), a heterobifunctional crosslinker, to modify MSCs by covalently binding to the amines on the cell membrane and subsequently decorating them with heterodimerizing leucine zippers. 235][26][27][28][29] In addition to NHS ester derivatives, other types of reagents have been developed for covalently binding to -NH 2 including cyanuric chloride and benzotriazole carbonate. 30,312 Thiol-mediated covalent modication strategy Thiol groups (-SH), mainly located on the cysteine residues of amino acids in proteins, are one of the most potent nucleophiles, stronger than amino groups.Thiol groups are frequently employed for the covalent modication of cell membranes.Maleimide derivatives, which form stable thioether bonds with thiol groups through an energetically favorable Michael addition reaction, are the most widely used reaction reagents because they exhibit high stability and chemoselectivity with thiol groups.The signicant advantage of this strategy lies in the extensive availability of commercially accessible reagents and linkers.
Early research was conducted by Irvine's team which focused on surface modication of various cell types containing thiols.3][34][35][36] Recently, Wang et al. developed PEGylated solid lipid nanoparticles functionalized with maleimide end groups (SLN-PEG-Mal). 36As is shown in Fig. 3B, by exploiting the reaction between maleimide and sulydryl groups on the surface of RBCs, the researchers successfully enhanced the adsorption of modied nanoparticles onto RBCs, leading to signicant alterations in the properties and morphology of RBCs.Moreover, these nanoparticle-loaded RBCs exhibited a remarkable ability to be engulfed by macrophages, thereby demonstrating promising potential for targeted drug delivery to macrophages.Wang et al. utilized 2-iminothiolane (Traut's agent), a thiolation reagent, to introduce extra free thiol groups by capping primary amines with thiol groups.This allows them to modify platelets with PD-L1 antibody, thereby reducing postsurgical tumor recurrence and metastasis. 35esearch has been conducted to combine maleimide with other functional components in order to develop multifunctional nanoparticles. 38For instance, Luo et al. synthesized double-bound magnetic nanoparticles (DBMN) containing PEG-Mal, hyaluronic acid (HA), and Fe 3 O 4 . 38Following a simple incubation, DBMN was able to anchor onto the cell membrane through a Michael addition reaction between the Mal component and sulydryl groups on the T cell surface, resulting in magnetized T cells (DBMN-T).Under external magnetic eld guidance, DBMN-T exhibited excellent targeting ability.Additionally, HA could bind to highly expressed CD44 on tumor cells, promoting recognition and killing of tumor cells.
0][41][42][43] Wayteck et al. incorporated thiol-reactive phospholipids into liposome bilayers with a pyridyldithiopropionate (PDP) head group, which was capable of forming reducible disulde bonds with thiol groups exposed on the cell surface, thereby enabling the covalent coupling of liposomes. 39

Vicinal diol-mediated covalent modication strategy
Vicinal diol groups are abundant on the cell membrane and primarily originate from sialic acid (SA), mannose, and galactose residues within glycoproteins and the extracellular matrix.Phenylboronic acid (PBA) derivatives form unique dynamic covalent bonds with vicinal diol groups of the cell membrane and are affected by pH, making them of substantial interest.However, only sialic acids can be efficiently coupled to PBA under physiological conditions, while other diol groups require alkaline reaction conditions with a pH value higher than the pK a of PBA.Therefore, most studies have focused on the reaction between PBA derivatives and SA on the cell membrane surface.Tao and colleagues reported a novel uorescent polymer containing PBA through the combination of multicomponent reactions (MCRs) with reversible addition-fragmentation chain transfer (RAFT) polymerization. 44,45Specically, the Hantzsch reaction, a classical four-component reaction, was carried out simultaneously with RAFT polymerization to create innately uorescent 1,4-dihydropyridine (1,4-DHP).The formed uorescent polymer is suitable for cell membrane conjugation and imaging through the interaction between phenylboronic acid and sialic acid on the cell membrane.In our laboratory, we utilized a copolymer containing PBA groups to modify silicon nanowire arrays which exhibited a high capture capacity for cells overexpressing SA on the membrane.This modication also allowed for high efficiency of intracellular delivery of diverse biomacromolecules. 46dditionally, overexpression of SA has been demonstrated in various tumors, including lung, melanoma, colon, and breast cancers. 479][50][51][52][53][54] Li et al. developed self-assembled nanorods of PBA-functionalized pyrene (Py-PBA NRs), which possess a highly efficient and specic imaging feature of SA on the cell membrane.Three cell lines with different expression levels of SA were utilized to demonstrate this imaging ability.Additionally, the nanorods exhibited efficient generation of 1 O 2 under two-photon irradiation, providing potential possibilities for tumor therapy (Fig. 3C). 37Furthermore, PBA derivatives with low pK a values have been developed to enhance the applicability of this strategy to cell species characterized by low expression levels of sialic acids.A series of PBAs with different substituents were synthesized, and it was demonstrated that the introduction of electron-withdrawing groups, such as uoro and nitro, effectively decreased the pK a even to 4.2. 55However, it should be noted that the optimal binding pH may not always exceed the pK a of PBAs, particularly in complex multicomponent systems. 56n addition to PBA derivatives, benzoxaborole (BA), a cyclic hemi-ester of boronic acid, can also be utilized for cell surface modication via the covalent reaction with vicinal diol. 57orgese et al. have successfully modied supramolecular polymers containing BAs onto the surface of human RBCs via the covalent reaction between BAs and SA. 58The specic interactions between functional copolymers and the cell surface were further visualized in real time using total internal reection uorescence microscopy.

