Article pubs.acs.org/cm
Functional PEG-Hydrogels Convey Gold Nanoparticles from Silicon and Aid Cell Adhesion onto the Nanocomposites Fang Ren, Cigdem Yesildag, Zhenfang Zhang,* and Marga C. Lensen* Technische Universität Berlin, Institut für Chemie, Nanostrukturierte Biomaterialien, Straße des 17. Juni 124, Sekr. TC 1, 10623 Berlin, Germany S Supporting Information *
ABSTRACT: A novel poly(ethylene glycol) (PEG)-based hydrogel has been synthesized and further functionalized by amine and thiol-ene Michael-type addition reactionsfirst with ammonia and then with dithiothreitol, respectivelyto yield hydrogels with thiol functions (8PEG-VS-SH), hence with high affinity for gold. Consequently, gold nanoparticles (Au NPs) can be firmly bound and immobilized on their surface. It is demonstrated that Au NPs with different sizes (20 and 42 nm, respectively) that decorate silicon surfaces in different densities can easily be transferred from silicon wafers to the surface of hydrogels, with a transfer efficiency of more than 98%. The novel Au NPs-PEG nanocomposite gels (Au NPs@Gel) have been investigated in a cell culture with murine fibroblasts L-929. Optical microscopy studies revealed remarkable cell adhesion on these nanocomposites. Apparently, the nonfunctionalized Au NPs on the intrinsically anti-adhesive PEG background do serve as anchoring points for cell adhesion. We propose that protein adsorption is enabled on these nanocomposite surfaces and this in turn results in the cell adhesion.
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proteins and cells usually needs to be avoided.19 PEG hydrogels, which possess an intrinsically inert and proteinrepellent surface, have demonstrated to be especially useful as a background platform for the in vitro investigation of cell behavior. In addition, the network properties, the swelling, and the elasticity of the gels can be controlled by tuning the length of the polymer chains, their architecture (e.g., linear, starshaped, or branched), and their functionalities.20 In our work, multivalent, 8-arm star-shaped PEG macromonomers (having a hexaglycerol core and a molecular weight of ∼15 000 Da; 8PEG) have been utilized for hydrogel preparation, offering some significant advantages over their linear counterparts, notably increased functionality (i.e., 8 instead of only 2 functional end-groups), and more variable physicochemical properties. Photoinitiated radical cross-linking and sulfur-based Michaeltype addition reactions are two typical methods for crosslinking PEG-macromonomers with functional CC double bonds to prepare hydrogels. Usually, all functional groups of the precursors are consumed through these methods.21,22 However, considering that any remaining functional CC double bonds could be further used for photoinitiated crosslinking and/or (bio)chemical functionalization, a new class of hydrogels formed by a modified amine Michael-type addition reaction between acrylate and amine functional groups has been developed in our lab.23 Nevertheless, due to the hydrolysis of
INTRODUCTION Functional nanostructured materials play an important role in many applications, such as biosensor development,1 catalysis,2 biomaterials design,3 and nanotechnology.4,5 Gold nanoparticles (Au NPs) have become a leading platform as the core structure of nanoconstructs by virtue of their chemical stability as well as facility to synthesize and functionalize their surface with biomolecules (e.g., proteins).6,7 However, there are issues of aggregation and nondispersibility of Au NPs in the desired solvent or film, which underlines the importance of the immobilization of Au NPs onto a support in a controlled manner.8 In the last two decades, many hard, inorganic supports, such as gold electrodes,9 glass,10 carbon,11 and silicon,12 have been employed for nanoparticle immobilization. Nevertheless, polymer-based supports are significantly more advantageous than inorganic supports in certain hindsights. Usually, polymeric substrates are more flexible, yet offering a wider range of rigidity, can be stretched dynamically, and are able to adopt different shapes.13,14 Moreover, the properties of polymer materials may be tailored to specific purposes, either by chemically modifying the (macro)monomers or by varying the polymerization and/or cross-linking conditions.15 Polymeric supports, especially those consisting of a highly hydrated material such as poly(ethylene glycol) (PEG)-based hydrogels, exhibit a number of advantageous properties such as being transparent, flexible, cytocompatible, and permeable to nutrients, solutes, and gases.16−18 In tissue engineering, biosensor systems, and other biomedical applications, the initial nonspecific adsorption of © 2017 American Chemical Society
