Cell Surface Engineering
Recent preclinical studies demonstrated the use of engineered cells as a potential way to treat many diseases and disorders. Tailoring the cell’s function and interactions using surface engineering methods is a very promising approach in developing novel cell-based therapeutics. For instance, cell surface modification has been used for the development of universal blood donor cells. In another example, it has been shown that surface modification of stem cells is a doable approach to regulate the fate of cells into specific phenotypes, which is necessary to regain function in specific environment such as different injury sites. Cell surface engineering using macromolecules/polymers could provide desired properties and functions to cells for applications in targeted delivery, biosensing, transfection, imaging techniques, and in the regulation of cell fate. This chapter will review the recent advancements in polymer-based cell surface engineering approaches for various applications. In terms of the cell types, we have chosen to focus, specifically, on red blood cells, lymphocytes, splenocytes, stem cells (multipotent and pluripotent), islet cells, endothelial cells, and hepatocytes as they offer the most promise in generating cell-based therapeutics. In terms of modification approaches, we mainly highlighted the literature associated with the use synthetic polymers via covalent conjugation and non-covalent bonding. We also discuss the future of such cell surface engineering methods for their potential clinical utility.
KeywordsCell surface engineering Polymers Bioconjugation Red blood cells Stem cells Islet cells Endothelial cells
Bovine aortic endothelial cells
Cell adhesion molecules
Central nervous system
Embryonic stem cells
Fibroblast growth factor 2
Hematopoietic stem cells
Intercellular cell adhesion molecule-1
Induced pluripotent stem cells
Mesenchymal stem cell
Neural stem cells
Polyelectrolyte multilayer film
Palmitated protein G
Pluripotent stem cells
Red blood cells
Vascular endothelial adhesion molecule
In this chapter, we will discuss cell surface engineering approaches using synthetic polymers for various applications. It will also address how chemical approaches including covalent and non-covalent methods are used to manipulate the cell surface effectively to enhance their therapeutic potential and other cellular functions. These include covalent conjugation of polymers to primary amine groups on cell surface proteins, incorporation of amphiphilic polymers into lipid membrane of cells via hydrophobic interaction, electrostatic binding between cationic macromolecules and negatively charged cell surface, and grafting from strategies and modifications through nonnative functional groups. In addition, this chapter also highlights a huge body of work on the engineering the surface of cells, including red blood cells (RBCs), white blood cells, multipotent and pluripotent stem cells, islet cells, endothelial cells, and hepatocytes for transplantation and transfusion applications. These cells are selected due to their promising therapeutic potential for various diseases. We review the advantages and challenges associated with these methods and how these approaches can be applied to improve the therapeutic applications of cells. Finally, conclusions about the current state of the field and insight into the future directions are given.
2 Methods for Cell Surface Engineering Using Polymers
Cell surface engineering has a pivotal role in tuning the cell function by controlling their biochemical interactions with their environments [2, 3, 4, 15, 18]. The availability of different functional groups on the cell surface will be an excellent opportunity for cell surface modification (Fig. 1). However, surface modification is quite challenging due to the fact that cell surface is not static, and also the modification should only have minimal effect on biological function of the cells [5, 7, 19, 20]. In the past two or three decades, researchers have focused on developing various tools for engineering the surface of cells [2, 9, 19, 21, 22]. A variety of functional groups and bioactive substances have been introduced onto cell surface by different biological transformations and physicochemical methods [9, 14, 21, 22]. Here, our intention is to focus on methods that are commonly used and utilize hydrophobic, electrostatic, covalent interactions and enzymatic approaches for cell surface modification.
2.1 Hydrophobic Insertion into the Cell Membrane
2.2 Electrostatic Interactions
2.3 Covalent Modification
Although a variety of functional groups are readily available on the cell surface, only a few functional moieties can be used to covalently modify the cell surface proteins due to the extreme complexity and heterogeneity of cell surface. This strategy is involved in a direct chemical reaction of functional groups on the cell surface such as amines, thiols, and carboxylic acids with proteins, polymers, nanoparticles, and other small molecules. The most commonly used functionality for cell surface modification is the amine groups originating from the lysine side chains present on cell surface proteins. The most easily and extensively used strategy is the treatment of amine groups on the cell surface with N-hydroxysuccinimide (NHS)-activated macromolecules at nearly physiological conditions. Although cell surface carbohydrates were used for covalent conjugation of small molecules on cell surface [37a], there is limited information available on such methods used for polymer conjugation.
