Surface Modification with Poly(sulfobetaine methacrylate-co-acrylic

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Surface Modification with Poly(sulfobetaine methacrylate-co-acrylic acid) To Reduce Fibrinogen Adsorption, Platelet Adhesion, and Plasma Coagulation Wei-Hsuan Kuo,† Meng-Jiy Wang,† Hsiu-Wen Chien,‡ Ta-Chin Wei,§ Chiapyng Lee,*,† and Wei-Bor Tsai*,‡ †

Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, Keelung Rd., Sec. 4, Taipei 106, Taiwan ‡ Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan § Department of Chemical Engineering, Chung Yuan Christian University, 200, Chung Pei Rd., Chung Li 320, Taiwan S Supporting Information *

ABSTRACT: Zwitterionic sulfobetaine methacrylate (SBMA) polymers were known to possess excellent antifouling properties due to high hydration capacity and neutral charge surface. In this study, copolymers of SBMA and acrylic acid (AA) with a variety of compositions were synthesized and were immobilized onto polymeric substrates with layer-by-layer polyelectrolyte films via electrostatic interaction. The amounts of platelet adhesion and fibrinogen adsorption were determined to evaluate hemocompatibility of poly(SBMA-co-AA)-modified substrates. Among various deposition conditions by modulating SBMA ratio in the copolymers and pH of the deposition solution, poly(SBMA56-co-AA44) deposited at pH 3.0 possessed the best hemocompatibility. This work demonstrated that poly(SBMA-co-AA) copolymers adsorbed on polyelectrolyte-base films via electrostatic interaction improve hemocompatibility effectively and are applicable for various substrates including TCPS, PU, and PDMS. Furthermore, poly(SBMA-co-AA)-coated substrate possesses great durability under rigorous conditions. The preliminary hemocompatibility tests regarding platelet adhesion, fibrinogen adsorption, and plasma coagulation suggest the potential of this technique for the application to blood-contacting biomedical devices. method.4,5 Besides, incorporation of phosphorylcholine moieties of cell membrane lipids also enhances protein resistance of substrates and improves blood compatibility. 6,7 Recently, polymers of zwitterionic molecules sulfobetaine methacrylate (SBMA) or carboxylbetaine methacrylate (CBMA), whose structures are similar to the head of phosphorylcholine, show high potential in improving the hemocompatibility of biomaterials.8−13 Poly(SBMA) has been proven to prevent protein adsorption and cell adhesion.8,14−17 Creating a highly hydrated surface is considered to be a key for the resistance of zwitterionic polymers to nonspecific protein adsorption. Zwitterions are capable of binding a significant number of water molecules and therefore exhibit excellent antifouling property. Furthermore, charge neutrality is another key factor for protein repulsion by reducing charge interactions with protein molecules. Many studies have been focused on deposition of a layer of zwitterionic polymers on biomaterials to enhance their

1. INTRODUCTION Synthetic biomaterials, lacking the antithrombogenic mechanism existing in the endothelial lining of blood vessels, usually exhibit poor hemocompatibility. Therefore, life-threatening biomaterial-induced thromboembolic complications remain in the deployment of blood-contacting devices. The adsorption of blood proteins, especially the clotting enzymes and fibrinogen, is recognized as the first event following blood−biomaterials contact and plays an important role in biomaterial-induced thrombus formation.1 Circulating platelets recognize and adhere to surface-bound plasma adhesive proteins (such as fibrinogen and vWF) via platelet membrane integrins such as GP IIb/IIIa.1 Platelet adhesion to biomaterials is often followed by activation of the adherent platelets, accelerating thrombosis by promoting thrombin formation and platelet aggregation.2 Therefore, prevention of protein adsorption and platelet adhesion has been a common strategy to improve hemocompatibility of biomaterials. A common approach to prevent thrombosis on biomaterials is to create a nonfouling surface to prevent plasma protein adsorption.3 In this context, modification of surface with poly(ethylene glycol) (PEG) has been the most used © 2011 American Chemical Society

Received: September 21, 2011 Revised: November 9, 2011 Published: November 13, 2011 4348

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diaphorase, 0.1 mg/mL BSA, and 4 mg/mL sucrose in PBS. Oxamate solution was prepared by adding 16 mg/mL of sodium oxamate in PBS. 2.3. Synthesis and Characterization of Poly(SBMA-coAA). SBMA/AA copolymers with five different monomer ratios of SBMA/AA (10/90, 25/75, 50/50, 75/25, and 100/0) were synthesized by free radical polymerization. The total molar concentration of the monomers was fixed at 0.75 M, and the monomers were degassed with nitrogen prior to reaction. Polymerization was initiated by addition of 5 mM ammonium persulfate in monomer solution, proceeded at 60 °C for 5 h with constant stirring (Scheme 1), and

