Acta Biomaterialia 7 (2011) 33703381

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Biocompatibility of modied polyethersulfone membranes by blending an amphiphilic triblock co-polymer of poly(vinyl pyrrolidone)b-poly(methyl methacrylate)b-poly(vinyl pyrrolidone)
Fen Ran a,b,c, Shengqiang Nie a,b, Weifeng Zhao a,b, Jie Li c, Baihai Su a, Shudong Sun a, Changsheng Zhao a,b,
a b c

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, Peoples Republic of China National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, Peoples Republic of China State Key Laboratory of Gansu Advanced Non-Ferrous Metal Materials, Lanzhou University of Technology, Lanzhou 730050, Peoples Republic of China

a r t i c l e

i n f o

a b s t r a c t
An amphiphilic triblock co-polymer of poly(vinyl pyrrolidone)b-poly(methyl methacrylate)b-poly(vinyl pyrrolidone) (PVP-b-PMMA-b-PVP) was synthesized via reversible additionfragmentation chain transfer (RAFT) polymerization. The block co-polymer can be directly blended with polyethersulfone (PES) using dimethylacetamide (DMAC) as the solvent to prepare at sheet and hollow ber membranes using a liquidliquid phase separation technique. The PVP block formed a brush on the surface of the blended membrane, while the PMMA block mingled with the PES macromolecules, which endowed the membrane with permanent hydrophilicity. After adding the as-prepared block co-polymer the modied membranes showed lower protein (bovine serum albumin) adsorption, suppressed platelet adhesion, and a prolonged blood coagulation time, and thereby the blood compatibility was improved. Furthermore, the modied PES membranes showed good cytocompatibility, ultraltration and protein anti-fouling properties. These results suggest that surface modication of PES membranes by blending with the amphiphilic triblock co-polymer PVP-b-PMMA-b-PVP allows practical application of these membranes with good biocompatibility in the eld of blood purication, such as hemodialysis and bioarticial liver support. 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 20 January 2011 Received in revised form 7 May 2011 Accepted 20 May 2011 Available online 27 May 2011 Keywords: Polyethersulfone membrane Amphiphilic triblock co-polymer Ultraltration Blood compatibility Cytocompatibility

1. Introduction The aim of this study was to nd an approach by which to modify polymeric hollow ber membranes with excellent biocompatibility, which would provide information for their practical application. Thus an amphiphilic triblock co-polymer was synthesized and directly blended with polyethersulfone (PES) to prepare at sheet and hollow ber membranes. It is well known that polymeric materials are widely used in the eld of blood purication for articial organs, medical devices and disposable clinical instruments, including hemodialysis, hemodialtration, hemoltration, plasmapheresis, and plasma collection [1,2]. Among the polymeric materials used in biomedical elds, PES is one of the most important and widely used. PES and PES-based membranes show outstanding oxidative, thermal and hydrolytic stability, as well as good mechanical and lm-forming properties

Corresponding author at: College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, Peoples Republic of China. Tel.: +86 28 85400453; fax: +86 28 85405402. E-mail addresses: zhaochsh70@scu.edu.cn, zhaochsh70@163.com (C. Zhao).

[3]. The membranes also showed high permeability for low molecular weight proteins when used as hemodialysis membranes [4]. However, the blood compatibility of PES membranes is inadequate, and injections of anticoagulants are needed during hemodialysis [5]. To improve the blood compatibility of biomedical materials many studies have focused on the development of new materials and the modication of conventional materials. The modication approaches mostly used for PES membranes include blending, coating, surface physical treatment, surface grafting, etc. [611]. Grafting anti-thrombogenic materials and/or hydrophilic polymers onto membrane material surfaces using chemical modication is the most efcient method, which can catalytically increase the formation rate of antithrombin III and inhibit thrombin and other coagulation proteases. However, the grafting method required a long reaction time and a rigorous reaction process [1214], and its use to modify hollow ber membranes is difcult. Blending, in contrast, is the simplest method to modify PES both at sheet and hollow ber membranes, and thus is widely used in industry. By directly blending hydrophilic polymers, such as poly(vinyl pyrrolidone) (PVP) [15], polyethylene glycol (PEG) [16] and poly(acrylic acidco-vinyl pyrrolidone) (PAA-co-PVP) [17,18], the

1742-7061/$ - see front matter 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.05.026

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surface hydrophilicity of the membranes could be increased; the anti-fouling properties and blood compatibility were also improved [19]. PVP is a non-ionic water soluble polymer and exhibits many fascinating properties [20,21]. It was initially used as a blood plasma substitute and later has been applied in a wide variety of applications, such as biomaterials and coatings, contact lenses, disinfectants, intra-ocular lenses, medicine, pharmacy, cosmetics and industrial production [22,23]. PVP has been used as a hydrophilic additive and membrane pore-forming agent to modify PES membranes [24,25]. However, hydrophilic polymers such as PVP and PAA-co-PVP are water soluble and elution of the polymers from the PES membranes is unavoidable [26]. Thus a novel hydrophilic silicaPVP nanocomposite additive was synthesized [27], and used to improve the surface coverage of PVP on PES membrane surfaces to enhance the anti-fouling properties. The results indicated that the antifouling ability of a PES membrane with a silicaPVP nanocomposite additive was better than that of a PES membrane with a PVP additive. To avoid elution acrylonitrile (AN) and methyl methacrylate (MMA) were used to synthesize co-polymers containing vinyl pyrrolidone (VP) chains by free radical solution polymerization in recent studies [28,29]; the co-polymers poly(acrylonitrilecoacrylic acidco-vinyl pyrrolidone) P(AN-co-AA-co-VP) [28] and poly(methyl methacrylateco-acrylic acidco-vinyl pyrrolidone) P(MMA-co-AA-co-VP) [29] were blended with PES to prepare the modied membranes. The terpolymers were water insoluble due to the AN (or MMA) chains. The acrylic acid (AA) and VP chains are hydrophilic. The terpolymers can be directly blended with PES as macromolecular additives using N-methyl-2-pyrrolidone (NMP) as the solvent to prepare the membranes. When the terpolymers were blended in the membranes protein adsorption decreased while the protein anti-fouling properties increased. The PVP-based co-polymer effectively improved the hydrophilicity and anti-fouling properties of the PES membranes. However, PVP and its co-polymers are synthesized by conventional free-radical polymerization techniques [30] and the need for further control over the constitution and functionality of polymeric materials, accessible through facile and general syntheses, remains a challenge in the polymer sciences, to prepare a regular structure block co-polymer. In fact, VP cannot easily undergo living/controlled polymerization, since it does not form stable radicals. In the past ingenuous alternative routes were explored to overcome this issue [31]. There are some recent reports referring to living radical polymerization of VP and the preparation of VP-based block co-polymers. Hadjichristidis et al. [32] reported that both nitroxide-mediated radical polymerization (NMRP) and additionfragmentation chain transfer (RAFT) polymerization techniques could be used for the controllable living polymerization of VP. In order to prepare PVP block co-polymers they combined anionic polymerization and NMRP. The copolymers had relatively broad molecular weight distributions [33]. Recently Yamago et al. [34] reported highly controlled living radical polymerization of VP. Based on organostibine-mediated living radical polymerization, PVP with the expected number average molecular weight was synthesized. Up to now several block co-polymers, such as poly(N-isopropylacrylamideco-hydroxylethyl methacrylate)b-poly(vinyl pyrrolidone) (P(NIPAAm-co-HEMA)-b-PVP) [35], poly(N-isopropylacrylamide)b-poly(vinyl pyrrolidone) (PNIPAAm-b-PVP) [36], poly(vinyl pyrrolidone)b-poly(methacryloylN0 -(a-naphthyl)-thiourea) (PVP-b-PMANTU) [37], poly(vinyl pyrrolidone)b-poly(vinyl pyrrolidonealt-maleic anhydride)bpoly(vinyl pyrrolidone) (PVP-b-P(VP-alt-MAn)-b-PVP) [38], poly(vinyl acetate)b-poly(vinyl pyrrolidone) (PVP-b-PVAc) [39], poly(e-caprolactoneb-vinyl pyrrolidone) (PCL-b-PVP) [40], polystyreneb-poly(vinyl pyrrolidone) (PS-b-PVP) [41,42], poly(vinyl pyrrolidone)b-poly(styrenealt-maleic anhydride) (PVP-b-PSMA) [43], poly(vinyl prrolidone)b-poly(N,N-dimethylaminoethyl

