Uniform zwitterionic polymer hydrogels with a nonfouling and functionalizable crosslinker using photopolymerization

Biomaterials 32 (2011) 6893e6899

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Uniform zwitterionic polymer hydrogels with a nonfouling and functionalizable crosslinker using photopolymerization Louisa R. Carr, Yibo Zhou, Jordan E. Krause, Hong Xue, Shaoyi Jiang* Department of Chemical Engineering, University of Washington, Box 351750, Seattle, WA 98195, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 May 2011 Accepted 4 June 2011 Available online 25 June 2011

We reported previously the design and synthesis of a zwitterionic carboxybetaine dimethacrylate crosslinker, and we showed that its use with zwitterionic carboxybetaine methacrylate led to nonfouling hydrogels with high mechanical properties and high hydration. Now, we use photopolymerization to improve the uniformity of the polymer network, resulting in drastically improved mechanical properties (compressive modulus up to 90 MPa). Furthermore, we designed and synthesized a new functionalizable carboxybetaine dimethacrylate crosslinker, enabling functionalization of the higher strength hydrogels against a nonfouling background. Additionally, the biostability of the carboxybetaine hydrogel systems was tested, and it was found that these hydrogels are stable in oxidative, acidic, and basic environments. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Hydrogel Crosslinking RGD peptide Compression

1. Introduction Hydrogels have long been of interest for biological and biomaterial applications due to their biomimetic high water content, high diffusive permeability, and mechanical strengths [1e6]. Particular interest has been given to PEG and poly(2-hydroxyethyl methacrylate) (pHEMA) [1] hydrogels because, in addition to the general properties of hydrogels, they are also commonly considered be low fouling, bioinert, and versatile. Hydrogels made from pHEMA have found use in and been studied for many applications [1,7e9], but their hydration is lower than that of native tissue, and their fouling, while low, is higher than other nonfouling materials. PEG hydrogels are routinely used, despite being subject to oxidative degradation [10e14]. The susceptibility of PEG to oxidative damage reduces its utility for applications that require long-term material stability. Furthermore, pHEMA and PEG functionalization via the hydroxyl group is generally difficult. The prominence and importance of zwitterionic materials has increased in recent years [15e19]. We have demonstrated that zwitterionic compounds, including poly(carboxybetaine methacrylate) (CBMA, Scheme 1), are ultra-low-fouling [20,21], meaning that surfaces coated with these polymers allow less than 5 ng/cm2 protein adsorption [22]. We also showed that pCBMA-coated surfaces are highly resistant to non-specific protein adsorption, even from undiluted blood plasma and serum [23,24], and prohibit long-term bacterial colonization for up to 10 days at room * Corresponding author. Tel.: þ206 616 6509; fax: þ206 543 3778. E-mail address: [email protected] (S. Jiang). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.06.006

temperature [25]. The ultra-low-fouling of zwitterionic materials results from the high hydration around the opposing charges and the high energy barrier required to remove that hydration layer [26]. Furthermore, CBMA is easily functionalized through conventional EDC/NHS chemistry [21,27]. Because of the high hydration and ultralow fouling properties of zwitterionic materials, zwitterionic materials are of interest as hydrogels with superior suitability for biomedical applications. Previously, we have demonstrated low protein adsorption on sulfobetaine methacrylate (SBMA) hydrogels [28], and low cell adhesion on CBMA, SBMA, and sulfobetaine vinylimidazole hydrogels [28,29]. The zwitterionic hydrogels studied so far, however, have shown low mechanical strength [28e30], which limits their potential biological uses. Another fundamentally limiting feature of these zwitterionic hydrogels is the dearth of hydrophilic crosslinkers. The most commonly used commercially available “hydrophilic” crosslinker is N,N’-methylenebis(acrylamide) (MBAA), but this crosslinker is only moderately soluble at crosslinker concentrations around 10%, and the acrylamide functionality incorporates poorly into the growing methacrylate polymer chains. Furthermore, MBAA is particularly illsuited for crosslinking zwitterionic hydrogels because it does not structure water. Structured water around the opposing charges in a zwitterionic material provides the nonfouling mechanism [26]; MBAA will disrupt the ordered water and present locations where proteins, bacteria, and even cells, may bind and foul the hydrogel. Previously, we reported a carboxybetaine (CB) crosslinker [31] (CBMAX-1, Scheme 1). Chemically, it is composed of the zwitterionic CB methacrylate with a second methacrylate replacing one of the methyl groups on the quaternary nitrogen. Due to its structural

