Covalent Deposition of Zwitterionic Polymer and Citric Acid by Click

Apr 23, 2014 - lant is that the citric acid can disrupt coagulation cascade by ..... analyzer CA-50 (Sysmex Co., Japan), and platelet-poor plasma (PPP...
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Covalent Deposition of Zwitterionic Polymer and Citric Acid by Click Chemistry-Enabled Layer-by-Layer Assembly for Improving the Blood Compatibility of Polysulfone Membrane Tao Xiang,† Rui Wang,† Wei-Feng Zhao,† Shu-Dong Sun,† and Chang-Sheng Zhao*,†,‡ †

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China ‡ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China S Supporting Information *

ABSTRACT: Development of blood compatible membranes is critical for biomedical applications. Zwitterionic polymers have been proved to be resistant to nonspecific protein adsorption and platelet adhesion. In this work, two kinds of zwitterionic copolymers bearing alkynyl and azide groups are synthesized by atom transfer radical polymerization (ATRP) and subsequent reactions, namely alkynyl-poly(sulfobetaine methacrylate) (alkynyl-PSBMA) and azide-poly(sulfobetaine methacrylate) (azide-PSBMA). The copolymers are directly used to modify azido-functionalized polysulfone (PSf-N3) membrane via click chemistry-enabled layer-by-layer (LBL) assembly. Alkynyl-citric acid is then clicked onto the membrane when the outermost layer was azide-PSBMA. The chemical compositions, surface morphologies, and hydrophilicity of the zwitterionic polymer and citric acid multilayer modified membranes are characterized. The composite multilayer is resistant to protein adsorption and platelet adhesion and also prolongs clotting times, indicating that the blood compatibility is improved. Moreover, after clicking the small molecule anticoagulant alkynyl-citric acid onto the outermost of the zwitterionic multilayer, the membrane shows further improved anticoagulant property. The deposition of zwitterionic polymer and citric acid via click chemistryenabled LBL assembly can improve the blood compatibility of the PSf membrane.

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INTRODUCTION For blood contacting devices such as hemodialyzer, hemofilter, artificial organs, and disposable clinical instruments, good blood compatibility is highly desirable. Polymers are widely applied in the blood contacting devices, such as cellulose acetate (CA), poly(vinyl alcohol) (PVA), polyacrylonitrile (PAN), polysulfone (PSf), and poly(ether sulfone) (PES).1−4 PSf has been widely used because of its mechanical strength, thermal stability, and chemical inertness. However, the modification for PSf is desired to improve the blood compatibility.5,6 In recent years, zwitterionic polymers are used to graft onto materials to fabricate superlow fouling surfaces to improve antifouling property and biocompatibility of materials,7−19 which is based on the conclusion that the normal conformations of biomacromolecules could be maintained by the zwitterionic structures. 14,20 In general, zwitterionic polymers are mainly introduced into materials by means of blending method10 and covalently grafting method, such as surface-initiated ATRP,13−15 O2 plasma surface grafting,17 atmospheric plasma-induced surface copolymerization,18 and so on. However, the designed material surfaces with nanoscale and microscale multilayers are of great significance for basic and applied studies in the biomedical field for the required surfaces,21 which could be realized by layer-by-layer (LBL) © 2014 American Chemical Society

assembly. Nanoscale multilayers are easily prepared using by the LBL assembly, while microscale multilayers can be prepared by the assembly using micrometer-sized particles and polymers.22 To our knowledge, the fabrication of zwitterionic polymer multilayer on material surface via layer-by-layer (LBL) assembly has not been reported. Recently, LBL assembly becomes a versatile method to fabricate designable multilayer on material surfaces.23−25 The multilayer is generally assembled by noncovalent electrostatic. However, it is difficult to prepare multilayer using zwitterionic polymer by the usual noncovalent electrostatic interaction due to the inner salt structure. In addition, covalently cross-linked multilayer under diverse environment conditions is more stable than those assembled by noncovalent electrostatic.26 The common click chemistry reaction, Cu(I)-mediated Huisgen 1,3dipolar cycloaddition reaction,27−29 can be used in the LBL assembly, and the click chemistry-enabled LBL assembly is an efficient method for deposition of diverse polymers, such as poly(ethylene glycol) (PEG),30 poly(acrylic acid) (PAA),31,32 and poly(2-(methacryloyloxy)ethyltrimethylammonium chlorReceived: January 14, 2014 Revised: March 22, 2014 Published: April 23, 2014 5115

