Cell-Adhesive Star Polymers Prepared by ATRP - Biomacromolecules

Biomacromolecules 2009, 10, 1795–1803

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Cell-Adhesive Star Polymers Prepared by ATRP Sidi A. Bencherif,† Haifeng Gao,†,‡ Abiraman Srinivasan,§ Daniel J. Siegwart,| Jeffrey O. Hollinger,*,§ Newell R. Washburn,*,† and Krzysztof Matyjaszewski*,† Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, Department of Chemistry, University of California, Berkeley, California 94720, Bone Tissue Engineering Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15219, and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received February 19, 2009; Revised Manuscript Received May 13, 2009

This study presents the synthesis and evaluation of cell adhesive poly(ethylene oxide) (PEO) star polymers for potential biomedical applications. Star polymers with a size of approximately 20 nm and with relatively low polydispersities (Mw/Mn e 1.6), containing GRGDS (Gly-Arg-Gly-Asp-Ser) segments, were prepared by atom transfer radical copolymerization of PEO methyl ether methacrylate macromonomer (MM), telechelic GRGDSPEO-acrylate MM, and ethylene glycol dimethacrylate (EGDMA). Results from 1H NMR spectroscopy confirmed the covalent incorporation of the peptide into the star periphery. In vitro cytotoxicity experiments showed star polymers to be cytocompatible (g95% cell viability) and GRGDS-star hybrid hydrogels supported the attachment of MC3T3.E1 (subclone 4) cells. Hybrid hydrogels were prepared by free radical photopolymerization based on 10% (wt/v) PEO dimethacrylates Mn ) 4000 g/mol with 1% (wt/v) GRGDS-star polymers having different peptide content. Cell adhesiveness was also determined from thin film coatings prepared with GRGDS-containing star polymers on nonadherent plastic plates. After 24 h incubation, phase contrast microscopy and scanning electron microscopy (SEM) images showed uniform cell adhesion and distribution over the film containing cell-adhesive star polymers. These results confirm that incorporation of RGD ligand-binding motifs into PEO-based star polymers is required to influence substrate-cell interactions.

Introduction Over the past few years, there has been considerable interest in developing highly branched nanoscale polymeric systems.1-6 These materials have unique properties and functionality that may be exploited for drug delivery and biological engineering. Moreover, highly branched (dendritic, stars, and hyperbranched) polymers have unique properties in solution, that is, low viscosity and higher solubility compared to their linear analogues, as well as versatile functionality options in solution and solid state (e.g., thin films). There are several advantages with nanocarriers for drug delivery. Nanoparticles can transit capillaries and become endocytosed by cells, thus permitting efficient and effective drug targeting to discrete sites in the body.7-9 Moreover, different hyperbranched nanoparticles, such as dendrimers, have been used to enhance drug delivery systems in the pharmaceutical fields.10-13 However, these systems present some limitations: the synthesis of dendrimers involves multiple steps and is complex and costly. Star polymer-based biomaterials are a profoundly compelling alternative to dendrimeric systems.14-18 They can still exploit polyvalency and a controlled environment but will reduce synthesis cost. The spherical shape of the star-based polymers and their functionality (both core and arms) will enable the design of a suite of biomaterials with tunable, unique properties. Star polymers have been prepared by numerous methods,4 but the advent of living polymerization techniques has enabled * To whom correspondence should be addressed. E-mail: km3b@ andrew.cmu.edu (K.M.); [email protected] (N.R.W.); hollinge@ cs.cmu.edu (J.O.H.). † Department of Chemistry, Carnegie Mellon University. ‡ University of California. § Bone Tissue Engineering Center, Carnegie Mellon University. | Massachusetts Institute of Technology.

researchers to synthesize well-defined molecular architectures, including star polymers. Star polymers are usually synthesized via one of three common strategies: “core-first” by growing arms from a multifunctional initiator, “coupling-onto” by attaching linear arm precursors onto a multifunctional core and “armfirst” by cross-linking preformed linear arm precursors using a divinyl compound.19 Recent efforts in our laboratory20-24 and by others25-29 have demonstrated that these macromolecules can be effectively synthesized by controlled/living radical polymerization (CRP) techniques, including atom transfer radical polymerization (ATRP).30-32 ATRP is based on the repetitive addition of monomers to propagating radicals that are generated from dormant alkyl halides in a reversible redox process.31 ATRP has been successfully used to prepare star polymers with a controlled architecture, narrow molecular weight distribution (MWD), and high chain-end functionality that allows further chemical modifications. Also, milder reaction conditions (lower temperatures) used are crucial when targeting polymer-peptide hybrid macromolecules. It is both challenging and important to control interactions between polymeric materials and biological systems by incorporating biomolecules in materials with defined molecular architectures. The surface of the particles can be designed to contain cell- or tissue-specific ligands. Surface modification may be accomplished by introducing specific ligand motifs in star particles. This effect can be accomplished through coupling the ligand and a neutral polymer spacer prior to polymerization, such as coupling the ligand at the free end of a macroinitiator and a macromonomer (MM). The success of this system is based on the star molecular architecture combined with the special properties of PEO chains. There are two main advantages of employing PEO in the design of star polymers for biomedical

