Multifunctional Polyampholyte Hydrogels with Fouling Resistance and

Aug 15, 2013 - to reduce the undesirable, but natural foreign body response. The focus of this investigation is to demonstrate that polyampholyte hydr...
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Multifunctional Polyampholyte Hydrogels with Fouling Resistance and Protein Conjugation Capacity Megan E. Schroeder,† Kevin M. Zurick,‡ Daniel E. McGrath,† and Matthew T. Bernards*,‡,† ‡

Departments of Chemical Engineering and †Biological Engineering, Columbia, Missouri 65211, United States S Supporting Information *

ABSTRACT: Materials that are resistant to nonspecific protein adsorption are critical in the biomedical community. Specifically, nonfouling implantable biomaterials are necessary to reduce the undesirable, but natural foreign body response. The focus of this investigation is to demonstrate that polyampholyte hydrogels prepared with equimolar quantities of positively charged [2-(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged 2-carboxyethyl acrylate (CAA) monomers are a viable solution to this problem. TMA/CAA hydrogels were prepared and their physical and chemical properties were characterized. The fouling resistance of the TMA/CAA hydrogels were assessed at varying cross-linker densities using enzyme-linked immunosorbant assays (ELISAs). The results clearly demonstrate that TMA/CAA hydrogels are resistant to nonspecific protein adsorption. A unique advantage of the fouling resistant TMA/CAA system is that bioactive proteins can be covalently attached to these materials using standard conjugation chemistry. This was demonstrated in this study through a combination of ELISA investigations and short-term cell adhesion assays. The multifunctional properties of the TMA/CAA polyampholyte hydrogels shown in this work clearly demonstrate the potential for these materials for use as tissue regeneration scaffolds for many biomedical applications.

1. INTRODUCTION

Zwitterionic functional groups including sulfobetaine (SB), carboxybetaine (CB), and phosphorylcholine (PC) have recently been proposed as alternative biocompatible, nonfouling chemistries.13−21 For example, PC-based hydrogels reduced unwanted cell interactions and the formation of a fibrous capsule around hydrogel-coated stainless steel implants as compared to high-density polyethylene control implants.22 Similarly, Yang and colleagues found similar performance improvements with CB-based hydrogels placed around glucose biosensors.23 However, as with PEG-based hydrogels, the nonfouling properties of most zwitterionic hydrogels are impacted when the chemistry is modified to conjugate bioactive signaling molecules. The one exception to this is CB-based materials, which have been shown to have a dual-functional capability.3,24 Polyampholyte hydrogels, which are comprised of a mixture of positively and negatively charged monomer subunits, have recently been proposed as nonfouling materials that mimic zwitterionic polymer systems.25 When the charged groups are homogeneously arranged, it produces an ideal environment for completely ionized charge groups through the electrical field influence of the surrounding ions. This minimizes the effects of any acid−base interactions with the material and is optimal for

A major challenge in the biomedical field is the development of materials that are resistant to nonspecific protein adsorption. It is ideal that an implantable biomaterial prevents nonspecific protein adsorption to the surface of the device upon contact with blood, as this is the first step in the foreign body inflammatory response that ultimately leads to encapsulation of the material, chronic inflammation, and the failure of the device.1 Nonfouling hydrogels provide a potential solution to this issue due to their ability to mimic natural tissue characteristics, their biocompatibility, and their versatility for biomedical applications.2 For instance, hydrogels have been used in various applications such as biosensors,3 drug delivery,4 and cell scaffolds for tissue regeneration,5,6 among others. Of the many synthetic materials that have been studied, poly(ethylene glycol) (PEG)-based polymers are the most widely investigated due to their ultralow fouling characteristics and their inert nature within the body.4,6 PEG hydrogels have been shown to be hydrolytically degradable7,8 and adept for drug release as a tunable “smart” material.8,9 However, PEG is vulnerable to oxidation damage, which limits its practical applications for long-term implants,10 and PEG-based hydrogels have a limited capacity for incorporating biologically active signaling molecules without impacting the underlying functional groups that are essential for the nonfouling properties.11,12 © 2013 American Chemical Society

Received: May 21, 2013 Revised: August 9, 2013 Published: August 15, 2013 3112

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forming a strongly hydrated surface layer.26 Our previous biosensor work has shown that these materials have excellent nonfouling characteristics only when the oppositely charged monomer subunits are present as a statistical copolymer mixture at the molecular level.27 Others have also completed surface-based investigations, confirming that polyampholyte polymer systems have nonfouling properties.28−31 As shown through biosensor-based investigations, nonfouling polyampholyte polymers can be synthesized with varying combinations of charged monomer subunits.27,32 The advantage to this approach is the fact that the materials can be designed to include pH-sensitive components.32 Furthermore, when carboxylic acid terminated monomers are included as the negatively charged monomer, the polyampholyte polymer systems have been demonstrated to have protein conjugation properties that mimic that of zwitterionic CB-based materials in biosensor investigations.33 Given our recent work demonstrating that polyampholyte hydrogels have nonfouling properties that mimic those of other known nonfouling materials,34 it is hypothesized that polyampholyte hydrogels can be formed with the ability to covalently attach proteins, within a fouling resistant background material, which is a multifunctional capability that is highly desirable for tissue engineering applications. To test this hypothesis and to demonstrate the viability of polyampholyte hydrogels as a novel tissue engineering scaffold, hydrogels were prepared from equimolar quantities of positively charged [2-(acryloyloxy)ethyl] trimethylammonium (TMA) and negatively charged 2-carboxyethyl acrylate (CAA) monomers with varying concentrations of a triethylene glycol dimethacrylate (TEGDMA) cross-linker. The molecular structures for all three of these monomers can be seen in Scheme 1. The physical and chemical properties of the TMA/