hemocompatibility. For example, surfaces decorated with brushlike poly(SBMA) or poly(CBMA) via surface-initiated atom transfer radical polymerization exhibited amazingly anticoagulant activity, indicated by very low protein adsorption from plasma and platelet adhesion.8 Furthermore, a block copolymer of poly(SBMA) and hydrophobic poly(propylene oxide) could physically adsorb on hydrophobic substrates and prevent fibrinogen adsorption.15 On the other hand, copolymer of AEDAPS (3-(2-(acrylamido)ethyldimethylammonio)propanesulfonate) and charged monomer such as acrylic acid (AA) could adsorb to a substrate with the opposite charges and prevent cell adhesion.18,19 Charged zwitterionic copolymers could be applied to a variety of biomaterials by combination with a simple surface modification technique, layer-by-layer polyelectrolyte deposition, which is based on the alternate adsorption of positively and negatively charged polyelectrolytes to build a ultrathin film with either positive or negative charges onto a substratum. One advantage of this technique is its applications with little limitation to the type, size, and shape of the substrates. Previously, Schlenoff and co-workers applied copolymers of AEDAPS and AA to adsorb onto layer-by-layer multilayer polyelectrolyte films, and found that cell attachment was inhibited at intermediate concentration of SBMA in the copolymers.18,19 In this study, similar approach was applied to improve biomaterials’ hemocompatibility. Particularly, we synthesized a series of poly(SBMA-co-AA) to evaluate the dependence of SBMA contents on the efficacy of inhibiting platelet adhesion and fibrinogen adsorption. Furthermore, the stability of multilayer polyelectrolyte films was provided by the integration of azide groups for UV-cross-linking, and only four-layer film was sufficient for the desired functions. Several materials such as tissue culture polystyrene, polyurethane, and polydimethylsiloxane were precoated with a trilayer polyelectrolyte base and then adsorbed with SBMA/AA copolymers at different ratios, followed by UV-cross-linking. The blood compatibility of poly(SBMA-co-AA)-modified surfaces was evaluated by platelet adhesion, fibrinogen adsorption, and plasma clotting time.

Scheme 1. Polymerization Reaction of Poly(SBMAx-co-AAy) Copolymer

terminated by placing the products at 4 °C for 10 min. The unreacted monomers and small oligamers are removed by dialysis (dialysis tubing cellulose membrane, MWCO 12 400), and the polymers were freezedried. The compositions of the products were analyzed by 1H nuclear magnetic resonance (1H NMR) (Avance-500 Hz, Bruker) with D2O as solvent (Supporting Information, Figure S1). The five different products were named according to their compositions: poly(SBMA), poly(SBMA80-co-AA20), poly(SBMA56-co-AA44), poly(SBMA28-coAA72, and poly(SBMA13-co-AA87) (Table 1).

2. EXPERIMENTAL SECTION 2.1. Materials. Sulfobetaine methacrylate (SBMA; 2-methacryloyloxyethyldimethyl-3-sulfopropyl, cat. #537284), acrylic acid (AA, cat. #147230), poly(acrylic acid) (PAA, cat. #523925), poly(ethylenimine) (PEI, Mw ∼ 750 kDa, cat. #P3143), 4-azidoaniline (Az, cat. #359556), and glass beads (borosilicate, 2 mm in diameter, cat. #Z273627) were received from Sigma-Aldrich (St. Louis, MO). Most of the other chemicals were purchased from Sigma-Aldrich unless specified otherwise. Citrated human plasma was prepared according to a procedure reported previously.20 2.2. Buffers and Solutions. Phosphate-buffered saline (PBS) was prepared as 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. PBS supplemented with 0.05% (v/v) of Tween 20 constituted phosphate-buffered saline Tween (PBST). Citrate phosphate-buffered saline (CPBS) was prepared by 10 mM Na2HPO4, 10 mM citric acid, 120 mM NaCl, pH 7.4. Platelet suspension buffer (PSB) was prepared by 137 mM NaCl, 2.7 mM KCl, 5.5 mM glucose, 0.35% (w/v) bovine serum albumin (BSA), 3 mM Na2HPO4, 3.5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1 U/mL apyrase, pH 7.4. Platelet suspension solution was supplemented with Ca2+ immediately before platelet adhesion studies. LDH (lactate dehydrogenase) reagent was composed of 1.33 mg/mL INT (iodonitrotetrazolium chloride), 12 mg/mL sodium L-lactate dissolved in Tris-base (1.21 mg Tris-base in DI H 2O, pH 8.5), 1 mg/mL NAD (β-nicotinamide adenine dinucleotie), 0.45 mg/mL

Table 1. Composition of Synthesized Poly(SBMAx-co-AAy) in Different Monomer Ratios and Calculated from the 1H NMR Results (Data from Figure S1) monomer feed ratio (mol %)

calculated composition (mol %)

sample

SBMA

AA

SBMA

AA

poly(SBMA13-co-AA87) poly(SBMA28-co-AA72) poly(SBMA56-co-AA44) poly(SBMA80-co-AA20) poly(SBMA)