Other functional groups-mediated covalent modication strategy
Carboxyl groups are abundantly present on the cell membrane, mainly distributed at the residues of aspartic acid (Asp) and glutamic acid (Glu) within membrane proteins, as well as at the C-terminus of polypeptide chains.However, the modication of membranes using carboxyl groups necessitates pre-activation of these groups, typically employing an activator known as 3-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-amine (EDC), which causes signicant harm to mammalian cell viability.Consequently, the utilization of carboxyl groups for cell modication is oen limited.Recently, Ma et al. developed a novel probe (3-phenyl-2H-azirine) that effectively labeled carboxyl groups on the surface of living cells.This presented new possibilities for chemical modication utilizing carboxyl groups present on the cell membrane. 59n addition to utilizing existing groups for direct modication of the cell membrane, strategies have been devised to convert commonly present but difficult-to-modify functional groups into easily modiable ones through mild oxidation or reduction reactions on the cell surface under gentle conditions.1][62][63][64][65] Recently, Liu et al. proposed a novel cell surface engineering platform using classical thiazolidine chemistry to combine small molecules containing aminothiol moieties with cells pretreated with aldehyde groups on their surface by NaIO 4 . 64fforts have also been devoted to converting disulde bonds (S-S) on cell membranes to thiol groups and tris(2-carboxyethyl) phosphine (TCEP) is a widely used mild reducing agent in this strategy.[68][69][70]

Physical modification
In addition to the chemical covalent binding strategy, physical approaches such as hydrophobic insertion, membrane fusion, electrostatic interaction, and layer-by-layer self-assembly offer versatile and easy ways to introduce synthetic molecules to the cell membrane while maintaining cellular physiology.

Hydrophobic insertion
0][101][102][103][104][105] They can be categorized into single and multiple anchors based on the number of hydrophobic anchors.Shi et al. constructed a polyvalent antibody mimic (PAM) for engineering NK cells with highly efficient targeting, adhesion and killing effects for tumor cells. 71The DNA initiator (DI) with a single anchor was displayed on the NK cell membrane by the hydrophobic insertion approach.Subsequently, a DNA scaffold was synthesized and hybridized with multiple aptamers in situ forming PAM-engineered NK cells.Sun et al. reported a DNA-assisted bottom-up self-assembly approach for achieving precise control over the lateral and vertical distributions of T cell activation ligands on RBCs and constructing RBCs-based articial antigen presenting cells (aAPCs) which could effectively activate and expand T cells. 107NA strands with a cholesterol end group were inserted into the membranes by hydrophobic interaction and then bound with T cell activation ligands through specic DNA hybridization as well as biotin-avidin interaction.The vertical distributions of T cell activation ligands can be easily manipulated by adjusting the length of DNA strands, while the lateral distributions were achieved through biotin-avidin interaction.The subsequent study shown in Fig. 4 employed the approach to construct lymphocyte-based aAPCs exhibiting homologous targeting functionality for personalized cancer immunotherapy.106 Zhao et al. developed a surface-anchored framework for sheltering the epitopes on Rhesus D (RhD)-positive RBCs.102 RBCs were modied with horseradish peroxidase containing a single oleyl chain via hydrophobic insertion, thereby catalyzing the reaction of H 2 O 2 to construct a polysialic acid (PSA)-tyramine framework on the RBC membrane.The crosslinking framework successfully achieved transfusion of the modied RBCs to RhDnegative recipients without eliciting immunogenicity, by effectively balancing the modied uidity of RBC membranes and shielding of RhD antigens.
In addition to the single anchor, hydrophobic insertion moieties with more anchors were developed.Niu et al. reported the rst effort for cytocompatible controlled radical polymerization (CRP) techniques. 72Chain-transfer agents

