Article pubs.acs.org/Langmuir
New Antifouling Silica Hydrogel Á ngela A. Beltrán-Osuna,† Bin Cao,‡ Gang Cheng,*,‡ Sadhan C. Jana,*,† Matthew P. Espe,§ and Bimala Lama§ †
Department of Polymer Engineering, ‡Department of Chemical and Biomolecular Engineering, and §Department of Chemistry, University of Akron, Akron, Ohio 44325, United States ABSTRACT: In this work, a new antifouling silica hydrogel was developed for potential biomedical applications. A zwitterionic polymer, poly(carboxybetaine methacrylate) (pCBMA), was produced via atom-transfer radical polymerization and was appended to the hydrogel network in a two-step acid−base-catalyzed sol−gel process. The pCBMA silica aerogels were obtained by drying the hydrogels under supercritical conditions using CO2. To understand the effect of pCBMA on the gel structure, pCBMA silica aerogels with different pCBMA contents were characterized using scanning electron microscopy (SEM), nuclear magnetic resonance (NMR) spectroscopy, and the surface area from Brauner−Emmet−Teller (BET) measurements. The antifouling property of pCBMA silica hydrogel to resist protein (fibrinogen) adsorption was measured using enzyme-linked immunosorbent assay (ELISA). SEM images revealed that the particle size and porosity of the silica network decreased at low pCBMA content and increased at above 33 wt % of the polymer. The presence of pCBMA increased the surface area of the material by 91% at a polymer content of 25 wt %. NMR results confirmed that pCBMA was incorporated completely into the silica structure at a polymer content below 20 wt %. A protein adsorption test revealed a reduction in fibrinogen adsorption by 83% at 25 wt % pCBMA content in the hydrogel compared to the fibrinogen adsorption in the unmodified silica hydrogel.
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INTRODUCTION Protein adsorption on solid surfaces is a critical issue in biomedicine and related fields. Because protein adsorption can lead to blood coagulation and associated complications, highly protein-resistant materials are of active interest in biomedical research.1 Inert or “antifouling” surfaces are desired in an array of applications (e.g., substrates for cell culture, protein purification, prostheses, contact lenses, catheters,2 tissue engineering, microfluidic and analytical systems,3 implantable devices, biosensors, and drug-delivery carriers4). An antifouling surface is characterized in terms of strong resistance to the nonspecific adsorption of proteins from an aqueous solution.2 Recently, zwitterionic polymers have attracted considerable attention because of their excellent antifouling properties (e.g., in the form of poly(sulfobetaine methacrylate), pSBMA,5 and poly(carboxybetaine methacrylate), pCBMA6). Both pSBMA and pCBMA coatings are found to be highly resistant to nonspecific protein adsorption7−9 and platelet adhesion.10,11 Zwitterionic materials are known to reduce bacterial adhesion dramatically12 and therefore biofilm formation.13 Among zwitterionic materials, pCBMA is considered to be one of the best candidates for rendering nonfouling surfaces. pCBMAcoated surfaces can prevent protein adsorption even when using undiluted blood plasma1 and serum,14 and protein adsorption on pCBMA-coated surfaces is less than 0.3 ng/cm.6 Most prior work involved the coating of flat, solid surfaces, but the use of gels has a promising future in solving some of the new challenges of biocompatibility requiring new materials, functions, and specific responses.15 Polymeric hydrogels have been extensively used in many biomedical and pharmaceutical © 2012 American Chemical Society
