Tunable Bioadhesive Copolymer Hydrogels of Thermoresponsive

Biomacromolecules 2010, 11, 1101–1110

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Tunable Bioadhesive Copolymer Hydrogels of Thermoresponsive Poly(N-isopropyl acrylamide) Containing Zwitterionic Polysulfobetaine Yung Chang,*,† Wetra Yandi,† Wen-Yih Chen,‡ Yu-Ju Shih,† Chang-Chung Yang,§ Yu Chang,| Qing-Dong Ling,⊥ and Akon Higuchi‡ R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan, Department of Chemical and Materials Engineering, National Central University, Jhong-Li, Taoyuan 320, Taiwan, Energy and Environmental Laboratories, Industrial Technology Research Institute, Hsinchu 300, Taiwan, Department of Obstetrics and Gynecology, Chung-Ho Memorial Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan, and Cell Biology Laboratory, Cathay Medical Research Institute, Cathay General Hospital, Hsi-Chi City, Taipei 221, Taiwan Received January 26, 2010

This work describes a novel tunable bioadhesive hydrogel of thermoresponsive N-isopropylacrylamide (NIPAAm) containing zwitterionic sulfobetaine methacrylate (SBMA). This novel hydrogel highly regulates general bioadhesive foulants through the adsorption of plasma proteins, the adhesion of human platelets and cells, and the attachment of bacteria. In this investigation, nonionic hydrogels of polyNIPAAm, zwitterionic hydrogels of polySBMA, and three copolymeric hydrogels of NIPAAm and SBMA (poly(NIPAAm-co-SBMA)) were prepared. The copolymeric hydrogels exhibited controllable temperature-dependent swelling behaviors and showed stimuli-responsive phase characteristics in the presence of salts. The interactions of these hydrogels with biomolecules and microorganisms were demonstrated by protein adsorption, cell adhesion, and bacterial attachment, which allowed us to evaluate their bioadhesive properties. An enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies was used to measure different plasma protein adsorptions on the prepared hydrogel surfaces. At a physiological temperature, the high content of the nonionic polyNIPAAm in poly(NIPAAm-co-SBMA) hydrogel exhibits a high protein adsorption due to the interfacial exposure of polyNIPAAm-rich hydrophobic domains. A relatively high content of polySBMA in poly(NIPAAm-co-SBMA) hydrogel exhibits reduced amounts of protein adsorption due to the interfacial hydration of polySBMA-rich hydrophilic segments. The attachment of platelets and the spreading of cells were only observed on polyNIPAAm-rich hydrogel surfaces. Interestingly, the incorporation of zwitterionic SBMA units into the polyNIPAAm gels was found to accelerate the hydration of the cell-cultured surfaces and resulted in more rapid cell detachment. Such copolymer gel surface was shown to be potentially useful for triggered cell detachment. In addition, the interactions of hydrogels with bacteria were also evaluated. The polySBMA-rich hydrogels exhibited evident antimicrobial properties when they were incubated with Grampositive bacteria (S. epidermidis) and Gram-negative bacteria (E. coli). This work shows that the bioadhesive properties of poly(NIPAAm-co-SBMA) hydrogels can be effectively controlled via regulated nonionic and zwitterionic molar mass ratios. The tunable-bioadhesive behavior of temperature-sensitive poly(NIPAAm-coSBMA) makes this biocompatible hydrogel appropriate for biomedical applications.

Introduction The development of nonbioadhesive surfaces is highly desired for various biomedical applications and for biomaterials used as antifouling membranes, antithrombogenic implants, and antimicrobial coatings.1-9 However, only a very limited number of synthetic biomaterials are regarded as being good candidates for nonbioadhesive surfaces. A good nonspecific protein-fouling resistance is one of the key requirements for nonbioadhesive materials or for superlow-bioadhesive materials. When a protein approaches an interface, electrical neutrality may be important in minimizing the electrostatic interactions, and the absence of hydrogen-bond donors may also be important for minimizing * To whom correspondence should be addressed. E-mail: ychang@ cycu.edu.tw. † Chung Yuan Christian University. ‡ National Central University. § Industrial Technology Research Institute. | Kaohsiung Medical University. ⊥ Cathay General Hospital.

the hydrogen bonding interactions. Therefore, the functional groups on low-bioadhesive surfaces are generally hydrophilic, electrically neutral, and are hydrogen-bond acceptors rather than hydrogen-bond donors.10 Zwitterionic polymers containing the pendant groups of phosphobetaine, sulfobetaine, and carboxybetaine have received growing attention for their use in the new generation of nonbioadhesive materials because of their good plasma protein resistance.6,10-15 Poly(sulfobetaine methacrylate) (polySBMA) with a methacrylate main chain and a pendant group consisting of an analogue of the taurine betaine (CH2CH2N+(CH3)2CH2CH2CH2SO3-) has become the most widely studied zwitterionic polymer because it is easily prepared synthetically.4-7,12-15 Our previous work reported that the zwitterionic polySBMA is an effective and stable nonbioadhesive material, potentially providing a surface appropriate for use in human blood and tissue implants.6 In general, intelligent polymers can respond strongly to large physical conformation changes in environmental stimuli that occur over a small scale, such as physical, chemical, or

10.1021/bm100093g  2010 American Chemical Society Published on Web 03/04/2010

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Table 1. Characteristics of Poly(NIPAAm-co-SBMA) Hydrogels reaction ratios of comonomersa (wt %)

compositions of hydrogelsb (mol %)

characterization of cross-linked hydrogelsc

characterization of uncross-linked hydrogelsd

sample ID

SBMA

NIPAAm

polySBMA

polyNIPAAm

xSBMA

swelling ratio

contact angle

x′SBMA

LCST (°C)

