Surface Engineering for Cell-Based Therapies: Techniques for

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Surface Engineering for Cell-based Therapies: Techniques for Manipulating Mammalian Cell Surfaces Srinivas Abbina, Erika Siren, Haisle Moon, and Jayachandran N Kizhakkedathu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00514 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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ACS Biomaterials Science & Engineering

Surface Engineering for Cell-based Therapies: Techniques for Manipulating Mammalian Cell Surfaces Srinivas Abbina,1,2,$ Erika Siren 1,3,$, Haisle Moon1,2 and Jayachandran N. Kizhakkedathu 1,2,3* 1

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver,

BC, Canada, 2Center for Blood Research, University of British Columbia, Vancouver, BC, Canada, 3Department of Chemistry, University of British Columbia, Vancouver, BC, Canada $-Equal contribution *Corresponding Author: Phone: 604-822-7085 Fax: 604-822-7742 Email: [email protected]

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Abstract The introduction of cell-based therapies has provided new and unique strategies to treat many diseases and disorders including the recent approval of CAR-T cell therapy for the leukemia. Cell surface engineering is a methodology in which the cell surface is tailored to modulate cellular function and interactions. In addition to genetic engineering of cell surface proteins, a wide array of robust, innovative and elegant approaches have been developed to selectivity target the cell surface. In this review, we will introduce the leading strategies currently used in cell surface engineering including broadly reactive chemical ligations and physical associations as well as more controlled approaches as demonstrated in genetic, enzymatic and metabolic engineering. Prominent applications of these strategies for cell-based therapies will be highlighted including targeted cell death, controlling stem cell fate, immunoevasion, blood transfusion and the delivery of cells to target tissues. Advances will be focused specifically on cells which are the most promising in generating cell-based therapeutics including red blood cells, white blood cells (lymphocytes, macrophages), stem cells (multipotent and pluripotent), islet cells, cancer cells and endothelial cells. Keywords: Cell surface engineering, Polymers, Bioconjugation, Red blood cells, Stem cells, Islet cells, Endothelial cells


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1. Background

The cell surface is a highly heterogeneous environment, displaying distinct types of proteins, glycans and lipids.1 These structures have a critical role in governing cell fate including regulating cell-cell interaction, cell-niche communication and intracellular signaling pathways 1b, 1f-h, 2

. An assortment of reactive functional groups including amines, thiols and carbonyls

decorate the cell surface, providing a fertile landscape for exogenous modification (Figure 1).1a As the cell surface is an integral aspect of cell function, controlling the biochemical and cellular functions of cells by engineering the cell membrane with biomaterials, proteins and nanoparticles allow new opportunities in drug delivery, cell-based therapeutics, transfusion, tissue engineering and exploring fundamental cell biology

1b-d, 2a, 3

. Currently, cell-based therapies are being

explored for the treatment of number of diseases including cancer, autoimmunity, and wound healing.4

With the recent clinical success of engineered T-cells for B cell hematological

malignancies, this field of research has yielded widespread interest and afforded a great degree of momentum in the development of cell-based therapeutics.5



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Figure 1. An illustration of the eukaryotic cell membrane with different functional moieties; amine, thiol and carbonyls in very small quantities are tethered to either membrane bound proteins, glycoproteins or the carbohydrate component of glycolipids. While there is a wide availability of different functional groups on the cell surface suitable for exogenous modification, surface modification is quite challenging in practice. The cell surface composition is not only dynamic making consistent modification difficult, but any successful modification should also not have any unintended effect on the normal biological function of the cell surface such as adhesion, proliferation and differentiation1e, 1g, 6. Here, our intention is to highlight the recent developments of robust methods for cell surface engineering which utilize either globalized, non-specific (hydrophobic insertion, electrostatic, and covalent) modification or site-specific, orthogonal approaches through genetic, enzymatic or metabolic engineering. These elegant strategies encompass both well-established chemistry/material sciences in addition to molecular biology based procedures.


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The therapeutic potential of cell surface engineering will also be explored with a focus on approaches which further enhance the therapeutic potential of cells and other cellular functions. Prominent applications of these strategies for cell based therapies will be highlighted including targeted cell death, controlling stem cell fate, immunoevasion and the delivery of cells to target tissues. Advances will be focused specifically on cells which are the most promising in generating cell-based therapeutics including red blood cells, lymphocytes, macrophages, stem cells (multipotent and pluripotent), islet cells, cancer cells and endothelial cells. Finally, conclusions about the current state of the field and insight into the future directions are given.

2. Non-Specific Strategies 2.1. Covalent modification While a variety of functional groups are readily available on the cell surface, only a few functional moieties are reactive in the extremely complex and heterogeneous environment of the cell surface. Lysine (amine, -NH2) and cysteine (thiol, -SH) side chains are the residues most often recruited for direct covalent attachment of proteins, polymers, nanoparticles, and other small molecules to the cell surface. This approach is considered simple and straightforward as cells do not require any chemical or genetic pre-conditioning. The exogenous ligating substrates, however, must be pre-activated before delivery to the cell surface. 2.1.1 Amine reactive strategies The covalent conjugation of cell surface amines is most often accomplished using substrates displaying an activated carboxylic acid. Ligation of amines through cyanuric acid activated carboxylic acids is a commonly used strategy and is well established in RBCs and T-cell surface engineering (Figure 2) 7.



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Figure 2. Overview of approaches currently used in cell surface engineering. Left: Non-specific strategies for globalized engineering of the cell surface including hydrophobic (biotinylation, hydrophobic insertion/liposome fusion), covalent (NHS esters, cyanuric acid, maleimides) and electrostatic approaches (poly-L-lysine). Right: site specific enzyme mediated strategies for cell surface engineering. One of the earlier approaches for modification of RBCs is by covalent grafting of bulky CmPEG (cyanuric chloride activated methoxy-PEG) on the surface of RBCs to mask the blood group antigens on the surface, a methodology pioneered by Scott and co-workers .8 Described as immunocamouflage, this strategy attenuates the immune response to foreign cells and is especially important in improving the outcomes for patients that require chronic blood transfusion regimes such as those afflicted with sickle cell anemia and thalassemia. The introduced polymer coating on the cell surface acts as a shield to sterically prevent antibody mediated recognition of cell surface antigens. Scott and co-workers have shown that engineering 6 ACS Paragon Plus Environment

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the surface of RBCs using methoxy PEG (mPEG) had decreased anti-blood group antibody binding to human RBCs an observation which was further enhanced with increasing polymer size (Figure 3).9 mPEG-modified sheep RBCs were then transfused into mice and exhibited improved survival in vivo when compared to the untreated RBC. Likewise, PEG modified human RBCs have also shown normal in vivo survival in murine models. 8c

Figure 3. Immunocamouflage of membrane antigens is a function of linker chemistry, polymer size, and polymer surface density. (A) Shown is a graphical representation of the RBC membrane and the topical distribution of the Rh (C/c), Kidd (Jka/b) and MNS (S/s) blood group antigens. The PEG exclusion layer is the physical entity which gives rise to the 7 ACS Paragon Plus Environment