applications such as controlled drug release and delivery, tissue engineering, surface coating for implantable biosensors, and regenerative medicine.16 Although hydrogels have been broadly used in biomedicine,17 the application of aerogels as a host matrix for biomaterials has attracted intense interest only in the past decade.18 Aerogels can be used in implantable devices15 or leaflet material for artificial heart valves,19 among many other applications.20,21 For example, silica aerogels are biocompatible and have been tested positively as potential drug-delivery systems due to their extremely large surface area.18,22,23 Also, other silicon-based materials (e.g. silica particles) have been used lately for many biomedical technologies.24,25 However, no previous work reported silica hydrogels with antifouling functions. It is perceived that this kind of material is highly desirable in biomedical applications. At the outset of this work, we show that silica hydrogels modified with pCBMA molecules have the potential to provide antifouling properties because of the presence of zwitterionic molecules. There are several potential applications for these materials (e.g., blood filtration, scaffold design, and carriers for drug delivery, among many others). For example, this new material can be used as a filter medium for the separation of bacterial impurities from air and water, where silica hydrogels fulfill the high-surface-area requirements for a good separation medium and the antifouling characteristics of the pCBMA brush can prevent biofilm Received: April 17, 2012 Revised: May 16, 2012 Published: May 18, 2012 9700
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Scheme 1. Synthesis Path of pCBMA by ATRP
Synthesis of BrTEOS. ATRP initiator BrTEOS was synthesized according to the chemical reaction shown in Scheme 1. The following procedure was used: APTES (22.14 g, 0.100 mol) was mixed with triethylamine (11.13 g, 0.110 mol) in 125 mL of dried THF, and the solution was added to a 500 mL three-necked flask. BIBB (25.29 g, 0.110 mol) was dissolved in 25 mL of dried THF, and the solution was added dropwise to the flask for 30 min with stirring, avoiding any trace of water. The reaction was carried out for 24 h with stirring under a nitrogen atmosphere in an ice/water bath at 4 °C. After 24 h, the precipitate was filtered off using a fritted funnel. After the removal of the solvent, the product obtained was a yellowish oil. The product was redissolved in toluene and washed a number of times with aqueous solutions of sodium bicarbonate (NaHCO3, 1 N) and a saturated solution of NaCl. The organic phase was dried completely with anhydrous CaCl2. After the removal of the solvent, the final product was obtained as a colorless oil with an approximate yield of 90%. Atom-Transfer Radical Polymerization (ATRP) Reaction. The synthesis of pCBMA was achieved by using ATRP as shown in Scheme
formation, thus extending the service life of the separation medium. This article describes the development of new antifouling silica hydrogels and aerogels with the aim of combining the antifouling characteristics of pCBMA with the high surface area and large porosity of silica hydrogels and aerogels. Some attributes of these materials are the ability to resist the nonspecific adsorption of proteins, a high surface area, and nanoporous structures. In this study, 3-aminopropyltriethoxysilane (APTES) was modified to obtain a functional group for the initiation of the atom-transfer radical polymerization (ATRP) process starting with carboxybetaine methacrylate (CBMA) as the monomer. The polymer product obtained, pCBMA, was mixed in different weight ratios with tetraethyl orthosilicate (TEOS) to obtain a modified silica substrate. The silica hydrogel was characterized using an enzyme-linked immunosorbent assay (ELISA) to evaluate the antifouling properties of the new material. Other characterization techniques involved nuclear magnetic resonance (NMR) spectroscopy, the surface area from the Brunauer−Emmett− Teller (BET) method, and scanning electron microscopy (SEM).
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Table 1. Reaction Conditions Used for pCBMA Synthesis by ATRP molar ratio
MATERIALS AND METHODS
Materials. The following materials were used in this work: (3aminopropyl) triethoxysilane (APTES, 99%), bromoisobutyryl bromide (BIBB, 98%), triethylamine (TEA), 2-(N,N′-dimethylamino)ethyl methacrylate (DMAEM, 98%), β-propiolactone (95%), copper(I) bromide (CuBr, 99.999%), copper(II) bromide (CuBr2, 99.999%), 2,2′-bipyridine (BPY, 99%), tetraethyl orthosilicate (TEOS, ≥ 99.0%), methanol (CH3OH, ≥ 99.5%), ethanol (CH3CH2OH, ≥ 99.5%), nitric acid (HNO3, 70%), ammonium hydroxide (NH4OH, 28%), fibrinogen (Fg, from human plasma), phosphate-buffered saline (PBS, 0.01 M phosphate, 0.138 M sodium chloride, 0.0027 M potassium chloride, pH 7.4), HNO3 