S#0 S#20 S#50 S#70 S#100

0 20 50 70 100

100 80 50 30 0

0.0 9.2 28.8 48.6 100.0

100.0 90.8 71.2 51.4 0.0

0.00 0.09 0.31 0.43 1.00

3.8 3.2 3.0 2.7 2.1

75 107 115 119 123

0.00 0.10 0.29 0.45 1.00

32 35 41

a Reaction mass ratios of SBMA and NIPAAm monomers used with fixed total monomer mass percentage of 20 wt % in the prepared reaction solution. The theoretical compositions of the poly(NIPAAm-co-SBMA) hydrogels were calculated based on reaction ratios and molecular weight of comonomers. c The mole fraction of polySBMA (xSBMA) in the cross-linked poly(NIPAAm-co-SBMA) hydrogels was determined by XPS in the dry state from the spectral area ratio of the atomic percentages based on the N 1s of the polyNIPAAm isopropyl groups and S 2p of the polySBMA side groups at the BE of approximately 399 and 168 eV, respectively. Swelling ratios of equilibrated hydrogel disks were estimated in deionized water at 25 °C. Surface contact angles of prepared hydrogels were measured with an angle-meter using diiodomethane in H2O at 37 °C. d The mole fraction of polySBMA (x′SBMA) in the uncross-linked poly(NIPAAm-co-SBMA) hydrogels was estimated by 1H NMR in D2O from the relative peak area of (CH3)2N+ proton resonance of the polySBMA side groups at δ ) 3.2 ppm and that of the methyl proton resonance of the polyNIPAAm isopropyl groups at δ ) 1.14 ppm. LCST were determined by reading the absorbance on a UV-visible spectrophotometer based on our previous work.25 b

biochemical stimuli. Poly(N-isopropylacrylamide) (polyNIPAAm) is the most widely studied thermoresponsive polymer.16-19 This nonionic polymer undergoes a sharp hydrophilic-hydrophobic transition in aqueous media at 32 °C, which is termed the lower critical solution temperature (LCST).17 In general, the solution properties of nonionic polyNIPAAm differ considerably from those of zwitterionic polymers. PolySBMA, like other zwitterionic polymers, exhibit an upper critical solution temperature (UCST) in aqueous solutions.20 Recently, studies have reported that physical micellization occurs in synthesized diblock copolymers with thermoresponsive and zwitterionic properties.21-24 Our previous work also reported a new class of intelligent statistical copolymers prepared from the combination of the zwitterionicpolySBMAandnonionicthermoresponsivepolyNIPAAm, exhibiting the controllable thermosensitive phase transition of LCST and UCST behaviors in an aqueous solution.25 The study suggests that a zwitterionic copolymer containing nonionic polyNIPAAm is a potential thermoresponsive biomaterial that can provide a coated surface with tunable stimuli for use in human physiologic environment. According to the dual-thermoresponsive phase behavior of nonionic-zwitterionic copolymers in an aqueous solution, a promising biomaterial of tunable-bioadhesive copolymer networks with thermosensitive properties could provide a wide range of biomedical applications. Potential applications include highly effective cell adhesion/detachment culturing and environmentally friendly antimicrobial coatings.25 In this work, we develop intelligent chemical hydrogels of poly(NIPAAm-coSBMA) copolymer networks having both tunable-bioadhesive and thermoresponsive functions. Herein, for the first time, we demonstrate bioadhesive studies for the interactions of nonioniczwitterionic hydrogel interfaces with biomolecules and microorganisms. We prepared nonionic hydrogels of polyNIPAAm, zwitterionic hydrogels of polySBMA, and three nonioniczwitterionic hydrogels of poly(NIPAAm-co-SBMA) having different monomer ratios of SBMA and NIPAAm. The effect of temperature on the swelling behavior and hydrophilic properties of these hydrogels in aqueous solutions were examined in detail. This study also demonstrates the adsorption of plasma proteins and platelets onto these hydrogel surfaces from human blood plasma and shows the bioadhesive activity of the prepared hydrogels by the tests of cell adhesion, cell detachment, and bacterial attachment. This work is mainly aimed at addressing two important issues of poly(NIPAAm-co-SBMA) hydrogels in five various nonionic-zwitterionic compositions; that is, (i) in situ measurement of the reversible swollen characteristics of temperature sensitive copolymeric hydrogels

in an aqueous solution and in the presence of salt ions and (ii) in vitro evaluation of the adsorption of plasma proteins, the adhesion of human platelets and cells, and the attachment of bacteria onto the copolymeric hydrogel surfaces at the physiological temperature.

Materials and Methods Materials. [2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)-ammonium hydroxide (sulfobetaine methacrylate, SBMA) macromonomer was purchased from Monomer-Polymer and Dajac Laboratories, Inc., U.S.A. N-Isopropylacrylamide (NIPAAm) from Sigma-Aldrich was recrystallized with hexane. Ammonium persulfate (APS), N,N,N′,N′tetraethylmethylenediamine (TEMED), N,N′-methylenebisacrylamide (MBAA), and diiodomethane were purchased from Sigma-Aldrich. Deionized water (DI water) used in experiments was purified using a Millipore water purification system with a minimum resistivity of 18.0 MΩ · m. Preparation of Poly(NIPAAm-co-SBMA) Hydrogels. A total of 20 wt % of different mass ratios of SBMA and NIPAAm (Table 1) were dissolved in an aqueous solution of 1.6 wt % MBAA, and nitrogen was bubbled to remove residual oxygen. The copolymerization of poly(NIPAAm-co-SBMA) networks was initiated using 0.2 wt % of APS and was cross-linked using 0.2 wt % of TEMED. The reaction was carried out between a pair of glass substrates and was separated with a silicone rubber spacer with a thickness of 0.2 mm at 25 °C for 5 h. After polymerization, the gel was immersed in a large amount of water for 48 h, and the water was changed every 6 h to remove the chemical residue. Poly(NIPAAm-co-SBMA) chemical hydrogels were punched (10 mm biopsy punch, Acuderm Inc., FL) into disks with a diameter of 10.0 mm and a thickness of 0.2 mm stored in DI water before use. In addition, the preparation of poly(SBMA-co-NIPAAm) copolymers without chemical cross-linking was described in our previous publication.25 All copolymer samples were controlled with a similar average molecular weight of approximately 6.0 kDa and the same molecular weight distribution (i.e., Mw/Mn ) 3.4-3.6). Characterization of Poly(NIPAAm-co-SBMA) Hydrogels. The chemical structure of dried poly(NIPAAm-co-SBMA) hydrogels was characterized using a Fourier-transform infrared spectroscopic (FT-IR) measurement (Perkin-Elmer Spectrum One) and using zinc selenide (ZnSe) as an internal reflection element. Each spectrum was captured by an average of 32 scans at a resolution of 4 cm-1. The compositions of the poly(NIPAAm-co-SBMA) hydrogels were also characterized by X-ray photoelectron spectroscopy (XPS). XPS analysis was performed using a Thermal Scientific K-Alpha spectrometer equipped with a monochromated Al K X-ray source (1486.6 eV photons). The energy of emitted electrons was measured with a hemispherical energy analyzer at pass energies ranging from 50 to 150 eV. All data were collected at a photoelectron takeoff angle of 45° with respect to the sample surface.

