phospholipid polymer having a 2-methacryloyloxyethyl phosphorylcholine (MPC) moiety was reduced compared to the amount of protein adsorbed onto poly[2- ...
Why do phospholipid polymers reduce protein adsorption? Kazuhiko Ishihara,1 Hiroto Nomura,1,2 Takashi Mihara,1,2 Kimio Kurita,2 Yasuhiko Iwasaki,1 Nobuo Nakabayashi1 1 Division of Organic Materials, Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan 2 Department of Engineering Chemistry, College of Science and Technology, Nihon University, 1-8-1 Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan Received 21 October 1996; accepted 26 February 1997 Abstract: The amount of plasma protein adsorbed on a phospholipid polymer having a 2-methacryloyloxyethyl phosphorylcholine (MPC) moiety was reduced compared to the amount of protein adsorbed onto poly[2-hydroxyethyl methacrylate (HEMA)], poly[n-butyl methacrylate (BMA)], and BMA copolymers with acrylamide (AAm) or N-vinyl pyrrolidone (VPy) moieties having a hydrophilic fraction. To clarify the reason for the reduced protein adsorption on the MPC polymer, the water structure in the hydrated polymer was examined with attention to the free water fraction. Hydration of the polymers occurred when they were immersed in water. The differential scanning calorimetric analysis of these hydrated polymers revealed that the free water fractions in the poly(MPC-co-BMA) and poly(MPC-con-dodecyl methacrylate) with a 0.30 MPC mole fraction were above 0.70. On the other hand, the free water fractions in the
poly(HEMA), poly(AAm-co-BMA), and poly(VPy-co-BMA) were below 0.42. The conformational change in proteins adsorbed on the MPC polymers and poly(HEMA) were determined using ultraviolet and circular dichroism spectroscopic measurements. Proteins adsorbed on poly(HEMA) changed considerably, but those adsorbed on poly(MPC-coBMA) with a 0.30 MPC mole fraction differed little from the native state. We concluded from these results that fewer proteins are adsorbed and their original conformation is not changed on polymer surfaces that possess a high free water fraction. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 39, 323–330, 1998.
INTRODUCTION
unique property of the hydrated MPC polymer is that the degree of hydration of poly[MPC-co-n-butyl methacrylate (BMA)] saturated with water increases with increasing temperature.8 With this phenomenon, the diffusion rate of the solutes in the MPC polymer also increase.9–11 This result suggests that the free water fraction increases with an increase in temperature.12 In normal amphiphilic polymers, including poly[2hydroxyethyl methacrylate (HEMA)], the degree of hydration decreases with increasing temperatures up to 60°C because the interaction between the hydrophobic part of the polymer strengthens and the polymer chains shrink.8 We suggest that the state of water molecules around the MPC polymer chains is quite different from that of the general amphiphilic polymers. In this study the water structure in hydrated MPC polymers was investigated with attention to the free water and compared with that in other nonionic amphiphilic polymers. The effects of water structure on protein adsorption, not only on the amount but also on the conformation of the proteins adsorbed on the MPC polymer, will be discussed.
We have reported that 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers show excellent blood compatibility.1–5 Suppression of clot formation following platelet adhesion and activation was observed even when the MPC polymer came in contact with human whole blood without anticoagulants.3 This is due to the reduced protein adsorption on the MPC polymer surfaces even from human plasma.2,3 Other research groups also have observed the protein adsorption-resistant properties of MPC copolymers.6,7 We hypothesized that these characteristics observed in the MPC polymer system are closely related to the state of water molecules around the polymer. A
Correspondence to: K. Ishihara Contract grant sponsor: The Cosmetology Foundation (1995) © 1998 John Wiley & Sons, Inc.
CCC 0021-9304/98/020323-08
Key words: phospholipid polymer; protein adsorption; conformational change; water structure; hydration; free water
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MATERIALS AND METHODS Materials MPC was synthesized by the method reported previously.8 BMA and n-dodecyl methacrylate (DMA) were purchased from Tokyo Kasei Industry, Co., Ltd., (Tokyo, Japan) and purified by distillation under reduced pressure. The fractions of BMA and DMA used in this study were 68.5°C/ 24 mmHg and 123°C/1.0 mmHg, respectively. Acrylamide (AAm, Kanto Chemical, Tokyo, Japan) was purified by recrystallization from benzene. N-Vinyl pyrrolidone (VPy, Nacalai Tesque, Tokyo, Japan) was purified by distillation under reduced pressure in argon atmosphere, and the fraction of bp 67.5°C/3 mmHg was used. Polymerization of MPC with BMA or DMA was carried out in ethanol using a, a8azobisisobutyronitrile (AIBN) as an initiator.8,13 The MPC polymers obtained were purified by the precipitation method using diethyl ether for poly(MPC-co-BMA) and acetone for poly(MPC-co-DMA) as precipitation solvents. Poly(AAm-co-BMA) (PAB70) and poly(VPy-co-BMA) (PVB90) were synthesized by the same method used for the MPC polymer synthesis. Poly(HEMA) was synthesized by the radical polymerization of HEMA (Nacalai Tesque, Tokyo, Japan; bp = 62°C/3.5 mmHg) in 2-propanol using AIBN as an initiator, and it then was purified by reprecipitation in diethyl ether from its 2-propanol solution.14 The chemical structure of these polymers is indicated in Figure 1. The structure of these polymers was confirmed by FTIR (Jasco FT/IR-500, Tokyo, Japan) and 1H-NMR (Jeol a-500, Tokyo, Japan). The molecular weight of the polymers was evaluated by gel-permeation chromatography (GPC, Tohso GPC system, Tokyo, Japan). The eluents were an ethanol/chloroform mixture (2/8) for the MPC polymers and N,N-dimethylformamide (DMF) for PAB70, PVB90, and poly(HEMA). Calibration was carried out from the elution time of the GPC curve using poly(oxyethylene) standards for the MPC polymers and polystyrene standards for the other polymers. The MPC unit mole fraction was determined by phosphorus analysis. The AAm and VPy unit mole fractions were deter-
mined by an elemental analysis of nitrogen. Results of the polymerization are summarized in Table I. Proteins, bovine serum albumin (BSA), and bovine plasma fibrinogen (BPF) were purchased from Sigma, Co., Ltd., USA, and used without further purification.
