Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24577−24587
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Photocleavable Peptide−Poly(2-hydroxyethyl methacrylate) Hybrid Graft Copolymer via Postpolymerization Modification by Click Chemistry To Modulate the Cell Affinities of 2D and 3D Materials Shin-nosuke Nishimura,† Naoki Hokazono,‡ Yukiko Taki,‡ Hideki Motoda,† Yusuke Morita,*,‡ Koji Yamamoto,‡ Nobuyuki Higashi,† and Tomoyuki Koga*,† Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering and ‡Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan
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ABSTRACT: Controlling the surface properties of engineered materials to enhance or reduce their cellular affinities remains a significant challenge in the field of biomaterials. We describe a universal technique for modulating the cytocompatibilities of two-dimensional (2D) and three-dimensional (3D) materials using a novel photocleavable peptide-grafted poly(2hydroxyethyl methacrylate) (PHEMA) hybrid. The reversible addition−fragmentation chain transfer copolymerization of HEMA and propargyl acrylate was successfully controlled. The resultant alkyne-containing PHEMA was then used to modify the azide-terminated oligopeptides [Arg-Gly-Asp-Ser (RGDS)] with a photolabile 3-amino-3-(2-nitrophenyl)propanoic acid moiety via the copper-catalyzed alkyne−azide click chemistry. This strategy was readily used to decorate the surfaces of both hydrophilic and hydrophobic materials with RGDS peptides due to the high film-forming abilities of the PHEMA unit. The resultant thin film acted as an effective scaffold for improving cell adhesion and growth of NIH/3T3 fibroblasts and MC3T3-E1 osteoblast-like cells in vitro. In addition, UV irradiation of the surface led to the detachment of cells from the material surface accompanied by the photocleavage of RGDS grafts and enabled the 2D-patterning of cells and cell sheet engineering. The applicability of this system to 3D materials was investigated, and the cell adhesion was remarkably enhanced on a 3D-printed poly(lactic acid) object. This facile, biocompatible, and photoprocessable peptide−vinyl polymer hybrid system is valuable for its ability to advance the fields of tissue engineering, cell chips, and regenerative medicine. KEYWORDS: peptide−polymer hybrid, photocleavable peptide, cell micropatterning, cell sheet engineering, postpolymerization modification, 2D and 3D coatings
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INTRODUCTION Significant efforts have been made recently toward designing functional scaffolds that control cell adhesion, shape, and proliferation for tissue engineering and regenerative medicine applications.1,2 The interaction strength between cells and a material’s surface can be controlled by the biochemical cues (i.e., cell-binding ligands, their densities and distributions, etc.) as well as the physical factors such as surface wettability, stiffness, and topographic and nanograined structures.3−8 Mechanical forces, which are generated from the extracellular microenvironment, can also substantially affect the cell response and development.9,10 In vivo, the extracellular matrix (ECM), which is consisted of proteins and polysaccharides with nano- and microscale structures, acts as a scaffold to regulate cell behavior and cell-to-cell communication.11 Fibronectin (FN) is one of the important ECM proteins that mediates the adhesion and spreading of many cells. The cellbinding activity of FN is attributed to the arginine-glycine© 2019 American Chemical Society
aspartic acid (RGD) sequence in the protein, which specifically binds to integrin receptors present on a cell’s surface.12−14 Chemically synthesized short RGD and RGD-serine (RGDS) peptides, which can be prepared easily using solid-phase peptide synthesis (SPPS) methods, can also function as cell recognition epitopes and have, therefore, been used to engineer ECM-mimicking surfaces.3,4,15−17 The hybridization of biofunctional oligopeptides and synthetic polymers having an appropriate biocompatibility and mechanical strength may offer a good material engineering strategy. One successful approach to preparing peptide−polymer hybrid materials involves combining SPPS and controlled radical polymerization (CRP) methods, including nitroxidemediated polymerization, atom transfer radical polymerization, Received: April 18, 2019 Accepted: June 12, 2019 Published: June 25, 2019 24577
DOI: 10.1021/acsami.9b06807 ACS Appl. Mater. Interfaces 2019, 11, 24577−24587
Research Article