Hydrogels Containing Zwitterionic Polysulfobetaine The binding energy (BE) scale is referenced by setting the peak maximum in the C 1s spectrum to 284.6 eV. The high-resolution C 1s spectrum was fitted using a Shirley background subtraction and a series of Gaussian peaks. The quantified mole fraction of polySBMA (xSBMA) in the cross-linked poly(NIPAAm-co-SBMA) hydrogels was determined by the spectral area ratio of the atomic percentages based on the N 1s of the polyNIPAAm isopropyl groups and S 2p of the polySBMA side groups at a BE of approximately 399 and 168 eV, respectively, as summarized in Table 1. The temperature sensitivity of swollen poly(NIPAAm-co-SBMA) hydrogels was determined using a temperature-dependent equilibrium swelling ratio. The swelling ratios of the samples were measured gravimetrically. Dried hydrogel disks were weighed (Wd) after the gel disks were dried to a constant weight under a vacuum at 25 °C for 3 days and at 60 °C for 1 day. The dried hydrogels were then immersed and equilibrated in DI water at a specific target temperature (4, 25, or 37 °C) for 48 h. The disks were weighed (Ww) after the excess water on the disk surfaces was wiped off with moisture filter papers. The weights from three measurements were averaged and the swelling ratios were determined by the ratio of the weight of water in the swollen gel to the dry gel weight using the following equation:

swelling ratio ) (Ww - Wd)/Wd Surface contact angles of prepared hydrogels were measured with an angle-meter (Automatic Contact Angle Meter, Model CAVP, Kyowa Interface Science Co., Ltd., Japan) at 25 and 37 °C. The diiodomethane was dropped on the sample surface under a water medium at three different sites. The average of the measured values for each of the prepared hydrogels from five independent disks was taken as its contact angle. The mole fraction of polySBMA (x′SBMA) in the uncross-linked poly(NIPAAm-co-SBMA) hydrogels was estimated by 1H NMR in D2O from the relative peak area of (CH3)2N+ proton resonance of the polySBMA side groups at δ ) 3.2 ppm. That of the methyl proton resonance of the polyNIPAAm isopropyl groups was estimated at δ ) 1.14 ppm, as summarized in Table 1. Molecular weights of prepared uncross-linked copolymeric hydrogels were determined by aqueous gel permeation chromatography (GPC), using 1 column of Viscogel TM GMPWXL K0105 (the range of molecular weight was from 500 Da to 800 kDa) connected to a model Waters 2414 refractive index detector at a flow rate of 1.0 mL/min and a column temperature of 23 °C. The eluent was an aqueous solution composed of 0.1 M NaCl at pH 7.4. Poly(ethylene oxide) (PEO) standards from Polymer Standard Service, Inc. (Warwick, U.S.A.) were used for calibration. Plasma Protein Adsorption. The adsorption of human plasma solutions of γ-globulin, fibrinogen, and human serum albumin (HSA) on the hydrogel disks was evaluated using the enzyme-linked immunosorbent assay (ELISA) method according to the standard protocol as summarized below. First, the hydrogel disks with a surface area of 0.785 cm2 were placed in individual wells of a 24-well tissue culture plate and each hydrogel disk was equilibrated with 1000 µL of PBS for 180 min at 37 °C. Then, the hydrogel disks were soaked in 1000 µL of 100% Platelet Poor Plasma (PPP) solution. After 180 min of incubation at 37 °C, the hydrogel disks were rinsed five times with 1000 µL PBS and then incubated in bovine serum albumin (BSA, purchased from Aldrich) for 90 min at 37 °C to block the surface areas unoccupied by proteins. The hydrogel disks were rinsed with PBS five more times before being transferred to a new plate and incubated in 1000 µL PBS solution. The hydrogel disks were incubated with a primary monoclonal antibody that reacted with the human plasma protein (i.e., HSA or Fg) for 90 min at 37 °C and were then blocked with 10 mg/mL BSA in PBS solution for 24 h at 37 °C. The hydrogel disks were subsequently incubated with the secondary monoclonal antibody, horseradish peroxidase (HRP)-conjugated immunoglobulins, for 60 min at 37 °C. The primary antibody was not used and only the secondary antibody (goat F(ab)2 antihuman immunoglobulin peroxidase conjugate antibody) treatment was performed for the assay of human