methacrylate) (PVP-b-PDMAEMA) [44] and poly(vinyl pyrrolidone)b-poly(D,L-lactide) (PVP-b-PDLLA) [45] have been synthesized by anionic polymerization, atom transfer radical polymerization (ATRP), RAFT, and NMRP. Micelles formed from these co-polymers were investigated for application as drug delivery systems. It is a great pity that none of the above research involved determining the biocompatibility of the products [46]. RAFT polymerization is tolerant of a wide range of functionalities on the monomer and solvent and can effectively control the rate and degree of polymerization [4749]. In this paper the polymerization of VP was controlled by RAFT polymerization using carboxyl-terminated trithiocarbonate as the RAFT agent. PMMA, which is often used as a biomaterial and medical material, was introduced as a hydrophobic block to the macro-RAFT agent PVP to synthesize the amphiphilic triblock co-polymer poly(vinyl pyrrolidone)b-poly(methyl methacrylate)b-poly(vinyl pyrrolidone) (PVP-b-PMMA-b-PVP) for the rst time. The block co-polymer was used as a macromolecular additive for direct blending with PES to prepare permanently hydrophilic PES membranes, and the contact angles, protein adsorption, blood compatibility (platelet adhesion and clotting time) and cytocompatibility (cell culture, cell morphology and MTT assay) of the membranes were investigated. In addition, block co-polymer modied PES hollow ber membranes were prepared, and ultraltration experiments were carried out to study the anti-fouling properties of the blended hollow ber membranes.

2. Materials and methods 2.1. Materials PES (Ultrason E6020P) was obtained from BASF, Germany. MMA (99.0% pure) was purchased from Uni-Chem, China. VP (99.0% pure) and tetrabutylammonium hydrogen sulfate were purchased from Alfa Aesar, USA. VP was pretreated with activated carbon before use. N,N-Dimethylacetamide (DMAC) (AR, 99.0% pure) and N,N-dimethylformamide (DMF) (99.0% pure) were purchased from Chengdu Kelong Inc. (Chengdu, China) and used as the solvents. Azo-bis-isobutryonitrile (AIBN) was purchased from Chengdu Kelong Inc., and 4,40 -azo-bis(4-cyanovaleric acid) (ACVA) was purchased from Alfa Aesar China (Tianjin, China), both of which were used as initiators. Bovine serum albumin (BSA) fraction V and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma, USA. All other chemicals (analytical grade) were obtained from Chengdu Kelong Inc., and were used without further purication.

2.2. Synthesis and characterization of poly(vinyl pyrrolidone)b-poly (methyl methacrylate)b-poly (vinyl pyrrolidone) 2.2.1. RAFT agent According to the literature [50] carbon disulde (27.4 g, 0.36 mol), chloroform (107.5 g, 0.9 mol), acetone (52.3 g, 0.9 mol), and tetrabutylammonium hydrogen sulfate (2.41 g, 7.1 mmol) should be mixed with 120 ml of mineral spirit in a 1 l jacketed reactor cooled with tap water under nitrogen. Sodium hydroxide (50%) (201.6 g, 2.52 mol) was added dropwise over 90 min to maintain the temperature below 25 C. The reaction was carried out overnight. Then 900 ml of water was added to dissolve the solid, followed by 120 ml of concentrated HCl to acidify the aqueous layer, and then the mixture was stirred for 30 min with nitrogen purging. After ltering and thoroughly rinsing with water the solid was dried to constant weight. 41.3 g of earth colored product was collected.

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Characterization. 1H NMR (DMSO-d6, p.p.m. from TMS): 1.59 (s, 12H, CH3), 12.91 (s, 2H, COOH). Fourier transform infrared (FTIR) spectrum (KBr, cm1): 1710 (s, m>C@O), 1060 (s, m>C@S). 2.2.2. Macro-RAFT agent (PVP) (see step 1 in Scheme 1) Polymerization of VP was carried out in a sealed tube. The general procedure is as follows. VP, the RAFT agent, ACVA, and H2O were added to a tube. After bubbling with nitrogen for 30 min the reaction mixture was allowed to warm to 80 C under a nitrogen atmosphere, and polymerization was carried out for 5 h. After precipitation in ethyl ether the product, PVP, was dried under vacuum at 50 C overnight. Characterization. 1H NMR (DMSO-d6, p.p.m. from TMS): 12.91 (s, 2H), 1.59 (s, 12H) for the RAFT agent terminated segment, and 3.51 (s, H, CHN), 3.13 (s, 2H, CH2N), 2.20 (s, 2H, CH2C@O), 2.04 (s, 2H, CH2CC@O), 1.86 (s, 2H, CH2CN) for PVP. FTIR spectra (KBr, cm1): 1060 (s, m>C@S) for the RAFT agent terminated segment, and 1668.2 (s, m>C@O) for PVP. 2.2.3. Amphiphilic triblock co-polymer (PVP-b-PMMA-b-PVP) (see step 2 in Scheme 1) Co-polymerization of MMA with PVP was carried out in a sealed tube. The general procedure is as follows. MMA, the macro-RAFT agent (PVP), AIBN, and DMF were added to a tube. After bubbling with nitrogen for 30 min the reaction mixture was allowed to warm to 80 C under a nitrogen atmosphere and polymerization was carried out for 5 h. After precipitation in ethyl ether the product was dried under vacuum at 50 C overnight. The product obtained was ground to a ne powder and immersed in water and THF for 1 week each. This was repeated three times, and then the product was dried under vacuum at room temperature for 24 h.

