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Tunable Blood Compatibility of Polysulfobetaine from Controllable Molecular-Weight Dependence of Zwitterionic Nonfouling Nature in Aqueous Solution Yu-Ju Shih and Yung Chang* R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Chung-Li, Taoyuan 320, Taiwan Received May 25, 2010. Revised Manuscript Received September 12, 2010 This work describes a tunable blood compatibility of zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymers at a wide range of high molecular weights from 50 kDa to 300 kDa controlled with a similar polydispersity via homogeneous free-radical polymerization. The control of molecular weights of polySBMA highly regulates the zwitterionic nonfouling nature to resist the adsorption of plasma proteins, the coagulant of human plasma, and the hemolysis of red blood cells. In this study, the upper critical solution temperatures (UCSTs) and hydrodynamic size of prepared polymers are determined to illustrate the correlations between intermolecular zwitterionic associations and blood compatibility of polySBMA suspension in human blood. The polySBMA exhibited clear shifts of UCSTs in the stimuli-responsive control of solution pH and ionic strength, which were strongly associated with the molecular weights of the prepared polymers. Plasma-protein adsorption onto the polySBMA polymers from single-protein solutions and the complex medium of 100% human plasma were measured by dynamic light scattering to determine the nonfouling stability of polySBMA suspension. It was found that the nonfouling nature as well as hydration capability of polySBMA can be effectively controlled via regulated molecular weights of zwitterionic polymers. This work shows that the polySBMA polymer with an optimized molecular weight of about 135 kDa at physiologic temperature is presented high hydration capability to function the best nonfouling character of anticoagulant activity and antihemolytic activity in human blood. The excellent blood compatibility of zwitterionic polySBMA along with their stimuli-responsive phase behavior in aqueous solution suggests their potential for use in blood-contacting targeted delivery and diagnostic applications.
*To whom correspondence should be addressed. E-mail: ychang@ cycu.edu.tw.
in providing resistance to nonspecific protein adsorption.8-11 Thus, it is generally recognized that hydrophilic surfaces are more likely to reduce protein adsorption, but these surfaces are often insufficient for preventing undesirable platelet adhesion or thrombogenic response. When a plasma protein approaches a polymeric interface, electrical neutrality may be important in minimizing electrostatic interactions, and the absence of hydrogen-bond donors may also be important for minimizing hydrogen-bonding interactions.2 For many nonfouling polymers, the general properties of the functional groups are that the polymer chains are hydrophilic, electrically neutral, and hydrogen-bond acceptors rather than hydrogen-bond donors.12 This set of properties has become a general consideration guiding the design of new nonfouling polymers that resist the adsorption of plasma proteins in human blood. One of the most widely studied nonfouling polymers is poly(ethylene glycol) (PEG).13,14 Although PEG exhibits an excellent nonfouling capability with nonspecific protein resistance, it faces the problem of chemical stability in the presence of oxygen and transition-metal ions, which are found in most biochemical
(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) Shen, M. C.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. Langmuir 2003, 19, 1692–1699. (5) Kwak, D.; Wu, Y. G.; Horbett, T. A. J. Biomed. Mater. Res. Part A 2005, 74A, 69–83. (6) Chang, Y.; Liao, S. C.; Higuchi, A.; Ruaan, R. C.; Chu, C. W.; Chen, W. Y. Langmuir 2008, 24, 5453–5458. (7) Chang, Y.; Chen, W. Y.; Yandi, W.; Shih, Y. J.; Chu, W. L.; Liu, Y. L.; Chu, C. W.; Ruaan, R. C.; Higuchi, A. Biomacromolecules 2009, 10, 2092–2100.
(8) Kane, R. S.; Deschatelets, P; Whitesides, G. M. Langmuir 2003, 19, 2388– 2391. (9) Zheng, J; Li, L; Chen, S.; Jiang, S. Langmuir 2004, 20, 8931–8938. (10) Morita, S; Tanaka, M; Ozaki, Y. Langmuir 2007, 23, 3750–3761. (11) He, Y.; Hower, J.; Chen, S.; Bernards, M. T.; Chang, Y.; Jiang, S. Langmuir 2008, 24, 10358–10364. (12) Holmlin, R. E.; Chen, X. X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841–2850. (13) Harris, J. M. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, 1st ed.; Springer: New York, 1992. (14) Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S. J. Phys. Chem. B 2005, 109, 2934–2941.