ACS Applied Materials & Interfaces and reversible addition−fragmentation chain transfer (RAFT) polymerization.18−21 Recent significant advances in CRP using peptide-based initiators, macromonomers, or chain transfer agents enables the sequence-controlled peptides to incorporate into well-defined vinyl polymers as block,22−25 multiblock,26−30 or graft31−33 segments. In fact, various RGD peptide-containing hybrids have been developed using this approach for use as artificial scaffolds for tissue engineering.15,22,31,33−37 An alternative approach to hybrid synthesis involves postpolymerization modifications,26,27,38−41 for example, via the alkyne−azide cycloaddition click chemistry. Click chemistry was first proposed in 2001 as an addition reaction that proceeds rapidly and selectively without side reactions.42 The Huisgen 1,3-dipolar cycloaddition is a representative example of click chemistry43 and proceeds between alkyne and azide groups via a copper catalyst, even in the presence of water.44 This method enables the versatile and selective modification of synthetic polymers using biomolecules without undesirable side reactions (e.g., chain transfer reactions during polymerization), even on a solid surface (i.e., in the film form), and provides more flexibility to material design.26,27,39,41,45 Hybrid polymer-based coatings that can form stable thin films with switchable cell−material interactions under the control of external stimuli are particularly valuable for the surface engineering of two-dimensional (2D) and threedimensional (3D) materials and for facilitating cell sheet engineering and cell micropatterning for cell-based sensing and drug discovery applications. Herein, we report the preparation of a graft-type photocleavable RGDS peptide−biocompatible vinyl polymer hybrid using a postpolymerization modification approach as a novel functional artificial ECM that enables spatial control over cell adhesion and detachment by photolithography. Several methods have been employed for cell micropatterning, including microcontact printing, inkjet printing, photolithography, and laser-guided writing.5,46 Among these, the photolithographic approach using photodegradable molecules, such as RGD with a photolabile linker or caged RGD,47−50 is especially attractive for constructing both RGD peptide micropatterned and gradient surfaces, permitting control over cell behavior, such as adhesion and migration, because light can be easily focused and its intensity controlled. Although cell adhesion has been successfully controlled by directly grafting light-sensitive RGD peptides onto engineered solid materials, in most cases, this approach is applicable only to a limited range of substrates, such as 2D glasses and silicon plates. The photodegradable peptide−polymer hybrid system described here overcomes limitations and will be a universal platform for decorating various materials, including simple 2D plates and complex 3D-printed objects. In this study, we designed an azide-terminated RGDS peptide with a photolabile 3-amino-3-(2-nitrophenyl)propanoic acid (ANP) linker that decomposed rapidly under mild UV light.51 Moreover, an alkyne-containing poly(2hydroxyethyl methacrylate) (PHEMA) prepared by RAFT polymerization was used as a base polymer to react with the peptide via a copper-catalyzed cycloaddition, leading to the desired photoprocessable hybrid graft copolymer (Figure 1). PHEMA is a biocompatible polymer commonly used in biomaterials for its ability to form films and hydrogels with sufficient mechanical strength and resist the nonspecific adsorption of proteins and cells.52,53 Spatially controlled UV
Figure 1. Chemical structure of the novel photocleavable and clickable oligopeptide (a) and alkyne-containing poly(HEMA-coPgA) (b) used as a biofunctional coating.
irradiation induces partial photocleavage of the RGDS grafts to generate regions with opposing surface properties: cell adhesive and nonadhesive (nonfouling) surfaces. To evaluate the versatility of this hybrid coating as a novel functional ECM, two different cells were employed as model cell lines: NIH/ 3T3 fibroblast cell, which has been widely used for soft tissue engineering including cell sheet, and MC3T3-E1 osteoblastlike cell, which has usually been used to assess cell−material interaction, including cell migration, as well as for bone tissue engineering. A detailed analysis of cell behavior on the hybrid film was conducted, especially in view of the effect of the photocleavage of the RGDS grafts. Furthermore, the applicability of this system to 3D-printed poly(lactic acid) (PLA) objects was confirmed. PLA is a biocompatible and biodegradable hydrophobic polymer derived from renewable biomass and has been used in biomedical applications, such as bone plates, surgical sutures, and drug delivery system carriers. We believe that this work provides a useful method to fabricate various scaffolds with a photocontrollable cell affinity for soft and hard tissue engineering.