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γ-globulin adsorbed on the hydrogel disks from the PPP for 90 min at 37 °C. The hydrogel disks were rinsed five times with 1000 µL PBS and transferred into clean wells, followed by the addition of 500 µL PBS containing 1 mg/mL 3,3′,5,5′-tetramethylbenzidine as a chromogen, 0.05 wt % Tween 20, and 0.05 wt % hydrogen peroxide. The enzymeinduced color reaction was controlled for approximately 5 min for Fg and HSA and 15 min for γ-globulin, respectively. After incubation at 37 °C, the reaction was stopped by adding 500 µL (or 1000 µL) of 1 mmol/mL H2SO4 to the solution in each well. Finally, the absorbance of light at 450 nm was determined using a microplate reader. Protein adsorption on the hydrogel disks was normalized with respect to that on the polyNIPAAm hydrogel disk as a reference. The ELISA measurement was repeated using six independent disks (n ) 6 in total) for each hydrogel substrate and the average result was reported. Blood Platelet Adhesion. The hydrogel disks of 0.785 cm2 surface area were placed in individual wells of a 24-well tissue culture plate and each well was equilibrated with 1000 µL of phosphate buffered solution (PBS) for 180 min at 37 °C. Blood was obtained from a healthy human volunteer. Platelet rich plasma (PRP) containing about 1 × 105 blood cells/mL was prepared by centrifugation of the blood at 20 Hz (1200 rpm) for 10 min. The platelet concentration was determined by a microscopy (NIKON TS 100F). A total of 1000 µL of the platelet suspension plasma was placed on the hydrogel surface in each well of the tissue culture plate and incubated for 120 min at 37 °C. After the hydrogel disks were rinsed twice with 1000 µL of PBS, they were immersed into 2.5% glutaraldehyde of PBS for 48 h at 4 °C to fix the adhered platelets and adsorbed proteins, then rinsed two times with 1000 µL of PBS and gradient-dried with ethanol in 95, 85, 75, 50, 25, 5, and 0% v/v PBS for 5 min in each step and dried in air. Finally, the samples were sputter-coated with gold prior to observation under JEOL JSM-5410 SEM operating at 7 keV. The number of adhering platelets on the hydrogels was counted from SEM images at a 500 magnification from five different places on the same hydrogel disks. The process was repeated using three independent hydrogel disks (n ) 15 in total). Cell Culture. Human HS-68 Fibroblasts were maintained in continuous growth in Dulbecco’s modified Eagle medium (DMEM, Gaithersburg, MD). The cells were then supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin streptomycin solution at 37 °C. This medium was maintained in a humidified atmosphere containing 5% CO2 on polystyrene tissue culture flasks. Before culturing the cells on the hydrogel surface, the hydrogel disks were incubated with 75 wt % ethanol for 1 h at 25 °C, and washed three times by PBS in a 24-well plate. Next, 1 mL of cell suspension at a final concentration of 104 cells/mL was added to each well. The cells were then incubated with the samples for 24 h at 37 °C in a humidified atmosphere of 5% CO2. The morphology and proliferation of the cells were observed using a Nikon TS100 phase contrast microscope equipped with a digital camera using a 10× objective lens. The number of cells at 1, 3, and 8 days were determined using an MTT assay. For the experiments of cell detachment, Human HT-1080 fibroblasts (ATCC, Manassas, VA) were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2. The plasmid pAAVluciferase-EGFP, used to engineer HT-1080 cells into stable luciferase and EGFP expression cells. A total of 1 mL of cell suspension at a final concentration of 2 × 104 cells/mL was added to hydrogel disk in each well. The cells were then incubated with the samples for 3 days at 37 °C in a humidified atmosphere of 5% CO2. The morphology and proliferation of the cells were observed using a Nikon TS100 microscope equipped with a digital camera using a 10× objective lens and a blue excitation fluorescence filter at the excitation range 450-490 nm. The percentage of cell attachment with the incubated temperature of 4 and 25 °C, respectively, at 0, 30, 60, and 120 min were determined using cell counting by fluorescence images, which were repeated using six independent disks (n ) 6 in total) for each hydrogel substrate and the average result was reported.

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Scheme 1. (a) Chemical Structure of the PolyNIPAAm, Poly(NIPAAm-co-SBMA), and PolySBMA Hydrogels; (b) Simplified Molecular Model of the Temperature Dependence of the Poly(NIPAAm-co-SBMA) Hydrogels

Bacterial Adhesion Assay. Two bacterial species, Staphylococcus epidermidis and Escherichia coli, were used to investigate bacterial adhesion behavior on the surface of poly(NIPAAm-co-SBMA) hydrogels. S. epidermidis and E. coli were cultured in a medium containing 3.0 mg/mL beef extract and with 5.0 mg/mL peptone. These cultures were incubated at 37 °C and were shaken at 100 rpm until the stationary phase was reached at a final S. epidermidis concentration of 109 cells/ mL for 15 h and a final E. coli concentration of 106 cells/mL for 12 h. The hydrogel disks were incubated with 75 wt % ethanol for 1 h at 25 °C and washed by PBS 3 times in a 24-well plate. A total of 1 mL of bacteria suspension was added to each well. The bacteria were then incubated with the samples for 24 h at 37 °C. The bacterial solution was removed after 24 h and each sample was then washed with PBS 3 times at 37 °C to remove the attached bacteria. Bacteria adhering to the sample surfaces were stained with 200 µL of Live/Dead BacLight for 10 min. After washing with PBS three times, samples with stained bacteria were observed with a CCD camera mounted on Olympus BX51 with a 10× objective lens. During observation, we used epifluorescent illumination through a blue excitation fluorescence filter at the excitation range 450-490 nm.