10.1021/bm900213u CCC: $40.75  2009 American Chemical Society Published on Web 06/11/2009

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applications.33 First, the protein-resistivity of PEO is a desirable attribute that may prevent adsorptive processes from blocking the chemical contribution of cell adhesion motifs. Second, the freedom of movement of PEO chains within an aqueous environment allows hydrophilic PEO chains to act as spacer groups to provide cell adhesion motifs with sufficient mobility for receptor-ligand interactions. Cellular adhesion ligands present in the extracellular matrix (ECM) play a critical role in controlling multiple aspects of cell phenotype, including morphology, motility, proliferation, and differentiation.34-39 There are several ligand-receptor interaction sites available in the ECM. The arginine-glycineaspartic acid (RGD) sequence is one attachment ligand motif extensively exploited in synthetic ECMs to regulate cellular responses.34,35,40 Cell adhesion plays an important role in a variety of basic biological processes, including guiding cells into their appropriate locations in the body. Consequently, RGD ligand-binding motifs can promote cellular uptake of nanoscale GRGDS-star polymers. Also, the design of a surface coated with cell-adhesive star polymers may improve tissue biocompatibility by limiting nonspecific protein adsorption and concurrently promoting cell-material interactions for implanted biomedical devices. Our objective was to synthesize well-defined cell-adhesive GRGDS-containing star polymers and to determine cellsubstrate interactions. The present study focused on the design of star-shaped nanomaterials for biomedical applications. We report for the first time the synthesis of novel cross-linked celladhesive star polymers prepared by ATRP based on the copolymerization of linear PEO MM with a divinyl compound using low molecular mass ATRP initiator. These star polymers may provide significant opportunities for a range of therapeutic applications, including surface modification of noncell adhesive surfaces in tissue engineering. Moreover, star polymers have a cross-linked core, which can be used as a drug reservoir, and the shell can be decorated with biomolecules to target the delivery of drugs to specific cells. The RGD amino acid sequence was incorporated into the star polymers using the “arm-first” approach, where PEO methyl ether methacrylate MM and GRGDS-PEO-acrylate MM are cross-linked with EGDMA to yield star polymers with a cross-linked core, statistically distributed radiating arms, and RGD-bound shell. The star polymers were characterized by several spectroscopic and chromatographic techniques and were subsequently evaluated in terms of their biocompatibility and ability to biomimetic microenvironments based on the formation of star polymerGRGDS hybrid macromolecules.

Experimental Section Materials. Poly(ethylene oxide) (PEO, Mn ) 4000 g/mol), PEO methyl ether methacrylate MM (Mn ) 2000 g/mol), methacrylic anhydride (MA), ethyl 2-bromoisobutyrate (EBiB), ethylene glycol dimethacrylate (EGDMA), 2,2′-bipyridine (bpy), copper(I) chloride (CuCl), and triethylamine (TEA) were purchased from Sigma-Aldrich. Dichloromethane was purchased from Sigma-Aldrich and dried over activated molecular sieves (4 Å) prior to use. Acryloyl-PEO-Nhydroxysuccinimide (acrylate-PEO-NHS, Mn ) 3400 g/mol) was purchased from Laysan Bio Inc. GRGDS peptide was purchased from Bachem Bioscience Inc. Photoinitiator Irgacure 2959 (I2959) was obtained from Ciba Specialty Chemicals and used as received. All other chemicals used were of reagent grade and were used without further purification. For in vitro cell culture, MC3T3.E1 (subclone 4) preosteoblast cells were obtained from American type Culture Collection (ATCC; Ma-

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Figure 1. Synthesis of GRGDS-(PEO)n-polyEGDMA star polymers by ATRP in the MM method.