2. MATERIALS AND METHODS 2.1. Materials. Phosphate-buffered saline (PBS, 150 mM, pH 7.4), TMA, CAA, TEGDMA, N-hydroxysuccinimide (NHS), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), o-phenylenediamine (OPD), phosphate-citrate buffer with urea and hydrogen peroxide (0.014%), bovine serum albumin (BSA; >96% pure), and LYZ from chicken egg white (≥90%) were purchased from Sigma-Aldrich (St. Louis, MO). Ethylene glycol, sodium chloride, paraformaldehyde, and sodium metabisulfite (SMS) were purchased from Fisher Scientific Inc. (Pittsburgh, PA). Horseradish peroxidase (HRP)-conjugated polyclonal anti-LYZ (chicken egg white) and HRPconjugated polyclonal anti-FBG (human plasma) were purchased from United States Biological (Swampscott, MA). N-(3-Sulfopropyl)-Nmethacryloxyethyl-N,N-dimethylammonium betaine (SBMA) was purchased from Monomer-Polymer and Dajac Laboratories (Trevose, PA). Ammonium persulfate (APS), sodium hydroxide, and hematoxylin were purchased from Arcos Organics (Pittsburgh, PA). FBG from human plasma (100%) was purchased from CalBiochem (San Diego, CA). Tris(hydroxymethyl)aminomethane (Tris−HCl) and NaCl were purchased from Thermo-Fisher Scientific (Waltham, MA). All other cell culture supplies (including α-minimum essential medium (αMEM), fetal bovine serum, penicillin−streptomycin, soybean trypsin inhibitor, and trypsin−ethylenediaminetetraacetic acid (trypsin− EDTA, 0.05%, 0.53 mM)) were purchased from Invitrogen (Carlsbad, CA). Ethanol was purchased from Decon Laboratories, Inc. (King of Prussia, PA). Drierite was purchased from W.A. Hammond Drierite Company (Xenia, OH). MC3T3-E1 osteoblast-like cells (subclone 14, ATCC# CRL-2594) were obtained from ATCC (Manassas, VA). Ultrapure water (18.2 MΩ·cm) was used for all experiments and it was taken from a Millipore Synergy UV water purification system (Billerica, MA). 2.2. Hydrogel Synthesis. The TMA/CAA hydrogels were synthesized in a 1:1 molar ratio of each monomer using procedures adapted from previous work.25,34 Briefly, 1.0 mmol of both the TMA and the CAA monomers were mixed and dissolved in 500 μL of solvent consisting of a 1.5:1:1.5 volume ratio of the following materials: ethylene glycol, ethanol, and 3 M NaOH. The cross-linker TEGDMA was then added to the solution in one of the following amounts: 0.076 mmol (1×), 0.152 mmol (2×), or 0.304 mmol (4×). This resulted in total monomer to cross-linker molar ratios of 26.3:1 (1×), 13.2:1 (2×), and 6.6:1 (4×). The polymerization was initiated with the addition of 8 μL of a 40% (w/w) APS solution in ultrapure water, followed by the addition of 15% (w/w) SMS in ultrapure water. The polymerization solution was well mixed and then inserted into a mold consisting of two microscope slides clamped around a 0.79 mm polytetrafluoroethylene spacer. The reaction was allowed to proceed at 60 °C for 1 h, after which the apparatus was removed from the oven and allowed to cool for 3 h to reach room temperature before being used in subsequent studies. The above procedures were also conducted by replacing the 3 M NaOH in the original solvent with an equal volume of DI water. Hydrogels comprised solely of each monomer were produced with the same method, using 2 mmol of each individual monomer in the procedure. The nonfouling control hydrogels comprised of SBMA were created with a similar process, consisting of a reaction mixture of 2 mmol SBMA and 0.076 mmol of TEGDMA in 500 μL of a 1:1 solvent solution of ethylene glycol and DI water. All other steps were identical to those used for the TMA/ CAA hydrogels. 2.3. Characterizations of the Physical and Chemical Properties. 2.3.1. Swelling. TMA/CAA hydrogels at all three cross-linker densities were prepared as described above and then they were removed from the hydrogel mold and cut into 1 cm2 sections using a scalpel. Samples were then placed in a solution of either PBS buffer or DI water. The hydrogel samples were removed from the solution and measured every 24 h and then they were placed in a fresh solution. The samples were monitored for a total of six days. The average percent swelling was determined by comparing the measured lateral surface area at each time point to the original dimensions and the

Scheme 1. Chemical Structures of the Monomers and CrossLinker Used in This Investigation

CAA hydrogels were assessed as a function of the TEGDMA cross-linker density and the fouling resistance was also characterized for both negatively charged fibrinogen (FBG) and positively charged lysozyme (LYZ). Then the potential for TMA/CAA hydrogels to be used as tissue engineering scaffolds was investigated through a combination of protein conjugation studies as well as cell adhesion assays. Our results clearly demonstrate the dual-functional properties and biocompatibility of TMA/CAA hydrogels, suggesting that they are a promising novel material for biomedical applications. 3113

