Route to hemocompatible polyethersulfone membranes via surface aminolysis and heparinization

Journal of Colloid and Interface Science 422 (2014) 38–44

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Route to hemocompatible polyethersulfone membranes via surface aminolysis and heparinization Linghui Wang, Yu Cai, Yihang Jing, Baoku Zhu ⇑, Liping Zhu, Youyi Xu Key Laboratory of Macromolecule Synthesis and Functionalization (MOE), ERC of Membrane and Water Treatment (MOE), Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

a r t i c l e

i n f o

Article history: Received 12 November 2013 Accepted 5 February 2014 Available online 13 February 2014 Keywords: Polyethersulfone membrane Surface modification Aminolysis Heparin immobilization Hemocompatibility

a b s t r a c t Polyethersulfone (PES) membranes with improved hemocompatibility were prepared via solid–liquid interface aminolysis and heparinization. Reactive amino groups were generated by immersing solid PES membranes in proper diamine solution. Heparin was covalent immobilized on the surface via amide bond. The feasibility of surface aminolysis for the introduction of amino groups and the effectiveness for further heparin immobilization were confirmed by surface group analysis. The effect of aminolysis time on surface amino group concentration and bulk mechanical properties was investigated. The surface amino group concentration determined the amount and bioactivity of immobilized heparin chains. SEM images suggested that both the aminolysis and heparinization reaction had little effect on the surface morphology of PES membranes. Contact angle measurement, surface charge analysis, protein and platelet adsorption/adhesion experiment were applied to study the surface properties. The results showed that the heparinized PES membranes displayed enhanced hydrophilicity and hemocompatibility, indicating potential application in blood purification and other blood contacting fields. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Polyethersulfone (PES) is a kind of engineering thermoplastics. Compared to traditional hemodialysis membrane materials, such as polyacrylonitrile, polymethylmethacrylate and polyester, PES possesses excellent thermal stability, mechanical strength and chemical inertness [1]. A key feature of PES membranes, in additional to their good transport properties, is their excellent biocompatibility. These membranes exhibit only slight complement activation [2], the drop in leukocytes is minimal [3], and the release of leukocyte elastase is low [4]. Therefore, PES is the most widely used membrane material for hemodialysis. Despite its advantages, the intrinsic hydrophobicity of PES often causes severe protein adsorption and platelet adhesion, decreasing the performance of PES membrane. A variety of studies have shown the effectiveness of immobilizing hydrophilic segments onto the surface to improve the hydrophilicity and hemocompatibility of materials [5–8]. Heparin is a kind of mucopolysaccharide, bearing abundant sulfate and carboxyl groups. Due to its excellent anticoagulation activity [9], heparin has been widely used as injected anticoagulant

⇑ Corresponding author. Fax: +86 571 87953723. E-mail address: [email protected] (B. Zhu). http://dx.doi.org/10.1016/j.jcis.2014.02.005 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.

in the hemodialysis process. However, the accumulated heparin in blood is detrimental to patients. Thus, immobilizing heparin onto hemodialysis membrane surfaces has attracted much attention. Electrostatic adsorption [10–12] and covalent bonding [13,14] are the two main methods. On consideration of stability, a lot of effort has been payed to covalent bonding strategies. Chemical immobilization is mainly achieved via the coupling reaction between the surface reactive groups and the carboxyl groups of heparin. While most materials that used for hemodialysis have few reactive groups, the generation of reactive groups is crucial. Many methods, including sulfonation and carboxylation in solution [15], plasma treatment [16], gamma radiation [17], UV radiation [18,19] and corona treatment [20], have been investigated to introduce reactive groups to PES material surface. However, drawbacks present in these methods, such as low modification efficiency, complex procedures, deterioration of mechanical strength, and damages of porous structure. Therefore, a simpler and more effective route to generate surface reactive groups is desirable for subsequent chemical modification. We have tested several methods to introduce reactive groups onto PES membrane surface for immobilization of heparin. After detailed anaylsis in theory and practice, a novel surface aminolysis route using diamine as reagent was proposed for the construction of aminated PES membranes. In this work, the surface aminolysis of PES membranes was studied in depth. Moreover, further

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heparinization of these aminated PES membranes was conducted, and improved performances were achieved and reported.