The covalent conjugation of cell surface amines with cyanuric acid containing polymers is an another commonly used strategy for various cell surface modifications, in particular RBCs and T cells [40, 41, 42, 43, 44, 45]. Hashemi-Najafabadi et al. developed a cell surface modification technique to mask the RBCs surface via covalent attachment of m-PEG to the cell membrane . They optimized PEGylation conditions in order to achieve the attenuated immunorecognition of RBCs in both organ transplantation and blood transfusion applications. The cyanuric acid derivative of m-PEG-OH was first synthesized under inert conditions; then, the Rh positive RBCs were suspended with cyanuric acid-PEG derivatives for different time intervals at different pH, temperature, and polymer concentrations. PEGylation, with linear PEG of molecular mass 5 kDa, of RBCs through this approach was quite successful at pH 8.7, temperature 14 °C, and reaction time 30 min. The polymer concentration was varied with molecular weight. The morphology of m-PEG-RBCs was intact, and it was further confirmed by light microscopy and scanning electron microscopy. Using similar approach, Scott and co-workers has shown that the grafting of m-PEG on peripheral blood mononuclear cell surface decreased the antibody recognition of different surface receptors involved in essential cell communication .
Connecting cell surface amines with aldehyde containing moieties (macromolecules, drugs) through Schiff base formation is another useful strategy. Tucaresol, a molecule bearing an aldehyde group, is an investigational drug as an immunopotentiator in chronic hepatitis B virus and HIV infections. Chen et al. conjugated the Tucaresol with T-cell surface amines via Schiff base formation to understand its immunoresponse mechanism. However, this method is not widely explored [47, 48, 49].
These strategies are very useful to enhance drug loading on the cell surface . In another report, Nacharaju et al. conjugated PEG-maleimides to the RBC surface to camouflage the RBC surface antigens from antibodies. This methodology worked well for different molecular weights of PEG, and also this linkage is stable at in vivo conditions .
Although the biotinylation strategy is very versatile and widely used for different applications, it has few limitations, for instance, when natural cell surface functionalities are targeted with highly reactive molecules, the cell surface might have over flooded with biotinylated products; this could generate some toxicity . In addition, the protein component of these techniques is of bacterial origin (e.g., streptavidin) which could generate immune reactions. This will be harmful especially for cell surface engineering for in vivo applications. Although covalent immobilizations/linkages of polymers or macromolecules to cell membranes were expected to be stable for chemical degradation for a long time, in few cases these non-covalent modifications disappeared from the cell surface over the time .
2.4 Modifications Through Nonnative Cell Surface Functional Groups
In 2010, Holden et al. also used a similar approach to coat the macrophage surface with polyamidoamine dendrimers through sialic acid modification. The geminal diol units of sialic acid on the cell surface are oxidized with sodium periodate to generate aldehydes, and the cells are dispersed with polymers to form a covalent bond between amine groups of polyamidoamine dendrimers and aldehyde groups on the cell surface. Finally, formed Schiff bases were further converted into stable secondary amine groups using sodium cyanoborohydride . However, the main limitation of this approach is that the reactive groups must be generated prior to the covalent grafting of cells with polymers.
Although various enzymatic treatments and metabolic approaches have been also employed to incorporate different functional groups such as biotin, alkyne, azide, thiol, and ketones into live cells surfaces, these technologies might influence cell physiology in the long run [81, 82].