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immunocamouflage of the membrane antigens. (B) Influence of polymer size of Rhc antigen camouflage. Imunocamouflage of the Rhc antigen is shown by the decrease in mean fluorescence of cells. While the modification on the same size polymer (5 kDa) has negligible effect, the molecular weight of the polymer has moderate effect on immunocamouflage of the Rhc antigen. (C) In contrast to Rh antigens, increase in polymer size enhances the immunocamouflage of the MNS and Kidd blood group antigens. The data point PEG 20 kDa showed superiority than PEG 5kDa.9 The advantages of immunocamouflage extends well beyond blood transfusion. Graftversus-host disease, is an immune mediated attack on implants, transplanted cells and donor organs triggered by circulating T-cells. The intense immune response to these therapeutics remains a significant challenge in translational medicine. Although few pharmacologic agents like azathioprine and methotrexate have been successfully used to inhibit T-cell activation, these drugs are highly toxic to kidney, liver and gastrointestinal glands.10 Scott and co-workers examined effect of covalently attaching CmPEG to allogeneic lymphocytes on minimizing the cell surface recognition which causes T-cell activation and eventually graft-versus-host disease.7a The PEG mediated camouflage of the cell surface dramatically attenuated allorecognition of cells as was evident by dramatic differences in T-cell proliferation between unmodified and m-PEGmodified versions in both one- and two-way mixed lymphocyte reactions and flow cytometric analysis. While cyuranic chloride modified residues show rapid attachment to cell surfaces, the side products can be harmful. A large excess of material is often needed to achieve the intended effect and these side products can accumulate in high concentrations, drastically affecting cell viability. An alternative for cyanuric chloride is N-hydroxysuccinimide (NHS) activated macromolecules,


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currently the gold standard in amine targeted modification of the cell surface. Like cyanuric chloride, NHS esters can react with any surface accessible primary amine and this technique can be used to modify many different cell lines, such as RBCs, primary T cells, and cardio myoblasts. Due to the issues related to the antibody generation associated with PEG 8, hyperbranched polyglycerol (HPG)

3b, 3c, 11 11b, 11d, 12

, has been investigated as an alternative to PEG mediated

immunocamouflage. Like PEG, HPG has a voluminous structure suitable for immunocamouflage but is less susceptible to undesirable effects such as protein aggregation. Chapanian et al. developed antigen protected RBCs by grafting NHS activated HPGs (HPG-succinate) onto the surface of RBCs (Figure 4).11a

OH RO

OR O OR O

HO

OH OR HO OH HO

O

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O O

HO

O

O OH

O

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O HO

O O

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Figure 4: Pictorial representation of covalent modification of cell surface with HPG via NHS ester -amine coupling. Reactions were performed under physiological conditions.12



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HPG modification resulted in significant reduction in binding of blood group antibodies to cell surface of engineered RBCs compared to control RBCs.

11a, 11b, 13

In comparison to PEG

molecules with a similar hydrodynamic radius, HPG provided significantly higher levels of CD47 self-protein accessibility and greater protection of certain antigens on the RBC surface without changing native properties of RBC. This effect was attributed to the advantageous architecture of the HPG structure, who’s compact dendritic structure provides a non-continuous exclusion layer on the cell surface compared to the linear PEG polymer grafts.11b Chapanian et al. further investigated the in vivo circulation of grafted RBCs with HPG in mice and showed a normal circulation behaviour for HPG modified RBCs within a certain range of polymer size and graft concentration (Figure 5).

3b,

14

polyethyloxazoline as replacement to PEG

Recent studies also tested polymers such as 11d, 15

, however, the immunocamouflage was lower

despite exhibiting improved cell morphology. Overall, cell surface engineering of RBCs using covalent grafting of polymers has shown promising results in production of antigen protected RBCs towards universal blood donor cells. In addition, these methods provide a general working principle for cell-surface engineering using simple non-nucleated cells that can be adapted for other type of complex cell types.


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Figure 5: Blood circulation of HPG grafted RBCs in mice at different graft concentrations. a) HPG 20 kDa , b) HPG 60 kDa. Polymer concentration played a significant role in tuning the blood circulation times of grafted RBCs.14 It is important to note that multiple factors contribute to successful covalent modification of the cell surface, especially in polymer mediated immunocamouflage. The grafting density and depth of m-PEG brushes on cell membrane are vital in controlling the efficacy of immunocamouflage of the grafted cells.9, 11b, 16 The density of polymer brush border on the cell membrane is highly dependent on linker chemistry, linker length and molecular weight, and the concentration of m-PEG derivatives that are being used.9 NHS chemistry can also be used to introduce new functionalities on the cell surface. Cheng et al. used this approach to couple the mesenchymal stem cell (MSC) surfaces with E-selectin targeting peptides to improve adhesion of such cells onto blood vessels (Figure 6).17 Using a bi11 ACS Paragon Plus Environment


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functional NHS-PEG-maleimide (MAL) linker, the peptides were delivered to the cell surface in a two-step approach.18 The NHS esters of the linker first reacted with accessible amines on the cell surface to decorate the MSCs with MAL functionalities. Cysteine terminated E-selectin peptides were then conjugated to the cell surface through thiol-MAL chemistry, covalently anchoring the biomolecule to the outside of the cell. The resulting engineered MSCs exhibited rolling on E-selectin without affecting MSC cell functions. This strategy was repeated with a related E-selectin binding peptide with a slower dissociation constant (koff ) to mediate strong cell adhesion without stem cell rolling. Subsequent in vitro results demonstrated that such engineered MSCs can be directly captured from the flow stream by selectin surfaces or selectin-expressing cells under flow conditions.


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Figure 6. Peptide-assisted stem cell rolling under flow conditions. NHS chemistry was used to engineer MSCs with E-selectin targeting peptides using an NHS-PEG2-maleimide linker. Depending on the type of peptide attached, MSCs can be engineered to induce strong adhesion (b) or increased rolling behavior (c) compared to unmodified cells (a).17 Cell surface amines can also react with aldehyde containing moieties (macromolecules, drugs) through Schiff base formation. Aldehydes are highly reactive to primary amines on the cell surface, and their rapid and widespread interaction with tissues have proven useful in the development of biological adhesives.19 Aldehyde based strategies have also found utility in drug 13 ACS Paragon Plus Environment


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delivery. The aldehyde bearing drug Tucaresol, is currently under investigation 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 used.1e, 20 Aldehydes can also be introduced on the cell surface through the mild oxidation of the terminal 1, 2 -diol units present on sialic acid residues in the extracellular matrix. De Bank et al. used sodium periodate to selectively oxidize the sialic acid residues of living L6 myoblast cells to induce cell aggregation (Figure 7).21 This approach showed high cell compatibility without any significant effect on cell morphology.