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L.R. Carr et al. / Biomaterials 32 (2011) 6893e6899 2. Experimental methods 2.1. Materials 2-(N-morpholino)ethanesulfonic acid (MES), methacrylic acid, ion exchange resin (IRA 400 OH form), iodomethane, triethyl amine, 2-hydroxy-2-methyl-propiophenone, papain, and phosphate-buffered saline were purchased from Sigma Aldrich (St. Louis, MO). Diethanolamine, tert-butyl acrylate, and methacryloyl chloride were purchased from Alfa Aesar; Acetonitrile, silica gel 60 (70e230 mesh), and diethyl ether from EMD Biosciences (Gibbstown, NJ); trifluoroacetic acid was purchased from Acros Organics (via Fisher Scientific, Pittsburg, PA); and t-butyl bromoacetate and N-cyclohexyl-2-aminoethanesulfonic acid (CHES) from TCI America (Portland, OR). Dulbecco’s Modified Eagle Medium, fetal bovine serum, nonessential amino acids, and penicillinstreptomycin were purchased from Invitrogen Corp (Carlsbad, CA). Cyclo(ArginineGlycine-Asparginine-D-Tyronsine-Lysine) peptide (cRGD) was purchased from Peptides International (Louisville, KY). Hydrogen peroxide and sodium chloride salt were purchased from J.T. Baker (Phillipsburg, NJ). COS-7 cells (African Green Monkey fibroblast cells) were purchased from the American Tissue Culture Collection (Manassas, VA). All water used had been purified to 18.2 mU on a Millipore Simplicity water purification system. 2.2. Synthesis of carboxybetaine monomer and carboxybetaine-based crosslinkers 2.2.1. Synthesis of the CBMA monomer 2-Carboxy-N,N,-dimethyl-N-(20 -(methacryloyloxy)ethyl) ethanaminium inner salt (carboxybetaine methacrylate, CBMA) was synthesized as previously reported [34]. 2.2.2. Synthesis of CBMAX-1 The crosslinker with 1 carbon between the quaternary amine and the carboxyl group, CBMAX-1, also called 1-Carboxy-N-methyl-N-di(2-methacryloyloxy-ethyl) methanaminium inner salt, was synthesized as previously reported [31].

Scheme 1. Chemical structures of CBMA monomer and crosslinkers with 1-carbon spacer (CBMAX-1) and 2-carbon spacer (CBMAX-2).

similarity to the CB monomer and high solubility in aqueous systems, improved incorporation of the crosslinker into the polymer mesh network was observed, and higher concentrations of crosslinker were achieved. A hydrogel made with 100% crosslinker had a compressive modulus of 8 MPa with 60% equilibrium water content. Importantly, this hydrogel exhibited the nonfouling property that is the hallmark of zwitterionic materials. Despite these attributes, however, the CB dimethacrylate reported previously had one key shortcoming: the CB group had only one methylene group separating the opposing charges, which limits its reactivity for functionalization [32,33]. In order to gain more reactivity for functionalization, and to make the crosslinker a true analog for the monomer, a new CB crosslinker was designed with two carbons between the opposing charges (CBMAX-2, Scheme 1). This two-carbon spacer makes the carboxylic acid functional group more reactive to carbodiimide chemistry like EDC-sulfoNHS zero-length crosslinker reactions [27,33]. Also in this study, photopolymerization was used rather than thermal polymerization used in the previous study [31], in order to further improve the uniformity of the growing polymer mesh network. This is due to the improved regularity of photon penetration relative to heat transfer through the polymerization medium, and results in network homogeneity that contributes to the bulk mechanics of hydrogels. Furthermore, polymerization by photo-irradiation is convenient due to the ability to control light exposure spatially and temporally, and does not require harsh conditions or long reaction times. Here, we designed and synthesized the more reactive crosslinker, and test the physical, mechanical properties, and functionalization properties of photopolymerized hydrogels made with 2-carbon spacer crosslinker (CBMAX-2), and compare these properties to those of photopolymerized hydrogels made with the 1-spacer crosslinker reported earlier (CBMAX-1). The stability of this new crosslinker was also evaluated in several bio-relevant media, such as oxidative, acidic, and basic solutions.

