Surface Modification of Poly(ether sulfone) - ACS Publications

Oct 27, 2014 - ABSTRACT: In this study, we provide a new method to modify poly(ether sulfone) (PES) membrane with good biocompatibility, for which ...
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Surface Modification of Poly(ether sulfone) Membrane with a Synthesized Negatively Charged Copolymer Wen Zou,† Hui Qin,† Wenbin Shi,† Shudong Sun,† and Changsheng Zhao*,†,‡ †

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

ABSTRACT: In this study, we provide a new method to modify poly(ether sulfone) (PES) membrane with good biocompatibility, for which diazotized PES (PES-N2+) membrane is covalently coated by a negatively charged copolymer of sodium sulfonated poly(styrene-alt-maleic anhydride) (NaSPS-MA). First, aminated PES (PES-NH2) is synthesized by nitro reduction reaction of nitro-PES (PES-NO2), and then blends with pristine PES to prepare PES/PES-NH2 membrane; then the membrane is treated with NaNO2 aqueous solution at acid condition; after surface diazo reaction, surface positively charged PES/PES-N2+ membrane is prepared. Second, poly(styrene-alt-maleic anhydride) (PS-alt-MA) is synthesized, then sulfonated and treated by sodium hydroxide solution to obtain sodium sulfonated (PS-alt-MA) (NaSPS-MA). Finally, the negatively charged NaSPS-MA copolymer is coated onto the surface positively charged PES/PES-N2+ membrane via electrostatic interaction; after UV-cross-linking, the linkage between the PES-N2+ and NaSPS-MA changes to a covalent bond. The surfacemodified PES membrane is characterized by FT-IR spectroscopy, X-ray photoelectron spectroscopy (XPS) analyses, and surface zeta potential analyses. The modified membrane exhibits good hemocompatibility and cytocompatibility, and the improved biocompatibility might have resulted from the existence of the hydrophilic groups (sodium carboxylate (−COONa) and sodium sulfonate (−SO3Na)). Moreover, the stability of the modified membrane is also investigated. The results indicated that the modified PES membrane using negatively charged copolymers had a lot of potential in blood purification fields and bioartificial liver supports for a long time. materials are ideal in a clinical situation.7−9 There are many methods to improve the materials with good blood compatibility; and an applicable choice is surface modification.10,11 It was reported that one of the most effective methods to improve blood compatibility of materials is surface heparinization.12 However, some inconveniences still exist for the methods, which strongly restrict the use of the heparinmodified materials. For instance, the long-term degradation and the serious loss of bioactivity in vivo may occur during the use of the heparinized materials.13 Thus, many investigators have tried many ways to mimic the activity of heparin by developing

1. INTRODUCTION Blood-contacting material has been widely used and plays an important role in biomedical fields, such as artificial lungs, vascular prosthesis, plasma separation, heart valve, hemodialysis, and artificial liver supports.1−5 Blood compatibility, which is defined as the compatibility of implanted material with blood, requires the material with low activation of blood coagulation system.6 If a blood-contacting material exhibits poor blood compatibility, it may give rise to thrombogenic formation and other tissue responses.6 Therefore, blood compatibility is the most significant aspect for blood-contacting materials. Many polymeric biomaterials have been developed for bloodcontacting devices, such as cellulose acetate (CA), poly(propylene) (PP), poly(methyl methacrylate) (PMMA), poly(vinyl alcohol) (PVA), polyethylene (PE), polysulfone (PSf), and poly(ether sulfone) (PES); nevertheless, not all of the © XXXX American Chemical Society