and NH4OH solutions, and bovine serum albumin (BSA, 98%). These materials were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as received without further purification. Deuterated chloroform (CDCl3) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Horseradish peroxidase conjugated antifibrinogen antibody (HRP-anti-Fg) was purchased from United States Biological (USA). Tetrahydrofuran (THF, HPLC grade) was obtained from Sigma-Aldrich and dried with sodium before use. 2-Carboxy-N,N-dimethyl-N-(2′-(methacryloyloxy)ethyl)ethanaminium inner salt (carboxybetaine methacrylate, CBMA) was synthesized by the reaction of DMAEM and β-propiolactone using a method published elsewhere.6,8
reaction
BrTEOS:
CBMA:
BPY:
CuBr:
CuBr2
Mn
PDI
1 2 3
1: 1: 1:
10: 10: 5:
2: 2: 2:
1: 0.9: 1:
0 0.1 0
24 310 68 980 55 440
2.3 3.2 3.6
1. Table 1 presents molar ratios of various components used in the synthesis of pCBMA. In a typical procedure, appropriate quantities of BrTEOS initiator, 2,2′-bipyridine (BPY), and copper bromide (I and II) (CuBr, CuBr2) were mixed in a 100 mL glass reactor and deoxygenated by five vacuum/nitrogen (>99.999%, Praxair, USA) cycles. The monomer, CBMA, was added to another flask and also deoxygenated by five vacuum/nitrogen cycles. The solvent, methanol or a mixture of methanol and DMF, was first bubbled with nitrogen gas for 30 min. Three mL of deoxygenated methanol was added to dissolve the reagents in the reactor, and another 7 mL of methanol was used to dissolve the monomer. The reaction started when the monomer/solvent solution was added at once to the reactor. The reaction solution was magnetically stirred under nitrogen protection at 30 °C for 18 h. After the reaction was stopped, the product was purified by dropping it slowly into ethyl ether. The polymer precipitate was separated by centrifugation at 12000g for 15 min and redissolved in methanol. The polymer was washed three times and dried under vacuum overnight, and a powder was obtained as the product. 9701
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Figure 1. 1H NMR spectrum for the BrTEOS initiator in CDCl3. refractive index detector and HR2 and HR4 Styragel columns (7.8 × 300 mm2). Samples were prepared by dissolving 20 mg of the polymer in 5 mL of the solvent and filtered with 0.2 μm PTFE filters. GPC was operated at a flow rate of 0.30 mL/min using DMF as the solvent and polystyrene standards to generate a universal calibration curve. Data were recorded and processed using the Breeze software package. SEM and BET. The morphology of the aerogels was examined by using a Hitachi S-2150 scanning electron microscope. The surface area of aerogels was obtained by following the BET method and using ASAP 2020 micromeritics equipment. The surface area was computed from N2 adsorption curves following the Barret−Joyner−Halenda (BJH) method using a bath temperature of 77.05 ± 0.02 K. Approximately 0.1100 g of an aerogel was used for this purpose with an automatic degassing option, a cold free space of 0.6104− 0.8228 cm3, a warm free space of 0.899−0.954 cm3, and equilibrium intervals of 45 s each. Protein Adsorption. Protein adsorption was characterized by using an enzyme-linked immunosorbent assay (ELISA). The samples used for this test were small disks of silica hydrogel with average measurements of 1 cm diameter and 5 mm height. Each sample was placed in a well of a 24-well plate, and 1 mL of 1 mg/mL human fibrinogen solution in PBS was added to each sample and held for 90 min. The samples were then incubated in a 1 mg/mL BSA solution in PBS for 90 min to block the areas not occupied by fibrinogen. Each sample was then kept for 30 min in 1 mL of a PBS solution containing 5.5 μg/mL of HRP-anti-Fg in PBS. The samples were rinsed five times with 1 mL of a PBS solution after each step, with 20 min of elapsed time after each wash. Later, the samples were placed into new wells, followed by the addition of 500 μL of a 0.1 M citrate-phosphate buffer at pH 5.0 containing 1 mg/mL o-phenylenediamine and 0.03% hydrogen peroxide, and the color developed with time. The enzyme activity was stopped any time by adding an equal volume of 2 N H2SO4 solution after 15 min at room temperature. The absorbance of the supernatant was measured at 492 nm.