nassas, VA) and cultured in RMEM, purchased from Invitrogen (Carlsbad, CA), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Nonadherent multiwell suspension culture plates were obtained from Greiner Bio-One Inc. (Germany). Phosphatebuffered saline (PBS) was purchased from Invitrogen and the Live/ Dead Viability/Cytotoxicity Kit was purchased from Molecular Probes, Inc. (Eugene, OR). Measurements. 1H NMR (300 MHz) spectra were taken on a Bruker Avance 300 spectrometer. Deuterium oxide (D2O) was used as solvent, and the polymer concentrations were varied between 0.5 and 3% by mass fraction. All spectra were run at room temperature, 15 Hz sample spinning, 45° tip angle for the observation pulse, and a 10 s recycle delay, for 128 scans. The standard relative uncertainty for calculation of reaction conversion via 1H NMR arises from the choice of the baseline and is estimated to be 8%. Molecular weights were determined by gel permeation chromatography (GPC; Polymer Standards Services (PSS); columns (guard, 105, 103, and 102 Å), with DMF eluent at 35 °C, flow rate ) 1.00 mL/min, and differential refractive index (RI) detector (Waters, 2410)). The apparent molecular weights and polydispersity (Mw/Mn) were determined with a calibration based on linear poly(methyl methacrylate) (polyMMA) standards using WinGPC 6.0 software from PSS. Conversion was also determined using GPC by monitoring the decrease of the MM peak area relative to the increase in the star polymer peak area. Particle size and size distribution of star polymers were measured by dynamic light scattering (DLS) on a high performance particle sizer, Model HP5001 from Malvern Instruments, Ltd. The sizes are expressed as Dav ( S (average diameter ( standard deviation). Synthesis of PEO Dimethacrylate (PEODM). PEODM was prepared as described previously.41 Briefly, the synthesis of PEODM 4000 g/mol was as follows. PEO 4000 g/mol (10 g, 0.0025 mol), 2.2 equiv of MA (0.85 g, 0.0055 mol), and TEA (0.4 mL) were reacted in 30 mL of dichloromethane over freshly activated molecular sieves (6 g) for 4 d at room temperature. The solution was filtered over alumina and precipitated into ethyl ether. The product was filtered and dried in a vacuum oven overnight at room temperature. Synthesis of GRGDS-PEO-acrylate MM. GRGDS-PEO-acrylate MM was prepared and characterized as described previously.42 Briefly, GRGDS peptide was dissolved in anhydrous DMF containing 4 molar excess of TEA. Acrylate-PEO-NHS was also dissolved in anhydrous DMF and immediately mixed with 1.1 molar excess of peptide. After incubating for 3 h at room temperature, GRGDS-PEO-acrylate was precipitated twice in cold anhydrous ether and dried in a vacuum oven overnight at room temperature. Preparation of PEO-Based Star Polymers by ATRP. As reported previously43 and shown in Figure 1, PEO-based star polymers containing cross-linked polyEGDMA as core and GRGDS as star shell, GRGDS-(PEO)n-polyEGDMA, were prepared by ATRP. Two concentrations of GRGDS-PEO-acrylate MM were used for the preparation of the cell adhesive star polymers (high and low peptide content). As a control reaction, PEO star polymers without GRGDS groups were also prepared. GRGDSHPC-(PEO)n-polyEGDMA Star Polymers with High Peptide Content (HPC, 8.2 mM). The procedure started with the ratio of reagents [PEO methyl ether methacrylate MM]/10 (Mn ) 2000 g/mol,