Received: June 21, 2014 Revised: October 11, 2014

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new antithrombogenic materials or synthetic anticoagulants with heparin-like structures. Because these materials contain sulfamide, sulfate, and carboxylic acid groups, it is believed that the existence of these negatively charged functional groups may act on the anticoagulant activity of heparin.14−16 For surface modification, we can apply negatively charged copolymers to several biomaterials by layer-by-layer polyelectrolyte deposition, for which an ultrathin film with either negative or positive charges is deposited onto a substrate by the alternative adsorption of negatively and positively charged polyelectrolytes due to the Coulombic interaction.17 One advantage of this method is that its application has nearly no restriction to the substrates, no matter the type, size, or shape. However, the stability between the substrate and the copolymer is usually not strong enough.18−20 In this study, we provided a new method to improve membrane biocompatibility and the coating stability between polymeric membrane and synthesized copolymer. We selected PES as a substrate material to be modified, because PES exhibited many good properties such as good thermal stability (the glass temperature of PES is about 225 °C), chemical resistance, mechanical and film-forming properties, and, in recent years, it had been applied in hemodialysis.7,8,21−23 Negatively charged sodium sulfonated poly(styrene-alt-maleic anhydride) (NaSPSMA) was used as the coating polymer. To prepare NaSPSMA, poly(styrene-alt-maleic anhydride) (PSMA) was synthesized via free-radical polymerization, then sulfonated by concentrated sulfuric acid to get a sulfonated poly(styrene-alt-maleic anhydride) (SPSMA); the SPSMA was then treated by sodium hydroxide solution to get a negatively charged copolymer. The negatively charged copolymer NaSPSMA could only be dissolved in water, and thus be applied on blood-contacting materials by coating or surface grafting method; however, it is difficult to modify polymeric membranes using the negatively polymer by a blending method. The as-prepared copolymer was coated onto diazotized PES membrane; after UV-cross-linking, the linkage between the NaSPSMA and PES membrane became stable. Moreover, blood compatibility (protein adsorption, platelet adhesion, and clotting time) and cytocompatibility (cell morphology and cell viability assay) for the membrane were investigated.

Scheme 1. (1) Synthesis of PSMA Alternating Copolymer, (2) Synthesis of SPSMA, and (3) Transformation of Anhydride Groups and Sulfo Groups to Sodium Carboxyl Groups and Sodium Sulfo Groups, Respectively, To Form SPSMA

of St and MA using AIBN as the initiator (see the Supporting Information). The prepared PSMA (10 g) then was thoroughly dissolved in DMF (200 mL), H2SO4 (50 mL) was slowly added, and the sulfonation reaction was left at 60 °C for 24 h; after the reaction, the product was precipitated by cold diethyl ether. After evaporating the diethyl ether from the filtrate by a rotary evaporator, the resulting sulfonated poly(styrene-alt-maleic anhydride) (SPSMA) was put into a vacuum oven (0.01 MPa) to be dried at 25 °C for 48 h. The last step was to dissolve SPSMA in 100 mL of NaOH solution (0.1 mol/L) with stirring, and then the solution was dialyzed in ethanol and double-distilled water for a couple of times to get the negatively charged copolymer of sodium sulfonated poly(styrene-altmaleic anhydride) (NaSPSMA). 2.3. Synthesis of Negatively Charged PES Membrane. First, aminated PES (PES-NH2) was synthesized by nitration reaction and reduction reaction (see the Supporting Information); then aminated PES membranes (PES/PES-NH2) were prepared by using spin-coating couple with a liquid−liquid phase separation technique, and the prepared aminated PES membranes were termed L-16-0 (PES 16%, PES-NH2 0%), L-12-4 (PES 12%, PES-NH2 4%), and L-8-8 (PES 8%, PES-NH2 8%), respectively (see the Supporting Information); and then diazotize PES membranes (PES/PES-N2+) were prepared by the surface diazo-reaction (see the Supporting Information). The positively charged PES/PES-N2+ membranes then were used as the substrates to prepare negatively charged PES membranes at room temperature in the dark. The PES/PES-N2+ membranes were immersed in the prepared NaSPSMA solution (negatively charged, 2 mg/mL) for 5 min, and then rinsed with double-distilled water; after drying in an oven at 50 °C for 5 min, the modified membranes were exposed to UV light for 20 s for cross-linking to make the linkages between the PES-N2+ and NaSPSMA change into covalent bonds, and the wavelength range of UV light was 280−380 nm. It had been reported17 that under UV irradiation, polyelectrolyte complexes formed from diazoresin as the cationic polyelectrolyte possessed a unique characteristic: the linkages in the complex could convert from ionic bonds to covalent bonds. Many other studies also proved the same conclusion, and the main function groups were diazo groups and anion groups (such as −COO− or −SO3−).24,25 In this study, the PES membrane containing abundant diazo groups (−N2+) on its surface was prepared, and then synthesized polyelectrolyte NaSPSMA was coated on the surface of the membrane. After UV irradiating, the linkages between the diazo groups and the coated NaSPSMA changed to covalent bonds, as shown in Scheme 2. Also, the membranes using different PES-N2+ amounts were termed M-16-0, M-12-4, and M-8-8, respectively. Moreover, the coating density of the copolymers onto the membranes was investigated, and the coating densities of the copolymers onto the membranes were 0.09 and 0.13 mg/m2 for the

