Dual-Thermoresponsive Phase Behavior of Blood ... - ACS Publications

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Biomacromolecules 2009, 10, 2092–2100

Dual-Thermoresponsive Phase Behavior of Blood Compatible Zwitterionic Copolymers Containing Nonionic Poly(N-isopropyl acrylamide) Yung Chang,*,† Wen-Yih Chen,‡ Wetra Yandi,† Yu-Ju Shih,† Wan-Ling Chu,† Ying-Ling Liu,† Chih-Wei Chu,§ Ruoh-Chyu Ruaan,‡ 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, and Research Center for Applied Sciences, Academia Sinica 128 Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Received February 17, 2009; Revised Manuscript Received May 29, 2009

Thermoresponsive statistical copolymers of zwitterionic sulfobetaine methacrylate (SBMA) and nonionic N-isopropylacrylamide (NIPAAm) were prepared with an average molecular weight of about 6.0 kDa via homogeneous free radical copolymerization. The aqueous solution properties of poly(SBMA-co-NIPAAm) were measured using a UV-visible spectrophotometer. The copolymers exhibited controllable lower and upper critical solution temperatures in aqueous solution and showed stimuli-responsive phase transition in the presence of salts. Regulated zwitterionic and nonionic molar mass ratios led to poly(SBMA-co-NIPAAm) copolymers having doublecritical solution temperatures, where the water-insoluble polymer microdomains are generated by the zwitterionic copolymer region of polySBMA or nonionic copolymer region of polyNIPAAm depending on temperature. A high content of the nonionic polyNIPAAm in poly(SBMA-co-NIPAAm) exhibits nonionic aggregation at high temperatures due to the desolvation of polyNIPAAm, whereas relatively low content of polyNIPAAm in poly(SBMA-co-NIPAAm) exhibits zwitterionic aggregation at low temperatures due to the desolvation of polySBMA. Plasma protein adsorption on the surface coated with poly(SBMA-co-NIPAAm) was measured with a surface plasmon resonance (SPR) sensor. The copolymers containing polySBMA above 29 mol % showed extremely low protein adsorption and high anticoagulant activity in human blood plasma. The tunable and switchable thermoresponsive phase behavior of poly(SBMA-co-NIPAAm), as well as its high plasma protein adsorption resistance and anticoagulant activity, suggests a potential for blood-contacting applications.

Introduction Blood compatibility is highly recommended for bloodcontacting materials in important biomedical applications, such as antithrombogenic implants, hemodialysis membranes, and biosensors.1-6 However, only a small number of synthesized biomaterials are regarded as good blood-compatible candidates. Zwitterionic polymers containing the pendant groups of phosphobetaine, sulfobetaine, and carboxybetaine have received growing attention for use in the new generation of bloodcontacting materials because of their good plasma protein resistance.4,7-12 In the last several years, poly(sulfobetaine methacrylate) (polySBMA) with a methacrylate main chain and an analogue of the taurine betaine pendant group (CH2CH2N+(CH3)2CH2CH2CH2SO3-) has become the most widely studied zwitterionic polymer due to its ease of synthetic preparation.4,8,10,12-16 It was reported that the surfaces grafted with polySBMA reduced fibrinogen adsorption to a level comparable with the adsorption on poly(ethylene glycol)-grafted films in our previous studies.8,15 We also grafted a dense polymer brush of polySBMA on a gold surface via surface-initiated atom transfer radical polymerization and suggested that zwitterionic * To whom correspondence should be addressed. E-mail: ychang@ cycu.edu.tw. † Chung Yuan Christian University. ‡ National Central University. § Research Center for Applied Sciences.

polySBMA is an effective and stable nonbiofouling material to provide a surface for use in human blood and implants.4 In general, intelligent biocompatible polymers can represent diverse forms, which might be dissolved as unimers or micelles in an aqueous medium, adsorbed or grafted on aqueous-solid interfaces, or cross-linked in the form of physical or chemical hydrogels.17-20 These polymers can undergo large physical chain conformation changes to small environmental stimuli of physical, chemical, or biochemical nature. Poly(N-isopropylacrylamide) (polyNIPAAm) is the most widely studied thermoresponsive polymer.20-27 This nonionic polymer undergoes a sharp hydrophilic-hydrophobic transition in water at 32 °C; this temperature is called the lower critical solution temperature (LCST).25 The solution properties of zwitterionic polymers differ considerably from those of nonionic polymers. In aqueous solution, polySBMA, like other zwitterionic polymers, exhibits an upper critical solution temperature (UCST) that increases with the molar content.28 This is attributed to the charge-charge or dipole-dipole interactions between the betaine groups. The combination of the UCST of the zwitterionic polymers with the LCST of the polyNIPAAm displays intriguing temperatureinduced self-assembly behavior of different types of polymeric aggregates in aqueous solution.24,29-34 To further develop the zwitterionic-based materials for biomedical applications, we were inspired to study smart polymer systems carrying both controllable biocompatibility and stimuli-responsive functions. Recently, some research works reported physical micellization of synthesized diblock copoly-

10.1021/bm900208u CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

Blood Compatible Zwitterionic Copolymers

Biomacromolecules, Vol. 10, No. 8, 2009

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Table 1. Characteristic Data of Poly(SBMA-co-NIPAAm) Statistical Copolymers reaction ratios of comonomersa (wt %)

compositions of copolymersb (mol %)

characterization of copolymersc (g/mol)

critical solution temperatured (°C)

sample ID

SBMA

NIPAAm

polySBMA

polyNIPAAm

Mw

Mw/Mn

UCST

S100-N0 S70-N30 S50-N50 S30-N70 S0-N100

100 70 50 30 0

0 30 50 70 100

100.0 45.3 29.0 15.0 0.0

0.0 54.7 71.0 85.0 100.0

6336 5818 6262 6649 6951

3.6 3.4 3.5 3.5 3.5

27 18 15

LCST

41 37 32

a Reaction mass ratios of SBMA and NIPAAm monomers used with fixed total monomer mass amount of 0.8 g in the prepared reaction solution. b The composition of the poly(SBMA-co-NIPAAm) copolymers 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. c Weight-average molecular weights (Mw) and molecular weight distributions (Mw/Mn) were estimated by GPC and calibrated with PEO. d UCST and LCST were determined by reading the absorbance at 230 nm on a UV-visible spectrophotometer.