Results and Discussion In this present work, nonionic hydrogels of polyNIPAAm (S#0), zwitterionic hydrogels of polySBMA (S#100), and three copolymeric hydrogels of poly(NIPAAm-co-SBMA) (S#20, S#50, and S#70) were prepared from various monomer compositions between NIPAAm and SBMA, as shown in Scheme 1a. To achieve similar cross-linking qualities with respect to mechanical strength and ductility, all hydrogels were prepared with the same quantity of 1.6 wt % cross-linker MBAA in total reaction compositions based on the previous study.26 The corresponding chemical composition of the poly(NIPAAm-coSBMA) hydrogels is shown in Table 1. The increasing amount of SBMA monomers in the reaction solution increased the molar mass ratio of polySBMA in the prepared hydrogel. The molar

ratio of polySBMA in the prepared uncross-linked gels was close to the molar ratio of polySBMA in the cross-linked gels, indicating the similar reactivity of SBMA and NIPAAm monomers during the polymerization. To determine the solubleinsoluble phase transition at LCST of poly(NIPAAm-co-SBMA) hydrogels without cross-linking, the optical absorbance of dilute uncross-linked gel solution was measured using a UV-visible spectrophotometer with precise temperature control from 25 to 70 °C. Characterizations of Temperature-Responsive Poly(NIPAAmco-SBMA) Hydrogels. The Fourier-transform infrared spectroscopic (FT-IR) measurement was used to characterize the chemical composition of the poly(NIPAAm-co-SBMA) hydrogel, and its typical spectrum is shown in Figure 1. The presence of the polySBMA segments could be ascertained from the ester carbonyl groups and the sulfonate groups observed from the bands of the -SO3 stretch at 1033 cm-1 and O-CdO stretch at 1727 cm-1, respectively. The intensity of -SO3 at 1033 cm-1 and O-CdO adsorption at 1727 cm-1 clearly increased as the starting SBMA concentration in the reaction solution increased from 20 to 100 wt %. It should be noted that the intensity ratio of 1033 cm-1 and 1727 cm-1 for S#20, S#50, S#70, and S#100 keeps almost similar due to the characteristic peaks contributed form the same polySBMA segments. Swelling behaviors at different temperatures and ionic strengths, as shown in Figure 2, illustrate the phase behaviors of the poly(NIPAAm-co-SBMA) hydrogels. In Figure 2a, the equilibrated swelling ratios of the nonionic polyNIPAAm gel and the poly(NIPAAm-co-SBMA) gels are temperature-dependent in an aqueous solution, which can be modulated by changing the zwitterionic polySBMA compositions in copolymeric hydrogels. However, polySBMA gels are not sensitive to the change in temperature from 4 to 37 °C in aqueous solution. Thus, as the molar mass of polySBMA increases in the

Hydrogels Containing Zwitterionic Polysulfobetaine

Figure 1. FT-IR spectra of the polyNIPAAm gel (S#0), the poly(NIPAAmco-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100).

Figure 2. Swelling ratios of the polyNIPAAm gel (S#0), the poly(NIPAAmco-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100) under the following conditions: (a) in H2O at 4, 25, and 37 °C and (b) in 0.0, 0.1, 1.0, and 2.5 M NaCl solution at 37 °C.

poly(NIPAAm-co-SBMA) gels, the decrease in the temperature sensitivity of swelling behavior for copolymeric hydrogels in aqueous solution are attributed to the less nonionic polyNIPAAm segment associations induced by hydrophobic interactions between isopropyl groups in polyNIPAAm side chains. At the


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physiologicaltemperature(37°C)abovetheLCSTofpolyNIPAAm, the swelling ratios of poly(NIPAAm-co-SBMA) gels are kept at a nearly constant value. This phenomenon maybe due to the fact that polyNIPAAm segments in poly(NIPAAm-co-SBMA) gels become more hydrophobic and that hydrogen bonding with water molecules is weakened, indicating the collapse of the polyNIPAAm domains and the deswelling of the hydrogel, as shown in Scheme 1b. As the temperature decreases, the swelling ratios of all of the copolymeric gels clearly increase. This could be attributed to the hydration of ordering water molecules around hydrophobic isopropyl moieties of polyNIPAAm segments in hydrogels below the LCST of polyNIPAAm. The effects of the ionic strength on the swelling behaviors of poly(NIPAAm-co-SBMA) hydrogels were further evaluated to investigate the phase characteristics at physiological temperature. The ionic strength of the aqueous medium was adjusted by dissolving sodium chloride (NaCl) into DI water in the designated concentrations ranging from 0.1 to 2.5 M (Figure 2b). Nonionic polyNIPAAm gels (S#0) deswelled, zwitterionic polySBMA gels (S#100) swelled in the presence of salt ions, and the swelling ratio varied with ionic strength. The swelling ratios of poly(NIPAAm-co-SBMA) hydrogels (S#20, S#50, and S#70) are dependent on both the salt concentration and the molar mass of polySBMA in the copolymeric gels. For the case of polyNIPAAm gels, almost no swelling was observed with the presence of 2.5 M NaCl at 37 °C. The dramatic decreasing trend in the swelling ratios of polyNIPAAm gels could be correlated with the ion-water interaction of salt ions, suggesting that the water molecules around the hydrophobic isopropyl moieties of polyNIPAAm segments affected by ions causes the dehydrated shift. In contrast, the swelling ratios of polySBMA gels gradually increased with an increase in ionic strength and approached their respective saturated values after using 1.0 M NaCl in DI water. In general, polySBMA gels have the ability to exhibit a unique antipolyelectrolyte behavior in the presence of salt ions, which increases notably with the ionic strength in the aqueous solution. The swelling process of polySBMA gels on the addition of NaCl electrolytes is attributed to the salt ion screening of the net attractive electrostatic interactions of ionic pairings of opposite charges between zwitterionic groups. This effect causes the zwitterionic polySBMA segments to swell. The dependence of the swelling behavior of the copolymeric gels containing polySBMA of three different molar masses on ionic strength can be explained by the competitive contribution of intramolecular hydrophobic interactions by NIPAAm segments of nonionic isopropyl groups and inter and/or intramolecular electrostatic interactions by SBMA segments of zwitterionic sulfobetaine groups. The swelling ratios of copolymeric gels containing polySBMA at 31 mol % (S#50) were found to be basically independent of the ionic strength over a wide range of concentrations of NaCl from 0.1 to 2.5 M at 37 °C. This finding is attributed to the balance between swelling and hydrating behaviors with water molecules due to the dehydration caused by the salting out effect around isopropyl groups and the hydrating effect of antipolyelectrolyte around zwitterionic moieties. Interestingly, based on our previous study, there appears to be both a UCST (15 °C) and a LCST (41 °C) of a doubly thermoresponsive poly(NIPAAm-co-SBMA) copolymer with a similar composition from the combination of 29 mol % polySBMA and 71 mol % polyNIPAAm in the same copolymer chain.25 For copolymeric gels containing polySBMA, less than 31 mol %, poly(NIPAAm-co-SBMA) hydrogels deswelled in the presence of salt ions and the swelling ratio decreased with ionic strength. This was similar to the case of the polyNIPAAm

