General and Facile Coating of Single Cells via Mild Reduction

Communication pubs.acs.org/JACS

Cite This: J. Am. Chem. Soc. 2018, 140, 1199−1202

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*,†

Downloaded via UNIV OF TOLEDO on June 17, 2018 at 21:28:49 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

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, Maryland 21205, United States of America S Supporting Information *

This conjugation-based surface engineering allows modifying individual cells uniformly and stabilizing them without aggregation, unlike electrostatically driven cell coating.2b,d 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 On the basis of the well-established bioconjugation techniques, the procedure is generally accessible without additional preparation steps or special equipment such as microfluidic devices.2i 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,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 non-natural 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

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.

C

ell-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. © 2017 American Chemical Society

Received: August 9, 2017 Published: December 27, 2017 1199

DOI: 10.1021/jacs.7b08440 J. Am. Chem. Soc. 2018, 140, 1199−1202

Communication

Journal of the American Chemical Society

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 outgrowth, respectively. hMSC was able to differentiate into osteoblast, and hiNSC differentiated into both astrocyte and neuron (Figures 1e and S7). We applied the method for coating cell surfaces with biopolymer including chondroitin sulfate (CS) and polyethylene glycol (PEG). The biopolymers grafted onto cell surface facilitated cell clustering, cell sheet construction, and manipulation of a scaffold−cell interaction. This modification technique promotes the formation of artificial tissues or complex scaffolds and is expected to induce cellular interplays and enhance adhesion onto polymeric matrices.7a,12 Reduced cell surfaces could be easily modified with maleimide-conjugated chondroitin sulfate (MCS), without compromising their viabilities (Figures S8 and S9). MCS on the cell could be observed by fluorescence microscopy using fluorescein isothiocyanate (FITC)-conjugated MCS on HeLa cells (Figures 2a and S9). MCS-coated HeLa cells

Scheme 1. Cell Surface Modification with Fluorescent Dye, Polymer, and Nanoparticles by Mild Reduction Using TCEP

over a wide range of pH, and resistant to air oxidation.10 It can selectively reduce disulfide bonds, but is essentially unreactive toward other functional groups in proteins.11 Compared to typical thiol-based reducing agents such as dithiothreitol and 2mercaptoethanol, TCEP does not react with maleimide groups. Fluorescent dye with a maleimide functional group (MFluor) was utilized to evaluate the coating efficiency (Figures 1a and

Figure 1. Cell surface modification by mild reduction. (a) Confocal microscopic images of cell surface modified with MFluor and PKH26labeled HeLa cells. (b) 3D-rendered image of MFluor-coated HeLa cells. (c) Viability of reduced cells and nontreated 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).

Figure 2. Coating of biomacromolecule and polymer. (a) Confocal microscopic image of the reduced cell coated with FITC-conjugated MCS. (b) Difference in ζ-potential between bare (Ctrl.) and MCScoated cells (**p < 0.01). (c) Facile development of cell clusters through electrostatic interaction between anionic MCS-coated cells and cationic PLL. Fluorescence images of cells treated with MCS and/or PLL (Green: DiO, Red: DiI), line graph showing average numbers of cells in a cluster, and bar graph showing percentage of clusters with different number of cells. (d) Schematic representation and confocal microscopic images of MPA-coated HeLa cells individually encapsulated in PEGdiacrylate gel in comparison to normal cell encapsulation (Blue: DAPI, Red: F-Actin).

1b). Fluorescence signals were distributed evenly over the cell surfaces without any evidence of internalization. When the cells were postlabeled 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. Dosedependent 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). Tthe coated nanohe 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. 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

retained a highly negative surface charge derived from the inherent characteristics of CS as confirmed by ζ-potential analysis (Figure 2b). When cells were additionally treated with poly-L-lysine (PLL), the extra negative surface charge derived from MCS induced rapid cell clustering through electrostatic forces, offering a favorable environment for cell−cell interaction by shortening the distances between the cells (Figures 2c and S10). In contrast, bare cells formed smaller clusters, with fewer cells being involved in the cluster formation. Without PLL, cells repelled each other via the highly negative MCS-derived charges, preventing aggregation. In addition, we were able to construct a 3-layered layer-by-layer structure by multiple rounds of MCScoated cell seeding and PLL covering as confirmed by quartz crystal microbalance analysis on gold substrate (Figures S11− S12). 1200