Figure 7: Selective modification of diol units of glycoproteins of cell membrane; Left: A mild sodium periodate oxidation of cell surface proteins generates the aldehyde groups at C-9 positions of the N-acetyl galactose. Right: Neuraminidase enzyme cleaves seductively terminal


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glyosidic linkages of protein, flowed by galactose oxidase treatment yields aldehydes at C-6 positions of the sugar molecules.21 In 2010, Holden et al. used a similar approach to establish a new drug delivery for cancer therapy by coating tumor targeting macrophage surfaces with drug loaded nanoparticles composed of polyamidoamine dendrimers.22 The aldehydes generated on the cell surface through periodate oxidation formed a semi-stable Schiff base with amine groups of polyamidoamine dendrimers. The Schiff bases were further converted into stable secondary amine groups using sodium cyanoborohydride. By coating the macrophages with drugs instead of direct cell loading, it was demonstrated that a high concentration of drug can be delivered to cancer cells without compromising macrophage viability. 2.1.2 Thiol reactive strategies At least 15 cell surface proteins on mammalian cells possess thiol groups in either oxidized (disulfide bridges) or reduced form (free thiols). Various research groups have made use of free thiol groups to engineer the cell surface for various biomedical applications.23 Maleimide functionalized probes are the most widely used reaction partner for thiol targeting as they are stable, light insensitive, and exhibit high chemoselectivity to thiols through an energetically favorable Michael addition reaction.24 A significant advantage of this strategy is the plethora of commercially available reagents and linkers. The free thiol groups on cell surface can be chemoselectively conjugated with maleimide containing nanoparticles, biopolymers, and dyes at neutral conditions (pH 6.5-7.5) to form stable thio-ether bridges. In addition, the fact that covalent linkage between the reduced form of the disulfide group and targeted moieties can be easily tuned by altering the reaction conditions.23b, 25 While not all cell types contain a large quantity of free thiols on the cell surface, hemopeotic stem cells (HSC) possess a substantial amount of these 15 ACS Paragon Plus Environment


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residues. Stephan et al. used this strategy to enhance the efficacy of HSC therapy by covalently attaching drug loaded lipid nanoparticles to the cell surface.23d Therapeutic cells often rely on the concurrent delivery of adjuvant drugs after cell transfer, to maintain viability after systemic delivery and prevent uncontrolled differentiation. These agents often need to be maintained at high and sustained systemic levels, which can lead to dose limiting toxicity if not delivered in a controlled and cell-specific manner. Using phospholipids containing a maleimide head group, maleimide decorated nanoparticles in the 100-300 nm range were successfully prepared, loaded with glycogen synthase kinase-3β (GSK-3β) inhibitor and grafted to thiol groups present on the surface of HSCs (Figure 8). GSK-3β has been proven to enhance the repopulation kinetics of donor HSCs which maintains cell viability following systemic injection.26 Once attached, the drug loaded nanoparticles released GSK-3β into the HSCs over the course of 7 days and demonstrated increased the self-renewal post engraftment compared to control HSCs without compromising normal HSC function. In addition to optimizing the efficacy of HSC therapies, this study also demonstrated that the simple maleimide chemical approach is can be used for large target structures such as liposomes, though a large abundance of available thiol residues is required.

O O O

O P O O ONa

O N H

O N O

O


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Figure 8. Schematic illustration of covalent attachment of phosphorous lipid maleimidefunctionalized nanoparticles with the free thiol group which is linked to the cell membrane proteins, at physiological pH. 23d Maleimide/thiol conjugation has also been successfully applied to islet engineering. Pancreatic islets are multicellular aggregates responsible for regulating blood glucose levels. There is a keen interest in using islets as a cell based therapeutic for diabetic patients, however, once administered in vivo, the transplanted cells are subjected to immune mediated rejection.3d, 27 To address these challenges, cell surface modification has been adopted as a strategy to evade the immune system. Using combination of heterobifunctional MAL-PEG-phospholipid (DMPE) derivatives and poly (vinyl alcohol) (PVA) modified with thiol groups, Teramura et al. established a layer-by-layer method for coating the cell surface with a polymer shield (Figure 9).27f MAL-PEG–DMPE was incubated with islet suspensions and hydrophobically inserted into the cell membrane, forming a thin layer on the islet surface. The first PVA layer was subsequently introduced via a maleimide/thiol reaction between maleimide group of the PEG layer and thiol groups on modified PVA. Additional PVA layers were added using thiol/disulfide exchange reactions between the PVA bound to the cell surface (disulfide) and the introduced PVA (free thiol).



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Figure 9. A schematic view of surface engineering of the islet cells by a multilayered PVA membrane approach.27f

2.2 Physical Associations

2.2.1 Biotinylation The process of biotinylation is a covalent attachment of biotin molecule, a water-soluble vitamin B7, to cell surfaces, biomaterials, small molecules and macromolecules. The high affinity biotin-avidin (as well as avidin analogs streptavidin and NeutrAvidin) interaction (Kd = 10-15 M) is highly resistant to heat, pH, organic solvents and other denaturing environments and as a result, has promising applications in different biotechnological and biomedical fields.28 Researchers have taken advantage of this extremely high affinity binding between biotin and its binding partners to expand the tool kit for cell surface modification. Commercially available heterobifunctional biotin terminated linkers that are for chemo selective for amines, thiols, and carbonyl groups are the most common used reagents for biotinylation of the cell surface. In some instances, enzymes such as biotin ligase can also be used as a biotinylation agent.29 Once the cell


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is labeled with biotin, it can be readily functionalized with a wide range of tagged small molecules, microspheres, polymers or proteins through an avidin bridge. 30 Surface modification of endothelial cells has been used as a tool to manipulate the properties of the vasculature to control cell-adhesion and cell-behavior. Endothelial cells form the outermost layer of blood vessels and provide the vasculature with antithrombotic and antiinflammatory properties as well as protection against blood flow mediated shear stress.31 One of the earliest studies demonstrating endothelial engineering employed avidin-biotin ligation approach. In this study, biotinylated endothelial surfaces were used to force strong cell adhesion onto synthetic surfaces coated with an avidin.32 This cell-surface modification enhanced the formation of lower affinity integrin-mediated focal adhesions and has been subsequently used to generate viable endothelial cell layers on polymeric vascular grafts. In another study on endothelial engineering, Deglau et al. used biotin-NeutrAvidininteractions for modelling site-specific targeted delivery to human coronary artery endothelial cells in ex-vivo conditions.33 Under flow conditions which mimic arterial shear stress, a solution of NHS-PEG-biotin was delivered to scrape-damaged bovine carotid arteries. Targeted delivery to damaged arteries was then modelled using fluorescently tagged NeutrAvidin-coated polystyrene microspheres which were flowed over the endothelial monolayers. A dense layer of styrene microspheres –almost 6-fold higher—was found on biotinylated bovine carotid arteries whereas control arteries showed minimal adhesion (Figure 10). Although the demonstrated approach has severe limitations and examined only in vitro conditions, this strategy might ultimately find applications in catheter-based or surgical procedures. Such studies are ongoing and have immense potential for clinical translation. It is worth mentioning that even though



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avidin–biotin was shown to significantly promoting initial endothelial cell adhesion, there is no report on the effects of avidin at longer adhesion times.32a

Figure 10: Schematic of the endothelial surface grafting with microspheres through biotinavidin bridge.33b While stem cell therapies have great potential in regenerative medicine, delivery to the affected tissues using systemic administration is extremely inefficient with only a small percentage of cells reaching the desired location.34 MSCs, in particular, have challenges in cellular homing due to insufficient expression of surface markers.35 Consequently, cell-surface modification approaches can play a key role in enhancing and presenting surface ligands onto the cell surface for the delivery of stem cells to their target tissues. For instance, Sarkar et al. presented a promising modification technique in which they modified MSCs with a nanometerscale polymer containing sialyl-Lewisx (SLeX), a carbohydrate structure found on the surface of leukocytes responsible for cell rolling and adhesion to endothelial surfaces.36 When present on cell surfaces, SLeX serves as homing ligands to areas of inflammation. Figure 11 presents the schematic of such engineering strategy for enhancing the rolling of MSCs in which streptavidin is used as a linker to bridge biotinylated Slex to the biotinylated MSC surface. It has been


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reported that the modified MSCs not only showed a great homing in vivo but also the MSC phenotypic properties including multi-lineage differentiation were conserved.