Characterization. 1H NMR (DMSO-d6, p.p.m. from TMS): 12.91 (s, 2H), 1.59 (s, 12H) for the RAFT agent terminated segment, 3.51 (s, H, CHN), 3.13 (s, 2H, CH2N), 2.20 (s, 2H, CH2C@O), 2.04 (s, 2H, CH2CC@O), 1.86 (s, 2H, CH2CN) for the PVP block, and 3.323.56 (s, 3H, CH3O), 1.76 (s, 2H, CH2CCH3), 0.730.93 (s, 2H, CH3CC@O) for the PMMA block. FTIR spectra (KBr, cm1): 1060 (s, m>C@S) for the RAFT agent terminated segment, 1668.2 (s, m>C@O) for the PVP block, and 1734.9 (s, m>C@O) for the PMMA block. 2.2.4. Characterization The FTIR spectrum of the co-polymer was measured using a FTIR Nicolet560 (Nicol America) instrument. To prepare FTIR samples the polymer was dissolved in NMP and cast on a potassium bromide (KBr) disk with the thickness of about 0.8 mm. The 1H NMR spectra were recorded on a Varian Unity Plus 300/54 NMR spectrometer using DMSO-d6 as the solvent at room temperature. The weight averaged molecular weight of PVP was measured with a multi-angle light scattering detector (BI-200SM, Brookhaven Instruments Co., Holtsville, NY) using H2O as the solvent at 25 C. 2.3. Preparation and characterization of co-polymer blended polyethersulfone membranes The PES/co-polymer membranes were prepared by a phase inversion technique. PES and the synthesized co-polymer were dissolved in the solvent DMAC by vigorous stirring until a clear homogeneous solution was obtained. The concentration of PES was 16 wt.%). In the experiments different membranes were prepared by changing the weight percentage of the co-polymer in the casting solutions. The content of co-polymer in the casting solutions was 0, 1, 3, and 5 wt.% (the maximum amount). After vacuum degassing the casting solutions were prepared as membranes by spin coating coupled with a liquidliquid phase separation technique at room temperature. The membranes were thoroughly rinsed with distilled water to remove the residual solvent. All the prepared membranes with PES/co-polymer ratios of 16/0, 16/1, 16/3, and 16/5 (wt.%) were of uniform thickness of about 60 70 lm, and were termed FSM-0, FSM-1, FSM-3 and FSM-5, respectively. Thermogravimetric analysis (TGA) (TA-500) was used to investigate the changes in thermal stability of the co-polymer and modied membranes at a heating rate of 10 C min1 under a dry N2 atmosphere. The morphologies of the membranes were observed by scanning electron microscopy (SEM) using an XL 30ESME scanning microscope. The membranes, frozen in liquid nitrogen, were broken and sputtered with a gold layer before SEM analysis. The structures and elements of the membrane surfaces were investigated by reected FTIR and X-ray photoelectron spectroscopy (XPS). The hydrophilicity of the membrane surface was characterized on the basis of contact angle measurements using a contact angle goniometer (OCA20, Dataphysics, Germany) equipped with video capture. A piece of membrane 2 2 cm was attached to a glass slide and mounted in the goniometer. For the static contact angle measurements 3 ll double distilled water was dropped onto the surface of the membrane at room temperature and the contact angle was measured after 10 s. At least eight measurements were averaged to obtain a reliable value. The measurement error was 3. 2.4. Ultraltration and anti-fouling properties of the modied polyethersulfone hollow bers 2.4.1. Preparation of the hollow ber membrane and lter PES/co-polymer hollow ber membranes were prepared using DMAC as the solvent. The PES/co-polymer solution (the contents

Scheme 1. Synthesis of the PVP-b-PMMA-b-PVP block co-polymer.

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of PES and co-polymer were 16 and 5 wt.%, respectively) was degassed to remove air bubbles and then a drywet spinning technique was used to fabricate the PES hollow ber membranes [29,51]. The membranes were immersed in water for >24 h to elute the residual DMAC, with the extraction water being changed every 3 h. Then the membranes were placed in 50 wt.% aqueous glycerol solution for 24 h to prevent collapse of the porous structures when they were dried. The resultant bers were then dried in air at room temperature. The hollow ber membrane lters were prepared by employing epoxy resin as the potting material, with an effective area of about 120 cm2. The prepared membranes with PES/copolymer ratios of 16/0 and 16/5 are termed HFM-0 and HFM-5.