10 25 50 75 100

90 75 50 25 0

13 28 56 80 100

87 72 44 20 0

2.4. Synthesis of Azide-Conjugated PAA. Poly(acrylic acid-gazidoaniline), abbreviated as PAA-g-Az, was synthesized by conjugating 4-azidoaniline on PAA via a carbodiimide reaction, according to a previously published protocol.21 The content of Az in PAA-Az was determined by 1H NMR (Avance-500 Hz, Bruker) according to the peak ratio of the azidophenyl protons at 6.5−7.5 ppm and the 4349

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platelet suspension was incubated with the plasma-preadsorbed samples at 37 °C for 1 h. The unattached platelets were rinsed away with PBS. The amount of adhered platelets was quantified by a lactate dehydrogenase method, as described previously.23,24 The morphology of platelet was analyzed by scanning electron microscopy (SEM, JEOL 6390, Japan), according to a previous procedure.23,24 2.7. Fibrinogen Adsorption and Plasma Clotting Time Tests. Fibrinogen adsorption was evaluated by enzyme-linked immunosorbent assay (ELISA). The samples were coated onto 96well plates and followed by the immersion of 1% (v/v) human plasma solution in CPBS (100 μL/well) at 37 °C for 1 h to allow protein adsorption. The ELISA procedure followed a previous procedure. 23,24 Glass beads were first coated with TLP films, followed by a layer of poly(SBMA-co-AA). The modified glass beads were dried at 50 °C and then exposed to UV light for cross-linking. The glass beads (0.5 g) with different modification were mixed with 1 mL of human plasma that was recalcified with 30 μL of 10% CaCl2. Plasma clotting time was determined by the time required for gelation of human plasma under shaking. 2.8. Statistical Analysis. The data were reported as mean ± standard deviation (SD) or standard error of the mean (SEM). The statistical analyses between different groups were determined using Student’s t-test. The probabilities of p ≤ 0.05 were considered as significant differences. All statistical analyses were performed by using GraphPad Instat 3.0 program (GraphPad Software).

methylene protons of the polymer main chain at 1.3−2.5 ppm. The coupling percentage of carboxylic groups in PAA-g-Az was estimated as 6.0 mol %. 2.5. Modification and Characterization of Poly(SBMA-coAA)-Modified Substrates. Three different substrates were modified by coating poly(SBMA-co-AA): tissue culture polystyrene (TCPS, Fisher Scientific, Leicestershire UK), polydimethylsiloxane (PDMS, Silgard 184, Dow Corning), and polyurethane (PU, Dow). PDMS mixture (precursor/curing agent in a 10/1 weight ratio) was coated into TCPS 96-well plate and allowed cross-linking at 70 °C for 4 h to form PDMS substrate. PU substrate was prepared by casting PU solution (100 μL/well, 0.25% (w/v) in DMF) into polypropylene 96well plates (Evergreen, CA) and dried at 50 °C. Both PDMS and PU substrates were treated by oxygen plasma (20 sccm, 100 mTorr, 30 W, 5 min) to create negatively charged surfaces for layer-by-layer polyelectrolyte deposition.22 In this study, substrates were first deposited with three layers of polyelectrolytes in the order of PEI, PAA-g-Az and PEI. Each polyelectrolyte was coated by 150 μL solution (1 mg/mL) per well for 20 min at room temperature in the dark for adsorption, followed by rinsing with deionized water twice. On the top of the trilayered polyelectrolyte (TLP) base, one layer of poly(SBMAx-co-AAy) was coated in the dark (150 μL of 1 mg/mL copolymer solution per well for 20 min). After dried in a oven at 50 °C, the PEM films were exposed to UV light (wavelength range 280−380 nm) for 20 s for cross-linking. The nomenclature of the prepared samples is composed of the TLP base and the topmost layer. For example, the TLP base coated with poly(SBMA80-co-AA20) and PAA are abbreviated as TLP/ poly(SBMA80-co-AA20) and TLP/PAA, respectively. The samples for surface characterization were prepared on TCPS. The wettability of the coating surfaces was evaluated by water static contact angle (WCA) measurement (Sindatek, Taipei, Taiwan) with deionized water. At least six droplets (1 μL) were measured for each sample. Surface chemical composition of each sample was characterized by electron spectroscopy for chemical analysis (ESCA). ESCA spectra were recorded on a VG ESCA Scientific Theta Probe (UK) with an Al Kα X-ray source radiation (1486 eV) at a takeoff angle of 53°. The atomic compositions of the surfaces were calculated from the high resolution spectrum of each element. Surface morphology was analyzed by using atomic force microscope (AFM) with a tapping mode (Digital Instruments, Nanoscope III, 125 μm AFM scanning head). The root-mean-square roughness of samples were calculated from three 2 μm × 2 μm images, n = 3. 2.6. Platelet Adhesion. Washed platelets were prepared from fresh blood from healthy human donors by venous phlebotomy according to a previous procedure.20 The concentration of platelet suspension was adjusted to 5 × 107 platelets/mL with PSB and supplemented with both Ca2+ (CaCl2, J.T. Baker) and Mg2+ (MgCl2) to a final concentration of 1 mM prior to platelet adhesion experiments. After soaked in PBS for 2 h, the samples were preadsorbed with 100 μL/well 1% (v/v) human plasma diluted with CPBS at 37 °C for 1 h and then blocked by 2% BSA for 1 h. 100 μL