Chemical Science
Review with a two-tailed hydrophobic anchor, 1,2-distearoyl-snglycero-3-phosphoethanolamine (DSEP), were successfully modied on the cell membranes with the hydrophobic insertion strategy, thereby realizing polymerization to be initiated directly in the live cell surface while maintaining high cell viability.The strategy effectively enhanced the efficiency of graing polymers compared to the traditional graing-to methods and offered novel possibilities for modulating cellular interactions.
Additionally, the method was also utilized for T cell modication with liposomal nanoparticles, as described in Hao et al.'s investigation. 73The tetrazine (Tre) groups with two-tailed lipids (DSPE) were inserted into T cells and subsequently drug liposomes with bicyclo nonyne (BCN) were modied on the cell membranes of T cells via click reaction while preserving the intact functionality of T cells.A platform (Fig. 5A) for cell membrane engineering with modular polymers was developed by our group. 91The study employed cholesteryl-methacrylate as one of the monomers for Fig. 5 Representative examples based on hydrophobic insertion and membrane fusion strategies.(A) Schematic illustration of the construction of a platform for the manipulation of cell behaviors, by using hydrophobic insertion to bind modular polymers to the cell surface. 91Copyright 2019, American Chemical Society.(B) Schematic showing the biomimetic LiFT approach to engineer the plasma membrane by membrane fusion. 108Copyright 2022, John Wiley & Sons, Inc.

Chemical Science Review
constructing modular polymers with multiple anchors through one-pot RAFT copolymerization, along with deoxy-2-(methacrylamido)glucopyranose (MAG) as a hydrophilic monomer and adamantane carbonyl methacrylate (Ada) as a guest monomer.In addition to the introduction of functional molecules through host-guest units, the residence time of the modular polymers could also be regulated on the cell membrane by adjusting the content of cholesterol modules.
The hydrophobic insertion strategy is considered a simple, powerful, and less invasive approach for cell surface modication.However, functional synthetic materials introduced to the cell membrane surface through hydrophobic insertion are prone to loss during membrane ow and endocytosis, thereby limiting their long-term presence on the cell membrane.The hydrophobic insertion moieties with multiple anchors may offer a promising strategy for achieving relatively stable and long-time modication.