Cell-Adhesive Star Polymers Prepared by ATRP 0.526 g) ) [GRGDS-PEO-acrylate MM]/0.5 (50 mg, Mn ) 3800 g/mol) ) [EGDMA]/12 ) [EBiB]/1.67 ) [CuCl]/2 ) [bpy]/4, in DMF (1.6 mL) at 60 °C. PEO methyl ether methacrylate MM, GRGDS-PEOacrylate MM, EGDMA, bpy, and DMF were sequentially charged to a 10 mL Schlenk flask. GRGDSLPC-(PEO)n-polyEGDMA Star Polymers with Low Peptide Content (LPC, 3.3 mM). The procedure started with the ratio of reagents [PEO methyl ether methacrylate MM]/10 (Mn ) 2000 g/mol, 0.526 g) ) [GRGDS-PEO-acrylate MM]/0.2 (20 mg, Mn ) 3800 g/mol) ) [EGDMA]/12 ) [EBiB]/1.67 ) [CuCl]/2 ) [bpy]/4, in DMF (1.6 mL) at 60 °C. PEO methyl ether methacrylate MM, GRGDS-PEO-acrylate MM, EGDMA, bpy, and DMF were sequentially charged to a 10 mL Schlenk flask. (PEO)n-polyEGDMA Star Polymers with No Peptide. The procedure started with the ratio of reagents [PEO methyl ether methacrylate MM]/ 10 (Mn ) 2000 g/mol, 0.526 g) ) [EGDMA]/12 ) [EBiB]/1.67 ) [CuCl]/2 ) [bpy]/4, in DMF (1.6 mL) at 60 °C. PEO methyl ether methacrylate MM, bpy, and DMF were sequentially charged to a 10 mL Schlenk flask. In each reaction, the flask was degassed by five freeze-pump-thaw cycles and filled with nitrogen. CuCl was quickly added to the frozen mixture. No special care was taken to avoid moisture condensation. The flask was sealed with a glass stopper and then evacuated and backfilled with nitrogen five times before being immersed in a 60 °C oil bath. The N2 bubbled initiator EBiB was injected into the reaction system, via a purged syringe, through the side arm of the Schlenk flask. The reaction was stopped at 15 h by exposure to air. The final star polymers were purified by dialysis against DI water for 4 d by using a dialysis bag with MWCO ) 50000 g/mol. Covalent Incorporation and Characterization of GRGDS(PEO)n-polyEGDMA Star Polymers into PEODM Hydrogel. PEODM (10 wt %; Mn ) 4000 g/mol), 1 wt % GRGDS-(PEO)npolyEGDMA star polymers, and aqueous I2959 (0.05% by mass fraction) were mixed in a phosphate buffered saline solution at pH 7.4. Cylindrical samples of 2 mm in height and 10 mm in diameter were cured with a long wavelength UV source (365 nm, 300 µW/cm2) for 10 min to obtain hydrogels. NMR spectroscopy was used to characterize vinyl group conversion of PEODM/GRGDS-(PEO)n-polyEGDMA star polymer hydrogels after photopolymerization. Photopolymerization was induced directly in an NMR tube. A total of 1 mL of 10% wt MM solution containing the photoinitiator (I2959) was transferred into the NMR tube and exposed to UV to obtain the hydrogel. 1 H and 1H 1D DOSY were performed at 300 K on a Bruker Avance DMX-500 NMR spectrometer operating at 500.13 MHz and equipped with a Bruker z-gradient probehead. 1H 1D DOSY experiment was performed using a bipolar pulse pair stimulated echo pulse sequence with 2 spoil bipolar gradient pulses of 2% strength with duration of 6 ms and a diffusion time of 0.2 s. The pulse sequence included a longitudinal eddy current delay of 5 ms. 1H 1D DOSY NMR spectroscopy was used to characterize the efficiency of vinyl group reactivity during photocross-linking. The conversion was evaluated by comparing the relative peaks of uncross-linked and cross-linked methylene protons. Preparation of GRGDSHPC-(PEO)n-polyEGDMA Star Polymer Surface Coating. The starting solution for the dip-coating process was prepared by dissolving GRGDSHPC-(PEO)n-polyEGDMA star polymers in phosphate buffer solutions (pH ∼ 7.4) at a concentration of 10 mg/ mL. Nonadherent 96 well culture plates (Greiner, Frickenhausen, Germany) were dipped for 5 min in the mixed solution. The dipping process was carried out once, and the plastic plates were dried at room temperature for 24 h. The thin films obtained were slightly opaque. Biocompatibility of GRGDS-(PEO)n-polyEGDMA Star Polymers. To determine the biocompatibility of GRGDS-(PEO)n-polyEGDMA star polymers, the materials were preconditioned in cell culture media with 10% fetal bovine serum at a concentration of 5 mg/mL. Preliminary assessment of cellular responses to the cell-adherent star polymers were carried out using MC3T3.E1 preosteoblast cells. The


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cells were directly cultured on standard aseptic tissue plates along with the star polymers dissolved in the culture media. Viability of MC3T3.E1 cells was measured by live/dead staining at 24 h postmixing with the polymer stars. For live/dead staining, the cell culture media was aspirated, the wells were rinsed with PBS, and live/dead stain was added (calcein 1:2000 and ethidium homodimer 1:500 diluted in PBS). Cells were incubated for 30 min at 37 °C in the dark. Images were captured using a monochrome CCD camera attached to an Axiovert 200 microscope (Zeiss). From the images, the number of pseudocolored cells and gross percent viability were calculated. Evaluation of Cell Attachment. Star polymer hydrogels, nonadherent plastic plates, plates coated with star polymer, and plates coated with GRGDS-modified star polymer were used for cells adhesion assay. The MC3T3.E1 cells were seeded in each gel or well plate at a density of 50000 cells/mL. The cells were cultured in an incubator (37 °C, 5% CO2) for 24 h, fixed with 3.7% formaldehyde, and stained with rhodamine-phalloidin and Hoechst blue to visualize the actin cytoskeleton and nuclei, respectively. The double-stain allowed us to adequately distinguish the cell cytoskeleton (i.e., actin filaments) and nucleus. All experiments were replicated three times. Cell attachment, morphology, and spreading were examined using phase-contrast and fluorescent light microscopy (Zeiss Axiovert 200). Characterization of Cells on GRGDSHPC-(PEO)n-polyEGDMA Coated Surface. Scanning electron microscopy (SEM) was used to study cell attachment and morphology on GRGDSHPC-(PEO)n-polyEGDMA star polymers coated on a nonadherent surface. Following cell culture after 1 d of incubation, the cell seeded surface was washed with PBS 3 times and fixed with 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer (pH 7.4; Sigma-Aldrich, St. Louis, MO) at 4 °C overnight and osmicated using 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4). After osmication the samples were thoroughly washed in PBS and the samples were dehydrated through ascending grades of alcohol (50, 70, 80, 90, and 100%) followed by critical point drying. Samples were mounted on metal stubs and sputter coated with gold-palladium (Denton Vacuum Desc II). Surface topographies were examined by scanning electron microscopy (SEM) using a JEOL JSM 5400 scanning microscope. Three runs of experiments were carried out, which included all different samples in 3-fold.