3. RESULTS 3.1. Surface Characterization of TLP/Poly(SBMAx-coAAy)-Modified TCPS. The deposition of the TLP base film increased the hydrophilicity of TCPS, indicated by the decrease of WCA from 80.4° to 42.5° (Table 2, p < 0.001). PAA coating on the TLP base further decreased WCA to 28.9° (p < 0.001 vs TLP). The WCA was varied by the deposition of poly(SBMAco-AA) with different compositions at pH 3.0. In general, the WCA decreased with increasing SBMA contents from ∼45° for poly(SBMA13-co-AA87) to ∼32° for poly(SBMA) (Table 2). Moreover, the surface hydrophilicity brought by the SBMA moiety depended on the pH of the poly(SBMA-co-AA). When poly(SBMA56-co-AA44) was deposited at different pHs, the WCAs increased with pHs from 38.1° (pH 3.0) to 66.9° (pH 10.0), which was even more hydrophobic than the TLP base (Table 2, p < 0.001 vs TLP). The ESCA analyses revealed that the deposition of the TLP base resulted in surface nitrogen (12.4 elemental %) in contrast to the absence of nitrogen on TCPS (Table 2), which is presumably contributed to PEI deposition. The deposition of PAA to the TLP base was verified by an increase in the surface oxygen content from 11.4% to 24.8%, accompanied by a corresponding decrease in the surface nitrogen content from

Table 2. ESCA Characterizations for the Elemental Composition (atomic %) and Water Contact Angle of TLP/Poly(SBMA x-coAAy)-Modified Surfaces elemental composition (At. %) surfaces

C

O

N

TCPS substrate PEI/PAA-Az/PEI (TLP) TLP/PAA TLP/poly(SBMA13-co-AA87) TLP/poly(SBMA28-co-AA72) TLP/poly(SBMA56-co-AA44) TLP/poly(SBMA80-co-AA20) TLP/poly(SBMA) TLP/poly(SBMA56-co-AA44)

91.4 76.2 69.5 68.8 68.9 66.7 74.8 77.9 75.4 80.4

8.6 11.4 24.8 26.5 24.3 25.4 17.1 14.6 16.2 11.6

12.4 5.7 3.0 4.2 4.3 5.7 5.6 7.6 7.3

pH 3.0

pH 6.5 pH 10.0

4350

S

1.7 2.6 3.6 2.4 1.9 0.8 0.7

S/N ratio

0.57 0.62 0.84 0.42 0.34 0.10 0.10

water contact angle (deg) 80.4 42.5 28.9 45.1 49.7 38.1 35.8 31.8 51.8 66.9

± ± ± ± ± ± ± ± ± ±

2.1 4.4 0.5 3.3 2.1 5.9 1.1 2.2 2.1 4.3

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12.4% to 5.7%. The deposition of SBMA-contained polymers was indicated by the appearance of sulfur atoms on the substrates (Table 2). The sulfur content was initially increased with increasing SBMA moieties of poly(SBMA-co-AA). The highest sulfur content (3.6%) was observed on the TLP base adsorbed with the polymer with an intermediate SBMA concentration, poly(SBMA56-co-AA44). Further increase in SBMA concentration decreased the surface sulfur contents to 2.4% and 1.9% when poly(SBMA80-co-AA20) and poly(SBMA) were adsorbed, respectively. Besides, the stoichiometric ratio of sulfur to nitrogen (S/N) in SBMA is unity (Scheme 1), so when surface coverage of SBMA increases, S/N ratio should approach to 1. We found that the S/N ratio was increased from 0.57 to 0.84 when the SBMA fraction in poly(SBMA-co-AA) was increased from 13% to 56% (Table 2), suggesting that surface SBMA concentration was increased with increasing SBMA fraction in the copolymers. However, further increase in SBMA fraction decreased the S/N ratio to 0.34 when 100% SBMA was adsorbed. The ESCA analyses also verified the pH dependence of poly(SBMA-co-AA) deposition. The sulfur contents (∼0.1%) on the substrates that were deposited with poly(SBMA 56-coAA44) at pH 6.5 and 10.0 were much less than that at pH 3.0 (Table 2). Actually, the deposition of poly(SBMA-co-AA) at pH 6.5 and 10.0 was the lowest among all the deposition conditions in this study. The surface topography of TLP and TLP/PAA, characterized by AFM, possessed extensive nanoscale texture with pits exhibiting a depth of 2−3 nm and a width ranged between 50 and 100 nm (Figure 1). Deposition of poly(SBMA13-co-AA87)