Received: August 23, 2016 Revised: February 4, 2017 Published: February 6, 2017 2008
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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Chemistry of Materials
solution preparations. All glassware was cleaned with Aqua Regia (VHNO3:VHCl = 1:3). Instrumental. Raman spectra were measured using a confocal Raman spectrometer (LabRam HR 800, Jobin Yvon) coupled to a liquid-nitrogen-cooled CCD detector. The spectral resolution was 1 cm−1 with an increment per data point of 0.28 and 0.15 cm−1 using a 413 nm laser excitation line. The laser power on the sample was 1.0 mW. The laser beam was focused onto the sample by a Nikon 20× objective with a numeric aperture of 0.35. Accumulation times of the SERR spectra were between 30 s. For SEM observation, the hydrogels were carbon-coated prior to the measurements, which were performed using an Inlens detector operating at 15.0 kV. UV spectra were recorded on a Cary 4000 by Agilent Technologies spectrophotometer with a 1 cm optical path quartz cuvette. Surface topography was analyzed using an atomic force microscope (AFM) Nanowizard II (JPK instruments, Germany). For imaging samples in the swollen state, the samples were submerged in distilled water and measured with silicon nitride cantilevers (PNP TR, k ≈ 0.08 N/m, f0 ≈ 17 kHz; Nanoworld Innovative Technologies, Switzerland) with a chromiumgold coating. Images were edited with Nano Wizard IP Version 3.3a (JPK instruments, Germany). Optical images were obtained using a fluorescence microscope (Carl Zeiss, Göttingen, Germany). Fluorescence images were obtained by fluorescence microscope using an inverted microscope (Axio Observer.Z1, Carl Zeiss) equipped with a light emitting diode source (Colibri, Carl Zeiss). Pictures were analyzed using the Axio Vision V4.8.2 software package (Carl Zeiss, Göttingen, Germany). Synthesis of 8-Arm PEG-Vinyl Sulfone (8PEG-VS) Macromonomers. 8-arm PEG-vinyl sulfone (8PEG-VS) macromonomer was synthesized following the method developed by Lutolf et al.16 PEG-OH (ca. 5 g) was used as received and dissolved directly in 300 mL of anhydrous dichloromethane (previously dried over molecular sieves). To the PEG dissolved in dichloromethane, NaH was added under nitrogen, at a 5-fold molar excess over −OH groups. Directly after hydrogen evolution, divinyl sulfone was added at a 50- to 100fold molar excess over −OH groups. The reaction was carried out at room temperature for 3 days under a nitrogen atmosphere with constant stirring. Afterward, the reaction mixture was neutralized with concentrated acetic acid, filtered through paper until clear, and reduced to a small volume (ca. 10 mL) by rotary evaporation. PEG was precipitated by adding the remaining solution dropwise into icecold diethyl ether. The polymer was recovered by filtration, washed with diethyl ether, and dried under vacuum. The dried polymer was then dissolved in 200 mL of deionized water containing ca. 5 g of sodium chloride and extracted three times with 200 mL of dichloromethane. This solution was dried with sodium carbonate, and the volume was again reduced by rotary evaporation. Finally, the product was reprecipitated and thoroughly washed with diethyl ether to remove all remaining vinyl sulfone. The final product was dried under vacuum and stored under argon at −20 °C. Derivatization was confirmed with 1H NMR (CDCl3): 3.6 ppm (PEG backbone), 6.1 ppm (d, 1H, CH2), 6.4 ppm (d, 1H, CH2), and 6.8 ppm (q, 1H, −SO2CH). The degree of end group conversion, as shown by NMR, was found to be at least 95%. Synthesis of 20 and 42 nm Au NPs. The different-sized spherical Au NPs were synthesized following the protocol of Bastús et 33 al. First of all, Au NPs seeds (∼10 nm) were synthesized. In the next step, the seeds were continuously grown to bigger particles; hereby, spherical particles from 20 to 200 nm were