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Scheme 1. Scheme Illustration for the Fabrication of Zwitterionic Polymer and Citric Acid Composite Multilayer Deposited PSf Membrane by Click Chemistry-Enabled LBL Assembly

ide (PMETA).26 In this study, click chemistry-enabled LBL assembly is utilized to deposit zwitterionic polymer poly(sulfobetaine methacrylate) (PSBMA) multilayer onto the PSf membrane for the first time. Citric acid (CA) is a common anticoagulant used in blood specimens required for tissue typing and plasmapheresis procedures instead of heparin.33 The mechanism of anticoagulant is that the citric acid can disrupt coagulation cascade by binding Ca2+ in the blood.34 Thus, it will lead to important advances in biomedical materials to fabricate CA into materials. Li et al.33 prepared modified membranes by blending citric acid grafted polyurethanes with PES, for which the CA was grafted onto the polyurethane through a covalent bond. The modified membrane showed good biocompatibility, especially the anticoagulant property. However, the amount of CA was quite small, since there were only two CA molecules in one polyurethane chain. Thus, directly graft CA onto material surfaces may be a good method to improve anticoagulant property, but few studies about it had been reported yet. In this study, cross-linked zwitterionic polymer multilayer and citric acid composite multilayer is fabricated via click chemistry-enabled LBL assembly on PSf membrane (Scheme 1). We hope that the zwitterionic PSBMA multilayer can improve the resistance of protein adsorption and platelet adhesion, while the grafted citric acid can improve the anticoagulant property of the modified membranes. Functionalized azido-polysulfone (PSf-N3) was prepared by chloromethylation and azidation reaction, and PSf-N3 membrane was obtained by a liquid−liquid phase inversion technique with the thickness of 60 ± 3 μm. Then, alkynyl-functionalized poly(sulfobetaine methacrylate) (alkynyl-PSBMA) and azide-

functionalized poly(sulfobetaine methacrylate) (azide-PSBMA) were alternately deposited onto the membrane surface via click chemistry-enabled LBL assembly. Finally, alkynyl-citric acid was deposited via click chemistry. The blood compatibility of the membranes, including protein adsorption, platelet adhesion, activated partial thromboplastin time (APTT), thrombin time (TT), and plasma recalcification time (PRT), was then investigated.

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EXPERIMENTAL SECTION

Materials. Monomers including 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%, Aladdin), glycidyl methacrylate (GMA, 97%, Aladdin), and propargyl methacrylate (PgMA, 98%, Alfa Aesar) were distilled under reduced pressure to remove inhibitor before use. CuBr (Aladdin, 98%) was purified by acetic acid and methanol.35 N,N,N′,N″,N″-Pentamethyldiethylenetriamine (PMDETA, 99%, Sigma), ethyl 2-bromoisobutyrate (EBiB, 98%, Aladdin), 1,3-propanesulfonate (99%, Aladdin), sodium azide (NaN3, 99.5%, Sigma), ammonium chloride (NH4Cl, 99.5%, Aladdin), copper(II) sulfate pentahydrate (99.5%, Aladdin), sodium ascorbate (99%, Aladdin), N,N-dimethylformamide (DMF, 99.8%, Kelong), and dimethyl sulfoxide (DMSO, 99.8%, Kelong) were used as received. Polysulfone (PSf, Udel P-1700) was purchased from Union Carbide. Bovine serum albumin (BSA, fraction V) and bovine serum fibrinogen (BFG) were obtained from Sigma Chemical Co. Micro BCA protein assay reagent kits were purchased from PIERCE. APTT and TT reagent kits were obtained from SIEMENS. Deionized water was used throughout the study. Polymer Synthesis. Poly(2-(dimethylamino)ethyl methacrylateco-propargyl methacrylate) copolymer (PDMAEMA-co-PgMA) was synthesized via ATRP using DMAEMA and PgMA as the monomers (Scheme 2). In a typical procedure, EBiB (195.1 mg, 1.0 mmol), CuBr (143.5 mg, 1.0 mmol), PMDETA (173.3 mg, 1.0 mmol), DMAEMA 5116

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Scheme 2. Schematic Illustration for the Preparation of alkynyl-PSBMA and azide-PSBMA by ATRP and Subsequent Reactions

(10.4 g, 66.0 mmol), PgMA (8.7 g, 33.0 mmol), and DMF (15 mL) were separately added to a glass tube. The solution was degassed by three freeze−pump−thaw cycles after stirring for 15 min under the nitrogen atmosphere. Then the tube was heated to 70 °C under constant stirring. After 12 h, the polymerization reaction was stopped by exposing to air, and the copolymer was purified through dialysis with water and dried by lyophilization. Zwitterionic polymer poly(sulfobetaine methacrylate-co-propargyl methacrylate) (PSBMAco-PgMA) was synthesized by the reaction of 1,3-propane sultone with the tertiary amino groups of DMAEMA in the copolymer PDMAEMA-co-PgMA. PDMAEMA-co-PgMA (0.5 g) was dissolved in 10 mL of DMSO, and 1,3-propane sultone (1.3 g, 11.0 mmol) in 10 mL of DMSO was added dropwise over 10 min. The reaction was kept at room temperature for another 24 h, and then the copolymer was purified through dialysis with water and dried by lyophilization. The resulting copolymer was denoted as alkynyl-PSBMA. Poly(2-(dimethylamino)ethyl methacrylate-co-glycidyl methacrylate) copolymer (PDMAEMA-co-GMA) was synthesized via ATRP using DMAEMA and GMA as the monomers (Scheme 2). The procedure was the same as the synthesis of PDMAEMA-co-PgMA, and

