Cytoprotective Self-assembled RGD Peptide Nanofilms for Surface

Article pubs.acs.org/cm

Cytoprotective Self-assembled RGD Peptide Nanofilms for Surface Modification of Viable Mesenchymal Stem Cells Daheui Choi,† Hwankyu Lee,‡ Hyun-Bum Kim,§ Miso Yang,† Jiwoong Heo,† Younsun Won,∥ Seung Soon Jang,⊥ Jong Kuk Park,# Youngsook Son,∥ Tong In Oh,∇,○ EunAh Lee,*,∥,○ and Jinkee Hong*,† †

School of Chemical Engineering & Materials Science, College of Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea ‡ Department of Chemical Engineering, Dankook University, 152 Jukjeon-ro, Suji-gu, Yongin-si, Gyeonggi-do 16890, Republic of Korea § Department of East-West Medical Science, Graduate School of East-West Medical Science, Kyung Hee University, 1732 Deogyeoung-daero, Giheung-gu, Youngin, Gyeonggi-do 17104, Republic of Korea ∥ Graduate School of Biotechnology, Kyung Hee University, 1732 Deogyeoung-daero, Giheung-gu, Youngin, Gyeonggi-do 17104, Republic of Korea ⊥ School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0245, United States # Department of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Nowon-ro 75, Nowon-gu, Seoul 01812, Republic of Korea ∇ School of Medicine, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea ○ Impedance Imaging Research Center, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea S Supporting Information *

ABSTRACT: Intravenous administration of mesenchymal stem cells (MSCs) has served as a clinical intervention for inflammatory diseases. Once entered to blood circulation, MSCs are exposed to a harsh environment which sharply decreases cell viability due to the fact that injected cells, being susceptible to shear stress, are subjected to the high velocities of the bloodstream and lack of proper mechanical support that keeping them in an attachment-deprived state. Here, we coated the nanofilm onto viable MSCs by depositing poly-L-lysine and hyaluronic acid molecules along with arginine-glycine-aspartic acid (RGD peptide) as building blocks to protect cells from shear stress and stabilize them in a single cell, suspension state. In this article, we found that nanofilmcoated cells showed significantly increased cell survival in vitro and in vivo, which was also supported by the activation of survival-related protein, Akt. The coated nanofilm did not interfere with the stemness of MSCs which was determined based on the colony forming unit-fibroblast (CFU-F) assay and in vitro differentiation potential. Because of the characteristics of films showing light molecular deposition density, flexibility, and looseness, application of nanofilms did not block cell migration. When the cells were administrated intravenously, the nanofilm coated MSCs not only prolonged blood circulation lifetime but also showed increased stem cell recruitment to injured tissues in the muscle injury in vivo model, due to prolonged survival. Surface modification of MSCs using nanofilms successfully modulated cell activity enabling them to survive the anoikis-inducing state, and this can provide a valuable tool to potentiate the efficacy of MSCs for in vivo cell therapy.

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and sepsis.12,13 In this case, direct intravenous injection of stem cells is the preferred method because of its noninvasive nature and systemic transplantability. However, upon systemic transplantation, MSCs are subjected to a situation that leads to

INTRODUCTION Translating stem cells’ regeneration capability into clinical application encountered with the need to precondition stem cells to secure a sufficient cell number and optimize their activity.1 In vitro-expanded MSCs are clinically used for several kinds of illnesses including stroke,2−4 arterial disease,5,6 paralysis,7 and inflammatory disease such as acute and chronic graft versus host disease (GVHD),8−11 autoimmune disease, © 2017 American Chemical Society

Received: September 25, 2016 Revised: January 19, 2017 Published: January 20, 2017 2055

DOI: 10.1021/acs.chemmater.6b04096 Chem. Mater. 2017, 29, 2055−2065

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Chemistry of Materials

Figure 1. Schematic illustration of the (a) chemical structures of materials used in this study (top), as well as the abbreviations of each film combination (bottom). (b) Schematic drawing indicating mesenchymal stem cells coated with nanofilms prepared using poly-L-lysine (PLL), hyaluronic acid (HA), and RGD and the functions exhibited by the nanofilm-coated cells during various in vitro and in vivo conditions.

hydrogel,25 bioinspired silication,26 photo-cross-linked hydrogel,27 polydopamine,28 graphene,29 and inorganic/organic shells30−32). These strategies resulted in better cell survival and enhanced differentiation potentials in vitro and in vivo for stem cell delivery. Hung et al. have reported that short-term culture of MSCs under hypoxia condition improves cell migration and engraftment in the response of chemokines which is involved in cell homing properties.33 Among these strategies, cell encapsulation especially showed better cell survival under mechanical disturbance. MSCs encapsulated with various hydrogels showed long-term survival for several weeks upon in vivo transplantation to the infarcted cardiac wall,34 prolonged trophic effect, and therefore, better wound regeneration.35 In addition to better survival, chemical functional groups tethered on the hydrogel effectively directed the differentiation tendency of MSCs.36 However, cell encapsulation prevents changes in cell morphology36 and also induces decreased cell metabolism based on the fact that cells in encapsulated state showed a lack of cell division.30 Therefore, for systemic delivery of functional stem cells, cell encapsulation needs to be flexible enough to allow cell morphology change that accompanies cell activities such as cell migration according to the physiological demand, proliferation, and direct communication with the surrounding tissue surface. Here, for the first time, we present layer-by-layer (LbL) assembly on individual MSCs’ plasma membrane surface and the effect of this nanofilms on cell survival and stability using nonadhesion culture condition and animal injury model. In the mouse muscle injury model, due to the prolonged duration of cell survival, the homing ability of nanofilms-applied MSCs to injury site was significantly increased. These results indicate that the application of nanofilms onto the MSCs’ plasma membrane can enhance the stability of MSCs in vitro and in vivo, without compromising the efficacy and homing ability of MSCs.