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ether sulfone) (PES, Ultrason E6020P) was purchased from BASF. Maleic anhydride (MA), styrene (St), azobisisobutryonitrile (AIBN), N,N-dimethylformamide (DMF; AR, 99.0%), diethyl ether, acetone, N,N-dimethylacetamide (DMAC; AR, 99.0%), concentrated nitric acid (HNO3, 65%), concentrated hydrochloric acid (HCl, 37%), concentrated sulfuric acid (H2SO4, 98%), sodium nitrite (NaNO2), and sodium hydroxide (NaOH) were purchased from Chengdu Kelong Chemical Reagent Co. Stannous chloride (SnCl2) was purchased from Tianjin Chemical Reagent Supply Co. Micro BCA Protein Assay Reagent kits were the products of PIERCE. Bovine serum fibrinogen (FBG), bovine serum albumin (BSA; fraction V), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, U.S. Styrene (St) was washed with 10% sodium hydroxide solution and then was distilled under reduced pressure before use. MA was recrystallized from chloroform and dried in a vacuum oven (0.01 MPa) at room temperature for 48 h. The other reagents were used without further purification. Double-distilled water was used throughout the studies. 2.2. Synthesis of Negatively Charged Copolymer. The synthetic route is shown in Scheme 1. Poly(styrene-alt-maleic anhydride) (PSMA) was synthesized via free-radical polymerization B

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Scheme 2. Modification of PES Membrane Using PES/PES-N2+ Membrane after Cross-Linking under UV Light or Heat; and Linkages Transform to Covalent Bonds in the Modified PES Membrane

anticoagulant) at 1000 rpm for 15 min. The membrane (1 cm × 1 cm) was immersed in PBS and incubated for 1 h at 37 °C. After that, the PBS solution was removed and 1 mL of PRP was added; after incubating with PRP for 30 min at 37 °C, the PRP was decanted off, then the membrane was rinsed using PBS at least three times. Finally, the membrane was treated using 2.5 wt % glutaraldehyde for 1 day at 4 °C, and then washed with PBS solution. The membrane then was passed through a series of graded alcohol−PBS solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) to be dried. The critical point drying of the specimens was done with liquid CO2. We used an S-2500C microscope (Hitachi, Japan) to investigate the platelet adhesion on the membranes. 2.5.3. Clotting Time. The anticoagulant activity of the membranes was tested by measuring activated partial thromboplastin time (APTT) using an automated blood coagulation analyzer (Sysmex Corp., Kobe, Japan), and the test method was shown as follows: first, platelet-poor plasma (PPP) was obtained by centrifuging healthy human fresh blood (man, 25 years old, containing sodium citrate as an anticoagulant) at 4000 rpm for 15 min. The membrane (three pieces, 0.5 cm × 0.5 cm) then was placed in 0.2 mL of PBS (pH 7.4) for 1 h; thereafter, the PBS was decanted off and 0.1 mL of PPP was added. After incubating for 0.5 h at 37 °C, 50 μL of the PPP was placed in a test cup, and then 50 μL of APTT agent (incubated for 10 min before use) was added and incubated for 3 min at 37 °C. After that, 50 μL of 0.025 M calcium chloride (CaCl2) solution was added. Finally, the APTT was measured using the automated blood coagulation analyzer; the results were analyzed by statistical method, and at least three measurements were averaged to get a reliable value, and were expressed as means ± SD. Moreover, platelet activation and complement activation of the modified membranes were also investigated, and the results are shown in the Supporting Information. 2.6. Cytocompatibility. 2.6.1. Cell Morphology on the Membranes. Hepatocyte was selected to investigate the cell morphology on the PES membranes. The method was shown as follows: first, hepatocytes were seeded onto the membranes and incubated for 6 days, and the hepatocyte density was about 2.5 × 104 cells/cm2. After that, the incubated membranes were slightly rinsed with PBS solution and then fixed with glutaraldehyde (2.5 wt %) for 12 h at 4 °C. Next, the incubated membranes were passed through a series of graded alcohol−PBS solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) and then isoamyl acetate to be dried. The critical point drying of the incubated membrane was done with liquid CO2. After sputter-coating with a gold layer, the incubated membranes were tested by a scanning electron microscope (JSM-5900LV, JEOL, Japan).