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results are expressed as percent swelling per day from three independently prepared samples for each cross-linker density (n = 3). 2.3.2. Weight Percentage Hydration. TMA/CAA hydrogels at all three cross-linker densities were prepared as described above and then they were removed from the hydrogel mold and cut into 0.5 cm wide × 2 cm long strips. These samples were immersed in ultrapure water for a minimum of 24 h. After immersion, the gel samples were removed, quickly patted dry, and weighed. Then the samples were placed into a desiccator containing Drierite and they were put under slight vacuum. The sample weights were monitored daily until they reached equilibrium and then the gels were removed and the final weight was determined. The change in sample weights from before and after desiccation was used to calculate the average mass percent of water present in each fully hydrated hydrogel sample. These experiments were conducted for three independently prepared gels at each cross-linker density (n = 3). 2.3.3. Compressive Strength. TMA/CAA hydrogels with crosslinker densities of 1×, 2×, and 4× were synthesized using the procedures described above, except that the synthesis was performed in 15 mL centrifuge tubes. To compensate, all of the volumes used in the synthesis were increased by a factor of 10 to produce a large hydrogel sample volume for mechanical testing. After cooling and removal from the 15 mL centrifuge tubes, the gels were soaked in PBS buffer overnight. The following day, the gels were cut into cylinders with a diameter of 1.8 cm and a height of 1.0 cm and used immediately for compression testing. Mechanical testing was performed on a TAHDi Texture Analyzer (Texture Technologies Corp, Scarsdale, NY). A 5 kg load cell was used for testing the 1× and 2× cross-linker density samples, and a 50 kg load cell was used for the 4× cross-linker density samples. A compression rate of 2 mm/sec was used for all of the hydrogels, and the samples were compressed until fracture. The fracture stress and strain were determined as the maximum values for each before fracture, and the Young’s modulus was determined as the slope of a linear fit to the stress−strain curve over the 0−0.1 strain ratios. Three independently prepared samples were analyzed for each cross-linker density (n = 3). 2.3.4. Surface Zeta Potential Measurements. TMA/CAA hydrogel samples were prepared as described above and then they were soaked for 2 days in DI water. The samples were then cut to fit into a flat sample zeta potential measurement cell for use with a DelsaNano HC instrument (Beckman Coulter, Brea, CA). The flow cell was thoroughly flushed with DI water, and then a 10 mM NaCl solution, pH 7.4, containing a standard particle for flat surfaces tracer solution (Otsuka Electronics Co., Japan) was injected into the flow cell. This solution contained a ratio of nanoparticles to buffer of 1:250. The surface was allowed to stabilize for 15 min and then the zeta potential was measured at 10 separate locations on each sample. This measurement was repeated three times. The average zeta potential for the sample was directly calculated and reported by the instrument. 2.3.5. X-ray Photoelectron Spectroscopy (XPS). Hydrogel samples were prepared as described above. However, the samples were swelled to their equilibrium size in DI water rather than PBS to reduce the presence of ions in the samples. Following two days of soaking, the samples were placed in a desiccator for 5 days to fully dehydrate the samples. Then the samples were loaded into a Kratos Axis 165 photoelectron spectrometer (XPS, Kratos Analytical, Inc., Chestnut Ridge, NY). XPS spectra were collected using a monochromatized Al Kα source with a source energy of 1486.6 eV, a spot size of ∼700 μm, and a takeoff angle of 60°. CasaXPS software was used to determine the peak areas, which were then used to determine the elemental compositions of the samples. The peak areas were normalized by the number of scans, points/electronvolt, Scofield photoionization cross section, and sampling depth for this calculation. Three samples were analyzed for each cross-linker density (n = 3). 2.4. Enzyme-Linked Immunosorbant Assay (ELISA) Studies. To test for nonspecific protein adsorption, TMA/CAA and control SBMA hydrogels were prepared for ELISA studies. The gels were prepared as described above and then the samples were soaked for 24 h in PBS buffer to swell them to their equilibrium size and to remove any unreacted initiators or monomers. The hydrogels were then cut

into 5 mm disks using a biopsy punch and the disk samples were placed in fresh PBS buffer to soak for an additional 24 h. TMA/CAA and SBMA samples were incubated in either 1 mg/mL of FBG or 1 mg/mL LYZ in PBS buffer for 1.5 h. As a positive fouling control, wells in a 24-well tissue culture polystyrene (TCPS) well plate were also incubated with FBG or LYZ for 1.5 h. Following the protein adsorption step, each of the incubated wells and hydrogel disks were rinsed 5 times with PBS buffer to eliminate any unbound protein. Each of the substrates were then incubated in a corresponding antibody solution for 1.5 h: the samples exposed to FBG were placed in solutions of 10 μg/mL HRP-conjugated anti-FBG while the samples exposed to LYZ were placed in 10 μg/mL HRP-conjugated anti-LYZ. The TCPS wells that were previously incubated with protein were also incubated with the corresponding antibody for 1.5 h. Following antibody adsorption, each of the samples and wells were rinsed 5 times with PBS buffer. Then each of the TMA/CAA and SBMA samples were placed into clean wells in the 24-well plate. A substrate solution containing 0.1 M citrate-phosphate buffer (pH 5.0) with 0.03% hydrogen peroxide and 0.1 g of OPD was prepared and 800 μL was added to each well of the well plate. The well plate was then immediately placed into a BioTek PowerWave XS2 multiwell plate reader (Winooski, VT). The absorbance was continuously monitored at 492 nm over 30 min using Gen5 1.07 software (BioTek). The final absorbance for each of the sample and control disks were averaged and normalized to the average final TCPS value. Three independent trials with three hydrogel disks each, for each cross-linker density and protein were investigated (n = 9). Propagation of experimental uncertainty was conducted throughout all of the trials and was used to represent the error in the experimental data. 2.5. Protein Conjugation. TMA/CAA hydrogels with both 1× and 4× cross-linker densities were synthesized, punched, and soaked as described above in the ELISA procedure. After the second 24 h soak, TMA/CAA hydrogel disks were placed into a solution of 0.5 M NHS and 0.2 M EDC for 7 min. One set of hydrogels from the EDC/NHS solution were immediately placed in a solution of 1 mg/mL FBG (activated sample) and one set of samples were placed in PBS buffer without proteins (control). These samples were soaked in their respective media for 15 min. The gels were then transferred into separate solutions of NaCl−PBS with a pH of 9.34 where they were soaked for 30 min. This was followed by a soak in separate solutions of PBS buffer for an additional 40 min. While the conjugation steps were being completed, a separate set of TMA/CAA disks and TCPS wells were soaked in 1 mg/mL FBG for 1.5 h as fouling resistant and positive fouling controls, using procedures identical to the ELISA studies. All of the samples were incubated in 10 μg/mL HRPconjugated anti-FBG in PBS buffer for 1.5 h, followed by five rinses with PBS buffer. Then the samples were placed into clean wells of the 24-well plate and 800 μL of a substrate solution containing pH 5.0, 0.1 M citrate-phosphate buffer with 0.03% hydrogen peroxide, and 0.1 g OPD was added to each well. The well plate was immediately placed into the BioTek PowerWave XS2 multiwell plate reader where the absorbance was measured at 492 nm over a period of 30 min. Due to the dramatic color change for the hydrogel samples with conjugated FBG, an extension of the ELISA procedure was implemented. The 24-well plate was removed from the plate reader after 30 min and 400 μL of substrate solution was removed from each well and transferred to a clean 24-well TCPS plate. The new plate was then reinserted into the plate reader and the absorbance was measured at 492 nm as a single test point read. The final signals for each of the sample and control disks were averaged and normalized to the averaged TCPS value. The relative level of protein adsorption was determined based on the final absorbance for each well after the 30 min interval and test point read. The averages were obtained from three samples over three independent trials for TMA/CAA hydrogels with cross-linker densities of 1× and 4× and normalized to the TCPS fouling control (n = 9). Propagation of experimental uncertainty was conducted throughout all of the trials and was used to represent the error in the experimental data. To verify that TMA/CAA hydrogels retain their fouling resistant properties following protein conjugation, hydrogels with a 4× cross3114