mers with thermoresponsive and zwitterionic properties.29,30,32 These block copolymers were found to exhibit double thermosensitive phase transition of LCST and UCST behaviors in water. However, these studies did not extend to the use or evaluation of these copolymers as biological or biomedical materials. An early work reported the biocompatible nature of SBMA-based statistical copolymer coatings as potential antibioadherent surface coatings.35 In this work, an interesting combination of the zwitterionic polySBMA and nonionic thermoresponsive polyNIPAAm was studied as an example of intelligent biocompatible polymers, especially for their humanblood-contacting properties. The effect of copolymer concentrations, solvent polarities, and ionic strengths on the LCST and UCST of poly(SBMA-co-NIPAAm) in aqueous solution with different monomer ratios of SBMA and NIPAAm are discussed in detail. This study also demonstrates the adsorption of plasma proteins on the surface coated with poly(SBMA-co-NIPAAm) from human blood plasma via surface plasmon resonance (SPR), and the anticoagulant activity of the copolymers in a plateletpoor plasma solution by recalcified plasma clotting tests. This work is aimed at addressing two important issues of poly(SBMAco-NIPAAm), that is, (i) systematic measurement of the LCST or UCST characteristics from various copolymer compositions at different copolymer concentrations, solvent polarities, and ionic strengths and (ii) in vitro evaluation of blood compatibility of the fully coated copolymer surface and copolymer suspension using human plasma solution.

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), copper(I) bromide (99.999%), 2-bromoisobutyryl bromide (BIBB, 98%), pyridine (98%), 2-hydroxyethyl acrylate (97%), 2,2-bipyridine (BPY, 99%), triethylamine (99%), tetrahydrofuran (THF HPLC grade), and ethanol (absolute 200 proof) were purchased from Sigma-Aldrich. 1-Undecanethiol (99+%), (1mercapto-11-undecyl)tetra(ethylene glycol) (99+%), and 11-mercapto1-undecanol (99+%) were purchased from Asemblon INC in Redmond, Washington, U.S.A. Fibrinogen (fraction I from human plasma), γ-globulin (fractions II, III, 99%), and human serum albumin (HSA, 96-99%) were purchased from Sigma Chemical Co. Acetone and methanol were of analytical grade, purchased from Sigma Chemical Co. Deionized water (DI water) used in experiments was purified using a Millipore water purification system with a minimum resistivity of 18.0 MΩ · m. THF for reactions and washings was dried by sodium before use. ω-mercaptoundecyl bromoisobutyrate was synthesized through a reaction of BIBB using a method published previously by

our group.4,101H NMR (300 MHz, CDCl3): 4.15 (t, J ) 6.9, 2H, OCH2), 2.51 (q, J ) 7.5, 2H, SCH2), 1.92 (s, 6H, CH3), 1.57-1.72 (m, 4H, CH2), and 1.24-1.40 (m, 16H, CH2). Preparation of Poly(SBMA-co-NIPAAm) in Aqueous Solution. A total solid content of 8 wt % for different mass ratios of SBMA and NIPAAm (Table 1) was dissolved in 10.2 mL of DI water, and nitrogen was bubbled through to remove residual oxygen. The copolymerization of poly(SBMA-co-NIPAAm) was initiated using 8.0 mg of APS and 8.0 mg (0.011 mL) of TEMED. The relative molar ratio of [APS]/[TEMED] was 1:2. The reaction was stirred under positive nitrogen pressure for 6 h at 23 °C. After polymerization, the resulting reaction solution was cooled to 4 °C for 3 h and then added slowly into acetone and redissolved into DI water repeatedly to precipitate the polymer out of the reaction solution and to remove residual chemicals. Finally, the copolymer was dried in a vacuum oven at room temperature (23 °C) to yield a white powder. Preparation of Self-Assembled Monolayers on Gold Surfaces. Two self-assembled monolayers (SAMs) were formed on the substrates: (1) methyl-terminated (CH3) and (2) initiator ω-mercaptoundecyl bromoisobutyrate (Br) SAMs. Glass chips were first coated with an adhesion-promoting chromium layer (thickness 2 nm) and a surface plasmon active gold layer (48 nm) by electron beam evaporation under vacuum. Before SAM preparation, the gold-coated glass substrate was cleaned by washing with pure ethanol and DI water in sequence, dried with N2, then left in a UV light cleaner for 20 min at a source power of 110 W, followed by rinsing with DI water and ethanol, and finally dried again by N2. For preparation of CH3-SAMs, the cleaned chip was soaked in a 2 mM ethanol solution of 1-undecanethiol or (1-mercapto-11-undecyl) and tetra(ethylene glycol) thiols for 24 h to form SAMs on the gold surface, and the chip was rinsed in sequence with ethanol and water and then dried in a stream of N2. For the preparation of an initiator SAM on a gold surface, the cleaned chip was soaked in a 2 mM ethanol solution of ω-mercaptoundecyl bromoisobutyrate for 24 h to form Br-SAMs on the gold surface and then rinsed with pure ethanol followed by THF and dried in a stream of N2.4,10 Preparation of SBMA and NIPAAm Polymer Brushes on Gold Surfaces. Dense polymer brushes of polySBMA and polyNIPAAm on an SPR sensor chip were achieved via the surface-initiated ATRP method, which were prepared by the following method, as reported previously.4,10,36 Polymer brushes were polymerized on gold substrates with immobilized initiators of Br-SAMs based on our previous reports.4,10 The reaction solutions of CuBr and BPY were first placed into a sealed glass reactor in a drybox under nitrogen atmosphere. 200 mM of degassed solution (pure water and methanol at a 1:3 volume ratio) with SBMA or NIPAAm monomers was transferred to the reactor, and the gold surface with immobilized initiators was then placed into the reactor under nitrogen. After polymerization, the substrate was removed and rinsed with ethanol and water, and the samples were kept in water overnight. The prepared substrates were usually rinsed with PBS buffer to remove unbound polymers before any experiments. The thickness of the substrates was measured by ellipsometry. The thickness