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Figure 3. Adsorption of γ-globulin, fibrinogen, and HSA from 100% human plasma on the surfaces of the polyNIPAAm gel (S#0), the poly(NIPAAm-co-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100) at 37 °C. Surface contact angles of prepared hydrogels were measured using diiodomethane in H2O at 25 and 37 °C.

gels. However, the swelling ratio of copolymeric gel containing polySBMA at 9 mol % (S#20) was found to be more significant change in low concentration of NaCl at 0.1 M. This might be due to the hydrating effect of antipolyelectrolyte around zwitterionic moieties is higher than the dehydration effect of water molecules around isopropyl groups in such low ionic strength. For copolymeric gels in high concentration of NaCl at 2.5 M, an increase in the molar mass of polySBMA led to an obvious increase in the swelling ratios of poly(NIPAAmco-SBMA) hydrogels, which was attributed to the salt-induced hydration of water molecules around the zwitterionic sulfobetaine groups. Interestingly, the swelling ratio of copolymeric gel containing polySBMA at 43 mol % (S#70) in such high ionic strength is higher than that the case of the polySBMA gels. This might be due to that the dehydration of water molecules from associated polyNIPAAm domains enhances the more hydrating effect of nonassociated polySBMA segments. Such copolymeric hydrogels were shown to have potential stimuli-responsive properties exhibiting not only temperature dependent but also ionic strength sensitive. Plasma Protein Adsorption and Blood Platelet Adhesion on Poly(NIPAAm-co-SBMA) Hydrogels. The adhesion of platelets from human blood was correlated with the adsorption of plasma proteins on surfaces and, particularly, fibrinogen adsorption.27,28 In this work, adsorption of human plasma proteins in blood plasma solutions onto copolymeric gel surfaces was measured using ELISA at 37 °C (the temperature of the human body). Three of the major plasma proteins, γ-globulin, human fibrinogen, and HSA, were selected for testing in this study. ELISA results for the relative protein adsorption from PPP solutions on different gel surfaces are shown in Figure 3. It was found that polySBMA gels are highly resistant to nonspecific adsorption for major plasma proteins at 37 °C, while hydrophobic polyNIPAAm gels show high protein adsorption. We observed significant decreases in the adsorption of plasma proteins on copolymeric hydrogel surfaces of S#20, S#50, and S#70 as compared to that on the surfaces of polyNIPAAm gels. The copolymeric hydrogel surfaces, even with a low molar ratio of 9 mol % polySBMA in S#20 gels, reduced all of the plasma protein adsorption to a level comparable to the adsorption on the polySBMA gel surface. The relative protein adsorption on all copolymeric hydrogel surfaces was effectively reduced below 12% of that on the polyNIPAAm gel surface.

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It is generally acknowledged that hydrophilic surfaces are more likely to reduce the nonspecific adsorption of proteins, when living systems encounter material surfaces. This study further examined the effect of the SBMA molar mass in poly(NIPAAm-co-SBMA) hydrogels on the change of surface contact angle at 25 and 37 °C. The increased values of the diiodomethane contact angle under a water medium in Figure 3 indicate that the surface hydrophilicity of prepared gels increased. The diiodomethane contact angle of the polyNIPAAm gels was found to be as low as approximately 75° at 37 °C, indicating an obvious decrease in surface hydrophilicity compared to the polyNIPAAm gels at 25 °C whose water contact angle was approximately 110°. We observed that plasma protein adsorption on the polyNIPAAm gel (S#0) at 37 °C is over 30% higher than that for polyNIPAAm gels at 25 °C, while the polySBMA gel (S#100) keeps its protein resistance in plasma solution at both 25 and 37 °C. The increased molar mass of SBMA in poly(NIPAAm-co-SBMA) at 25 and 37 °C was found to cause its hydrogel surface to be more hydrophilic as shown by the increase in diiodomethane contact angle. The hydrophilicity of gel surface was shown to be correlated with the nonspecific adsorption of proteins on copolymeric hydrogels, indicating their ability to inhibit plasma proteins can be related to their surface hydration ability. These results also strongly support the hypothesis that hydrated surfaces with overall charge neutrality formed from zwitterionic sulfobetaine components can present nonfouling characteristics for resisting nonspecific protein adsorption. Figure 4 shows SEM images, at a magnification of 1000×, of the prepared substrates in contact with platelet-rich plasma solutions prepared from human whole blood for 120 min at 37 °C in vitro. The SEM results showed that the platelet adherence was remarkably suppressed on copolymeric hydrogel surfaces compared to that on surfaces of polyNIPAAm gels. It is clearly observed that the platelets have spread on polyNIPAAm gels at 37 °C, which indicates the activation of the platelet. However, there is still a small quantity of slightly activated platelets on gel surfaces of S#20. The excellent performance of gel surfaces containing polySBMA above 31 mol %, with no obvious adhesion of blood platelets, is due to the ability of these surfaces to highly resist nonspecific protein adsorption from blood plasma. No platelets were found to adhere to the gel surfaces of S#50 and S#70 as compared with S#20 gels. This might be attributed to the reduced relative plasma protein adsorption levels below 10% on S#50 and S#70 gels, while the relative adsorbed amount of plasma proteins is above 10% on S#20, as shown in Figure 3. On the basis of previous reports from Horbett et al., it is believed that reducing plasma protein adsorption levels on biomaterial surfaces below 10 ng/cm2 can effectively prevent the adhesion and activation of platelets from the bloodstream.27,28 These results indicate that the reduced relative plasma protein adsorption of 10% on gel surfaces is comparable to a level of 10 ng/cm2. These results also confirmed the previous hypothesis that even a small quantity of proteins adsorbed on the surface can lead to the adhesion and activation of platelets from human blood. Measurements of both the plasma protein adsorption and blood platelet adhesion in vitro demonstrated for the first time that copolymeric hydrogels of poly(NIPAAm-co-SBMA) with tunable molar mass of zwitterionic SBMA can be used to achieve high compatibility with human blood. Cell Adhesion and Growth. Human fibroblasts (HS-68) were cultured on hydrogel surfaces in 24-well tissue culture polystyrene (TCPS) plates at 37 °C for 3 days, and the samples were then observed under a microscope equipped with a digital