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linker density were prepared and conjugated to FBG as described above. Following the conjugation procedures, the samples with conjugated FBG were exposed to LYZ and HRP-conjugated antiLYZ using procedures that were identical to the LYZ ELISA procedures described above. Following the final PBS buffer rinse, the samples were placed into clean wells of the 24-well plate and 800 μL of a substrate solution containing pH 5.0, 0.1 M citrate-phosphate buffer with 0.03% hydrogen peroxide and 0.1 g OPD was added to each well and the well plate was immediately placed into the multiwell plate reader. The absorbance was measured again at 492 nm over a period of 30 min. Following 30 min of analysis, the 24-well plate was removed from the plate reader and 400 μL of substrate solution was removed from each well and transferred to a clean 24-well TCPS plate. The new plate was then reinserted into the plate reader and the absorbance was measured at 492 nm as a single test point read for consistency with the conjugation procedures. The final signals for each of the sample and control disks were averaged and normalized to the averaged TCPS value. The relative level of protein adsorption was determined based on the final absorbance for each well after the 30 min interval and test point read. The averages were obtained from three samples over three independent trials for TMA/CAA hydrogels with a cross-linker density of 4× and normalized to the TCPS fouling control (n = 9). Propagation of experimental uncertainty was conducted throughout all of the trials and was used to represent the error in the experimental data. 2.6. Cell Culture. MC3T3-E1 cells were continuously grown on TCPS flasks in α-MEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS), as previously published.35,36 To passage, cells were rinsed twice with 10 mL of PBS buffer and then they were exposed to 2 mL of trypsin-EDTA. After the cells detached, they were resuspended in the supplemented α-MEM and replated onto new flasks. The cells were passaged once per week and passages 5−10 were used for experiments. 2.7. Cell Adhesion Assay. The cell adhesion assay is similar to that used previously.36 Briefly, 1× TMA/CAA hydrogels were prepared both with and without conjugated FBG using the procedures described above. Following the hydrogel sample preparation, the hydrogel disks were then placed in a 24-well TCPS plate where they were rinsed three times using 1 mL of NaCl-Tris buffer. At the same time, three wells in the 24-well culture plate were exposed to 1 mL of either 1 mg/mL of FBG or 1 mg/mL heat denatured bovine serum albumin (BSA) in NaCl-Tris buffer for 30 min. In the meantime, freshly confluent MC3T3-E1 cells were detached using 2 mL of trypsin-EDTA and resuspended in 5 mL of 5 mg/mL soybean trypsin inhibitor in PBS buffer. The cells were then centrifuged at 1000 rpm for 5 min, followed by supernatant removal. Then the cells were washed twice with 10 mL of 5 mg/mL BSA in serum-free α-MEM. The cells were resuspended in serum-free α-MEM and diluted to a final concentration of 1 × 105 cells/mL, determined with a hemocytometer. The cells were incubated for 15 min in an α-MEM before being used in the cell adhesion assay. Once the protein adsorption to the well plates was completed, the FBG and BSA solutions were removed, and the wells were rinsed three times using 1 mL of NaCl-Tris buffer. Then, 1 mL of cell solution was added to each well, including wells with TMA/CAA hydrogel samples with and without conjugated FBG, and the well plate was incubated for 2 h in a humidified atmosphere with 5% CO2 at 37 °C. Three samples of each type of substrate were prepared for each assay and the assay was performed three times (n = 9). 2.8. Fixing, Staining, and Light Microscopy. After the cell adhesion assays, the cell solution was removed from the wells and the wells were washed three times with warm (37 °C) NaCl-Tris buffer to remove loosely bound cells. After this, the cells were fixed by adding 0.5 mL of 4% paraformaldehyde solution to each well for 5 min. The samples were then rinsed three times with 1 mL of warm NaCl-Tris buffer and then stained with 1 mL hematoxylin for 5 min. Next, the samples were rinsed extensively with ultrapure water and exposed to 1 mL of warm NaCl-Tris buffer for 3 min. The samples were then rinsed three times with ultrapure water and dried in air. Three 10× brightfield images from each sample were randomly selected and captured using a

Nikon Eclipse Ti optical microscope (Shinjuku, Tokyo, Japan) equipped with a Nikon DS-2MBW camera and NIS Elements-BR3.1 software (Nikon). Each of the images were analyzed to determine the total number of adherent cells in each (9 samples × 3 images = 27 total images). 2.9. Data Analysis. All of the data are presented as the average of all of the measurements that were taken. The average normalized nonspecific protein adsorption and conjugation sample results from each trial (n = 3) were analyzed using one-way analysis of variance (ANOVA) and they were considered statistically significant when they had a probability value less than 0.05 (p < 0.05). Similarly, the number of adherent cells per unit area determined from the microscope images were analyzed using one-way ANOVA with the same probability value. Statistical analysis was performed using OriginPro 9.0 (OriginLab Corporation, MA).

