Advanced Materials for Membrane Separations - American Chemical

Chapter 25

Downloaded by UNIV MASSACHUSETTS AMHERST on September 12, 2012 | http://pubs.acs.org Publication Date: April 20, 2004 | doi: 10.1021/bk-2004-0876.ch025

Chemically Modified Polysulfone Hollow Fibers with Zwitterionic Sulfoalkylbetaine Group Having Improved Blood Compatibility 1

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Akon Higuchi , Hirokazu Hashiba , Rika Hayashi , Boo Ok Yoon , Mitsuo Hattori , and Mariko Hara 2

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Department of Applied Chemistry, Seikei University, Tokyo 180-8633, Japan Health Care Center, Seikei University, Tokyo 180-8633, Japan

Hydrophilic polysulfone membranes (SPE-PSf) were prepared by covalently bonding poly(sulfoalkylbetaine) (SPE) on the surface. The immobilized amount of SPE on SPE-PSf membranes was controlled by the SPE monomer concentration in the reaction solution. The SPE-PSf membranes were significantly more hydrophilic than unmodified polysulfone or other surface-modified polysulfone membranes due to the long hydrophilic side chain of SPE, which contributes to the hydrophilic nature of the modified PSf membranes. SPE-PSf membranes showed lower protein adsorption from a plasma solution than polysulfone and other surface-modified membranes due to the highly hydrophilic surface of the SPEPSf membranes. The SPE-PSf membranes showed significantly lower number of adhering platelets on its surface than polysulfone and other surface-modified membranes. The hydrophilic surface of SPE-PSf membranes may suppress of platelet adhesion on the SPE-PSf membranes.

366

© 2004 American Chemical Society

In Advanced Materials for Membrane Separations; Pinnau, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


367 Polysulfone hollow fibers blended with poly(vinylpyrrolidone) [PVP] have been widely used as hemodialysis membranes, because the pore size and pore distribution of the polysulfone membranes can be easily controlled by changing the composition of the polysulfone casting solution. These polysulfone membranes can effectively remove low-molecular weight proteins, such as ffemicroglobulin ( M W = 11,500 g/mol) and endotoxin ( M W = 5,000 - 20,000 g/mol). Because polysulfone itself has poor blood compatibility, modification of the polysulfone membranes is required for use as hemodialysis membranes. Polysulfone membranes blended with P V P are currently used as hemodialysis membranes in the medical industry, because P V P is a hydrophilic polymer having neither a hydroxyl group nor ionically charged groups and, therefore, shows good blood compatibility (7). Other types o f modification have also been reported for improving the blood compatibility of polysulfone membranes (2-4). Ishihara et al. prepared a phospholipid polymer having a 2-methacryloyloxyethyl phosphorylcholine (MPC) unit (2). The M P C polymer containing a zwitterionic group could be blended with polysulfone by a solvent evaporation method during membrane processing. The number of platelets adhering to the polysulfone membranes blended with the M P C polymer was reduced, and the change in the morphology of adherent platelets was suppressed (2). Another typical zwitterionic group is iV,iV-dimethyl-iV-methacryloxyethyli^-(3-sulfopropyl)ammonium betaine (SPE). Viklund and Irgum prepared a porous sulfoalkylbetaine polymer with zwitterionic S P E , which showed reversible protein adsorption relative and was used as a chromatographic monolith for protein separation (5). Surface-modified polysulfone hollow fibers having several hydrophilic groups were previously prepared in our laboratory (6-9) and by other researchers (3,4,10). A variety of hydrophilic groups such as - C H 2 C H 2 C H 2 S O 3 " , - C H ( C H ) C H O H , - C H N ( C H C H ) 3 , - C H 2 N H C H 2 C H 2 N H 2 and - C H O H were introduced on the surface of the hydrophobic polysulfone membranes by chemical reaction (6-9). We recently succeeded in introducing an aliphatic double bond on the surface o f polysulfone hollow fibers using iV-succinimidylacrylate (77). In this study, the polymerization of zwitterionic monomer of SPE on the surface of polysulfone hollow fibers is reported. Platelet adhesion on polysulfone membranes conjugated with SPE polymer on the surface in human plasma was investigated. The blood compatibility of the modified membranes was evaluated by comparison of unmodified and other surface-modified polysulfone membranes. +