Introduction Nonfouling polymers have important medical uses as bloodcontacting materials to prevent thrombogenicity by implanted devices, drug-delivery carriers, and diagnostic biosensors.1-7 It is generally acknowledged that nonspecific protein adsorption is the first interaction event occurring in the interface between many materials and human blood.1 Some plasma proteins, such as fibrinogen, are known to be important in material-induced blood clotting. For example, even a small amount of fibrinogen adsorbed on a surface (10 ng/cm2) may induce a full-scale bloodplatelet adhesion, leading to thrombosis and embolism at the blood-contacting side of material interfaces in the bloodstream.4,5 Thus, a good nonspecific plasma-protein-fouling resistance is one of the most important requirements for blood-contacting polymers. On the basis of the current studies of general nonfouling mechanisms, it is reasonable to assume that the water molecules around the pendent groups of the nonfouling chains play a key role
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solutions.15-17 Another class of nonfouling polymers is the biomimetic materials containing zwitterionic phosphatidylcholine (PC) headgroups for resisting nonspecific protein adsorption. Recently, synthetic polymers containing zwitterionic structures similar to PC, such as phosphobetaine, sulfobetaine, and carboxybetaine, have received growing attention for use in a new generation of blood-contacting materials because of their good plasmaprotein-fouling resistance.18-24 Surface-packing density and film thickness are important for zwitterionic surface resistance to nonspecific protein adsorption.25,26 In the past few years, poly(sulfobetaine methacrylate) (polySBMA), with a methacrylate main chain and an analogue of the taurine betaine pendent group (CH2CH2Nþ(CH3)2CH2CH2CH2SO3-), has become the most widely studied zwitterionic polymer due to its ease of synthetic preparation.6,7,20,22,25-29 Previous studies have shown that surfaces grafted with polySBMA polymer brushes are ideal for resisting nonspecific protein adsorption when the surface density and chain length of the zwitterionic groups are controlled, yielding excellent properties with respect to antithrombogenic response. Our previous work also reported that grafted dense polymer brushes composed of zwitterionic polySBMA formed an effective and stable nonfouling surface, potentially enabling practical use in human blood-contacting devices and implants.6,7,29 On the basis of the intended medical applications, zwitterionic polymers such as polySBMA can present diverse forms; they might be dissolved as unimers or micelles in aqueous medium, adsorbed or grafted onto aqueous-solid interfaces, or cross-linked in the form of physical or chemical hydrogels. While the singleprotein adsorption resistance of grafted zwitterionic polySBMA brushes on aqueous-solid interfaces has been studied in great depth,6,20,26-29 little is known about how polySBMA polymer conformations, such as zwitterionic chain lengths or associations, would influence the correlations between solution properties and blood compatibility. It is also important to understand the effects of stimuli-responsive signaling on the solution properties of polySBMA polymers, and the results from such studies would directly enable the rational design of zwitterionic polymers for blood-contacting targeted delivery applications. Recently, it has been reported that the solution properties of polySBMA polymers exhibit an upper critical solution temperature (UCST) in an aqueous solution that is attributed to the charge-charge or (15) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605–5620. (16) Luk, Y. Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604–9608. (17) 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. (18) Iwasaki, Y; Ishihara, K. Anal. Bioanal. Chem. 2005, 381, 534–546. (19) Chen, S.; Zheng, J.; Li, L.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473– 14478. (20) Chang, Y.; Chen, S. F.; Zhang, Z.; Jiang, S. Y. Langmuir 2006, 22, 2222– 2226. (21) Feng, W.; Brash, J. L.; Zhu, S. P. Biomaterials 2006, 27, 847–855. (22) Zhang, Z.; Chen, S. F.; Chang, Y.; Jiang, S. Y. J. Phys. Chem. B 2006, 110, 10799–10804. (23) Chen, S. F.; Jiang, S. Y. Adv. Mater. 2008, 20, 335–338. (24) Zhang, Z.; Vaisocherova, H.; Cheng, G.; Yang, W.; Xue, H.; Jiang, S. Biomacromolecules 2008, 9, 2686–2692. (25) Chang, Y.; Chen, S.; Yu, Q.; Zhang, Z.; Bernards, M.; Jiang, S. Biomacromolecules 2007, 8, 122–127. (26) Yang, W.; Chen, S.; Cheng, G.; Vaisocherova, H.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Langmuir 2008, 24, 9211–9214. (27) Zhang, Z.; Chao, T.; Chen, S. F.; Jiang, S. Y. Langmuir 2006, 22, 10072– 10077. (28) Zhang, Z.; Zhang, M.; Chen, S. F.; Horbett, T. A.; Ratner, B. D.; Jiang, S. Y. Biomaterials 2008, 29(32), 4285–4291. (29) Chang, Y.; Shu, S. H.; Shih, Y. J.; Chu, C. W.; Ruaan, R. C.; Chen, W. Y. Langmuir 2010, 26, 3522–3530. (30) Schulza, D. N.; Agarwala, P. K.; Larabeea, J.; Kaladasa, J. J.; Sonia, L.; Handwerkera, B.; Garnera, R. T. Polymer 1986, 27, 1734–1742. (31) Mary, P.; Bendejacq, D. D.; Labeau, M. P.; Dupuis, P. J. Phys. Chem. B 2007, 111, 7767–7777.
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Article Table 1. Characteristic Data of Zwitterionic PolySBMA Homopolymers characterization of polymers reaction molar ratiosa Mw hydrodynamic sample [SBMA] / (kDa)b Mw/Mn [APS] size (nm)c ID
critical solution temperature CUST (°C)d
S250 250/1 ∼56 1.85 ∼12 28 S350 350/1 ∼82 2.00 ∼13 30 S450 450/1 ∼106 1.93 ∼15 33 S550 550/1 ∼135 1.62 ∼17 35 S750 750/1 ∼180 2.02 ∼19 43 S1000 1000/1 ∼256 1.93 ∼22 50 S1250 1250/1 ∼307 1.86 ∼24 52 a Reaction molar ratios of SBMA monomer and APS initiator used with fixed total solid content of 15 wt % in the prepared reaction solution. b Weight-average molecular weights (Mw) and molecular weight distributions (Mw/Mn) were estimated by GPC and calibrated with PEO. c Hydrodynamic diameter of suspended polySBMA polymers in water at 70 °C were estimated by dynamic light scattering. d UCST was determined by reading the absorbance at 550 nm on a UV-visible spectrophotometer.
dipole-dipole interactions among the zwitterionic sulfobetaine groups.7,30,31 Herein, for the first time, we report a systematic study of the molecular-weight dependence of blood compatibility associated with the solution properties of zwitterionic polySBMA polymers. In this work, we prepared a set of zwitterionic polySBMA polymers at varying molecular weights with similar molecular-weight distributions. The effects of solution pH and ionic strengths on the UCST of these polymers of various molecular weights in aqueous solutions were examined in detail. We also demonstrated the adsorption of plasma proteins onto the zwitterionic polySBMA suspension from human blood plasma and the anticoagulant activity of the polymers in a platelet-poor plasma solution in recalcified plasma-clotting tests. This study not only introduces a fundamental understanding of the aqueous solution behavior of polySBMA polymers but also provides a physical insight into blood compatibility depending on polySBMA molecular weights correlated with zwitterionic polymer hydrations and intermolecular associations.