3. RESULTS AND DISCUSSION 3.1. Hydrogel Physical Characterizations. To compare protein adsorption levels on various substrates using ELISA, it is essential to ensure that the samples have identical surface areas being exposed to protein. Therefore, the first characterization that was completed was to determine the swelling characteristics of the TMA/CAA hydrogels at all three crosslinker densities in both DI water and PBS buffer. As shown in Figure 1a,b, the results of the swelling examinations demonstrate that all of the hydrogels equilibrated to their maximum swelling within 24 or 48 h of being placed in PBS buffer or DI water, respectively. Based on these results, a swelling time of 24 h in PBS buffer was selected for the subsequent ELISA experiments. As expected, the TMA/CAA hydrogel with the lowest cross-linker density (1×) showed the greatest increase in size due to the least amount of cross-links being present. When the swelling properties of the TMA/CAA polyampholyte materials are compared to those recently obtained for TM/SA polyampholyte hydrogels it can be seen that the TMA/CAA has a smaller range of swelling behaviors. This is based on the swelling differences between the 1× and 4× cross-linker densities in both solutions, which is larger for TM/SA polyampholyte hydrogels.34 It is widely believed that a strongly hydrated surface layer is critical for a hydrogel to be resistant to nonspecific protein adsorption. Weight percent hydration characterizations were conducted to determine the water composition of the TMA/CAA hydrogels at varying cross-linker densities and the results are presented in Table 1. It was determined that the weight percent hydration did not vary as a function of the cross-linker density, with all of the weight percent values being greater than 90.7%. This suggests that the TMA/CAA hydrogels are a highly hydrated system that is appropriate for biological applications. To complete the physical characterizations of the TMA/CAA hydrogel samples across a range of cross-linker densities, the compressive properties of the materials were determined. Figure 2 shows representative stress−strain curves for each of the three cross-linker densities that were investigated, and the results of replicate trials are summarized in Table 1. The strength of the hydrogel increases as a function of cross-linker density, with a maximum fracture stress of 554.6 ± 104 kPa, as determined for the 4× cross-linker density. The results also demonstrate that there is only a minimal decrease in the elasticity of the hydrogels, as indicated by the modulus of the materials. It is also interesting to compare the mechanical properties of the TMA/CAA polyampholyte hydrogel to the TM/SA polyampholyte hydrogel.34 The TMA/CAA samples had a much wider range of fracture stress values and had a 3115

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Figure 2. Representative compressional stress−strain curves for TMA/ CAA hydrogels with varying concentrations of TEGDMA cross-linker.

charged TMA and CAA monomer subunits must be present in equal quantities within the final polymer chemistry. If either monomer is enriched, the overall material would have a surface charge that would facilitate the adsorption of oppositely charged proteins.27 Surface zeta potential measurements were completed on TMA/CAA hydrogels with all three cross-linker densities at pH 7.4 to verify that the monomers are present in a 1:1 ratio and the results are shown in Table 1. As a control, a poly(methyl methacrylate) calibration sample was also analyzed under identical conditions. This control does not have a surface charge and it has a calibration zeta potential of −16.3 ± 10.0 mV. When the control was analyzed at pH 7.4, a value of −16.4 mV was determined. As can be seen in Table 1, all three of the TMA/CAA hydrogel systems had measured surface potentials whose absolute values were less than the neutral control and less than ±10.0 mV from a value of zero. These results demonstrate that there is no measurable surface charge present on any of the TMA/CAA hydrogel systems at neutral pH, suggesting that the TMA and CAA monomers are present in a 1:1 ratio on the surface. The underlying polymer chemistry was examined in further detail using XPS and the results were compared to the theoretical polymer composition based on the monomer and cross-linker ratios fed in the polymerization solution. A representative broad survey spectrum from the XPS analysis can be seen in Figure 3a, and the results of the elemental analysis can be seen in Table 2. It can be seen that the amount of carbon present in the hydrogel samples is greater than the theoretical levels across all of the cross-linker densities, while the amount of nitrogen and oxygen are below the theoretical levels. This suggests that there is some carbon contamination on the samples. In addition to the expected elements (C, N, and O), there are trace levels of sodium and sulfur found in the hydrogels. These elements are below 0.3 atomic percent and they can be attributed to the SMS (Na and S) and APS (S)

Figure 1. Mean ± standard deviation of the swelling behavior of TMA/CAA hydrogels with varying concentrations of TEGDMA crosslinker in (a) PBS buffer and (b) DI water. SBMA hydrogels (1× crosslinker density) are included as a reference (n = 3).

maximum fracture stress that was ∼3.7 times greater than that found for the TM/SA materials with identical TEGDMA concentrations. However, the opposite trend was true for the elasticity of the materials as the TM/SA hydrogels had a much larger range and had a maximum modulus that was ∼3.6× greater than that found for the TMA/CAA samples. The results of this study, combined with our previous work,34 suggest that it should be possible to design polyampholyte hydrogels with controlled mechanical properties by combining multiple positively and negatively charged monomer subunits (for example a TM/SA/CAA system). 3.2. Hydrogel Chemical Characterizations. For the TMA/CAA hydrogels to be fouling resistant, the oppositely

Table 1. Physical Properties of TMA/CAA Hydrogels with Varying Cross-Linker Densities sample

hydration (wt%)

zeta potential (mV)

fracture stress (kPa)

fracture strain

modulus (kPa)

1× TMA/CAA 2× TMA/CAA 4× TMA/CAA

90.9 ± 2.5 90.8 ± 0.9 90.7 ± 2.4

3.60 7.19 8.98

19.3 ± 5 140.5 ± 34 554.6 ± 104

0.89 ± 0.02 0.82 ± 0.10 0.83 ± 0.03

4.5 ± 0.2 20.2 ± 11.6 35.4 ± 14.7

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Figure 3. Representative XPS spectra for TMA/CAA hydrogels taken from samples with a 2× cross-linker density showing a (a) broad survey spectrum, (b) high resolution carbon spectrum, (c) high resolution oxygen spectrum, and (d) high resolution nitrogen spectrum. Each high resolution spectrum includes curve fits for each of the functional groups, along with the overall curve fit (dashed line).