membranes M-12-4 and M-8-8, respectively (see the Supporting Information). 2.4. Characterization. To prepare FT-IR sample, the polymer was dissolved thoroughly in solvent DMAC, and then cast on potassium bromide (KBr) disc with the thickness of about 0.8 mm, and afterward dried by an infrared light. The sample then was investigated by reflected FT-IR with a FT-IR Nicolet 560 (Nicol American). The 1HNMR spectra were recorded on a Varian Unity Plus 300/54 NMR spectrometer using deuteroxide (H2O-d) or deuterated acetone (acetone-d) as the solvent at room temperature. GPC measurement was carried out with tetrahydrofuran (THF) and H2O as the eluent by a PL220 GPC analyzer (Britain). Polystyrene was used as the standard polymer to calibrate the test. To observe the morphology of the membrane, an XL 30ESME scanning microscope was used. Before the analysis of scanning electron microscopy (SEM), the membrane was frozen in liquid nitrogen, and then broken and sputtered with a gold layer to prepare the sample. X-ray photoelectron spectroscopy (XPS) was used to investigate the elements and the structures of the modified membranes. Surface zeta potentials were determined using a Beckman Coulter, DelsaTM NanoC Particle Analyzer and an accessory (a sample cell) to investigate the surface of the negatively charged PES membranes (with the area of 1 × 3 cm2) at 37 °C. To detect the zeta potentials of the PES membrane surface, the membrane was put into a sample cell; then standard electrolyte solution was injected into the sample cell; finally, the surface zeta potential of the membrane was detected. 2.5. Blood Compatibility. 2.5.1. Protein Adsorption. Protein adsorption experiments were carried out under static condition using FBG and BSA solutions. The method was shown below: the membrane (1 cm × 1 cm) was immersed in BSA or BFG solution (phosphatic buffer solution, PBS, pH = 7.4, 1 mg/mL) and incubated for 1 h at 37 °C; and then the incubated membrane was rinsed with double-distilled water and PBS slightly. Afterward, the membrane was immersed in a washing fluid that consisted of 0.05 M NaOH and 2% sodium dodecyl sulfate (SDS), and then shaken at 37 °C for 2 h to remove the adsorbed protein. The adsorption and desorption conditions were carefully determined in preliminary experiments, and the concentration of the protein was determined using a Micro BCATM Protein Assay Reagent Kit (PIERCE). At last, the amount of the adsorbed protein was calculated, and the results were expressed as means ± SD. 2.5.2. Platelet Adhesion. In this study, platelet-rich plasma (PRP) was used to study the platelet adhesion on the surfaces for the membranes. The PRP was obtained by centrifuging healthy human fresh blood (man, 25 years old, containing sodium citrate as an C

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2.6.2. MTT Assay. MTT assay was used to investigate the survivability of the hepatocytes cultured on the PES membranes. The main process was shown as follows: first, the hepatocytes were cultured on the membranes with the density of about 2.5 × 104 cells/ cm2. Simultaneously, the cells cultured without membrane were used as the control. After cell culturing for 2, 4, or 6 days, respectively, 45 μL of MTT solution with a concentration of 1 mg/mL was introduced in each well and incubated at 37 °C for 4 h. Mitochodrial dehydrogenases of viable cells cleave selectively to the tetrazolium ring, yielding blue/purple formazan crystals. The formazan crystals then were dissolved in ethanol (400 μL). After that, the activity of the cell metabolism could be reflected by determining the quantity of the formazan crystals that dissolved in the ethanol. The dissolvable solution then was homogeneously jogged using a shaker for 15 min. After aspirating the solution of each sample into a microtiter plate, the Microplate reader (model 550, Bio-Rad) was used to determine the optical density of the dissolvable solution at a wavelength of 492 nm. The experiments were repeated three times, and the results were expressed as means ± SD. The statistical significance was assessed by Student’s t-test with the level of significance chosen as P < 0.05.