obtained. For the seed synthesis, 150 mL of aqueous solution of sodium citrate (2.2 mM) was boiled for 15 min. Then, 1 mL of HAuCl4 solution (25 mM) was injected. The color of the solution changed from yellow to bluish-gray and then finally to soft pink within 10 min. For growing bigger-sized particles, the seed solution was cooled down to a temperature of 90 °C. Into that solution, 1 mL of sodium citrate (60 mM) and 1 mL of HAuCl4 solution (25 mM) were injected, and 30 min later, again HAuCl4 solution (25 mM) was added. After another 30 min, the solution was diluted via taking out 27.7 mL of the Au NPs solution (20 nm) and adding 27.6 mL water. By repeating this process (sequential addition of 1 mL of 60 mM sodium citrate and 1 mL of 25
ester groups, it was found that such hydrogels formed via crosslinking reaction between acrylate and amine groups may degrade within 1 day during incubation, making them not suitable for cell adhesion tests that typically span from a few hours up to several days. In the present work, in order to avoid the degradability of hydrogels that are cross-linked by acrylate and amine groups, and to provide stable substrates for short and medium term cell tests, instead of acrylate groups, vinyl sulfone groups have been utilized to form hydrogels, 8PEG-VS. The gels are partly crosslinked via the same amine Michael-type addition chemistry, yet the resulting gels lack the hydrolyzable ester groups at the cross-linking points. After gelation, the residual functional (vinyl) groups can be converted to thiol groups via a second thiol-ene Michael-type addition reaction, providing the resulting 8PEG-VS-SH gels with affinity for Au NPs. By a soft lithographic procedure, coined “nano-contact deprinting”, Au NPs with diameters of 20 and 42 nm are transferred from silicon to 8PEG-VS-SH hydrogels, displaying variable densities. To mimic the cellular environment of soft tissue analogues, a transfer technique was utilized to convey gold nanostructures from a solid substrate to PEG-based hydrogels, which is the platform for investigating in vitro cellular responses, such as cell adhesion.24 In vivo, cell adhesion is a crucial process for the assembly of individual cells into three-dimensional tissues, which are regulated by the extracellular matrix (ECM).24−26 Many other cell activities, such as migration, proliferation, and morphogenesis, are initiated by cell adhesion.27,28 The most commonly used method to control cell adhesion is to functionalize substrates with the tripeptide arginine-glycineaspartate (RGD) or the whole protein fibronectin.29−31 Hence, PEG-based hydrogels decorated with Au NPs assemblies functionalized with biomolecules are interesting mimics of natural ECM with hierarchically organized nanostructures for studying cell adhesion.32 In the present work, a series of Au NPs have been synthesized by the seeded growth method, and two generations of Au NPs, with a diameter of 20 and 42 nm, respectively, have been selected for further application. The Au NPs are immobilized in a controlled manner and in different densities on silicon wafers and subsequently transferred to the functional hydrogel. While SEM is employed to analyze the novel nanocomposite surfaces and determine the transfer efficiency of Au NPs, UV−vis spectroscopy is utilized to characterize the stability of Au NPs binding with the hydrogels. The novel nanocomposite biomaterials are cultured with cells, and the influence of different densities and diameters of Au NPs on fibroblasts adhesion and cytocompatibility has been investigated.
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EXPERIMENTAL SECTION
Materials. Silicon wafers (polished on one side) were obtained from Microchemicals. Isopropanol, acetone, (3-aminopropyl)triethoxysilane (APTES), ammonia (25%), hydrogen peroxide (H2O2 30%), concentrated sulfuric acid (H2SO4 98%), and toluene were purchased from Carl Roth. Acryloyl chloride, sodium hydride (NaH), 2-iminothiolane hydrochloride, vinyl sulfone, DL-dithiothreitol (DTT), and fluorescein diacetate (FDA) were purchased from SigmaAldrich. RPMI 1640, fetal bovine serum (FBS), 1% penicillin/ streptomycin, and trypsin-EDTA were purchased from PAA Laboratories GmbH. Propidium iodide (PI ≥ 94%) and phosphatebuffered saline (PBS) containing K2HPO4 and KH2PO4 were purchased from Fluka. Ultrapure deionized water was used for all 2009
