Surface modification of polyethersulfone membranes by blending triblock copolymers of methoxyl poly(ethylene glycol)–polyurethane–methoxyl poly(ethylene glycol)

Colloids and Surfaces B: Biointerfaces 88 (2011) 315–324

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Surface modification of polyethersulfone membranes by blending triblock copolymers of methoxyl poly(ethylene glycol)–polyurethane–methoxyl poly(ethylene glycol) Jingyun Huang a , Jimin Xue a , Kewei Xiang a , Xu Zhang a , Chong Cheng a , Shudong Sun a , Changsheng Zhao a,b,∗ a b

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

a r t i c l e

i n f o

Article history: Received 7 April 2011 Received in revised form 28 June 2011 Accepted 4 July 2011 Available online 13 July 2011 Keywords: Surface modification Polyethersulfone membrane mPEG-PU-mPEG copolymer Anti-protein-fouling Platelet adhesion Cytocompatibility

a b s t r a c t The surface of polyethersulfone (PES) membrane was modified by blending triblock copolymers of methoxyl poly(ethylene glycol)–polyurethane–methoxyl poly(ethylene glycol) (mPEG-PU-mPEG), which were synthesized through solution polymerization with mPEG Mns of 500 and 2000, respectively. The PES and PES/mPEG-PU-mPEG blended membranes were prepared through spin coating coupled with liquid–liquid phase separation. FTIR and 1 H NMR analysis confirmed that the triblock copolymers were successfully synthesized. The functional groups and morphologies of the membranes were studied by ATR-FTIR and SEM, respectively. It was found that the triblock copolymers were blended into PES membranes successfully, and the morphologies of the blended membranes were somewhat different from PES membrane. The water contact angles and platelet adhesion were decreased after blending mPEG-PU-mPEG into PES membranes. Meanwhile, the activated partial thromboplastin time (APTT) for the blended membranes increased. The anti-protein-fouling property and permeation property of the blended membranes improved obviously. SEM observation and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) assay proved the surfaces of the blended membranes promoted human hepatocytes adhesion and proliferation better than PES membrane. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Due to the excellent mechanical strength, thermal tolerance, chemical resistance and membrane forming properties, polyethersulfone (PES) membranes have been extensively applied in many fields. However, because of its intrinsic poor hydrophilic property, when contacting with blood, proteins will rapidly adsorb onto the surface of PES membrane and the adsorbed protein layer may lead to further undesirable results, such as platelet adhesion, aggregation and coagulation, which restrict the application of PES in blood-contacting environment. Therefore, it is necessary to modify PES membrane surface to improve its hydrophilicity and biocompatibility [1]. Blending technology is an effective way to obtain new materials with optimized properties without complicated synthesis process. Many efforts have been made to improve the hydrophilicity and biocompatibility of PES membrane by blending with other polymers directly [2–5]. PEG has been extensively investigated for

∗ Corresponding author. Tel.: +86 28 85400453; fax: +86 28 85405402. E-mail address: [email protected] (C.S. Zhao). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.07.008

biomedical applications, and the immobilization of PEG onto membrane surface has been known to decrease protein adsorption and platelet adhesion [6–10]. However, if PEG was blended into PES casting solution directly, the elution was unavoidable for its watersoluble nature when preparing membranes. The same problem also exists in other water-soluble additives. In order to avoid the elution and improve the compatibility of hydrophilic segments with PES, amphiphilic copolymers were synthesized recently and blended with PES to prepare membranes [11–13]. The hydrophobic segments in amphiphilic copolymers had good compatibility with PES, and acted as anchors in the matrix to prevent the hydrophilic branches from being eluted. Zhu et al. [12] synthesized amphiphilic graft copolymers poly(styrene-alt-maleic anhydride) (SMA) backbones and mPEG grafts, and then blended into PES membranes. The SMA-g-mPEG could be well preserved in the membrane nearsurface and not lost during membrane washing due to their high molecular weight and comb-like architecture. Wang et al. [13] prepared amphiphilic Pluronic polymers with different contents and poly(ethylene oxide) (PEO) chain lengths and blended with PES for improving fouling-resistant ability. By increasing Pluronic content or PEO chain length, the fouling-resistant ability of the blended membranes was improved obviously.