GMA (4.7 g, 33.0 mmol) was used. Then, poly(sulfobetaine methacrylate-co-glycidyl methacrylate) (PSBMA-co-GMA) was synthesized in the same procedure as PSBMA-co-PgMA. PSBMA-co-GMAN3 copolymer with azide groups (denoted as azide-PSBMA) was synthesized by the reaction of sodium azide with the pendant oxirane rings in the GMA units of the random copolymer. NaN3 (375.5 mg, 5.5 mmol) and NH4Cl (294.3 mg, 5.5 mmol) were added to 10 mL aqueous solution of PSBMA-co-GMA (0.5 g, 1.1 mmol of oxirane moieties). The reaction was kept at 60 °C for 24 h, and then the copolymer was purified through dialysis with water and dried by lyophilization. Polymer Multilayer Prepared by Click Chemistry-Enabled LBL Assembly. The functionalized azido-polysulfone (PSf-N3) was synthesized by chloromethylation and azidation reactions,28,29 and PSf-N3 membrane was obtained by a liquid−liquid phase inversion technique with compact double skin layers (see Supporting Information). According to our previous study,36 membranes prepared using a liquid−liquid phase inversion technique are porous structure, while those prepared using the evaporation method are dense structure. The pore size of the active surface (skin layer) of the 5117

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phase-inversion membrane was about 4 nm depending on the concentration of the casting solution.37 Because of the skin layers and the small pore size of the active surface, it was difficult for the modifier polymer to penetrate from the surface into the porous structure. The alkynyl-PSBMA and azide-PSBMA were alternately deposited on the functionalized PSf-N3 membrane by LBL assembly using Cu(I)-mediated click chemistry reaction. The membrane was alternately exposed to alkynyl-PSBMA and azide-PSBMA solutions at room temperature for 1 h and rinsed in water after each layer was deposited.31 The dipping solution consisted of alkynyl-PSBMA (or azide-PSBMA) (2 mg/mL, 9 mL), copper(II) sulfate (0.72 mg/L, 3 mL), and sodium ascorbate (1.76 mg/mL, 3 mL). The polymer multilayer deposited PSf-N3 membrane was denoted as PSf-n, where n represents the number of the clicked layers. For the PSf-n membranes with the outermost layer of azide-PSBMA, the anticoagulant alkynylcitric acid38−40 (the synthesis consisted of three reactions as shown in the Supporting Information) was then deposited by click chemistry at room temperature for 2 h, and the dipping solution consisted of alkynyl-citric acid (5 mg/mL, 9 mL), copper(II) sulfate (0.72 mg/L, 3 mL), and sodium ascorbate (1.76 mg/mL, 3 mL). The citric acidgrafted PSf-n membrane was denoted as PSf-n-CA. All the PSBMA and citric acid functionalized membranes were thoroughly washed with deionized water by using ultrasonication to eliminate the residual copper catalyst. Moreover, in order to change the carboxyl groups to carboxylate ions, the PSf-n-CA membrane was incubated in alkaline solution with pH = 12 for 24 h and washed with deionized water. To measure the thickness of the polymer multilayer films, spectroscopic ellipsometry was utilized and the silicon wafer was chosen as the substrate for its smooth surface. The pretreatment of silicon wafer was carried out according to the published procedure.41 PSf-N3 (5 wt %) dissolved in DMF was spun on the silicon wafer at high speed (about 8000 rpm) and was dried in vacuo at 50 °C for 48 h to obtain a thin film (about 22 nm). Then the PSf-N3 deposited silicon wafer was alternately exposed to alkynyl-PSBMA and azide-PSBMA solutions containing copper(II) sulfate and sodium ascorbate at the same conditions as those for the PSf-N3 membranes. The polymer multilayer deposited silicon wafer was denoted as silicon-n, where n represents the clicked layer number. Polymer Characterization. FTIR spectra were recorded on Nicolet 560 instrument (Nicolet Co., American). Each spectrum was scanned and collected over the range of 500−4000 cm−1 at a resolution of 4 cm−1. 1H NMR measurements were recorded on a Bruker AVII-400 MHz spectrometer (Bruker Co., Germany) with deuterated water for all the polymers. The molecular weight was measured on a gel permeation chromatography (GPC) instrument, which was performed on a Waters-1515 against poly(ethylene oxide) (PEO) standards with water as an eluent. The sample concentration was 2−3 mg/mL, and the flow rate was 1.0 mL/min at 40 °C. Surface Characterization. Ellipsometry measurements were performed on a spectroscopic Sentech SE850 ellipsomter (Sentech, Japan). The spectroscopic data were obtained between 400 and 800 nm with a 2 nm increment, and the thicknesses were extracted with the integrated software by fitting with a classical wavelength dispersion model. Morphology images were obtained by field emission scanning electron microscopy (JSM-7500F, JEOL, Japan). Atomic force microscope (AFM) images of the polymer multilayer assembled membranes were obtained by a Multimode Nanoscope V scanning probe microscopy (SPM) system (Bruker Co., Germany). The rootmean-square roughness (Rq) and mean roughness (Ra) of the samples over 5 μm × 5 μm were calculated from the AFM images. The surface compositions of the samples were acquired by a Kratos AXIS ULTRADLD XPS, using Al Kα excitation radiation. Water contact angle (WCA) was measured by a goniometer (Dataphysics OCA20, Germany) in static mode. 3 μL of water was dropped on the membrane surface by an automatic piston syringe and photographed by a video capture. Blood Compatibility. Plasma Collection. Healthy human fresh blood (man, 24 years old) was collected by using vacuum tubes (6 mL, Terumo Co.), containing citrate/phosphate/dextrose/adenine-1 mix-