extremely low cell survival, and therefore, they show poor retention at the intended target site. Numerous studies on direct stem cell injection have focused on the role of the host environmental condition as a crucial factor resulting in low viability. First, right upon intravenous transplantation, the cells are subjected to shear stress induced by fluid flow in the venous system. Second, MSCs eventually undergo apoptosis caused by an attachment-deprived state.14 To accelerate the prospective translation of emerging stem cell therapies to successful clinical trials, the viability of intravenously injected MSCs must be improved by mitigating the mechanical disruption applied to the injected stem cells. To ensure increased efficacy of injected MSCs, we suggest to modify cell surface to mimic signals coming from extracellular matrix (ECM) molecules to induce better cell survival. The ECM is a complex of molecules surrounding cells, mostly composed of proteins and proteoglycans, although the composition is dependent on cell or tissue types. ECM not only provides mechanical support to cells but also affects their morphology, cell movement, and direct interaction with other proteins.15 Being adherent cells, MSCs require cell−ECM interactions to survive; upon adhesion of cells to the matrix substrate, an ECM receptor integrin transduces the intracellular signaling pathway to promote the survival state of the cell.16 When cell−ECM interactions are disrupted for a prolonged period of time, apoptotic signaling pathways get activated to lead the cells to anoikis, the type of cell death induced by detachment from the ECM substratum.17 Therefore, to increase stem cell survival right upon intravenous injection, mimicking cell−ECM interaction could be a good strategy to maximize stem cell survival in the bloodstream. While many previous studies mainly focused on connection between cells or the supply of nutrient and oxygen,18 some recent literature suggests that the viability of adherent stem cells or cell delivery can be improved by applying various strategies such as preconditioning of culture condition (hypoxia1,19 and mechanical stimulation20), pretreatment of proteins (FGF221 and VCAM-122 (vascular cell adhesion protein 1)), genetic engineering (integrin-alpha 423 and CXCR424 (CXC chemokine receptor type 4)), and cell encapsulation for the single cell or multicellular state (silica

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EXPERIMENTAL SECTION

Materials. Poly-L-lysine (PLL-HCl, Mw 15,000−30,000), poly-Llysine solution (0.01 wt %, Mw 150,000−300,000), HA sodium salt (from Streptococcus equi), PLL-fluorescein isothiocyanate isomer I labeled (PLL-FITC, Mw 30,000−70,000), HA-fluorescein isothiocyanate isomer I (HA-FITC, Mw 800,000), 2-(N-morpholino) ethane2056


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number of film layers by fluorescence-activated cell sorting analysis (FACSort Flow Cytometer, BD Biosciences, Franklin Lakes, NJ, USA). PLL-FITC solution was used with 1 mg/mL concentration containing 10% of PLL-FITC. Film Fabrication onto MSCs. For film preparation on cells, the MSCs were treated with 0.25% of trypsin-EDTA to detach from the plastic culture dishes. Film preparation was performed as follows: 1 or 0.01 mg/mL of positively charged polymer dissolved in cell culture media was added to the centrifuged cell pellet. The low concentration of positively charged materials was chosen depending on the in vitro assay to prevent excessive endocytosis into MSCs. In general, for LbL assembly, the high excess amount of polymer solutions was conducted for adsorption of layers on a certain substrate until the thermodynamically equilibrium state. In the case of a cell, especially, LbL coating with hyper-concentration of polymer solution could cause an addition effect due to the existence of non- or weakly bonded PLL or excessive endocytosis. After mixing of the solution by pipetting for approximately 1 min, the mixture was centrifuged at 1500 rpm for 5 min at 4 °C. Rinsing was conducted twice by using cell culture media. The negative charge of materials sequentially were introduced to MSCs at a concentration of 1 mg/mL (concentration of RGD peptide was 0.25 mg/mL) and washed with cell culture media as described above. We fabricated 4 different types of films onto MSCs: NF1, NF2, NF3, and NF4. The schematic illustration of procedure for LbL assembly on MSCs is shown in Figure S1. Cell Culture. Human MSCs (MSCs) were isolated from bone marrow aspirates of patients in Kang-Nam St. Peter’s Hospital. All tissues were obtained according to an Institutional Review Boardapproved protocol after patients had signed an informed consent agreement. The multicolony-derived MSC culture was established and maintained as described previously.37 Briefly, bone marrow aspirates were added to plastic conical tubes containing Ficoll (density 1.077 g/ L, GE Healthcare, Little Chalfont, UK) and centrifuged at 2200 rpm for 20 min. Mononuclear cells at the buffy coat layer were collected and washed with PBS before being suspended in cell culture media. The single cell suspension of MSCs was seeded at a density of 5 × 106 cells per 100 mm plastic culture dish. Nonadherent cells were removed 1 day after seeding by extensive washing with medium. The cells were maintained at 37 °C and 5% CO2. The medium was changed three times per week. Upon reaching confluency, MSCs were subcultured by treatment with 0.25% trypsin and 1 mM EDTA in HBSS. Test of Cell Survival in Agitation Culture. MSCs with or without nanothin films were seeded into 2 mL cryogenic vials at a density of 1 × 104/mL. The nonadherent cryogenic vials containing the single cell suspension in an upright position were subjected to continuous agitation on an orbital shaker at 65 rpm. At least three tubes were collected from each group, and viable cells were counted based on the exclusion of trypan blue dye. The concentration of PLL solution was used 0.01 mg/mL. Colony-Forming Unit Fibroblast (CFU-F) Analysis and Differentiation Assay. For CFU-F analysis, 1 × 103 MSCs with or without nanothin films were seeded to each 25T flasks and maintained for 12 days without changing media. Colonies composed of more than 50 cells were considered for counting. For osteogenic differentiation, MSCs with or without nanothin films were seeded onto 6-well plates at a density of 2 × 104 cells/well and maintained in osteogenic differentiation media supplemented with 10−8 M dexamethasone, 0.2 nM ascorbic acid, and 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA). For chondrogenic differentiation, MSCs with or without nanothin films were seeded into 15 mL tubes at a density of 1 × 105 cells/well and centrifuged at 1,500 rpm for 5 min and maintained in chondrogenic differentiation media consisting of highglucose DMEM containing 1% penicillin/streptomycin and supplemented with 10−7 M dexamethasone, 1 mM sodium pyruvate, 0.35 mM proline (Sigma-Aldrich), 1× ITS-3, 0.3 mM ascorbic acid, and 10 ng/mL Transforming growth factor-β3 (Peprotech, Rocky Hill, NJ, USA). Differentiated cultures were washed with PBS and fixed with 4% paraformaldehyde in PBS. Test of in Vivo Cell Survival and Homing to Tissue Injury. Green fluorescent protein (GFP) (+) MSCs with or without nanothin