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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Chemistry of Materials mM HAuCl4), up to 4 generations of Au NPs were progressively performed to obtain Au NPs with a diameter of 42 nm. Synthesis and Characterization of 8PEG-VS-SH Hydrogel. Different amounts of ammonium solution (30% NH3 in H2O) were added to the precursor solution of 8-arm poly(ethylene glycol) vinyl sulfone (8PEG-VS) with 50% water content at room temperature under vigorous magnetic stirring until the solution turned to a viscous liquid. Compositions were set in order to receive 20, 10, 5, and 2.5 wt % NH3-8PEG by weight. The resulting liquids were deposited on a glass slide and covered with a glass coverslip. After 30 min, the 8PEGVS hydrogels were formed. After gel formation, the colorless polymeric films formed with 5% NH3 were peeled off mechanically. The stand-alone films (250−300 μm in thickness) were handled with tweezers. These hydrogels were immersed in DTT solution (5 mg/ mL) for 60 min. Afterward, these hydrogels were washed thoroughly with water for several times and stored in water before use. Preparation of Triethoxysilane (APTES) Modified Silicon Wafer. After ultrasonication in a mixture of acetone and water (v/v = 1:1) for 20 min, the silicon wafers were immersed in Piranha solution (mixture of H2SO4 and H2O2 with v/v = 7:3) for 30 min. They were thoroughly washed with deionized water and isopropanol, and then dried under a stream of pure nitrogen gas. Afterward, the as-prepared silicon wafers were placed inside a small Teflon chamber filled with a solution of APTES (100 μL). APTES was then introduced into the sealed chamber as the pressure of the deposition chamber was raised. The surface reacted with the gas phase adsorbate for 2 h; then, the wafers were washed with anhydrous toluene (3 times) and isopropanol, and dried with nitrogen gas. Deposition of Au NPs onto Silicon Wafers. The concentration of Au NPs was tuned by diluting the original Au NPs dispersion with water (in the case of 20 nm particles by 2×, 4×, 8×, and 16× and in the case of 42 nm Au NPs by 2×, 4×, 8×, 16×, and 32×), respectively). A drop of 100 μL of homogeneously dispersed Au NPs was placed on the APTES modified silicon wafer. After incubation for 60 min, the silicon wafers were washed thoroughly with deionized water for 8 times in order to remove nonadsorbent and aggregated Au NPs and then dried with nitrogen gas. They were kept in a glovebox to avoid oxidization before use. Transfer of Au NPs in Different Densities from Silicon Wafers to Hydrogels. The as-prepared 8PEG-VS-SH hydrogels were firmly contacted with the silicon wafers for about 30 s before they were withdrawn from their surface. At last, they were washed gently for 3 times with deionized water in order to remove any nonadsorbent Au NPs. They were kept in water in a swollen state for cell culture, or dried at room temperature for 12 h prior to the SEM measurements. The Au NPs density on the sample is given by the number of Au NPs on 1 μm2 area of sample, which is counted from 3 randomly selected regions. Incubation of Hydrogels with Fibroblasts L-929. After spraying ethanol (70% v/v) on both sides of hydrogels, they were washed carefully with deionized water and dried in the sterile bench. Afterward, they were put into a μ−Slide 8-well plate, each with 300 μL of a cell suspension containing 30 000 cells/mL L-929 cells and incubated at 37 °C, 5% CO2 atmosphere, and 100% humidity. Following incubation for 24 h, the adhered cells were observed by microscopy. Viability staining of the cells was carried out according to protocol with a LIVE/DEAD staining kit; cells were washed with Dulbecco’s PBS and then stained with 100 μL of a vitality staining solution containing FDA (stock solution 0.5 mg/mL in acetone) and PI (stock solution 0.5 mg/mL in DPBS). Live and dead cells were detected by a fluorescence microscope (Carl Zeiss, Göttingen, Germany).
content and an ammonium hydroxide solution (5%, compared with 8PEG-VS) via amine Michael-type addition between vinyl sulfone and amine groups (Figure 1). In this reaction, NH3 acts
Figure 1. Schematic representation of novel hydrogel prepared through amine Michael-type addition chemistry.