Table 2. Chemical Compositions of TMA/CAA Hydrogels with Varying Cross-Linker Densitiesa 1:1

1×

2×

4×

element

TMA/CAA

theory

actual

theory

actual

theory

actual

C N O Na S Si

66.7 4.8 28.6

66.8 4.6 28.6

81.7 ± 6.0 0.7 ± 0.6 13.1 ± 3.8 0.0 ± 0.0 0.2 ± 0.1 4.3 ± 2.1

66.9 4.4 28.7

75.3 ± 3.5 2.1 ± 0.6 17.9 ± 1.9 0.2 ± 0.2 0.0 ± 0.0 4.6 ± 1.6

67.1 4.2 28.8

73.6 ± 1.4 3.1 ± 0.9 19.1 ± 1.0 0.1 ± 0.1 0.0 ± 0.0 4.1 ± 0.3

a

Theoretical values given as atomic concentration excluding hydrogen, based on the monomer and cross-linker concentrations fed to the polymerization solution. The total value may exceed 100% due to rounding.

antifouling performance of the hydrogels based on the results discussed below. High resolution XPS spectra were collected to gain further insight into the carbon, nitrogen, and oxygen functional groups present in the materials. The theoretical changes in the concentration of the different functional groups with increasing cross-linker density are less than 3%, which falls below the expected resolution of the XPS analysis and curve fitting techniques. Therefore, Figures 3b−d show representative high resolution spectrum for carbon, oxygen, and nitrogen obtained

initiators. As can be seen in Table 2, there is also a significant level of silicon present in all of the samples. This contamination is attributed to the microscope slides which were used as the hydrogel mold, as it is the only source for silicon in this system. This contamination appears to be constrained to the outer levels of the hydrogel mold, as the silicon contamination significantly decreases when the surface of the hydrogel is cleaned with 60 s of ion sputtering (Supporting Information, Table S.1). However, it does not appear to impact the 3117

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from TMA/CAA hydrogels with a 2× cross-linker density. In the high resolution carbon spectrum, shown in Figure 3b, all four of the expected functional groups can easily be seen. The hydrocarbon functional group accounts for a greater percentage of the overall composition, as compared to the theoretical composition, but this is likely due to surface contamination as indicated by the higher atomic percentage of carbon overall. The other functional group contributions suggest that there are greater levels of TEGDMA in the final hydrogel composition than were fed to the polymerization vessel. This is based on the relative sizes of the peaks associated with the acid/ester content as compared to the alcohol/ether peak, and because the total contribution from the alcohol/ether peak is greater than the theoretical contribution. This is also supported by the relative sizes of the functional group contributions seen in the high resolution spectrum for oxygen, shown in Figure 3c. In this figure, it is apparent that the contributions from the alcohol/ ether peak and the ester peak are similar, even though the theoretical contributions show significantly more ester. This confirms that there is an enrichment of the TEGDMA in the final hydrogel because it is the primary source for ether contributions. In the nitrogen high resolution spectrum, shown in Figure 3d, it can be seen that there are two functional groups present. The amine functional group contribution, indicating the TMA monomer, is the more significant contributor to the overall nitrogen signal. The second, smaller peak can be attributed to trace amounts of the APS initiator, as suggested by its binding energy location. 3.3. Protein Adsorption Characterization. The antifouling characteristics of the TMA/CAA hydrogels were assessed through ELISA techniques using both negatively charged FBG and positively charged LYZ. All of the protein adsorption results were normalized to a TCPS fouling control due to the fact that it is not possible to interpret ELISA as a specific adsorbed amount of protein per unit surface area. TCPS is considered to have a complete monolayer of adsorbed protein under the conditions investigated here. In addition to a fouling control, it is important to interpret the ELISA results with respect to a known nonfouling control. SBMA hydrogels were used here because of their well-characterized nonfouling properties.25,37 ELISA depends on the reaction between an enzyme (HRP) and a substrate (OPD) to produce 2,3diaminophenazine, which is directly detected by absorbance, therefore, it is both time- and concentration-dependent. To determine the best experimental time point for analysis, the ELISA assay was continuously monitored over a period of 30 min. Following 30 min, the measured absorbance value for the TCPS controls as well as all of the other samples significantly leveled off (data not shown). Based on this response, an analysis time period of 30 min was selected and used for all of the nonspecific protein adsorption investigations. The results of the ELISA investigation into the antifouling properties of the TMA/CAA hydrogels are summarized in Figure 4. The nonfouling control SBMA samples exhibited absorbance levels of less than 10% of that measured for the TCPS for both FBG and LYZ, confirming its known nonfouling properties. The results shown in Figure 3 also demonstrate that the TMA/CAA hydrogels have protein adsorption levels that are on par with those seen for the SBMA control sample across all of the cross-linker densities. A one-way ANOVA also confirms that there are no statistically significant differences between the TMA/CAA samples and the SBMA control at a 95% confidence interval (p > 0.05 for all samples).

Figure 4. Mean ± propagated error of the nonspecific protein adsorption to various hydrogel substrates (n = 9). The results are presented as the average absorbance measured after 30 min and normalized to TCPS.