Results and Discussion Synthesis and Characterization of (PEO)n-polyEGDMA and GRGDS-(PEO)n-polyEGDMA Star Polymers. In this study, we report the synthesis of (PEO)n-polyEGDMA star polymers via copolymerization of linear MM with a divinyl compound using an ATRP initiator. PEO MM (Mn ) 2000 g/mol) containing a methacrylate chain-end group and EGDMA were copolymerized by ATRP using EBiB as an initiator and CuCl/bpy as a catalyst.43 In a similar way, cell-adhesive GRGDS-(PEO)n-polyEGDMA star polymers were prepared by ATRP via copolymerization of PEO MM and GRGDS-PEOacrylate MM (Mn ) 3800 g/mol containing an acrylate chainend group), with EGDMA using EBiB as an initiator and CuCl/ bpy as a catalyst. The characterization of purified star polymers by 1H NMR spectroscopy confirmed the incorporation of the peptide into the star polymers. The proton peaks from pendant vinyl groups were identified, indicating that only one methacrylate group reacted in a small amount of EGDMA (Figure 2). In this arm-first method, the numbers of initiating sites and arms are independently controlled. The number of initiating sites in the star core was decreased by using a lower molar ratio of initiator to MMs, which limited the extent of star-star reactions and resulted in star polymers with relatively low polydispersities. Figure 3 shows the evolution of the GPC traces for the produced GRGDS-containing star polymers. As reaction time

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Figure 2. 1H NMR (300 MHz) spectrum of GRGDS-(PEO)n-polyEGDMA star polymers in D2O, confirming covalent incorporation of peptide into star shell architecture. Methacrylated protons (a,b) were identified showing incomplete conversion of vinyl groups from EGDMA cross-linker.

Figure 3. Evolution of GPC traces during synthesis of GRGDSHPC(PEO)n-polyEGDMA star polymers by linear macromonomer method. Experimental conditions: [PEO methyl ether methacrylate MM]0/ [GRGDS-PEO-acrylate MM]0/[EGDMA]0/[EBiB]0/[CuCl]0/[bpy]0 ) 10/ 0.5/12/1.67/2/4; [PEOMM]0 ) 0.16 M; in DMF at 60 °C. Linear PEO standards were used for calibration of the DMF GPC.

increased, the star yield increased and the apparent molecular weight of the star polymers (Mw,RI, based on linear polyMMA standards) shifted to the higher molecular weight region. The MM peaks in the GPC traces decreased but did not disappear, indicating fractional MM conversion. The polymerization was stopped after 24 h to target stars with relatively narrow MWD. The star yield was approximatively 77%, determined by the multipeak splitting of the GPC curve using Gaussian function. The unreacted MMs were removed by dialysis against water. After dialysis, the purified star polymers were lyophilized and characterized by GPC again. As shown in Figure 3, unreacted MMs were essentially removed, the apparent Mn for GRGDSHPC(PEO)n-polyEGDMA star polymers was 65000 g/mol, and the MWD was Mw/Mn ) 1.54. The apparent molecular weight, MWD, and average hydrodynamic diameter of (PEO)n-polyEGDMA and GRGDS-(PEO)n-polyEGDMA star polymers are summarized in Table 1. The incorporation of GRGDS-PEO-acrylate moiety, regardless of the concentration used, did not significantly alter the Mn of cell-adhesive star polymers relatively to (PEO)n-polyEGDMA stars. Star polymers with cell-adhesive GRGDS segment had diameters of approximatively 20.0 nm, similarly to the size of (PEO)n-polyEGDMA stars. The terminal GRGDS motifs serve as binding sites for the promotion of receptor-mediated cell adhesion. The high density