ture solution (CPDA-1) as the anticoagulant (anticoagulant to blood ratio, 1:9).33 The blood was centrifuged at 1000 rpm (or 4000 rpm) for 15 min to obtain platelet-rich plasma (PRP) (or platelet-poor plasma (PPP)). All the blood compatibility experiments were performed in compliance with the relevant laws and institutional guidelines. Protein Adsorption. BSA and BFG were used in the protein adsorption experiments. The protein was dissolved in isotonic phosphate-buffered saline solution (PBS, pH 7.4, 1 mg/mL). The membrane (1 × 1 cm2) was incubated in PBS for 24 h and then immersed in the protein solution for 2 h at 37 °C. After the adsorption process, the membrane was slightly rinsed by PBS and placed in SDS solution (2 wt %) for 1 h at 37 °C under constant agitation for 1 h to remove the adsorbed protein. The protein concentration into the SDS solution was determined by using the Micro BCA protein assay reagent kits (the standard curve is shown in Figure S1). More than 95% of the adsorbed protein could be eluted into the SDS solution, eliminated, and then the adsorbed protein amount was calculated by using the standard curve. Platelet Adhesion. To eliminate the effect of other components in blood on the platelet adhesion test, platelet-rich-plasma (PRP) was used in the experiment.42 Membrane (1 × 1 cm2) was equilibrated at 37 °C for 1 h. Then PBS was decanted off, and 1 mL of fresh PRP was added. After the membrane was incubated at 37 °C for 2 h, the PRP was removed and the membrane was rinsed PBS. Then, the membrane was immersed in 2.5 wt % glutaraldehyde solution at 4 °C for about 1 day. The membrane was then subjected to a drying process by a series of graded alcohol−PBS solutions (25%, 50%, 70%, 75%, 90%, 95%, and 100%) and isoamyl acetate−alcohol solutions (25%, 50%, 75%, and 100%) for 15 min each time. The number of the platelets adhered on the membrane was calculated from five SEM images at 1000× magnification from different places on the same membrane. APTT and TT. Activated partial thromboplastin time (APTT) and thrombin time (TT) were used to evaluate the antithrombogenicity of the modified membranes by using an automated blood coagulation analyzer CA-50 (Sysmex Co., Japan), and platelet-poor plasma (PPP) was used.43 The APTT was measured as follows: the membrane with an area of 0.5 × 0.5 cm2 (4 pieces) was incubated in 0.2 mL PBS at 37 °C for 1 h, then the PBS was decanted off, and 100 μL of fresh PPP was introduced. After being incubated at 37 °C for 30 min, 50 μL of the incubated PPP was added into a test cup, and then 50 μL of APTT agent (incubated 10 min before use) was added and incubated at 37 °C for 3 min; then 50 μL of 25 mM CaCl2 solution was added, and the APTT was measured. For the TT test, 100 μL of TT agent was added into the test cup, in which 50 μL of the incubated PPP added, and then the TT was measured. At least three measurements were conducted to get a reliable value. To explore the effect of calcium ion on the clotting time, CaCl2 solutions (containing 1−5 times calcemia concentration) were added into the PPP before the test.33 Then the APTT was measured in the same procedure as above. Plasma Recalcification Time (PRT). The membrane (1 × 1 cm2) was immersed and equilibrated in PBS at 37 °C for 1 h. Then the PBS was decanted off, and 100 μL of fresh PPP was introduced. The membrane was statically incubated at 37 °C; 100 μL of 25 mM CaCl2 aqueous solution was added to the solution. Then the solution was monitored by manually dipping a stainless-steel hook (coated with silicone) to detect fibrin threads. The clotting time was recorded as the time when the first fibrin thread was formed on the hook. At least three measurements were conducted to get a reliable value.