2.5. Blood compatibility 2.5.1. Protein adsorption The protein adsorption experiments were carried out with BSA solution (1 mg ml1 in PBS, pH 7.4). The membrane, with an area of 1 1 cm, was incubated in PBS for 24 h and then immersed in the protein solution for 2 h at 37 C. After protein adsorption the membranes were gently rinsed with PBS and then immersed in glass tubes containing 2 wt.% aqueous sodium dodecyl sulfate (SDS) solution for 1 h at 37 C to remove the proteins adsorbed onto the membranes. The amount of protein eluted into the SDS solution was quantied with a Micro BCA protein assay reagent kit. More than 95% of the adsorbed protein could be eluted into the SDS solution. Then the amount of adsorbed BSA was calculated. 2.5.2. Platelet adhesion Healthy fresh human blood (male, 25 years old) was collected using vacuum tubes containing sodium citrate as an anti-coagulant; the concentration of sodium citrate was 3.8 wt.%, the ratio of anticoagulant to blood was 10:90 vol.%. The blood was centrifuged at 1500 r.p.m. for 15 min to obtain platelet-rich plasma (PRP) or at 4000 r.p.m. for 15 min to obtain platelet-poor plasma (PPP). The PES and modied PES membranes were immersed in PBS and equilibrated at 37 C for 1 h. The PBS was removed and then 1 ml of fresh PRP was introduced. The membranes were incubated with PRP at 37 C for 2 h. Then the PRP was decanted off and the membranes were rinsed three times with PBS. Finally, the membranes were treated with 2.5 wt.% glutaraldehyde in PBS at 4 C for 1 day. The samples were washed with PBS, subjected to a drying process by passing them through a series of graded alcoholPBS solutions (25%, 50%, 75%, and 100%) and isoamyl acetatealcohol solutions (25%, 50%, 75%, and 100%). Platelet adhesion was observed using an S-2500C microscope (Hitachi, Japan). The number of adherent platelets on the membranes was calculated from ve SEM pictures at 500 magnication from different places on the same membrane. 2.5.3. Clotting time To evaluate the anti-thrombogenicity of the membranes the activated partial thromboplastin time (APTT) was measured with an automated blood coagulation analyzer CA-50 (Sysmex Corp., Kobe, Japan). To measure APTT samples (1 1 cm, one piece) were incubated with 0.15 ml of healthy human blood plasma at 37 C for 30 min and then the APTT was measured. 2.6. Cytocompatibility 2.6.1. Cell culture Human embryonic hepatocytes (LO-2) were grown in R1640 medium supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), 2 mM L-glutamine and 1 vol.% antibiotics mixture (10,000 U penicillin and 10 mg streptomycin). Cultures were maintained in a humidied atmosphere of 5% CO2 at 37 C (Queue Incubator, Paris, France). Conuent cells were detached from the culture ask with sterilized PBS and 0.05% trypsin/EDTA solution. The culture medium was changed every day. The PES and modied PES membranes were cut into 1 1 cm to t 24-well cell culture polystyrene plates and pre-wetted by immersion in culture medium for 3 h in a 37 C incubator. Then the membranes were placed in the cell culture plates, rinsed with PBS and sterilized by c-irradiation. 2.6.2. Cell morphology on the membranes For SEM observation hepatocytes were seeded onto the membranes at a density of approximately 2.5 104 cells cm2. After

2.4.2. Ultraltration of the PEG solution The permeability of the hollow ber membranes was investigated using polyethylene glycol (PEG-10000). The feed solutions were prepared by dissolving PEG-10000 in double distilled water at a concentration of 0.1 g l1. The PEG-10000 solution was applied to the membrane using the apparatus described in our earlier study [52]. The permeant solution was collected and the ux calculated using Eq. (1).

Fluxml=m mmHg h1 V =StP

where V is the volume of the permeant solution, S is the effective membrane area, t is the time of solution collection, and P is the pressure applied to the hollow ber membrane. The concentration of PEG was determined using a UVVIS spectrophotometer (model 756, Shanghai Spectrophotometer Instrument Co., Shanghai, China) at a wavelength of 510 nm after reaction with Ninhydin. The observed sieving coefcient (S0) of PEG was dened as follows:

S0 C p =C b

where Cp is the permeant concentration of PEG and Cb is the bulk concentration.

2.4.3. Ultraltration of the protein solution Ultraltration of bovine serum albumin (BSA) solution was carried out to study the anti-fouling properties of the modied PES hollow ber membranes. BSA solution was prepared by dissolving BSA in phosphate-buffered saline (PBS), adjusted to pH 7.4 with hydrochloric acid, at a concentration of 1.0 mg ml1. The hollow bers under test were pre-compacted at an internal pressure of 135 mm Hg and external pressure of 100 mm Hg for 30 min under PBS ow to obtain a steady state, then the internal pressure was reduced to 70 mm Hg and the external pressure was reduced to 40 mm Hg, and the PBS ux was measured. After 30 min ltration the feed solution was switched to 1.0 mg ml1 BSA solution (pH 7.4) and the ux again measured. Finally, the lter and the solution reservoir were fully emptied and relled with deionized water. The hollow ber membranes were washed with deionized water for 30 min. Then the PBS solution ux was again measured. The uxes of PBS and BSA solution through the hollow bers were calculated using Eq. (1). In order to evaluate the fouling-resistance ability of the membranes the ux recovery ratio (FRR) was calculated using the expression:

F RR % F 2 =F 1 100

where F1 and F2 (ml/(m2 mmHg h)1) are the PBS solution ux before and after the protein ultraltration experiment, respectively.

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4 days the seeded membranes were rinsed with PBS and xed with 2.5 wt.% glutaraldehyde in PBS at 4 C for 12 h. For morphology observation the xed samples were subjected to a drying process by passing them through a series of graded alcoholPBS solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%, 15 min each) and then dehydrated through isoamyl acetate. Critical point drying of the specimens was carried out with liquid CO2. The specimens were sputter coated with a gold layer and examined in an S-2500C microscope (Hitachi, Japan).

2.6.3. MTT assay After cell culture for 2, 4, and 7 days viability of the hepatocytes was determined by MTT assay. The hepatocytes were seeded onto the membranes at a density of approximately 2.5 104 cells cm2. Cells cultured in wells without membranes served as controls in this study. After predetermined time intervals 45 ll of MTT solution (1 mg ml1 in the test medium) was added to each well and incubated for 4 h at 37 C. Mitochodrial dehydrogenases of viable cells selectively cleave the tetrazolium ring, yielding blue/purple formazan crystals. Then 400 ll of ethanol was added to dissolve the formazan crystals. Thus the quantity of formazan dissolved in the ethanol reects the level of cell metabolism. The solution was shaken homogeneously for about 15 min. The sample solutions were aspirated into microtiter plates and the optical density read in a Microplate reader (model 550, Bio-Rad) at 492 nm. All experiments were repeated three times, and the results are expressed as means SD. The statistical signicance was assessed by Students t-test, with the level of signicance set at P < 0.05.

which was repeated three times alternately. The remaining white powder was the triblock co-polymer of PVP-b-PMMA-b-PVP. The structure of the puried PVP-b-PMMA-b-PVP triblock copolymer was demonstrated by FTIR and 1H NMR, and is specied in Section 2.2.3. The co-polymer compositions and molecular weights, determined from the 1H NMR spectra and multi-angle light scattering, are summarized in Table 1. The PVP content (wt.%) estimated from 1H NMR spectra and the amount added before polymerization were almost identical. On the basis of the ratio of the signal intensities of the peaks at d 1.00.51 p.p.m. to those at d 3.252.91 p.p.m. the degrees of polymerization of the PVP and PMMA blocks were about 62 and 285. In addition, according to a previous study, it is easy to control the molecular weights and compositions of the co-polymers by changing the molecular weight of PVP or the ratios of MMA to PVP, and thus to design different well-dened triblock co-polymers for biomedical applications [54].