features were observed. On the surface of poly(SBMA80-coAA20), islet-like morphology was found with a length of ∼500 nm, width of ∼200 nm, and depth of ∼5 nm. It is interesting to note that when poly(SBMA56-co-AA44) was deposited at pH 6.5 or 10.0, surface roughness was increased with scattered islets compared to that at pH 3.0 (Figure 1). 3.2. Platelet Adhesion vs Fibrinogen Adsorption. Platelet adhesion on the various substrates deposited with poly(SBMA-co-AA) at pH 3.0 was first investigated. Platelet adhesion on the two control samples, TCPS and TLP, reached 3.2 × 105 and 3.8 × 105 platelets/cm2, respectively (Figure 2A), while the deposition of PAA and poly(SBMA13-co-AA87) greatly reduced platelet adhesion by 85−89% (p < 0.001). The best inhibition in platelet adhesion occurred on TLP/poly(SBMA28co-AA72) and TLP/poly(SBMA56-co-AA44) with more than 94% reduction in comparison with TCPS (p < 0.001), suggesting that 28−56% SBMA moieties in copolymers possess a better ability in inhibiting platelet adhesion. On the other hand, the deposition of poly(SBMA80-co-AA20) blocked platelet adherence less than 29% of that on TCPS, while deposition of poly(SBMA) did not show any significant inhibitory effect on platelet adhesion (p = 0.6989 vs TCPS). When platelets are activated by a surface, they progress through a morphological transition from discoid to round, extension of pseudopodia, and spreading.25,26 Furthermore, platelet spreading is partially correlated with platelet procoagulant activity.27 Thus, the morphology of adherent platelets was further analyzed from SEM images (Figure 2B). The platelets adhering on TCPS and TLP showed spreading morphology with extended pseudopodia (Figure 2B). Although the numbers of platelet adhesion on TLP/PAA and TLP/ poly(SBMA13-co-AA87) were limited, the adhered platelets displayed dendritic morphology, showing a certain degree of activation. On the other hand, platelets were barely found on TLP/poly(SBMA28-co-AA72) and TLP/poly(SBMA56-co-AA44) surfaces. On the surfaces with higher SBMA fraction (80% and 100%) in the copolymers, the adhered platelets with dendritic morphology increased. In particular, many adhered platelets on TLP/poly(SBMA) exhibited spread morphology with extended pseudopodia, suggesting lack of platelet resistance. Fibrinogen (FGN) is the major plasma protein in mediating platelet adhesion and activation on biomaterials.28 Therefore, fibrinogen adsorption on the PEM substrates was evaluated by using ELISA in this study. Fibrinogen adsorption was significantly decreased by the deposition of PAA and poly(SBMA-co-AA) at pH 3.0 compared to TCPS and TLP (p < 0.001, Figure 2A), although the deposition of poly(SBMA) did not reduce fibrinogen adsorption. Among all types of poly(SBMA-co-AA), the deposition of poly(SBMA56-co-AA44) resulted in the lowest fibrinogen adsorption, only ∼3% of that on TLP. When the platelet adhesion data are plotted against the fibrinogen adsorption data, it is obvious that platelet adhesion increases with fibrinogen adsorption (Figure 2C). Such correlation suggests that the reduction in platelet adhesion by deposition of poly(SBMA-co-AA) is due to blocking fibrinogen adsorption. As previously shown, the deposition of poly(SBMA-co-AA) is affected by deposition pHs, which may in turn affect subsequent platelet adhesion. When the pHs of poly(SBMA56-co-AA44) solution varied, the amount of adherent platelets to the substrate deposited at pH 6.5 was about 5-fold that at pH 3.0 (p < 0.01, Figure 3). Furthermore, the surface coated with poly(SBMA56-co-AA44) at pH 10.0 did not show

Figure 1. AFM images of TLP, TLP/poly(SBMA13-co-AA87) with pH 3.0, TLP/poly(SBMA28-co-AA72) with pH 3.0, TLP/poly(SBMA56-coAA44) with pH 3.0, TLP/poly(SBMA80-co-AA20) with pH 3.0, TLP/ poly(SBMA) with pH 3.0, TLP/poly(SBMA56-co-AA44) with pH 6.5, and TLP/poly(SBMA56-co-AA44) with pH 10.0 (all images are in 2 μm × 2 μm scale).

and poly(SBMA28-co-AA72) seemed to flatten the surface and resulted in smaller pit features (∼0.6 nm in depth). A further increase in SBMA ratio in copolymer to 56% brought significant different surface morphology that almost no pit 4351