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of polySBMA brushes and polyNIPAAm brushes was able to be controlled from 10 to 15 nm, as measured by ellipsometry. The chemical characterization of polymer brushes is described in detail via X-ray photoelectron spectroscopy and surface contact angle measurements in our previous publications.4,10 Characterization of the Copolymers. The structure of poly(SBMAco-NIPAAm) statistical copolymers was characterized by 1H NMR spectra using a 500 MHz spectrometer and D2O as the solvent. The composition of the poly(SBMA-co-NIPAAm) copolymers 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. Molecular weights of prepared statistical copolymers were determined by aqueous gel permeation chromatography (GPC) using one column of Viscogel TM GMPWXL K0105 (the molecular weight range was from 500 Da to 800 kDa) connected to a Waters 2414 refractive index detector at a 1.0 mL/min flow rate 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. Determination of UCST and LCST for the Copolymers. The phase transition temperatures (UCST or LCST) of the aqueous soluble and insoluble copolymers at various copolymer concentrations, solvent polarities, and ionic strengths of the solution conditions were determined by reading the absorbance at 230 nm on a UV-visible spectrophotometer using SpectraMax M5 from Molecular Devices. A given concentration of prepared copolymer was dissolved in an aqueous solution at 23 °C. The temperature of polymer solution in a well was controlled using a heating circulator and a cooler. The temperature was first cooled from 23 to 1 °C and then gradually raised from 1 to 70 °C, and the absorbance value was read for every 1 °C increment of each sample after a 10 min thermal equilibration. The UCST or LCST in a particular solution condition was defined as the temperature where the maximum slope for the absorbance versus temperature curve occurs. The confidence in the accuracy of the measured values of UCST or LCST at 230 nm was justified by the phase transition temperatures of polyNIPAAm determined at 600 nm. It was shown that the LCST values of polyNIPAAm determined by reading the absorbance at 230 and 600 nm were identical over a range of concentrations of sodium chloride from 0.01 to 2.0 M, consistent with the observation by Hoffman et al. that an increase in the sodium chloride concentration led to a decrease in LCST values of polyNIPAAm.42 Plasma Protein Adsorption. A custom-built SPR biosensor with a four-channel Teflon flow cell, designed by the Institute of Photonics and Electronics Academy Sciences (Prague, Czech Republic) based on wavelength interrogation, was used to monitor protein adsorption on the coated substrate.37 An SPR chip was attached to the base of the prism, and optical contact was established using a refractive index matching fluid (Cargille). A protein solution of 1.0 mg/mL human fibrinogen in a phosphate buffer saline (PBS, 0.15 M, pH 7.4) was delivered to the surfaces at a flow rate of 0.05 mL/min at 37 °C. In this study, platelet poor plasma (PPP) solution containing plasma proteins was also tested on the coated substrate. A surface-sensitive SPR detector was used to monitor protein-surface interactions in real time. The wavelength shift was used to measure the change in the surface adsorption amount (mass per unit area). The calibration of the wavelength shift from SPR data associated with the amount of adsorbed protein was calculated based on equations established by Campbell and co-workers.38 The calibration follows the standard calculation for the same custom-built SPR system, with a 1 nm wavelength shift resulting in an SPR response equivalent to about 15 ng/cm2 of adsorbed proteins.7,38 Plasma Clotting Time. The anticoagulant activity of prepared copolymers in this work was evaluated by testing plasma clotting time in human plasma solution. PPP solution was prepared by centrifugation of the human blood at 3000 rpm for 10 min at 23 °C. Next, 160 µL of

Chang et al. the human PPP solution was mixed with the polymer solution (10 mg/ mL, 46 µL) in a 96-well plate. The solution was then recalcified by addition of calcium (1 M CaCl2, 4 µL) and agitation for 30 s. Two incubation temperatures were tested, 23 and 37 °C. The clotting time of the plasma was determined at the time when the onset of the absorbance transition occurs by reading the absorbance at 660 nm using a PowerWave microplate spectrophotometer with programmed temperature control. Each clotting time is an average value of five samples from repeated measurements.