3.2. Membrane preparation and characterization PVP-b-PMMA-b-PVP modied PES membranes were prepared by a liquidliquid phase separation technique at room temperature with 16% PES and 05% block co-polymer. In order to evaluate the composition of the modied membrane the thermal degradation of the co-polymer PVP-b-PMMA-b-PVP and PES/PVP-b-PMMA-b-PVP (16/5) membranes was determined by TGA. As shown in Fig. 1A, the modied PVP-b-PMMA-b-PVP/PES membrane had two obvious weight loss peaks, below about 450.3 C, where the co-polymer degraded, and above about 450.3 C, where the PES matrix degraded. From the relative weight losses it could be calculated that the ratio of the co-polymer to PES was about 1:3.8, which was close to the feed ratio of 5:16. These studies provide evidence that the modifying additive PVP-b-PMMA-b-PVP was incorporated into the PES membrane, with almost no elution during the membrane preparation and processing procedures. Fig. 1B shows cross-sectional SEM micrographs of the PES and PES/PVP-b-PMMA-b-PVP membranes. The characteristic morphology of asymmetric membranes, consisting of a dense top layer and porous bottom layer with a nger-like structure, was observed for all membranes [55,56]. However, the porous bottom layer for the PES/PVP-b-PMMA-b-PVP membrane was more established and obvious than that of the PES membrane. For the modied membrane there were large numbers of micropores on the surface of the macrovoids, and a few self-assembled microspheres were observed, embedded in the pores. The SEM images suggest that the structure of the modied membrane had been altered by the block co-polymer additive. To understand how the block co-polymer is distributed inside the PES membrane and why the structure is formed during the phase separation process the mechanism of formation can be summarized as follows. The initial solution, including PES, the copolymer, and DMAC, formed a homogeneous phase, with the co-polymer macromolecules homogeneously dissolved in the solution. Because of the amphiphilic nature of the block co-polymer [57,58], when phase separation began PVP-b-PMMA-b-PVP rose

3. Results and discussion 3.1. Synthesis and characterization of poly(vinyl pyrrolidone)bpoly(methyl methacrylate)b-poly(vinyl pyrrolidone) amphiphilic triblock co-polymer The schematic procedure for the synthesis of PVP-b-PMMA-bPVP is shown in Scheme 1. As shown in the scheme, the copolymer retained the carboxyl-terminated structure of the RAFT agent, which helps to improve, or at least not decrease, the amphiphilicity of the co-polymer compared with other RAFT agents [53]. For the facile polymerization of VP trithiocarbonate was selected as the RAFT agent, and the PVP synthesized was further used as a macro-RAFT agent for the co-polymerization of MMA with PVP. The chain transfer reaction between the propagation radical and the macro-RAFT agent happens randomly and identically. That is to say the MMA or PMMA propagation chains insert not only in the left side between the PVP (M1) and the S atom but also in the right side between the PVP (M1) and the S atom, resulting in ABBA or ABA block co-polymers rarely containing fractions of diblock species. Meanwhile, it is expected that more PMMA homopolymer will be produced than PVP homopolymer. To extract the homopolymers of PVP and PMMA which are probably produced by bi-radical termination the prepared product was ground to a ne powder and immersed in water and THF for 1 week each,

Table 1 Composition and molecular weight data of the copolymer.a Feed ratio (PVP wt.%) 25
a b c d

Calculated (NMR) (PVP wt.%)b 19.5

Mw 6.9

PVP

(103)

Mn

copolymer

(103)

35.4

The polymerization was conducted at 80 C for 12 h using DMF as the solvent. Estimated from the 1H-NMR Spectrum of the copolymer. Weight-average molecular weight measured by multi angle light scattering detector using H2O as solvent at 25 C. Molecular weight calculated by comparing the features peaks of PMMA methyl (1.000.51 ppm) and PVP methylene (3.252.91 ppm) from 1H-NMR spectrum.

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Fig. 1. (A) The thermal degradation arcuation of PVP-b-PMMA-b-PVP block co-polymer and the co-polymer modied PES membrane by TGA. (B) SEM images of crosssectional views of the FSM-0, FSM-1, FSM-3, and FSM-5 membranes. (C) Schematic formation mechanism of the as-prepared block co-polymer modied membrane: (a) before phase separation; (b) after phase separation.

to the surface of the membrane and assembled with the hydrophilic VP block directed to the surface and the hydrophobic MMA block directed into the membrane, as shown in Fig. 1C. Thus a PVP brush layer formed on the surface of the membrane, including the membrane top surface and the pore surfaces. In addition, with increasing block co-polymer content the concentration of the copolymer on the surface of the blended membrane increased. In these circumstance the co-polymer partially forms aggregates or micelles of spheres during phase separation [59,60]. In fact, polymer miscibility, surface segregation, and surface composition are important aspects for successful implementation of the surface strategy. The binary matrix composed of PMMA blocks and PES, based on the principle of similar solubility parameters (dPMMA = 22.7, dPES = 21.9), results in good dispersion [61,62]. PVP could be directly blended with PES to prepare membranes and the adoption of PVP as a hydrophilic membrane block could provide a miscible polymer system during membrane formation due to the strong donor/acceptor interaction between the O@CN functional groups of the PVP block and the O@S@O and/or benzene ring of the PES polymer [63,64]. Surface segregation will be inuenced by the relative surface energies of the two blocks and the PES substrate. To determine the surface energies of PVP and PVP-b-PMMAb-PVP/PES the static contact angles of water, diiodomethane and ethylene glycol on each substrate were rst measured and then the energies were calculated using the van OssChaudhuryGood

Table 2 Contact angles and surface energies for the membranes. Substrate Surface energy (mJ/m )
a 2

PVPa 48.8

PMMAb 41.1

PESb 46

FSM-5c 47.3

Evaporated lm was used and surface energy was calculated with the van Oss ChaudhuryGood method. b Literature values. c Phase inversion membrane was used and surface energy was calculated with the van OssChaudhuryGood method.