2. Experimental 2.1. Materials PES (Veradel PES3000P) powder was provided by Solvay and dried at 100 °C for 12 h before use. Dipropylenetriamine (DPTA) was obtained from TCI. toluidine blue (TB) and N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDCHCl) were purchased from Aladdin. Dimethyl acetylamide (DMAc), poly (ethylene glycol) (PEG400, Mn = 400 g/mol), 4-morpholineethanesulfonic acid (MES), dimethyl sulfoxide (DMSO), ninhydrin, heparin sodium and bovine serum albumin (BSA) were purchased from Sinopharm Chemical Reagent Co., Ltd. All other reagents were analytical grade and used without further purification.

2.2. Preparation of PES membranes Original PES membranes were prepared via non-solvent induced phase separation using PEG400 as pore forming agent. The cast solution for preparing PES membranes was prepared by dissolving 40 g PES and 20 g PEG400 in 140 g DMAc at 80 °C. The PES solution was cast on a clean glass and then immersed into a water coagulation bath at 30 °C for 5 min. The solid PES membranes were washed in water at 60 °C for 24 h to extract residual PEG400 and DMAc. The obtained PES membranes were stored in 1% sodium bisulfite solution for further modification. The specific area of PES membranes determined by mercury intrusion porosimetry (Autopore IV 9500, Micromeritics) was 57.6 m2/g.

2.3. Surface amination of PES membranes PES membranes were immersed in aminolysis solution (10.0 wt% DPTA in water) at 90 °C for a prefixed reaction time, then thoroughly washed with copious ethanol to remove physically adsorbed DPTA. The aminated PES membranes (NH2-PES membranes) were stored in deionized water before use. The surface amino group concentration (CNH2, mol/cm2), defined as the mole number of amino group on per square centimeter of surface (include the inner pore walls and the top surface of membrane), was determined by a modified ninhydrin reaction analysis [21]. Briefly, Dry membrane sample (1.5 cm in diameter) was weighted and placed in a 20 mL test tube, 20 lL ninhydrin solution (2 wt% in DMSO) was added, and heated at 80 °C for 30 min to accelerate the chromogenic reaction between ninhydrin and amino group, 5 mL DMSO was added to the test tube to dissolve the purple substance (Ruhemann’s purple) and the optical density (O.D.) of the solution was measured at 598 nm using a spectrophotometer (UV-1601 Shimadzu, Kyoto, Japan). NH2-PES powder was prepared via solution aminolysis of PES with DPTA, with amino group concentration determined by 1H NMR. The standard curve of the ninhydrin reaction analysis was obtained by using NH2-PES powder, as shown in Fig. 1. The optical density showed good linearity with amino group amount. Then, CNH2 was calculated as follow:

cNH2 ¼

nNH2 mmembrane Sspecific

where nNH2 is the molar amount of amino group that determined through the ninhydrin reaction, mmembrane is the mass of membrane sample, and Sspecific is the specific area of PES membrane.

Fig. 1. Calibrating curve of the ninhydrin reaction analysis.

The mechanical properties were analyzed on Reger-RWT10 instrument using sample membranes (10  40 mm2) under the stretching rate of 10 mm/min. 2.4. Heparinization of PES membranes The covalent immobilization of heparin onto PES membrane surface was realized via the coupling reaction between the amino groups on NH2-PES membranes and the carboxyl groups on heparin chains under the catalysis of EDC. NH2-PES membranes were immersed in heparin solution (2.0 wt%, 0.05 M MES buffer solution as solvent), then the coupling catalyst EDCHCl (molar ratio of EDC:–COOH was 1:1) was added. After reacting at room temperature for a prefixed reaction time, the membranes were washed with PBS (pH 7.4) thoroughly and heparin immobilized PES membranes (Hep-PES membranes) were obtained. The concentration of immobilized heparin (Cheparin, lg/cm2), defined as the weight of heparin per square centimeter of the membrane surface, was determined by a modified colorimetric method based on toluidine blue complexation [22]. Briefly, the membranes (1.5 cm in diameter) were dried and incubated in 2.5 mL TB solution (0.005 wt%, pH 2.07, 0.2 wt% NaCl as solvent) for 2 h, the solution was then diluted with 0.2 wt% NaCl solution to a total volume of 5 mL. The TB solution was sampled and diluted 1:10 with absolute ethanol. Then, the absorbance at 631 nm was measured using a UV–vis spectrophotometer. The calibrating curve was obtained using heparin solution of known concentration. Cheparin was calculated by the following equation:

C heparin ¼

mheparin S  ð1  pÞ

where mheparin is the amount of immobilized heparin, S is the surface area of membrane sample, and p is the surface porosity of PES membrane that determined from surface SEM image using image pro-plus software (Media Cybernetics, Atlanta, GA). 2.5. Characterization of membrane surface properties The surface morphology of the membranes was observed by field emitted scanning electronic microscopy (FESEM, S-4800, Hitachi) after being coated with a gold layer. The water contact angles on membranes were measured at 25 °C using a contact angle meter system (OCA20, Dataphysics, Germany). The zeta potentials were evaluated with the SurPASS Electrokinetic Analyzer (Anton Paar GmbH, Graz, Austria) equipped with an adjustable gap cell following a standard operation [23].

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Protein adsorption experiment was conducted by immersing membrane samples (diameter of 1.5 cm) into BSA solution (2 mL, 1 mg/mL in PBS, pH 7.4) at 25 °C for 6 h. The amount of adsorbed BSA was calculated by comparing the concentration of BSA solution before and after incubation. The BSA concentration was determined by the Bradford method. The platelet adhesion on membranes was observed by SEM (FESEM, S-4800, Hitachi) after incubating the membranes in platelet rich plasma (PRP) for 60 min at 37 °C and washed with PBS (pH 7.4) for 3 times. The plasma recalcification time (PRT) of membrane samples was measured by dropping 50 lL platelet-poor plasma and 50 lL CaCl2 solution (0.025 M) on the membrane surface in sequence, and recording the time needed for the fibrin to be detected [24]. 3. Results and discussion PES is synthesized by polycondensation of bisphenol S and dichlorodiphenylsulfone via the aromatic nucleophilic substitution mechanism [1]. Unlike other polycondensation polymers, such as polyester and polyamide, the aryl ether bonds in the main chain endow PES with excellent chemical inertness. Therefore, the modification of PES by chemical reactants is usually thought to be difficult. However, due to the strong electron-drawing effect of the sulfonyl groups, the phenylene rings are highly electron-deficient, which enhances the nucleophilic-substitution reaction on the aryl ether bonds. So we proposed the surface aminolysis route for the introduction of reactive groups. The reactivity of PES and amine was tested using ethylenediamine (EDA) in DMAc solution. The structure of purified product was characterized by 1H NMR spectroscopy (data not shown here). Two new peaks were observed at 3.05 and 2.68 ppm, corresponding to the residual ethylene group of EDA. This confirmed the formation of C–N bonds after the reaction of aryl ether bond and amino group. Thus, it was reasonably expected that primary amino groups were introduced onto the PES membrane surface by using excessive diamine. Following this strategy, hemocompatible PES membranes were obtained by covalent immobilization of heparin, as illustrated in Fig. 2. 3.1. Surface amination of PES membranes During the aminolysis reaction, the reactant solution would penetrate into the inner membrane pores. Therefore, the aminolysis reaction occurred on both the top surface and the pore walls of the membrane. This provided possibility for the functionalization of membrane pore walls. Fig. 3 showed the effect of aminolysis time on CNH2. During the first 48 h, CNH2 increased gradually with aminolysis time and then a plateau value of about 92.0 pmol/cm2 was observed. The well controllability of CNH2 provided good reliability for the subsequent functionalization reaction. The effect of aminolysis on mechanical properties of PES membranes was investigated via tensile test (Fig. 3). The tensile strength of the original PES membrane was about 3.8 MPa, and only a slight decline of 0.3 MPa was observed after being aminolyzed for 50 h at 90 °C. The drop in mechanical strength was the major drawback of the high energy radiation method [20]. Generally, the deterioration of mechanical properties is owed to the degradation of matrix polymer chains [25,26]. While for the PES/DPTA reaction system, the relative hydrophobicity of PES material prevented the diffusion of DPTA into the bulk and avoided the aminolysis of matrix chains. This means the aminolysis reaction of PES by DPTA dominates on the surface. From the aminolysis kinetics and the mechanical properties, it could be reasonably concluded that the surface aminolysis of PES

Fig. 2. Schematic diagram illustrating the surface heparinization of PES membrane.