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Figure 2. (A) Platelet adhesion and fibrinogen adsorption from plasma onto different surfaces: bare TCPS and TLP modified surfaces are the control groups, TLP with a top layer of PAA or poly(SBMAx-co-AAy) in different SBMA ratios is abbreviated as x%. (pH 3.0, concentration of 1 mg/mL). The surfaces were preadsorbed with 1% plasma. The antibody binding was determined by ELISA, and the OD value was obtained by subtracting the background value 0.053. Value = mean ± standard deviation, n = 6. (B) SEM photographs of platelets adhered onto (a) TCPS substrate, (b) TLP, (c) TLP/PAA, (d) TLP/poly(SBMA13-co-AA87), (e) TLP/poly(SBMA28-co-AA72), (f) TLP/poly(SBMA56-co-AA44), (g) TLP/poly(SBMA80-coAA20), and (h) TLP/poly(SBMA) (all poly(SBMAx-co-AAy) at pH 3.0) modified surface. All images were with magnification 1000×. Scale bar = 10 μm. (C) Cross-plots of fibrinogen adsorption against platelet adhesion.

any resistance to platelet adhesion compared to the control groups (p = 0.2795 vs TCPS). Similarly, fibrinogen adsorption was the lowest on the substrates deposited at pH 3.0, followed by pH 6.5 and 10.0 (Figure 3), in accordance with platelet adhesion. These results all point to the same conclusion that the inhibition of platelet adhesion by deposition of poly(SBMA-co-AA) is mainly due to the repellence to fibrinogen adsorption. 3.3. Applicability of Poly(SBMA56-co-AA44) on Different Substrates. One of the advantages of layer-by-layer polyelectrolyte deposition is its applicability to almost all types of substrates. Therefore, we also evaluated the efficacy of poly(SBMA56-co-AA44) on improving hemocompatibilty of PU and PDMS, which are widely applied to blood-contacting devices.29,30 Deposition of both PAA and poly(SBMA56-coAA44) reduced more than 80% of platelet adhesion on PU (p < 0.001 vs pristine PU, Figure 4). It is interesting to note that while only poly(SBMA56-co-AA44) effectively blocked more than 90% of platelet adhesion on PDMS (p < 0.001, Figure 4),

PAA did not show any inhibitory effect. These results indicated that TLP/poly(SBMA56-co-AA44) is effective against platelet adhesion on PU and PDMS substrates. 3.4. Stability of TLP/Poly(SBMA56-co-AA44)-Modified Surfaces. In this study, the TLP base was cross-linked via PAA-g-Az to enhance its stability on substrates. The stability of deposited poly(SBMA56-co-AA44) under various acidic (HCl) or basic (NaOH) conditions was evaluated regarding its ability to inhibit platelet adhesion. We found that TLP/poly(SBMA56co-AA44) was stable under acidic conditions (0.1 or 1 N HCl), and its efficacy in inhibiting platelet adhesion was not affected comparing to that on the untreated poly(SBMA56-co-AA44) layer (p = 0.8551 vs untreated, Figure 5). Nevertheless, the efficacy of TLP/poly(SBMA56-co-AA44) was reduced after alkaline treatment (0.1 and 1 N NaOH) but still remained more than 65% on 1 N NaOH-treated substrates. The unstability of poly(SBMA-co-AA) in the alkaline condition may be due to that the SBMA moiety might disintegrate at high pH, especially with pH ≥12.31 These results indicated that the 4352

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3.5. Plasma Clotting Time Tests. Plasma clotting is another aspect of hemocompatibility of biomaterials. The plasma clotting time was employed to evaluate the efficacy of zwitterionic moieties for hemocompatibility. The pristine glass beads and those coated with TLP and TLP/PAA required about 11 min for coagulation (Figure 6). On the other hand, it

Figure 3. Platelets adhesion and fibrinogen adsorption from plasma on TCPS, TLP, and TLP/poly(SBMA56-co-AA44) with different pH values (at the concentration of 1 mg/mL). The surfaces were preadsorbed with 1% plasma. The antibody binding was determined by ELISA, and the OD value was obtained by subtracting the background value 0.053. Value = mean ± standard deviation, n = 6.

Figure 6. Plasma clotting time of recalcified platelet poor plasma (PPP) in the presence of TLP, TLP/PAA, and TLP/poly(SBMA 56-coAA44)-modified surface. Value = mean ± standard deviation, n = 6 (*** p < 0.001; ** p < 0.01 vs TLP/poly(SBMA56-co-AA44)).

took a significant longer clotting time (18 min) for the beads coated with TLP/poly(SBMA56-co-AA44) (p < 0.001 vs untreated glass beads). This result indicates that the highly pro-coagulant activity of glass beads was greatly depressed by the TLP/poly(SBMA56-co-AA44) surface coating.