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Therefore, in this study, a copolymer of PU and PEG was considered. PU has been reported to be used in blood-contacting application due to its excellent mechanical properties and relatively good blood compatibility [14–16]. Moreover, the PEG can be blocked with PU chain successfully, and the hydrophobicity of PU will ensure PEG not to be eluted from the blended PES membranes. In the present study, a simple method was designed to synthesize novel triblock copolymers of mPEG-PU-mPEG and blended with PES membranes. The morphologies and contact angles of the membranes were investigated. The water flux, protein antifouling property, and flux recovery ratio of the membranes were also extensively investigated. The blood compatibility (platelet adhesion and APTT) of the membranes was examined. The cytocompatibility of the membranes was also investigated to estimate whether the modified membranes had potential to be used in biomedical fields. 2. Experimental 2.1. Materials and reagents All the reagents used in the study were of reagent grade and obtained from Chengdu Kelong Chemical Reagent, China unless otherwise described. 4,4-Diphenylmethane diisocyanate (MDI), 1,4-butanediol (BDO), methoxyl poly(ethylene glycol) (mPEG, Mns: 500 and 2000, Sigma–Aldrich, USA) and N,N-dimethylacetamide (DMAC, as the solvent), which were distilled before use, were used to synthesize mPEG-PU-mPEG. Polyethersulfone (PES, Ultrason E6020P, BASF, Germany) was used to prepare membranes. Bovine serum albumin (BSA, fraction V) and 3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma, USA. Micro BCATM Protein Assay Reagent kits were purchased from Pierce Biotechnology, USA. Sodium dodecylsulfate (SDS) was obtained from Rong Hai Chemical Ltd (P.R. China). Double distilled water was used throughout the study. Healthy human fresh blood (man, 25 years old) was collected using vacuum tubes, containing acid-citrate-dextrose (ACD) as anticoagulant (anticoagulant to blood ratio, 1:9). The blood was centrifuged at 1500 rpm for 15 min to obtain platelet-rich plasma (PRP) and at 4000 rpm for 15 min to obtain platelet-poor plasma (PPP). The fresh PRP and PPP samples were used in the study. 2.2. Synthesis and characterization of the triblock copolymer mPEG-PU-mPEG MDI and BDO (molar ratio of 10:9) were dissolved in DMAC with the total monomer concentration of 20 wt.%. The initiator, stannous octoate, was introduced into the solution with about 1 wt.‰ of the total weight of the monomers. Polymerization was carried out in airtight equipment at 75 ◦ C for 2 h with passing nitrogen. Then, mPEG (the mole ratio of mPEG to the initial MDI was 0.2) was added to the mixed solution, and the reaction system was maintained at 80 ◦ C for 4 h. Thereafter, the temperature was raised to 90 ◦ C and maintained for 24 h before the reaction system was cooled to room temperature. The route for the synthesis was illustrated in Fig. 1. The product was extracted with methanol and hot de-ionized water for several times to remove the residual monomers, initiators and solvent thoroughly, which were confirmed by UV scanning. The UV adsorption band to detect the reaction residuals was 200–400 nm; and it was regarded that there was no residuals when the absorbance was lower than 0.005. The obtained copolymers were dried completely at 60 ◦ C in a vacuum oven for 72 h. In this study, mPEG with molecular weights of 500 and 2000 were selected, thus two copolymers were obtained in different molecular weights. M1 and M2 represent the copolymers of

Table 1 Compositions of different membranes used in this study. Samples

PES (wt.%)

PES PES-M1-1% PES-M1-2% PES-M1-4% PES-M2-1% PES-M2-2% PES-M2-4%

16 16 16 16 16 16 16

M1 (wt.%)

M2 (wt.%)

DMAC (wt.%)