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Figure 4. SEM photographs of human blood platelets adhered onto the surfaces of the polyNIPAAm gel (S#0), the poly(NIPAAm-co-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100). All images are shown at a magnification of 1000×.

Figure 5. Cell culture of HS-68 adhering onto the surfaces of TCPS, poly(NIPAAm-co-SBMA) gels (S#20, S#50, and S#70), and polySBMA gel (S#100) for 3 days. All images are shown a magnification of 100×.

camera (NIKON CCD) using a 10× objective lens. The images are shown in Figure 5 at a magnification of 100×. HS-68 cell adhesion and growth on the gel surfaces was compared with a flat TCPS surface. For the bare TCPS plate, HS-68 cells adhered and spread over the TCPS surface into a confluent-like layer. The cell image on the gel surface of polyNIPAAm (S#0) is not shown in Figure 5 due to opaque S#0 hydrogels, which did not allow us to capture a cell image. Thus, the number of cells on polyNIPAAm gel surface was determined using an MTT test. In Figure 5, there appeared to be a small number of cells that adhered to gel surfaces of S#20 containing polySBMA at 9 mol %. Poly(NIPAAm-co-SBMA) hydrogel surfaces (S#50 and S#70) containing polySBMA above 31 mol % showed no obvious adhesion of cells, which was correlated with its ability to highly resist nonspecific protein adsorption and platelet adhesion from blood plasma. No cells were found on the zwitterionic polySBMA gel surfaces. This, along with results from a previous study,6,10,14 supports the hypothesis that zwitterionic surfaces with an overall charge neutrality formed from ion pairings between the ammonium cation and the sulfoanion can present nonfouling characteristics. The results show that the increase of SBMA molar mass in poly(NIPAAm-co-

SBMA) hydrogels could inhibit cell adhesion on gel surfaces. Figure 6 shows changes from days 1 to 8 in the number of live HS-68 cells grown on the TCPS plate and on five gel surfaces at 37 °C in a long-term culture, which were determined by an MTT test. Cell growth on the poly(NIPAAm-co-SBMA) gel surfaces (S#20, S#50, and S#70) was suppressed compared with that on the TCPS plate and on polyNIPAAm gel surfaces. No cell growth can be observed on the copolymeric gels as an SBMA molar ratio above 43 mol %, even at a culture time of up to 8 days. Thermal Stimulus of Cell Detachment. Human fibroblasts (HT-1080) carrying the enhanced green fluorescent protein (EGFP) was used in this work to observe thermal stimulus of cell detachment from the prepared hydrogels. HT-1080 cells were cultured on hydrogel surfaces in 24-well TCPS plates at 37 °C for 3 days, and the samples were then observed under the temperature transition as the cultured temperature decreased from 37 °C to the incubated temperature of 4 and 25 °C for a period of 120 min, respectively. In Figure 7, HT-1080 cell detachment was observed in the samples of S#0, S#20, and S#50 as the temperature decreased to 25 °C, which is below the LCST

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Figure 6. Cell growth of HS-68 adhering onto the surfaces of the TCPS, the polyNIPAAm gel (S#0), the poly(NIPAAm-co-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100) after 1, 3, and 8 days. Cell culture was performed at an initial concentration of 104 cells/mL.

of prepared samples. We observed that HT-1080 cell attachment on the S#0 gels is obviously reduced during the incubation period of 120 min at 25 °C, while blank TCPS surface still keeps its cell adhesion without any detachment. This was attributed to the hydration of polyNIPAAm segments with water molecules, which formed hydrophilic gel surfaces as shown in Figure 3. Interestingly, the incorporation of zwitterionic SBMA units into the polyNIPAAm gels, such as S#20, was found to accelerate the hydration of the cell-cultured surfaces and resulted in more rapid cell detachment during the incubation period of 60 min at 25 °C. However, it should be noted that the introduction of highly increased zwitterionic components in the poly(NIPAAm-co-SBMA) hydrogels, such as S#50, resulted in a dramatic decrease in cell adhesion and growth at 37 °C. As shown in Figure 8, the quantification of cell detachment for the

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Figure 8. Normalized cell attachment of HT-1080 on the surfaces of the polyNIPAAm gel (S#0) and the poly(NIPAAm-co-SBMA) gels (S#20 and S#50) with incubated temperature of 4 and 25 °C at 0, 30, 60, and 120 min, respectively. The standard deviation for all data is below 5% (n ) 6).

incubated temperature of 4 and 25 °C at 0, 30, 60, and 120 min was normalized by the numbers of cell attachment cultured for 3 days at 37 °C in each individual sample of S#0, S#20, and S#50. The dynamic cell detachment was shown to be correlated with the incubated temperature and the composition of polySBMA in copolymeric hydrogels. For the tested hydrogels of S#0, S#20, and S#50, the incubated temperature at 4 °C resulted in more rapid cell detachment than that at 25 °C. The decrease in incubated temperature induces a conformation change of polyNIPAAm segments in the tested hydrogels to produce an expanded, swollen and hydrophilic surface as shown in Figure 2a and Figure 3. This change in gel surface property weakens cellular adhesion, resulting in cell detachment from the copolymeric hydrogels. This unique property indicates that thermoresponsive polyNIPAAm hydrogels incorporating zwitterionic

Figure 7. Fluorescence microscopic images of HT-1080 cell attachment on the surfaces of the TCPS, the polyNIPAAm gel (S#0), the poly(NIPAAmco-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100) with incubated temperature of 25 °C at (a) 0, (b) 30, (c) 60, and (d) 120 min. Cell culture was performed at an initial concentration of 2 × 104 cells/mL.