Membrane fusion
Unlike the strategy of hydrophobic insertion into the cell membrane through hydrophobic anchors, cell surface modication is achieved through liposomes loaded with synthetic materials or functional groups diffusing and mixing with the cell membrane in the membrane fusion strategy.
Sarkar et al. developed a versatile platform technology for the modication of cell membranes. 109Biotinylated lipid vesicles were utilized for the incubation with MSCs, leading to the attachment of biotin on the cell surface via vesicle fusion.The biotin moieties serve as binding sites for subsequent ligands.1][112][113][114][115][116][117][118] The prepared lipid, containing either ketone or oxyamine molecules, underwent spontaneous insertion and fusion into the cell membrane, resulting in the modication of cells with either ketone or oxyamine molecules for subsequent bio-orthogonal ligation reactions. 110Additionally, the bioorthogonal molecules, ketone or oxyamine, could also be modied onto different populations of cells using the same method to regulate the cell-cell interaction and generate 3D tissue-like structures. 111Following this, the researchers employed a membrane fusion approach to create and modify cell membrane surfaces with bioorthogonal chemical molecules possessing diverse characteristics, including photoresponsive and redox-responsive cleavage.The primary emphasis of their investigation was on the utilization of these modied cells in the eld of three-dimensional tissue engineering.Membrane fusion strategies have recently been extensively used as a potent tool for modifying cell membranes in various investigations.
Zheng et al. designed core-shell membrane-fusing liposome (MFL) containing NK cell-activating glycans, Lewis X trisaccharide (LeX), and loaded it into a thermosensitive hydrogel which could be released responsively through the tumor microenvironment.Subsequently, the released MFL was fused with tumor cell membranes, realizing the modication of tumor membranes with Lex which could enhance the anti-tumor effects. 119Shi et al. designed T-celltargeting fusogenic liposomes by conjugating ROSscavenging groups, 2,2,6,6-tetramethylpiperidine (TEMP) and T-cell-targeting anti-CD3 F(ab ′ ) 2 fragments to the surface of liposomes, in which TEMP groups were designed for neutralizing ROS and protecting T cells from an oxidationinduced loss of activity.In the meantime, the procedure would result in paramagnetic transition of TEMP to TEMPO molecules, allowing for the measurement of the in situ activity of T cells, enabling a better understanding of engineering T cells for cancer treatment. 120Lin et al. reported a liposomal fusion-based transport (LiFT) strategy to anchor functional DNA strands on the inner face of the cell membrane, addressing the previous lack of suitable synthetic tools to engineer the intracellular interior (Fig. 5B). 108In subsequent studies, the group combined membrane-anchored catalysts with the previously reported LiFT strategy, through which they were able to prepare the corresponding fusion liposome catalyst through a simple strategy, achieving precise position control of the catalyst on the cell membrane. 121The drug molecules generated by this method may have higher drug delivery efficiency than traditional methods using drug delivery vehicles.Furthermore, by integrating targeting motifs into the outer surface of liposomes, cell-specic membrane engineering can be achieved for potential targeted drug delivery.

Electrostatic interaction
Cell surface modication through electrostatic interactions is an appealing strategy that capitalizes on the negative charge conferred mainly by sialic acid residues in the carbohydrate layer, along with the phosphatidylserine on the plasma membrane.3][124][125][126] For instance, Choi and colleagues achieved the development of silica coating on mammalian cells by modifying PEI on the cell membrane through electrostatic interactions, serving as a catalytic template for silicication. 126In a subsequent study, TiO 2 shells were developed for the cytoprotective encapsulation of Jurkat T cells. 122This method could effectively protect the T cells in the shell while simultaneously preserving their functionality, including cell division, juxtacrine interactions and cytokine secretion.Upon administration into the organism, the lymphocytes' therapeutic capabilities are effectively reinstated through the rupture of the protective shell.The TiO 2 -inducing peptide, (RKK) 4 D 8 (R: arginine, K: lysine, D: aspartic acid), was deposited on the surface of Jurkat cells via electrostatic interactions to facilitate the formation of bioinspired TiO 2 using titanium bis(ammonium lactato)dihydroxide (TiBALDH) as a precursor.However, interactions with most cationic polymers readily lead to the destruction of the cell membrane, resulting in pronounced cytotoxicity and cellular damage.To address this issue, cationic polymers can be modi-ed with biocompatible molecules, such as graing PEG or alginate, to mitigate the detrimental effects on cell viability. 127espite the overall negative charge of the cell membrane surface, a few cationic sites on the plasmalemma still exist which can be modied with negatively charged materials. 130,131r instance, Thomsen et al. modied T cells with negatively charged degradable poly(lactic acid) (PLA) nanoparticles with electrostatic adsorption. 125Furthermore, the modication of Fig. 6 Methods based on layer-by-layer self-assembly.(A) Schematic illustration of MSCs nanofilms prepared using positively charged PLL, negatively charged HA and RGD, and the functions exhibited by the modified cells. 128Copyright 2017, American Chemical Society.(B) Schematic illustration of mammalian cell nanoencapsulation conducted by LBL self-assembly between GA and GB, and subsequent thiol-maleimide reaction.GSH could be added for on-demand release. 129Copyright 2017, Elsevier.

Chemical Science Review
3][134] It is important to note that the presence of a negatively charged cell membrane hinders the uptake of negatively charged nanoparticles by cells.