2.2.3. Synthesis of CBMAX-2 Compound 6 (Scheme 2), the crosslinker with 2 carbons between the quaternary amine and the carboxyl group, CBMAX-2, was synthesized as follows: N,N-Bis(2-hydroxyethyl)-3-aminopropionic acid tert-butylester 3: A sample of 12.25 g (95.5 mmol) of tert-butyl acrylate was added to 8.77 g (83.4 mmol) of diethanolamine. The mixture was stirred at room temperature overnight. The excess tert-butyl acrylate was removed in vacuo, yielding 19.4 g (100%) of 3 as a colorless oil of purity sufficient for further reactions. 1H NMR (CDCl3, 500 MHz): d 3.61 (t, 4H, J ¼ 8.5 Hz), 3.12 (s, 2H), 2.80 (t, 2H, J ¼ 10.5 Hz), 2.63 (t, 4H, J ¼ 8.5 Hz), 2.40 (t, 2H, J ¼ 10.5 Hz), 1.46 (t, 9H). N,N-Bis(2-methacryloyloxyethyl)- 3-aminopropionic acid tert-butylester 4: To a solution of 3 (19.4 g, 83.4 mmol) in dry CH2Cl2 (300 mL) was added 58 mL (0.42 mol) of triethylamine at 0  C, and to this mixture was then added a solution of methacryloyl chloride (18 mL, 0.18 mol) in dry CH2Cl2 (50 mL) dropwise at 0  C. After addition, the mixture was allowed to warm to room temperature and stirred overnight until the reaction completed. 300 mL of water was added to the mixture and the organic layer was separated. The aqueous layer was then extracted with CH2Cl2 (2  150 mL), and the combined organic layers were washed with brine and dried with anhydrous sodium sulfate. After removal of the solvent, the residue was purified by column chromatography (silica gel 60, eluent: hexanes/ethyl acetate 5:1) to afford 26.2 g (85% yield) of product 4 as a pale brown liquid. 1H NMR (CDCl3, 500 MHz): d 6.10 (s, 2H), 5.56 (s, 2H), 4.21 (t, 4H, J ¼ 6.0 Hz), 2.90 (t, 2H, J ¼ 7.0 Hz), 2.85 (t, 4H, J ¼ 6.0 Hz), 2.38 (t, 2H, J ¼ 7.0 Hz), 1.94 (s, 6H), 1.44 (s, 9H) ppm. N-Methyl-N,N-di(2-methacryloyloxyethyl)-N-2-(tert-butyloxycarbonylmethyl) ammonium iodide 5: Compound 4 (22.64 g, 61.3 mmol), iodomethane (5.7 mL, 92 mmol), hydroquinone (2.0 g, as a stabilizer), acetonitrile(150 ml) were mixed in a flask and stirred at 50  C for 2 days. The solvent was evaporated by rotary evaporation, and the crude product was directly used for next step without further purification. 1H NMR (CD3OD, 500 MHz): d 6.15 (s, 2H), 5.66 (s, 2H), 4.72 (m, 4H), 4.35e4.10 (m, 4H), 3.87 (t, 2H), 3.55 (s, 3H), 2.97 (t, 2H), 1.94 (s, 6H), 1.44 (s, 9H) ppm. 2-Carboxy-N-methyl-N,N-di(2-methacryloyloxyethyl) methanaminium inner salt 6: The crude product 5 above was treated with trifluoroacetic acid (TFA, 137 ml) in dichloromethane (700 ml) for 1 day at room temperature. The solvent was removed by rotary evaporation. The residue was dissolved in water and neutralized over an ion exchange resin (IRA 400 OH form), subsequently concentrated, precipitated into ether, and finally vacuum dried to obtain compound 6 (15.5 g, 77% yield from compound 4 through 2 steps). 1H NMR (CD3OD): d 6.18 (s, 2H), 5.74 (s, 2H), 4.66 (m, 4H), 3.88 (m, 4H), 3.80 (t, 2H, J ¼ 8.0 Hz), 3.26 (s, 3H), 2.70 (t, 2H, J ¼ 8.0 Hz), 1.99 (s, 6H) ppm. 2.3. Hydrogel Preparation Hydrogel solutions were prepared in 1M NaCl, with a total concentration of monomer and crosslinker, either CBMAX-1 or CBMAX-2, of 65% (wt/vol). The different formulations were made by substituting monomer for crosslinker in increasing amounts, starting at 2% by mole. The 100% crosslinker hydrogels were

