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General and Facile Coating of Single Cells via Mild Reduction Hyunbum Kim, Kwangsoo Shin, Ok Kyu Park, Daheui Choi, Hwan Drew Kim, Seungmin Baik, Soo Hong Lee, Seung-Hae Kwon, Kevin J. Yarema, Jinkee Hong, Taeghwan Hyeon, and Nathaniel Suk-Yeon Hwang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08440 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 27, 2017
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Journal of the American Chemical Society
General and Facile Coating of Single Cells via Mild Reduction Hyunbum Kim,†,|| Kwangsoo Shin,†,‡,|| Ok Kyu Park,‡,|| Daheui Choi,§ Hwan D. Kim,† Seungmin Baik,†,‡ Soo Hong Lee,†,‡ Seung-Hae Kwon,# Kevin J. Yarema┴, Jinkee Hong,§ Taeghwan Hyeon,*,†,‡ and Nathaniel S. Hwang*,† †
School of Chemical and Biological Engineering, and Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea
‡
Center for Nanoparticle Research, Institute of Basic Science (IBS), Seoul 08826, Republic of Korea
§
School of Chemical Engineering and Material Science, Chung-Ang University, Seoul 06974, Republic of Korea Division of Bio-imaging, Korea Basic Science Institute (KSBI), Chun-Cheon 24341, Republic of Korea ┴ Department of Biomedical Engineering and, Translational Tissue Engineering Center, The Johns Hopkins University, Baltimore, MD 21205, United States of America #
Supporting Information Placeholder ABSTRACT: Cell surface modification has been extensively studied to enhance the efficacy of cell therapy. Still, general accessibility and versatility are remaining challenges to meet the increasing demand for cell-based therapy. Herein, we present a facile and universal cell surface modification method that involves mild reduction of disulfide bonds in cell membrane protein to thiol groups. The reduced cells are successfully coated with biomolecules, polymers, and nanoparticles for an assortment of applications, including rapid cell assembly, in vivo cell monitoring, and localized cell-based drug delivery. No adverse effect on cellular morphology, viability, proliferation, and metabolism is observed. Furthermore, simultaneous coating with polyethylene glycol and dexamethasone-loaded nanoparticles facilitates enhanced cellular activities in mice, overcoming immune rejection.
Cell-based therapies involving transplantation and direct injection have provided a viable solution for the treatment of congenital defects and damaged tissues.1 However, decline in survival rate and therapeutic effect of administered cells due to allogeneic host immune rejection substantially limits the extensive applications of cell-based therapy. Consequently, a suitable method is required to track and monitor the administered cells to evaluate the efficacy of cell therapy. Hence, incorporation of biomaterials and nanomaterials in cells has been spotlighted in cell-based therapies as a strategy to provide therapeutic cells with a protective layer or to tag them with imaging probes.2 In addition, incorporating drug-loaded nanoparticles is expected to enhance the efficiency of drug delivery because of their targeting capability.3 One of the main approaches for the incorporation of exogenous materials is cell surface modification via chemical conjugation to functional groups in the cell membrane proteins.2c,3d,4 Compared to other cell surface engineering methods, this approach enables direct engraftment of various materials, and guarantees their stable attachment to cells when they are implanted into the complex biological environment. This conjugation-based surface engineering can modify individual cells uniformly and stabilize them without aggregation, unlike electrostatically driven cell
coating.2b,2g Because the approach does not involve hydrophobic interaction, it broadens the selection of available materials without compromising the solubility and stability of the exogenous materials.5 Based on the well-established bioconjugation techniques, the procedure is generally accessible without additional preparation steps or special equipment such as microfluidic devices.2m Despite the advantages of conjugation-based modification, the introduction of active functional groups on the cell surface remains challenging because most cell surfaces do not contain chemically reactive moieties.6 Although amide coupling has been employed for cell surface modification,7 cross-coupling between carboxylate and amine groups potentially decreases both efficiency and specificity of the reaction between coating materials and cell surface. There are several reported methods to introduce nonnatural functional groups such as ketone and azide on mammalian cell surfaces via glycoengineering.8 However, this process takes several days to express these functional groups and to confirm their expression. Herein, we report a facile and universal method for cell surface engineering that involves mild reduction of disulfides in cell surface proteins9 with tris(2-carboxyethyl)phosphine (TCEP) and subsequent thiol-maleimide conjugation. A variety of cell types can be coated without any adverse effect on cell functions. This method can coat biomolecules and polymers to demonstrate rapid formation of multicellular assembly and facilitation of cell adhesion to a polymeric scaffold. Multifunctional nanoparticles can be attached to cell surface for tracking the administered cells and simultaneously delivering adjuvant drugs. Finally, synergistic enhancement of cellular activity is achieved through a dual coating of polymer and nanoparticles. Scheme 1 describes the surface modification method that consists of mild reduction of disulfides in cell surface protein with TCEP and subsequent thiol-maleimide conjugation. TCEP is nonvolatile and stable in aqueous solution at room temperature over a wide range of pH, and resistant to air oxidation.10 It can selectively reduce disulfide bonds, but is essentially unreactive towards other functional groups in proteins.11 Compared to typical thiolbased reducing agents such as dithiothreitol and 2mercaptoethanol, TCEP does not react with maleimide groups.