Layer-by-layer (LBL) self-assembly
LBL self-assembly strategies have been developed on the basis of electrostatic interaction and widely employed for the construction of cell coatings, in which oppositely charged materials are sequentially deposited onto the cell membrane through electrostatic interaction along with hydrogen bonding, van der Waals forces, etc. 21,127,128,[135][136][137][138][139][140][141][142][143][144][145][146][147] As shown in Fig. 6A, Hong and colleagues developed LBL self-assembled nanolms for cell surface modication of viable MSCs.Positively charged PLL was layer-by-layer assembled with negatively charged hyaluronic acid (HA) and arginine-glycine-aspartic acid (RGD) to fabricate nanolms, which not only provided biochemical signals but also offered mechanical support for MSCs without interfering with the stemness of MSCs. 128Subsequent studies have demonstrated the successful construction of nanolms on the surface of human induced pluripotent stem cells (iPSCs) and immune cells such as AML-12 cells and peripheral blood mononuclear cells (PBMCs) via the LBL self-assembly strategy. 140,148Gels can be formed via LBL to coat or encapsulate cells.Chen and colleagues proposed a gentle approach (Fig. 6B) to achieve the nanoencapsulation of individual mammalian cells. 129The gelatin coatings, which mimic the extracellular matrix (ECM), are formed through LBL selfassembly between positively charged gelatin type A (GA) and negatively charged gelatin type B (GB) on the cell membrane surface.Additionally, the outer layer of PEG was further constructed using thiol-maleimide click chemistry which could be degraded on-demand by the addition of the reducing agent glutathione (GSH).Subsequent studies involved the development of an enzyme-responsive nano-coating for encapsulating individual living cells, which was prepared through layer-bylayer self-assembly of oppositely charged gelatin-poly(ethylene glycol)maleimide and the incorporation of cysteineterminated peptide sequences (CGGPLGLAGGC) via click reaction. 135Moreover, the peptide chain could undergo enzymolysis upon exposure to high concentrations of matrix metalloproteinase-7 (MMP-7), which is frequently overexpressed in tumors, leading to the release of encapsulated cells.

Bioorthogonal modification
Despite the abundance of functional groups on the surface of the cell membrane that are amenable to chemical covalent modication, the utilization of non-specic covalent modication strategies may have detrimental effects on the viability and functionality of normal cells.Additionally, physical strategies are limited by the short residence time of synthetic molecules.In contrast, bioorthogonal chemistry offers a highly efficient and selective approach that takes place within a mild physiological environment, without disrupting intrinsic biochemical processes.This strategy represents a substantial advancement in terms of both cell viability and the stability of modications.Here, we provide an overview of recent developments in the integration of metabolism with copper-free click chemistry, Halo-Tag proteins, and enzyme-mediated approaches.