ELISA investigations were also conducted on two additional sets of control hydrogels. The first of these were TMA/CAA hydrogels with a 1× cross-linker density that were formed with DI water in the synthesis solution, rather than 3.0 M NaOH. The results for this control sample are shown in Figure 3. As it can be seen, there is a significant increase in the FBG adsorption to the TMA/CAA hydrogel formed in the absence of NaOH based on the absorbance levels. This suggests that the hydrogel has a net positive charge based on related biosensor studies conducted with polyampholyte materials.27 The positive charge is likely a direct result of the pH sensitivity of the CAA monomer.32,33 Under acidic conditions, the CAA monomer becomes protonated, making it a neutral monomer. When combined with TMA, the resulting hydrogel has a net positive charge. The addition of base to the synthesis solution ensures that the CAA monomers are deprotonated and negatively charged, facilitating the electrostatic interactions that are believed to greatly aid in the formation of a molecular level homogeneous mixture of TMA and CAA monomers. The second set of control hydrogels that were investigated were hydrogels formed from the individually charged monomers themselves (data not shown). However, these hydrogels had significantly weaker mechanical properties and they were unable to withstand handling during the ELISA procedure. It is believed that the electrostatic repulsions that are present with the individually charged monomers disrupt the hydrogel polymerization reaction by increasing the spacing between the monomer subunits. The hydrogel fragments that did survive through ELISA investigations did have significantly greater levels of nonspecific protein adsorption as compared to the TMA/CAA polyampholyte hydrogels, despite the fact that they often had smaller surface areas due to sample damage. These results, combined with those discussed above, strongly suggest that TMA/CAA polyampholyte hydrogels have antifouling properties when the individual monomer subunits are present in a 1:1 ratio and they are present in their oppositely charged states. 3.4. Conjugation Characteristics. The TMA and CAA monomers were selected for this investigation with the 3118

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intention to design a multifunctional hydrogel capable of protein conjugation within a fouling resistant background for tissue engineering applications. As discussed in the Introduction, TMA/CAA polyampholyte polymer brushes have recently been demonstrated to have protein conjugation capacity.33 To conjugate proteins to the TMA/CAA polyampholyte hydrogels, standard EDC/NHS conjugation chemistry was used to react −COOH groups in the CAA monomers to primary amines present in FBG. However, in order to activate the CAA monomer to facilitate this conjugation approach, the TMA/ CAA hydrogels were first exposed to an acidic solution to ensure that the −COOH groups were fully protonated. The advantage to this system is the fact that if a −COOH group is not reacted to a primary amine through the EDC/NHS chemistry, it can be returned to −COO− with a neutral or slightly basic pH buffer rinse. This returns the TMA/CAA hydrogel to its neutral, fouling resistant state in areas away from conjugated proteins. The presence of conjugated FBG on TMA/CAA hydrogels with both a 1× and 4× cross-linker density was confirmed using similar ELISA procedures to those used to assess the nonspecific protein adsorption to the hydrogels and the results are shown in Figure 5a. As seen in this figure, the samples that underwent the full conjugation procedure (activated) had 2.5× the absorbance measurement as compared to the fouling resistant TMA/CAA hydrogel control samples, which was statistically significant for both cross-linker densities. However, this result does not adequately represent the amount of protein that is covalently attached to the surface of the TMA/CAA hydrogels. The inset images in Figure 5a show representative pictures of 1× TMA/CAA hydrogel materials themselves following the conjugation and ELISA procedures. As seen in these inset images, the conjugation procedure was successful to the point that the HRP-OPD reaction caused a significant color change to the hydrogel samples themselves. It appears that the colored reaction product is unable to fully diffuse out of the hydrogel and homogenize throughout the liquid in the sample well. This resulted in the conjugated sample becoming black after 30 min of reaction while the fouling resistant control samples remained clear. The color change was so drastic that it interfered with the absorbance readings, resulting in initially inconsistent results. To address this issue the ELISA procedures were modified (as described in Materials and Methods) to analyze the absorbance for an aliquot of the supernatant rather than all of the supernatant. This retention of colored product within the conjugated samples is the primary reason why the quantitative assessment suggests there is only a 2.5-fold increase in the amount of FBG present between the conjugated and fouling resistant samples, which is not an accurate representation of the differences in the amount based on the images. Figure 5a also provides the results that were obtained for the TMA/CAA control samples, which were exposed to all of the conjugation procedure steps with the substitution of a PBS buffer soak in place of the FBG exposure. This control was conducted to examine the impact that the EDC/NHS procedures have on the antifouling properties of the TMA/ CAA hydrogels based on anti-FBG adsorption alone. As seen in Figure 5a, the control TMA/CAA samples have a statistically significantly lower level of adsorbed protein than that seen for the samples with conjugated FBG for both cross-linker densities, and no statistically significant difference from the fouling resistant controls. At the same time, an edge effect is clearly discernible in the inset picture of the control hydrogel in

Figure 5. Mean ± propagated error of the (a) anti-FBG response to 1× and 4× TMA/CAA hydrogels following EDC/NHS conjugation and (b) anti-LYZ response to 4× TMA/CAA hydrogels following FBG conjugation and exposure to LYZ (n = 9). The dashed line in (b) represents the value determined for fouling resistant control samples from one of the repeat trials (n = 3). * Represents a statistically significant difference between the samples being compared (p < 0.05).