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Experimental Materials The membranes used for chemical modification were commercially available polysulfone (PSf) ultrafiltration hollow fibers (SI-1, Asahi Chemical


368 Co., Ltd.). The inside and outside diameters of the fibers were approximately 0.75 mm and 1.3 mm, respectively. The zwitterionic monomer JViJV-dimethyl-JVmethacryloxyethyl-iV-(3-sulfopropyl) ammonium betaine (SPE) was kindly suppliedfromDr. P. Koeberle (Rasching Chemie GmbH). Mouse monoclonal anti-human albumin (300-06551, Nippon Bio-Test Laboratories, Inc., Tokyo), mouse monoclonal anti-human fibrinogen (F4639, Sigma-Aldrich, Inc., MO), and goat anti-human fibronectin (F1509, SigmaAldrich, Inc., MO) were used as primary antibodies. Goat F(ab) anti-human immunoglobulin peroxidase conjugate antibody (AHI1304, Biosource International, CA), rabbit H+L anti-mouse immunoglobulin peroxidase conjugate antibody (014-17611, Wako Pure Chemical Industries, Ltd., Tokyo), and rabbit anti-goat immunoglobulin peroxidase conjugate antibody were used as secondary antibodies. Block Ace™ (UK-B80) was purchased from Funakoshi Co., Ltd. TMB (3,3,5,5-tetramethylbenzidine) peroxidase substrate (TMB Microwell Peroxidase Substrate System, 50-76-00, Kirkegaard & Perry Laboratories, Guildford UK) was used as received. Other chemicals were of reagent grade and were used without further purification. Ultrapure water was used throughout the experiments.


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Synthesis ofNSA The active ester of Af-succinimidylacrylate (NSA) was synthesized from Nhydroxysuccinimide and acryloyl chloride following the method of Adalsteinsson et al (12-14). The recovered yield was 65±8%.

Surface Chemical Modification of PSf Membranes Chloromethylation of the PSf membranes (Cl-PSf) was performed by dipping in a solution of chlorodimethyl ether, hexane, and SnCU as the FriedelCrafts catalyst at 25 °C for 15 min as described previously (9,11) (Scheme 1). The apparatus for the chemical modification of the membranes was described recently (6,9\ and the same procedures for the chemical modification were employed in this study. Ethylenediamination of the chloromethylated PSf membranes (i.e., Scheme 1) was performed by dipping the chloromethylated membranes into ethylenediamine for 15 min at 25 °C. Ethylenediaminated PSf membranes (EDA-PSf, fiber length = 100 cm) were immersed in 250 ml of phosphate buffer solution (0.02 M , PBS, pH 7.4) containing 0.327 g of NSA for the preparation of NSA-PSf-1 membrane, 0.1635 g of NSA for NSA-PSf-2 membrane, and 0.109 g of NSA for NSA-PSf-3 membrane at 37 °C. The reaction was then incubated at 37 °C for one hour to



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25%, 15 min

EDA

C H O C H C l , SnCU, Hexane 25%, 15 min

Polysulfone (PSf)

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CH NHCH CH NH

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Scheme 1

Ethylenediamination of chloromethylated polysulfone (EDA-PSf)

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CHi ( a Chloromethylated polysulfone (Cl-PSf)


On SO

In Advanced Materials for Membrane Separations; Pinnau, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004. 2

f

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/==\

2

H

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Sulfoalkylbetaine (SPE)

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2

h

C

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(CH2) SO3"

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(C»2)2 H3C-N-CH3

: H N H C H CH2N-C-S CH N CH2NHCH HO 2

H6

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6 ° NSA

:H NHCH CH NC C=CH

Scheme 2

H3C 0 CH H C=C-C-0-(CH^ -N-(CH2)3-S03 CH

AAm, 0.1M APS 0.8M TEMED

SPE, 2S"C, 3hrs

PBS(0.02M,pH7

3tfC, lhr

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CH NHCH CH NH

CH 2

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so;