1 2 4

84 83 82 80 83 82 80

1 2 4

mPEG(Mn500)-PU-mPEG(Mn500) and mPEG(Mn2000)-PU-mPEG (Mn2000), respectively. The structures of the copolymers were analyzed by FTIR (coated on a KBr pellet, 64 scans at a resolution of 1 cm−1 ) with FTIR Nicolet 560 (Nicol American) instrument and 1 H NMR (400 MHz, using tetramethylsilane (TMS) as the internal standard in dimethylsulfoxide (DMSO)-d6 ) at room temperature). Gel permeation chromatography (GPC) measurement, which is based on the liquid chromatography analysis using an aqueous gel permeation column, was performed by using a PL220 GPC analyzer (Britain), N,N-dimethylformamide (DMF) was chosen as the eluent and polystyrene (PS) as the reference. 2.3. Membrane preparation and characterization In this study, membranes were prepared by a phase inversion technique (Pure water as non-solvent phase and glass slice as the substrate). 16 wt.% PES and various contents of the synthesized mPEG-PU-mPEG were dissolved into DMAC with vigorous stirring until clear homogeneous solutions were obtained. The contents of mPEG-PU-mPEG in the casting solutions were 0, 1, 2 and 4 wt.%, respectively (as shown in Table 1). After vacuum degassing, the casting solutions were converted into membranes by spin coating coupled with a liquid–liquid phase separation technique at room temperature as the method mentioned in literatures [17,18]. The obtained membranes were rinsed with double distilled water to remove the residual solvent, which was confirmed by UV scanning at the wavelength of 200–400 nm. All the prepared membranes were in a uniform thickness of about 75–80 ␮m. The cross-section morphologies of the membranes were observed by SEM. Before SEM observation, the membranes were dried in a vacuum oven, and then quenched by liquid nitrogen and cut with a single-edged razor blade, attached to the sample supports and coated with a gold layer. The SEM images were recorded by an S-2500 C microscope (Hitachi, Japan). Images shown in this paper were the representatives of the cross-sections of the membranes. ATR-FTIR spectra for the membrane surfaces were obtained using a Fourier-transform infrared spectrometer (Nicolet 560, America), and were carried out with 64 scans at a resolution of 1 cm−1 at room temperature. The hydrophilicity of the membrane surfaces was characterized by means of a contact angle goniometer (OCA20, Dataphysics, Germany) equipped with a video capture. A piece of about 2 cm × 4 cm membrane was stuck on a glass slide and mounted on the goniometer. A total of about 3 ␮l of double distilled water was dropped on the airside surface of the membrane at room temperature, and the contact angle was measured after 10 s. At least eight measurements were averaged to get a reliable value. The measurement error was ±2◦ . 2.4. Ultrafiltration experiments The water flux, protein antifouling property, and water flux recovery ratio of the membranes were assessed by ultrafiltration experiments. The dead-end ultrafiltration cell was used, in which the effective membrane area was 13.8 cm2 .

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317

Fig. 1. Route for the synthesis of mPEG-PU-mPEGs.

Firstly, the membranes were pre-compacted by double distilled water for 30 min to get steady filtration. Then, the water flux (F) was determined at room temperature and calculated using the equation: F = V/SPT, where V is the volume of the permeated solution (ml); S is the effective membrane area (m2 ); P is the pressure applied to the membrane (mmHg) and T is the time (h). After the filtration of water, the feed was switched to 1 mg/ml BSA/water solution to determine protein rejection ratio (R), which was defined as follows: R (%) = (1 − Cp /Cb ) × 100, where Cp and Cb (mg/ml) are the BSA concentrations in the permeated and bulk solutions, respectively. The BSA concentrations were measured with BCATM by a UV–vis spectrophotometer at the wavelength of 562 nm. The flux of BSA solution was also measured. After BSA filtration, the membranes were rinsed with flowing double distilled water for 30 min. Then, the water flux recovery ratio (FRR ) of the membrane was measured again and calculated using the following expression: FRR (%) = (F2 /F1 ) × 100, where F1 and F2 (ml/(mmHg h m2 )) are the water flux before and after BSA filtration, respectively. The tests were repeated three times for each sample to get a reliable value, and the results were expressed as means ± SD. 2.5. Biocompatibility experiments 2.5.1. In vitro blood compatibility experiments 2.5.1.1. Platelet adhesion. Before the experiments, all the appliance, reagents, and membranes (1/2 cm × 1/2 cm) were sterilized by 75% alcohol for 3 h, and then the membranes were immersed into isotonic (pH 7.4) phosphate buffer solution (PBS) for 24 h. The membranes were placed in disposable plastic tubes. 200 ␮l of fresh PRP was dropped in each tube and then incubated with the mem-

brane at 37 ◦ C for 90 min. Then the membranes were rinsed for three times with PBS (37 ◦ C). The adhered platelets were fixed with 2.5 wt.% glutaraldehyde/PBS solution (1 ml) at 4 ◦ C for 2 h, and then the samples were rinsed with PBS for 5 min. The dehydration was carried out through a series of ethanol/PBS mixtures with increasing ethanol concentrations (25, 50, 75, and 100 wt.%, 15 min for each mixture), and then through isoamyl acetate/ethanol mixtures with increasing isoamyl acetate concentrations (25, 50, 100 wt.%, 15 min for each mixture). After freeze-drying with FD-1C-50 lyophilizer (Boyikang, China), the platelet-attached membranes were coated with a gold layer and the SEM images were recorded using an S2500 C microscope (Hitachi, Japan) at the magnification of 5000× [1]. The number of the adherent platelets onto the membranes was calculated from five SEM pictures at 500× magnification from different places on the same membrane. 2.5.1.2. Activated partial thromboplastin time (APTT) test. In this work, APTT test was used for the evaluation of anticoagulant property of the membranes. The APTT was determined by an automated blood coagulation analyzer (CA-50, Sysmex Corp., Kobe, Japan). The APTT was measured as follows: firstly, all the samples (1/2 cm × 1/2 cm) were immersed into PBS for 24 h, and then incubated in PPP at 37 ◦ C for 30 min. Afterward, the APTT was recorded automatically. The tests were repeated four times for each sample to get a reliable value, and data were expressed as means ± SD. 2.5.2. Cytocompatibility experiments 2.5.2.1. Cell culture. Human hepatocytes LO-2 were grown in R1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), 2 mM l-glutamine and 1% (v/v) antibiotics mixture (10,000 U penicillin and 10 mg streptomycin). Cultures were