Meanwhile, the molecular weight and its dispersity were decreased due to the increased chain transfer in solution polymerization. The above results were caused because both the gel effect and the chain transfer occurred at the same time. After the sulfonation using H2SO4, as for the NaSPSMA, the molecular weight decreased and the molecular weight distribution increased, because the molecular chains of the copolymer PSMA might be broken during the chemical reaction. 3.2. Characterization of Aminated PES and Negatively Charged PES Membrane. 3.2.1. FT-IR Spectra Analysis. The spectra for PES, PES-NO2, PES-NH2, and PES-NaSPSMA are shown in Supporting Information Figure S3. For the PES-NO2, the peak at 1536 cm−1 was assigned to the asymmetric stretching vibration of −NO2. Also, the peak at 1536 cm−1 disappeared after the nitro reduction reaction, and new peaks appeared at 1625, 3422, and 3377 cm−1, which were ascribed to the N−H stretching. The modified PES membrane (PESNaSPSMA) was characterized by reflected FT-IR spectrum. It was found that there was a very small peak at 1728 cm−1, which was ascribed to the carboxyl groups in NaSPSMA; however, as the peak was so small, it was uncertain whether or not the NaSPSMA was grafted onto the PES membrane by the reflected FT-IR spectrum only, so more analysis on the surfacemodified PES membrane should be carried out. 3.2.2. XPS Analysis. The surface compositions of the modified membrane were tested by XPS. As shown in Supporting Information Figure S4, a new element content of Na appeared, which was attributed to the existent of NaSPSMA. With the increase of the content of PES-N2+, the content of Na increased from 0.74% (mol) to 1.26% (mol). 3.2.3. Morphology of the Modified Membranes (SEM Micrographs). The cross-sectional and surface SEM micrographs for the membranes are shown in Supporting Information Figure S5. For the membranes, a dense top skin layer and a finger-like structure were observed.30 After the PESNH2 was blended, the membrane morphology was changed slightly, and the PES-NH2 was well incorporated into the PES matrix. For the modified membranes, the top skin of the membranes became denser; the formation mechanism was summarized in our previous study9 (see the Supporting Information). Also, with increasing the PES-NH2 amounts in the PES membranes, a slight change of the finger-like structure was observed, as shown in Supporting Information Figure S5a− c. Furthermore, we could also find that after the UV-crosslinking,18,31 the membrane surfaces slightly changed, as shown in Supporting Information Figure S5A,a. 3.3. Blood Compatibility. 3.3.1. Protein Adsorption. As is known to all, one of the significant factors for evaluating the blood compatibility of materials is the adsorbed protein amount on the material surface;32 furthermore, platelet GP IIb/IIIa receptor can combined with fibrinogen in blood plasma, so fibrinogen is particularly important for platelet adhesion.33 Thus, to improve blood compatibility, low protein adsorption is necessary.33,34 The amounts of BSA and FBG that adsorbed on the surfaces membrane are shown in Figure 1. We could observe that the amounts of BSA and FBG adsorbed on the NaSPSMA grafted PES membranes decreased as compared to the PES membranes, and with the increasing of the NaSPSMA amounts grafted onto the PES membranes, the values decreased due to the improved wetting property;35 another reason might be the electrostatic repulsion between the modified PES membrane