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Scheme 2. Synthesis of the CBMAX crosslinker (6) with 2 carbons between the quaternary amine and the carboxylic acid groups.

made from 65% (wt/vol) solutions of the crosslinker. The solutions were mixed by sonication in an ice bath to prevent premature polymerization. After sonication, the photoinitiator, 2-hydroxy-2-methylpropiophenone, was added at 1% (wt/wt) to the solutions and homogenized by gentle mixing. Finally, the solutions were polymerized between glass microscope slides separated by 0.5 or 1.5 mm-thick polytetrafluoroethylene (PTFE) spacers with 362 nm UV light. The solutions cast at 0.5 mm thickness were polymerized for an hour, with the side exposed to the light switched every 30 min. The solutions cast at 1.5 mm thickness were polymerized for 2 h, and the light switched similarly. The gels were then removed from the slides and immersed in phosphate-buffered saline (PBS) to hydrate. This hydration water was changed daily for 5 days to remove unreacted chemicals and excess salt. Biopsy punches were used to punch hydrated hydrogels into 5 mm-diameter disks. 2.4. Hydration properties of hydrogels Hydrogels were allowed to swell to equilibrium in PBS for five days. Disks with 0.5-cm diameter were cut from the swollen gel cast at 0.5 mm thickness. The disks were weighed and then dehydrated under vacuum at 50  C and 30 in. Hg vacuum for 3 days. Dried hydrogel disks were weighed. The equilibrium water content values were determined as 100(%)*(mw-md)/mw, where mw is the mass of the wet hydrogel and md is the mass of the dry hydrogel. All samples were measured in triplicate. 2.5. RGD functionalization via EDC/sulfoNHS Chemistry EDC/sulfoNHS chemistry was used to functionalize CBMA hydrogels with cRGD. Hydrogels with very low crosslinking were used to demonstrate the functionalizability of the CBMA monomer while 100% CBMAX-1 and 100% CBMAX-2 were used to demonstrate the functionalizability of the respective crosslinkers themselves. For each formulation, six hydrogel disks of 0.5 cm diameter (0.5 mm thickness when cast) were placed individually in the wells of a plasma-treated tissue culture polystyrene 48-well plate. The hydrogels were incubated in 500 ul MES buffer solution (pH ¼ 5.5, 10 mM MES, 100 mM NaCl) overnight. The MES was then removed from three disks of each formulation and replaced with 500 ml of an MES-based buffer solution containing 5 mM sulfoNHS and 100 mM EDC, to activate the surface, for 2 h at room temperature. The EDC/sulfoNHS and sulfoNHS solutions were then removed from the wells, and the hydrogel disks were washed three times with MES buffer, allowing at least 20 min for each wash. To the activated hydrogels was next added 500 ml 1.4 mM cRGD in CHES buffer (pH ¼ 9, 50 mM CHES, 100 mM NaCl), and the reaction was allowed to proceed at room temperature. After 3 h, the hydrogels were washed three times with PBS, allowing 10 min per wash. Finally, the hydrogels were sterilized with UV light for 30 min, followed by penicillin-streptomycin in sterile PBS overnight, and cell adhesion was performed the next day. 2.6. Cell adhesion to hydrogels After sterilization with penicillin-streptomycin in PBS, the antibiotics were removed and the hydrogels were washed with sterile PBS in preparation for cell culture. Three functionalized hydrogel disks and three nonfunctionalized hydrogel disks of each formulation were used. COS-7 cells (p ¼ 6) were seeded onto the hydrogels at a concentration of 105 cells/ml in supplemented Dulbecco’s Modified Eagle Medium. Cells were allowed to grow for 72 h at 37  C, 5% CO2, and 100%