DOI: 10.1021/jacs.7b08440 J. Am. Chem. Soc. 2018, 140, 1199−1202

Communication

Journal of the American Chemical Society

endure multiple washing steps (Figure S15). Even after the cell surface modification, the accessibility to cellular surface and attached glycoproteins are retained (Figures S16−18), suggesting that functionality of the surface ligands and receptors is preserved. Nanoparticle coating did not compromise cell viability, metabolism, pluripotency, and differentiation capacity (Figures S19−21). The coated nanoparticles were distributed on cell surface for 9 h in vitro (Figure S22), and taken up by the cells via endocytosis. Using the MSN-coated cells, we performed two animal experiments including in vivo fluorescence imaging to track the location of cells and intravital microscopic imaging to visualize the early stages of cell therapy. Luciferase-producing HeLa cells were coated with near-infrared fluorescent MSNs and injected subcutaneously in the dorsal region of nude mice (Figure S23). Strong fluorescence from the nanoparticles was observed where the cells were injected, colocalizing with the cellular luminescence, and it lasted for 8 days. Along with the early stage intravital imaging of cells, we studied localized drug delivery using the drug-loaded MSNs to investigate the potential application of the MSN-coated cells for drug delivery. To study drug diffusion in vivo, rhodamine was loaded into the MSNs and analyzed using fluorescence correlation spectroscopy (Figures S24−25). After coating HeLa cells with the rhodamine-loaded MSNs (Figure S26), the cells were administered subcutaneously to nude mice and imaged with intravital confocal microscope. The red signal from the fluorescent MSNs delineates the round shapes of the coated surfaces, while green fluorescence shows rhodamine released from the MSNs (Figure 3d). Initially, most of the rhodamine molecules were distributed in the HeLa cells, but as time passed, they gradually diffused to neighboring immune cells, upper muscle fibers, and finally lower muscle fibers. The MSN-derived red signal on the cell surface sustained over 18 h, clearly distinguishing the administered cells from the nearby immune cells and tissues (Figures 3e, S22, and S27). Some of the cells were destroyed by immune rejection, losing their red round shape. Finally, we demonstrated that dual coating of drug-loaded MSNs and protective polymer (PEG) can enhance the activity of the implanted cells. MSNs loaded with dexamethasone immunosuppressant and PEG were simultaneously coated on the surface of luciferase-expressing HeLa cells. The cells were subcutaneously injected to mice with normal immunity. Cellular activity was evaluated based on luminescence of the implanted cells. The PEG- or MSN-coated cells showed enhanced activities as compared to the uncoated cells. Notably, the MSN-PEG dualcoated cells showed higher activity than the other groups, suggesting the synergistic effect of the dual coating (Figure 4). In conclusion, we demonstrated a universal and innocuous method for cell surface modification to impart various properties of exogenous materials to cells. As this method does not require any additional stabilization and culture step and no adverse effect was observed, it is expected to be used for cell therapy using primary cells, such as cancer immunotherapy and hematopoietic stem cell transplantation. An examination of surface ligands and targeting capacity of cells will be necessary prior to practical therapeutic applications, considering the nonspecificity of the reduction process using TCEP. Given the need for incorporation of various materials onto cell surface to monitor cell therapy and to produce artificial tissue, this versatile technique is anticipated to play a key role in next-generation cell-based therapies.

Cells can similarly be coated with functionalized PEG to enhance the cell−material interaction in a PEG hydrogel that is biologically inert, allowing efficient transport of external nutrients to the encapsulated cells.13 PEG itself does not provide sufficient cell adhesion to the substrate because of the lack of cellspecific binding activity.14 Therefore, maleimide-PEG-monoacrylate (MPA) was applied onto the cell surface and the cells were incorporated into a conventional PEG diacrylate (PEGDA)-based hydrogel. During scaffold polymerization, the MPA on the cell surface participates in polymerization and induces cell−material interaction. Uncoated cells exhibited globular morphology, whereas MPA-coated cells showed cellular protrusions into the surrounding PEG matrix promoted by the interaction between the acrylates of the MPA and PEGDA (Figure 2d). Not only single modification but also secondary modification could be achieved via specific bonding between the coated anterior material and the additional posterior material (Figure S13). Nanoparticles have been employed in a wide range of biomedical applications, such as bioimaging probes and drug delivery vehicles.3d,15 In this study, we functionalized cells by coating with nanoparticles for tracking, imaging, and localized drug delivery. As one of the representative nanomaterial candidates, we chose fluorescent mesoporous silica nanoparticles (MSNs) because of their facile surface modification, high drugloading capability, and good biocompatibility.2a,16 Maleimideconjugated MSNs were prepared and conjugated to the surfacereduced cells (Figures 3a and S14). The nanoparticles were aligned around the cell surface (Figures 3b and c). As shown by the flow cytometry, a significant number of nanoparticles were attached to the surface-reduced cells. Compared to nonspecific adsorption of nanoparticles to the cell surface, the MSNs covalently bound to the cells, thus providing sufficient stability to