sulfonic acid (MES hydrate), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), microparticle size standard based on polystyrene (PS) (2 μm), PKH26 red fluorescent cell linker kit, and poly-2-hydroxyethyl methacrylate (poly-HEMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RGD-peptide was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PLL-RGD was synthesized by EDC-NHS reaction. The detailed procedure for the synthesis is indicated in Supporting Information. Cell culture media consisted of alpha-MEM (Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine (WelGene, Daegu, South Korea), 100 U/mL penicillin (WelGene, Daegu, South Korea), 100 μg/mL streptomycin sulfate (Invitrogen, Carlsbad, CA, USA), 10−8 M dexamethasone (Sigma), 10−4 M L-ascorbic acid (Sigma), and 20% fetal bovine serum (Lonza, Walkersville, MD, USA). Vectashield was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA), and 0.25% trypsin containing 1 mM EDTA in HBSS was purchased from Welgene, Inc. (Daegu, South Korea). Film Fabrication on Flat Substrate and PS Particles. All polymers and peptides were dissolved in culture media to adjust the solutions to the same conditions before LbL encapsulation. The concentration of each solution was 1 mg/mL for HA, PLL, and PLLRGD. For RGD-peptide, a concentration of 0.25 mg/mL was used. Prior to deposition, silicon wafers were treated with O2 plasma to confer a negative charge. The negatively charged silicon wafers were dipped into PLL or PLL-RGD solution for 10 min and then moved to culture media for 2 and 1 min each to remove weakly bound polymers (Rinsing step). Immediately, the wafers were deposited into negatively charged HA or RGD-peptide solution for 10 min. The rinsing step was performed as described above. We deposited 4 different types of films onto the wafers, including wafer/(PLL/RGD)3 (NF1), wafer/(PLL/ HA)/(PLL/RGD)2 (NF2), wafer/(PLL/HA)3 (NF3) and wafer/ (PLL-RGD/HA)3 (NF4). The materials and film combinations we used are shown in Figure 1a. For quartz crystal microbalance (QCM; QCM200, Stanford Research Systems, Sunnyvale, CA, USA), we used an Au−Cr electrode (5 MHz) to analyze the adsorption or desorption quantities of each building material. The electrode was treated with piranha solution for 5 min to clean and activate the surface to negative charge (H2SO4/H2O2 = 3:1). Next, the electrode was dipped into the positively charged solution for 10 min and then rinsed thoroughly with culture media for 2 min. To eliminate cell culture media components on the electrode, the electrode was soaked in pH-adjusted deionized (DI) water (pH 7.4) for 1 min. After deposition, the electrode was dried with N2 gas. We also carried out the preparation of LbL films (NF1−4) on polystyrene (PS) microparticles. The LbL films were prepared by centrifugation as follows: 0.5 mL of PLL solution dissolved in culture media was added to 2 μm of PS particles. The mixture was deposited for 5 min with vigorous vortexing and sonication. After mixing, the suspension was centrifuged (over 12,000 rpm), and the supernatant was removed. To eliminate unbound or additional adsorbed polymers, we conducted 2 rinsing steps with 1.0 mL of cell culture media and pure DI water for 1 min each. The negatively charged material was deposited as described above. Film Characterization. Film thicknesses and morphologies were measured using a profilometer (Dektak 150, Veeco, Plainview, NY, USA) and an atomic force microscope (AFM) (X-10, Park Systems, Santa Clara, CA, USA), respectively. To confirm film deposition on the PS particle, we evaluated the surface charge of the PS particle covered with film using a zeta-potential analyzer (Nano Partica SZ100, Horiba, Kyoto, Japan). Film morphologies were also confirmed using a field-emission scanning electron microscope (EF-SEM) (Carl Zeiss, Oberkochen, Germany). To visualize the films on the cells, MSCs were coated with PLL-FITC and HA-FITC multilayers. The concentration of PLL-FITC and HA-FITC solutions were 0.01 and 1 mg/mL, respectively, with 10% of FITC-labeled polymers. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI: blue fluorescence) by mounting on Vectashield with DAPI after FITC labeled-film fabrication for confocal laser microscopy (LSM710, Zeiss). We also measured green fluorescence intensity depending on the 2057