humidity, after which time the hydrogels were photographed at 10magnification on a Nikon Eclipse TE2000-U microscope. Photographs were taken at five predetermined areas on the surface of the hydrogel, for a total of fifteen images per hydrogel formulation, and the number of adherent cells from the images was totaled. 2.7. Mechanical strength of hydrogels At least five 0.5 cm diameter disks of each formulation (1.5 mm thickness when cast) were compressed to failure at a rate of 1 mm/min using an Instron 5543A mechanical tester (Instron Corp., Norwood, MA) with a 10 kN load cell. The Young’s modulus was calculated from 3 to 13% strain to avoid any complications in the instance where the top platen might not be completely engaged with the specimen when compression begins. 2.8. Long-term stability assay To test the ability of the CB hydrogels to resist oxidative degradation, lightly crosslinked (5%) hydrogels and 100% CBMAX-2 hydrogels were cut into 5-mm disks. Three disks of each formulation were submerged in 18.2 mU water, three were submerged in a solution of 5% hydrogen peroxide, and the final three were submerged in a solution of 10% hydrogen peroxide. The hydrogels were incubated at 37  C in their respective solutions for 24 h, after which they were washed thoroughly with 18.2 mU water. After overnight incubation in 18.2 mU water, they were compressed to failure as described above. To test the ability of the CB hydrogels to resist degradation in physiological acidic and basic conditions, lightly crosslinked (5%) hydrogels and 100% CBMAX-2 hydrogels were cut into 5-mm disks. Disks were incubated at 37  C in buffered solutions at pH ¼ 5, 7, or 9. At 20-day intervals, three disks of each formulation were removed from each condition and compressed to failure as described above.

3. Results and discussion 3.1. Hydration properties The equilibrium water content of CB hydrogels prepared by photopolymerization and thermal polymerization [31] are shown in Fig.1. As expected, the hydrogels with lower crosslinker content have higher hydration, and all systems exhibit an exponentially decaying relationship between crosslinker content and equilibrium water content. There are two important differences in the profiles, however, each with significant meaning. Firstly, the overall curves for CB hydrogels are markedly smoother when the hydrogels were polymerized with photoinitiators, indicating that photopolymerization is more controlled than thermally initiated polymerization. This is not a new observation, but its manifestation in hydrogels creates more uniform and complete crosslinked networks, and is further demonstrated by the fact that the error bars of each datum are smaller for the

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Fig. 1. Hydration of hydrogels as a function of crosslinker content. The equilibrium water content of thermally polymerized CBMAX-1-crosslinked hydrogels (circles with solid line to guide the eye, from reference 31) is compared to photopolymerized CBMAX-1 and -2-crosslinked hydrogels (triangles and diamonds, respectively, with dashed lines to guide the eye).

photopolymerized hydrogels. By contrast, the error bars of the equilibrium water content of thermally polymerized hydrogels are relatively large, which reveals that there are many more imperfections in the hydrogel polymer network and that spatial variations are prevalent. The second important difference in the hydration-crosslinker content profiles is that the thermally polymerized hydrogels have higher equilibrium water content than photopolymerized hydrogels of the same nominal crosslinker content. This is attributed to a more erratic polymer mesh network, which, again, results from the unreliable chain initiation and propagation characteristic of less controlled polymerization techniques. Imperfections in the mesh network permit more water infiltration. Both of the photopolymerized hydrogel systems, CBMAX-1 and CBMAX-2, had lower equilibrium water content values than thermally polymerized hydrogels, but of these two CBMAX-1 had the lowest values. At lower crosslinker content, the CBMAX-1 and CBMAX-2 hydrogels exhibited identical equilibrium water contents, but the relationships diverge when the crosslinker becomes the dominant component of the hydrogel formulation. It is widely accepted that longer side-chains result in lower degrees of polymerization, and it is interesting to note that the same appears to hold true for the CBMAX-1 and CBMAX-2 crosslinkers: the CBMAX-2 has a slightly longer side-chain than the CBMAX-1. Thus, because the equilibrium water contentcrosslinker content relationship of CBMAX-2 hydrogels was also very smooth, its higher values relative to CBMAX-1 hydrogels at higher crosslinker content can attributed to lower degree of crosslinker polymerization, and not to a less uniform polymer network. 3.2. Mechanical properties We next tested the effect of switching from a thermally initiated hydrogel system to a photoinitated system on the mechanical properties of the CBMAX-1 crosslinked hydrogels. Fig. 2 shows the compressive modulus as a function of crosslinker content for these systems. In keeping with the well-understood trade-off between hydration and mechanical strength, the compressive moduli of photopolymerized hydrogels, ranging from 0.43 MPa for 2% crosslinker to nearly 90 MPa for 100% crosslinker, were higher than those of their thermally polymerized counterparts [31], which only attained approximately 9 MPa for 100% crosslinker. The order-ofmagnitude increase was greater than expected, and highlights just how sensitive the mechanical properties are on the uniformity of the polymer mesh network. Photopolymerization, which promotes