Preparation of Silica Hydrogels and Aerogels. The preparation of silica hydrogels requires four reagents: TEOS, water, alcohol, and the pCBMA polymer. A two-step acid−base-catalyzed sol−gel process was used for the production of the gels. In this method, the molar ratio of water/TEOS was 5:1 and methanol was used as the solvent, whereas the concentration of Si from the silane precursor, TEOS, was 1 M, and the concentration of pCBMA was varied between 0 and 50 wt % in the gel. First, HNO3 was added as the catalyst, and the mixture was hydrolyzed for 1 h at pH 2. NH4OH was then added to the solution to obtain a pH of 9. The reacting solution was quickly poured into the mold for gelation. The mold was a plastic syringe with the top cut off and the plunger pulled back along the inside tube to make the desired volume for the sol. After gelation, the gel was easily pushed out of the syringe, thus allowing the production of cylindrically shaped gels with an 8 mm diameter and the length varying from 3 to 4 mm (small disks) to 4 cm (complete monoliths). The silica hydrogels obtained were blue because of trace amounts of copper present in them from the ATRP reaction. The color was removed by soaking the gels several times in a 50 mM EDTA solution. Monolith aerogels were obtained after drying the gels in a supercritical dryer using liquid CO2. The supercritical dryer consisted of a chamber where the gels were placed while the pressure (P) and the temperature (T) were controlled. First, the gels were washed four times by soaking in liquid CO2 at 1000 psi. At least 3 h was allowed between washes to reach steady and homogeneous conditions inside the chamber. In the second step, the temperature was raised to 48 °C while the pressure reached a value of 1500 psi. The temperature and pressure were kept constant in the system for 1 h, after which the chamber was depressurized very slowly at constant T. To remove any water adsorbed by the hydrophilic surface of the aerogels, the samples were always oven dried at 30 °C under vacuum overnight before any characterization was performed. NMR. 1H solution NMR was carried out on a Varian Mercury 300 MHz spectrometer for the BrTEOS initiator in CDCl3, and pCBMA polymers and pCBMA aerogels were analyzed by solid-state 13C and solid-state 29Si NMR. Solid-state 13C NMR spectra at 100.5 MHz were collected on a Varian INOVA 400 MHz (9.4 T) spectrometer using a Varian 4 mm DR-T3 probe. Samples were packed into 4 mm ceramic rotors, and all spectra were collected with magic-angle spinning (MAS) at a spinning speed of 12 kHz. Cross-polarized 1H, 13C, and 29Si data were acquired using 90° pulse widths of 4.0 and 5.0 μs. 13C chemical shifts were referenced to hexamethylbenzene (17.3 ppm, methyl), and 29 Si chemical shifts were referenced to 2,2-dimethyl-2-silapentane sulfonate (1.46 ppm) using a recycle delay of 2 s for all experiments. Gel Permeation Chromatography (GPC). GPC was used to determine the molecular weight and polydispersity index, PDI, of the polymer in a Waters 515 series GPC system with a Waters 2414
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RESULTS AND DISCUSSION Polymer Synthesis. The ATRP initiator, BrTEOS, was successfully synthesized and characterized by 1H solution NMR, as shown in Figure 1. The pCBMA polymer was synthesized using ATRP. The molecular weight (Mn) and polydispersity index (PDI) of pCBMA were measured via GPC and summarized in Table 1. The measured molecular weights for pCBMA polymers do not match the expected values, and the PDI values obtained are broad (Table 1). A narrow PDI (1.02−1.05) is usually expected for the ATRP reaction from the literature,26 but it may range widely depending on each system. 9702
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presence of the aerogel. Remaining carbons I and b from the CBMA unit give rise to the broad peak at 20 ppm. The 13C spectrum for the aerogel with the highest pCBMA content, 50 wt % (Figure 2b), shows a mixture of sharp peaks and broader peaks. These results indicate that two populations of polymer chains are present in the polymer-doped aerogel materials. In the aliphatic region of the 13C spectrum of the aerogel at 20 wt % pCBMA, the sharp peak at 62 ppm is assigned to the CH2− carbons (e, c) attached to the nitrogen atom of the CBMA unit on the basis of solution 13C NMR. The peak at 58 ppm is due to the CH2− carbon of the polymer backbone (j) and the carbon in COO−CH2 (f). The two CH3 carbons (d) attached to the nitrogen atom give rise to the peak at 52 ppm. The peak near 45 ppm is from carbons h, k, and o. Because the APTES content is very low in