Results and Discussion Three zwitterionic-based copolymers with differing SBMA molar contents of copolymers (S70-N30, S50-N50, and S30N70) were prepared from various monomer compositions in the reaction solution. Molecular weight distributions of the synthesized poly(SBMA-co-NIPAAm) copolymers from GPC were calculated using Empower Pro from Waters, as shown in Table 1. All copolymer samples were controlled with a similar average molecular weight of about 6.0 kDa and the same broad molecular weight distribution (i.e., Mw/Mn ) 3.4-3.6). The increasing amount of NIPAAm monomers in the reaction solution increased the molar mass ratio of polyNIPAAm in the prepared copolymer. The composition of the poly(SBMA-coNIPAAm) copolymers was estimated by 1H NMR in D2O. A typical spectrum for S50-N50 is shown in Figure 1. Results showed that a pure poly(SBMA-co-NIPAAm) copolymer was obtained. It is noted that the molar ratio of polySBMA in the prepared copolymers is only 42 mol % even though the amount of SBMA monomers used in the reaction solution is as high as 70 wt %, indicating higher polymerized reactivity of NIPAAm monomers than of SBMA monomers in water. For comparison, two homopolymers of polySBMA (S100-N0) and polyNIPAAm (S0-N100) were also synthesized as references. To determine the soluble-insoluble phase transition of zwitterionic-based copolymers, the optical absorbance of dilute copolymer solution was measured using a UV-visible spectrophotometer with precise temperature control from 1 to 70 °C. All prepared samples exhibited clear phase transitions associated with the compositions of copolymers, even though their molecular weight distributions were poorly controlled by conventional free radical polymerization. It is generally acceptable that self-assembled monolayers (SAMs) presenting hydrophobic methyl groups usually induce large amounts of protein adsorption. Therefore, a self-assembly method was used to create one dense surface with CH3-SAMs as references for the SPR study. To characterize the blood compatibility of zwitterionic-based copolymers, prepared copolymers were first physically adsorbed onto the SPR sensor surfaces covered by CH3-terminated SAMs, followed by the in situ evaluation of plasma protein adsorption on the surfaces with self-assembled poly(SBMA-co-NIPAAm) copolymers. For comparison, the surface-initiated atom transfer radical polymerization (ATRP) method was also used to create two well-packed grafted surfaces with polySBMA and polyNIPAAm polymer brushes as references. Phase Transition Temperatures of Poly(SBMA-coNIPAAm) Copolymers in Aqueous Solution. The phase transition temperature of the copolymer solution was measured using UV-visible spectrophotometer coupled to a temperature controller at 1 °C/10 min. No thermal hysteresis was observed at equilibrium temperature from heating or cooling the copolymer solution. The maximum slope of a phase transition curve from the change of absorbance (A) signal as a function of temperature (d2A/dT2 ) 0) was determined by referring to the UCST or LCST that was recorded for each sample. As shown



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Figure 1. 1H NMR spectrum of the copolymer S50-N50 in D2O.

Figure 2. Absorbance of copolymer solutions as a function of temperature for the various samples of (a) S100-N0, (b) S70-N30, (c) S50-N50, (d) S30-N70, and (e) S0-N100 at the polymer concentration of 5 wt %.

in Figure 2, zwitterionic polySBMA exhibited a UCST in water at 27 °C, and nonionic polyNIPAAm exhibited an LCST in water at 32 °C, as reported previously.29 At temperatures below the UCST, polySBMA (S100-N0) is considered to exist as a collapsed coil and precipitates in water due to strong mutual intra- and intermolecular associations of the zwitterionic groups by electrostatic interaction.40,41 At temperatures above the LCST, polyNIPAAm (S0-N100) chains become more hydrophobic, and the hydrogen bonds with water molecules weaken, indicating the collapse of the polyNIPAAm coils and the precipitation of the polymer.25,43 The soluble-insoluble phase transitions of copolymers S70-N30, S50-N50, and S30-N70 in dilute aqueous solution were studied as functions of temperature to observe the influence of differing molar contents of polySBMA and polyNIPAAm combinations on the UCST and LCST of copolymer solutions. Interestingly, there appear both a UCST (15 °C) and an LCST (41 °C) of a doubly thermoresponsive copolymer S50-N50 from the combination of 29.0 mol % polySBMA and 71.0 mol % polyNIPAAm in the same

copolymer chain. This indicates that copolymer S50-N50 in aqueous solution is soluble from 15 to 41 °C but insoluble below 15 °C and above 41 °C at a copolymer concentration of 5 wt %. It should be noted that all prepared samples exhibit a clear shift of phase transitions strongly associated with the compositions of copolymers, even though their molecular weight distributions are poorly controlled (Table 1). As illustrated in Scheme 1, based on the results from the UV-visible spectrophotometer, the dependence of the solubility and insolubility of the copolymers on temperature can be explained by intraand intermolecular interactions between copolymer chains. In the case of the homopolymer polySBMA, copolymers S70-N30 and S50-N50 also have the ability to exhibit a UCST in water, which decreases with decreasing molar content of polySBMA in the copolymer chain. This is attributed to the interaction level of mutual electrostatic attraction by ion pairings between the ammonium cation and the sulfo-anion of the zwitterionic sulfobetaine groups.42 Thus, the copolymer precipitates with the zwitterionic polySBMA segment associations as temperature falls below the UCST. In contrast, in the case of the homopolymer polyNIPAAm, copolymers S50-N50 and S30-N70 exhibit an LCST in water that also notably depends on the molar content of polyNIPAAm in the copolymer chain. It was observed that the LCSTs of the copolymers are obviously higher than that of the homopolymer polyNIPAAm, as listed in Table 1. As the molar content of polyNIPAAm decreases in the copolymer, the collapse temperature increases for copolymer precipitates with the nonionic polyNIPAAm segment associations in water. This is attributed to the less hydrophobic interactions between polyNIPAAm side chains induced by the isopropyl groups. In the intermediate temperature range, that is, above the UCST and below the LCST, copolymers were observed to exist as soluble unimers in water, but beyond this range, copolymers were considered to precipitate as collapsed associations in water. As shown in the simplified model proposed in Scheme 1, we believe that this doubly thermoresponsive solubility behavior of the prepared copolymer results from the formation of interand intramolecular electrostatic interactions by SBMA segments