3. RESULTS AND DISCUSSION 3.1. Characterization of Negatively Charged Copolymer. 3.1.1. FT-IR Spectra Analysis. The FT-IR spectra for PSMA and NaSPSMA are shown in the Supporting Information. As shown in Supporting Information Figure S1, the peaks at 1632, 1495, 1455 cm−1 were attributed to the C C (aromatic) stretching vibrations; the peaks at 734 and 706 cm−1 reflected the C−H stretching in the monosubstituent aromatic ring, and the peaks at 1854 and 1780 cm−1 corresponded to the C−O stretching in the anhydride groups. After sulfonation and transformation, a new peak at 1029 cm−1 appeared, which was assigned to the −SO3− groups in the aromatic ring; moreover, the peak at 1632 cm−1 was changed to 1568 cm−1, which was also influenced by the −SO3− groups. In addition, the peak of the carboxyl groups was hidden in the peak at 1568 cm−1. 3.1.2. 1H NMR Spectra of the PSMA and the NaSPSMA. As shown in Supporting Information Figure S2, for the PSMA, the peaks of the backbone hydrogen for the St and MA (δ = 0−3 ppm, 2H from MA R−CH−COO−, 3H from St R−CH2−R′, R−CH−Ar, peaks b and c) and the aromatic peaks of the St (δ = 6−7.5 ppm, 5H, a) were the markers to determine the copolymer composition. The NMR results coincided with those in ref 26. According to Supporting Information Figure S2, we could calculate that the molar ratio of the St to MA in the PSMA was approximately 1:1, as expected from the tendency of the monomers to polymerize together as one unit.27 Comparing the NMR results between the PSMA and NaSPSMA, the molar ratio of the Ha′ to Hb′ and c′ for the NaSPSMA was calculated to be about 4.3:5, indicating the sulfonation degree of the copolymer was nearly 70%. 3.1.3. GPC Measurement. Through GPC measurement, the molecular weights and molecular weight distributions of the prepared copolymer PSMA and NaSPSMA were obtained. For PSMA, the number-average molecular weight (Mn) and weightaverage molecular weight (Mw) were 3.00 × 104 and 1.72 × 105, respectively; and the molecular weight distribution was 5.73. For NaSPSMA, the Mn and Mw were 2.47 × 104 and 1.54 × 105, respectively; and the molecular weight distribution was 6.23. For PSMA, the molecular weight was very large, while the dispersity was wide. The reason might be the gel effect and chain transfer.28,29 In solution polymerization, the gel effect was significant as the monomer concentration was considerably large, so the molecular weight and its dispersity increased. D

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Figure 1. BSA and BFG adsorbed amounts on PES and modified PES membranes. (Values are expressed as mean ± SD, n = 3.)

and BSA or FBG. Because the modified PES membrane and BSA or FBG were negatively charged, with increasing the amount of the NaSPSMA grafted onto the modified PES membrane, the electrostatic repulsion between the modified PES membrane and BSA or FBG increased and the adsorption of BSA or FBG onto the modified PES membrane decreased.36 Thus, the blood compatibility of the modified PES membranes might be improved by the improvement of antiprotein adsorption (especially fibrinogen). In addition, for negatively charged surface, proteins with different isoelectric points (pI’s) show different adsorption behaviors (amount, kinetics, etc.). Generally, when pH increased within a reasonable range, the adsorption of proteins onto the substrate decreased because of the increased electrostatic repulsion; in contrast, as the pH value was decreased, the charge of the substrate was reduced because of the partial protonation, the adsorption of proteins onto the substrate increased due to the reduction of the repulsive electrostatic interactions, and the impact of proteins with different pI’s was different.36 In this study, we mainly investigated the biocompatibility of the modified PES membrane, which was used as blood-contacting material, and the pH value of human blood ranges from 7.35 to 7.45; thus protein adsorption experiments were carried out at pH 7.4 using PBS. 3.3.2. Platelet Adhesion. The adsorbed proteins, especially fibrinogen, can be combined with integrins, which is on the platelet surface, causing platelet adhesion and further platelet activation. Thrombin formation and platelet aggregation then can be promoted due to the activated platelets, and thrombosis is accelerated.34,37,38 Thus, another important factor to evaluate blood compatibility is platelet adhesion. In this study, SEM was used to investigate the morphologies of the platelets adhered on the surfaces of the PES and the modified PES membranes, as was shown in Figure 2. By comparing pictures with the same amplification multiple, large amounts of platelets were observed, adhering and aggregating on the pristine PES membrane surfaces, and the shapes of the adhered platelets were irregular and flattened. On the contrary, few platelets were found on the modified membranes, and the platelet expressed a rounded morphology with little deformation and pseudopodium. Comparing Figure 2B and C, it was found that, with the increase in the content of the NaSPSMA, the platelet adhesion decreased. Supporting Information Figure S6 shows the numbers of the adhered platelets on the membranes. As shown in Supporting Information Figure S6, the numbers of the adhered platelets on the modified membranes were decreased significantly as

Figure 2. Typical SEM of the platelets adhering on the membranes. (A) and (a) are M-16-0; (B) and (b) are M-12-4; (C) and (c) are M8-8.