as the cross-linker and, at the same time, serves as a catalyst because of its very basic nature. The advantage of using NH3 as cross-linker is the ease to remove its excess after the reaction, by evaporation. Moreover, due to the inefficient chemical reactivity of NH3 with vinyl sulfone in aqueous solution, a significant fraction of vinyl sulfone groups remain for further functionalization. In order to follow the progress of the reaction, Raman spectroscopy was utilized for monitoring the disappearance of the vinyl groups. The Raman spectra (Figure 2b) have been normalized according to the intensity of the peak at 1480 cm−1 that corresponds to the C-O vibrations, because the content of C-O groups in the hydrogels is constant. As can be observed, the intensity of the peak at 1612 cm−1, which is attributed to the CC vibrations of the unreacted vinyl sulfone groups, decreases with increasing amount of NH3. Consequently, the content of the unreacted vinyl sulfone groups was determined to be 59, 31, and 24% when 5, 10, and 20% of NH3 was used, respectively. When only 2.5% of NH3 is used, more than 70% of the unreacted vinyl sulfone groups are left after the formation of hydrogels, which is very useful for the next functionalization step, i.e., the conversion of vinyl sulfone groups into thiol functions. By virtue of the multifunctionality of the 8PEG macromonomers, these only partly cross-linked gels possess mechanical integrity; they can be conveniently prepared as stand-alone films and handled mechanically (with tweezers). After the addition of DTT, the absence of the peak at 1612 cm−1 shown in Figure 2c indicates complete consumption of vinyl sulfones due to the second Michael-type addition reaction, in this case between thiols and vinyl sulfones. This observed difference in conversion of the two Michael-type addition reactions further highlights that thiol groups are more reactive than amine groups, which is in accordance with the literature reporting the quantitative conversion of vinyl sulfones by reaction with thiols in a 1:1 ratio.21 The strategy of transferring Au NPs from silicon wafers onto 8PEG-VS-SH hydrogels is schematically depicted in Figure 3.
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RESULTS AND DISCUSSION In the present work, PEG hydrogels are used because of their renown bioinertness and biocompatibility.34 Novel hydrogels composed of star-shaped PEG molecules (having 8 arms with vinyl sulfone end groups; 8PEG-VS) are readily formed in situ by mixing of two aqueous solutions: 8PEG-VS with 50% water 2010
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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Figure 2. Schematic representation of the synthesis of 8PEG-VS-SH hydrogel (a). Raman spectra of residual vinyl sulfone groups of 8PEG-VS hydrogels with different amounts of NH3 addition (b) and Raman spectrum of 8PEG-VS-SH hydrogel (c).
Figure 4. SEM images of Au NPs transferred from the surface of silicon wafers to the surface of hydrogels. (a) 20 nm Au NPs, (b) 42 nm Au NPs.
Figure 3. General scheme of transferring Au NPs from APTES modified silicon wafer to the surface of 8PEG-VS-SH hydrogels.
than they used to be in the hydrated state during the actual transfer process. Further quantification of the SEM results (Figure 4b) revealed that hardly any remaining Au NPs can be observed on the silicon wafer, indicating that the vast majority of Au NPs, up to 98%, are transferred from silicon wafer to hydrogel by the nanocontact deprinting method. In comparison, the transfer efficiency of Au NPs with a diameter of 20 nm from silicon to hydrogel is about 83% (Figure 4a). Since the only difference between those two samples is the size of the particles (20 nm in Figure 4a and 42 nm in Figure 4b, respectively), this implies that the larger particles are more effectively transferred than the smaller ones. One possible reason for this is that the smaller Au NPs with their smaller surface area benefit less of the chemical interaction (i.e., Au−S) that is responsible for the transfer. In order to verify if a nonfunctional gel (8PEG-VS hydrogel without SH groups) can also transfer Au NPs from silicon to the hydrogel surface, a nonthiolated gel film was contacted with a Au NPs-decorated silicon wafer and peeled
First, Au NPs are immobilized on a silicon wafer via electrostatic interactions between positively charged amino groups on the silicon wafer and negatively charged citratestabilized Au NPs (Figure 3a). The amino groups have been introduced onto silicon wafer through silanization of APTES via chemical vapor deposition (CVD), which is the most potentially reproducible method for producing high density, homogeneously functionalized silane monolayers on silicon surfaces. In order to investigate the transfer efficiency of our novel deprinting method, SEM was utilized to characterize and count Au NPs on the silicon before and after transfer, as shown in Figure 4. It should be noted that the density of Au NPs on the hydrogel appears to be 3 times higher than that on the silicon wafer (i.e., 2.4·103 versus 0.77·103 Au NPs/μm2). This is because the hydrogel nanocomposites were dried for the SEM measurement, which made the gels shrink and become smaller 2011
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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Chemistry of Materials
Figure 5. Representative SEM images of Au NPs immobilized onto APTES modified silicon wafers and thiolated hydrogels (same scale in all images). 1/2, 1/4, 1/16, and 1/32 denote the dilution factor of the original dispersion of Au NPs.