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Scheme 1. Simplified Model of the Temperature Dependence of the Copolymer Solubility and Insolubility in Aqueous Solution for the Case of Copolymer S50-N50

of zwitterionic sulfobetaine groups and intramolecular hydrophobic interactions by NIPAAm segments of nonionic isopropyl groups. We further studied the dependence of UCSTs and LCSTs on copolymer concentration in aqueous solution at a concentration of e5 wt %. In Figure 3, UCSTs of homopolymer S100-N0 and copolymer S70-N30 were found to be dependent on both the polymer concentration and molar ratio of polySBMA and polyNIPAAm. In general, as the concentration increases, the distance between polymer chains is reduced, which contributes to the formation of more intermolecular electrostatic attractions of ionic pairings of opposite charges between zwitterionic groups. Therefore, in the case of a high concentration of polymer solution, a high temperature is required to provide thermal

Figure 3. Effect of copolymer content on the UCST or LCST of the poly(SBMA-co-NIPAAm) solution for the samples of (a) S100-N0, (b) S70-N30, (c) S30-N70, and (d) S0-N100, where UCST or LCST was obtained from the absorbance transition with temperature.

energy sufficient to break the electrostatic bonding between interchain ionic pairs and to form the soluble polymer chains dispersed in water. In contrast, curve (d) in Figure 3 shows that LCSTs of homopolymer S0-N100 are basically independent of the polymer concentration. The same tendency in aqueous solution at a concentration of e1 wt % was also found in the LCST of the homopolymer of polyNIPAAm reported by Narain and his co-workers.44 It is well established that the origin of the LCST behavior of polyNIPAAm arises from the release of ordering water molecules and the formation of intrachain hydrophobic interactions associated with the side chain isopropyl moieties as the temperature increases above a critical point. Thus, the results indicate that the intramolecular hydrophobic interaction by nonionic isopropyl groups in the same polymer chain becomes so dominant that polyNIPAAm precipitation occurs at a high temperature. Interestingly, it was found that LCSTs of copolymer S30-N70 exhibit polymer concentration dependence, decreasing with an increase in polymer concentration. This might be attributed to the zwitterionic sulfobetaine groups contributing to the increase in the hydration capacity of a copolymer chain while the concentration is below 5 wt %. Effect of Solvent Polarity and Ionic Strength on the UCSTs and LCSTs of Poly(SBMA-co-NIPAAm) Copolymer Solutions. The effects of solvent polarity on the UCSTs and LCSTs of prepared poly(SBMA-co-NIPAAm) solutions were investigated using the addition of methanol to water as a case study to regulate the order of decreasing polarity at a fixed polymer concentration of 5 wt %. The results are shown in Figure 4. It was found that UCST and LCST strongly depended on the polarity of the solvent used. UCSTs of S100-N0 and S70-N30 increased with a decrease in solvent polarity (i.e., increase in methanol content). In general, as the solvent polarity is reduced, the surrounding dielectric property of the solution medium decreases, which enhances the formation of stronger intra- and intermolecular electrostatic interactions of opposite


Figure 4. Effect of methanol content on the UCST or LCST of the poly(SBMA-co-NIPAAm) solution for the samples of (a) S100-N0, (b) S70-N30, (c) S30-N70, and (d) S0-N100 at the polymer concentration of 5 wt %.

charges between zwitterionic groups. Thus, the results of curves (a) and (b) in Figure 4 show that a high temperature is required to provide high thermal energy to disrupt the intra- and interchain electrostatic interactions of polySBMA associations in a less polar environment. This also explains why the soluble zwitterionic polymers can be generally achieved by the addition of a stronger polar solvent such as water. In contrast, the LCSTs of S30-N70 and S0-N100 gradually decreased with a decrease in solvent polarity. It was found that the LCST of aqueous polyNIPAAm solutions shifts to a lower temperature when methanol is added, which was observed in previous studies.45,46 This enhanced phase separation of polyNIPAAm in watermethanol mixtures is known as cononsolvency. This property was retained at the lower SBMA molar content of S30-N70 but was lost at the higher SBMA molar content of S70-N30. On the basis of the hydration of polymer chains, the dependence of LCST on solvent polarity can be explained by the following mechanism. The insoluble polyNIPAAm in water occurs as the temperature increases above a LCST due to the thermally induced disruption of H-bonding water molecules hydrated around isopropyl groups. Thus, it is reasonable to consider that less hydration around polymer chains in a less polar aqueous solution results in a decrease in the LCST of the polymer solution. The effects of the ionic strength on the UCSTs and LCSTs of poly(SBMA-co-NIPAAm) solutions were further evaluated to investigate the solubility characteristics at a fixed polymer concentration of 5 wt %. The ionic strength of the aqueous medium was adjusted by dissolving the ionic salt NaCl into DI water at appropriate concentrations ranging from 0.01 to 2.0 M. The degree of diminution of the UCSTs of homopolymer S100-N0 and copolymer S70-N30 are dependent on both the salt concentration and the molar content of polySBMA in the polymer chain, as shown in Figure 5. Below the UCST, the formation of intra- and interchain ionic contacts between zwitterionic groups causes the polymer chains to collapse and precipitate from the solution. The variation in the UCSTs of the prepared polymer solution is found to be sensitive to the slight change of low salt concentrations from 0.01 to 0.07 M in DI water. A good linear relationship can be obtained between the critical solution temperature and the sodium chloride concentration. PolySBMA has the ability to exhibit an unusual antipolyelectrolyte behavior in the presence of salt ions that


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Figure 5. Effect of NaCl content on the UCST or LCST of the poly(SBMA-co-NIPAAm) solution for the samples of (a) S100-N0 and (b) S70-N30 at the polymer concentration of 5 wt %, and (c) S30N70 and (d) S0-N100 at the polymer concentration of 3 wt %.