Figure 4. Vinyl conversion of PEODM/star polymer hybrid hydrogels monitored by NMR. 1H NMR of uncross-linked (a) and 1H DOSY 1D of photocross-linked (b) macromonomers (PEODM and (PEO)npolyEGDMA star polymers) in D2O at 10% mass fraction. Photopolymerization is induced directly in an NMR tube. Macromonomer solution (1 mL) containing the photoinitiator (I2959) was transferred into the NMR tube before curing with long wavelength UV source (365 nm, 300 µW/cm2) to obtain hydrogel. The vinylic peaks (between 5.5-6.5 ppm) for both PEODM (9) and (PEO)n-polyEGDMA star polymers (0) disappeared after 10 min irradiation.

of RGD groups at the periphery of the macromolecules enables them to bind efficiently to MC3T3.E1 cells. In the arm-first method, the length of each star polymer arm can be controlled by the molecular weight of the linear MM. The PEO MM used has a Mn of 2000 g/mol. The PEO chains, which repel adsorbing proteins, may sterically interfere with the binding of cell-surface receptors to the star polymer-bound ligands. For this reason GRGDS was coupled to a PEO linker with a higher Mn of 3800 g/mol to avoid steric congestion between cell adhesion motifs and PEO MM arms. This approach allows the formation of welldefined star polymers with distributed arms radiating the RGDbinding integrins from the core.34-36 GRGDS-(PEO)n-polyEGDMA Star Polymer Hydrogels. As discussed previously, characterization of star polymers by 1 H NMR showed a small fraction of unreacted vinyl groups from the cross-linker (Figure 2). The remaining vinyl-groups are not toxic toward the cells, but they can be still used for further reactions. Thus, GRGDS-(PEO)n-polyEGDMA star polymers could be used for covalent incorporation into threedimensional (3-D) hydrogels to provide mechanical support for cell-star polymer interactions. The gels were prepared by free radical photopolymerization using a photoinitiator (I2959). Covalent incorporation of star polymers into a scaffold was achieved by mixing 10 wt % PEODM (Mn ) 4000 g/mol) and

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Figure 5. SEM micrographs of uncoated (a) and coated (b) nonadherent surfaces. Arrows show roughness on coated surface with polymeric film. Scale bars are (a) 500 nm and (b) 1 µm.

Figure 6. In vitro viability assay of MC3T3.E1 cells mixed in suspension with GRGDSHPC-(PEO)n-polyEGDMA star polymers. Live (left, green) and dead (right, red) stains of cells exposed to the polymeric material (a,b) and control (c,d) after 24 h of incubation. Table 1. Molecular Weight (Mn), Molecular Weight Distribution (Mw/Mn), and Size Expressed as Davg ( SD (Average Diameter ( Standard Deviation) Results for (PEO)n-polyEGDMA and GRGDS-(PEO)n-polyEGDMA Star Polymers

(PEO)n-polyEGDMA GRGDSLPC-(PEO)n-polyEGDMA GRGDSHPC-(PEO)n-polyEGDMA

[GRGDS] (M)

MnGPC (g/mol)

Mw/MnGPC

sizeDLS (nm)

3.3 × 10-3 8.2 × 10-3

69.2 × 103 63.4 × 103 65.0 × 103

1.62 1.58 1.54

22.0 ( 0.1 19.4 ( 0.1 20.9 ( 0.1

1 wt % GRGDS-(PEO)n-polyEGDMA star polymers at both peptide content (low and high) in a deuterated phosphate buffered saline solution at pH 7.4. 1H NMR spectroscopy was used to characterize the conversion of vinyl groups from both PEODM and GRGDS-(PEO)n-polyEGDMA star polymers. A solution of 1 mL was transferred and sealed in an NMR tube before being exposed under a UV light for 10 min to form a cross-linked network. The conversion and star polymer incorporation into the hydrogel was confirmed by the disappearance of methacrylate groups after network formation. As shown in Figure 4, the disappearance of the vinyl protons (between 5.9-6.5 ppm) from PEODM and star-polymers indicated nearly full vinyl conversion after the photopolymerization process.

GRGDS-(PEO)n-polyEGDMA Star Polymer-Coated Thin Film. In this experiment, a nonadherent plastic dish that has a positive charge on the surface was used. The dip-coating method was applied to obtain a cell-adhesive polymer film. This technique was used to modify the biomaterial surface with bioactive ligands. Covalent immobilization of biologically active molecules onto polymer surfaces can often lead to a significant reduction in the activity of the immobilized molecules. To avoid this problem, a PEO spacer, known to have low nonspecific protein adsorption properties, was inserted between the star core surface and GRGDS, as discussed previously. Dip-coating was used as a simple and inexpensive method that does not require a sophisticated technique to modify the

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Figure 7. Cell adhesivity of GRGDS-(PEO)n-polyEGDMA star polymers covalently incorporated into PEODM-based hydrogel. 50000 cells/gel were cultured for 24 h and fixed with formaldehyde. Phase contrast picture of MC3T3.E1 cells cultured in a series of three types of hydrogels: (control, a) PEODM, (b) PEODM × GRGDSLPC-(PEO)n-polyEGDMA, and (c) PEODM × GRGDSHPC-(PEO)n-polyEGDMA.