Fig. 3. Surface amino group concentration and tensile strength of PES membranes after different aminolysis time.

by diamine was a feasible and effective route for the preactivation of PES membranes. 3.2. Heparinization of PES membranes The heparinization kinetics of virgin PES membrane (CNH2 = 0 pmol/cm2) and three different NH2-PES membranes (CNH2 = 2.3, 11.6 and 41.1 pmol/cm2, respectively) were shown in Fig. 4(a). When CNH2 was 0 pmol/cm2, no immobilized heparin was detected on the membrane surface even after reacting for 12 h. While for NH2-PES membranes, the heparin immobilization occurred obviously. In the first 1 h, Cheparin increased quickly, showing a fast reaction rate. Then, Cheparin levelled off to a plateau value after about 4 h. The heparinization kinetics of NH2-PES membranes suggested the heparin immobilization reaction was highly efficient. Fig. 4(a) also showed that the plateau Cheparin was greatly dependent on CNH2, larger CNH2 always resulted in higher Cheparin. The influence of CNH2 on the maximum Cheparin was further studied and displayed in Fig. 4(b). The maximum Cheparin increased rapidly at low CNH2, and levelled off as CNH2 exceeded about 30 pmol/cm2. This phenomenon can be explained by comparing the size of hep-

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Fig. 4. Kinetic plots for the heparinization of PES membranes having different surface amino group concentration (a) and the relationship between maximum immobilized heparin amount and surface amino group concentration (b).

arin chains (hydrodynamic radius was 2.1–3.6 nm [27]) with the distance between amino groups. When CNH2 was low, amino groups were far apart and the distance between adjacent amino groups was larger than the size of heparin chains. Under this condition, heparin chains were more likely to be immobilized via one covalent bond as shown in Fig. 4(b), and there was still much space for new heparin chains. So the new introduced amino groups mainly caused the immobilization of new heparin chains, and a quick increase in Cheparin was observed. When CNH2 was high enough, most of the surface was occupied by heparin chains, it’s difficult for the new heparin chains to reach the amino groups. In this situation, the new introduced amino groups mainly reacted with the immobilized heparin chains. This mean the further increase in CNH2 did not elevate Cheparin, but restrained the mobility of heparin chains. The observation of saturated immobilization amount was very common for other heparin-immobilized materials [28–30]. While the activity of the surface layer is not only determined by heparin amount, but also relied on the bioactivity of each chain. So it is important to verify the binding status of the heparin chains. Theoretically, the best surface is with high heparin concentration but low binding point density. However, it is difficult to determine the binding point density via experimental technology. So the suppositional heparin binding status profile illustrated in Fig. 4(b) will provide important evidence for the performance behaviors analysis. Comparing with electrostatic adsorption, the covalently bonded heparin possess greatly improved stability. The stability of the covalently immobilized heparin on PES membranes was investigated by incubating Hep-PES membranes (Cheparin, 9.2 lg/cm2) in PBS and measuring the change of Cheparin with incubation time. As given in Fig. 5, little decrease in Cheparin was observed even after incubating in PBS for 7 days, indicating excellent immobilization stability. Meanwhile, the stability of surface immobilized heparin was further investigated by incubating Hep-PES membranes in platelet-poor plasma. Fig. 5 indicated the good stability of surface immobilized heparin in plasma. The hydrolytic stability of the amide linkage bonds was believed to contribute to the immobilization stability. The observed long-term stability of the Hep-PES membranes revealed the advantages of the covalent bonding route in practical applications.

Fig. 5. Surface heparin amount of Hep-PES membranes after being incubated in PBS (pH 7.4) and plasma for different time period.

influence on the surface structure of PES membranes. Many reports have shown increased roughness and appearance of cracks during the surface aminolysis of conventional polymer membranes, e.g. polycaprolactone (PCL), polylactic acid (PLA) or polyethylene terephthalate (PET) [31,32]. This adverse effect is mainly attributed to the semi-crystalline property of these polymers [33]. The amorphous phase is much more susceptible to reaction than the crystalline phase. The different degradation rate of these two phases causes increased roughness. Meanwhile, the stress between the two phases also increases as reaction proceeds, and cracks finally emerge after the stress exceeding critical value. PES material is, however, totally amorphous, so the aminolysis reaction takes place uniformly, suppressing the increase in roughness and cracks. This result also supposed that the diffusion of DPTA into PES bulk was neglectable, which was in agreement with the tensile test. After heparinization reaction, the pore blockage of PES membrane was not obvious. That is partially due to the smaller hydrodynamic radius of heparin as compared to the pore size of PES membrane. On the other hand, the high negative charge density of heparin generates repulsive force among heparin chains, leaving the immobilized heparin chains sparsely separated. The preservation of pore structure was an important advantage in preserving the separation performances of PES membranes.