2.5 Grafting from Strategies
Surface-initiated atom transfer radical polymerization, for instance, on solid surfaces, nanoparticles, and metallic surfaces, has been extensively studied. Taking advantage of this, recently Kim et al. reported a yeast cell surface modification with synthetic polymers (“grafting from” approach) using highly cytocompatible surface-initiated activator regenerated by electron transfer, atom transfer radical polymerization (SI-ARGET-ATRP) . They coated the cell surface with polydopamine by dispersing the cells in a solution containing dopamine-bearing ATRP initiator. The polydopamine-coated cell surface was treated with ARGET-ATRP solution for different time intervals to optimize the density of polymer chains on the cell surface. Such polymer-live cells hybrid structures have enormous potential for different applications and might serve as cell-based sensors, biomotors, and diagnostics [83, 90, 91].
3 Cell Surface Modification of Different Cell Types and Applications
The reasoning behind the cell surface engineering is to bring new advances in cell therapies which would eventually lead to control the fate and function of therapeutic cells. Engineered cells will be potentially used for their enhanced survival, proliferation, or differentiated function. In the following section, we will detail various cell surface modification methods applied to different cell types and applications associated with. Our main focus will be on RBCs, stem cells (multipotent and pluripotent), islet cells, endothelial cells, lymphocytes and splenocytes, and hepatocytes. Since cell surface engineering has emerged as a promising method for applications such as tissue replacement, regenerative medicine, transfusion, and transplantation medicine, the aforementioned cell types play key roles in these fields. Such approaches often involve the use of combinations of materials and cells to create functional structures that can be used in place of the original tissue. For instance, stem cells have the unique property of pluripotency, the ability to differentiate into any cell type making them particularly useful in tissue engineering and regenerative medicine. Here, we aim to summarize the benefit of cell surface engineering of these cells and report the current challenges associated with their application.
3.1 Antigen Protected Red Blood Cells
3.2 Lymphocytes and Splenocytes
Due to the complexity of human immune system and antigenic diversity of the human cells, rejection of biomaterial, foreign tissues, and donor organs is still a great challenge in translational medicine. This questions the long-term compatibility of transplanted tissues and quality of the recipient’s life. Especially, graft-versus-host disease causes significant morbidity and mortality among the transplanted patients. Although few pharmacologic agents, for instance, azathioprine and methotrexate, have been successfully used to inhibit T-cell activation, these drugs are highly toxic to the kidney, liver, and gastrointestinal glands [99, 100, 101]. Surface engineering of the cells with biomaterials may provide a viable solution to reduce the rejection of allografts. Scott and co-workers examined the covalently m-PEG (5 kDa)-grafted allogeneic lymphocytes in minimizing of allorecognition necessary for T-cell activation and graft-versus-host disease both in vitro and in vivo in murine models. The masking of cell surface dramatically attenuated allorecognition of cells, and it was evident by dramatic differences in T-cell proliferation between unmodified and m-PEG-modified versions in both one- and two-way mixed lymphocyte reactions and flow cytometric analysis. Further, the in vivo murine models, graft-versus-host disease is induced in immunocompetent as well as immunocompromised mice via transfusion of allogeneic splenocytes which are collected from murine major histocompatibility complex disparate mice, further validate the effect of m-PEG derivatization in attenuation of allorecognition and subsequent reduction of the risk of graft-versus-host disease in mice .
The efficiency of induced immunocamouflage of grafted murine splenocytes was examined using three different modified PEGs and covalent linkages. Two different molecular weights of PEG were used 
Immunoquiescence, a state of low baseline immune activation, is another parameter to evaluate the efficiency of immunocamouflaged cell surface. Wang et al. studied whether m-PEG-modified allogeneic human peripheral blood mononuclear cells (PBMC) or murine splenocytes can produce immunoquiescence or tolerance in both in vitro and murine in vivo models . Lymphocyte proliferation, differentiation, and cytokine production was verified in mixed lymphocyte reactions and conditioned media experiments. The studies demonstrated that PEG grafting does not have any significant effect on cell viability and immunomodulatory response and cytokine production, whereas the controls demonstrated significant (p < 0.001) effect on pro-proliferative potential and enhancing interleukin-2, tumor necrosis factor alpha, and interferon gamma levels.