notably increases with the ionic strength in the aqueous solution, as in the case of other zwitterionic polymers.40 The dissolution process of polySBMA upon the addition of electrolytes is attributed to the salt ions screening the net attractive electrostatic interactions between the zwitterionic polymer chains, which leads to polymer chain expansion. It can be seen that for each prepared sample, the LCSTs of the polymer solution sharply decrease at a certain sodium chloride concentration, which is a similar tendency found in polyNIPAAm.42 The mechanism responsible for the salt-induced changes in the soluble-insoluble properties of nonionic polyNIPAAm solutions has been reported by Hoffman and his co-workers.42 They showed that the decreasing trend in the transition temperature was correlated to the ion-water interaction of salt ions, the so-called salting out effect, suggesting that the water structure around the polymer chains of polyNIPAAm segments affected by ions results in the LCST shift.47 Blood Compatibility of Coated Poly(SBMA-co-NIPAAm) Copolymers in Human Plasma Solution. Horbett et al. showed that the adhesion and activation of platelets from the bloodstream was correlated with the adsorption of proteins on surfaces, especially fibrinogen adsorption.5,6,43 For example, for surfaces in contact with blood, even 10 ng/cm2 of adsorbed fibrinogen may introduce a full-scale blood platelet adhesion and lead to thrombosis and embolism at the blood contact side of implant devices. The extent of this effect was evaluated by the inspection of blood compatibility of the surface coated with poly(SBMAco-NIPAAm) copolymer. The physical adsorption of the prepared copolymers onto hydrophobic CH3-SAM surfaces was performed to study the antibiofouling characteristics of copolymer-coated surfaces. Nonionic isopropyl groups in the polyNIPAAm chains were used as the hydrophobic moiety of the copolymers that mediate hydrophobic interaction with CH3-SAM surfaces. It was then followed by the in situ evaluation of plasma protein adsorption on the surfaces with coated copolymers by SPR measurements. Figure 6 shows a typical SPR sensorgram in the case of the adsorption of S30-N70 copolymers, followed by the in situ evaluation of human fibrinogen adsorption. In the first stage of the copolymer coated surface formation, the amount of adsorbed copolymer is defined as the SPR wavelength difference (∆n1, nm) between the two baselines established before and after

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Figure 6. Schematic illustration of the adsorption of S50-N50 copolymers onto a CH3-terminated SAM surface followed by an in situ evaluation of fibrinogen adsorption on the surface with self-assembled poly(SBMA-co-NIPAAm).

copolymer adsorption. The saturated adsorbed amounts for each prepared copolymer on the surfaces can be obtained by the control of mass concentrations of copolymer solution above 0.1 mg/mL. In the second stage of the biofouling evaluation of fully coated copolymer surface, the amount of protein adsorption is defined as the SPR wavelength difference (∆n2, nm) between the two baselines established before and after protein adsorption. Real-time adsorption of human fibrinogen in buffered aqueous solutions and human plasma proteins in blood plasma solutions onto copolymer coated surfaces was monitored using SPR at 37 °C (human body temperature). CH3-SAMs and two homopolymer brushes of polyNIPAAm and polySBMA were used as references. The polymer brushes were prepared on gold surfaces via surface-initiated atom transfer radical polymerization following the method reported previously.4,10 A diluted solution containing 10% (v/v, in PBS) plasma proteins from platelet poor plasma was used in this measurement to reduce the effects of plasma viscosity in the laminar flow channel. The adsorption amounts from the SPR for the protein adsorption on different surfaces are shown in Figure 7. It is known that CH3-SAMs presenting hydrophobic methyl groups usually induce large amounts of protein adsorption,4,8 which can also be observed in Figure 7. It was found that polySBMA brushes are highly resistant to nonspecific adsorption for human fibrinogen and human plasma proteins at 37 °C, while hydrophobic CH3-SAMs and polyNIPAAm brushes show high protein adsorption. We observed significant decreases in adsorption of proteins on copolymer coated surfaces with S30-N70, S50-N50, and S70-N30 as compared to those on surfaces of CH3-SAMs and polyNIPAAm brushes. The copolymer-coated surfaces, even with a low molar ratio of 15% polySBMA in S30-N70 copolymers, reduced the protein adsorption to a level comparable with the adsorption on the surface grafted with polySBMA homopolymer brushes. The adsorbed amounts of plasma proteins on all copolymer-coated surfaces are found to be less than 5 ng/cm2. On the basis of previous reports from Horbett et al., it is believed that reducing plasma protein adsorption levels to below 10 ng/cm2 on biomaterial surfaces can effectively prevent the adhesion and activation of platelets from the bloodstream.6 This result suggests that a surface coated

Figure 7. Adsorption of 1 mg/mL fibrinogen and 10% human plasma in PBS buffer on CH3-SAMs, surfaces grafted with polyNIPAAm brushes and polySBMA brushes, and surfaces coated with S30-N70, S50-N50, and S70-N50 at 37 °C. A 1 nm wavelength shift in SPR is equivalent to 15 ng/cm2 adsorbed proteins.

with a copolymer prepared from a combination of the zwitterionic polySBMA and nonionic thermoresponsive polyNIPAAm has the potential to provide excellent anticoagulant activity in human blood. Poly(SBMA-co-NIPAAm) was directly incubated with human plasma to inspect the effect of direct contact activation on copolymer-induced plasma clotting by the evaluation of their recalcified plasma clotting time. Normal human PPP solution was used in this work to minimize the effects of pro-coagulant activation. All polymer samples were inserted into the recalcified human PPP solution in a 96-well plate at a room temperature of 23 °C and at human body temperature of 37 °C. Commercial PEG with a molecular weight of 4.2 kDa and a polydispersity of 1.1 was used for comparison due to the lack of anticoagulant activity induced by PEG, as reported in a previous study,39 with the results shown in Figure 8. It is generally known that the human plasma clotting time will be prolonged with a decrease in environmental temperature control, which is consistent with our results, shown in Figure 8. The plasma clotting time for the recalcified plasma solutions in blank wells was detected to have an upper limit value of about 40 min at 23 °C and of about


Figure 8. Plasma clotting time of recalcified platelet-poor plasma in the presence of different polymers at 10 mg/mL. Clotting time for blank PS wells was 40 ( 2 min at 23 °C and 8 ( 0.5 min at 37 °C. Each clotting time is an average value of five samples.