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Scheme 1. Cell surface modification with fluorescent dye, polymer, and nanoparticles by mild reduction using TCEP.
Fluorescent dye with a maleimide functional group (MFluor) was utilized to evaluate the coating efficiency (Figures 1a and 1b). Fluorescence signals were distributed evenly over the cell surfaces without any evidence of internalization. When the cells were post-labeled with a membrane dye (PKH-26), MFluor signals colocalized with those of PKH-26, confirming that the conjugation takes place solely on the cell surface. Dose-dependent effects of TCEP on HeLa cells were evaluated by flow-cytometric analysis. The reduction reaction dramatically increased the fluorescence of attached MFluor on the cells, which was saturated after treatment with 1 mM TCEP (Figure S1). In quantitative analysis, the ratio of the amount of MFluor to cellular surface area is nearly identical between attached and detached state, indicating that the conjugation evenly occurs regardless of the cellular morphology. (Figures S2 and S3). Most importantly, no adverse effect on cellular morphology, viability, proliferation, and metabolism was observed for TCEP concentrations equal or below 1 mM (Figure S4), and the reduced thiols are recovered in a single day (Figure S5). Thus, 1 mM of TCEP was designated as the optimal concentration for cell surface reduction.
Figure 1. Cell surface modification by mild reduction. (a) Confocal microscopic images of cell surface modified with MFluor and PKH26-labeled HeLa cells. (b) 3D-rendered image of MFluorcoated HeLa cells. (c) Viability of reduced cells and non-treated cells. (d) Confocal microscopic images of the indicated cell types coated with MFluor. (e) Functionality analysis of cells. Quantification of multinucleated cells for C2C12, nuclear-axon distance for N2A, calcium content for osteoblasts differentiated from hMSC, and pluripotency-related RNA expression for hiNSC. Controls are the results prior to (N.Ctrl) and after (P.Ctrl) differentiation (n=3 for c and e). We further examined the universal and innocuous character of the coating method in other cell types such as Jurkat T, C2C12, Neuro-2a (N2A), human mesenchymal stem cells (hMSC), and human induced neural stem cells (hiNSC) with different cellular morphology, potency, and tissue origins. All cell types were efficiently labeled with MFluor, and no sign of cytotoxicity was observed (Figures 1c, d, and S6). All cell lines retained their potency, differentiation capability, and functionalities during reduction and labeling steps as optimized for the HeLa cells. The C2C12 and N2A cells cultured in differentiation-inducing media were capable of forming multinucleated myotubes and neurite out-
growth, respectively. hMSC was able to differentiate into osteoblast, and hiNSC differentiated into both astrocyte and neuron. The potencies of both stem cell types were evaluated by qPCR, and no significant differences in the extent of differentiation as compared to the control groups were observed (Figures 1e and S7).
Figure 2. Coating of biomacromolecule and polymer. (a) Confocal microscopic image of the reduced cell coated with FITCconjugated MCS. (b) Difference in ζ-potential between bare (Ctrl.) and MCS-coated cells (**p