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Figure 9. Fluorescence microscopic images of S. epidermidis attachment on the surfaces of the TCPS, the polyNIPAAm gel (S#0), the poly(NIPAAm-co-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100) after 24 h.

Figure 10. Fluorescence microscopic images of E. coli attachment on the surfaces of the TCPS, the polyNIPAAm gel (S#0), the poly(NIPAAmco-SBMA) gels (S#20, S#50, and S#70), and the polySBMA gel (S#100) after 24 h.

polySBMA can be used to control the attachment or detachment of cells on the surfaces. Bacterial Attachment and Biofilm Formation. The control of biofilm formation onto the surface of a biomaterial is a critical issue for many biomedical and engineering applications. It was generally believed that the initial step in biofilm formation is the nonspecific, reversible attachment of bacteria to substratum surfaces.29 Some studies have suggested that surfaces must be able to resist protein adsorption to resist bacterial adhesion.30 This hypothesis was tested in this study. Two bacterial species, Gram-positive S. epidermidis and Gram-negative E. coli, were used to investigate the ability of bacteria to adhere to the surface of poly(NIPAAm-co-SBMA) hydrogels. The long-term accumulation of both of the bacteria on prepared gel surfaces in a 24 h period at 37 °C was characterized using Live/Dead BacLight assay and was then observed using a fluorescent microscope. Hydrophobic surfaces of 24-well TCPS plates were used as references. The qualitative images of accumulated S.

epidermidis and E. coli on sample surfaces are shown at a magnification of 100× in Figures 9 and 10, respectively. The hydrophobic TCPS exhibited the highest biofilm accumulation in the bacterial culture media after 24 h. This could be attributed to the initial stage of nonspecific protein adsorption and subsequent bacterial adhesion onto its hydrophobic surface, especially in terms of the protein content on the outer cell membrane of S. epidermidis. S. epidermidis and E. coli appear to have different morphologies on polyNIPAAm gel surfaces. S. epidermidis tend to form biofilm accumulation with a fullscale bacterial adhesion, while E. coli tend to form a networklike structure. Significant qualitative decreases in the adhesion of both Gram-positive and Gram-negative bacteria were observed on poly(NIPAAm-co-SBMA) and polySBMA hydrogels in comparison to TCPS and polyNIPAAm gels. The results showed that the reduced protein adsorption levels lead to a lower adhesion of bacteria or accumulation of biofilm on these hydrogel surfaces as indicated in Figure 3. In this study, the

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results support the hypothesis that the adhesion of S. epidermidis and E. coli might be correlated with the adsorption of proteins on gel surfaces. Thus, the molar mass of zwitterionic SBMA in poly(NIPAAm-co-SBMA) hydrogels could provide tunable control of bacterial attachment and biofilm formation on a biomaterial surface. As a result, this study shows for the first time that the right combination of nonionic polyNIPAAm and zwitterionic polySBMA can produce an excellent smart hydrogel system for achieving tunable bioadhesive characteristics. The bioadhesive characteristics can be used for a wide range of biofoulant species including proteins, cells and bacteria while maintaining controllable thermoresponsive functions.

Conclusions In this study, a controllable-thermoresponsive and tunablebioadhesive hydrogel was obtained in the cross-linked chemical networks with a regulated ratio of nonionic polyNIPAAm and zwitterionic polySBMA. We found that the balance of hydrophobic or electrostatic intra- and intermolecular interactions between polymer segments in poly(NIPAAm-co-SBMA) hydrogels was dominated by the portion of polyNIPAAm or polySBMA. The polyNIPAAm or polySBMA determine stimuliresponsive swelling characteristics, which depend strongly on the temperature and ionic strength. It is worth emphasizing that the appropriate control of nonionic and zwitterionic molar mass ratios leads to poly(NIPAAm-co-SBMA) hydrogels containing polySBMA at 31 mol % that exhibit a balanced swelling behavior in a steady swollen gel state, which is independent of the ionic strength over a wide range of concentrations of NaCl from 0.1 to 2.5 M at the physiological temperature. The ELISA test and contact angle measurements revealed that the hydrating effects on the gel surface is associated with the nonfouling characteristics for resisting nonspecific protein adsorption. The results showed that copolymeric hydrogels containing even a low polySBMA content of approximately 31 mol % can be obtained with extremely low plasma protein adsorption and high anticoagulant activity from human blood plasma. Furthermore, it was found that poly(NIPAAm-co-SBMA) hydrogels can be used to control the attachment or detachment of cells on the surfaces using the switchable temperatures above or below the LCST of copolymeric gels. The excellent performance of poly(NIPAAm-co-SBMA) and polySBMA hydrogels in dramatically reducing the accumulation of both S. epidermidis and E. coli is due to their ability to resist nonspecific protein adsorption and bacterial adhesion. This study suggests that the copolymeric hydrogel from nonionic polyNIPAAm incorporating zwitterionic polySBMA is a potential stimuli-responsive biomaterial providing a tunable bioadhesive function for use in a wide range of general biomedical applications. Acknowledgment. The authors express their sincere gratitude to the Center-of-Excellence (COE) Program on Membrane Technology from the Ministry of Education (MOE), R.O.C., to the project Toward Sustainable Green Technology in the Chung Yuan Christian University, Taiwan (CYCU-98-CR-CE), and to

Chang et al.

the National Science Council (NSC 98-2221-E-033-023 and NSC 98-2120-M-008-002) for their financial support.

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BM100093G

Tunable Bioadhesive Copolymer Hydrogels of Thermoresponsive

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