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RESULTS AND DISCUSSION Synthesis of alkynyl-PSBMA and azide-PSBMA. To covalently deposit polymer multilayer onto the PSf-N3 membrane by click chemistry, alkynyl (PgMA) and azide (GMA-N3) groups were introduced on the side chains of the copolymers. Random copolymer PDMAEMA-co-PgMA was first synthesized by ATRP using DMAEMA and PgMA as the monomers, and the copolymer alkynyl-PSBMA was subse5118

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quently synthesized by the reaction of 1,3-propane sultone with the tertiary amino groups of the DMAEMA in the copolymer PDMAEMA-co-PgMA10 (Scheme 2). The random copolymer PDMAEMA-co-GMA was also synthesized by ATRP using DMAEMA and GMA as the monomers, and the copolymer PSBMA-co-GMA was subsequently synthesized by the reaction with 1,3-propane sultone. Subsequent reaction of sodium azide with the pendant oxirane rings in the GMA units of the copolymer introduced the azide groups,44,45 and the final copolymer was denoted as PSBMA-co-GMA-N3 (azidePSBMA) (Scheme 2). As shown in Table S1, the ATRP method provides the zwitterionic copolymers with controlled molecular weights (Mn) and narrow polydispersities (Mw/Mn = 1.1−1.2), and the narrow polydispersities indicate the wellcontrolled polymerization using the ATRP method. The copolymers were characterized by FTIR and 1H NMR as shown in Figures 1 and 2. For the copolymers PDMAEMA-

Figure 1. FTIR spectra for (a) PDMAEMA-co-PgMA, (b) alkynylPSBMA, (c) PDMAEMA-co-GMA, (d) PSBMA-co-GMA, and (e) azide-PSBMA.

co-PgMA and alkynyl-PSBMA, the obvious band at 1730 cm−1 was contributed to the ester carbonyl groups, and the absorption band at 2123 cm−1 corresponded to the alkynyl groups, indicating that the copolymers were successfully synthesized. The 1H NMR results are shown in Figure 2a. The chemical shift at δ = 2.51 ppm (g and g′) is attributed to the alkynyl groups. The chemical shifts at δ = 4.21 (c) ppm and δ = 2.80 ppm (d), which were attributed to the −CH2− disappearing after that the tertiary amino groups were changed to quaternary ammonium groups, and a new shift at δ = 3.30 (e′) appeared, illustrating that the tertiary amino groups were all converted into sulfobetaine and the conversion was 100% completed. Through calculating the ratio of the integrals at δ = 3.27 ppm (e′) to that at δ = 2.51 ppm (g′ and j′), the molar fraction of PgMA in the copolymer alkynyl-PSBMA was 12.9 mol % (Table S1). The copolymers PDMAEMA-co-GMA, PSBMA-co-GMA, and azide-PSBMA were also characterized as shown in Figures 1c−e and 2b. The band at 2106 cm−1 corresponded to the azide groups, indicating that the copolymer was successfully synthesized. The chemical shifts at δ = 4.19 (c) ppm and δ =

Figure 2. 1H NMR spectra for alkynyl-PSBMA and azide-PSBMA and their precursors.

2.78 ppm (d), which were attributable to the −CH2− disappearing after that the tertiary amino groups were changed to quaternary ammonium groups, and a new shift at δ = 3.30 (e′) appeared, illustrating that the tertiary amino groups were all converted into sulfobetaine and the conversion was 100% completed. Through calculating the ratio of the integrals at δ = 3.30 ppm (e′) to that at δ = 1.73 ppm (m′), the molar fraction of GMA in the copolymer alkynyl-PSBMA was 15.5 mol % (Table S1). Surface Characterization. Compositions of Membrane Surfaces. The chemical compositions of the polymer multilayer assembled membranes were first investigated by ATR-FTIR, as shown in Figure S2. After that the PSBMA multilayer was deposited on the PSf-N3 membrane, a new band at 1722 cm−1 appeared, attributed to the ester carbonyl groups O−CO. It was also observed that the intensity increased with the increase 5119

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Figure 3. XPS spectra for the modified membranes: (a) wide scan, (b) C 1s, and (c) N 1s.