surface. In comparison, the characteristic peak is absent in the spectrum of glass with Au NPs after ultrasonication for 5 min, which means that the Au NPs are detached from the glass. The results underline that the attachment of Au NPs to the PEGVS-SH hydrogel is strong and stable. This substrate for immobilizing Au NPs is thus very useful in our further experiments when incubated with L-929 cells in aqueous solution. It has been documented that the surface of gold may be functionalized with cell adhesion-mediating proteins and then facilitate the adhesion of cells.35,36 In our present study, however, the Au NPs are “naked”, without any specific (bio)functionalization. Taking this into account, and based on the quality of PEG-hydrogels as being anti-adhesive, cell adhesion could hardly be expected on these novel nanocomposite hydrogels. Nevertheless, the as-synthesized Au NPs@Gel samples with a series of nanoparticle densities were investigated in cell culture with mouse fibroblasts (L-929) cells. Optical microscopy was used to monitor any cell adhesion, and cytotoxicity tests were carried out to verify the cytocompatibility (vide inf ra). Figure 6 depicts the representative bright-field micrographs of L-929 cells after incubation with Au NPs@Gels for 24 h. Much to our surprise, we observed significant cell adhesion on the supposedly nonadhesive biomaterial. Visual inspection of the micrographs in Figure 6a already revealed that, in some cases (notably the sample with the highest density of the small Au NPs; denoted “1/2 20 nm”), the number of adhered cells on Au NPs composite hydrogels was even significantly larger than on the “golden standard” TCPS. Further quantification of the number of cells (Figure 6b) demonstrates a clear trend; i.e., the number of cells increases with the increasing density (corresponding to the lower dilution factor) of Au NPs. This clear and positive correlation strongly implies that the Au NPs are in fact adhesive or at least attractive to the fibroblasts. It should be noted that the number of particles in the stock solutions of the 20 nm- and 42 nm-sized Au NPs are not the same; thus the dilution factors alone are not enough to compare the surface density of the particles. On top of that, due to their different sizes, the Au NPs exhibit different surface areas of gold per particle. In Table SI 1, all of these parameters are listed for a more meaningful comparison. The calculations reveal that, although the density of 42 nm Au NPs is much
off. Figure SI 2 in the Supporting Information shows that some Au NPs are indeed transferred by the soft gel alone, but that the transfer efficiency is obviously greater when the gel is thiolated. Due to the high transfer efficiency and strong Au−S bonds, the density of the Au NPs (first on silicon and consequently on the hydrogel) can be controlled. In order to prepare a series of samples with different densities, the original Au NPs suspension was diluted several times, resulting in dispersions with 1/2, 1/4, 1/8, 1/16, and 1/32 times the original concentration. Immobilization of the series of dispersions on APTES modified silicon wafers then leads to Au NPs with different densities adsorbed on silicon (Au NPs@Si) as shown in Figure 5. After transfer, the densities of Au NPs on the surface of 8PEG-VS-SH hydrogels (Au NPs@Gel) are changed accordingly. The quantification of Au NPs with different densities (#/μm2) on silicon and gels can be found in Table SI 1. In order to distinguish the difference between various densities of Au NPs on the surface of 8PEG-VS-SH hydrogels optically, UV−vis spectroscopy was used to characterize hydrogels with different densities of Au NPs (Figure SI 2a). As the density of Au NPs deposited on the hydrogels decreases, the intensity of the peak for Au NPs correspondingly decreases as well. In addition, the SPR peaks of Au NPs on hydrogels are blue-shifted, due to the enlarged interparticle distance, leading to diminished plasmon coupling between the neighboring Au NPs. After Au NPs have been transferred onto hydrogels, UV−vis spectroscopy was also utilized to characterize the stability of Au NPs binding with the hydrogels (Figure SI 2b). Absorption spectra were recorded before and after treatment of the nanocomposite films with ultrasound. For comparison, we wanted to measure the stability of Au NPs on hard substrates as well. However, since silicon wafers are nontransparent, but glass is, we investigated Au NPs modified on glass, prepared by the same procedure as described for silicon wafers, for the UV−vis absorption test. In Figure SI 2b, solid lines represent the immobilized Au NPs on hydrogel (red line) and glass (blue line), and dotted lines stand for the corresponding samples after sonication treatment for 5 min. After the hydrogels had been ultrasonicated for 5 min, the same characteristic peaks at ∼535 nm can still be observed in the UV−vis spectrum, indicating that the Au NPs are still bound on the hydrogel 2012