3.3. Surface morphology of PES membranes 3.4. Hydrophilicity and charge properties The surface morphologies of original PES, NH2-PES and Hep-PES membranes imaged by SEM were given in Fig. 6. Clearly, neither the aminolysis nor the heparinization reaction had obvious

Water contact angle (CA) is an effective piece of data for analyzing the hydrophilicity of solid materials. For membranes with

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Fig. 6. Surface SEM images of original PES membrane (a), NH2-PES membrane (CNH2, 24.5 pmol/cm2) (b) and Hep-PES membrane (Cheprin, 9.2 lg/cm2) (c).

similar pore structures, theoretically, the better hydrophilicity, the smaller CA and the easier for water to penetrate into membrane pores. The contact angles of PES membranes before and after modification were shown in Fig. 7(a). For original PES membrane, the initial CA was as high as 80°, and only a decrease of about 10° was found in 120 s, revealing the intrinsically hydrophobic nature of PES material. The CA profile did not changed apparently for the aminated membrane surface (CNH2 = 24.5 pmol/cm2). While for Hep-PES membrane (Cheparin, 9.2 lg/cm2), the initial CA decreased to 65°, showing largely improved surface hydrophilicity. In addition, the CA decreased much more quickly with time, indicating the excellent hydrophilicity of membrane inner pore surface. This result suggested that the heparinization not only happened on the top surface, but also on the pore walls of the PES membranes. TB adsorption was conducted to prove this supposition. After TB treatment, the cross-section of Hep-PES membranes was found to be dark blue, indicating the existence of heparin on the inner pore walls. The immobilization of heparin on the pore walls was possible, for the molecular size of heparin was smaller than the surface pore size. The hydrophilization of the inner pore can reduce the

transmembrane resistance, thus improving the separation efficiency. The surface charge property is also very important for the blood contacting materials. The zeta potential profiles of PES membranes were measured and displayed in Fig. 7(b). For the original PES membrane, the isoelectric point was around pH 4.5, which was obviously lower than 7. This phenomenon was common for polymeric materials, owing to adsorption of hydroxide ions in aqueous electrolyte solution [34]. For NH2-PES membrane, the amino groups tended to be protonated, thus higher positive zeta potentials and higher isoelectric points were detected. The Zeta potential profile of Hep-PES membrane was apparently different from that of PES and NH2-PES membranes. In the pH range of 2–10, all the zeta potentials were negative and the curve profile was much more flat. This was ascribed to the dissociation of the large quantity of acid groups, e.g. carboxyl and sulfate groups, that from the immobilized heparin molecules. Strangely, the absolute zeta potential value of Hep-PES membrane was lower than that of original PES membrane in basic solution. This phenomenon was also reported previously [35] and was due to the swelling of the highly hydrophilic heparin

Fig. 7. Contact angles (a) and Zeta potentials (b) for original PES, NH2-PES (CNH2, 24.5 pmol/cm2) and Hep-PES (Cheparin, 9.2 lg/cm2) membranes.

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Fig. 8. BSA adsorption and plasma recalcification time of PES membranes with different immobilized heparin amount.

layer. During the measurement, the shear plane moved toward the solution as the heparin chains extended to the solution, then a lower zeta potential was obtained. 3.5. Protein adsorption and anticoagulation properties The adsorption of proteins on materials surface is usually considered as the premonition of thrombogenicity generation [36]. Meanwhile, the adsorbed proteins will block the membrane pores, thus bring down the membrane performances. Using the widely adopted BSA as indicative substance, the protein adsorption behavior on PES and Hep-PES membranes was investigated (Fig. 8). The BSA adsorption amount on the original PES membrane was as high as 178.8 lg/cm2. The poor protein adsorption resistance of PES membrane was due to the hydrophobicity of PES material. The BSA adsorption on NH2-PES membranes was also investigated (data not shown here), and no apparent decrease as compared to original PES membrane was found. For Hep-PES membranes, the amount of adsorbed BSA decreased gradually with increased Chepa2 rin. When Cheparin reached 9.2 lg/cm , the adsorbed protein amount 2 was as low as 36.3 lg/cm . The good protein adsorption resistance ability was due to the strong hydration of heparin chains. That’s