Figure 4. (a) Typical AFM images for the membranes. (b) Water contact angles (WCAs) for membranes with different layer numbers. (c) BSA adsorption by membranes with different layer numbers. (d) BFG adsorption by membranes with different layer numbers. Values are expressed as means ± SD, n = 3.

spectrum were appeared, illustrating that the alkynyl-citric acid was successfully clicked onto the PSf-N3 membrane. After that the alkynyl-PSBMA and azide-PSBMA were clicked on the substrates (such as PSf-2 and PSf-10), the XPS C 1s spectra were curved-fitted into four peaks with BEs at 284.4, 284.8, 285.5, 286.2, and 288.5 eV, which were attributed to C−H, C− N, C−O, C−N+, and O−CO, respectively; the XPS N 1s spectra were curved-fitted into four peaks with BEs at 399.6, 401.1, 402.0, and 404.0 eV, which were attributed to the negatively charged nitrogen (N−), the imine ((C)−N) moiety of the triazole rings, the positively charged nitrogen of N(CH3)3+, and the positively charged nitrogen (N+) in the residual azide groups, respectively. Through analyzing the peaks

of the layer number, indicating that the thickness of the polymer multilayer increased. To further confirm the polymer multilayer assembly on the PSf-N3 membranes, XPS was applied as shown in Figure 3a−c. For the PSf-N3 membrane, the XPS C 1s spectrum (Figure 3b) was curved-fitted into three peaks with binding energies (BEs) at 284.6, 285.2, and 286.1 eV, which were attributed to C−H, C−N, and C−O, respectively; the XPS N 1s spectrum (Figure 3c) was curved-fitted into three peaks with BEs at 399.6, 400.3, and 404.1 eV, which were attributed to the (C)−N, N−, and N+, respectively. For the PSf-CA (alkynyl-citric acid was directly grafted on PSf-N3 by click chemistry), a new BE at 288.5 eV in XPS C 1s spectrum and a new BE at 400.7 eV in XPS N 1s 5120

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WCAs were slightly increased compared with those of the precursor PSf-n membranes, indicating that the clicked citric acid slightly decreased the hydrophilicity of membranes. Thus, the membranes modified by the zwitterionic polymers and alkynyl-citric acid were more hydrophilic than the PSf and PSfN3 membranes. Blood Compatibility of the Modified Membranes. Protein Adsorption. Protein adsorption on biomaterial surfaces is thought to be the first step of many undesired bioreactions and bioresponses, followed by platelet adhesion and activation of coagulation pathways, leading to thrombus formation.48−51 Thus, the protein adsorption on material surface is one of the most important factors to evaluate the blood compatibility of materials.5 Though the membrane prepared by a liquid−liquid phase inversion technique was porous (as shown in Figure S3), the skin layers of the prepared membrane were compact and the protein adsorption occurred on the surface. Thus, the porosity of the membrane had little influence on the protein adsorption. Because the fibrinogen in blood plasma can bind to the platelet GP IIb/IIIa receptor,52 the fibrinogen adsorption is particularly significant to evaluate blood compatibility of biomaterials. In this study, both BSA and BFG adsorptions were studied, and the results are shown in Figure 4c,d. It showed that after the zwitterionic multilayer was clicked on the membrane, even for two layers, the values for both BSA and BFG adsorptions were obviously decreased compared with those of the PSf and PSf-N3 membrane, and the adsorbed amounts of BSA and BFG were about 4 and 2 μg/cm2 for all the PSBMA clicked membranes. For the PSf-n-CA (n > 1) membranes, the adsorbed amounts of both BSA and BFG were almost the same as those of the precursor PSf-n membranes. The results were consistent with our previous study,13 in which PSBMA brush was grafted from PSf membrane via surfaceinitiated ATRP, and the adsorbed amounts of BSA and BFG were about 3.5 and 2 μg/cm2, respectively. Generally, protein adsorption can be measured by many methods, such as surface plasmon resonance (SPR), ELISA, and 125I notation methods, and the precision is ng/cm2. Micro BCA is another convenient and cheap method, and the precision is μg/cm2. Furthermore, many factors affect the protein adsorption amounts and the interaction between material surface and protein, such as surface free energy and topological structure, surface charged character, physical property of solution (e.g., pH, ionic strength), and protein characters.42 In this study, we used the micro BCA method to measure protein adsorption, and the values for both BSA and BFG adsorptions (about 4 and 2 μg/cm2) were reasonable compared with other studies in which the micro BCA method was used. Simultaneously, the protein adsorption values of PSBMA multilayer deposited membranes were obviously decreased compared with those for the pristine PSf membrane (about 17.8 and 15.8 μg/cm2). Thus, we can make a conclusion that after that PSBMA multilayer was deposited on the membranes, the antifouling property (protein adsorption) was significantly improved. The protein adsorption data were similar to those in our previous study.13 The antifouling property might help to improve the blood compatibility, which will be discussed in the following sections. Platelet Adhesion. For blood contacting biomaterials, the adhesion and activation of platelets to the interface is a crucial event in thrombus formation, which can further activate many kinds of coagulation factors, resulting in thrombus formation.53