1/2 method: cS = cSLW + 2c , which leads to a Youngs equation s cs 1/2 1/2 of the form: cL (1 + cosh) = 2[(cSLW cLLW)1/2 + c + c ] s cL S cL (Table 2) [65]. As shown in Table 2, PMMA and PES lms have similar surface energies, which are lower than that of PVP. For the blended membranes the polymers with higher surface energy are usually expected to be buried below the lower surface energy PMMA block and PES substrate [6669]. The afore-mentioned data and analysis reveal that the PVP blocks, PMMA blocks and PES substrate could provide a miscible polymer system during membrane formation with PVP dispersed in the PES matrix. However, to impart biocompatibility to the PES membranes it is important that the hydrophilic block (PVP) be present at the surface of the membrane (and the surface of the membrane pores). Special techniques are generally required to drag high surface energy blocks to the surface of a coating [70]. In this paper a liquid liquid phase separation method was used [55,56]. During phase separation hydrophilicity was the greatest driving force for migration and self-assembility of the block co-polymer. Thus, phase separation occurs at the interface between PES and water in seconds, which may be the underlying reason for orientation of the block co-polymer with the PVP block present at the surface and the PMMA block embedded in the substrate. There may be a high copolymer concentration and a PVP brush layer formed on the surface of the membrane, conrmed by FTIR and XPS analysis. Fig. 2A shows the FTIR spectra of the membrane surfaces for PES and PES/PVP-b-PMMA-b-PVP membranes. It can be seen that the most signicant changes were the appearance of the peaks at 2960, 1734.8 and 1668.2 cm1, which are the characteristic peaks of the block co-polymer PVP-b-PMMA-b-PVP, as mentioned above. Fig. 2B presents the XPS spectrum of the PES/PVP-b-PMMA-b-PVP membrane surface at an X-ray incidence angle of 85 (X-ray incidence depth 9 ). The elemental content of N in the blended membrane surface was 5.29%, which is close to the N content of PVP. Angle-resolved XPS data (Table 3) shows that the S concentration (at.%) increases and N concentration decreases with increasing XPS probe depth. Thus examination of the surfaces by contact

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Fig. 2. (A) FTIR spectra for the PES, PVP-b-PMMA-b-PVP and PES/PVP-b-PMMA-b-PVP membranes. (B) XPS spectrum of the PES/PVP-b-PMMA-b-PVP membrane surface. (C) Static contact angles for the membranes (n = 8).

Table 3 Angle-resolved XPS results for the modied membrane surfaces. Depth (k) 9 21 57 80 C (%) 48.75 60.59 60.89 62.62 O (%) 45.89 34.84 34.83 33.41 N (%) 5.29 4.24 3.75 3.39 S (%) 0.07 0.33 0.53 0.58

angle measurement and XPS revealed that the PVP blocks migrate to the surface of the membrane, and that there is a composition gradient at the surface of the membrane. These results are proof that the co-polymer moved to the surface of the membrane and self-assembled during preparation of the blended membranes. That there was an abundance of the aggregated and self-assembled amphiphilic co-polymer and a PVP brush layer on the surface of the membrane may improve many properties of the membrane, especially the hydrophilicity. Contact angle is a convenient way to assess the hydrophilic/hydrophobic properties of membrane surfaces [71]. Fig. 2C shows the contact angles of the prepared PES and PVP-b-PMMA-b-PVP blended membranes. As shown in Fig. 2C, when the co-polymer was blended into the membranes the water contact angle decreased signicantly. The PES membrane possessed the highest contact angle of about 73, corresponding to the lowest surface hydrophilicity. The values gradually decreased to within the range 6065 with increasing co-polymer content in the blended membranes. 3.3. Blood compatibility 3.3.1. Protein adsorption Protein adsorption on the material surface is a common phenomenon during thrombogenisis. The amount of protein adsorbed on the membrane is reported to be one of the important factors in

evaluating the blood compatibility of materials [7]. It is well known that hydrophobic interactions between the material surface and protein plays an important role in the non-selective adsorption of protein. Materials possessing a hydrophilic surface normally show relatively low non-selective adsorption of proteins and cells [72]. In the present work the surfaces of the membranes were studied in relation to the adsorption of BSA in vitro, as shown in Fig. 3A. It was found that all the PES/PVP-b-PMMA-b-PVP membranes had lower BSA adsorption than the PES membranes, and the values decreased with increasing block co-polymer content in the membranes. In a word, the hydrophilicity of the PES/PVP-bPMMA-b-PVP membranes was improved and, accordingly, BSA adsorption was decreased. 3.3.2. Platelet adhesion The adhesion of platelets to blood-contacting medical devices is a key event in thrombus formation on material surfaces. In this study platelet adhesion on the membrane surface was investigated. Both spreading of and pseudopodium formation by the platelets were obviously suppressed, which indicated lower platelet activation by and improved blood compatibility of the block copolymer modied membranes. Fig. 3B shows typical scanning electron micrographs of platelets adhering to the PES and PVP-b-PMMA-b-PVP blended PES membranes. Comparing the gures at the same magnication it can be observed that numerous platelets aggregated and accumulated on the PES membrane surface. These platelets had a attened and irregular shape. However, on the blended membranes few platelets were observed and the platelets had a rounded morphology with almost no pseudopodium formation. Fig. 3B(h) shows the number of adherent platelets on the PES and block co-polymer modied membranes after immersion in platelet-rich plasma. The blended membranes showed a much lower number of adherent platelets on the surface compared with the PES membranes. This should

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Fig. 3. (A) Protein adsorption by the modied membranes. (B) SEM images of platelets adhering to the membranes (h, number of the adherent platelets on the membranes adsorbed from platelet-rich plasma estimated from the SEM pictures). (C) Activated partial thromboplastin time (APTT) for the modied PES membranes.

be attributed to the difference in the PVP brush layer on the surface of the modied membranes. 3.3.3. Clotting time To further study the blood compatibility of the modied membranes the APTT was measured, as shown in Fig. 3C. The APTT test determines the bioactivity of intrinsic blood coagulation factors. It was found that the APTT of the block co-polymer blended membranes increased compared with the PES membrane, and with increasing block co-polymer content the APTT of the modied membranes increased, being nearly twice that of the PES membrane at 5% co-polymer. The enhancement in anticoagulant activity might have resulted from partial contributions of surface hydrophilicity, lower protein adsorption, and depressed platelet adhesion and activation. Compared with others methods to improve the blood compatibility of bulk materials, such as grafting heparin and insulin onto poly(ethylene terephthalate) lms [73] or grafting poly(ethylene glycol) onto silicon and glass surfaces [74], the enhancement of APTT after grafting was lower than that of the block co-polymer modied membranes produced in this study. However, Lin et al. [75] reported that the enhancement of APTT increased sharply with increasing graft density of heparin on a polyacrylonitrile surface. Nevertheless, grafting, in contrast to blending, is a complex method to modify bulk materials, especially hollow bers, and is hard to use in an industrial setting. Recently Wang et al. [19] reported that blending PVP as an additive in a PES membrane improved the blood compatibility, and the APTT increased signicantly. However, the elution of PVP was unavoidable. On the other hand, compared with others studies [75], almost no adherent platelets were observed when 5% co-polymer was blended in, as shown in Fig. 3B(h). Thus, this is a simple and effective way to improve the blood compatibility of the matrix. As discussed above, the modied PES membrane showed good blood compatibility. As we know, most of the membranes used