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because expulsion of water molecules from both surface and protein is the first and obligatory step to facilitate protein adsorption by reducing free energy barrier arising from dehydration entropic effects [37]. The anticoagulation property of PES membranes was characterized by PRT. The PRT basically has the same clinical significance as the whole blood coagulation time (CT). The PRT is generally more sensitive and accurate than CT, and is closer to the in vivo experiment [24]. As shown in Fig. 8, the PRT of original PES membrane was about 7 min. With the immobilized heparin amount increased, the PRT also increased. When Cheparin was 9.2 lg/cm2, the PRT prolonged to about 14 min, indicating good anticoagulation property. Because the membranes were thoroughly washed before each test, the anticoagulation activity of the surface was merely due to the immobilized heparin chains. While further elevating Cheparin from 9.2 lg/cm2 to 11.2 lg/cm2, the bonding points of each heparin chain were also increased, as demonstrated in Fig. 4(b). Therefore, the mobility of heparin chains was restrained, leading to lower bioactivity. As a result, the enhancement of anticoagulation was not apparent when Cheparin exceeded 9.2 lg/cm2. The immobilized heparin layer also inhibited the adhesion of platelet as imaged in Fig. 9. Clearly, there were a large number of platelets adhering on the surface of original PES membrane. The adhesive number of platelet decreased dramatically with increased immobilized heparin amount. The improved anti-adhesion property was due to the excellent hydrophilicity of heparin, as well as the negative–negative repulsion force between heparin and platelet. Meanwhile, the deformation and pseudopod growth of platelet were greatly suppressed for Hep-PES membranes. When Cheparin reached 11.2 lg/cm2, nearly no growth of pseudopod was found, indicating the good hemocompatibility of the heparin layer. 4. Conclusions Following the nucleophilic-substitution reaction between the aryl ether bonds of PES and amine groups, aminated PES membranes were obtained by immersing PES membranes in DPTA

Fig. 9. SEM images of original PES membrane (a), and Hep-PES membranes with Cheparin of 4.0 (b), 9.2 (c) and 11.2 lg/cm2 (d) after platelet adhesion.

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solution. Results show the liquid–solid interface aminolysis approach is effective and controllable for the introduction of amino groups. Moreover, the mechanical properties of the PES membranes are substantially reserved even after long time aminolysis. The surface aminolysis route opens a door for the modification or functionalization of PES materials. Heparin chains are covalently bonded onto the surface of PES membranes via amidation reaction, and high efficiency and excellent stability are observed. The control of surface amino group concentration is crucial, for it determines the quantity and bioactivity of immobilized heparin chains. Both the top surface and the inner pore walls of the PES membranes are heparinized, which increases the wettability remarkably. Due to the intrinsic hydrophilicity and electro-negativity of heparin chains, the Hep-PES membranes exhibit strong resistance against protein and platelet adsorption/adhesion. The bioactivity of heparin is reserved after covalent bonding, rendering the Hep-PES membranes with improved anticoagulation property. The enhanced hemocompatibility of PES membranes suggests their potential practical applications in blood purification and other blood contacting fields. Acknowledgments The authors would like to gratefully acknowledge the financial support of the National 973 program (Grant Number 2009CB623402) and the Nature Science Foundation Committee (Grant Number 20974094) of China. References [1] R.N. Johnson, A.G. Farnham, R.A. Clendinning, W.F. Hale, C.N. Merriam, J. Polym. Sci. Part A-1: Polym. Chem. 5 (1967) 2375–2398. [2] S. Stannat, J. Bahlmann, D. Kiessling, K. Koch, H. Deicher, H. Peter, Contrib. Nephrol. 46 (1985) 102–108. [3] E. Streicher, H. Schneider, Contrib. Nephrol. 46 (1985) 1–13. [4] R. Schaefer, A. Heidland, W. Hörl, Contrib. Nephrol. 46 (1985) 109–117. [5] M. Amiji, K. Park, J. Biomater. Sci., Polym. Ed. 4 (1993) 217–234. [6] H. Wang, T. Yu, C. Zhao, Q. Du, Fibers Polym. 10 (2009) 1–5. [7] L.-P. Zhu, J.-Z. Yu, Y.-Y. Xu, Z.-Y. Xi, B.-K. Zhu, Colloids Surf., B 69 (2009) 152– 155.

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