Materials and Methods Materials. [2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide (sulfobetaine methacrylate, SBMA) macromonomer was purchased from Monomer-Polymer & Dajac Laboratories, Inc., United States. Ammonium persulfate (APS) and ethanol (absolute; 200 proof) were purchased from SigmaAldrich. Poly(propylene oxide) (PPO) with an average molecular weight of 1 kDa and poly(ethylene glycol) (PEG) with an average molecular weight of 4 kDa were also purchased from SigmaAldrich. Fibrinogen (fraction I from human plasma) was purchased from Sigma Chemical Co. Deionized water (DI water) used in the experiments was purified using a Millipore water-purification system to a minimum resistivity of 18.0 MΩ 3 m. Phosphate-buffered saline (PBS) was purchased from Sigma.
Preparation of Zwitterionic PolySBMA Polymers in Aqueous Solution. A total solids content of 15 wt % for the different molar ratios of SBMA monomer and APS initiator (Table 1) was dissolved in 15 mL of deionized water, and nitrogen was bubbled through to remove residual oxygen. The reaction was stirred under positive nitrogen pressure for 6 h at 70 °C. After polymerization, the resulting reaction solution was cooled to 4 °C for 3 h and then added slowly into ethanol and redissolved into deionized water repeatedly to precipitate the polymer out of the reaction solution and to remove residual reagents. The copolymer was dried in a freeze-dryer at -45 °C to yield a white powder. DOI: 10.1021/la103186y
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Characterization of Zwitterionic PolySBMA Polymers. The structure of polySBMA polymers was characterized by their 1 H NMR spectra using a 500-MHz spectrometer and D2O as the solvent. The chemical compositions of the polySBMA polymers were estimated from the peak of the (CH3)2Nþ proton resonance of the sulfobetaine groups at δ = 3.2 ppm. The molecular weights of the prepared zwitterionic polymers were determined by aqueous gel-permeation chromatography (GPC), using two Viscogel columns, a G4000 PWXL and a G6000 PWXL (the range of molecular weight was from 2 kDa to 8000 kDa) connected to a model Viscotec 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 NaNO3 at pH 7.4. Poly(ethylene oxide) (PEO) standards from Polymer Standard Service, Inc. (Warwick, USA) were used for calibration.
Determination of UCST for the Zwitterionic PolySBMA Polymers. The phase-transition temperatures of the aqueous soluble and insoluble polySBMA polymers at various molecular weights, solution pH, and ionic strengths of the solutions were determined by reading their absorbance at 550 nm on a UV-visible microplate spectrophotometer using the PowerWave XS software from BioTek. A given molecular weight of prepared polymer was dissolved in an aqueous solution at a polymer concentration of 5 mg/mL at 70 °C. The temperature of the polymer solution in the microplate was controlled using a heating circulator and a cooler. The temperature was first reduced from 70 to 4 °C and then gradually raised from 4 to 70 °C. The absorbance value was read at every 1 °C increment for each sample after 120 min of thermal equilibration. The UCST for a particular solution condition was defined as the temperature where the maximum slope for the absorbance versus temperature curve occurred.
Hydrodynamic Sizes of Zwitterionic PolySBMA Polymers in a Plasma-Protein Solution. In general, aggregation due to colloidal polymer association and/or protein adsorption onto suspended polymers will result in an increase of the measured polySBMA hydrodynamic size. The dynamic measurement of hydrodynamic size using dynamic light scattering (DLS) was applied to monitor nonspecific plasma-protein adsorption from a single-protein solution and from a plasma solution onto the prepared polySBMA polymers. A single-protein solution of 1.0 mg/mL human fibrinogen in phosphate buffer saline (PBS, 0.15 M, pH 7.4) was prepared at 37 °C. A platelet-poor plasma (PPP) solution was prepared by centrifugation of human blood at 3000 rpm for 10 min at 37 °C. A volume of 100 μL of the fibrinogen solution (1.0 mg/mL) or PPP solution (100%) was mixed with 100 μL of polymer solution (10 mg/mL) at 37 °C. The hydrodynamic diameter of polySBMA in the protein solutions was determined by DLS using a Malvern ZETA-SIZER Nano S90, and the measured value was read at 2-min increments for each sample over a period of 40 min at a constant temperature of 37 °C. Plasma-Clotting Time. The anticoagulant activities of the prepared copolymers was evaluated by testing plasma-clotting time in human plasma. A volume of 160 μL of the 100% PPP solution was mixed with each polymer solution (10 mg/mL, 46 μL) in a 96-well plate. The solution was then recalcified by the addition of calcium (1 M CaCl2, 4 μL) and shaken for 30 s at 37 °C. The clotting time of the plasma was determined as the time where the onset of the absorbance transition occurred by reading the absorbance at 660 nm using the PowerWave microplate spectrophotometer with programmed temperature control. Each clotting time is reported as the average value of repeated measurements of six samples (n = 6). Red Blood Cell Hemolysis. The membrane disruption of red blood cell (RBC) was estimated to determine the nonfouling nature of prepared polySBMA polymers using a RBC hemolysis assay. The protocol used to isolate and purify the red blood cells (32) Murthy, N.; Robichaud, J. R.; Tirrell, D. S.; Stayton, P. S.; Hoffman, A. S. J. Controlled Release 1999, 61, 137–143.