Figure 11: Engineering the MSCs surface using the conjugation of SLeX by covalent biotinylation and a streptavidin-biotin bridge to improve their rolling interactions in vitro.36 While the biotinylation strategy is versatile and widely used for different applications, it comes with limitations. For instance, overcrowding the surface with biotin can inhibit proper cell function and even lead to cell death.11a In addition, the protein component of this technique is of bacterial origin (e.g. streptavidin) which can generate an immune response. The increased risk of immune mediated rejection of biotinylated cells makes this cell surface engineering strategy unrealistic for in vivo applications.37 2.2. Electrostatic interactions The outer surface of the cell possesses significant native negative charges due to the presence of sialic acid residues on glycoproteins anchored to the cell membrane. This charge plays a 21 ACS Paragon Plus Environment


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crucial role in preventing cell aggregation.38 By exploiting the charged nature of cell membrane, various electrostatic methods have been developed to engineer the cell surface. Over the two past decades, layer-by-layer assemblies of polyelectrolytes to form polyelectrolyte multilayer (PEM) films have been well optimized. PEM films are formed by the alternate assembly of polycations and polyanions, and represent an innovative strategy for engineering the cell surfaces at the molecular level.39 Cationic polymers, such as chitosan, poly-(allylamine hydrochloride), poly-Llysine (PLL), and poly(ethyleneimine) (PEI), strongly interact with the negatively charged mammalian cell surface and have been widely used for this approach .39 However, the cytotoxicity of the polycations is one of the main limitation of this approach. To circumvent this issue, several groups have used an inert PEG spacer between the polycation and the cell surface to avoid the direct contact which may trigger apoptosis.40 Recently, Wilson and coworkers utilized this approach in making more biocompatible PEM films using poly(L-lysine)-graftpoly(ethylene glycol) (PLL-g-PEG) copolymers.41 Pancreatic islets were encapsulated in a PEM shell through the layer-by-layer (LbL) assembly of PLL-g-PEG and negative charged alginate (Figure 12).


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Figure 12: Graphical illustration of coating of a pancreatic islet cell surface by layer-by-layer self-assembly of poly(ethyleneimine) films. Appropriate combination of poly-L-lysine-g-PEG copolymer and poly(alginate) were used. 41 In another effort to overcome the cytotoxic effects of using polycations, Brooks and Kizhakkedathu groups developed neutral polymers capable of cell-surface modification. Hyperbranched polyglycerols (HPGs) were decorated with choline phosphate (CP), a neutral zwitterionic polymer which electrostatically interacts with the phosphatidyl choline end groups on cell-surface lipids in the RBC membrane (Figure 13).42 Various groups have adopted this methodology to modify the cell surface for different applications.39, 41, 43



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Figure 13: An illustration representing the structure of polyvalent choline phosphate molecules contained hyperbranched polyglycerol (HPG-CP) and schematic representation of adsorption of HPG-CP on the RBC membrane.42

2.3 Hydrophobic insertion into the cell membrane

Hydrophobic interactions are frequently exploited for cell surface engineering, as therapeutic molecules containing lipid tails and cholesterol anchors can easily insert into the cell membrane.44 Hydrophobic insertion is also considered less harmful to cells compared to covalent modification as the potential for cross-linking and irreversible loss of protein function is prevented. The type of lipid chain is an important consideration, with long and saturated lipid chains increasing the likelihood of establishing a relatively stable molecule attachment on cell membranes.44 While hydrophobic regions can be genetically or non-genetically incorporated into target substrates, covalent attachment of lipids is the most common strategy for polymers, carbohydrates and proteins alike. In an effort to improve MSC homing, Won et al. used a MAL-


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PEG-DMPE phospholipid to incorporate a lipid anchor into a recombinant version of the protein CXCR4 (rCXCR4).45 rCXCR4 is a chemokine receptor found on stem cells in vivo that specifically binds to stromal-derived factor-1 (SDF-1), a protein released by myocardial tissue to recruit MSCs.46 The receptor is lost, however, when MSCs are subjected to in vitro cell expansion as is often done in stem cell based therapies.47 In as little as 10 minutes of incubating expanded MSCs with rCXCR4-PEG-DMPE, the cells exhibited a recovered response to SDF-1 with a 2 fold enhanced migration toward an SDF-1 gradient surface. In a subsequent study, the homing of rCXCR4 enriched MSC’s were tested in vivo, in which an enhanced homing and retention of the MSC’s were observed compared to the control up to 3 days post-injection in a rat model.48 Hydrophobic insertion can also be used to attach polymers to the cell surface. Bioactive HPGs decorated with vasculature binding peptides (VBP) were conjugated with octadecyl chains and incubated with MSCS, generating a surface coating suitable for targeted delivery to the vascular endothelium (Figure 14).49 The bioactive polymer coating significantly enhanced the cellular affinity for the vascular endothelial adhesion molecule (VCAM), a protein which is over expressed in inflamed blood vessels.



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Figure 14: Stem cell modification via hydrophobic insertion of modified hyperbranched polyglycerol (HPG). A bioactive HPG modified with octadecyl chains and vasculature binding peptides (VBPs) was utilized for the modification of stem cells as a novel cell-guidance molecule and guide them to defective vasculature. In vitro studies demonstrated the proof-of-concept.49 Glycans are considered as one of the most important components of stem cells as they are essential in signaling between the cell and the external environment. In embryonic stem cell (ESC) differentiation, modulation is mainly controlled by signaling molecules such as fibroblast growth factors 2 (FGF2), Wnt, Notch and other lineage-specific and stage-specific embryonic antigens (Lewis X/SSEA-1, SSEA3–4). Like proteins, glycans can be engineered using different techniques to direct cell behavior. Through hydrophobic insertion, Huang et al. demonstrated 26 ACS Paragon Plus Environment

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that the installation of synthetic lipid anchored neoproteoglycans (neoPGs) on the surface of mouse ESCs resulted in an enhanced affinity to FGF2. ESC differentiation was subsequently directed towards a neural phenotype following neoPG surface modification.50

2.4 Liposome fusion

Liposome fusion is a special case of hydrophobic insertion used to deliver a large number of non-endogenous substrates to the cell surface. In this process, lipid vesicles composed of phospholipids containing unique functionalities dock onto the cell surface and are subsequently triggered to fuse with the cell membrane. This process mimics pre-existing cellular processes in which contents encapsulated by lipid vesicles are delivered to tissues via membrane fusion. An essential aspect of this process is triggering membrane fusion, first demonstrated by Dutta

et al.51 In this study, 60 nm large unilamellar vesicles (LUV) displaying ketone

functionalities were prepared by mixing together dodecanone with egg palmitoyl-oleoyl phosphatidylcholine (POPC) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) at a ratio of 5:93:2 (Figure 15). The cationic nature of DOTAP was responsible for the induction of membrane fusion due to the electrostatic destabilization of the negatively charged cell surface.52 Fibroblasts were incubated with ketone-LUV’s for 2 hours followed by the addition of Rhodoxyamine, used to visualize the successful introduction of ketone functionalities onto the cell surface. Furthermore, no change in cell behavior or morphology was observed following liposome fusion demonstrating the non-invasive nature of the liposome fusion strategy and ketone-oxime ligation.