compared to those of the pristine PES membrane. For pristine PES membrane, the number of the adhered platelets was about 4.3 × 107/cm2, while those for the modified PES membranes M-12-4 and M-8-8 were 0.7 × 107 and 0.2 × 107/cm2, respectively. The above results indicated that the blood compatibility of the modified PES membranes was improved due to the low adsorption of protein and platelet adhesion. 3.3.3. Clotting Time. Coagulation abnormalities in the intrinsic pathway can be evaluated by APTT.39,40 When blood is contacted with an anticoagulative material, the coagulation factors might be combined or reacted with the anticoagulative material, and the APTT could be prolonged. Thus, the anticoagulation activity of blood-contacting materials could be evaluated by APTT. Figure 3 shows the APTTs results (significant difference, p < 0.05) for the pristine PES and modified PES membranes. The APTTs increased with the increase of the NaSPSMA amounts grafted onto the PES membranes. The prolonged APTTs of the modified membranes might be resulted from the interaction between the coagulation factors (such as factor II, V, X) and the NaSPSMA.40 It could be concluded that the low BFG protein adsorption and low platelet adhesion might have a positive influence to prolong the APTT of the modified membranes. 3.4. Cytocompatibility. 3.4.1. Cell Morphology Analysis. PES-based membranes have been widely used in the field of blood purification due to their significant physic-chemical and morphological properties, as well as good blood compatibility. In addition, PES membranes have also been used in tissue engineering and artificial organs due to its good cytocompatibility.41−44 Thus, in this study, human hepatocytes LO2 were E

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of the hepatocytes adhered on the negatively charged surfaces increased, and the hepatocytes grew better. In addition, we could find that the cells covered the entire surfaces of the negatively charged PES membranes with extending pseudopodia as compared to that of the cells cultured and adhered on the pristine PES membrane. The results indicated that, with the grafting of NaSPSMA, the cell growth and cell adhesion had been promoted, and the cytocompatibility of PES had been improved. 3.4.2. MTT Assay. Figure 5 shows the results of the MTT assay. The formazan absorbance indicated that the cells seeded Figure 3. APTT for PES and the negatively charged PES membranes. (M-0-0 is the control sample, values are expressed as mean ± SD, n = 3; *p < 0.05 as compared to the values for the control sample.)

selected to evaluate the cytocompatibility of the modified membranes. It is well-known that when cells contact biomaterials, the morphologies of the cells will be changed to achieve the integration of the biomaterials. 45 Figure 4 shows the

Figure 5. MTT tetrazolium assay; formazan absorbance is expressed as a function of time from hepatocytes seeded on different membranes and the controls. (Values are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.05, and ***p < 0.05 as compared to the values for the M-16-0 membrane at 2, 4, and 6 days, respectively; #p < 0.05 between days for the same sample.)

onto the samples were able to convert MTT into a blue formazan product.. It could be seen that with increasing the culture time (p < 0.05), the survivability of the hepatocytes cultured on the membranes increased. It was also found that the viability of the cells cultured on the second, fourth, and sixth day for the modified PES membranes was increased as compared to the pristine PES membrane, and the M-8-8 exhibited the best cytocompatibility among the membranes (p < 0.05). In addition, the modified membranes kept a better viability, which indicated that the NaSPSMA might have positive influence on the cytocompatibility and could prevent the inflammatory risks.46 3.4.3. Zeta Potential Analysis. Figure 6 shows the surface zeta potentials of the pristine PES membrane, the aminated PES membrane, and the negatively charged PES membrane. The zeta potential was −6.15 mV for the pristine PES membrane; after being blended with the aminated PES, zeta potentials of the prepared aminated PES membranes (L-12-4 and L-8-8) increased to −4.27 and −3.65 mV, respectively. Surface diazotized PES membranes then were prepared through surface diazo-reaction; however, the −N2+ could not exist for a long time, and we could not characterize the zeta potential of the surface diazotized PES membranes directly. After UV-crosslinking, the zeta potentials of the prepared negatively charged membranes (M-12-4 and M-8-8) decreased to −17.86 and −20.37 mV, respectively. It was reported that the interaction between cell and substrate was related to the surface charge,47 and the suitable zeta potential for cell proliferation ranged from about −30 to −20 mV. Because both the cell surface and the NaSPSMA were negatively charged, therefore, no direct electrostatic interaction

Figure 4. Scanning electron micrographs of LO2 human liver cells cultured on the PES and modified PES membranes after 6 days. (A), (a) are M-16-0; (B), (b) are M-12-4; (C), (c) are M-8-8.