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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hydrophilic, and that is the main reason for its excellent antiadhesiveness to cells, besides the high water content. The present 8PEG-VS-SH hydrogel is not as hydrophilic (although, because of the water uptake, it is practically impossible to determine reliable WCA values on the dry surface of these gels), which may help to explain the observation that cells adhere to it. Nevertheless, Figure 6 shows that the pure gel itself is not adhesive. Thus, the argument of wettability is not sufficient to explain the result. The Au NPs are coated with citrate molecules, which represent negative charges that can lead to electrostatic interactions with proteins and cells. From literature reports on self-assembled monolayers (SAMs) on gold, it is known that cell-adhesive proteins may displace small surface-bound molecules and thereby induce cell adhesion.42 In order to verify if indeed cell-adhesive proteins (from the serum in the cell culture medium) adsorb in significant amounts onto the gold structures, we have investigated the “adsorption” of fluorescently labeled serum proteins, the abundant bovine serum albumin (BSA) and the cell adhesive fibronectin (Fn). We observed that both proteins diffused into the gels and the whole gel exhibited fluorescence. Thus, protein adsorption located at the interface could not be verified. . In addition, the remaining −SH groups on the hydrogel surface might interact with (exposed cysteine residues on) proteins in the cell culture medium; even chemical fixation cannot be excluded. However, as stated above, the pure 8PEG-VS-SH hydrogel does not support cell adhesion itself. The second important property to discuss is the effect of surface topography. We and others have recognized the importance of topographic structures or mere roughness in mediating cell adhesion.43−45 For example, we discovered that pure PEG-based hydrogels, which are intrinsically anti-adhesive to cells, lose their inertness when topographic (or elastic) patterns are present.43 Teixeira et al. reviewed cellular adhesion and “contact guidance” on nano- and microtopographies and concluded that cells may sense topographic structures with feature sizes down to 70 nm.34 In our case, the nanoparticles are even smaller, and isolated Au NPs might not be recognized by the cells. The actual surface topography and roughness were determined by atomic force microscopy (AFM) under water (Figure SI 3). AFM analysis revealed that the surface of the 8PEG-VS-SH hydrogels is quite smooth under water (i.e., 3−4 nm, Figure SI 3a), while the presence of Au NPs at the interface increases the rms roughness, as expected (to 7 nm for 20 nm Au NPs and 13 nm for 42 nm Au NPs, Figures SI 3b and 2c, respectively). It should be noted that the swollen, soft hydrogels are very difficult to image. The pure gels are featureless, and it is a great challenge to resolve the nanofeatures at the interface of the gels with Au NPs. The third and final aspect that we consider in this discussion is the substrate’s elasticity. Since a few decades only, it has been realized that cells sense the stiffness of the substrate and respond to it.46,47 This so-called “mechanosensing” and the following responses, e.g., preferential cell adhesion on and cell migration toward stiffer regions, which is denoted “durotaxis”, are intriguing processes, and there are many biophysical questions unanswered so far.47−49 In line with the tunable stiffness of our recent, other hydrogel system that was crosslinked by ammonia,23 the stiffness of the present 8PEG-VS-SH gels also increases with the concentration of ammonia, as expected. Table SI 2 and Figure SI 6 depict the results of the AFM measurements to analyze the Young’s modulus of the
Figure 6. (a) Optical images of L-929 cells and (b) number of L-929 cells on the surface of TCPS, pure hydrogel, and Au NPs@Gels.