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Figure 15: A representative scheme of cell surface engineering based on liposome fusion. Top (left to right): Egg palmitoyl-oleoyl phosphatidylcholine (POPC), dodecanone and 1,2-dioleoyl3-trimethylammoniumpropane (DOTAP) are combined to generate large unilamellar vesicles (LUV) displaying ketone functionalities (blue). Bottom: Ketone-LUV’s are fused with the cell membrane and subsequently tagged with Rhod-oxyamine to confirm cell surface ketone display.51 This versatile strategy has recently been incorporated with orthogonal click chemistry in a study used to generate 3D cell cultures in the absence of scaffolds. The generation of complex three-dimensional (3D) tissues with multiple cell types in vitro is a major area of research in regenerative medicine dedicated to the in vitro generation of artificial organs. The proper functioning of organs is extremely sensitive to minor changes in tissue structure and organization. As such, control over cellular assembly would be a huge advantage in streamlining the generation of artificial organs.

Rogozhinikov et al. demonstrated such

control by using liposome fusion mediated click chemistry (ketone-oxime) to construct complex cardiac tissue composed of cardiomyocytes, cardiac fibroblasts and human umbilical 28 ACS Paragon Plus Environment

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vein endothelial cells (HUVECs) (Figure 16).53 Ketone and oxyamine presenting cells were rapidly clicked together via the stable oxime ligation and assembled into spheroids and then tissues. When added in sequential order, assembled tissues displayed defined layers allowing for strict pattern control. The various tissue constructs were stable and viable for several weeks.

Figure 16: Bio-orthogonal assembly of 3D cardiac tissue. Cardiomyocytes, HUVEC and fibroblast cells are represented in blue, green and red respectively. Top: Ketone and oxyamine groups are installed on cell surface through liposome fusion and rapidly click cells together via oxime linkages. Bottom: 3D projection of fluorescently labelled cardiomyocytes, HUVEC and fibroblast cells as (A) a single monolayer of non-engineered cells (10 µm thick) (B) A random mixture of engineered cells clicked together to form a multi-layer cardiac tissue (55 µm thick). (C) As an oriented 3-dimensional cardiac tissue following sequential addition 29 ACS Paragon Plus Environment


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of engineered cells displaying clickable bio-orthogonal groups on the cell surface (20 µm thick). Scale bar = 60 µm.53 There are some limitations to hydrophobic approaches. Although introducing therapeutic molecules into cell membrane may not perturb cellular physiology, the inserted molecules may get transferred to the inner leaflet facing cytoplasmic side of the cells by flippases or get distributed to the membranes of other cellular organelles which may affect cell function. In addition, the half-life of lipid conjugated proteins is relatively short usually ranging from one to two hours.32b, 54 Serum proteins present in cell media are also able bind to the lipid portion of these molecules. This interaction can often reduce the insertion efficiency into cell membranes unless buffer conditions are used exclusively.44

3. Orthogonal Strategies

One major disadvantage of direct modification of cell surface membrane is the lack of specificity. Any free amino (-NH2) or thiol (-SH) groups presented on the cell membrane/protein is susceptible for modification which can result in alteration of the natural membrane/protein functions. It is also difficult to impart specificity in hydrophobic insertion, as lipid anchors traditionally interact with the cell surface in a random fashion though there has been progress in directing hydrophobic insertion using photolithographic patterning techniques.55 To impart reaction specificity towards cell surface modification, it is desirable to generate cell surface functional groups that not normally present on cell surface which can be utilized as functional groups for covalent grafting. These non-native substrates are termed as bioorthogonal, meaning they have no natural reaction partner in vivo, do not interfere with normal cell processes, and their ligation does not generate side reactions or side products which may be


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harmful to the cell. The introduction of non-native functional groups on the cell surface with exogenous materials is not only advantageous towards developing cell-based therapeutics, but also for live cell imaging, cell separation and cell-based censors.56 In addition to genetic engineering, these attempts have utilized either enzymatic or metabolic labelling of the cell surface –and in some occasions, both.

3.1 Genetic engineering

The genetic engineering of living cells is a well-established technique used to upregulate (or downregulate) protein expression. Using viral vectors as a delivery vehicle for new genetic information, the subsequent changes in protein expression can be used to manipulate cell behaviour. This strategy is particularly useful in understanding the mechanisms of cells undergoing the drastic, unregulated changes often seen in cancer progression. To provide insight into the role of the cell surface in cancer metastasis, Shurer et al. genetically manipulated the glycocalyx of HEK cells, a carbohydrate brush structure on the cell surface, which plays a major role in homeostasis.57 By upregulating the Muc1 glycostructures, cellular adhesion was inhibited, highlighting the importance of Muc1 in metastatic cancer progression. In another effort, Levy et al. engineered MSCs through mRNA-transfection to upregulate the expression of PSGL-1 and SLeX on the MSC surface to enhance cellular homing.58 Like SLeX, PSGL-1 serves as homing ligands to the areas of inflammation. In vivo, the upregulation of PSGL-1 and SLeX on MSCs enhanced homing into mouse inflamed ear vascular endothelium by 30 percent compared to native MSCs. The engineered MSCs also improved homing to the mouse bone marrow.



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There are risks, however, when it comes to genetic engineering in vivo. The genomic location of the inserted genes is not tightly regulated with vector based techniques and the risk of insertional mutagenesis is present when this technique is applied to clinical applications.59 In addition, not all cell types can be easily transfected such as erythrocytes and T cells.60 In these systems, fusion proteins can be used to introduce non-native proteins on the cell surface. While some applications can successfully generate fusion proteins in situ to display non-native substrates on the cell surface, often the fusion protein is expressed outside the cell, purified and then added to the target cells. For instance, the glycosylphosphatidylinositol (GPI) anchor is a glycolipid naturally abundant on the cell surface. It was found that some proteins genetically engineered to contain a GPI moiety can naturally anchor to cell membranes and ‘paint’ cell surfaces.1d Recently, this approach has been used to deliver the cancer suppressing protein, TIMP-1 to SW480 cells, a colon carcinoma cell line. Following insertion of the TIMP-1-GPI fusion protein into the cell membrane, cell proliferation was significantly attenuated. This effect was further illustrated in vivo, as the peritumoral injection of TIMP-1-GPI fusion protein caused an ~8 fold decrease in tumor growth.61 Antibody fragments can also be used to specifically attach proteins to the surface of a given cell or tissue. The single-chain antibody fragment of TER-119 (ScFv), binds to glycophorin A, a glycoprotein widely expressed on cell surfaces.62 ScFv mediated targeting has been used successfully in supressing immune mediated lysis of mismatched erythrocytes through the display of a ScFv fused decay-accelerating factor (DAF).63 DAF is a complement regulatory protein (CRP) known to downregulate the cytokine signalling responsible for clearing foreign substances from systemic circulation.64