morphologies of the hepatocytes cultured on the membranes for 6 days. As shown in Figure 4, the cells grew well on all of the membrane surfaces with pseudopodia. Comparing the negatively charged PES membranes with the pristine PES membrane, we could observe that the amounts of the hepatocytes increased dramatically on the negatively charged PES membrane surfaces. Moreover, we could also observe that a majority of the cells were gathered together and formed a lamellar structure on the negatively charged membrane surfaces. With the NaSPSMA amount increasing, the amounts F

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The M-12-4 and M-8-8 (washed by SDS aqueous solution, respectively) were then washed by double-distilled water and dried; the treated modified membranes showed nearly the same blood compatibility and cytocompatibility as did the untreated modified membranes. These results indicated that the elution of NaSPSMA does not occur. The zeta potentials of the modified cross-linked negatively charged membranes were also investigated, and no significant increase in the zeta potential was observed. This higher stability was ascribed to the formed covalent bond linkage of the PES-N2+ and NaSPSMA after cross-linking.18

4. CONCLUSIONS In the study, negatively charged copolymer NaSPSMA was synthesized and coated onto PES membrane by the combination with PES-N2+ via UV-cross-linking to improve hemocompatibility. The modified membranes showed increased blood compatibility and cytocompatibility as compared to the pristine PES membrane, for instance, lower BSA and FBG adsorption, lower platelet adhesion, prolonged APTT, and lower activation of platelets and complements because of the −COO− and the −SO3− groups on the modified membranes surfaces. Furthermore, the stability between the negatively charged copolymer and PES membrane was enhanced significantly. The results indicated that the surface-modified PES membranes had a lot of potential in blood-contact fields and bioartificial liver supports for a long time; in addition, this study also provided useful information on preparing stable coating membranes.

Figure 6. Zeta potentials for pristine PES membrane, aminated PES membrane, and negatively charged PES membrane. (Values are expressed as mean ± SD, n = 3.)

existed between the cell and negatively charged PES membrane. It is known that cell spreading and proliferation were significantly influenced by adhesive protein. As an amphoteric polyelectrolyte, a protein has both acidic and basic peptide, and could act as a bridge between positive or negative groups on cells or the surface of the negatively charge PES membrane. Thus, the negatively charged PES membrane might have interacted with serum proteins in the well by electrostatic interactions, and the serum proteins can behave as bridges.48 3.5. Stability of the Modified PES Membranes. In this study, the modified PES membrane was cross-linked via PESN2+ and NaSPSMA to enhance its stability. To investigate the stability of the modified PES membranes, the cross-linked membranes (M-12-4 and M-8-8; 6 pieces, 5 × 5 cm2 for each piece) and the uncross-linked membranes (M′-12-4 or M′-8-8; 6 pieces, 5 × 5 cm2 for each piece) were immersed in SDS aqueous solution (200 mL, 2 wt %), respectively. The absorption of the SDS solutions then was measured at 224 nm, and then the NaSPSMA concentrations eluted in the SDS solutions were calculated; data are shown in Figure 7. As shown

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ASSOCIATED CONTENT

S Supporting Information *

ATR-FTIR spectra, 1HNMR spectra, XPS spectra, and SEM images of the modified membranes, figures of the number of the adhering platelets onto the membranes, platelet activation and complement activation of the modified PES membranes, and complement activation for various membranes. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-28-85400453. Fax: +86-28-85405402. E-mail: [email protected], [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially sponsored by the National Natural Science Foundation of China (nos. 51173119 and 51225303). We also thank our laboratory members for their generous help, and gratefully acknowledge the help of H. Wang, of the Analytical and Testing Center at Sichuan University, for the SEM, and Liang, of the Department of Nephrology at West China Hospital, for the human fresh blood collection.

Figure 7. NaSPSMA concentration (in 2 wt % SDS aqueous solution, 200 mL) versus etching time for the cross-linked and uncross-linked membranes.

in the figure, for the cross-linked membranes (M-12-4 and M-88), there was almost no NaSPSMA eluted. However, for the uncross-linked membranes (M′-12-4 and M′-8-8), large amounts of NaSPSMA were eluted into SDS solution; and after about 20 min, the elution became steady. Furthermore, the elution amount for the M′-8-8 was larger than that for the M′-12-4 due to the larger coated amounts of the NaSPSMA on the membrane surface.

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REFERENCES

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