Although various reports provided the detailed understanding of late events in T-cell activation of allografts such as T-cell proliferation and cytokine secretion, a better understanding of initial triggering events/molecular mechanisms is limited. Scott research group, in their subsequent studies, monitored the initial triggering events by examining the effect of PEGylation of cells in initial cell-cell interactions, changes to activation pathways, and apoptosis [101, 104, 105]. The role of these events in minimizing proliferative response is observed in modified cells during mixed lymphocyte reactions. The m-PEG-engineered cells exhibited significant global immunocamouflage of surface proteins of lymphocytes and also minimized interactions with antigen-presenting cells and other intracellular signaling process. And also, the reported PEG approach is nontoxic. Due to the global immunocamouflage of this approach, it overcomes the biological redundancy inherent to surface adhesion, costimulatory, and growth receptors and shows no evidence of systemic toxicity.
3.3 Modification of Stem Cells
Stem cells are currently known as one of the most promising candidates for developing novel and clinically translatable cell therapy [106, 107, 108, 109]. In particular, focuses on multipotent and pluripotent stem cells (PSCs) have been significantly increasing [108, 109]. PSCs are characterized by immortality – the ability to continuously self-renew – and pluripotency, the ability to differentiate into all somatic cell types . PSCs include both embryonic stem cells (ESCs) and induced PSCs (iPSCs). Both pluripotent and multipotent stem cells can generate the necessary quantities of cells required for transplantation due to their ability to continuously divide. These cells can then be differentiated into desired phenotypes for therapeutic applications. HSC’s transplantation is also known as one of the most commonly used cells for clinical trials [18, 39, 110, 111, 112].
Cell surface engineering approaches have been used to modify the cells to provide them with a desired property. Stephan et al. have proposed the conjugation of drug-loaded liposome nanoparticles onto the surface of HSCs . Not only such approach did compensate normal HSC’s function, but they also increased the self-renewal durability of such cells. In addition to self-renewal and differentiation capability of stem cells, one of the current importance of stem cell therapy is the safe and efficient delivery of such cells into the desired tissue without losing their prominent properties such as proliferation or differentiation. For the delivery of stem cells into their desired tissues, the cell surface can be engineered to enhance the homing properties. In particular for MSCs, due to insufficient expression of surface markers, these cells would not present efficient homing properties. Consequently, cell surface modification approaches can play a key role in enhancing and presenting surface ligands onto the cell surface in order to address such issues. For instance, Sarkar et al. presented a promising modification technique in which they modified MSCs with a nanometer-scale polymer containing SLeX which has been found to be present on the surface of leukocytes and regulate the cell rolling of MSCs .
In another study, improving the homing transplantation of MSCs were carried out by using engineered mRNA-transfected MSCs which highly expressed homing ligands, such as P-selectin glycoprotein ligand-1 (PSGL-1) and sialyl-Lewisx (SLeX), leading eventually to enhancing homing of such cells into mouse’ inflamed ear vascular endothelium. Lecy et al. have shown that engineered MSCs with the homing ligands PSGL-1/SLeX via mRNA transfection significantly improved their homing to the mouse bone marrow . Their results confirmed that mRNA-transfected MSCs have enhanced homing to inflamed ear 2 h after injection by 30% compared to native MSCs.
So far, we have explored most studies that investigated the effects of cell membrane modification on regulating the fate of stem cells. Glycans are considered as one of the most important cellular component of stem cells where they are in charge of cell signaling communications to their exterior environment. Therefore, the glycan engineered using different techniques to stimulate the fate of stem cells into their desired properties accordingly. Such modulation would be mainly controlled by the signaling molecule transmission such as fibroblast growth factor 2 (FGF2), Wnt, and Notch and other lineage-specific signatures such as the stage-specific embryonic antigens (Lewis X, stage-specific embryonic antigens-1, stage-specific embryonic antigens 3 and 4). For instance, Huang et al. have shown that by using synthetic neoproteoglycans (neoPGs) they were successful in engineering the surface of mouse ESCs to further enhance their affinity to bind to FGF2 . FGF2 is widely used for culturing many stem cell types including ESCs and PSCs. Using such synthetic approach, these researchers could remodel the glycocalyx of mouse ESCs which eventually lead to enhance the neural differentiation of such cells. In another study on engineering the glycans of stem cells, Pulisipher et al. have focused on how to regulate the fate of ESCs into neural phenotypes . They have used HaloTag proteins (HTPs) to present heparin sulfate glycosaminoglycan (HS-GAG) anchor onto the membrane of mouse ESCs. It has been shown that remodeling the glycocalyx of ESCs with such strategy could accelerate the self-renewal exit and eventually promote neural lineage commitment and their differentiation into mature neuronal cells. Pulisipher et al. showed that, consistent with an accelerated loss of pluripotency, transcription factor NANOG levels in heparin sulfate chloroalkane-treated cells was decreased which then accompanied by a corresponding increase in the neuroectoderm-specific marker SOX1. Overall, bioengineering approaches to regulate the fate of stem cells toward their desired application is becoming very promising for preclinical and clinical researchers in the field.