Fig. 2. Mechanical properties of hydrogels as a function of crosslinker content. The compressive modulus of thermally polymerized CBMAX-1-crosslinked hydrogels (circles with solid line to guide the eye, from reference 31) is compared to photopolymerized CBMAX-1 and -2-crosslinked hydrogels (triangles and diamonds, respectively, with dashed lines to guide the eye).

more controlled, and thereby more uniform, polymerization is thus a powerful tool for creating stronger polymer networks. Photopolymerized hydrogels crosslinked with CBMAX-2 also demonstrated vastly improved mechanical properties relative to the thermally polymerized hydrogels [31], but they are unable to attain moduli quite as high as the photopolymerized CBMAX-1 crosslinked hydrogels. This can also be attributed to the lower degree of crosslinker polymerization, which results in shorter polymer chains within the uniform polymer mesh network. Again, the importance of a uniform mesh network is highlighted by comparing the hydration and mechanical properties of the thermally polymerized CBMAX-1 hydrogels to those of photopolymerized CBMAX-2 hydrogels: at 100% crosslinking, both hydrogels have the same approximate equilibrium water content (z60%) and yet very different compressive moduli (9 MPa for thermally polymerized and 60 MPa for photopolymerized). This implies that the structural strength imparted by the uniformity of the photopolymerized network is more than enough to overcome the shorter polymer chain lengths that result from lower a degree of polymerization. 3.3. Functionalization One of the prime motivations to introduce the CBMAX-2 crosslinker is that the 2-carbon spacer will serve to separate the electron withdrawing quaternary amine from the carboxylic acid of the zwitterionic group. When the amine is too close to the carboxyl group, the carboxyl group is less easily protonated. Protonation is an essential step for simple EDC-NHS chemistry, and protonation under mild conditions is desirable. EDC-NHS chemistry was performed on hydrogels of very low crosslinker content (i.e. mostly monomer, which itself has 2 carbons between the amine and the carboxyl group) and of 100% crosslinker, either CBMAX-1 or CBMAX-2, to attach the cell-binding peptide cyclicRGD. The results of this study are shown in Fig. 3. Because the zwitterionic hydrogels have been shown to have very low non-specific cell binding [28,29], no cell binding was expected on the unfunctionalized hydrogels, and the low cell binding levels observed here are consistent with previous studies. The CBMAX-1 hydrogel, with its 1-carbon spacer, showed no change in cell adhesion, indicating that EDC-NHS chemistry was unable to successfully functionalize it with cRGD. The CBMAX-2 hydrogel and the low-crosslinker hydrogel both have 2 carbons separating the functional group from the electron-withdrawing group, and are thus functionalizable. Both hydrogels exhibited

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Fig. 3. Functionalization of CB crosslinkers (CBMAX-1 and CBMAX-2 representing crosslinker with 1-carbon spacer and 2-carbon spacer, respectively) and monomer (CBMA, which has a 2-carbon spacer). Gray bars represent non-specific cell adhesion per untreated hydrogel, while black bars represent integrin-mediated cell adhesion on cyclicRGDfunctionalized hydrogels. Figures b and c are representative images of hydrogels either functionalized (c) or not (b). CBMA hydrogels are 95% monomer, and are lightly crosslinked with 5% crosslinker.

a drastic increase in cell binding upon EDC-NHS chemistry with cRGD. Importantly, the cells adhered to the functionalized hydrogels are bound through integrin-mediated binding, which demonstrated the ability to gain control over cell behavior via hydrogel functionalization. This can be seen in Fig. 3b and c, which shows representative microscope images of the surfaces of hydrogels untreated (b) or functionalized (c) with cRGD. The cells that do remain attached to the untreated hydrogel surface after a PBS wash exhibit a rounded morphology, indicative of poor adhesion, whereas the vast majority of the many cells on the surface of cRGD-functionalized hydrogels exhibit the flattened and spread morphology that is the hallmark of healthy cells that are strongly attached through multiple integrinadhesion foci. This suggests that the level of functionalization is high enough to effectively influence the hydrogels’ interaction with a biological interface. 3.4. Long-term stability To test the ability of these hydrogels to resist degradation in an oxidative environment, hydrogel disks were incubated for 24 h at 37  C in pure water, 5% hydrogen peroxide, or 10% hydrogen peroxide. After 24 h, they were washed overnight in pure water to remove any degradation products. The compressive modulus of each disk was measured. Due to the zwitterionic nature of the hydrogels, the compressive modulus will be a sensitive means of analyzing the integrity of the side-chains as well as the structural integrity of the hydrogel in general. Cleavage of the polymer backbone would fragment the hydrogel and reduce the compressive modulus, while cleavage of the methacrylate ester would release the zwitterionic group into solution. Depending on the pH of the solution and the pKa of the resulting carboxyl groups, methacrylate ester cleavage could