Figure 3. Cell surface modification with nanoparticles for localized drug delivery. (a) Schematic representation of administration of MSN-coated cells, and early stage imaging of localized drug delivery in vivo. (b) Confocal image of cells coated with fluorescent MSNs. (c) Transmission electron microscopic image of MSNs attached on the cell surface. (d) Time-lapse intravital images of rhodamine(green)-containing MSNcoated cells administered on the muscle under subcutaneous layer. XZplane images of administered cells and neighboring tissues. (e) Timedependent variation of the rhodamine concentration in each cell and tissue. 1201

DOI: 10.1021/jacs.7b08440 J. Am. Chem. Soc. 2018, 140, 1199−1202

Communication

Journal of the American Chemical Society

Song, Y.; Tsukruk, V. V.; Chaikof, E. L. J. Am. Chem. Soc. 2011, 133, 7054−7064. (e) Kim, J. Y.; Lee, B. S.; Choi, J.; Kim, B. J.; Choi, J. Y.; Kang, S. M.; Yang, S. H.; Choi, I. S. Angew. Chem., Int. Ed. 2016, 55, 15306−15309. (f) Fakhrullin, R. F.; Zamaleeva, A. I.; Minullina, R. T.; Konnova, S. A.; Paunov, V. N. Chem. Soc. Rev. 2012, 41, 4189−4206. (g) Lee, J.; Choi, J.; Park, J. H.; Kim, M. H.; Hong, D.; Cho, H.; Yang, S. H.; Choi, I. S. Angew. Chem., Int. Ed. 2014, 53, 8056−8059. (h) Niu, J.; Lunn, D. J.; Pusuluri, A.; Yoo, J. I.; O’Malley, M. A.; Mitragotri, S.; Soh, H. T.; Hawker, C. J. Nat. Chem. 2017, 9, 537−545. (i) Mao, A. S.; Shin, J. W.; Utech, S.; Wang, H.; Uzun, O.; Li, W.; Cooper, M.; Hu, Y.; Zhang, L.; Weitz, D. A.; Mooney, D. J. Nat. Mater. 2017, 16, 236−243. (3) (a) Chen, W.; Fu, L. W.; Chen, X. Y. J. Controlled Release 2015, 219, 560−575. (b) Stephan, M. T.; Irvine, D. J. Nano Today 2011, 6, 309− 325. (c) Fliervoet, L. A. L.; Mastrobattista, E. Adv. Drug Delivery Rev. 2016, 106, 63−72. (d) Stephan, M. T.; Moon, J. J.; Um, S. H.; Bershteyn, A.; Irvine, D. J. Nat. Med. 2010, 16, 1035−1041. (4) (a) Rossi, N. A.; Constantinescu, I.; Brooks, D. E.; Scott, M. D.; Kizhakkedathu, J. N. J. Am. Chem. Soc. 2010, 132, 3423−3430. (b) Teramura, Y.; Iwata, H. Soft Matter 2010, 6, 1081. (c) Takaoka, Y.; Ojida, A.; Hamachi, I. Angew. Chem., Int. Ed. 2013, 52, 4088−4106. (d) Hayashi, T.; Yasueda, Y.; Tamura, T.; Takaoka, Y.; Hamachi, I. J. Am. Chem. Soc. 2015, 137, 5372−5380. (5) Rabuka, D.; Forstner, M. B.; Groves, J. T.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 5947−5953. (6) Sampathkumar, S. G.; Li, A. V.; Jones, M. B.; Sun, Z.; Yarema, K. J. Nat. Chem. Biol. 2006, 2, 149−152. (7) (a) Dutta, D.; Pulsipher, A.; Luo, W.; Yousaf, M. N. J. Am. Chem. Soc. 2011, 133, 8704−8713. (b) Rossi, N. A.; Constantinescu, I.; Kainthan, R. K.; Brooks, D. E.; Scott, M. D.; Kizhakkedathu, J. N. Biomaterials 2010, 31, 4167−4178. (c) Cheng, H.; Kastrup, C. J.; Ramanathan, R.; Siegwart, D. J.; Ma, M.; Bogatyrev, S. R.; Xu, Q.; Whitehead, K. A.; Langer, R.; Anderson, D. G. ACS Nano 2010, 4, 625− 631. (8) (a) Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125−1128. (b) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664−667. (c) Kang, K.; Joo, S.; Choi, J. Y.; Geum, S.; Hong, S. P.; Lee, S. Y.; Kim, Y. H.; Kim, S. M.; Yoon, M. H.; Nam, Y.; Lee, K. B.; Lee, H. Y.; Choi, I. S. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E241−248. (9) Metcalfe, C.; Cresswell, P.; Ciaccia, L.; Thomas, B.; Barclay, A. N. Open Biol. 2011, 1, 110010. (10) Han, J. C.; Han, G. Y. Anal. Biochem. 1994, 220, 5−10. (11) Kirley, T. L. Anal. Biochem. 1989, 180, 231−236. (12) Gong, P. Y.; Zheng, W. F.; Huang, Z.; Zhang, W.; Xiao, D.; Jiang, X. Y. Adv. Funct. Mater. 2013, 23, 42−46. (13) Hwang, N. S.; Kim, M. S.; Sampattavanich, S.; Baek, J. H.; Zhang, Z.; Elisseeff, J. Stem Cells 2006, 24, 284−291. (14) Kim, H. D.; Heo, J.; Hwang, Y.; Kwak, S. Y.; Park, O. K.; Kim, H.; Varghese, S.; Hwang, N. S. Tissue Eng., Part A 2015, 21, 757−766. (15) (a) Moon, J. J.; Huang, B.; Irvine, D. J. Adv. Mater. 2012, 24, 3724−3746. (b) Chen, G.; Qiu, H.; Prasad, P. N.; Chen, X. Chem. Rev. 2014, 114, 5161−5214. (16) (a) He, Q.; Shi, J. Adv. Mater. 2014, 26, 391−411. (b) Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. J. Am. Chem. Soc. 2012, 134, 5722−5725.