of the imine ((C)−N) moiety and the positively charged nitrogen (N+), the amount of the residual azide group could be calculated. For the PSf-2, 42 mol % of the azide groups were reacted in the click chemistry-enabled LBL assembly, and 58 mol % of those were available for the subsequent reaction. For the PSf-10, through the same calculation method, 57 mol % of the azide groups was reacted in the click chemistry-enabled LBL assembly, and 43 mol % of those was available for the subsequent reaction. These results indicated that there were nearly half amount of the azide groups that were available for the subsequent reaction, even for the PSf-10 membrane. The data of the chemical compositions for the membranes are shown in Table S2. After that the alkynyl-PSBMA and azidePSBMA were clicked on the PSf-N3 membranes (for PSf-N3, PSf-2, and PSf-10), the contents of the element C and S decreased and those of the element N and O increased. However, for the membranes with the same layer number, the contents of the element N, C, and S decreased and those of the O increased when the CA is clicked on the outermost layer. These results demonstrated that the alkynyl-PSBMA and azidePSBMA were successfully clicked onto the PSf-N3 membrane, and the alkynyl-citric acid was also clicked onto the outermost layer. Morphologies and Hydrophilicity of Membrane Surfaces. SEM and AFM were used to investigate the morphologies and microstructures of the PSBMA deposited membranes as shown in Figure S3 and Figure 4a. It was observed that all the membrane surfaces (as shown in the SEM images) were smooth and showed no distinct difference. AFM was applied by using a tapping mode over an area of 5 μm × 5 μm. The 3D images of the membranes with different layer numbers are shown in Figure 4a. The surface roughness Ra and Rq for the PSf-N3 membrane were about 7 and 9 nm, respectively. After the polymer multilayers assembling, the Ra and Rq for the samples decreased to about 5 and 6 nm, which indicated that the surface was successfully modified by the click chemistry-enabled LBL assembly method, and the modified membranes were smoother than the PSf-N3 membrane. In order to measure the thickness of the polymer multilayers using ellipsometry, silicon wafer was chosen as the substrate, since ellipsometry works only for samples on reflecting substrates. To accurately simulate the click chemistry-enabled LBL assembly process for PSf-N3 membrane, a primer layer of PSf-N3 was deposited on the silicon wafer by spinning coating. Then, alkynyl-PSBMA and azide-PSBMA were clicked on the PSf-N3 deposited silicon wafer in the same way as that for PSfN3 membrane, and the thicknesses are shown in Figure S4. With the exclusion of the primer layer (PSf-N3), an average thickness increase of about 4.1 nm per bilayer was observed and the thickness showed evident linear relation with the layer number. Water contact angle (WCA) is usually utilized to detect the hydrophilicity property of materials surface, which provides information on the wettability property of the material surface and the interaction energy between the material surface and the liquid.46 As shown in Figure 4b, the WCA of PSf-CA (about 49°) was obviously decreased than that of the PSf-N3 (about 82°), indicating that grafting of citric acid could effectively improve the hydrophilicity of the modified membranes. When the zwitterionic polymer multilayer was deposited, the WCAs of PSf-n membrane were decreased to about 38°, since the sulfobetaine groups could form a hydration layer via electrostatic interaction.47 For the PSf-n-CA (n > 1) membranes, the 5121

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Therefore, in vitro platelet adhesion test was applied to investigate the blood compatibility of biomaterials. The numbers and shapes of the adhered platelets could be confirmed by SEM images. Once the platelets were activated, “pseudopods” would stretch out, followed by the aggregation of the platelets.54 Figure 5 shows the typical SEM images of the platelets adhered onto the PSf, PSf-N3, and PSBMA multilayer clicked