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maintained in humidified atmosphere of 5% CO2 at 37 ◦ C (Queue Incubator, Paris, France). Confluent cells were detached from the culture flask with sterile PBS and 0.05% trypsin/EDTA (Ethylene Diamine Tetraacetic Acid) solutions, and the culture medium was changed every day. The membranes (1 cm × 1 cm) were placed into 24-well and 96-well polystyrene (PS) cell-culture plates, and pre-wetted by immersion in the culture medium for 3 h in the incubator at 37 ◦ C. Then the membranes were placed into the cell-culture plates, rinsed with PBS and sterilized by ␥-radiation, and the dose of ␥radiation was 25 kGy. 2.5.2.2. Cell morphology observation. Human hepatocytes LO-2 were seeded onto the membranes at a density of approx 2.5 × 104 cells/cm2 . Four hours later, the seeded membranes were rinsed immediately with PBS and fixed with 2.5 wt.% glutaraldehyde/PBS solution at 4 ◦ C for 2 h. For morphology observation, the fixed samples were dehydrated through gradient alcohol solutions (30, 50, 70, 80, 90, 95 and 100%, 30 min for each time) and then dehydrated through iso-amyl acetate. The critical point drying of the specimens was done with liquid CO2 . After freeze-drying with FD-1C-50 lyophilizer (Boyikang, China), the membranes were coated with a gold layer and the SEM images were recorded using an S-2500 C microscope (Hitachi, Japan) at the magnification of 500×. 2.5.2.3. MTT assay. After cell culture for 2, 4 and 6 days, the viability of the hepatocytes was determined by MTT assay. The hepatocytes were seeded onto the membranes at a density of approx 2.5 × 104 cells/cm2 . The cells cultured in the wells without the membranes were served as the control. After various time intervals, 20 ␮l of the MTT solution (1 mg/ml in the test medium) was added to each well and incubated for 4 h at 37 ◦ C. Mitochondrial dehydrogenases of viable cells cleaved selectively to the tetrazolium ring, yielding blue/purple formazan crystals. 200 ␮l of DMSO was added to dissolve the formazan crystals. The quantity of the formazan crystals dissolved in the DMSO could reflect the level of the cell metabolism. The dissolvable solution was jogged homogeneously for about 15 min by a shaker. The solution of each sample was aspirated into a microtiter plate and the optical density of the formazan solution was read on a microplate reader (Model 550, Bio-Rad) at 492 nm. All the experiments were repeated for six times, and the results were expressed as means ± SD. The statistical significance was assessed by Student’s t-test, and the level of significance was chosen as P < 0.05. 3. Results and discussion 3.1. Characterization of the triblock copolymer mPEG-PU-mPEG The FTIR spectra of the triblock copolymers M1 and M2 are shown in Fig. 2. The peaks at 2270 cm−1 (–NCO stretching vibration from MDI) and 3440 cm−1 (O–H stretching vibration from BDO and mPEG) disappeared in the products and switched to 3317 cm−1 , which belonged to the urethane linkage. The strong adsorption band at 1704 cm−1 attributed to the C O stretching vibration in the urethane linkage, and the peaks at 1533 and 1536 cm−1 derived from N–H bending vibration. The results indicated that a typical PU structure might be formed. What’s more, the event that mPEG blocked into PU chain could be deduced from the adsorption bands at 2958, 2955, 2894 and 2891 cm−1 , which were from CH3 bending vibrations at the end of mPEG chains. These results suggested that the triblock copolymers might be synthesized. The chemical structures of the triblock copolymers were also analyzed by 1 H NMR as shown in Fig. 3. The corresponding chemical shifts were as follows: ı = 3.310 and 3.321 ppm (a, –CH3 ), ı = 3.516 and 3.514 ppm (e, –OCH2 –), ı = 7.096 and 7.099 ppm (d,

Fig. 2. FTIR spectra of mPEG-PU-mPEGs.