in blood purication are hollow ber type membranes. To provide information on real application of the modied PES membranes in blood purication co-polymer modied hollow ber PES membranes were prepared, the permeability and protein anti-fouling properties of which are discussed below. 3.4. Ultraltration and antifouling properties of modied PES hollow bers 3.4.1. Preparation and characterization of the hollow ber membrane The block co-polymer modied PES hollow ber membrane was spun by a drywet spinning method. SEM images of cross-sectional views of the hollow ber membranes are shown in Fig. 4A. The prepared blended membranes (FSM-5) were opaque in appearance and satisfactorily strong for high pressure applications. The wall thickness of the hollow ber was about 110 lm, and the inner diameter was about 400 lm. A skin layer was found on both sides of the membrane wall, under which there was a nger-like structure layer, and then a porous layer. Furthermore, it was clearly observed that the nger-like structure was in the middle of the membrane. This was caused by the exchange between NMP and water during membrane formation. There were no obvious differences between HFM-0 and HFM-5 except for the greater porosity of the modied membrane. This indicated that small amounts of the co-polymer did not inuence the main structure of the PES membrane, and the co-polymer was well incorporated into the hollow ber membrane. A schematic of the mechanism of formation of the modied membrane (Fig. 1C) also indicated that PVP brushes could be formed on the surface of the modied hollow ber membranes. 3.4.2. Permeability to PEG solution For end-stage renal disease patients hemodialysis is widely used as a life sustaining treatment. However, it is difcult to remove middle molecular weight toxins (such as b2-microglobulin

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shown in Fig. 4B, the ux recovery ratio of the modied hollow bers increased from 50.6% (PES hollow bers) to 96.6% due to blending with the amphiphilic block co-polymer. The ux decreased, but not dramatically, in the initial stage of BSA solution ultraltration due to membrane fouling caused by the deposition and adsorption of protein molecules on the membrane surface and in the membrane pores. When the adsorption of protein molecules became saturated a relatively steady ux was reached during the nal stage of BSA solution ultraltration. After 30 min protein ultraltration the membranes were cleaned with owing double distilled water; PBS solution ux of the membranes recovered to some extent. The modied PES membranes showed a large ux recovery ratio of nearly 100%, which could be explained by the fact that VP chains increase the hydrophilicity of hollow ber membranes, passivity the membrane surface, and lower protein adsorption [79,80]. Recently Zhao et al. [81] investigated the anti-fouling properties of a poly(ethylene glycol)-based diblock co-polymer modied PES hollow ber; the ux recovery ratio increased from 56.3% to 86.5% after blending in the diblock co-polymer. However, it was lower compared with the ux recovery ratio of the PVP-based triblock co-polymer modied PES hollow ber (96.6%) prepared in this study, which might be attributed to the PVP brush layer that formed on the surface of the membrane, as discussed above. 3.5. Cytocompatibility It is well known that hollow ber membranes have been widely used in blood purication therapies, including plasmapheresis, hemodialysis, hemodialtration, and bioarticial liver support [82]. The efciency of hollow ber-based bioarticial livers depends greatly on the choice of membrane in terms of permeability, cut-off and biocompatibility [83]. Thus the cytocompatibility of the modied PES membranes was investigated. In this study LO2 human hepatocytes were selected to evaluate the cytocompatibility of the modied membranes, which could possibly be used for bioarticial liver support. 3.5.1. Cell morphology Generally speaking cells will undergo morphological changes to stabilize the cellmaterial interface after contacting a biomaterial. The whole process of adhesion and spreading consists of cell attachment, lopodial growth, cytoplasmic webbing, attening of the cell mass and rufing of the peripheral cytoplasm progressing in a sequential fashion [84]. Fig. 5A shows the morphology of hepatocytes cultured for 4 days on the PES and modied PES membranes. It can be seen that the hepatocytes extended pseudopodia to adhere onto the materials. The number of hepatocytes on the PES membrane was least. With increasing co-polymer content the number of adherent hepatocytes increased. On the surfaces of the modied membranes the cells spread, with rufing of the peripheral cytoplasm, and almost covered the whole surface, and had been become attened with a larger area of attachment compared with cells cultured on the PES membrane, especially on FSM-5, which indicated that addition of the co-polymer could promote better cell attachment and growth. Furthermore, spheroids of hepatocytes, formed by the rearrangement and compaction of cell aggregates, have been advocated as a highly useful culture mode for hepatocytes instead of the traditional monolayer culture, since their tissue-like structure could promote cell proliferation and differentiation and maintain higher level liver-specic functions over a long period [85,86]. Thus spheroids of hepatocytes are ideal for bioarticial liver support. From Fig. 5A it can be observed that there were some spheres or balling, especially on FSM-3 and FSM-5, in some areas. This indicates that the block co-polymers could induce hepatocytes to

Fig. 4. (A) SEM images of cross-sectional views of the PES hollow ber membranes. (B) Time-dependent uxes of the modied membranes during the ultraltration process for the PES and modied PES membranes: (a) HFM-5; (b) HFM-0. Distilled water and protein solution were used alternately in ultraltration (n = 3).

(b2-M)) during conventional hemodialysis. b2-M, which is considered one of the middle molecular weight toxins for end-stage renal disease patients [76], is a non-glycosylated protein with a molecular weight of 11,800 Da. Free b2-M is found in the body uids as a result of shedding from cell surfaces or intracellular release [77,78]. Generally the content of b2-M in the body of end-stage renal disease patients is higher than that in a healthy one. Thus it is necessary to remove b2-M during hemodialysis. Now, most high ux hemodialysis membranes have been designed to remove b2-M during hemodialysis. To investigate whether the modied hollow ber membrane could effectively remove b2-M the observed sieving coefcient (So) for polyethylene glycol (PEG) was evaluated. PEG-10000 was selected in order to simulate the molecule weight of b2-M. PEG has been widely used as a standard macromolecule in ultraltration experiments to determine the sieving coefcient. The observed sieving coefcients for PEG-10000 of the PES and modied hollow ber membranes were 28.64% and 38.38%, respectively. So for the modied PES hollow ber membrane increased after blending with the co-polymer. The results also suggest that the modied hollow ber membranes could effectively remove b2-M, and can be used as high ux hemodialysis membranes. 3.4.3. Membrane anti-fouling properties Ultraltration experiments were also carried out to study the protein anti-fouling properties of the blended hollow ber membranes to provide further information as to practical applications. Fig. 4B shows the time-dependent ux during ultraltration. As