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Shih and Chang and to quantitate hemolysis is described in previous work.32 RBCs were isolated by blood centrifugation and washed three times with a 0.15 M saline solution. In each hemolysis experiment, 108 RBCs were suspended in 500 μL of PBS. A 500 μL portion of RBC solution was followed by the addition of 500 μL of polySBMA solution, which was prepared in PBS at a concentration of 20 mg/ mL. The polySBMA and RBC mixtures were incubated in a 37 °C water bath for 60 min. The mixed solution was then centrifuged for 5 min at 2000 rpm to separate intact RBCs and disrupted membranes from the solution. The absorbance of the supernatant containing the released hemoglobin (Hb) was then measured at 541 nm using the PowerWave microplate spectrophotometer. Hemolysis of 100% was determined by measuring the absorbance of 108 RBCs with complete lysis by suspending them in DI water. The control was 108 RBCs in PBS. Each hemolysis is reported as the average value of three repeated measurements with eighteen samples (n = 18).
Water Sorbed onto Zwitterionic PolySBMA Polymers at Equilibrium. Previous studies have pointed out that the nonfouling characters of polymers are associated with their hydration capabilities.9-11 To observe the hydration of the prepared polySBMA polymers, the relative molarity of water sorbed onto the polymer powders in contact with liquid vapor was examined.33-35 A total of 10 mg of each polySBMA powder was dried in a 0.2-cm2 dish plate in a freeze-dryer at -45 °C for 3 days. The dried polySBMA powders were first allowed to hydrate by contact with water vapor in a constant-relative-humidity chamber (RH = 70% ( 5%) and allowed to equilibrate for 24 h under 1.1 bar at 37 °C. The change of molar water content between the hydrated polymers and the dried powders divided by the mass of the dried powders was recorded as the molarity of equilibrium-trapped water in the polymers. The hydrated polymers were then dehydrated to remove free water from the polymer matrix by contact with dry air at the low pressure of 0.1 bar and equilibrated for 24 h at 37 °C in a vacuum oven. The change of molar water contents in the dehydrated polymers and the dried powders divided by the mass of the dried powders was recorded as the molarity of equilibrium-retained water in the polymers. Each water sorption is reported as the average value of repeated measurements of six samples (n = 6).
Results and Discussion For the characterizations of the polySBMA polymerization in aqueous solution, the integrated data of 65 polymer samples are shown in Figure 1. These results showed that controllable molecular weights were obtained for the polySBMA polymers over a wide range, from 1.6 to 450 kDa. The hydrodynamic diameter of the polymers in aqueous solution was estimated by DLS. Increasing the ratio of SBMA monomer to APS initiator in the reaction solution increased the molecular weight and hydrodynamic size of the prepared zwitterionic polymers. To study blood compatibility associated with the solution properties of the polymers, seven samples with different molecular weights (S250, S350, S450, S550, S750, S1000, and S1250) were prepared from various molar ratios of monomer to initiator in the reaction solutions. The molecular-weight distributions of the synthesized polySBMA polymers were calculated from the GPC data using the OmniSEC software from Viscotek and are shown in Table 1. The polymer samples have similar molecular-weight distributions (i.e., Mw/Mn = 1.8 ( 0.2). To characterize the soluble-insoluble phase transitions of the polymers, the optical transmittance of dilute polymer solutions was measured using a UV-visible (33) He, Y.; Chang, Y.; Hower, J. C.; Zheng, J.; Chen, S. F.; Jiang, S. Y. Phys. Chem. Chem. Phys. 2008, 36, 5539–5544. (34) Bajpai, A. K.; Bajpai, J.; Shunkla, S. React. Funct. Polym. 2001, 50, 9–21. (35) Ide, M.; Mori, T.; Ichikawa, K.; Kitano, H.; Tanaka, M.; Mochizuki, A.; Oshiyama, H.; Mizuno, W. Langmuir 2003, 19, 429–435.
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Figure 1. Evolution of molecular weights (Mw), molecular-weight distribution (Mw/Mn), and hydrodynamic size of polySBMA polymers versus molar ratio of SBMA monomer to APS initiator.
spectrophotometer with precise temperature control from 4 to 70 °C. All the prepared samples exhibited clear solution-phase transitions associated with the molecular weights of the polySBMA polymers. It is recognized that PEG, presenting hydrophilic polymer chains, usually reduces protein adsorption. For comparison, PEG was used as a reference material to characterize the blood compatibility of the zwitterionic polySBMA polymers. This study aimed to address two important issues with sulfobetaine polyzwitterions: (i) a fundamental understanding of the correlation between blood compatibility and solution phase behavior of polySBMA polymers depending on their molecular weights and (ii) a physical insight into blood compatibility of polySBMA correlated with zwitterionic polymer hydrations and intermolecular associations. Critical Temperatures of PolySBMA Polymers in Aqueous Solution. To investigate the solution properties of the prepared zwitterionic polymers, the critical temperatures of polySBMA polymer solutions were measured using a UV-visible spectrophotometer coupled to a temperature controller set to heat or cool at 1 °C/120 min. There was no thermal hysteresis observed at the equilibrium temperatures during the heating or cooling of the polymer solutions. Scheme 1 shows two typical phase-transition curves, with the transmittance of the polymer solution and the hydrodynamic size of the polymer suspension as functions of temperature for the case of the polySBMA polymer S550 (with a molecular weight of ∼135 kDa) at a polymer concentration of 5 mg/mL in aqueous solution. In a dilute zwitterionic polymer solution at temperatures below 15 °C, polySBMA (S550) was observed to exist as aggregated gel precipitates with a transmittance of 20%, which indicated the formation of a translucent physical gel state with a self-sustaining water content. A phasetransition region was observed between 15 and 50 °C in which the transmittance of the S550 polymer solution exhibited temperature dependence, increasing with an increase in solution temperature. This may be attributable to the increase in available thermal energy contributing to a decrease in the polymer chain associations as well as the polymer hydrodynamic size as the solution temperature was increased from 15 to 50 °C. At temperatures above 50 °C, polySBMA (S550) polymers existed as a transparent soluble unimer state with a hydrodynamic size of 18.8 ( 1.3 nm, indicating the suspension of the polySBMA without polymer chain associations in water. As illustrated in Scheme 1, based on the DLS results the temperature dependence of the hydrodynamic Langmuir 2010, 26(22), 17286–17294