aromatic ring), ı = 7.360 and 7.347 ppm (c, aromatic ring), ı = 9.530 and 9.535 ppm (b, –NH–). By integration treatment, the integration ratio of the signals e to b in M1 was 6.4, and that was 18.5 in M2, which were approximately agreed with the expected values. Combining the FTIR spectra, it could be concluded that the triblock copolymers M1 and M2 had been synthesized successfully. The weight average molecular weights (Mw) of M1 and M2 from GPC were 5075 and 9302, respectively; and the molecular weight distribution were 1.64 and 1.95, respectively. 3.2. Characterization of the membranes 3.2.1. SEM observation SEM micrographs of PES and PES/mPEG-PU-mPEG blended membranes are shown in Fig. 4. It was indicated that the general structures of all the membranes were similar, consisting of a thin dense skin layer at the air-facing side, a finger-like structure and porous architecture at the bottom. Furthermore, with the increase of the contents of the copolymers and mPEG chain lengths in the copolymers, the finger-like structure gradually disappeared, and the pores became larger, which indicated that the introduction of the amphiphilic copolymers could affect the morphology of PES membrane to some extent. 3.2.2. ATR-FTIR analysis ATR-FTIR spectra for the membrane surfaces of PES and PES/mPEG-PU-mPEG membranes are shown in Fig. 5. The most significant changes between PES and PES/mPEG-PU-mPEG blended membranes were the peaks at 1728 cm−1 and 1529 cm−1 appeared in the blended membranes, and neither of them was observed in the PES membrane. The two peaks were the characteristic peaks of the C O stretching vibration and N–H bending vibration, respectively. In addition, the areas of the two peaks increased with the increasing contents of the copolymers. Therefore, a conclusion could be drawn that mPEG-PU-mPEG with different contents had been blended into PES membranes successfully. 3.2.3. Water contact angle analysis Water contact angle is a convenient parameter to assess the hydrophilic/hydrophobic property of membrane surface, which provides information on the interaction energy between the surface and liquid [19–21]. Increased hydrophilicity of membranes could improve the flux and anti-fouling property of membranes. The water contact angles of the PES and PES/mPEG-PU-mPEG blended membranes are shown in Fig. 6. As shown in Fig. 6, after blending the copolymers into the PES membranes, the water contact angles decreased. PES membrane

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Fig. 3.

1

319

H NMR spectra of mPEG-PU-mPEGs in DMSO-d6 .

had the highest contact angle of about 87.1◦ , corresponding to the lowest surface hydrophilicity. For the PES membranes blended with M1, the water contact angles were slightly decreased from 87.1◦ to a range from 81.4 to 74.4◦ with the increase of the contents of M1; for the PES membranes blended with M2, the water contact

angles were decreased from 87.1◦ to a range from 68.2◦ to 58.0◦ with the increase of the contents of M2. In addition, for the triblock copolymer M1, the mPEG molecular weight is 500; for M2, the molecular weight is 2000. Thus, the total content of the mPEG segments in the membranes PES-M1-4% and PES-M2-1% are the same,

Fig. 4. SEM images of the PES and PES/mPEG-PU-mPEG blended membranes (magnifying 2000×).

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Fig. 5. ATR-FTIR spectra of the PES and PES/mPEG-PU-mPEG blended membranes. A: PES membrane; B: PES-M1-1% membrane; C: PES-M1-2% membrane; D: PES-M1-4% membrane; E: PES-M2-1%; F: PES-M2-2%; G: PES-M2-4%.

resulting in the contact angles for the PES-M1-4% and PES-M2-1% membranes were similar. Some other properties, such as antifouling property and platelet adhesion for the two membranes were also similar, which would be discussed in the following sections. These results indicated that the hydrophilicity of the PES membrane could be improved by blending mPEG-PU-mPEG copolymers. Due to the hydrophilic chain of mPEG, a higher molecular weight of mPEG led to a higher hydrophilicity and lower water contact angle. The water flux, protein rejection ratio and flux recovery ratio were all related to the hydrophilicity of the membranes, which would be discussed in the following sections. 3.3. Permeation and antifouling property analysis The protein antifouling properties of PES and PES/mPEGPU-mPEG blended membranes were studied by ultrafiltration experiments. The time-dependent fluxes during ultrafiltration operation are shown in Fig. 7. As shown in Fig. 7, the average water fluxes ranged from 406.5 (PES-M2-4%) to 51.7 (PES) ml/mmHg h m2 . Compared with the PES membrane, the fluxes of the blended membranes were higher. These could be explained by the increased hydrophilicity caused by the mPEG-PU-mPEG. Under a constant pressure filtration, a considerable reduction in the flux with ultrafiltration time was observed, which was the membrane