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Chemistry of Materials film (0.01 mg/mL of PLL solution was utilized) were injected to nude mice at a number of 5 × 105 cells/mice in a volume of 100 μL. Mice were sacrificed at the indicated interval, and mononuclear cells in blood was collected using Ficoll-Paque PLUS (GE Healthcate BioSciences, Uppsala, Sweden) to analyze the percentage of GFP (+) cells in the circulation by FACS analysis (FACSort Flow Cytometer, BD Biosciences, Franklin Lakes, NJ, USA). To test the homing ability of injected MSCs, muscle injury was made at 12 h upon stem cell injection, by inserting 26G needle in the middle of Rectus femoris muscle under the skin and dragging toward the knee joint without skin incision. To detect the presence of GFP (+) cells, injured muscle tissues collected and fixed in 4% paraformaldehyde for 2 days after muscle injury were sliced to the thickness of 1 mm and observed under a two-photon microscope (TPM) (Olympus FV-1000) with a 20× objective lens (UPLSAPO20X, NA:0.75). The acquired TPM images were analyzed and processed using Olympus Fluoview (Ver. 1.7a) software. All animal studies were performed in accordance with NIH guidelines and were approved by IACUC of Kyung Hee University (Approval number KHUASP07-004).

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RESULTS AND DISCUSSION Preparation of LbL Films in Model Systems. On the basis of the hypothesis that the loss of cell attachment in intravenous circumstances may provide a pivotal reason for cell loss after in vivo transplantation, we developed an ideal design of nanofilm that can be applied on the membrane of MSCs to increase cell stability and survival. By taking full advantage of the LbL assembly method,38−40 we prepared a series of nanofilms assembled using RGD, PLL, and HA by varying the film composition, structure, and function (Figure 1a,b). Molecules were dissolved in standard cell culture media to simulate the physiological conditions during the assembly process. The main driving force of the nanofilm formation is electrostatic interaction. In cell culture medium condition, the pH is around 7.4 with high salt concentration. The pKa values of PLL and HA are 9.36 and 2.99, respectively, and the degree of ionization of each molecule at pH 7.4 is close to 100%.41,42 However, high amounts of salt could shield the ionic portion of the polymers and provide an opportunity to absorb onto the previous layer with less tendency of repulsion.43 Moreover, the pI of the RGD is approximately 7; therefore, it carries a slightly negative charge in the growth medium condition. Positively charged PLL and RGD-conjugated PLL are electrostatically assembled with negatively charged HA and RGD to provide biochemical signal and mechanical support to MSCs (Figure 1b and Figure S1). We have prepared 4 kinds of multilayer films, which are simply named and ordered as Nano Film No. (NFχ) indicated by Figure 1a. Nanofilm thicknesses were easily tuned by increasing the number of assembled layers which shows linear fashion (Figure 2a). The thickness of the molecular assembly of the nanofilm is investigated by profilometer, and the measured values of thickness are 3.55 ± 0.09 nm (NF1), 11.65 ± 1.66 nm (NF2), and 10.48 ± 2.60 nm (NF3) (Figure 2a). The continual decrease in stepwise frequencies measured by QCM revealed that multilayer films were successfully deposited onto the substrate and showed a linear build-up mechanism for each layer. Decrease in the frequency was proportional to the thickness of the multilayer film, confirming that the nanofilms were successfully prepared as designed at the molecular level. The NF3 and NF4 films may have exponential build-up curves because of polymer diffusion in and out of the whole nanofilm during assembly.44 However, molecules that are negatively charged at pH 7.4 such as L-glutamine, albumin, and biotin are present in α-MEM and FBS and may act as ionic salts (including calcium chloride, sodium bicarbonate, and sodium

Figure 2. (a) Growth curves of electrostatically assembled nanofilms in terms of the number of bilayers. (b) Frequency shift confirmed by quartz crystal microbalance in the stepwise assembly of the nanofilm fabricated using the culture medium as a solvent.

chloride in α-MEM) in the polymer solution, resulting in an increase of the ion concentration. Thus, the electrostatic interaction between molecules was screened, and the exponential growth regime disappeared.45 To compare the effectiveness of cell culture media impacts on thickness or film morphologies, we carried out LbL deposition by using pH-adjusted (pH 7.4) DI water-dissolved polyelectrolytes and RGD solution (Figure S2). The frequency shift decreased continuously as the number of layers increased, indicating that all material candidates successfully formed multilayer films. Specifically, RGD, a small molecule (Mw 614.61 g/mL), consistently assembled on the previous layer (for NF1 and NF2). The RGD may have diffused in and out of the multilayer film, brining RGD to the top of the film during LbL formation. The remaining charge that arises from upper side of RGD allows for deposition of PLL on the previous layer.46 However, for NF1, the slope of the growth curve decreased after formation of 15 bilayers due to the lack of charge (Figure 2a). Previous studies examined LbL multilayer film formation using the small molecule gentamicin, an antibacterial agent (Mw 477.6 g/mol), and revealed that the LbL multilayer film was formed to a thickness of approximately 5 μm.47 Additionally, as shown in Figure 4 and Figure S2, the quantities of absorbed molecules in media-based film were 2fold higher than that in DI water-based film. This indicates that either the increased coil and tail portions of polymers or the other components in the media (vitamins, amino acids, and proteins) would show higher attachment on the film layers during deposition. In the case of NF3 and NF4 film, the trends for layer deposition were completely different between standard mediabased films and DI water-based films. For water-based films, we observed an increased frequency shift for polycation layers, 2058