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size of polySBMA can be explained by intra- and intermolecular interactions between polymer chains. In general, at temperatures below the UCST, zwitterionic polymers are considered to exist as a collapsed coil and precipitate in water owing to strong mutual intra- and intermolecular associations of the zwitterionic groups due to electrostatic interaction. In aqueous solution, polySBMA, like other zwitterionic polymers, exhibits an UCST.30 In a case study of polySBMA polymers in deionized water, the hydrodynamic size of the polymer S550 decreased with increasing solution temperature, which was attributed to the different interaction level of mutual electrostatic attraction in intrachain and/or interchain associations by ion pairings between the ammonium cation and the sulfo-anion of the zwitterionic sulfobetaine groups.29 The zwitterionic polymers S250, S350, S450, S550, S750, S1000, and S1250 were studied in dilute aqueous solution by varying the temperature and inspecting the influence of their differing molecular weights on the soluble-insoluble phase transitions of the polymer solutions. The maximum slope of a phasetransition curve from the change in the transmittance (Λ) signal as a function of temperature (d2Λ/dT2 = 0) was determined by referring to the cloud point (UCST) recorded for each sample. As shown in Figure 2, the zwitterionic polySBMA polymers at a concentration of 5 mg/mL in deionized water exhibited UCSTs depending on their molecular weights. The data show an obvious correlation between the increase in the molecular weight of polySBMA and the increase in UCST values from 28 to 52 °C, as shown in Table 1. In general, as the molecular weight increases, the increased hydrodynamic size of a zwitterionic polymer coil contributes to the formation of more intramolecular electrostatic attractions, that is, ionic pairings of opposite charges between zwitterionic groups. Therefore, there is no obvious increase of hydrodynamic size as the molecular weight of polySBMA increased from ∼200 to ∼400 kDa. In the high molecular weight of polySBMA, the high density of the polymer chain inside the unimer coil structure might have partial intrachain association, resulting in the contraction of some domain inside the unimer coil structure. In the case of a high-molecular-weight polySBMA polymer solution, a high temperature is required to provide thermal energy sufficient to break the electrostatic bonding between interchain ionic pairs and to form soluble polymer chains dispersed in aqueous solution. Effect of Solution pH and Ionic Strength on the UCSTs of PolySBMA Polymer Solutions. The effect of solution pH on the UCSTs of the prepared polySBMA polymers was investigated using the addition of HCl or NaOH to deionized water to adjust the pH to values from 1 to 12 at a fixed polymer concentration of 5 mg/mL, and the results are shown in Figure 3. At room temperature (25 °C), all tested polySBMA polymers were insoluble in deionized water but soluble in aqueous solutions containing a critical concentration of added HCl or NaOH. It was found that the variation of solution pH may affect the UCSTs of the prepared polySBMA with respect to the equilibrium temperature when cooling the polymer solutions from 70 to 4 °C at 1 °C/ 120 min. It is known that changes in solution pH can act to screen the repulsive electrostatic forces between charged groups along the polymer chain of polyelectrolytes, resulting in shrinkage of the polymer coil. In contrast, as HCl or NaOH was used to control the solution pH, the UCSTs of the zwitterionic polySBMA polymers decreased depending on their molecular weights when the pH values were adjusted to below or above the physiological pH of 7.4. This indicates that an aqueous solution containing acidic or basic ions might introduce antipolyelectrolyte effects and thus activate intra- and intermolecular electrostatic interactions of opposite charges between zwitterionic groups and promote the DOI: 10.1021/la103186y
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Shih and Chang Scheme 1.
a
a Shown are (a) chemical structures of associated and nonassociated polySBMA homopolymers; (b) simplified model of the temperature dependence of the zwitterionic polymer solubility in aqueous solution for the case of S550 with an Mw of about 135 kDa. In phase transition region, there is a formation of colloidal gel state of polySBMA micelles.
formation of soluble polySBMA as hydrated polymer chains dispersed in water. Interestingly, the pH-induced changes in the soluble-insoluble phase transitions appear to be very sharp in a narrow temperature range between 10 and 12 °C in dilute aqueous solution in a strongly acidic or basic medium. We observed the polySBMA polymer collapse sharply at a UCST of about 11 °C at a solution pH below 3 or above 12 in a manner independent of the molecular weight over the range of 50-300 kDa. In general, chain expansion of a zwitterionic polymer in aqueous solution occurs upon the addition of electrolyte, exhibiting the so-called antipolyelectrolyte effect. This behavior is also typical of most zwitterionic polymers composed of sulfobetaines with three methylene units between the cationic and anionic groups.7,29 The effects of the ionic strength on the UCSTs of polySBMA solutions 17290 DOI: 10.1021/la103186y
were evaluated to investigate the solubility characteristics associated with polymer molecular weight at a fixed polymer concentration of 5 mg/mL. The ionic strength of the aqueous medium was adjusted by dissolving the electrolyte NaCl in deionized water at concentrations ranging from 0.005 to 0.5 M. The degree of diminution of the UCSTs of the tested zwitterionic polySBMAs of three different molecular weights (S250, S550, and S1250) was dependent on both the electrolyte concentration and the polymer molecular weight, as shown in Figure 4. As expected, polySBMA polymers exhibited an unusual antipolyelectrolyte behavior in the presence of salt ions that notably increased with the ionic strength of the aqueous solution. The dissolution process of polySBMA on the addition of electrolytes was attributed to the screening of the net attractive electrostatic interactions between the zwitterionic Langmuir 2010, 26(22), 17286–17294
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Figure 2. Phase transitions of polySBMA polymer in aqueous solution as functions of temperature for the samples of the S250, S350, S450, S550, S750, S1000, and S1250 polymers at a concentration of 5 mg/mL.