Fig. 7. Fluxes of the PES and PES/mPEG-PU-mPEG blended membranes. () PES membrane; () PES-M1-1% membrane; () PES-M1-2% membrane; () PES-M1-4% membrane; () PES-M2-1% membrane; () PES-M2-2% membrane; (䊉) PES-M2-4% membrane.

fouling caused by pore blocking, concentration polarization and cake formation takes place [22,23]. The fluxes decreased dramatically when the solution changed from water to BSA solution due to the membrane fouling caused by deposition and adsorption of the protein molecules onto the membrane surfaces or in the membrane pores, with a average range from 71.1 (PES-M2-4%) to 7.32 (PES) ml/mmHg h m2 . Under a constant pressure, equilibrium fluxes were obtained as the adsorption and deposition of protein molecules became saturated. After 60 min ultrafiltration, the BSA concentrations in the permeated and bulk solutions were measured by UV–vis spectrophotometer, and the BSA rejection ratios were calculated, as shown in Table 2. It was found that the BSA rejection ratios were similar for all the membranes, proving there was no significant difference in the dense layers among the membranes, which controlled the flux and sieving performance. Of course, the morphology (large pores) might slightly affect the water flux, BSA rejection ratio and flux recovery ratio. However, the water flux and flux recovery were mainly affected by the hydrophilicity and antifouling. It was a good performance for the blended membranes to keep a superior R in the practical application. The protein antifouling property could be expressed by water flux recovery ratio (FRR ), which reflected the extent of cleaning efficiency of the membranes, and the data are shown in Table 2. In Table 2, the FRR values were also similar. These results indicated that the blended membranes were much more easily recovered with washing compared with PES membrane for the introduction of the hydrophilic mPEG segments [24], which was a desirable performance [25]. 3.4. Biocompatibility analysis 3.4.1. In vitro blood compatibility 3.4.1.1. Platelet adhesion. Platelet adhesion and activation may lead to occurrence of thrombus when material surfaces contact Table 2 Rejection ratios (R) and flux recovery ratios (FRR ) of different membranes.

Fig. 6. Water contact angles of the PES and PES/mPEG-PU-mPEG blended membranes. Data are expressed as means ± SD, n = 8.

Samples

R (%)

PES PES-M1-1% PES-M1-2% PES-M1-4% PES-M2-1% PES-M2-2% PES-M2-4%

98.13 96.14 94.22 91.07 95.19 94.88 94.76

FRR (%) ± ± ± ± ± ± ±

1.27 1.45 2.32 2.55 1.23 2.66 1.49

61.32 98.29 99.77 100.01 101.23 102.57 103.14

± ± ± ± ± ± ±

0.87 0.12 0.78 0.29 0.63 0.22 0.57

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321

Fig. 8. SEM images (magnifying 5000×) and the amount of the platelets adhered on the PES and PES/mPEG-PU-mPEG blended membranes. Control sample: The virgin membrane.

with blood. The diversity among different membrane surfaces for platelet adhesion, activation and aggregation and the adhesion amounts were investigated by SEM, as shown in Fig. 8. It was found that the platelet adhesion amount of blended membranes were much lower than that of PES membrane. It could be observed that numerous platelets adhered to the surface of PES membrane as aggregates; the platelets spread flattened into irregular shapes, indicating the activation of the platelets. Meanwhile, some of the adhered platelets were cumulated in a degree for PESM1-1% and PES-M1-2% membranes, demonstrating the activation of the platelets partly. In contrast, platelets were rarely observed on the membranes with higher contents of mPEG-PU-mPEG copolymers, and the adhered platelets kept their round shape. The results of platelet adhesion revealed that the modified membranes might have good blood compatibility featured by the low platelet adhesion and the fact that the initial shapes of platelets were kept. This might be due to the low interfacial energy attributed to mPEG, and to the high mobility of mPEG in aqueous environments, which resulted in the improved resistance to platelet [26,27]. For the PES-M1-1% and PES-M1-2% blended membranes, the relatively worse performance might be related to less mPEG contents and shorter mPEG chain lengths, while the anti-platelet adhesion property of PU segment was not so outstanding [28,29]. For the PES-M1-4% and PES-M2-1% blended membranes, the similar platelet adhesion amount and morphology might attribute to the same mPEG content of them. Pits and settlement craters were observed on the membranes. The pits and settlement craters might be resulted from the phase separation, which was led by PU segments in the copolymers