10 min at 37 °C. Almost no change in clotting time was observed with the absence or presence of 10 mg/mL PEG at 23 °C and at 37 °C, which is in agreement with the reported results.39 It was found that S30-N70, S50-N50, and S70-N30 copolymers exhibit prolonged plasma clotting time as well as anticoagulant activity in comparison to PEG for the plasma clotting test at 23 °C, while polyNIPAAm (S0-N100) and polySBMA (S100-N0) generate faster activation than do the presence of other polymers or the absence of any polymers in plasma solution. The decrease in clotting time for S0-N100 might be associated with the high fibrinogen adsorption on the polyNIPAAm brush surface by the SPR measurement at 37 °C. We observed that plasma clotting time for S100-N0 is 75% higher than that for blank PS wells at 37 °C, while S100-N0 loses its anticoagulant activity in plasma solution at 23 °C. This indicates that polySBMA has anticoagulant activity for preventing clotting of plasma from human blood at 37 °C. The distinction in the dependence of anticoagulant activity on temperature can be explained by the UCST of S100-N0 in aqueous solution, as shown in Table 1. Based on aforementioned phase behavior of polySBMA, a possible reason is that S100N0 at 23 °C, which is below its UCST, exists as a collapsed coil and precipitates out of polySBMA chains in plasma solution due to strong mutual intra- and intermolecular associations of the zwitterionic groups by electrostatic interaction. Thus, the loss of anticoagulant activity for polySBMA at 23 °C is attributed to dehydration of the zwitterionic moieties to form a hydrophobic domain, leading to plasma clotting from contact activation in human plasma solution. However, poly(SBMAco-NIPAAm) exhibits much higher anticoagulant activity in comparison to PEG and polySBMA, which depended on the temperature of human blood plasma. The results support that a judicious combination of the zwitterionic polySBMA and nonionic thermoresponsive polyNIPAAm leads to a new generation of blood-compatible biomaterial. As a result, the study shows for the first time that zwitterionic-based copolymers containing polySBMA and polyNIPAAm can be used to achieve high blood compatibility while maintaining controllable thermoresponsive functions.

Conclusions We have demonstrated the soluble-insoluble phase transition of poly(SBMA-co-NIPAAm) in aqueous media. We have found that the level of electrostatic or hydrophobic intra- and


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intermolecular interactions between polymer chains is dominated by the SBMA content. Both UCST and LCST behavior can be obtained, which depend strongly on the copolymer composition, solution concentration, solution polarity, and ionic strength. It is worth emphasizing that appropriate control of zwitterionic and nonionic molar mass ratios leads to poly(SBMA-coNIPAAm) copolymers that exhibit double-critical solution temperature in water, which means that the water-insoluble polymer-associated microdomains with zwitterionic or nonionic characters can be switched by a thermal stimulus. It was found that different ionic strengths, as well as their solution polarities, have different solubility-promoting effects on the prepared copolymers. Furthermore, results showed that there is a remarkable reduction in the adsorption of plasma proteins from human blood plasma onto the zwitterionic-based copolymer coated surface. Recalcified plasma clotting tests showed that the prepared copolymers exhibit an anticoagulant activity on human blood plasma, which depends on the composition of copolymers and the temperature of the medium. It is demonstrated that copolymers containing a relatively low polySBMA content of about 29 mol % can lead to extremely low plasma protein adsorption and high anticoagulant activity in human blood plasma. This study suggests that a zwitterionic polySBMA copolymer containing nonionic polyNIPAAm is a potential thermoresponsive biomaterial to provide a coated surface with tunable stimuli for use in human blood and biomedical implants. 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-97-CR-CE), and to the National Science Council (NSC 97-2120-M-008-002) for their financial support.

References and Notes (1) Hoffman, A. S. AdVances in Chemistry Series; American Chemical Society: Washington, DC, 1982; p 3. (2) Ratner, B. D.; Hoffman, A. D.; Schoen, F. D.; Lemons, J. E. Biomaterials Science, an Introduction to Materials in Medicine, 2nd ed.; Elsevier: Amsterdam, 2004. (3) Ratner, B. D. Biomaterials 2007, 28, 5144–5147. (4) Chang, Y.; Liao, S. C.; Higuchi, A.; Ruaan, R. C.; Chu, C. W.; Chen, W. Y. Langmuir 2008, 24, 5453–5458. (5) Shen, M. C.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. Langmuir 2003, 19, 1692–1699. (6) Kwak, D.; Wu, Y. G.; Horbett, T. A. J. Biomed. Mater. Res. 2005, 74A, 69–83. (7) Chen, S.; Zheng, J.; Li, L.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473–14478. (8) Chang, Y.; Chen, S. F.; Zhang, Z.; Jiang, S. Y. Langmuir 2006, 22, 2222–2226. (9) Feng, W.; Brash, J. L.; Zhu, S. P. Biomaterials 2006, 27, 847–855. (10) Zhang, Z.; Chen, S. F.; Chang, Y.; Jiang, S. Y. J. Phys. Chem. B 2006, 110, 10799–10804. (11) Chen, S. F.; Jiang, S. Y. AdV. Mater. 2008, 20, 335–338. (12) Zhang, Z.; Vaisocherova, H.; Cheng, G.; Yang, W.; Xue, H.; Jiang, S. Biomacromolecules 2008, 9, 2686–2692. (13) Chang, Y.; Chen, S.; Yu, Q.; Zhang, Z.; Bernards, M.; Jiang, S. Biomacromolecules 2007, 8, 122–127. (14) Yang, W.; Chen, S.; Cheng, G.; Vaisocherova, H.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Langmuir 2008, 24, 9211–9214. (15) Zhang, Z.; Chao, T.; Chen, S. F.; Jiang, S. Y. Langmuir 2006, 22, 10072–10077. (16) Higuchi, A.; Hashiba, H.; Hayashi, R.; Yoon, B. O.; Hattori, M.; Hara, M. ACS Symposium Series 876; American Chemical Society: Washington, DC, 2004; Chapter 25. (17) Hoffman, A. S.; Stayton, P. S.; Bulmus, V.; Chen, G. H.; Chen, J. P.; Cheung, C.; Chilkoti, A.; Ding, Z. L.; Dong, L. C.; Fong, R.; Lackey, C. A.; Long, C. J.; Miura, M.; Morris, J. E.; Murthy, N.; Nabeshima, Y.; Park, T. G.; Press, O. W.; Shimoboji, T.; Shoemaker, S.; Yang,