3.4 Surface Modification of Islets
Teramura and Iwata have reviewed various cell surface engineering methods which can be applied to provide non-recognizable surfaces against the immune system. Such methods have been challenged for immune evasion: PEG23, multilayered PVA-PEG-lipid24, hyperbranched alginate-poly(amidoamine) dendrimer complex, hyperbranched polyglycerol, complement receptor 1-heparin layer-by-layer assembly, factor H-binding peptide, and apyrase [14, 26].
3.5 Endothelial Cell Engineering
3.6 Hepatocytes Modification
Hepatocyte-based therapies have immense potential to be alternatives to liver transplantations in many liver-related diseases including liver failures and other liver disorders. However, the sufficient grafting of hepatocytes and their viability is highly essential for the success of both hepatocyte-based therapies and liver tissue engineering applications. A detailed understanding of the surface modification/interaction of hepatocytes with biomimetic materials is another crucial factor for the success of this therapeutic approach [25, 125, 126]. To better understand the hepatic cell interactions with biomaterial surface, recently, Kojima et al. coated hepatic cells with poly(lactic acid) through avidin-biotin binding system without losing crucial metabolic functions such as serum protein secretion and metabolic capacity . Initially, hepatic cells were attached with sulfo-NHS-biotin, and these modified cells were grafted on an avidin-adsorbed flat poly(l-lactic acid) surface. The adhesion process is completed in less than 10 min. The proliferation of these modified cells was intact and almost comparable with cells cultured in collagen plates.
4 Summary and Future Prospective
The attachment of polymers onto the surface of different cell types opens a new and exciting avenue in the field of cell-based therapy which needs to be further explored in clinical studies. In fact, cell surface engineering enhances the therapeutic potential of cells used for transfusion and transplantation applications. Cell surface engineering explores how manipulation of cell fate can present a dominant innovative technology that will likely find wide applications in cell therapy, tissue engineering, drug delivery, and biosensing/bioimaging.
Here, we have described three major types of modification used for cell surface engineering including hydrophobic insertion, electrostatic, and covalent modification, along with the modifications through nonnative cell surface functional groups, and graft from strategies for most clinically important cells such as RBCs, stem cells, islets, lymphocytes/splenocytes, endothelial cells, and hepatocytes. Although we have highlighted the importance of polymer-based methods, advances in enzyme engineering can enhance the efficiency of cell surface engineering approaches by introducing new biomolecules that are designed for specific targets and applications. In addition to the benefits of enzymatic approaches, metabolic strategies to engineer the cell surface glycans are very promising as well, but considerable progress still needs to be achieved using the various pathways. For instance, unlike enzymatic approaches, which, for example, delete entire saccharides from cell surface proteoglycans , metabolic strategies can modify these structures in a manner to engineer the cell surface to eventually regulate the function of cells.
Overall, advances in cell surface engineering approaches, especially in the design of new polymers for modifying the cell surface with specific biological functions combined with either enzymatic approach or metabolic modification, will hold great promises in the fields of bioengineering and transplantation medicine.
The authors acknowledge the funding by the Canadian Institutes of Health Research (CIHR) and from the Natural Sciences and Engineering Research Council (NSERC) of Canada to JNK. JNK holds a Career Investigator Scholar award from the Michael Smith Foundation for Health Research.
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