and the poor miscibility between the copolymers and PES chains. The mechanism for the structure formation in the phase separation process between copolymers and PES chains was believed to be as follows: during the liquid-liquid phase separation, as the solvent was escaping, the polymer remained in the continually diminishing liquid phase, which led to the increase of the bulk concentration of the blends and thus increased the non-favourable interactions between the minor component block mPEG-PU-mPEG and the major component block (PES). These interactions actively promoted the displacement of the copolymer towards the nascent, and then matured at the polymer–solvent interface, resulting in a higher localized concentration of the copolymer at these interfaces. This dynamic process thus resulted in the formation of the pits and settlement craters structure on the membrane surface (as shown in Fig. 8) [30]. On the other hand, the thermodynamic incompatibility of hard and soft segments in the copolymer which led to microphase separation could also form the pits and settlement on the membrane surface [31–33]. 3.4.2. APTT analysis APTT is usually used to examine mainly the intrinsic pathway [34]. The clotting times of PES and PES/mPEG-PU-mPEG blended membranes are shown in Fig. 9. It could be seen that the clotting times of the blended membranes were longer than that of the PES membrane, which suggested that the copolymer might be prolonged the blood clotting time These agreed with the fact that mPEG had gained recognition as a biocompatible material since it appeared to interact minimally with proteins, reduce platelet adhesion and improve anticoagulation effect. Therefore, the blood

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Fig. 9. APTT of the PES and PES/mPEG-PU-mPEG blended membranes. Data are expressed as means ± SD, n = 4.

compatibility of the PES membrane might be improved through blending mPEG-PU-mPEG. In addition, the APTT results showed slightly longer clotting time for the PES than that for the plasma. PES is a commercially available material with good properties. PES-based materials are already used as high-flux membranes for hemodialysis and hemodiafiltration [34–37]. In fact, some researches [37–40] have confirmed that PES has some anticoagulant property. However, the anticoagulant property is not enough, and injections of anticoagulants are needed during hemodialysis [41].

3.4.3. Cytocompatibility 3.4.3.1. Cell morphology analysis. In practice application of artificial organs and tissue engineering, some biomaterials will contact cells directly for a relatively long time. Therefore, cytocompatibility is another important parameter for biomaterials. Porous PES membranes could constitute a good mechanical and chemical support for cell adhesion because of their physico-chemical and morphological properties. Therefore, PES membranes have been widely used in artificial organs and tissue engineering, and showed good cell compatibility [40,42–45]. In this study, human hepatocytes LO2 were selected for the evaluation of the cytocompatibility of the blended membranes, which might be used for bioartificial liver support. Generally speaking, the cells will change their morphology to stabilize themselves onto material surface after contacting biomaterials. The whole process of adhesion and spreading, consisting of cell attachment, filopodial growth, cytoplasmic webbing, flattening of cell mass and ruffling of peripheral cytoplasm progressing occurred in a sequential fashion [45]. The morphologies of the hepatocytes cultured for 4 h on the PES and PES/mPEG-PU-mPEG blended membranes are shown in Fig. 10. It could be seen that the hepatocytes had the morphologies of triangle, spindle and roundness, and they extended pseudopodia to adhere onto the materials and grew by monolayer. The amount of hepatocytes grown on the PES membrane was the least. With the increase of the contents and the molecular weights of mPEGPU-mPEG copolymers, the adhesion amount of the hepatocytes increased. On the surfaces of the blended membranes, especially the PES-M1-4%, PES-M2-1%, PES-M2-2%, PES-M2-4% membranes, the cells spreading with ruffling of peripheral cytoplasm, and almost had covered the whole surfaces and had been flattened

Fig. 10. SEM images of human hepatocytes LO2 cultured on the PES and PES/mPEG-PU-mPEG membranes after 4 h (magnifying 500×).

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Fig. 11. Formazan absorbance expressed as a function of time from hepatocytes seeded onto different membranes and the control sample. Data are expressed as means ± SD, n = 6.