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Figure 4. Effect of ionic strength on the UCST of the polySBMA solutions of polymers S250, S550, and S1250 at a concentration of 5 mg/mL.
Figure 3. Effect of solution pH on the UCST of the polySBMA solution for the samples of the S250, S350, S450, S550, S750, S1000, and S1250 polymers at a concentration of 5 mg/mL.
polymer chains by salt ions, and this effect led to polymer chain expansion. It was found that the increasing trend in the transition temperature was correlated with the increased molecular weight of polySBMA, indicating that the intra- and intermolecular associations of opposite charges between zwitterionic sulfobetaine groups affected by polymer chain length resulted in the UCST shift. Nonfouling Stability of PolySBMA Polymers in Plasma Protein Solution. Nonspecific protein adsorption has been recognized as the first event in blood-polymer interactions, and blood proteins such as fibrinogen play an important role in polymer-associated clotting.4,5 Thus, polymers with proteinresistant properties are highly desired to reduce blood clotting. The stability of polySBMA polymers of three different molecular weights (S250, S550, and S1250) was first evaluated in 1 mg/mL solution of human fibrinogen (340 kDa, pI = 5.5) at 37 °C to examine nonspecific protein adsorption onto these polymers, using the hydrophobic PPO (1 kD) and hydrophilic PEG (4 kDa) polymers as references for comparison. Aggregation by colloidal polymer association and/or protein adsorption on the surface of the polymers results in an increase of the measured hydrodynamic size using DLS. As shown in Figure 5a, after mixture with singleLangmuir 2010, 26(22), 17286–17294
Figure 5. Hydrodynamic size of polySBMA polymers versus time with different polymer molecular weights in a single-protein solution and in plasma at 37 °C. PPO (1 kDa), PEG (4 kDa), and polySBMA (S250, S550, and S1250) in (a) 1.0 mg/mL human fibrinogen solution and (b) 100% blood plasma.
protein fibrinogen in PBS solution the hydrodynamic diameter of protein-adsorbed PPO particles increased to ∼1000 nm after 25 min, indicating a dramatic aggregation which was attributed to the hydrophobic interaction between PPO and protein. As DOI: 10.1021/la103186y
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expected, the PPO polymer, presenting hydrophobic methyl groups, induced large amounts of protein adsorption. Interesting phenomena were observed for the three tested polySBMA polymers when mixed with the single-protein solution. Similar to the stability of PEG in the fibrinogen solution, the hydrodynamic diameter of the polymer S550 remained nearly constant at 16.7 ( 0.2 nm, with no obvious size increase during the test period of 40 min. This indicates that polymer S550 is quite stable in the presence of a single-protein solution under physiological conditions. Contrary to the stable behavior of S550, polymer S1250, with the relative large molecular weight of about 300 kDa, showed a size increase of ∼600 nm by the end of 20 min of incubation, indicating significant protein adsorption and particulate aggregation. However, it was also found that a 30 mol % polymer S250 suspension was not stable in such a single-protein solution, which was determined by DLS analysis. However, it was found that a 30 mol % of polymer S250 still kept stable unimers in such a single-protein solution, but a 70 mol % of polymer S250 forms protein-adsorbed polySBMA particles by the end of 40 min of incubation, which was determined by DLS analysis. The stability of polySBMA polymers in undiluted (100%) human (platelet-poor) blood plasma was tested to elucidate the plasma-protein adsorption characteristics. It should be noted that the adsorption of components from PPP solution onto the dispersed polymer surface occurs not only with the major protein components of plasma but also other small biomolecules such as amino acids, lipids, urea, fats, and polysaccharides. The adsorption test in a multicomponent solution of 100% plasma is more complex and challenging than that with single-protein solutions. As shown in Figure 5b, the PPO polymer with hydrophobic methyl groups induced nonspecific protein adsorption in plasma due to hydrophobic interactions. PEG, polymer S250, and polymer S1250 were also unstable in the extreme situation of contact with 100% plasma. Their hydrodynamic diameter increments were ∼200 and ∼300 nm, respectively, after an incubation period of 40 min. Aggregates of colloidal polymer associated with protein nonspecifically adsorbed on their surfaces could be observed in the above solutions. Surprisingly, polymer S550 exhibited almost the same hydrodynamic size without nonspecific protein adsorption in 100% plasma, indicating its excellent nonfouling stability. The above results demonstrate that the nonspecific proteinadsorption characteristics of polySBMA polymers are strongly associated with their molecular weights in single-protein solution or 100% plasma under physiological conditions. Anticoagulant Activity of PolySBMA Polymers in Human Plasma Solution. In general, nonspecifically adsorbed plasma proteins interact in a serious of reactions leading to plasma clotting.4,5,7,28 Among plasma proteins, fibrinogen plays a leading role in mediating surface-induced activation as polymeric materials contact human blood plasma under static conditions. The measurement of plasma clotting has already become a recognized test to estimate the blood compatibility of a prepared material.28 The prepared polySBMA samples of S250, S350, S450, S550, S750, S1000, and S1250 were directly incubated with human plasma to inspect the effects of direct-contact activation on polymer-induced plasma clotting as evaluated by their recalcified-plasma-clotting times. Commercial PEG with a molecular weight of 4.2 kDa and a polydispersity of 1.1 was used for comparison owing to the lack of anticoagulant activity induced by PEG as reported in the previous study.28 All polymer samples at 10 mg/mL were added to recalcified human PPP solution in a PS 96-well plate at physiologic temperature of 37 °C. In Figure 6, the plasma clotting for the recalcified plasma solutions in blank PS wells was determined to have an upper limit of plasma-clotting 17292 DOI: 10.1021/la103186y
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Figure 6. 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 9 ( 1.0 min at 37 °C. Each clotting time is an average value of six samples.