Metabolic glycan labeling strategy
Since the pioneering work of Bertozzi and colleagues, who introduced exogenous glycans into the cell membrane glycocalyx, there has been a gradual development of strategies for metabolic glycan labeling to modify the cell membrane. 149,1501][152][153][154][155][156] In recent years, glycans containing azide groups represented by N-azidoacetylmannosamine-tetraacetate (Ac 4 -ManNAz) have gained the most widespread adoption with the development of copper-free "click" azide-alkyne reactions due to their high selectivity, synthetic simplicity and commercial availability.8][159] In a recent study, Tomas et al. who proposed the "engineering cells to capture polymers" strategy incubated tumor cells with Ac 4 -ManNAz for 96 hours to obtain azido-modied cancer cells which could capture chemotherapeutic polymers covalently and this strategy signicantly augmented the concentration specifically targeted towards the tumor cell membrane whilst optimizing therapeutic efficacy by reducing systemic toxicity and enhancing selectivity. 1581][162] Zhou et al. successfully modied an oligomeric proanthocyanidin loaded liposome on the membrane of MSCs (MSC-Lipo-OPC) via metabolic labeling combined with the click chemistry strategy (Fig. 7A). 160The MSC-Lipo-OPC could control the progression of inammation due to the excellent abilities to scavenge free radicals and effectively prevent the formation of radiation-induced pulmonary brosis.Chen et al. developed polyvalent spherical aptamer (PSA) engineered macrophages which could effectively recognize tumor cells and inhibit tumor growth. 161PSA which has superior affinity and specicity to tumor cells was constructed through covalent reaction of gold nanoparticles (AuNPs) with AS1411 aptamer and DBCO groups, and was subsequently modied on macrophage membranes via metabolic labeling.Moreover, Lamoot et al. developed a 2-step click strategy for achieving highly specic cell surface conjugation of nanoparticles.In the study, cells were incubated with Nazidoacetylmannosamine-tetraacetylated (Ac 4 ManN 3 ) to present azido groups on cell membrane (Fig. 7B). 162Subsequently, sulfo-6-methyl-tetrazine-dibenzyl cyclooctyne (Tz-DBCO) was exploited as a "bridge" between azide-modied cells and trans-cyclooctene (TCO) functionalized nanoparticles to realize nanoparticle-engineered cells exhibiting extremely low non-specic background binding.
In addition to in vitro applications for modifying cell membranes, researchers have also conducted studies to achieve this process in vivo. 163,164   dendritic cells.Dibenzocyclooctyne (DBCO)-labelled immunomodulatory agents, such as tumor antigens, adjuvants, and cytokines could be modied on the DC membrane via click chemistry in vivo thereby effectively enhancing the subsequent T cell activation and tumor killing process.Additionally, Tu et al. employed Ac 4 ManAz nanoparticles for in situ labeling of tumor cell membranes with azido groups, followed by the binding of chlorin e6 (Ce6), a commonly used photosensitizer, via click chemistry. 164This approach effectively enhanced the therapeutic efficiency of photodynamic therapy.Recently, Chen and colleagues reported a cell-type-specic labeling approach in vivo. 165In this study, the cardiomyocyte was specically labeled without any interference from other cardiac cell types, which provided a powerful tool for cell-type selective modication.Gong et al. accomplished in situ PEGylation of CAR-T cells through the utilization of the metabolic glycan labeling strategy. 166When the molecular weight of PEG reached 600 000, it effectively hindered the intercellular interactions among CAR-T cells, tumor cells, and monocytes, thereby attenuating the secretion of cytotoxic cytokines and ameliorating the symptoms associated with cytokine release syndrome (CRS).
Compared to the direct covalent binding with functional groups on the cell membrane, the metabolic glycan labeling strategy effectively enhances the density of reactive sites on cells, but it is a time-consuming process that can take several days.The signicant advantage of metabolic glycan labeling combined with bioorthogonal reactions is that it transits cell surface modication from nonspecic to specic, enabling cell surface modication in situ and in vivo.This is still an emerging eld, with immense potential for further development and expansion.

Halo-Tag protein
Halo-Tag protein (HTP) is an engineered protein derived from the bacterial haloalkane dehalogenase, which selectively reacts with alkanes containing a terminal chloride group (chloroalkanes) forming a covalent bond. 167,168Similar protein recognition tags, such as SNAP tags 169 and ACP tags, 170 could also be utilized in cell surface modication, but they will not be extensively discussed in this section.A two-step approach was utilized in the HTP strategy: the expression of HTP on the cellular membrane is achieved via genetic engineering methods and further combined with cargoes containing the chloroalkane.HTP is commonly used in protein isolation and puri-cation, molecular imaging, molecular interactions etc. in most reported studies and was rst utilized for cell surface Fig. 8 Methods based on other bioorthogonal strategies.(A) Schematic illustration of displaying synthetic glycopolymers on HeLa cell membranes using HTP anchors. 172Copyright 2019, American Chemical Society.(B) Schematic illustration of Kell C-terminal sortase labeling with GGG-carrying antigen peptides. 173Copyright 2017, National Academy of Sciences.(C) Schematic illustration of transferring biomacromolecules to glycocalyx on the surface of living cells with fucosyltransferase. 174Copyright 2018, American Chemical Society.