of the adherent platelets could be easily counted and which were significantly decreased as shown in Figure 5b. The numbers of the adherent platelets were almost the same for the PSf-2, PSf-6, and PSf-10 membranes, and the platelets expressed a rounded morphology with nearly no pseudopodia and deformation. The decreased platelet adhesion was ascribed to the improved hydrophilicity and antifouling property after that PSBMA multilayer was deposited. For the PSf-n-CA membranes, the numbers of the adherent platelets were slightly increased compared with those of the precursor PSf-n membranes, which was not consistent with the results of protein adsorption experiments. The reason might be that the high concentration of the charge on the membranes led to the activation of platelets and then led to the higher adhesion and aggregation.55,56 APTT and TT. APTT and TT are usually used to examine mainly the intrinsic pathway and exhibit the bioactivity of intrinsic blood coagulation factors,57,58 and the results of APTT and TT for the modified membranes are shown in Figure 6a,b. The APTTs of zwitterion PSBMA clicked membranes were prolonged about 20 s compared with that of the pristine PSf membrane (50.2 s). Meanwhile, the APTTs of the PSf-n-CA membranes were slightly increased compared with the precursor PSf-n membranes (about 75 s). The results demonstrated that the PSBMA multilayer improved the anticoagulant property of the modified membranes (PSf-n). However, the TTs for all the membranes were almost the same with the value of about 20 s. In this study, since citric acid was used as the anticoagulant in the blood collection process, the APTT was slightly increased for the citric acid grafted membranes. Thus, a modified APTT test was conducted, in which CaCl2 solutions with different concentrations were added into the plasma before the APTT test.33 The typical results for PSf, PSf-6, and PSf-6-CA membranes are shown in Table 1. The APTT for PSf membrane was decreased from 50.8 to 44.5 s after the incubation for 30 min in the plasma with 2.5 mmol/L Ca2+; when Ca2+ with higher concentration was added, the plasma coagulated during the incubation. For the PSf-6 membrane, when the Ca2+ concentration of the solution added into the plasma was higher than 7.5 mmol/L, the plasma coagulated during the incubation. Surprisingly, for PSf-6-CA membrane, the APTTs were slightly decreased for all the concentrations (0−12.5 mmol/L). The results indicated that the citric acid grafted membranes could efficiently combine Ca2+, and the anticoagulant property was further improved compared with the precursor PSf-n membranes. Plasma Recalcification Time (PRT). Coagulation occurs in three stages. The first stage occurs via two distinct pathways, i.e., intrinsic and extrinsic pathways. This phase can be contributed to a cascade of interactions between the clotting factors, resulting in the formation of enzyme prothrombinase.59 Plasma recalcification profiles can be used to mimic the intrinsic coagulation system in vitro and the results are shown in Figure 6c. The PRTs for the PSBMA and citric acid composite multilayer clicked membranes were prolonged about 100 s compared with that (492 s) for the PSf membrane, indicating that the anticoagulant property of the modified membranes was improved than that of the PSf membrane. For the PSf-n-CA membranes, the PRTs (about 600 s) were slightly increased than those for the precursor PSf-n membranes. Compared with our previous study,13 in which PSf-g-PSBMA membranes were prepared via surface-initiated ATRP, the

Figure 5. SEM images of the platelets adhered onto the membranes with the magnification of 1000× (b is the number of the adhered platelets estimated by SEM images).

membrane surfaces. For the PSf-N3 membrane, the platelet aggregation was serious and a biofilm was observed; thus, we had to roughly estimate the number of the adherent platelets through counting the adherent platelets with clear shape. After PSBMA multilayer was clicked on the membranes, the numbers 5122

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Figure 6. (a) Activated partial thromboplastin times (APTTs), (b) thrombin times (TTs), and (c) plasma recalcification times (PRTs) for the modified membranes. Values are expressed as means ± SD, n = 3.

precursor PSf-n membranes, alkynyl-citric acid modified membranes (PSf-n-CA) showed similar protein adsorption results. More importantly, the PSf-n-CA exhibited better anticoagulant property, which could be confirmed by the APTT test with added Ca2+, although the grafted citric acid had effect on the morphology of platelets. These results indicated that the zwitterionic PSBMA and citric acid clicked membranes had potential to be used in biomedical fields, such as blood purification.

Table 1. APTTs for the Membranes PSf, PSf-6, and PSf-6CA, Ca2+ with Different Concentrations Added in the PPP APTT (s) PPP (μL)

Ca2+ concn (mmol/L)

PSf

PSf-6

PSf-6-CA

100 100 100 100 100 100

0 2.5 5 7.5 10 12.5

50.8 44.5

76.1 59.2 50.1

76.8 70.5 62.5 55.5 53.4 50.7

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ASSOCIATED CONTENT

* Supporting Information S

blood compatibility of the click chemistry-enabled LBL assembly PSBMA multilayer deposited membranes was improved. The protein (BAS and BFG) adsorption results were similar, but the anticoagulant property (prolonged APTT, TT, and PRT) was obviously improved.

Synthesis details for the azido-functionalized polysulfone and alkynyl-citric acid, ATR-FTIR spectra, and the SEM images for the modified membranes. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSIONS Two kinds of zwitterionic copolymers bearing alkynyl and azide groups were prepared and characterized. The copolymers alkynyl-PSBMA and azide-PSBMA were then deposited onto the surface of azido-functionalized polysulfone (PSf-N3) membrane via click chemistry-enabled LBL assembly. The deposited multilayer improved the hydrophilicity and blood compatibility (low protein adsorptions, suppressed platelet adhesion, and prolonged clotting time compared with the pristine PSf membrane) even when two layers of PSBMA were deposited, and more layers of PSBMA showed similar results. Alkynyl-citric acid was then clicked onto the membranes when the outermost layer was azide-PSBMA. Compared with the

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] or [email protected]; Tel +86-28-85400453; Fax +86-28-85405402 (C.-S.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially sponsored by the Natural Science Foundation of China (No. 51173119 and 51225303) and Program for Changjiang Scholars and Innovative Research Team in University (IRT1163). We also thank the help of Ms. Hui. Wang for the SEM. 5123

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