(CHj) H3C-N-CH3 (CHj)3

o

CHA


OH


371 introduce vinyl groups onto the surface of the membranes (Scheme 2). After the reaction, the membranes conjugated with N S A were washed in a phosphate buffer solution for one hour and then in water for one hour. The N S A conjugated membranes (NSA-PSf) were immersed in 150 ml of phosphate buffer solution containing 10, 200, 500, 1,000, 2,000 or 3,000 moles of SPE to N S A moles of the N S A - P S f membranes together with 0.01 ml of 0.1 M aqueous ammonium persulphate (APS) and 0.01 ml of 0.8 M aqueous n,n,n\n'~ tetramethylethylenediamine ( T E M E D ) as redox initiators. Polymerization of SPE was performed at 25 °C on the surface of the membranes for 3 hours (SPEPSf, see Scheme 2). SPE-PSf-X-Y ( X = l , 2 or 3 and Y=10, 200, 500, 1000, 2000 or 3000) indicates SPE-PSf membranes prepared from N S A - P S f - X membranes using Y moles of SPE to N S A moles of the membranes.

Characterization of Surface-modified PSf Membranes The degree of chloromethylation of the C l - P S f membranes was measured by ' H - N M R (400 M H z , J N M G X - 4 0 0 , J E O L , Ltd.). The degree of ethylenediamination of the E D A - P S f membranes was estimated using a standard titration method ( i i ) . The advancing and receding waterlcontact angles (11,15) were measured in air at 25±2 °C using a Langmuir-Blodgett trough ( N L LB200S-NWC, Nippon Laser & Electronics Lab.) as described previously ( i i ) .

Protein Adsorption Assay on the Membranes Small samples of PSf membranes and surface-modified P S f (Cl-PSf, E D A PSf, N S A - P S f and SPE-PSf) membranes were cut into separate membranes and were immersed in a phosphate buffer solution (PBS, 0.02M, p H 7.4) of (1) 5,000 ppm bovine serum albumin (BSA), (2) 5,000 ppm bovine v-globulin, (3) 300 ppm fibrinogen or (4) 50% platelet-poor plasma (PPP) containing 0.15 mol/L N a C l for 120 min at 37 °C. The membranes were then rinsed five times with PBS. The membranes were then inserted into a glass tube containing 1 wt% aqueous solution of sodium dodecyl sulfate (SDS) and the glass tubes containing the membranes and SDS solution were shaken for 60 min at room temperature to remove the proteins adsorbed on the membranes. The amount of proteins adsorbed on the membrane surface was calculated from the concentration o f proteins in the SDS solution using a protein analysis kit (Micro B C A protein assay reagent kit) (2). Specific protein adsorption from human plasma on the membranes was evaluated using the antigen-antibody reaction using enzyme-immunoglobulin conjugate as described previously ( i i ) .


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Platelet Adsorption on the Membranes A 35 ml sample offreshhuman blood (woman, 32 years old) was collected using five vacuum tubes (7 ml, Venoject II, Terumo, Co.) containing 10.5 mg of EDTA*2Na and centrifuged at 1000 rpm for 10 min to obtain platelet-rich plasma (PRP) or at 2800 rpm for 15 min to obtain platelet-poor plasma (PPP) (2,16,17). Fresh PRP or PPP samples were used in all the studies. PSf and the modified membranes were cut, placed into 24-well tissue culture plates and equilibrated with 0.15 mol/L NaCl solution at 37 °C for one hour. The 0.15 mol/L NaCl solution was removed and then 1 ml of fresh PRP was introduced. The membranes were incubated with PRP at 37 °C for two hours. PRP was decanted, and the membranes were rinsed again with 0.15 mol/L NaCl solution. Finally, the membranes were treated with 3 wt% glutaraldehyde in 0.15 mol/L NaCl solution for two days at 4 °C. The samples were washed again with 0.15 mol/L NaCl solution, subjected to a drying process by passing them through a series of graded alcohol-NaCl solutions (0, 25, 50, 75, and 100 %) and dried in vacuum for 10 hours at room temperature. The dried membranes were sputtercoated with gold and were examined using a JSM-5200 scanning electron microscope (SEM, JEOL, Ltd.). The number of adhering platelets on the membranes was calculated from 4 SEM pictures at a magnification of 500 from different places on the same membranes. These procedures were performed on each membrane using four independent membranes (totally n = 16), and the results were averaged to obtain reliable data.