Figure 5a and this was typical of all of the control samples with a 1× cross-linker density. The TMA/CAA samples with a 4× cross-linker density also appeared to have an edge effect, although it was to a lesser degree than that seen in the 1× samples. This edge effect is attributed to physical damage caused to the hydrogel samples because of the extra handling required during the conjugation and ELISA procedures, which was not needed in the fouling resistant control samples. This was supported by light microscopy images that were taken of samples following the procedures which indicated microscale damage to the edges of the hydrogels (data not shown). However, despite this edge effect, the results of the ELISA investigation confirm that the TMA/CAA samples that were exposed to EDC/NHS and FBG have significantly more FBG present than both the control and fouling resistant samples. As discussed above, it was hypothesized that the TMA/CAA hydrogels could maintain their antifouling properties away from any covalently attached proteins, as long as the carboxylic acid functional groups were fully deprotonated following conjuga3119

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Figure 6. Optical microscopy images (10×) of MC3T3-E1 cell adhesion to the following substrates: (a) TCPS with adsorbed BSA; (b) TMA/CAA hydrogels without conjugated proteins; (c) TCPS with adsorbed FBG; and (d) TMA/CAA hydrogels with conjugated FBG. The scale bar represents 100 μm.

conjugated FBG. MC3T3-E1 cells have previously been demonstrated by us to have RGD-dependent cellular adhesion behaviors, making them a good candidate as a model cell line.35,36,39 The control surfaces in the cell adhesion assay were TCPS wells with adsorbed FBG (positive control) and heat denatured BSA, which is known to block MC3T3-E1 adhesion to TCPS (negative control).36 Figure 6 shows representative light microscopy images of the cells following the 2 h cell adhesion assay. The fouling resistant properties of the TMA/CAA polyampholyte hydrogels without conjugated proteins are demonstrated with the cell adhesion results shown in Figure 6a,b. It is clearly evident that there is minimal or no cell attachment to the TMA/CAA hydrogel and the cellular response is equivalent to that seen for TCPS with adsorbed BSA. At the same time, the bioactivity of the conjugated FBG, along with the biocompatibility of the TMA/ CAA hydrogel, is clearly demonstrated in Figure 6d. There is approximately 50% surface coverage of adherent cells in the presence of conjugated FBG. Furthermore, when this image is compared to the positive control, TCPS with adsorbed FBG (Figure 6c), there is clearly a greater level of adherent cells on the TMA/CAA sample with conjugated FBG. While these differences are easy to see qualitatively with the light microscopy images, it is also important to verify the results

tion. This was verified in this study by examining the adsorption of LYZ to 4× TMA/CAA hydrogels following FBG conjugation and the results are shown in Figure 5b. In this figure it can be seen that the level of LYZ adsorbed to the TMA/CAA hydrogels with conjugated FBG is nearly identical to that adsorbed to control samples that were activated and deactivated, but not exposed to FBG. In one of the replicate trials, a set of 4× TMA/CAA fouling resistant controls were also included, and the results for that trial are indicated with the dashed line in Figure 5b. Again, the levels of adsorbed LYZ were nearly identical, confirming the hypothesis that the underlying antifouling properties of the TMA/CAA hydrogels are not impacted by protein conjugation. 3.5. Cell Adhesion. The conjugation studies clearly indicate that there are significant increases in the amount of protein present on the TMA/CAA hydrogel materials. However, if this multifunctional property is to be utilized for tissue engineering applications, it is essential to verify the bioactivity of the attached molecules. FBG is well-known to have a cell binding arginine-glycine-aspartic acid (RGD) sequence that facilitates cell binding through specific integrin interactions.38 Therefore, the activity and accessibility of this cell binding sequence was tested in this investigation using short-term cell adhesion assays with MC3T3-E1 cells as a probe of the bioactivity of the 3120

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Conjugated FBG was also shown to retain its cell attachment bioactive property. This represents a unique approach for the development of tissue regeneration scaffolds from polyampholyte materials that combines specific bioactive signaling molecules within a fouling resistant background material.

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ASSOCIATED CONTENT

* Supporting Information S

Additional details from the XPS characterization of the TMA/ CAA hydrogels is available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

Figure 7. Average number of MC3T3-E1 cells (cells/mm2) that adhered to TCPS and TMA/CAA hydrogels with or without adsorbed or conjugated proteins. The data are presented as the mean ± standard error of the mean from nine samples completed over a total of three separate occasions. Three optical microscopy images were collected and analyzed for each sample completed (n = 27). * Represents a statistically significant difference between the surfaces being compared (p < 0.05).

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Dr. Fu-hung Hsieh and Harold Hess for their assistance with the compression testing, Brian Porter from the Materials Research Center at Missouri Science and Technology for assistance with the XPS characterization, and Joseph Mathai and the Macro/Nano Technology Facility for assistance with the surface zeta potential measurements. This study was supported by the MU College of Engineering and by the National Natural Science Foundation of China through Grant #31350110223.

quantitatively. The results of the quantitative analysis across multiple images and samples are shown in Figure 7. As anticipated from the images, the quantitative assessment confirms that there are a statistically significantly greater number of cells on the TMA/CAA hydrogels with conjugated FBG as compared to both the TMA/CAA hydrogel alone and TCPS with adsorbed FBG. It is interesting to note that there are approximately 2-fold more cells on the TMA/CAA hydrogel with conjugated FBG as compared to TCPS with adsorbed FBG. The exact cause of this is unknown at this time, but it is possible that during conjugation the underlying positive charge of the TMA/CAA hydrogel imparts a favorable orientation or conformation to FBG. The influence of charge on the cell adhesion capabilities of proteins has been demonstrated before,40−43 and we are currently investigating this possibility further. More importantly, these results clearly demonstrate that the bioactivity of the FBG is maintained following the EDC/NHS conjugation. They also demonstrate that TMA/CAA hydrogels are fouling resistant even in more complex environments, as indicated by the lack of cell attachment in the absence of conjugated FBG. These combined results suggest that TMA/CAA polyampholyte hydrogels have a unique multifunctional nature that allows for bioactive proteins to be conjugated within a fouling resistant background. This represents a promising platform for providing tissue specific cues within a fouling resistant tissue regeneration scaffold to promote an improved wound healing response.

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REFERENCES

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4. CONCLUSIONS In this work, multifunctional polyampholyte hydrogels were formed from mixtures of positively charged TMA and negatively charged CAA monomer subunits and characterized. These hydrogels were shown to have resistance to nonspecific protein adsorption from both FBG and LYZ. Furthermore, these TMA/CAA hydrogels were shown to have pH-dependent protein conjugation capabilities, while retaining their fouling resistant properties away from the attached proteins. 3121

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