comparison to the CBMA repeat unit content, there is very little contribution from carbons k and o. The other APTES carbons (p, q, and l) are very difficult to see and have not been identified in the 13C spectrum. Chemical shifts of the three carbonyl groups are found at COO− (172 ppm, g), CO−NH (172 ppm, m), and COO− (178 ppm, a). In the case of the solid polymer, only a single broad peak is observed at 178 ppm (Figure 2c). Again, the broad peak is indicative of substantial structural disorder at the carbonyl sites. The peak from the carbonyl carbons in the aerogel at low pCBMA content (Figure 2a) is sharp and shifted to 172 ppm. The upfield shift is consistent with the COO− group being involved in a charge neutralization interaction, such as hydrogen bonding. Because this shift is observed only in the presence of the aerogel, the most likely hydrogen bond donors are the surface Si−OH groups that are present in high abundance. In the 13C spectrum of pCBMA/aerogel at 50 wt % (Figure 2b), the carbonyl region shows two peaks corresponding to two polymer morphologies. Thus, the broad peak, with a line width and chemical shift that are the same as that observed for the neat polymer, arises from polymer domains with bulk polymer morphology. Similar results are also observed in the aliphatic carbon region of the spectrum from pCBMA/aerogel at 50 wt %, confirming the presence of two polymer domains at high doping levels. 29 Si solid-state NMR was also used to characterize aerogels containing different concentrations of pCBMA; the results are shown in Figure 3. The silica aerogel with no pCBMA was used as a reference. The aerogel network produces peaks in the 29Si NMR spectra at −94, −104, and −115 ppm from [Si(−OSi)2(−OH)2, Q2], [Si(−OSi)3(−OH), Q3], and [Si-
For example, PDI varies from 1.02 to 2.97 for PDMA grown on a charged surface,27 but also values of 2.89−3.97 are found for thermoresponsive polymers of IPPAm-co-BMA grafted on silica beds28 and are usually around 1.2−1.5 for amphiphilic and zwitterionic polymer brushes produced via ATRP.29−31 It is very challenging to control and measure the molecular weight and polydispersity index of the pCBMA polymers with a terminal triethoxylsilane group. Because the CBMA monomer and pCBMA polymer are not soluble in a majority of the organic solvents, in this study we use methanol or mixed methanol/DMF as solvents. In an aqueous solvent or polar organic solvent with high water content, the terminal triethoxysilane group in pCBMA will hydrolyze and polymerize, thus leading to an increase in molecular weight and polydispersity. Although the pCBMA polymer is very soluble in water, aqueous GPC cannot be used to measure the molecular weight of the pCBMA polymer with the terminal triethoxylsilane group. Because triethoxysilane will react with hydroxyl-based aqueous GPC columns, in this work we use an organic-phase GPC with DMF as the solvent. We believe that the high molecular weight and polydispersity index are caused by the trace amount of water in the reaction solution and GPC solvent. Solid-State NMR Analysis. Aerogels were characterized by 13 C CP/MAS solid-state NMR, as shown in Figure 2. The 13C
Figure 2. 13C CP/MAS solid-state NMR spectra of (a) a silica aerogel with 20 wt % pCBMA, (b) a silica aerogel with 50 wt % pCBMA, and (c) unreacted pCBMA.
NMR spectrum from unreacted pCBMA (Figure 2c) consists of four broad peaks at 20, 45, 55, and 178 ppm. The peak at 20 ppm ranges from 10 to 30 ppm, including carbons s, q, p, l, and i. The carbon peak at 45 ppm is from carbons b, d, h, k, and o. The broad peak at 55 ppm is due to carbons c, e, f, j, and r. The polymer contains three carbonyl groups, COO− at 172 ppm (g), CO−NH at 172 ppm (m), and COO− at 178 ppm (a), giving rise to only one broad peak at 178 ppm. In polymer samples, broad peaks arise from the structural heterogeneity present in amorphous materials. Because the peaks are broad, it is not possible to assign these chemical shifts more specifically to each type of carbon present in the polymer. However, in the 13 C spectrum from the lowest pCBMA content in the aerogel at 20 wt % (Figure 2a), sharper and much better resolved peaks are observed. This change in line width implies that the polymer has adopted a more uniform structure when in the
Figure 3. 29Si CP/MAS solid-state NMR spectra of (a) the silica aerogel with no pCBMA and the silica aerogel with pCBMA at (b) 20, (c) 33, and (d) 50 wt % and (e) unreacted pCBMA. 9703
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Figure 4. SEM pictures of silica aerogels with (a) 0, (b) 20, (c) 33, (d) 43, and (e) 50 wt % pCBMA.