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Figure 3. (a) Snapshots of the side view at the beginning (0 ns; left) and the end (130 ns; right) of P4R32−3. Thick blue and red lines represent PLLand RGD, respectively, while thin red and green lines represent lipid heads (phosphates) and tails in the membrane, respectively. Water molecules are shown in light blue, and counterions (Cl−) are omitted for clarity. Images were generated using Visual Molecular Dynamics.48 (b) Mass density profiles of palmitoyloleoylglycerophosphocholine (POPC) phosphates, PLL, and RGD, and (c) radial distribution functions between anionic POPC phosphates and cationic residues of PLL and RGD (left) and between charged residues of RGD and PLL (right).

as the number of bilayers increased because of the formation of polymer complexes (Figure S4). Figure S3b also shows the morphologies of LbL films based in DI water, and roughness results on NF1, NF2, NF3, and NF4 were 0.707, 1.158, 2.001, and 0.889 nm, respectively. Some islets and dots formed a polymer complex, coacervated on the surface.49 Molecular Dynamics Simulation for Adsorption Mechanism of LbL Films on Cell Membrane. Formation of the NF1 and NF3 layers on the cell membrane was analyzed using molecular dynamics simulation on model cell membranes (Figures S6−8). We chose the NF1 layer, and each layer of PLL and RGD was simulated near the palmitoyloleoylglycerophosphocholine (POPC) lipid membrane with a different number of layers for 130 ns. Simulated systems are shown in Table S1. The initials “P” and “R” indicate PLL and RGD, respectively, which are followed by the number of each polymer per bilayer. The last number designates the number of bilayers. Figure 3a and Figure S6a show the initial and final snapshots of the simulations. Starting with the position of layers above the membrane surface, PLL and RGD interact with the membrane surface or with each other. PLL and RGD remain on the membrane surface and form stable layers for systems composed of layers with 4 PLLs. All simulation results showed that although the PLL and RGD layers become irregularly mixed, the membrane surface interacted with PLLs rather than with RGDs, indicating the presence of electrostatic interactions between cationic PLLs and anionic phosphates of lipid head groups. Particularly, multiple bilayers of P4R32-3 were nearly completely mixed, indicating interlayer diffusion of PLL and RGD. These configurations were confirmed by calculating their density profiles. In Figure 3b and Figure S6b, P4R32-1 and P4R32-3 show that PLL and RGD were close to the membrane

whereas when large amounts of polyanions were adsorbed on the surface, the frequency changes decreased (Figure S2). This occurred because of film erosion during assembly as indicated in a previous study.45 The stronger electrostatic interaction between polycations in solution and lower side of complexed film layers may induce detachment of the superficial complex layers, increasing the frequency of alternation. In addition, fully charged PLL and HA molecules were present at pH 7.5, and slight detachment occurred because of the repulsion force. In contrast, the films using cell culture media as a solvent showed continuous growth regimes regardless of the number of layers (Figure 2b). This may be because charged proteins or molecules were present in the cell culture media. In cell culture media, negatively charged molecules may dominantly bind with polycations or adsorb onto polycation layers through electrostatic interactions, partial hydrogen bonding, or entanglement with polymers, decreasing the frequency of the PLL and PLL-RGD layers for QCM (Figure 2b). Thus, adsorbed polycation layers remained in the lower portion of the charged area, resulting in slower growth of HA layers compared to that of the DI water-based HA layer. We also measured the surface morphologies of each type of film (Figure S3). After the deposition of 3 bilayers, for NF1, the topographical image appeared to be similar to the deposition of PLL layers on the silica substrate, which was highly smooth according to a previous study;49 the RGD had little or no impact on surface morphology (Rq = 0.643 nm). NF2, NF3, and NF4 layers showed low surface roughness (values of roughness (Rq) for NF2, NF3, and NF4 were 1.031, 1.013, and 1.403 nm, respectively). Visible dots were regarded as aggregation of polymers, RGD, or complexes of cell culture media components. Additional dots were generated for all film types 2059


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Chemistry of Materials surface, which is consistent with a visual molecular snapshot and video (Figure 3a, Figure S6a, and Videos S1 and S2). PLL was closer to the lipid headgroup region than was RGD, indicating the presence of electrostatic interactions between PLL and lipid phosphates. Interestingly, although PLL and RGD of P4R32-3 were broadly positioned above the membrane surface, high peaks for PLL and RGD were alternately observed, indicating that the layers of PLL and RGD were mixed but still formed clusters. To examine this, we calculated the number of “clusters” as a function of time, where a “cluster” was either a complex of any size or a free molecule. If the distance between any two atoms of different molecules was less than 0.4 nm, the molecules were considered to be in a cluster. Other criteria using distances in the range of 0.35−0.5 nm showed similar qualitative trends. Figure S7 shows that the number of clusters of PLL increased from 3 to up to 5−6, while that of RGD decreased from 3 to 1−2, indicating that 5−6 aggregates of PLL interact with RGD. To understand the interactions among PLL, RGD, and the membranes, radial distribution functions among anionic POPC phosphates, cationic residues of PLL, and RGD as well as cationic PLL and charged (either anionic or cationic) residues of RGD were calculated. Figure 3c shows a much higher radial distribution function peak for POPC-PLL than for POPC-RGD, indicating stronger electrostatic interactions between lipid head groups and PLLs, as observed in the simulation results. For the PLLRGD interaction, anionic residues of RGD (Asp, C-terminal) showed a much higher peak than did cationic residues of RGD (Arg, N-terminal), as expected. This indicates that PLL electrostatically interacted with the membrane surface as well as with the anionic residues of RGD. These strong electrostatic interactions may influence the structural behavior of flexible PLL and RGD chains. To resolve this, the root-mean-squared end-to-end distances (⟨h2⟩1/2) and radii of gyration of PLL and RGD were calculated. Table S2 shows the comparison of the values in water and in layer systems; both ⟨h2⟩1/2 and the radii of gyration changed very little. Although PLL and RGD formed the layers above the membrane surface, they behaved much like an isolated chain in solution, likely because PLL and RGD interacted with water molecules inside multilayers. These simulation findings indicate that bilayers of PLL and RGD remained on the membrane surface while continuing to diffuse between layers, leading to the development of an irregularly mixed conformation. PLLs formed aggregates and interacted with membrane head groups and RGD, as they electrostatically interacted with anionic residues of RGD and lipid phosphates. In particular, PLL and RGD chains in layers behaved as isolated chains in solution, indicating that layer formation does not influence the conformation of the random-coil polymer. Note that the simulation and experimental environments as well as the mass transport conditions were different; hence, quantitative comparison with the experiments could not be achieved. For example, experiments performed in culture media showed that the thickness of one layer was ∼1 μm by CLSM results (Figure 4a,b), which is beyond the simulation size and time scale. Additionally, it cannot be ruled out that the charged components in media significantly modulated layer formation and interlayer diffusion. We demonstrated layer formation and interlayer diffusion, supporting our experimental observations regarding stable layers on cell membranes, and revealed atomicscale insights into the interaction between molecules and membranes. We have also conformed 3 bilayers of NF3, which