Review
Chemical Science modication by Pulsipher et al. 171 They proposed a long-lived cell membrane engineering strategy utilizing HTP as an anchor for modifying embryonic stem cells (ESCs) with heparan sulfate (HS), which was covalently modied on the ESC membrane and stayed for more than one week.Subsequently, our group developed a series of studies via the HTP strategy. 172,175,176Tumor cells were modied with specic glycopolymers via the HTP fusion technique combined with RAFT polymerization (Fig. 8A).The glycopolymers that were modied on tumor cells could bind to lectins on dendritic cells or macrophages which effectively enhanced the tumor immune response. 172An interesting discovery was that the migration of the tumor cells modied with glycopolymers could be affected.Specically, compared with the unmodied tumor cells, the migration direction was altered and diffusion slowed down which offered novel insights pertaining to the management of cancer metastasis. 175A following study was carried out and we constructed glycopolymers modied DCs via the HTP strategy.Enhanced interactions were discovered between glycopolymer modied DCs and T cells which effectively promoted the T cell activation and proliferation, providing a novel approach to designing more efficient DC vaccines. 176he HTP strategy for cell surface modication is still in its infancy.It is noteworthy due to the strong stability of binding between HTP expressed on the cell membrane and its corresponding ligand, thereby enabling sustained modications that persist for over a week, offering a suitable method for long-time and stable cell surface modication.However, the implementation of HTP expression necessitates the manipulation of gene transfection, a process that is intricate and timeconsuming, thereby inapplicable to certain challenging-totransfect cell types such as primary cells.

Enzyme-mediated strategy
Enzyme-mediated modication of cell membranes represents a novel approach for in situ modication of candidate materials, reacting with pre-existing structures on the cell surface under the specic catalysis of enzymes.Specically, certain enzymes such as oxidoreductases (galactose oxidases 177 ), glycosyltransferases (sialyltransferases, 178,179 galactosyltransferases, Nacetyl-glucosaminyl transferases and fucosyltransferases 174,180 ), transpeptidases (butelases and sortases 173,181,182 ), transglutaminases (TGases 183 ) etc. have been utilized for the modi-cation of cell membranes, representing an appealing approach due to their remarkable specicity and high yield.For example, galactose oxidases can specically convert the endogenous terminal galactoses or N-acetylgalactosamine residues on the cell surface into aldehyde groups, facilitating subsequent reactions between aldehyde groups and aminooxy-functional molecules. 177Glycosyltransferases are primarily utilized for the modication of pre-existing sugars on cell membranes, thereby facilitating the introduction of non-natural sugars.Moreover, it's worth noting that in comparison to metabolic engineering approaches, glycosyltransferases, particularly sialyltransferases and fucosyltransferases, offer a novel method for introducing greater kinds and intricacy of sugars on the cell membrane surface.In the presence of transpeptidases, molecules bearing recognition motifs can be directly conjugated to either the N or C termini of membrane proteins.For example, as shown in Fig. 8B, Pishesha et al. reported a strategy for inducing antigen-specic tolerance by utilizing the transpeptidase sortase to covalently conjugate disease-associated autoantigens onto red blood cells (RBCs), thereby attenuating the contribution of major subsets of immune effector cells to immunity in an antigen-specic manner. 173Li et al. focused on fucosyltransferase and transferred bio-macromolecules to the glycocalyx on the surface of living cells, which represented faster speed, better biocompatibility, and less interference to cells (Fig. 8C). 174Through this method, they constructed two antibody-cell conjugates, which exhibited signicant improvements in the process of targeting and killing anti-cancer immune responses.

Conclusion and outlook
Modifying cell surfaces with tailor-made and well-characterized synthesized molecules can effectively introduce novel functionalities or manipulate cells.This offers a powerful tool to overcome challenges encountered in cell-based biomedical applications.In this review, we present a comprehensive overview of the latest advances in cell surface modication using synthetic molecules.We summarize the typical strategies, including chemical covalent modications, physical alterations, and bioorthogonal approaches (Table 1), along with the advantages, disadvantages, and applicable conditions of each strategy.The chemical covalent strategy offers a straightforward and versatile approach for achieving stable and long-lasting surface modication. 184However, the strategy has the potential to adversely impact cell activity and functionality.The physical modication strategy provides a non-invasive and cytocompatible approach.However, modications achieved through physical interactions, such as electricity and hydrophobicity, are relatively short-term and unstable.It is important to note that the two methods mentioned above are non-specic, lacking precision in cell surface modication and potentially increasing the risk of adverse effects during practical applications.Therefore, bioorthogonal chemistry provides a valuable strategy for the selective and highly biocompatible incorporation of synthetic molecules onto cell surfaces, even enabling cell surface modication in vivoa remarkable development.However, the approaches used to introduce bioorthogonal groups, whether via genetic engineering or metabolic engineering, are time-consuming.
Despite notable advancements in the utilization of synthetic compounds for cell surface modication, there remain unresolved challenges and prospects for further investigation.One such challenge pertains to the inherent detrimental impact of exogenous synthetic compounds bound to the cell surface on cellular functionality, albeit with varying degrees of severity.Hence, it is of utmost importance to meticulously choose a suitable strategy for modifying cells, taking into consideration the particular cell type and application scenarios.Subsequently, it becomes imperative to assess and describe the condition of