lower than that of 20 nm Au NPs at the same dilution factor (e.g., 1/8), the corresponding gold surface areas are in fact very similar. Considering that, at the same dilution factor (hence similar Au surface area), more cells adhere on the gels with 20 nm Au NPs (Figure 6b), this implies that the smaller ones are better able to induce cell adhesion. It could also indicate that the exposed gold surface area is actually smaller than calculated, for example because the Au NPs are slightly embedded in the gel. In any case, when comparing now the number of cells on surfaces at the same density of Au NPs instead of at the similar dilution factor, it becomes evident that cell adhesion is larger on the 42 nm-sized NPs, because the gold surface area is larger. As far as the morphology of the cells is concerned, the cells are spindle-shaped when cultured on Au NPs@Gel with a high density of Au NPs, while they are round-shaped when cultured on Au NPs@Gel with a low density of Au NPs. This further indicates that the nonfunctionalized nanoparticles not only induce cell adhesion but also aid cell spreading, when the density of Au NPs is sufficiently high. In order to understand such remarkable cell adhesion on these composite hydrogels (Au NPs@Gel), we must consider a number of possible explanations. The following factors are discussed: surface chemistry and reactivity, topography and elasticity. Some parameters depend more on the Au NPs and others more on the 8PEG-VS-SH hydrogel. First of all, it should be noted that we are very aware that cell adhesion, spreading, and migration heavily depend on the chemistry, topography, and elasticity of the biointerface, as we have studied these relationships in great detail during the last several years.37−40 For example, on the aspect of surface chemistry, we have observed that there is an optimal, intermediate wettability (water contact angle, WCA roughly between ∼55° and 85°) that supports the adhesion of L-929 fibroblasts, and we compared our results with literature reports.41 Pure PEG is very 2013
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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8PEG-VS-SH hydrogel interface with and without Au NPs under water. Interestingly, the presence of Au NPs seems to increase the stiffness as well. Macroscopic measurements, e.g., rheology, however, should be employed to verify this trend. Nevertheless, the novel nanocomposite gels are in fact orders of magnitude softer than TCPS (Table SI 2), so this argument completely fails to explain the observed enhanced cell adhesion on the Au NPs@Gel substrates when compared with TCPS. A final property that is related to the hydrogel’s elasticity is the porosity. The surface of the hydrogel is not compact, like a solid surface, but the interface is defined by rather long, loose polymer chains that are protruding into the medium. Proteins that are diffusing through this “forest” experience less of the hydrodynamic effects that would wash them away. The resulting, prolonged residence time of weakly bound proteins may be sufficient in aiding the establishment of focal contacts and adhesion complexes. In fact, the soft nature and the diffusability of the hydrogels play a very significant role in aiding cell adhesion. We have performed cell adhesion studies on glass surfaces that were coated with APTES and decorated with Au NPs. Cell adhesion on the substrates with Au NPs was slightly, but not significantly, larger than on the substrates without Au NPs, and still lower than on TCPS (Figure SI 5). Taking everything into consideration, it is clear that none of the individual arguments convincingly explain the observed cell adhesion. Yet, these factors in combination might result in an environment, where proteins from the medium can accumulate and accommodate themselves on or between the nanoparticles, slightly embedded in the porous, soft surface of PEG chains. While the cell adhesion studies already indicated that fibroblasts are viable in cell culture with the novel nanocomposite biomaterials, the Live/Dead staining assay was used to verify the cytocompatibility of Au NPs@Gel, using fluorescein diacetate (FDA) and propidium iodide (PI) to stain viable (green) and dead (red) cells, respectively. Fluorescence micrographs (Figure SI 4) reveal that red, dead cells can hardly be observed on any of the hydrogels, demonstrating that no cytotoxicity to L-929 cells originates from 8PEG-VS-SH hydrogels with either high or low density Au NPs on the surface.
Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b03548. Optical images of the gels, quantification data, UV−vis spectra, atomic force microscopy (AFM), Young’s modulus, and optical and fluorescence microscopy images of L-929 cells (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (M.C.L.). *E-mail:
[email protected] (Z.Z.). ORCID
Marga C. Lensen: 0000-0002-5448-6291 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS F.R. acknowledges financial support from China Scholarship Council in the form of a CSC scholarship. REFERENCES
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CONCLUSIONS In this study, novel 8PEG-VS-SH hydrogels have been synthesized and further functionalized by amine Michael-type addition reactionsfirst with ammonia and then with DTT to yield hydrogels with high affinity for gold. Furthermore, Au NPs with different densities have been transferred from silicon wafers to the surface of hydrogels with a transfer efficiency of up to 98% through the strong Au−S bond between Au NPs and hydrogels. The stability of the Au NPs on the hydrogels has been proved by UV−vis spectroscopy after sonication treatment for 5 min; the Au NPs still remain on the surface of hydrogels. More importantly, Au NPs@Gels offer a substrate to control the cell adhesion and spreading of fibroblast L-929 cells. The results implied that Au NPs played an important role in cell adhesion, with a clear, positive correlation between the density of the Au NPs on hydrogels and the number of adherent cells. Therefore, these novel Au NPs-PEG nanocomposite materials represent an attractive and versatile biomaterial with great potential for tissue engineering and biomedical applications. 2014
DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
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DOI: 10.1021/acs.chemmater.6b03548 Chem. Mater. 2017, 29, 2008−2015