2100

(18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33)


H. J.; Monji, N.; Nowinski, R. C.; Cole, C. A.; Priest, J. H.; Harris, J. M.; Nakamae, K.; Nishino, T.; Miyata, T. J. Biomed. Mater. Res. 2000, 52, 577–586. Qiu, Y.; Park, K. AdV. Drug DeliVery ReV. 2001, 53, 321–339. Jeong, B.; Gutowska, A. Trends Biotechnol. 2002, 20, 305–311. Gil, E. S.; Hudson, S. A. Prog. Polym. Sci. 2004, 29, 1173–1222. Chilkoti, A.; Dreher, M. R.; Meyer, D. E.; Raucher, D. AdV. Drug DeliVery ReV. 2002, 54, 613–630. Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32, 7370–7379. Sershen, S. R.; Westcott, S. L.; Halas, N. J.; West, J. L. J. Biomed. Mater. Res. 2000, 51, 293–298. Schilli, C. M.; Zhang, M. F.; Rizzardo, E.; Thang, S. H.; Chong, Y. K.; Edwards, K.; Karlsson, G.; Muller, A. H. E. Macromolecules 2004, 37, 7861–7866. Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249. Pelton, R. AdV. Colloid Interface Sci. 2000, 85, 1–33. Uchida, K.; Sakai, K.; Ito, E.; Kwon, O. H.; Kikuchi, A.; Yamato, M.; Okano, T. Biomaterials 2000, 21, 923–929. Schulza, D. N.; Agarwala, P. K.; Larabeea, J.; Kaladasa, J. J.; Sonia, L.; Handwerkera, B.; Garnera, R. T. Polymer 1986, 27, 1734–1742. Arotcarena, M.; Heise, B.; Ishaya, S.; Laschewsky, A. J. Am. Chem. Soc. 2002, 124, 3787–3793. Virtanen, J.; Arotcarena, M.; Heise, B.; Ishaya, S.; Laschewsky, A.; Tenhu, H. Langmuir 2002, 18, 5360–5365. Cai, Y. L.; Armes, S. P. Macromolecules 2004, 37, 7116–7122. Maeda, Y.; Mochiduki, H.; Ikeda, I. Macromol. Rapid Commun. 2004, 25, 1330–1334. Weaver, J. V. M.; Armes, S. P.; Butun, V. Chem. Commun. 2002, 2122–2123.

Chang et al. (34) Butun, V.; Liu, S.; Weaver, J. V. M.; Bories-Azeau, X.; Cai, Y.; Armes, S. P. React. Funct. Polym. 2006, 66, 157–165. (35) Lowe, A. B.; Vamvakaki, M.; Wassall, M. A.; Wong, L.; Billingham, N. C.; Armes, S. P.; Lloyd, A. W. J. Biomed. Mater. Res. 2000, 52, 88–94. (36) Plunkett, K. N.; Zhu, X.; Moore, J. S.; Leckband, D. E. Langmuir 2006, 22, 4259–4266. (37) Boozer, C.; Ladd, J.; Chen, S.; Yu, Q.; Homola, J.; Jiang, S. Anal. Chem. 2004, 76, 6967–6972. (38) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636–5648. (39) Zhang, Z.; Zhang, M.; Chen, S. F.; Horbett, T. A.; Ratner, B. D.; Jiang, S. Y. Biomaterials 2008, 29, 4285–4291. (40) Lowe, A. B.; McCormick, C. L. Chem. ReV. 2002, 102, 41774189. (41) Dimitrov, I.; Trzebicka, B.; Muller, A. H. E.; Dworak, A.; Tsvetanov, C. B. Prog. Polym. Sci. 2007, 32, 1275–1343. (42) Park, T. G.; Hoffman, A. S. Macromolecules 1993, 26, 5045–5048. (43) Shen, M. C.; Martinson, L.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. J. Biomater. Sci., Polym. Ed. 2002, 13, 367– 390. (44) Housni, A.; Narain, R. Eur. Polym. J. 2007, 43, 4344–4354. (45) Winnik, F. M.; Ringsdorf, H.; Venzmer, J. Macromolecules 1990, 23, 2415–2416. (46) Schild, H. G.; Muthukumar, M.; Tirrell, D. A. Macromolecules 1991, 24, 948–952. (47) Schild, H. G.; Tirrell, D. A. J. Phys. Chem. 1990, 94, 4352–4356.

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