Results and Discussion Chemical Modification of PSf Chloromethylation and ethylenediamination of the PSf membranes were performed as previously reported (see Scheme 1) (9). The degree of chloromethylation was estimated to be 2.6% of PSf repeat unitsfromthe peak of 'H-NMR spectra at 6=4.5 ppm. The degree of ethylenediamination was estimated from the ion-exchange capacity of the EDA-PSf membranes and was 0.8% of PSf-repeat units. The immobilized amount of NSA on PSf (NSA-PSf, see Scheme 2) membranes was estimated from the amount of NSA consumed in the NSA reaction solution detected by U V absorption at 275 nm and was 0.88±0.26 ^mol/cm (NSA-PSf-1), 0.18±0.05 *imol/cm (NSA-PSf-2) and 0.10±0.03 (imol/cm (NSA-PSf-3) depending on the composition of NSA in the reaction solution (i.e., 0.13, 0.065, and 0.044 g/100 ml of PBS, respectively). The immobilized amount of SPE on PSf (SPE-PSf, see Scheme 2) membranes was also estimated from the amount of SPE consumed in the SPE reaction solution detected by UV absorption at 260 nm. The immobilized amount of SPE 2

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373 on PSf (i.e., SPE-PSf) membranes was controlled by the amount of its monomer (i.e., vinylpyrrolidone) in the reaction solution. Figure 1 shows the dependence of the polymerized number of SPE (JV) on the molar ratio of SPE to N S A [C(SPE)/C(NSA)] of NSA-PSf-1 in the reaction solution when the reaction time was 3 h. The polymerized number of SPE was calculated from the immobilized SPE amount divided by the immobilized N S A amount on the SPE-PSf membranes and was an average number for the polymerization of SPE with the SPE-PSf membranes. The polymerized number of S P E increased with the concentration of S P E in the reaction solution. However, the polymerized number of S P E tended to reach a saturated polymerization number at a high molar ratio of SPE to N S A .

Water Contact Angles of Surface-modified PSf Membranes The advancing and receding water contact angles on P S f and surfacemodified PSf membranes were measured. The advancing water contact angle of SPE-PSf was the same as that of PSf or other surface-modified PSf membranes (i.e., 89-90±3 degrees), while the receding water contact angle of SPE-PSf (i.e., 26±2 degrees) was smaller than that o f P S f or the other surface-modified P S f membranes (i.e., 3 2 - 3 4 ± 2 degrees). This result indicates that the SPE-PSf membranes were more hydrophilic than P S f and other surface-modified membranes prepared in this study. This can be explained by the long hydrophilic side chain of SPE, which contributes to formation of the hydrophilic surface of the PSf membranes.

Protein Adsorption on Surface-modified PSf Membranes Plasma protein adsorption on materials is a key phenomenon during thrombogenic formation. The amount of plasma proteins adsorbed on the materials has been reported to be one of the most important factors in evaluating the blood compatibility of materials (2). Figure 2 shows the adsorption of single proteins on P S f and surfacemodified P S f membranes from albumin, y-globulin or fibrinogen solution containing 0.15 mol/L N a C l at p H 7.4. It was found that the SPE-PSf membranes had a lower protein adsorption o f albumin, Y-globulin, and fibrinogen than the PSf, Cl-PSf, EDA-PSf, and N S A - P S f membranes. This result can be attributed to the hydrophilic surface of the SPE-PSf membranes, because a hydrophilic surface is known to reduce the protein adsorption on the membranes (5-5). SPE-PSf-1-500 showed the best characteristics for antiprotein adsorption. This is because SPE-PSF-1-500 (27 p m o l / c m ) had significantly more immobilized S P E than SPE-PSF-2-500 (1.3 pmol/cm ) or SPE-PSF-3-500 (0.62 pmol/cm ). 2

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0 . 1 1 — — — i — — • ••••• i . . . . I 0 1000 2000 3000 4000

C(SPE)/C(NSA-PSf) Figure I. Dependence ofpolymerized number of SPE on the concentration molar ratio of SPE to NSA [C(SPE)/C(NSA)] of NSA-PSf 1 in the reaction solution for a reaction time of 3 hr. Data are expressed as the means±S.D. of four independent measurements.

Figure 2. Adsorption of single proteins on PSf and some surface-modified PSf membranesfromBSA, y-globulin orfibrinogen solutions containing 0.15 mol/L NaCl at pH 7.4. Data are expressed as the means±S.D. of four independent measurements.