with a larger attachment area compared to the cells cultured on the PES membrane, which indicated that the blended membranes could promote cell attachment and growth better. PEG is widely reported to decline bio-adhesion. But in this study, we think that the morphological structures of the modified membranes and the PU segments are critical to affect the cell adhesion. According to the literatures [46–49], the non-uniform and rough surface could enlarge the contact areas and humidification of cell and surface, which could promote cell adhesion. As shown in Fig. 8, pits and settlement craters could be observed. The pits and settlement craters were regarded as non-uniform and rough structure to promote cell adhesion. The different adhesion between PES-M1-4% and PES-M2-1% could also prove the point: different PU segment and different miscibility led to different non-uniform and rough structure, showing different cell adhesion. The good cell adhesion might be mainly attributed to the surface morphology in the study. 3.4.3.2. MTT analysis. The MTT data for the membranes and control samples are illustrated in Fig. 11. The formazan absorbance indicated that the hepatocytes seeded onto the control (the PS cellculture plate) and different membranes were able to convert the MTT into a blue formazan product. In Fig. 11, on the day 2, 4 and 6, with increasing culture time, the viability of cells on each membrane increased; and from the slope data, the viability of the cells on the modified membranes was more superior compared to the control sample and the pure PES membrane, for a high slope indicated a high proliferation rate. The relatively small slope for the membrane PES-M2-4% might be caused by the large amounts of the adhering cells at the beginning. These results indicated that the modified membranes promoted cell proliferation better. It was noted that the viability of the cells seeded on all the membranes were better than that on the control sample, which indicated that PES, even without modification, was a better material for cell culture compared to PS, and these had been demonstrated by an earlier report [45]. Therefore, it could be concluded that the addition of the copolymers M1 and M2 into the PES membranes would improve the cytocompatibility and the modified membranes had the potential to be used as bioartificial liver supports. 4. Conclusion The goal of this study was to improve the hydrophilicity and biocompatibility of PES membranes. This has been successfully accomplished by blending triblock copolymers mPEG-PU-mPEG

with PES. The triblock copolymers were synthesized through a two-step solution polymerization, and the method could also be used for the synthesis of a series of triblock copolymers containing PU with different soft segments and hard segments, which will show different properties. After blending the copolymers, the hydrophilicity and protein antifouling property increased. With the increase of the contents of the copolymers and the molecular weights of mPEG in the copolymers, the platelet adhesion and anticoagulant performances of the membranes were enhanced, and the cytocompatibility of the membranes also increased. These results indicated that the blended membranes showed good biocompatibility, and were potential to be used in biomedical fields. Acknowledgements This work was financially sponsored by the National Natural Science Foundation of China (No. 50973070, 51073105 and 30900691), and Sichuan Youth Science and Technology Foundation (08ZQ026-038). We should also appreciate the Blood Transfusion Section Office of West China Hospital for the help of collecting fresh blood, and gratefully acknowledge the help of Ms. Wang (The Analytical and Testing Center at Sichuan University) for the SEM observation. Moreover, we would thank our laboratory members for their generous help. References [1] B.H. Fang, Q.Y. Ling, W.F. Zhao, Y.L. Ma, P.L. Bai, Q. Wei, H.F. Li, C.S. Zhao, J. Membr. Sci. 329 (2009) 46–55. [2] H.T. Wang, T. Yu, C.Y. Zhao, Q.Y. Du, Fiber Polym. 10 (2009) 1–5. [3] A. Higuchi, K. Shirano, M. Harashima, B.O. Yoon, M. Hara, M. Hattori, K. Imamura, Biomaterials 23 (2002) 2659–2666. [4] M. Wang, L.G. Wu, X.C. Zheng, J.X. Mo, C.J. Gao, J. Colloid Interface Sci. 300 (2006) 286–292. [5] Y.Q. Wang, T. Wang, Y.L. Su, F.B. Peng, H. Wu, Z.Y. Jiang, J. Membr. Sci. 270 (2006) 108–114. [6] J.H. Lee, B.J. Jeong, H.B. Lee, J. Biomed. Mater. Res. 34 (1997) 105–114. [7] M.A. Dyer, K.M. Ainslie, M.V. Pishko, Langmuir 23 (2007) 7018–7023. [8] R. Murthy, C.E. Shell, M.A. Grunlan, Biomaterials 30 (2009) 2433–2439. [9] D. Li, H. Chen, W. Glenn McClung, J.L. Brash, Acta Biomater. 5 (2009) 1864–1871. [10] Z.F. Li, E. Ruckenstein, J. Colloid Interface Sci. 269 (2004) 62–71. [11] W.J. Chen, J.M. Peng, Y.L. Su, L.L. Zheng, L.J. Wang, Z.Y. Jiang, Sep. Purif. Technol. 66 (2009) 591–597. [12] L.P. Zhu, Z. Yi, F. Liu, X.Z. Wei, B.K. Zhu, Y.Y. Xu, Eur. Polym. J. 44 (2008) 1907–1914. [13] Y.Q. Wang, Y.L. Su, X.L. Ma, Q. Sun, Z.Y. Jiang, J. Membr. Sci. 283 (2006) 440–447. [14] S.A. Guelcher, K.M. Gallagher, J.E. Didier, D.B. Klinedinst, J.S. Doctor, A.S. Goldstein, G.L. Wilkes, E.J. Beckman, J.O. Hollinger, Acta Biomater. 1 (2005) 471–484.

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