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Fig. 5. (A) SEM images of LO2 human liver cells cultured on the PES and PES modied membranes after 4 days. (B) MTT tetrazolium assay. Formazan absorbance expressed as a function of time from hepatocytes seeded onto different membranes and the controls. Values are expressed as means SD (n = 3). P < 0.05, P < 0.05 and P < 0.05 compared with the values for the FSM-0 membrane at 2, 4 and 7 days, respectively; #P < 0.05 between days for the same sample.

agglomerate and form spherical multicellular aggregates (spheroids), and the extent of aggregation increased with increasing copolymer content. 3.5.2. MTT assay Fig. 5B shows the MTT data for the control sample and the membranes. The formazan absorbance indicates that the hepatocytes seeded onto the control sample (the polystyrene cell culture plate) and different membranes were able to convert MTT into a blue formazan product. It was observed that on days 2, 4, and 7 the viability of the cells on all the modied membranes was increased compared with the PES membrane (P < 0.05). Moreover, the FSM-5 membrane exhibited the best cytocompatibility among all the membranes. It

was found that with increasing culture time the viability of cells on each membrane increased (P < 0.05), as shown in Fig. 5B. Furthermore, the viability of cells on the modied membranes cultured for 7 days was nearly the same as that of the cells on the control sample, especially for FSM-5 (P < 0.05). Therefore, it can be concluded that addition of the co-polymer to PES membranes could improve the cytocompatibility, and the modied membranes have the potential to be used as bioarticial liver supports. 4. Conclusion A good approach to modify polymeric membranes with excellent biocompatibility has been provided by blending them with a

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F. Ran et al. / Acta Biomaterialia 7 (2011) 33703381 [12] Dattatray S, Ellen W, Fisher R. Hydrophilic modication of polyethersulfone membranes by low temperature plasma-induced graft polymerization. J Membr Sci 2002;209(1):25569. [13] Bernacca GM, Gulbransen MJ, Wilkinson R, Wheatley DJ. In vitro blood compatibility of surface-modied polyurethanes. Biomaterials 1998;19(13):115165. [14] Yuan J, Zhang J, Zang XP, Shen J, Lin S. Improvement of blood compatibility on cellulose membrane surface by grafting betaines. Colloid Surfaces B Biointerf 2003;30(1/2):14755. [15] Su BH, Fu P, Li Q, Tao Y, Li Z, Zao HS, et al. Evaluation of polyethersulfone highux hemodialysis membrane in vitro and in vivo. J Mater Sci Mater Med 2008;19(2):74551. [16] Wang YQ, Wang T, Su YL, Peng FB, Wu H, Jiang ZY. Protein-adsorptionresistance and permeation property of polyethersulfone and soybean phosphatidylcholine blend ultraltration membranes. J Membr Sci 2006;270(1/2):10814. [17] Liu ZB, Deng XP, Zhao CS. BSA hybrid synthesized polymer. Chin Chem Lett 2006;17(11):151922. [18] Liu ZB, Deng XP, Wang M, Chen JX, Zhang AM, Gu ZW, et al. BSA-modied polyethersulfone membrane: preparation, characterization and biocompatibility. J Biomat Sci 2009;20(3):37797. [19] Wang HT, Yu T, Zhao CY, Du QY. Improvement of hydrophilicity and blood compatibility on polyethersulfone membrane by adding polyvinylpyrrolidone. Fiber Polym 2009;10(1):15. [20] Henry M, Bertrand P. Surface composition of insulin and albumin adsorbed on polymer substrates as revealed by multivariate analysis of ToF-SIMS data. Surf Interface Anal 2009;41(3):10513. [21] Zhang LF, Liang Y, Meng LZ, Wang C. Characterization of complexation of PVP copolymer with DNA. Polym Adv Technol 2009;20(4):4105. [22] Haaf F, Sanner A, Straub F. Polymers of N-vinylpyrrolidone: synthesis, characterization and uses. Polym J 1985;17(1):14352. [23] George KA, Wentrup-Byrne E, Hill DJT, Whittaker AK. Investigation into the diffusion of water into HEMA-co-MOEP hydrogels. Biomacromolecules 2004;5(4):11949. [24] Barzin J, Feng C, Khulbe KC, Matsuura T, Madaeni SS, Mirzadeh H. Characterization of polyethersulfone hemodialysis membrane by ultraltration and atomic force microscopy. J Membr Sci 2004;237(1/2): 7785. [25] Mosqueda-Jimenez DB, Narbaitz RM, Matsuura T. Effects of preparation conditions on the surface modication and performance of polyethersulfone ultraltration membranes. J Appl Polym Sci 2006;99(6):297888. [26] Wan LS, Xu ZK, Wang ZG. Leaching of PVP from polyacrylonitrile/PVP blending membranes: a comparative study of asymmetric and dense membranes. J Polym Sci Part B Polym Phys 2006;44(10):14908. [27] Sun MP, Su YL, Mu CX, Jiang ZY. Improved antifouling property of PES ultraltration membranes using additive of silica-PVP nanocomposite. Ind Eng Chem Res 2010;49(2):7906. [28] Li LL, Yin ZH, Li FL, Xiang T, Chen Y, Zhao CS. 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novel amphiphilic triblock co-polymer PVP-b-PMMA-b-PVP, which was synthesized via RAFT polymerization. The block co-polymer can be directly blended with PES to prepare PES at sheet and hollow ber membranes with a PVP block brush layer on their surface. The blended membranes have better blood compatibility (BSA adsorption, platelet adhesion, and blood coagulation time) compared with the PES membrane due to modication on blending with the amphiliphilic block co-polymer. The modied membranes also showed good ultraltration and protein anti-fouling properties. Furthermore, the cytocompatibility of the modied membranes was improved. These results indicate that the modied membranes have the potential to be used in blood purication, including hemodialysis and bioarticial liver support, and the study has provided useful information for practical application of the membranes. Acknowledgements This work was nancially sponsored by the National Natural Science Foundation of China (grants nos. 50973070, 51073105 and 30900691) and Sichuan Youth Science and Technology Foundation (08ZQ026-038). We would also like to thank our laboratory members for their generous help, and gratefully acknowledge the help of Ms H. Wang, of the Analytical and Testing Center at Sichuan University, for the SEM, and Ms Liang, of the Department of Nephrology at West China Hospital, for the human fresh blood collection.

Appendix A. Figures with essential colour discrimination Certain gures in this article, particularly Figure 4, is difcult to interpret in black and white. The full colour image can be found in the on-line version, at doi:10.1016/j.actbio.2011.05.026. References
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