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EXPERIMENTAL SECTION
Materials. Analytical grade solvents were used as received. N,NDimethylformamide (DMF), methanol, 2,2,2-trifluoroacetic acid (TFA), dichloromethane, diethylether, hexane, ethyl acetate, acetonitrile, hydrochloric acid (12 M HCl), triethylamine, N-methylmorpholine (NMM), piperidine, 2,2,2-trifluoroethanol (TFE), 2,2′-azobisisobutylonitrile (AIBN), sodium sulfate anhydrous (anhydrous Na2SO4), sodium azide (NaN3), ammonia water (25 vol % NH3), dimethyl sulfoxide (DMSO)-d6, chloroform-d, and D2O were purchased from Nacalai Tesque. 2-Hydroxyethyl methacrylate (HEMA), rhodamine B (RhB), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium chloride (DMT-MM), N,N′-diisopropylcarbodiimide (DIPC), N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu), CuBr2, N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA), and LiBr were purchased from Wako Pure Chemical (Japan). Fmoc-L-Arg(Pbf), Fmoc-L-Asp(OtBu), Fmoc-β-Ala, FmocGly, Fmoc-L-Lys(Mtt), Fmoc-L-Ser(tBu), Fmoc-NH-SAL 4-methylbenzhydrylamine (MBHA) resin (0.67 mmol/g), and 1-hydroxybenzotriazole anhydrous (HOBt) were purchased from Watanabe Chemical Industries. 2,5-Dihydroxybenzoic acid (DHBA), 4-cyano(4-thiobenzoylthio)pentanoic acid (CTPA), propargyl acrylate (PgA), and 6-bromohexanoic acid were purchased from Sigma-Aldrich. Ascorbic acid (AsAc) was purchased from Tokyo Chemical Industries. 3-Amino-3-(2-nitrophenyl)propanoic acid (ANP) was purchased from Alfa Aesar. Poly(lactic acid) (PLA) (MakerBot PLA filament 1.75 mm) for 3D printing was purchased from B&H 24578
DOI: 10.1021/acsami.9b06807 ACS Appl. Mater. Interfaces 2019, 11, 24577−24587
Research Article
ACS Applied Materials & Interfaces
Figure 2. (a) SEC traces (DMF containing LiBr, 40 °C) of poly(HEMA-co-PgA) obtained after RAFT polymerization for 1, 3, 6, 13, or 24 h. (b) 1 H NMR spectrum of poly(HEMA-co-PgA) obtained after polymerization for 13 h in DMSO-d6 at 25 °C. (c) Plots of Mn (blue circles), Đ (red squares), and FPgA (green triangles) as a function of monomer conversion. The solid line represents the calculated theoretical Mn as a function of the monomer conversion, assuming a constant FPgA feed composition. Foto & Electronics Corp. HEMA and PgA were purified by passing through an activated alumina prior to use. Measurements. 1H NMR spectra were recorded using a JEOL JNM-ECA500 (JEOL Resonance) spectrometer (500 MHz). The values of polydispersity indexes (Đ, Mw/Mn) of the polymers were evaluated by size exclusion chromatography (SEC) using a JASCO LC-net II/AD (JASCO Ltd.) with a refractive index detector. DMF (containing 10 mM LiBr) was used as an eluent (0.6 mL/min, 40 °C, TSKgel α-4000). Poly(methyl methacrylate)s (GL Sciences Inc.) was used as the calibration standard. Matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS) analyses were performed on an Autoflex speed (Bruker Daltonics). DHBA was used as a matrix. Fourier transform infrared (FTIR) spectra were recorded on FT/IR-4600 (JASCO Ltd.) using a deuterated L-alanine triglycine sulfate detector fitted with an attenuated total reflection (ATR) accessory that used a germanium internal reflection element (resolution: 4 cm−1, 32 scans). Absorption spectra were recorded by a V-650 spectrophotometer (JASCO Ltd.). AFM images were measured at room temperature on an SPM9700 (Shimazu Co.) by contact mode (OMCL-TR800PSA-1, tip radius