4. DISCUSSION In this study, we demonstrated that the hemocompatibility of biomaterials can be greatly improved by the deposition of poly(SBMA-co-AA) via a three-layer positively charged polyelectrolyte film. The stability of the prepared copolymer coatings was improved by the cross-linking of polymers under UV irradiation due to the generation of highly reactive nitrene from the Az groups in PAA-g-Az. The cross-linked polymers containing zwitterionic groups also revealed electrostatic interactions which enhanced the stability of poly(SBMA-coAA) deposited films and also further maintained the resistance to platelet adhesion under both acidic and basic conditions. The efficacy of poly(SBMA-co-AA) in improving hemocompatibility depends on the composition of SBMA/AA copolymers and deposition pH. Prior to discussion on the above observation, we would like to first discuss an interesting finding on PAA coating. The deposition of PAA greatly reduces platelet adhesion and fibrinogen adsorption, almost as good as poly(SBMA28-coAA72) and poly(SBMA56-co-AA44). The resistance of PAA to fibrinogen adsorption and platelet adhesion might be due to two factors: highly negative charges and hydration. It has been long found that some substrates possessing negatively charged groups tends to be antithrombogenic, while those possessing positively charged groups are thrombogenic.32 It is suggested that a surface bearing negative charges repels negatively charged platelets and clotting factors such as fibrinogen. Therefore, researchers incorporate negatively charged groups such as sulfonate or carboxyl groups into polyurethanes for improvement of

Figure 4. Effect of substrates: platelets adhesion onto TLP, TLP/PAA, and TLP/poly(SBMA56-co-AA44) modified PDMS and PU substrates (n = 6).

Figure 5. Platelet adhesion on TLP/poly(SBMA56-co-AA44)-modified surfaces treated with acidic (0.1 N, 1 N HCl) or basic (0.1 N, 1 N NaOH) solutions (n = 6).

cross-linked TLP/poly(SBMA56-co-AA44) coating possesses a degree of durability under rigorous conditions, showing its potential for biomedical applications. 4353

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blood compatibility.33−38 Second, the deposition of PAA greatly enhanced the hydrophilicity of the substrate, indicated by a decrease in static water contact angles compared to the controls (Table 1). It is interesting to note that although the deposition of PAA effectively reduced platelet adhesion to TCPS and PU, it did not work on PDMS, which the underlying mechanism is still unclear. On the other hand, the platelets adhered on TLP/PAA presented dendritic morphology, an indication of activation, in contrast to those on TLP/ poly(SBMA56-co-AA44) maintaining round shape (Figure 2B). Platelet spreading is suggested to be partially correlated with platelet procoagulant activity,27 inducing thrombin generation. The observation suggested that although PAA greatly reduces platelet adhesion, but the adherent platelets are prone to be activated. Previous studies also indicated that negatively charged surfaces (e.g., carboxylic SAMs) strongly induced contact activation of platelets.39 Therefore, although only a few platelets adhered to PEM/PAA, the clotting time on PEM/PAA was comparable to that on the untreated glass beads samples (p = 0.0639 vs untreated glass beads, Figure 6). Nevertheless, the extent of surface activation of platelets needs further investigation on other activation events such as the release of factors in alpha and dense granules (i.e., β-thromboglobulin, platelet factor 4, and serotonin).40 The resistance of poly(SBMA-co-AA) deposition to platelet adhesion is apparently due to its ability to withstand the adsorption of fibrinogen, which is the major plasma protein mediating platelet adhesion under static or shear conditions.1,41−44 We showed that platelet adhesion to the poly(SBMA-co-AA)-deposited substrates is highly correlated to the antibody binding to the adsorbed fibrinogen (Figure 2C). The detection of fibrinogen by ELISA does not provide a quantitative value of the total amount of adsorbed fibrinogen, but an index for antibody-recognizable conformation. Several studies indicated that platelet adhesion to plasma-coated surfaces depends on the conformation/orientation of the adsorbed fibrinogen, which is presented by the binding of antifibrinogen antibodies.23,45,46 Although the binding of the polyclonal antibodies used in the study does not represent the exposure of platelet binding sites of adsorbed fibrinogen, platelet adhesion was highly correlated to the antibody binding. The surface coverage of SBMA after deposition should be a decisive factor to determine the antifouling property of poly(SBMA-co-AA). Chang et al. suggested that higher surface SBMA coverage exhibited lower protein adsorption.15 The efficacy of the strategy used in this study relies on the ratio of SBMA/AA in the copolymers. We found that the maximal SBMA coverage appears at an intermediate SBMA concentration (∼56%), which is corresponding to the maximal ability to resist platelet adhesion. Previous studies also showed that poly(AEDAPS-co-AA) with intermediate AEDAPS contents possessed better resistance to protein adsorption and cell adhesion.18,19 The SBMA moieties in poly(SBMA-co-AA) provide the antifouling properties, while the AA moieties link copolymers to positively charged PEI of the TLP base. The surface SBMA content reflects the balance of these two moieties. High AA portion facilitates the attachment of copolymers to TLP but may sacrifice the total amount of SBMA. On the other hand, high SBMA portion enhances SBMA surface content once copolymers adsorb to TLP, but the attractive forces may not be sufficient to form stable interaction. The surfaces of PEM/poly(SBMA-co-AA) copolymers with lower SBMA composition (13, 28, and 56%) resisted both