(−OSi) 4 ,Q 4 ] groups, respectively (Figure 3a−d). The population of Q2 and Q3 sites is much larger than that of Q4 sites because of the high surface area of the material. Thus, only a small population of silica is involved in making a silica network, and a large fraction of silica is on the surface as expected. Because pCBMA contains APTES ends, peaks at −46, −50, −57, and −65 ppm in the 29SiNMR spectra are due to the presence of R−Si(−OR)3, R−Si(−OSi)OR2, R− Si(−OSi)(OH)2, and R−Si(−OSi)2OH, respectively (Figure 3e). The high concentration of species such as R−Si(−OSi)2OH and R−Si(−OSi)(OH)2 also shows that APTES ends of the polymer are interacting with each other. Hydrolysis and condensation reactions form Si−O−Si bonds between two APTES molecules, which gives the peaks at −57 and −65 ppm in 29Si NMR spectra.32−34 These reactions may occur during the purification and storage of the polymer. The peaks at ∼−70 ppm (Figure 3b−d) are due to the APTES end group of pCBMA. The chemical shift is consistent with the structure of R−Si(−OSi)3 and shows that for low pCBMA content (20 wt %) all or nearly all of the APTES from pCBMA was integrated into the aerogel network. As the fraction of polymer in the samples increases, an additional peak is observed at −65 ppm in the 29Si NMR. This peak is assigned to APTES end groups that have not undergone complete hydrolysis and produce the species R−Si(−OSi)2(OH). Morphology, Density, and BET Surface Area. SEM images of the aerogels are shown in Figure 4. The particle size and porosity show two different trends. At low pCBMA content (0−20 wt %), the presence of the polymer in the aerogel decreases the particle size of the silica network in comparison to that in the unmodified silica aerogel, whereas at higher pCBMA content (33−50 wt %) the particle size increases. This dramatic change in the morphology is evident from the large particle agglomerates (Figure 4e). These agglomerates can be attributed first to the formation of polymer brushes on the silica networks and second to the collapse of the silica network at a critical concentration of polymer brushes. In addition, the low solubility of the pCBMA in liquid CO2 may have also contributed to the collapse of the silica networks during the supercritical drying process. Therefore, SEM images clearly show a change in the porous structures of the aerogels in the presence of pCBMA. Bulk densities of the silica aerogel samples are calculated on the basis of the weight and volume of each sample. The density of the silica aerogels as a function of the polymer content is shown in Figure 5, where it is clear that the presence of pCBMA in the aerogel caused a significant modification of the porous structure, especially at low pCBMA content. The surface area of aerogels as a function of pCBMA content is shown in Figure 6. A third-order polynomial regression equation (y = 0.0031x3 − 2.0163x2 + 94.15x + 1257.6) was used to interpolate the data points. This correlation was
Figure 5. Bulk density of silica aerogels as a function of pCBMA content in the gel.
Figure 6. Surface area of silica aerogels vs pCBMA content in the gels. The surface area of the silica aerogels is measured by BET.
computed with the aim of comparing the BET surface area with the ELISA test values obtained for the gels, as explained below. It is noticed from Figure 6 that the addition of pCBMA opened up the network structure of the aerogel, thus explaining the increase of porosity and reduction of the surface area. This trend was true for aerogels with polymer content ranging from 33 to 50 wt %, as was confirmed from SEM images (see Figure 4c-e). It is also clear from Figure 6 that aerogels with low polymer content (below 20 wt %) behaved differently, reaching a suggested maximum value of surface area at about 25 wt %. It is evident that the surface area increased by 91% over that of unmodified silica aerogels in the presence of pCBMA of up to 25 wt %. Protein Adsorption Test. A low protein adsorption value was expected for a less-fouling material, indicating that less protein adsorbed on the surface. The ELISA method was used to measure the protein adsorption on the pCBMA silica hydrogel. Three pCBMA silica hydrogel specimens in the form of disks were tested at each polymer content to calculate the 9704
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The minima in the protein adsorption value as a function pCBMA concentration may be explained on the basis of the kinetics of the sol−gel process. It appears that the reaction rate of pCBMA on the silica networks may have been reduced by steric effects between the polymer molecules at higher concentrations of pCBMA. This possibly reduced the packing density of the polymer and led to a reduction of its antifouling properties. In view of this argument, an increase in the pCBMA content above a critical value (e.g., 25 wt %) cannot further reduce the adsorption of the protein on the materials. Further investigation is necessary to obtain a better understanding of the surface distribution of the polymer on the hydrogel and to study how this can quantitatively affect the protein adsorption values.