Figure 4. Nanofilm on MSCs. The cells were coated with PLL-FITC (a and b) or (PLL-FITC/HA-FITC)2 (c and d). The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The fluorescent image of the nanofilm coated on the cell membrane and the cell nuclei stained with DAPI were overlaid with a differential interference contrast (DIC) image (a and c) and a cross-section of each cell (b and d). (e) FITC intensity increased depending on the number of deposited layers.

was successfully constructed on the cell membrane under both hydrate and anhydrate conditions (Figure S8). Deposition of Nanofilm on MSCs’ Cell Surface. To visualize the nanofilm coated on the cell membrane, MSCs were coated with fluorescein conjugated PLL (PLL-FITC) (Figure 4a,b) or (PLL-FITC/HA-FITC and observed with confocal microscopy (Figure 4c,d). In MSCs covered with 1 layer, PLL-FITC molecules were localized to the plasma membrane, indicating that the applied materials remain attached to the cell surface. For the first layer, PLL in its free form attached to the cell surface and formed a stable layer. Compared with the cell size shown in the DIC image, localization of fluorescein indicated that the nanofilm was formed on the boundary of the cell membrane. MSCs that were detached from the plastic culture dish exhibited a round morphology, and the fluorescein-conjugated nanofilm conformed to the size and shape of the MSC plasma membrane. Fluorescein intensity increased as the number of LbL assembly on the cell membrane increased (Figure 4e and Figure S9), which was also numerically compared by FACS analysis based on comparison of the number and fluorescence intensity of nanofilm-coated cells (Figure S10). Thickness values estimated from the microscopic images were 0.89 μm for 1 layer and 1.62 μm for 4 layers. On the basis of comparative simulation on hydrated LbL and dehydrated LbL, the thickness value from microscopic images (Figure S8) and profilometery data (Figure 4b) might reflect the swollen state of the LbL on the plasma membrane under hydrated conditions, which are significantly different compared with the thickness of the dried layers. Increasing fluorescence intensity according to the number of layers indicates that PLL can attract HA, resulting in the accumulation of building blocks on the cell membrane when these components were reacted with cells in an alternative sequence. However, it is likely that the nanofilm did not completely coat the entire surface of the cells because the cell surface has many filopodia and a ruffled membrane, as shown in Figure 4d and Figure S9. Analysis of Cell Survival and Signal Transduction under Agitation Culture. For mimicking the situation in blood vessels that MSCs encounters after systemic transplantation, MSCs with or without nanofilms were subjected to continuous agitation culture. Typically, the average peak and 2060


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Figure 5. (a) Illustration showing different responses of MSCs with or without nanofilms after being subjected to spinner culture in nonattachment condition (top). Effect of the nanofilm on stem cell survival (bottom) (**, P < 0.01; *, P < 0.05). (b) Illustration of the signaling pathway of ErK1/2 and Akt after interaction with the nanofilms. (Top) Activation status of the survival-related signaling components on MSCs measured by Western blot analysis (bottom).

mean flow of the ascending aorta are approximately 60 and 11 cm/s, and those of the superior vena cava are approximately 28 and 12 cm/s, respectively.50 The agitation speed was calculated to be 30.6 cm/s, which is similar to the values observed in the bloodstream. After trypsin treatment, MSCs with or without nanofilms were subjected to agitating culture in a tube with nonadherent surface, which offered physical stress to MSCs. In agitation culture, the survival fraction of the control MSCs on day 1 was 10.6 ± 6.02%, indicating attachment-deprived cell death (Figure 2f). In Figure 5a, nanofilm-coated MSCs showed a significantly higher survival rate compared with that of the control group (296 ± 132.66%, 316 ± 75.7%, and 127 ± 42.3% for NF1, NF2, and NF3-coated cells, respectively). Among the nanofilm-coated groups, cells coated with the RGD-containing film showed a higher survival rate (p = 0.0194), while cells coated with NF3 film, which does not have the RGD motif, showed the lowest survival rate. However, the survival rate of cells coated with NF2 was consistently slightly higher than that of cells coated with NF1, despite the lack of RGD in the bottom layer. It is speculated that the PLL/HA, the bottom layer in NF2, provided structural support in its hydrated state, which provided structural integrity to MSCs. Also, previous reports showed that HA transduces the signal via its receptor CD44 and that this leads to activation of focal adhesion kinase (FAK) and Akt, key components in survival signaling.51,52 Continuous agitation induced severe cell death on day 2, showing 2.35 ± 2.4%, 28.8 ± 25.08%, 57 ± 47.63%, and 7.8 ± 7.5% of survival rates compared with the number of cells at day 0. In the nanofilm-coated groups, the cell number actually increased on day 1 and then decreased on day 2. Cells coated with nanofilm proliferated until day 1 even in agitation culture conditions, indicating that the application of nanofilm not only transiently inhibited anoikis but also allowed cells to proliferate, albeit for a limited period of time. The decreased cell viability observed on day 2 may have been induced by the loss of the nanofilm due to continuous agitation or cell division. A similar tendency was observed for cell survival when MSCs with or without nanofilms were subjected to static adhesion-deprived culture conditions using a polyhydroxyethyl methacrylate