375 Figure 3 shows the amount of total plasma proteins on P S f and surfacemodified P S f membranes from platelet-poor plasma. Clearly, the SPE-PSf membranes showed lower protein adsorption than the PSf, Cl-PSf, E D A - P S f and N S A - P S f membranes. The specific plasma protein adsorption on the membranes was also investigated using E L I S A assay. Figure 4 shows the standardized amount of adsorbed proteins (albumin, globulin, fibrinogen, and fibronectin) on P S f and surface-modified PSf membranes from platelet-poor plasma. The adsorption of specific plasma proteins on the membranes from PPP (i.e., Figure 4) showed a different amount measured from a single protein solution (Figure 2). This is because the specific protein adsorption on the membranes is influenced by other proteins co-existing in the plasma solution, and co-operative binding of each protein on the membranes from PPP. SPE-PSf membranes showed significantly lower adsorption of albumin from P P P , while slightly reduced adsorption of globulin, fibrinogen and fibronectin on SPE-PSF-1-500 from PPP was observed relative to that on PSf, Cl-PSf, E D A - P S f and N S A - P S f membranes. In summary, the SPE-PSf membranes showed lower protein adsorption of each of the proteins in PPP evaluated from single protein solution (Figure 2) and from PPP (Figures 3 and 4).

Platelet Adhesion on Surface-modified PSf Membranes Platelet adhesion on the membranes from human plasma is an important test for the evaluation of the blood compatibility of the membranes. Platelet adhesion on PSf and surface-modified PSf membranes from platelet-rich plasma was investigated. A typical scanning electron micrograph of platelets adhering to PSf, EDA-PSf, NSA-PSf-1, and SPE-PSf-1-500 membranes is shown in Figure 5. Platelets were rarely observed on SPE-PSf-1-500 membranes, while numerous platelets were observed on P S f and E D A - P S f membranes. The activated platelets on E D A - P S f membranes showed spread and deformed shapes. Figure 6 shows the number of platelets adhering to PSf, SPE-PSf, and other surface-modified P S f membranes using platelet-rich plasma. The SPE-PSf-1500 (immobilized amount o f S P E = 27 jimol/cm ), SPE-PSf-1-1000 (immobilized amount of SPE = 35 pmol/cm ), SPE-PSf-1-2000 (immobilized amount of SPE = 45 ^mol/cm ), SPE-PSf-1-3000 (immobilized amount of SPE = 130 jimol/cm ), SPE-PSf-2-500, and SPE-PSf-3-500 membranes showed a reduced number of platelets adhering to the surface compared to PSf, Cl-PSf, EDA-PSf, and N S A - P S f membranes. More than 27 Mmol/cm of SPE should be immobilized on the SPE-PSf membranes in order to show the characteristic surfaces for extensively suppressed adhesion of platelets, although SPE-PSf-2500 and SPE-PSf-3-500 showed suppressed platelet adhesion and have only 0.6 - 1.3 jtmol/cm of SPE. The different synthesis conditions of SPE on the SPE2

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Figure 3. Adsorption of total plasma proteins on PSf and some surfacemodified PSf membranesfromPPP at pH 7.4. Data are expressed as the means±S.D. of four independent measurements.



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Figure 4. Standardized amount of adsorbed proteins (albumin, globulin, fibrinogen and fibronectin) on PSf and surface-modified PSf membranes from PPP evaluatedfrom ELISA assay. Data are expressed as the means±S.D. of five independent measurements.



(a) PSf (b) EDA-PSf


QO


(d) SPE-PSf-1-500

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Figure 5. Scanning electron micrograph of platelets adhering to (a) PSf (b) EDA-PSf, (c) NSA-PSf-1 and (d) SPE-PSf-1-500 membranes.

(c) NSA-PSf. 1


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Figure 6. The number of adhering platelets on PSf SPE-PSf and other surfacemodified PSf membranesfromplatelet rich-plasma estimated by SEM. Data are expressed as the means±S.D. of 16 measurements.