Figure 8. Fluorescence micrographs showing cytoskeletal organization (actin staining, red) and labeled nuclei (Hoechst dye, blue) of MC3T3.E1 cells seeded to polystyrene cell culture well-plate, used as control (a), and to PEODM × (PEO)n-polyEGDMA star polymers hydrogel (b).

Figure 9. Phase contrast photomicrographs of MC3T3.E1 cells after 24 h of incubation on nonadherent (a, magnification 10×) and nonadherent GRGDSHPC-(PEO)n-polyEGDMA star polymers coated culture plates (b and c with a magnification of 10× and 20×, respectively).

interface properties. Despite the disadvantage of irregular film thickness, dip-coating can coat a substrate on a smooth surface. As shown in Figure 5, the outer surface of the nonadherent plate is smooth and uniform. However, films deposited by dip-coating exhibit coating with some surface roughness and nonuniform thickness, presumably due to nonuniform deposition of the polymer solution onto the surface of the plate. The stability of the coated polymer film on the nonadhesive surface results from the combination of gravity, capillary force, viscous force, and surface absorption. Biocompatibility of GRGDS-(PEO)n-polyEGDMA Star Polymers. GRGDS-containing star polymers were evaluated for in vitro cytotoxicity. Unreacted components, such as vinyl group moieties from the star polymers and remaining copper from the catalyst system, were investigated for their effects on cell viability using MC3T3.E1 cells. The materials were dissolved in media and directly incubated in suspension with MC3T3.E1 cells and were examined after 24 h incubation with star polymers using phase contrast and fluorescent microscopy. As shown in Figure 6, live/dead cell staining indicates that the star polymers were biocompatible and nontoxic with more than 95% viable cells. This outcome is comparable to that of the positive control tissue culture polystyrene surface. GRGDS-(PEO)n-polyEGDMA Star Polymers Promoting Cell Attachment. The targeted attachment of MC3T3.E1 cells on hybrid hydrogels through surface presentation of

GRGDS-specific ligands was studied. The strategy is based on the covalent attachment of GRGDS-(PEO)n-polyEGDMA star polymers via photopolymerization and cross-linking of the unreacted vinyl groups from the star polymers and PEODM. The result is a 3-D scaffold to support cells, thus enabling cellpolymer interactions and attachment. High cell viability was observed for all cross-linked hybrid GRGDS-(PEO)n-polyEGDMA star PEODM hydrogels with successful cell attachment within 24 h of incubation (Figure 7). Cell attachment was observed for hybrid hydrogels with higher peptide content, while no cell attachment was observed for PEODM hydrogels (control). Phalloidin TRITC staining was used to visualize actin organization and cell anchorage to the polymer surface, and cell nuclei were stained with Hoescht blue. Star polymers without GRGDS did not support cell adhesion after 24 h of culture and cells aggregated (Figure 8b). This observation suggests that grafting GRGDS peptides into PEO star polymers is critical for mediating adhesion of cells through their integrin receptors on the cell membrane.44 The peptides available on the star shells retained their bioactivity after the two-polymerization processes, ATRP followed by free radical photopolymerization initiated under a ultraviolet light. These results suggest that GRGDS was not deteriorated during the polymerization and the cell-adhesive star polymers can potentially be used as a drug delivery system.

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Figure 10. Fluorescence micrographs showing cytoskeletal organization (actin staining, red) and labeled nuclei (Hoechst dye, blue) of MC3T3.E1 cells seeded to surfaces with (a) and without (b) GRGDSHPC-(PEO)n-polyEGDMA star polymers coating.

Figure 11. SEM micrographs of MC3T3s grown on uncoated (a) and coated (b-d) nonadherent surfaces with GRGDSHPC-(PEO)n-polyEGDMA star polymers after 1 d of incubation. Note the abundant presence of extracellular matrix (d). Scale bars are (a,b) 20 µm and (c,d) 10 µm.