(HEMA)-coated culture dish to inhibit cell adhesion to a substrate (Figure S11). In the case of static culture, the cell survival fraction did not abruptly decrease on day 2 and day 3, indicating that the shear stress greatly compromised cell viability. Thus, it is expected that if nanofilm-coated cells are transplanted by local injection rather than by intravenous transplantation, the nanofilm exhibits a cytoprotective and cellstabilizing effect for a longer period of time. In normal tissue environments, cells bind to the ECM through integrin, which activate FAK to transduce a intracellular survival signal leading to Akt phosphorylation. To understand the mechanism underlying the cytoprotective effect exerted by nanofilms on MSCs, a key component of the survival signal transduction pathway was examined by Western blot analysis (Figure 5b). Compared with the basal level of betatubulin, activation of Erk1/2 was not affected by nanofilm application and stayed nearly at the same level. However, activation of Akt was significantly increased for all types of nanofilm-coated MSCs. Activation of Akt in nanofilm-coated MSCs indicates that the presence of nanofilm on the plasma membrane transduced and activated the intracellular survival signal. This might be induced by the interaction of RGD and HA included in the LbL assembly with their corresponding cell surface receptor which subsequently activated their downstream signaling components such as the FAK-PI3K-Akt axis thereby providing a cytoprotective effect from anoikis.53 In the case of the NF3 film, which lacks the RGD motif, it also induced Akt activation, indicating that the ECM-like soft multilayer matrix may have been involved in activation of the survival signaling pathway.54 On the basis of these results, we found that the RGD- and HA-incorporated LbL film activated intracellular survival signal successfully. Furthermore, we also examined the effectiveness of nanofilms on stem cell migration as shown in Figure S12; the film coated cells slightly decreased their migration property but still have almost 80% migration potential compared with that of the control group. Effect of Nanofilm Application on MSCs’ Stemness and Differentiation Potentials. Through the comparison of cytoprotective property, the NF2 film was selected for its 2061


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Figure 6. (a) CFU-Fs assay of MSCs for 10 days. Statistical analysis was performed by unpaired t test. (n = 5). (b) Effect of cell surface-applied nanofilm on the differentiation potential of MSCs. MSCs with or without nanofilm were maintained in osteogenic (top) or chondrogenic (bottom) differentiation induction media for 3 or 5 weeks as indicated in the figure. Stained area and normalized pellet size were calculated and graphically analyzed (n = 3).

the duration of in vivo survival and the clearance profile of injected stem cells, GFP (+) MSCs with or without nanofilms were administered to nude mice intravenously by tail vein injection (Figure 7a). The in vivo cytoprotective effect of nanofilms was confirmed by comparing the percentage of GFP (+) donor cells in circulation between nanofilm-applied and nonapplied groups by fluorescence-activated cell sorting (FACS) analysis. While the nanofilm-applied group already seemed to reach a peak increase at 6 h upon injection showing 4.95-fold higher than the control, the nonapplied control group showed delayed peak increase in circulation which was at about 48 h upon injection and always stayed at a lower level compared with that of MSCs with nanofilms (Figure 7b−d). MSCs show a homing effect that cells can home or migrate to the damaged tissue site or tumor site. The MSCs express many receptors and adhesion molecules that aid migration toward the target site. The chemokine receptors and integrins, ECM receptors, play a significant role in the homing effect. Systemically injected MSCs exert homing behavior through the active chemokine receptors and ECM receptors, and therefore, the cells can attach to and migrate through endothelial cells to get to the target tissue.58 To investigate the homing activity of nanofilm-applied MSCs compared with control MSCs, mice injected with MSCs were subjected to muscle injury 12 h upon injection (Figure 7a,e). After 2 days of muscle injury, the injured muscle tissues were subjected to twophoton fluorescence microscopy to detect the presence of GFP (+) cells (Figure 7f). Two tiny clusters of GFP (+) cells were detected from only one mouse out of 5 mice in the control group, while bigger clusters of GFP (+) cells were detected in 4 mice out of 5 mice injected with nanofilm-applied MSCs indicating the nanofilm-coated MSCs postponed clearance time in circulation and increased accumulation of cells in the muscle injury site.