381 PSf membranes might be contributing to the homogeneous or heterogeneous distribution of the length and location of S P E on the surface of SPE-PSf membranes. It is suggested that the hydrophilic surface of SPE-PSf membranes caused the suppression of platelet adhesion on SPE-PSf membranes. The suppression of platelet adhesion on membranes is generally believed to be due to the reduction in protein adsorption, especially the suppression of fibrinogen adsorption (2,18). The fibrinogen adsorption from plasma onto membranes is known to regulate the adhesion of platelets because the fibrinogen causes the glycoprotein, GP Ilb-IIIa, of the platelet membranes to bind (2,19). SPE-PSf membranes showed a slightly lower amount of fibrinogen adsorption compared to PSf membranes based on the evaluation of the E L I S A assay of PPP (i.e., Figure 4). The platelets are less adsorbed on SPE-PSf membranes than on PSf and other surface-modified membranes.

Conclusions Polysulfone membranes covalently conjugated with zwitterionic sulfoalkylbetaine on the surface were prepared. The immobilized amount of SPE on the SPE-PSf membranes could be controlled by the concentration of SPE monomer in the reaction solution. The SPE-PSf membranes were significantly more hydrophilic than P S f and other surface-modified P S f membranes. This results from the long hydrophilic side chain of SPE which determines the hydrophilic nature of the modified P S f membranes. The SPEPSf membranes had a lower protein adsorption from plasma solution than PSf, Cl-PSf, EDA-PSf, and N S A - P S f membranes. This can be attributed to the hydrophilic surface of the SPE-PSf membranes (5-8). The SPE-PSf membranes showed a lower number of platelets adhering on the surface than the PSf, C l PSf, EDA-PSf, and N S A - P S f membranes. It is suggested that the hydrophilic surface of the SPE-PSf membranes caused suppression of platelet adhesion.

References 1. 2. 3. 4. 5. 6.

Tanaka, K . ; Kobayashi, T. Japanese Patent (Tokkaihei) 4-300636, 1992. Ishihara, K . ; Fukumoto, K . ; Iwasaki, Y . ; Nakabayashi, N . Biomaterials 1999, 20, 1553-1559. Ulbricht, M . ; Belfort, G. J. Membrane Sci. 1996, 111, 193-215. Pieracci, J.; Crivello, J. V.; Belfort, G . J. Membrane Sci. 1999, 156, 223240. Viklund, C.; Irgum, K . Macromolecules 2000, 33, 2539-2544. Higuchi, A.; Iwata, N . ; Tsubaki, M . ; Nakagawa, T. J. Appl. Polym. Sci. 1988, 36, 1753-1767.


382 7. 8. 9.


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Higuchi, A.; Iwata, N.; Nakagawa, T. J. Appl Polym. Sci. 1990, 40, 709717. Higuchi, A.; Nakagawa, T. J. Appl. Polym. Sci. 1990, 41, 1973-1979. Higuchi, A.; Koga, H.; Nakagawa, T. J. Appl. Polym. Sci. 1992, 46, 449457. Nabe, A.; Staude, E.; Belfort, G. J. Membrane Sci. 1996, 133, 57-72. Higuchi, A.; Shirano, K.; Harashima, M . Yoon, B.-O.; Hara, M.; Hattori, M.; Imamura, K. Biomaterials, 2002, 23, 2659-2666. Miyata, T.; Asami, N.; Uragami, T. Nature, 1999, 399, 766-769. Shoemaker, S. G.; Hoffman, A. S.; Priest, J. H. Appl. Biochem. Biotech., 1987, 15, 11-24. Adalsteinsson, O.; Lamotte, A.; Baddour, R. F.; Colton, C. K.; Pollak, A.; Whitesides, G. M. J. Mol. Cat. 1979, 6, 199-225. Marchant, K. K.; Veenstra, A. A.; Marchant, R. E. J. Biomed. Mater. Res. 1996, 30, 209-220. Bahulekar, R.; Tamura, N.; Ito, S.; Kodama, M . Biomaterials 1999, 20, 357362. Kawakami, H.; Nagaoka, S.; Kubota, S. ASAIOJ. 1996, 42, M871-M876. Park, K.; Mao, F.W.; Park, H. J. Biomed. Mater. Res. 1991, 25, 407-420. Phillips, D. R.; Charo, I.F.; Parise, L. V.; Fitzgerald, Blood 1988, 71, 831843.


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