Cell attachment was also determined from thin film coatings prepared with GRGDS-containing star polymers on nonadherent plastic plates. The generation of round spheroid shaped cells is due to the aggregation of cells on the nonadherent culture plate (Figure 9). However, cell adhesion on the surfaces with star polymers containing GRGDS peptides was significantly higher than with nonmodified star polymers. In Figure 9, the phase contrast photomicrographs show that the surface coated with GRGDSHPC-(PEO)n-polyEGDMA star polymers had an effect on the attachment and morphology of MC3T3.E1 cells (Figures 9b,c) when compared to the control (Figure 9a). Furthermore, phase contrast microscopy images clearly show good cell attachment and homogeneous distribution

over the coated well plate, as a result of receptor-ligand interactions. These observations suggest that GRGDSHPC(PEO)n-polyEGDMA star polymers facilitate cell adhesion through integrin mediated receptors coupling with the GRGDS ligands available on the star shells. As a final measurement of the bioactivity and adhesivity of GRGDS fragments grafted on the star periphery, MC3T3.E1 attachment, spreading, and cytoskeleton organization on coated and uncoated nonadherent surfaces were examined after 24 h of incubation. Representative fluorescence images of surface attached cells with stained actin and nuclei are shown in Figures 10a,b. No organization of actin fibers was observed on the uncoated surface (Figure 10b) due to the lack of integrin-binding

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domains. In contrast, microfilament bundles with focal adhesions were seen in MC3T3.E1 cells attached to the coated surfaces with GRGDSHPC-(PEO)n-polyEGDMA star polymers (Figure 10a). In addition to the cell adhesive property of GRGDS(PEO)n-polyEGDMA star polymer hydrogels shown previously, these results confirm that these star polymers promote cell attachment of cells in 2- and 3-D environments. In addition, after incorporating RGD sequences, star polymers exhibited greater cell adhesion and spreading compared to samples not containing RGD. Further cell attachment to coated and noncoated GRGDS-containing PEO star polymer surfaces was validated by SEM. MC3T3.E1 cells seeded on the nonadherent surface demonstrated random orientation with a minimal cell attachment and spreading (Figure 11a). Moreover, the observed globular-shaped aggregations were attributed to nonspecific cellsubstrate interactions, whereas MC3T3.E1 cells seeded on the coated surface showed good cell attachment, retained their osteoblast-like morphology, and were confluent (Figures 11b-d). Purportedly, cells closest to the surface are stimulated to produce ECM proteins. This observation is an indication of cell response to the surface modifications, that is, ECM production by the adherent cells. Furthermore, an interconnecting network of capillary tubes on a layer of cells with identifiable lumens was observed by day 1 (Figure 11c,d). In addition, there was a prominent amount of fibrillar ECM that covered the surface of the coated GRGDSHPC-(PEO)n-polyEGDMA star polymers as well as the surface of migrating cells. The fibrillar ECM plays an important role in morphogenetic cell movement during early development and it was also deposited on the surface of migrating cells. The fibrils, which are components of the meshwork, and fibril surfaces had a granular appearance due to granules about 0.5-2 µm in diameter (Figure 11d). Polymeric coatings may improve the properties of materials for biomedical applications as well as enhance interfacial interactions between the medical device and the biological system. However, the adhesion of the polymeric coating to the substrate may be problematic as a consequence of inert surface chemistries with insufficient cell-substrate interactions. To mitigate against such problems, we have described a compelling new approach, which consists of depositing a cell adhesive GRGDSHPC-(PEO)n-polyEGDMA star polymers film onto an organic substrate.

Conclusion This study reports the synthesis of novel cross-linked celladhesive star polymers that contain signaling molecules to control and direct cell responses. The covalent incorporation of adhesion peptide sequences (such as RGD) on the periphery of PEO-star polymers was achieved by ATRP using GRGDSfunctionalized MM. 1H NMR spectroscopy and GPC confirmed the formation of (PEO)n-polyEGDMA star polymers with GRGDS peptides. Cytotoxicity assays revealed g95% cell viability after 24 h when MC3T3.E1 cells were exposed to star polymers at a concentration of 5 mg/mL. To assess the bioactivity of GRGDS, two different methods were used to provide mechanical support for cells based on the star polymers: covalent attachment into a 3-D PEO network by photocrosslinking, and depositing a thin film onto a nonadherent polystyrene culture plate by dip-coating. Both techniques validated that GRGDS-(PEO)n-polyEGDMA star polymers supported cell attachment within 24 h when incubated with MC3T3.E1, confirming their cell-adhesiveness capacity. Furthermore, SEM indicated that GRGDSHPC-(PEO)n-polyEGDMA star polymers promoted cell-substrate and cell-cell interactions with an interconnected network of capillary tubes. Deposition of ECM was observed in surface grooves as well as between cell layers.

Bencherif et al.

On the basis of the current study, we conclude that the incorporation of the RGD ligand-binding motif into (PEO)npolyEGDMA star polymers is required to enhance substrate-cell interactions in various ways, that is, by determining the cellular extensions, altering cell adhesions and spreading, and altering ECM production. Acknowledgment. Partial support was provided from NIDCR DE R01-15392-4 (J.O.H.), NSF DMR 05-43953, and U.S. Army DAMD 17-02-1-0717 (N.R.W.).

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