significantly high cytoprotective property and proceeded for verification of stemness and differentiation potential (Figure 6). In terms of colony forming ability, the application of nanofilm significantly increased the number of CFU-Fs (Figure 6a). It is not likely that the nanofilm changed the clonogenic potential of MSCs but the cytoprotective effect coming from nanofilm potentiated colony-forming ability. This also indicates that the application of nanofilm did not interfere with cell attachment to the surface. In terms of differentiation potential, there also was no significant difference between MSCs with or without nanofilms (Figure 6b). There was only a slight decrease of calcium deposition and an increase in cartilage pellet size in the nanofilm-applied group after 5 weeks of differentiation induction. These data might indicate that the nanofilm influenced the differentiation activity of MSCs favoring chondrogenic differentiation rather than osteogenic differentiation. This tendency was shown in previous reports,36,55,56 which showed that the carboxylic acid group could induce chondrogenesis in MSCs. Further, when MSCs with or without nanofilms were subjected to spontaneous adipogenesis by culturing beyond confluence for 4 weeks and visualizing lipid droplets by Oil red-O staining, adipogenesis was suppressed in nanofilm-coated MSCs compared with what was observed in uncoated MSCs (Figure S13). Specifically, the extent of adipogenic differentiation was found to be dependent on the number of RGD peptide-containing layers.57 Therefore, these results indicate that the application of nanofilm did not compromise the characteristics of MSCs including the differentiation potential, albeit suggesting the possibility of modulation on differentiation tendency. Effect of Nanofilm on in Vivo Survival of MSCs after Systemic Injection. The ultimate purpose of stem cells’ surface modification is to enhance survival of MSCs after in vivo systemic transplantation. To examine the effect of nanofilms on 2062


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Figure 7. (a) Schematic illustration of intravenous administration of GPF (+) MSCs with or without nanofilms into nude mice. At determined time points after injection, whole blood was collected, and the percentage of GFP (+) cells in circulation was analyzed by FACS analysis. (b−d) Effect of nanofilm on in vivo stem cell survival after systemic transplantation. 5 ×105 GFP (+) MSCs with or without nanofilms were injected to each immunocompromised mice via the tail vein, and whole blood was withdrawn at the indicated time intervals to examine the proportion of GFP (+) MSCs in circulation (n = 4). Mononuclear cells isolated from uninjected mice and GFP (+) MSCs served as the negative control and positive control, respectively (b). (e,f) Investigation of stem cell recruitment in muscle injury model. (e) Images of muscle with or without injury. (f) Twophoton microscopy images of recruited GFP (+) MSCs (FITC) in injured muscles. The muscle tissues were collected 2 days after muscle injury and were sliced to the thickness of 1 mm for detecting GFP (+) cells (n = 5). The white bar indicates 100 μm.

moderate binding to each other to produce a flexible multilayer, rather than elaborately packed encapsulation around the cells, thereby expressing that molecules or receptors involved in stem cell homing could exert its effect. Furthermore, the cytoprotective effect of nanofilms potentiated cell survival rate upon injection, increasing the chance to get delivered to injured target tissue. These results demonstrate that the nanofilm increased the functionality of MSCs in vivo by supporting cell survival in circulation and readiness to recruitment.

Cell movement is mediated by membrane ruffles, a structure formed as protruded plasma membrane on the cell surface. Because of the irregularity of the ruffled structure, we expect that the coating cannot entirely cover the cell plasma membrane but that the protruded membrane ruffles remain naked from LbL layers. Through those naked surfaces, the cells could sense and react toward the environment. It is very likely that the main spots containing receptors for ECM molecules are covered with LbL assembly and transduce the intracellular signal to activate the survival signal transduction pathway, while membrane ruffles were free from the coating so that the cell migration was not entirely blocked. In a recent publication, HA was reported to mediate the chemotactic signal and facilitate the migration potency of MSCs in vitro and in vivo environments. In rats, HA and CD 44 interaction induces MSCs to migrate and home toward the kidney, and the anti-CD 44 antibody blocked the migration of MSCs.59 In our results, the migration of cells with LbL assembly was reduced to the 72−80% level compared with that of the control group (Figure S12), and the reason is because HA itself is functioning as a chemotactic agent. However, the cell migration was not entirely blocked, possibly because there are various agents inducing chemotactic movement of cells, and HA is one of them. On the basis of the result showing that the MSCs with LbL layer still showed migration activity, we could conclude that during film formation, each material produced

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CONCLUSION In conclusion, MSCs coated with an LbL assembly film on the plasma membrane showed enhanced stability under harsh conditions. For LbL assembly, RGD-peptide was used as the building block to activate integrin. Nanofilm containing RGDpeptide on the plasma membrane of MSCs was prepared under normal culture conditions using culture media, and this achieved higher survival rates under agitation culture conditions. Additionally, LbL film-coated MSCs showed significantly activated Akt, the signaling component of survival-promoting pathway, and thereby revealed that the LbL film stabilized MSCs under harsh conditions. Although application of LbL films slightly decreased cell movement, considering the extent of increased cell survival, the net outcome is highly beneficial. Thus, the LbL assembly is a suitable technique for the preparation of films for highly 2063


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Chemistry of Materials sensitive stem cell operations because the LbL film confers stability to MSCs under harsh conditions without affecting nutrition and down-regulating cell activity. This technique can be a promising application in next-generation single cell manipulation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b04096. Experimental description, AFM images, molecular dynamic simulation for films, and supplemental data of in vitro assays (PDF) Molecular dynamic simulation for formation of PLLRGD film on cellular membrane (MPG) Molecular dynamic simulation for formation of PLLRGD film on cellular membrane (MPG)

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AUTHOR INFORMATION

ORCID

Jinkee Hong: 0000-0003-3243-8536 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Korean Government (Grants 2012M3A9C6050104 and 2015R1A5A1037656); the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grants HI14C-3266, HI15C-1653, and HI16C1010); “Cooperative Research Program for Agriculture Science & Technology Development (Grant PJ00998601)” Rural Development Administration, Republic of Korea; Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2014R1A1A2054016 and 2015R1D1A1A01059702), Midcareer Research Program (NRF-2015R1A2A2A04006172), and by DGIST Supercomputing & Big Data Center for the allocation of the supercomputing time. J.H. is thankful to Dr. Kahee Kwon for her thoughtful help with this research.

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REFERENCES

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Cytoprotective Self-assembled RGD Peptide Nanofilms for Surface

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