fibrinogen adsorption and platelet adhesion effectively. Similarly, previous studies reported that poly(AEDAPS-coAA) with 10, 25, and 50 mol % of AEDAPS moieties resisted protein adsorption and cell adhesion effectively. 18,19 The maximal inhibitory effect on platelet adhesion appears at the copolymer with intermediate SBMA concentration (56%). We suggest that platelet repellent ability of these substrates should be correlated to surface SBMA concentrations. Since sulfur atoms only appear on SBMA, surface sulfur contents should reflect the amount of surface SBMA. As shown in Table 2, sulfur contents also appear a maximum on poly(SBMA56-coAA44) and decrease when the SBMA compositions decrease or increase. One observation should be noted that the sulfur contents on poly(SBMA13-co-AA87) and poly(SBMA28-co-AA72) are correspondent with those on poly(SBMA) and poly(SBMA80-coAA20), respectively (Table 2), but their resistance to platelet adhesion is quite different (Figure 2A). We conjecture that two factors might contribute to this phenomenon. First, the surface sulfur contents around 1.7−2.6% may not be sufficient to resist platelet adhesion. However, since AA also resist platelet adhesion, poly(SBMA13-co-AA87) and poly(SBMA28-co-AA72) inhibit platelet adhesion better than poly(SBMA) and poly(SBMA80-co-AA20). Second, several studies indicate poly(SBMA) with high molecular weights form aggregates in solution. Yang et al. suggested that surface-grafted poly(SBMA) with longer brushes may self-condense and weaken surface hydration, resulting in the reduction of the antifouling performance of the zwitterionic polymer. 17 A study on zwitterionic copolymer of acrylamide and 4-vinylpyridine propylsulfobetaine suggests that the copolymers aggregated due to the strong electrostatic interactions between the dipolar groups of zwitterionic moieties.47,48 Another study suggests that the size of aggregated zwitterionic copolymers is increased with increasing zwitterionic contents.47,48 Two consequences may occur due to aggregation of poly(SBMA-co-AA). One is that the AA portion of the copolymer may be buried, which reduces the amount of adsorbed poly(SBMA-co-AA). Another is that the aggregates may retard the packing of poly(SBMA-co-AA) on a surface. Chang et al. previously showed that the antifouling efficacy of diblock copolymers of poly(SBMA) and poly(propylene oxide) was decreased when the molecular weight of poly(SBMA) reached a certain value.15 They suggest that large SBMA segments form aggregates and create cavities among themselves when they are adsorbed to a substrate. Therefore, the surface cannot be fully covered, leading to an increase in protein adsorption. In this study, a similar observation was found. The AFM images (Figure 1) showed a smooth and uniform coating upon the deposition of poly(SBMA56-co-AA44) but aggregation of copolymers and the formation of islets on poly(SBMA80-co-AA20) and poly(SBMA) substrates. Incomplete surface coverage of SBMA functionality results in failure in inhibiting platelet adhesion. Deposition pH strongly affects the adsorption of poly(SBMA-co-AA) onto the TLP base. The pH of polyelectrolyte solutions affects the degree of ionization of polyelectrolytes and subsequent adsorption of polyelectrolytes.49,50 Deposition of poly(SBMA-co-AA) was retarded at neutral or basic conditions (pH 6.5 and 10.0) compared to at pH 3.0. Previous studies reported that weak polyelectrolyte branched PEI possesses more positively charges under pH 3.0, and the degree of ionization decreases significantly at pH 6.5 and pH 10.50 This explained that the poly(SBMA-co-AA) prepared under pH 3.0 4354

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might possess higher degree of ionization and therefore provides more adsorption sites with PEI layer.

5. CONCLUSION In the study, zwitterionic copolymers of SBMA and AA were applied to a variety of biomaterials by combination with layerby-layer polyelectrolyte deposition technique to improve hemocompatibility. We demonstrated that poly(SBMA-coAA) with an appropriate composition could effectively inhibit platelet adhesion and fibrinogen adsorption when adsorbed to a positively charged substrates. The efficacy in inhibiting platelet adhesion was correlated with the surface content of SBMA moieties. Among all samples, the surfaces coated with TLP/ poly(SBMA56-co-AA44) revealed uniform coating and high surface SBMA density and possessed the best anticoagulant performance. The preliminary hemocompatibility tests regarding platelet adhesion, fibrinogen adsorption, and plasma coagulation suggest the potential of this technique for the application to blood-contacting biomedical devices. More aspects of hemocompatibility should be tested in the future.

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ASSOCIATED CONTENT

S Supporting Information *

NMR results of the synthesized zwitterionic copolymers poly(SBMA-co-AA) with different SBMA ratios were analyzed. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *Tel +886-2-3366-3996, Fax +886- 2-362-3040, e-mail [email protected] (W.-B.T.); Tel +886-2-2733-6623, Fax +886-2-2737-6644, e-mail [email protected] (C.L.).

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ACKNOWLEDGMENTS The authors express great appreciation to the National Science Council for the financial supports (99-2221-E-011-121-MY2). REFERENCES

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