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practical scenarios, the presence of synthetic molecules on cellular surfaces carries the potential for immune activation and subsequent clearance by the immune system, thereby considerably restricting their capacity to modify cell surfaces in vivo.Consequently, it becomes crucial to implement suitable adjustments to synthetic molecules to ensure their compatibility with in vivo applications.Additionally, a signicant hurdle lies in selecting and designing molecules that possess both biocompatibility and augmented functionality for specic applications.Determining the optimal chemical group, structure, and sequence becomes essential in this regard.Therefore, the availability of databases serving as a toolbox for researchers to facilitate informed molecule selection is highly desirable.There are several possible avenues for future research.One potential area of exploration is the development of more precise and targeted methods for modifying cell surfaces.There is a need to develop more procient and potent methodologies for the selective and highly biocompatible integration of synthetic molecules onto cell surfaces.Furthermore, additional research is necessary to gain a better understanding of the inuence of synthetic molecules on cellular functionality and to optimize modication strategies tailored to specic cell types and applications.An additional area of research that holds promise for the future is the advancement of synthetic molecules that possess improved biocompatibility and biofunctionality, enabling their application in the modication of cell surfaces.Notable examples of these molecules encompass functional nucleic acids, targeting aptamers, and polymers characterized by well-dened structures and chain sequences.The utilization of articial intelligence (AI) can be facilitated by the establishment of databases containing comprehensive information regarding ligand-receptor interactions specic to cells, as well as the attributes associated with each modication technique.This integration of AI can aid in the design of optimal, customized molecules and the selection of appropriate methods for modication.

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Review

Fig. 2
Fig. 2 Illustration of chemical covalent modification through the reactions between functional groups on cells and synthetic molecules.

Fig. 4
Fig. 4 Construction of LC-aAPCs with lymphocytes from peripheral blood. 106(A) Schematic diagram of constructing lymphocyte-based aAPCs from peripheral blood for personalized tumor immunotherapy.(B) Schematic illustration of DNA-mediated bottom-up assembly of pMHC-I and aCD28 on lymphocytes, including hydrophobic insertion and specific DNA hybridization as well as biotin-avidin interaction.(C) Confocal microscope images showing distribution of pMHC-I and aCD28 on the surface of lymphocytes.Copyright 2022, John Wiley & Sons, Inc.
conducted an interesting study by labeling and modulating DCs and regulating DC-T cell interactions in vivo.163They synthesized Ac 4 ManAz nanoparticles overcame the limitations of Ac 4 ManAz utilized in vivo such as poor encapsulation and water solubility.The Ac 4 ManAz nanoparticles and granulocyte-macrophage colony-stimulating factor (GM-CSF) were loaded into an injectable alginate gel for the purpose of realizing in situ recruitment and azide labeling of

Fig. 7
Fig. 7 Methods based on the metabolic glycan labeling strategy.(A) Schematic illustration of binding Lipo-OPC to MSCs which were preincubated with Ac 4 ManNAz and presented azido groups on the cell membrane. 160Copyright 2023, Elsevier.(B) Overview of the 2-step click strategy for achieving highly specific cell surface conjugation of nanoparticles. 162Copyright 2020, John Wiley & Sons, Inc.

Table 1
A summary of the advancements in strategies for the cell surface modification with synthetic molecules in this review © 2023 The Author(s).Published by the Royal Society of Chemistry Chem.Sci., 2023, 14, 13325-13345 | 13339 Review Chemical Science the modied cells, encompassing cell viability, phenotype, and associated functionalities.It is noteworthy that the cell surface constitutes a dynamic membrane structure, wherein synthetic molecules may undergo endocytosis or excretion by the cell.Consequently, it is crucial to monitor the destiny of synthesized molecules during and post cell surface modication.In