time of about 10 min at 37 °C for the protocol used. When the hydrophobic PPO (1 kDa) was put into the recalcified PPP solution, the clotting time decreased to ∼7 min. The result indicates that hydrophobic PPO is a highly activating polymer which activates plasma clotting through the intrinsic coagulation pathway. Almost no change in plasma clotting time of the absence or presence of PEG was observed in a 96-well plate at 37 °C. The results indicate that PEG polymers do not activate plasma clotting through the intrinsic coagulation pathway, which is in agreement with the reported results.28 In the absence or presence of SBMA monomer, again no change in clotting time was detected. Similar to PEG, SBMA monomer did not activate plasma clotting and exhibited no anticoagulant activities at 37 °C. It is interesting to observe that when polymer S250 was added into PPP solution, the average clotting time increased to ∼12 min, indicating an anticoagulant activity of polySBMA. In the case of polymer S550, the plasma-clotting time was further prolonged to ∼20 min at 37 °C. Plasma-clotting time as well as anticoagulant activity was maximized when the molecular weight of polySBMA was about 135 kDa. Above this molecular weight, plasma clotting time decreased as the polymer molecular weight increased. In the case of polymer S1250, with a molecular weight of ∼300 kDa, the plasma-clotting time was shortened to ∼5 min at 37 °C; it generated faster activation in plasma than the absence or presence of PEG and other polySBMA polymers. The decrease in clotting time for S1250 might be associated with fibrinogen adsorption onto the high-molecular-weight polySBMA in single-protein solution revealed by the DLS measurement at 37 °C, as shown in Figure 5a. In physiological conditions at 37 °C, we observed that the plasma-clotting time for S550 was much higher than that for blank PS wells, while the SBMA monomer exhibited no anticoagulant activity and S1250 lost its anticoagulant activity in 100% plasma. The polySBMA with an Mw of about 135 kDa presented the best anticoagulant activity in 100% blood plasma, which is because the S550 polymer is highly stable and resistant to nonspecific protein adsorption from fibrinogen solution and 100% plasma. This clearly indicates that polySBMA has a molecular-weight dependence with respect to anticoagulant activity or contact activation for preventing or activating plasma clotting in human blood. The inflection point in the dependence of anticoagulant activity on polySBMA molecular weight may be Langmuir 2010, 26(22), 17286–17294
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Figure 7. Hemolysis of RBC solution in the presence of different polySBMA at a polymer concentration of 10 mg/mL. Hemolytic activity is normalized relative to that observed from the positive control (DI water) and negative control (PBS) at 37 °C. Each hemolysis result is representative data from an average value of 18 samples.
Figure 8. Relative molarity of water sorption in the different polymers. The measurement of (a) equilibrium-absorbed water and (b) equilibrium-retained water in polySBMA polymers equilibrated for 24 h at 37 °C. Each relative molarity of water sorption is an average value of six samples.
due to the zwitterionic association or nonassociation of polymer chains in aqueous solution, as shown in Scheme 1a. One possible interpretation is that a high-molecular weight polySBMA below its UCST, such as S1000 and S1250 at 37 °C, exists as a collapsed coil and precipitates out of polySBMA chains in solution due to strong mutual intra- and intermolecular associations of the zwitterionic groups by electrostatic interaction. Thus, the loss of anticoagulant activity for high molecular weights of polySBMA at 37 °C is attributed to the dehydration of their zwitterionic moieties to form a hydrophobic domain, leading to clotting from contact activation in human plasma. Furthermore, it was found that the anticoagulant activity of S1000 at 50 °C gives better results than a specific molecular weight of S550 and S1250. As illustrated in Scheme 1b, the results support the proposed model in the phase transition region of polySBMA suspension that the good accessibility of the zwitterionic groups in polySBMA polymers above its UCST or the negatively charged groups around polySBMA micelles at its UCST that introduce good anticoagulant activity. However, most biomaterials are usually used in biological environment at 37 °C. Thus, it is important to control specific molecular weight of polySBMA to give blood compatibility in human blood. Antihemolytic Activity of PolySBMA Polymers in RBC Solution. To further evaluate the influence of polymer molecular weights on blood compatibility of prepared polySBMA, a RBC hemolysis assay was performed. The observed hemolysis of RBCs in DI water and PBS solutions at 37 °C were used as positive and negative controls, respectively. The observed hemolytic activity of polySBMA at a given molecular weight at 37 °C was normalized to that of the positive control, DI water. The hemolytic activity of hydrophobic PPO (1 kDa), hydrophilic PEG (4 kDa), and heparin were also tested as references for comparison. Figure 7 shows the molecular weight dependent hemolytic activity of the prepared polySBMA polymers in RBC solution. In general, hydrophobic polymers are capable of interacting with biological membranes, causing disruption. Thus, it was observed that PPO exhibited ∼12% hemolytic activity. No apparent hemolytic activity was observed for the references of PEG and heparin (less than 1%). It is interesting to observe that hemolytic activity exhibits a minimum for the S550 at a specific molecular weight of