3.2 Enzyme mediated cell surface engineering 32 ACS Paragon Plus Environment

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Fusion proteins offer a simpler approach to modifying the cell surface compared to direct genetic engineering, however, developing and purifying these proteins is a time intensive process when a wide array of candidates are required. In addition, the ligation of large proteins to target molecules can often compromise the function of the substrate.65 Enzyme mediated cell surface attachment is a new technique with allows for the in situ ‘fusion’ of candidate proteins/molecules to pre-existing structures. Enzymes often recognize a specific peptide sequence within the protein substrate referred to as a ‘tag’ in literature (Figure 2). These tags are either covalently attached a polymer or genetically engineered to be incorporated into either the C or N terminus of the target protein. As the tag accounts for a small mass percentage of the candidate macromolecule, this process is much less likely to interfere with protein function compared to fusion proteins. Most enzymes are inherently site specific, and exhibit high conversion rates under physiological conditions. Cofactors may also be used to easily improve enzyme reaction rates without drastically changing the tag structure or requiring a large excess of substrate. This technique is already well established in the preparation of protein-protein or protein drug conjugates and continues to be a rapidly developing field.29c 3.2.2 Sortases Sortase A (SrtA) is a calcium dependent transpeptidase derived from Staphylococcus aureus which catalyzes the ligation between a LPXTG recognition motif and substrates bearing a series of glycine repeats.66 The enzyme contains a catalytically active cysteine residue, which cleaves between the Thr and Gly resides of the LPXTG recognition motif to form a thioester intermediate that subsequently reacts with the N-terminus of oligoglycines. While SrtA is specific to the LPXTG motif, the enzyme has a broad nucleophilic scope beyond oligoglycines. Known in literature as ‘sortagging’, a wide array of proteins, lipids, sugars, polymers and



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surfaces can be used as SrtA substrates by simply labelling the macromolecules with the LPXTG recognition motif. Tanaka et al. first demonstrated this approach on cell surfaces by engineering the SrtA recognition tag LPETG to the osteoclast differentiation factor (ODF) cell surface marker transfecting the protein into HEK cells.67 Using Alexa-633 streptavidin as a probe, it was shown that SrtA was successfully able to modify the cell surface when co-incubated with biotin containing a three-glycine oligomer. They further demonstrated that this approach can work in multiple cell types, including CHO and HeLa cells. Recently, this strategy has been expanded for use in controlling cell behavior. Using LPETG-tagged tumor specific antibodies (VHH7), non-genetically engineered CD8+ T cells were subjected to sortagging.68 Within one hour at room temperature, ~1 million tumor specific antibodies were conjugated per cell using the sortagging approach. When incubated with a mixture of cells, only cells containing the VHH7 antigen were targeting by the engineering Tcells, demonstrating cell specific killing in a fashion analogous to, but conceptually distinct from the well-established genetically prepared CAR T cells.69 In an alternative approach to sortagging, cell surfaces themselves have been engineered to display LPTXG tags. Using CRISPR/Cas9 technology, Shi et al. modified the cell surface receptors Kell and Glycophorin A on erythrocytes to express LPETG tags on their extracellular domains (the N and C termini for Kell and Glycophorin A respectively).70 The in vivo survival of the sortagged cells was not affected indicating that the display of the tag itself does not cause premature removal of the cells. The study further demonstrated successful ligation to LPTEG tagged erythrocytes via the attachment of glycine terminated biotin.


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It is important to note that a major drawback of sortagging is its susceptibility to reaction reversibility. The glycine residue cleaved from the LPXTG tag in the first step can compete as a nucleophile to reform the original species and an excess of nucleophile on the cell surface is needed to drive the labelling reaction. As such, the total percentage of cell surface residues that can be modified is limited. While strategies to overcome the inherent reversibility of the reaction have been explored, studies have been limited to solution based protein ligations and have yet to be demonstrated on the cell surface.71 3.2.2 Transglutaminases Transglutaminases (TGases) are a class of calcium dependent enzymes ubiquitous in nature with roles in pathways related to wound healing as well as apoptosis and migration.72 TGases catalyze cross-linking reactions between the ε-amino group of lysine and the terminal amide on glutamine residues, forming a ε-(γ-glutaminyl) lysine isopeptide bond that is highly resistant to proteolysis. Like sortases, TGases operate through the formation of a thioester intermediate with glutamine which is subjected to nucleophilic attack from the lysine substrate. While TGases are specific towards glutamine substrates, the enzyme active site accepts a wide array of amine containing compounds, making the enzyme an attractive catalyst for preparing protein conjugates.72a Microbial transglutaminases in particular have found widespread use in developing antibody-drug conjugates.73 In cell surface engineering, however, tissue transglutaminases (tTGases) are the type of TGase most often employed in modifications. This is because tTGase has a number of known amine substrates found in the ECM of cells including collagen II, fibronectin, osteopontin and osteonectin.74 The enzyme also demonstrates good activity at low temperatures and a reduced antigenicity in cell and tissue cultures.75



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tTGase was first utilized for modifying the cell surface by Lin et al.

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76

A series of

glutamine tags (Q-tags) were identified and expressed on the N terminus of PDGF receptor of HeLa cells, which displayed the Q tag on the cell surface. In the presence of tTGase and biotin containing a terminal amine handle, HeLa cells were biotinylated exclusively on the cell surface in as little as 30 minutes. Messersmith et. al. further expanded on this concept by attaching Q tags to the terminus of polyethylene glycol and using tTGase to ligate the polymer to the endogenous amine substrates on cartilage tissue.74 While still largely unexplored, this approach has the potential to be broadly applicable to a variety of tissues for delivering therapeutic agents or controlling adhesion at the tissue surface. 3.2.3 Halotag proteins The enzyme mediated strategies for engineering the cell surface discussed so far have used proteins with endogenous cross-linking function to covalently attach and release two substrates. Halotag protein (HTP) mediated cell surface modification is a departure from this behavior as the catalytically active HTP is a partially inactive derivative of a bacterial hydrolase that is irreversibly incorporated into the crosslink itself.77 Traditionally, HTP’s are responsible for the removal of halides from aliphatic hydrocarbons through hydrolytic cleavage. In its inactive form however, a mutation in the hydrolytic active site of HTP traps the enzyme substrate intermediate, preventing the originally intended hydrolysis and instead forms a highly stable covalent adduct.78 In this sense, the enzyme itself is a tag which orthogonally reacts with alkane substrates containing a terminal chloride (chloroalkanes). Halotagging is accomplished using a two-step approach: the genetic fusion of a 33 kDa HTP to the protein of interest followed by capture and covalent modification of the HaloTag ligand.79 Most reported applications have used HTP methods to append molecules that serve as detection or capture agents but when fused to cell


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surface proteins, this platform can also be amended to modulate biological processes in living cells.80 The HTP platform was first used for cell surface engineering by Pulisipher et al. They used HTPs to regulate the fate of ESCs into neural phenotypes by engineering the glycans displayed on stem cell surfaces.81 HTPs were used to present chloroalkane terminated heparan sulfate (HSCL) glycosaminoglycans (GAGs) onto the membrane of mouse ESCs (Figure 17). The remodelled glycocalyx of ESCs triggered an accelerated exit from the self-renewal and promoted differentiation into mature neuronal cells.

Figure 17: A) Halotag strategy for attaching HS GAGs to cell membranes using HaloTag protein to control stem cell differentiation. CL = chloroalkane linker, HTP=Halotag Protein. B)



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Molecules used in this study.