Figure 4. Enhancement of cellular activity of implanted cells attached with drug delivery vehicle (DDV) and protective polymer coatings. Maleimide-PEG was used for polymer coating, and immunosuppressant (dexamethasone)-loaded MSN for DDV. (a) Luminescence images showing the cellular activity after transplantation of nontreated, MPEGcoated, MSN-coated, and MPEG/MSN dual-coated HeLa cells. (b) Comparison of luminescence intensity obtained at the indicated time points after cell transplantation (n = 5, *p < 0.01 versus control group at the same time point).

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b08440. Experimental details and Figures S1−S27 (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Kwangsoo Shin: 0000-0001-5337-0679 Jinkee Hong: 0000-0003-3243-8536 Nathaniel S. Hwang: 0000-0003-3735-7727 Author Contributions #

H.K., K.S., and O.K.P. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT Grant (NRF-2017M3A9C6029699, NRF-2016R1E1A1A01943393) (N.S.H.) and Institute for Basic Science (IBS) in Korea (IBSR006-D1) (T.H.).

■

REFERENCES

(1) (a) Mangi, A. A.; Noiseux, N.; Kong, D.; He, H.; Rezvani, M.; Ingwall, J. S.; Dzau, V. J. Nat. Med. 2003, 9, 1195−1201. (b) Laflamme, M. A.; Chen, K. Y.; Naumova, A. V.; Muskheli, V.; Fugate, J. A.; Dupras, S. K.; Reinecke, H.; Xu, C.; Hassanipour, M.; Police, S.; O’Sullivan, C.; Collins, L.; Chen, Y.; Minami, E.; Gill, E. A.; Ueno, S.; Yuan, C.; Gold, J.; Murry, C. E. Nat. Biotechnol. 2007, 25, 1015−1024. (2) (a) Huang, X.; Zhang, F.; Wang, H.; Niu, G.; Choi, K. Y.; Swierczewska, M.; Zhang, G.; Gao, H.; Wang, Z.; Zhu, L.; Choi, H. S.; Lee, S.; Chen, X. Biomaterials 2013, 34, 1772−1780. (b) Wilson, J. T.; Krishnamurthy, V. R.; Cui, W.; Qu, Z.; Chaikof, E. L. J. Am. Chem. Soc. 2009, 131, 18228−18229. (c) Singh, A.; Corvelli, M.; Unterman, S. A.; Wepasnick, K. A.; McDonnell, P.; Elisseeff, J. H. Nat. Mater. 2014, 13, 988−995. (d) Wilson, J. T.; Cui, W.; Kozlovskaya, V.; Kharlampieva, E.; Pan, D.; Qu, Z.; Krishnamurthy, V. R.; Mets, J.; Kumar, V.; Wen, J.; 1202

DOI: 10.1021/jacs.7b08440 J. Am. Chem. Soc. 2018, 140, 1199−1202