convert the formerly zwitterionic hydrogel to an anionic hydrogel, which would be indicated by dramatic swelling and reduced compressive modulus. Alternatively, side-chain cleavage would result in a switch from a zwitterionic to a hydrophobic polymer network. The latter would be characterized by a drastic reduction in swelling and a corresponding increase in compressive modulus. As indicated in Fig. 4a, hydrogels incubated for 24 h at 37  C in hydrogen peroxide solutions exhibited no change in compressive modulus relative to the compressive modulus of hydrogels incubated in pure water under the same conditions. Thus, the hydrogels appear to be impervious to oxidative degradation after 24 h in harsh oxidative environments. We also tested the ability of these hydrogels to withstand degradation in acidic and basic conditions over long periods of time. Every 20 days for 100 days, the compressive modulus of disks of either 95% monomer (5% crosslinker) or 100% crosslinker was measured. These data are shown in Fig. 4b. In this figure, the 95% monomer hydrogel is shown with a solid line and the 100% crosslinker hydrogel is shown with a dashed line. The different environmental conditions are distinguished by the shape of the markers: acidic condition (pH ¼ 5) is represented by gray diamonds, neutral condition (pH ¼ 7) is represented by black squares, and basic condition (pH ¼ 9) is represented by white triangles. In order to be shown on the same scale, the values have been normalized to the initial modulus for each respective hydrogel. As can be seen from this figure, the hydrogels were able to retain their compressive modulus relative to their initial value in neutral conditions. Under harsher biologically relevant conditions, the hydrogels all show good long-term stability, either by remaining very close to their initial values or by drifting slightly and leveling off for the remainder of the assay. These data show that the

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and demonstrated good stability in oxidative, acidic, and basic environments.

Acknowledgement This work was supported by the Office of Naval Research (N000140910137).

References

Fig. 4. Resistance of hydrogels to oxidative degradation in 5% and 10% hydrogen peroxide (a). The compressive modulus of 95% CBMA (gray bars) and 100% CBMAX (black bars) hydrogels after overnight incubation at 37  C was used as an indicator. Resistance to degradation in acidic and basic conditions is shown in (b), where 95% CBMA is shown with solid lines and 100% CBMAX is shown with dashed lines. Acidic condition (pH ¼ 5) is represented by gray diamonds, neutral condition (pH ¼ 7) is represented by black squares, and basic condition (pH ¼ 9) is represented by white triangles For comparison of the different hydrogel formulations, absolute compressive modulus values were normalized to the respective pure water value for (a) and the day 0 value for (b). For the absolute values of these moduli, please refer to Fig. 2.

hydrogels are able to maintain their bulk properties very well over very long periods of time in harsh conditions. 4. Conclusions The new zwitterionic carboxybetaine dimethacrylate crosslinker with two carbons separating the opposing charges is easily functionalizable. This means that, unlike in CBMAX-1 systems, there is no trade-off between mechanical strength and functionalizability. In addition, zwitterionic CBMA hydrogels photopolymerized with carboxybetaine dimethacrylate crosslinkers show a dramatic increase in network homogeneity, as evidenced by the order-of-magnitude improvements in their compressive modulus relative to thermally polymerized hydrogels with the same crosslinker contents. Varying the crosslinker content enables hydrogels to be made with compressive moduli ranging from 0.5 to 90 MPa, which is a wide enough range to include many native tissue properties. Thus, with this photopolymerizable system, hydrogels can be made with controllable bioactivity on a nonfouling background and a versatile range of mechanical properties. Finally, all hydrogels exhibited nonfouling behavior,

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