average protein adsorption on the gels. Then, the optical density of the supernatant at 492 nm for each sample was obtained with a UV−vis spectrophotometer and normalized by the area of each specimen and also the value obtained for the unmodified sample (i.e., a silica aerogel with no pCBMA). The protein adsorption value reported in this article was obtained from eq 1. relative protein adsorption ⎡ ODsample /A sample ⎤ ⎥× 100% =⎢ ⎣ ODunmodified /A unmodified ⎦
(1)
where OD denotes the optical density at 492 nm and A denotes the surface area of the specimen (Figure 6). Note that this differs from the conventional data treatment for ELISA, where usually flat surfaces are used and where the area involved in the calculation is the area of the surface of the specimen. However, in this research, it was imperative to take into account the BET surface area of the porous structures as the entire area accessible for protein adsorption. Thus, data correlations were required to calculate densities and BET surface areas from Figures 5 and 6 and apply those to the normalization of the values obtained for the ELISA adsorption test. The results of this calculation are shown in Figure 7.
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CONCLUSIONS A water-soluble material, poly(carboxybetaine methacrylate) (i.e., pCBMA), was incorporated into silica hydrogels to obtain antifouling materials with high surface areas. NMR results for the aerogels showed that pCBMA was successfully incorporated into the gel structure at low concentration. At higher concentration (above 33 wt %), the polymer does not entirely interact with the silica network and instead interferes with the formation of gel networks. SEM images of the aerogels qualitatively showed that for concentrations below 20 wt % the presence of the polymer actually reduced the silica particle size compared to those seen in the unmodified gel. However, for pCBMA content above 33 wt %, an increase in the concentration of polymer in the formulation led to an increase in the particle size, thus considerably modifying the porous structure of the network. The BET surface area values quantitatively corroborated the observations made from SEM images. Thus, for the silica aerogel with a low pCBMA content (below 20 wt %), the presence of pCBMA generated an increase in the surface area of the aerogel, whereas at high pCBMA content (above 33 wt %) the polymer molecules opened up the network structure, but at the cost of a reduction in surface area. Our data suggested that the maximum antifouling performance can be obtained from the silica hydrogel with 25 wt % pCBMA, corresponding to a 91% increase in the surface area and an 83% reduction in fibrinogen adsorption on the material in comparison to that on the unmodified hydrogel.
Figure 7. Protein adsorption results for silica hydrogels as a function of pCBMA content. Protein adsorption on all surfaces was normalized to a silica hydrogel with no pCBMA. Each result is the average of three replicate measurements.
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The results in Figure 7 show that hydrogels with pCBMA effectively reduce protein adsorption. The optimal antifouling property was obtained from the hydrogel with 25 wt % pCBMA, reducing the fibrinogen adsorption by 83%. Figure 7 shows that increasing the pCBMA content to over 25 wt % will not further improve the antifouling performance of silica hydrogels. This behavior may be explained taking into account that the polymer was not incorporated into the network structure at above 33 wt % of pCBMA. The polymer interacted with the silica hydrogel only at a relatively low polymer content (below 20 wt %), as was found from the NMR results. Thus, no improvement may be expected from increasing the pCBMA content in the gels if the polymer did not partially consist of the porous structures. These findings are also in agreement with the observations made from SEM images where the particle size and porosity of the sample were noticeably smaller for the aerogel with a lower pCBMA content (20 wt %), compared to the unmodified aerogel (Figure 4a,b).
AUTHOR INFORMATION
Corresponding Author
*(G.C.) E-mail:
[email protected]. (S.C.J.) E-mail: janas@ uakron.edu. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Steven Chuang of the University of Akron for allowing the use of the BET setup. This work was partially supported by NSF grant CMMI-1129727.
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REFERENCES
(1) Zhang, Z.; Zhang, M.; Chen, S.; Horbett, T. A.; Ratner, B. D.; Jiang, S. Blood compatibility of surfaces with superlow protein adsorption. Biomaterials 2008, 29, 4285−4291. (2) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Zwitterionic SAMs that resist nonspecific
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dx.doi.org/10.1021/la301561j | Langmuir 2012, 28, 9700−9706