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Biotinylated heparan sulfate (right) was functionalized with

chloroalkane linker 1 using reductive amination chemistry.81 While the HTP approach to cell surface engineering is still in it’s infancy, the simple chloroalkane moiety required for cross-linking is advantageous from a synthetic perspective compared to more complex peptide and protein tags. It is also important to note that the HTP adducts are extremely stable, and have been shown to persist on the cell surface for 8 days following attachment. With this, the halotagging strategy may be ideal for cell surface engineering techniques that require prolonged effects.81 3.2.1 Glycotransferases and hydrolases In some instances, cell behavior can be modified by removing functionalities from the cell surface. Regular remodelling of the cell surface already exists in nature on the cell surface glycocalyx. Sialic acids and other sugars which decorate the glycocalyx exist in dynamic equilibrium and are regularly modified by a range of enzymes including sialosides and glycotransferases in response to a range of exogenous effectors.82 As the majority of the glycostructures play an important role in a cell’s ability to trigger or evade immunological recognition, glycocalyx remodelling of pre-existing cell surface molecules may be used as an additional strategy to control the immune response.83 To convert erythrocytes into universal donor, antigen-null stealth cells which, Kwan et al. used carefully selected galactosidase from high-throughput screen to remove blood group antigens from cell surfaces (Figure 18).84 Using an engineered hydrolase from Streptococcus pneumoniae or Elizabethkingia miricola or Bacteriodes fragilis, enzymatic removal of the


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terminal N-acetylgalactosamine or galactose of A- or B-antigens respectively yielded cells which mimic O-type erythrocytes. 11c

Figure 18: Structures of AB antigenic carbohydrates on the RBC surface and their enzymatic conversion to O-type blood group. Structures represented in red indicate the sugar moieties removed by the enzyme to generate universal RBC’s.11c Precise glycocalyx editing has also opened a promising avenue for immunotherapy applicable to cancer therapeutics. Successful tumors evade immune cell recognition by overexpressing inhibitory ligands. Sialylation of cancer cell glycans provides immune protection to cancer cells by disrupting the interaction between the natural killer (NK) cell activating receptor natural killer group 2D (NKG2D) and its corresponding ligands on the tumor surface.85 To achieve targeted glycocalyx editing to cancer cells, Xiao et al. synthesized tumor-specific 39 ACS Paragon Plus Environment


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antibody-enzyme conjugates by chemically fusing a recombinant sialosides to the anti-human epidermal growth factor receptor 2 (HER2) antibody trastuzumab (Tras).86 The antibody-enzyme conjugates desialylated cancer cells in a HER2 dependent manner, allowing increased NKG2Dligand binding and restoring NK cell killing of the cancer cells. Although existing enzymatic approaches are still subjected to certain restraints, this innovative, non-genetic approach allows for the rapid and stable engineering of membranes. Additionally, the orthogonality of this technique may allow for one or more ligands to be used.

3.3 Metabolic Engineering

Enzyme engineering has introduced orthogonality into cell surface engineering, imparting spatial control over modification in a gentle, but catalytic manner. However, much of enzyme mediated labelling still requires some aspects of time intensive genetic engineering for tagged substrates. The continued pursuit of imparting orthogonality without the use of genetic manipulation has ignited the field of metabolic engineering. A technique first developed by Bertozzi and co-workers, metabolic engineering utilizes the endogenous machinery of the cell to incorporate tags into biomolecules using tagged metabolic precursors.87 As metabolic precursors are often small molecules, the tags must represent only a small portion of the structure and so peptide and protein tags cannot be used. Instead, simple chemicals able to undergo ‘click’ reactions are used. Transformations classified as ‘click’ reactions are fast, chemoselective, occurs at physiological conditions (37 °C, pH 7) and have no side reactions or side products.88 While there are many types of click reactions, the ones most often used in biology are azidealkyne Huisgen [3 + 2] cycloaddition promoted by either copper (CuAAC) or ring strain (SPAAC) and the Staudinger ligation.89 Both processes require azide (-N3) moieties as a click 40 ACS Paragon Plus Environment

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reaction partner and the azide is the most popular tag used for metabolic labelling. Metabolic labelling is particularly advantageous in engineering cell surface proteoglycans, as glycostructures in various forms compose a significant portion of the extracellular matrix and the rapid turnover of these structures ensure rapid metabolic incorporation.90 This was first demonstrated by Saxon et al. in which azides were installed on the cell surface using the synthetic azido sugar precursor N-azidoacetylmannosamine (Ac4GalNAz) .91 The precursors were incorporated into mucin-type O-linked glyco-proteins in jurkat cells via the Nacetylgalactosamine (GalNAc) salvage pathway. After 3 days of incubation, the glycostructures were the orthogonally labelled with biotinylated triarylphosphine via click-based Staudinger ligation to produce stable cell-surface adducts. This strategy was also used to manipulate cell-cell interactions on peripheral blood mononuclear cells (PBMCs) to achieve targeted apoptosis.92 MCF-7 human breast cancer cells were cultured with Ac4GalNAz to enrich the cell surface with azido groups followed by the conjugation of alkynyl and PEG-modified β-cyclodextrins (alkynyl-PEG- β -CD) via a bioorthogonal copper(I)-catalysed azide-alkyne Huisgen [3 + 2] cycloaddition (CuAAC). In a size-selective process known as host-guest complexation β-cyclodextrins specifically and irreversibly bind to molecules containing azobenzene moieties.93 The β -CD decorated MCF-7 cells were then co-incubated with azobenzene aptamer decorated PMBCs. Compared with unmodified PBMCs, aptamer-modified PBMCs effectively targeted MCF-7 cells and induced cell apoptosis as measured by lactate dehydrogenase release assay. While most cell therapies rely on cell surface engineering in vitro followed by in vivo administration, metabolic engineering can label cells in vivo without ex vivo or in vitro interventions. This has been most recently demonstrated by Wang et al. in an elegant strategy 41 ACS Paragon Plus Environment


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used to selectively label cancel cells in vivo.94 Taking advantage of the enhanced enzyme activity exhibited in cancerous cells, the study utilized a ‘caged’ Ac4ManAz that can be selectively cleaved by cancer-overexpressed enzymes (Figure 19). Once decorated with azide moieties, an ‘active’ tissue targeting via anchored click chemistry (ATTACK) strategy was employed to deliver alkyne labelled dibenzocyclooctyne–doxorubicinconjugate to cancer tissue via click chemistry. The ATTACK strategy was deemed effective against MDAMB-231 triplenegative breast cancer, LS174T colon cancer, and 4T1 metastatic breast cancer in mice. Substantially reduced toxicity on non-cancerous cells was also observed with the use of ATTACK therapy.


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Figure 19: Tumor targeted metabolic labelling. a) ‘Caged’ Ac4ManAz is delivered to cells and selectively uncaged by enzymes present in cancerous cells which ‘trigger’ the continued metabolism of Ac3ManAz derivatives. P (blue) represents the trigger-removable protecting group phosphoenolpyruvic acid (PEP). b) Schematic illustration of ATTACK (active tissue targeting via anchored click chemistry) strategy in vivo.94 It is important to note that although various enzymatic treatments and metabolic approaches have been also employed to incorporate different functional groups such as biotin,



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alkyne, azide, thiol and ketones into living cell surfaces, these technologies might influence cell physiology in long term studies.95