about 135 kDa, which is comparable with heparin. However, all of polySBMA in RBC solution at physiologic conditions showed very little hemolysis (less than 2%), indicating good nonfouling nature of zwitterionic polymers with antihemolytic activity to resist the disruption of blood cell membranes. Correlation of Nonfouling Nature and Hydration Capability of PolySBMA Polymers. Recent studies have shown that the nonfouling properties of PEG polymers are attributed to their strong hydration capabilities and conformational structures.9,10 It is accepted that the formation of a hydration layer around polymer chains results in a nonfouling character. In general, while hydrophilic and neutral PEG polymers form a hydration layer via hydrogen bonds, zwitterionic polySBMA forms a hydration layer via electrostatic interactions.11,33 Therefore, it is expected that polySBMA polymers are capable of binding significant quantities of water, which is correlated to their blood compatibility. To quantify the hydration capacity of zwitterionic polySBMA, its molar water sorption was determined as the molar change between water in hydrated polySBMA and dry polySBMA divided by the mass of fully dried polySBMA.34,35 The measured hydration capacity may consist of the following two types of contributions: (1) equilibrium-absorbed water, which is measured from the amount of water vapor sorbed onto polySBMA at 1.1 bar and 37 °C after 24 h of controlled humidity (RH ≈ 70%) and (2) equilibrium-retained water, which is measured as the residual of trapped water desorbed from polySBMA at 0.1 bar and 37 °C after 24 h in a vacuum oven. The results of the relative molar water-sorption measurements in different molecular weights of polySBMA at 37 °C are shown in Figure 8, with hydrophobic PPO (1 kDa) and hydrophilic PEG (4 kDa) polymers used as references for comparison. The relative molarity of water sorption in polymer at a given molecular weight at 37 °C was normalized to that of water sorption in SBMA monomer. In the water-vapor sorption trials, the increased molecular weight of polySBMA promoted the hydration capacity of equilibrium-absorbed water in the prepared polymers, which mainly consists of the confined free space between polySBMA chains and water-binding sites around polySBMA pendent groups. The measured water sorption of equilibrium-retained water was proportional to the quantity of tightly bound water molecules around the zwitterionic sulfobetaine
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structure. We observed that the equilibrium-retained water for the zwitterionic SBMA monomer with a measured water sorption of ∼2 mmol/g at 37 °C was similar to that for the hydrophilic PEG polymers, and neither activated plasma clotting nor exhibited anticoagulant activities in physiological conditions. Interestingly, it was found that the increase in molecular weight of polySBMA from 55 to 135 kDa gave the polymer more capacity for binding water molecules as evidenced by the increase in equilibriumretained water, indicating the formation of a strong hydration layer from the zwitterionic sulfobetaine groups. In the range of polySBMA molecular weight without polymer chain associated by ion pair (such as S250, S350, S450 and S550), the increased molecular weight of polySBMA in the aqueous solution promoted the increased volume of polySBMA random coil to retain more bound and free water from within the hydrated polySBMA coils. However, the capacity for binding water molecules decreased as the molecular weight of polySBMA increased from 135 to 300 kDa. This could be due to the enhanced interaction level of mutual electrostatic attraction by ion pairing between the ammonium cation and the sulfo-anion of the zwitterionic sulfobetaine groups with the increased molecular weight of polySBMA. In the case of polymer S1000 and S1250, similar to the hydrophobic PPO polymers, it was observed that the measured relative molarity of water sorption for equilibrium-retained water was below 5% at 37 °C, indicating the low hydration capacity of polySBMA chains with high molecular weight due to the strong intra- and intermolecular associations of opposite charges between sulfobetaine groups. Thus, the polymer S1000 and S1250 precipitated with the zwitterionic polySBMA segment associations at 37 °C, resulting in the activation of coagulant properties in contact with human blood plasma. There appeared to be an inflection point in the equilibrium-retained water at a molecular weight of ∼135 kDa for the polymer S550. It was shown that polymer S550 had the strongest hydration capability for binding water, resulting in the best anticoagulant activity and antihemolytic activity in physiological conditions, which was attributed to the formation of a highly hydrated, nonassociated polySBMA conformational state, as shown in Scheme 1. As a result, this study showed for the first time that the nonfouling nature of sulfobetaine polymers as well as the water-binding
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capabilities of zwitterionic polySBMA are strongly associated with their molecular weights.
Conclusions A tunable blood compatibility of polysulfobetaine was observed in a controllable range of molecular weights with a regulated zwitterionic nonfouling nature in aqueous solution. The soluble-insoluble phase transitions of polySBMA in aqueous media were determined to illustrate intermolecular associations of prepared polyzwitterions, which depend strongly on the stimuliresponsive control of solution pH and ionic strength. We found that polySBMA polymers of increasing molecular weights had enhanced mutual intra- and interchain associations of the sulfobetaine groups in aqueous solution, resulting in the increase of their UCSTs associated with the nonfouling nature of polySBMA suspension. The blood compatibility of the polySBMA polymers in human blood was characterized with respect to plasma-protein adsorption from a pure fibrinogen solution as well as from human plasma solution and to recalcified plasma-clotting time and RBC hemolysis at the physiological temperature. The results showed that polySBMA polymers exhibited an anticoagulant activity in 100% human plasma and antihemolytic activity in RBC solution that depended on the molecular weights of the prepared polyzwitterions. Importantly, the polySBMA polymer with a molecular weight of about 135 kDa presented an excellent nonfouling character in human blood for plasma-protein resistance, anticoagulant activity, and antihemolytic activity, which was attributed to the formation of a strong hydration layer due to the binding of water molecules around sulfobetaine groups. This study suggests that that the tunable hemocompatible nature of polySBMA polymers by controlling molecular weights gives them great potential in the molecular design of antithrombogenic materials for use in human blood. 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., and to the National Science Council (NSC 99-2221-E-033-001-MY2) for their financial support.
Langmuir 2010, 26(22), 17286–17294