4. ‘Grafting from’ strategies

Surface initiated polymerizations such as atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain-transfer (RAFT) polymerization have been intensely studied for different applications including the generation of non-biofouling surfaces, cellselective adhesiveness, and stimuli-response materials.96 In a similar manner, the grafting of synthetic polymers on cell surface offer unique advantages over proteins, especially in terms of changing the physical properties of modified surface, increasing the functional groups for secondary interactions, and providing opportunities to generate cell-polymer hybrid structures.97 Although the cell surface possesses an abundance of different functional groups suitable for surface initiated polymerizations, the reaction conditions required for polymerizations which are very toxic to live cells. Early polymerization studies performed on RBCs using either a low molecular weight ATRP catalyst or macromolecular ATRP catalyst gave promising data on ATRP-mediated polymerizations on the cell surface. The catalyst concentration required however, greatly interfered with proper RBC function and viability.98 In a significant advance to this approach, Niu et al. modified Jurkat cells by a novel ‘grafting from approach’ in which polymerizations occurred starting from the cell membrane to form very finely distributed polymers on the cell surface.99

Niu. et al. inserted chain transfer reagents through the

hydrophobic insertion of molecules on Jurkat cell membrane using lipid anchors (Figure 20). These initiators were used for subsequent visible light mediated photoinduced electron transferreversible addition fragmentation chain-transfer polymerization (PET-RAFT) with Eosin Y


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(triethanolamine) as a catalyst, methoxy-PEG acrylamide-1k (PEGA-1k) as a monomer and initiation by a light emitting diode source (465 nm). In this study, molecular weights of up to 10 kDa were observed in in PBS buffer (pH 7.4) in as little as 30 minutes. The ‘grafting from’ approach was additionally compared to traditional NHS estermediated chemical ligation of PEG. As the ‘grafting from’ approach begins by adhering less bulky small molecules to the cell surface, followed by polymerization into larger structures, the approach exhibited a marked increase in the number of grafted chains that successfully attached to the cell surface compared to NHS mediated interventions.

Figure 20: A scheme showing non-covalent attachment of chain transfer agents on the cell surface and polymer brushes generation on the mammalian Jurkat cell by PET-RAFT process.99

Summary and future prospective

The attachment of small molecules, macromolecules (peptides, proteins, glycans etc.) onto the surface of different cell types opens a new and exciting avenue in the field of cell-based therapy. Cell surface engineering enhances the therapeutic potential of cells used for transfusion, 45 ACS Paragon Plus Environment


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transplantation, targeted cell delivery and drug delivery 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, regenerative medicine, drug delivery and bio sensing/ bioimaging. Here, we have described the major types of modification used for cell surface engineering which are able to engineer the cell surface in either a globalized or site-specific manner (Table 1).

Globalized or non-specific approaches include hydrophobic insertion, electrostatic, and

covalent modification while orthogonal approaches include modifications through genetic, enzymatic or metabolic engineering. Finally, a new area of cell surface engineering was discussed in which polymers are synthesized on the cell surface itself. These strategies have been demonstrated on a range of clinically relevant cells such as RBCs, stem cells, islets, lymphocytes, endothelial cells, and cancer cells. While some more well-established strategies have reached early stage clinical trials, much of the cell surface engineering toolkit is still in its infancy. To ensure these strategies develop into robust, biocompatible techniques, the mechanism of ligation as well as any post engineering impact on cell behavior in vitro and in vivo must be more aggressively studied.

Table 1. Methods to engineer cell membranes Conjugation Strategy Chemical

Method(s)

• Amine-reactive (Cyuranic Chloride, NHS esters, aldehydes) • Thiol-reactive (maleimides)

Advantages

• Amines/thiols are abundant on the cell surface allowing for widespread and extensive cell surface functionalization • Direct attachment without an genetic or chemical preconditioning of the cell surface • Commercial reagents widely available

Disadvantages

Cell Types Modified

• Exogenous substrates must be pre-activated before delivery to the cell surface. • Accumulation of cyuranic chloride side products can be cytotoxic • Potential for cell surface crosslinking and irreversible loss of protein function

RBCs9, 11b, 100, lymphocytes7a, 101, macrophages22, stem cells (MSC’s1f, 17, 36, HSC’s23d), L6 myoblasts21, pancreatic islets27f


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Physical

• Electrostatic (PLL,PEI,CP) • Biotin/Avidin

• Spontaneous, strong and stable associations with the cell surface • Commercial reagents widely available for biotinylation • Electrostatic interactions require no pre-activation of the cell surface

• Polycations can be cytotoxic if charges are in too close proximity to the cell surface • Cell surface must be preactivated for biotinylation strategy • Extensive biotinylation can impair cell function • Bacterial origin of streptavidin susceptible to immune rejection in vivo

Pancreatic Islets102, RBC’s42, Endothelial cells32a, 33, MSC’s36

Hydrophobic

• Lipid anchors • Aliphatic chains • Liposome fusion

• Less harmful than covalent modification • Liposome fusion allows for large quantities of exogenous substrates to be delivered to the cell surface • Fast modification process (~10 minutes)

• Lifetime on the cell surface is limited due to enzyme mediated transfer to the inner leaflet of the cell surface (t1/2 ~1-2 hours) • Hydrophobic insertion is not specific to the outer cell surface and may modify organelle membranes, impairing proper functioning • Presence of serum proteins in cell media can limit insertion efficiency

Endothelial cells49, 53 , stem cells (ESC’s50, MSC’s45), fibroblasts51, cardiomyocytes53

• Highly site selective technique (spatial/temporal control) • Useful for both upregulation and downregulation of proteins displayed on the cell surface

• Some cell types not susceptible to non-invasive genetic manipulation • Limited to the introduction of proteins only • Genetic manipulation may lead to uncontrolled mutagenesis • Fusion constructs of proteins may cause loss of protein function due to steric bulk of protein • Time intensive process

MSC’s58, RBC’s63 T-cells69, HEK cells57, SW480 cells61

• Small peptide tags do not compromise function of the attached substrate • High conversion rates under physiological conditions • HTP mediated ligations can persist over multiple days for long-term effects • Hydrolases can engineer surfaces via the removal of structures

• Fusion proteins/tags still required for some enzymes (sortases, HTP’s) • Sortase mediated ligation is subject to reversibility

T-cells68, 86, HEK cells67, RBC’s11c, 70, 84 CHO cells, HeLa cells76, ESC’s103

PBMC’s92, cancer cells (MCF7. MDAMB-231, LS174T)92, 94, 104, Jurkat cells91

Genetic Engineering

Enzyme Mediated Engineering

mRNA transfection generating fusion proteins

• Sortases • Tranglutaminases • Halotag Proteins, (HTP’s) • Glycotransferases, Hydrolases

Metabolic Engineering

Click labelling using Azido (-N3) Sugars and Alkyne/PPh3 labelled surfaces

• Site-specificity without the need for genetic engineering • Click labelling is fast and produces no side products • Highly orthogonal click reactions are suitable for engineering in vivo

• Genetic or chemical preconditioning required to introduce non-native azide/alkyne moieties on the cell surface • Limited to modification of ECM glycostructures • Limited to nucleated cells

‘Graftingfrom’ Engineering

PET-RAFT polymerization

• Greater extent of modification on the cell surface compared to traditional polymer attachment

• Limited to polymer attachment exclusively • Polymerization requires UV irradiation and high catalysts loads which may cause cytotoxicity


RBC’s98, Jurkat cells99


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Overall, advances in cell surface engineering approaches 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. Acknowledgements: 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 (MSFHR). SA acknowledges MSFHR postdoctoral fellowship. ES acknowledges graduate scholarship from Centre for Blood Research. HM acknowledges fellowship from Canadian Blood Services, NSERC CREATE NanoMat Program and MITACS Canada.


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TOC Figure.


Surface Engineering for Cell-Based Therapies: Techniques for

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