Preparation of polymer membrane and determination of water fraction in the membrane Polymer membranes were prepared by a solvent evaporation method.8,13 The MPC polymers were dissolved in ethanol at 10 wt% concentration, and the polymer solution was spread on a polyethylene (PE) plate to allow the solvent to evaporate at room temperature. In the case of PAB70 and PVB90, DMF was used as a solvent instead of ethanol, and the solvent evaporation temperature was 40°C. Every membrane obtained was dried under reduced pressure to eliminate residual solvent. The polymer membrane was immersed in water to equilibrate at 25°C, and the hydration degree in the membrane was determined from an increment of the membrane weight. The equilibrated degree of hydration (Heq) of the polymer membrane saturated with water was determined by the following equation: Heq =
(weight of water in the polymer membrane) (weight of polymer membrane saturated with water) (1)
The water structure in the saturated polymer membrane was estimated by differential scanning calorimetry (DSC, DSC-100, Seiko Instruments, Tokyo, Japan). DSC analysis was carried out between 50°C and −50°C at a cooling rate of 2.5°C/min. About 100 mg of hydrated polymer membrane equilibrated in water was placed in an aluminum pan for DSC analysis. The pan was sealed tightly to prevent water evaporation during the measurement. The exothermal peak of the hydrated membrane of around 0°C, which is attributed to melting of the frozen water, was compared with that of pure water, and the free water fraction was calculated. To adjust the degree of hydration of the polymer membrane at 0.36, 64 mg of polymer membrane was placed in the aluminum pan and 36 mg of water was added to the polymer membrane. The pan was sealed tightly and stored at 25°C for 10 days to swell the polymer membrane. The DSC measurement then was carried out. We confirmed that the polymer membrane was saturated within 7 days and that under this condition the degree of hydration became 0.36.
Determination of amount and conformation of adsorbed protein
Figure 1. study.
Structure of amphiphilic polymers used in this
The proteins were dissolved in phosphate-buffered solution (PBS, pH 7.4; ion strength, 0.15M). The concentrations of BSA and BPF were 0.45 g/dL and 0.03 g/dL, respectively, which is approximately 10% of their plasma level. In Figure 2 the procedure of protein adsorption measurement using both circular dichroism (CD) and ultraviolet
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TABLE I Synthetic Results of Polymers Used in This Study
Code PMB10 PMB30 PMD10 PMD30 PAB70 PVB90 Poly(HEMA)
Hydrophilic Monomer
Hydrophobic Monomer
MPC MPC MPC MPC AAm VPy HEMA
BMA BMA DMA DMA BMA BMA
Mole Fraction of Hydrophilic Unit
Time*
Conversion†
Mw‡
In Feed
In Polymer
(h)
(%)
105
0.10 0.30 0.10 0.30 0.70 0.90 1.0
0.07 0.28 0.12 0.31 0.58 0.81 1.0
10 10 6 6 4 7 2
40.1 52.6 41.1 51.7 30.7 22.9 18.7
1.3 2.1 2.0 3.1 1.8 1.1 4.2
*[Monomer] = 1.0M, [AIBN] = 5 mM at 60°C; †polymerization was carried out in ethanol for PMB10, PMB30, and PMD30; in chloroform/ethanol (3/7) for PMD10; in DMF for PAB70 and PVB90; and in 2-propanol for poly(HEMA); ‡determined by gel-permeation chromatography with polyoxyethylene standards for the MPC polymers, with polystyrene standards for PAB70, PVB90, and poly(HEMA). (UV) spectroscopies is demonstrated.15 Test polymers were coated on a quartz plate (40 mm × 9.5 mm × 1.0 mm) by a solvent evaporation method. The quartz plate was immersed in one of the test polymer solutions at 0.5 wt%. Ethanol was used as the coating solvent for PMB10, PMB30, and PMD30; 2-propanol for PMD10; DMF for PAB70 and PVB90; and methanol for poly(HEMA). Solvents were evaporated carefully under the same solvent vapor atmosphere at a given temperature, and the samples then were dried in vacuo at room temperature. The evaporation temperature of DMF was 40°C, and that of the other solvents was 25°C. The polymer-coated quartz plate was transparent and did not show UV absorption in the range between 190 nm and 600 nm except for PAB70 and PVB90. Though PAB70 and PVB90, unfortunately, showed considerable UV absorption below 240 nm, they were characterized by UV spectroscopy as having only an equilibrium amount of adsorbed protein. The polymer-coated quartz plate was immersed into the protein solution for 60 min at 37°C and rinsed five times using 5 mL of PBS for each rinse. After protein adsorption
the sample plate was placed in the CD spectrometer (Jasco J-720W, Tokyo, Japan), and the CD spectrum was recorded in the range between 205 and 350 nm. The spectrum was recorded 5 times for each sample plate and averaged automatically on a personal computer system. The UV absorbance of the protein-adsorbed sample plate then was measured immediately after CD spectroscopic measurement using a Jasco V-560 spectrophotometer with a double monochrometer (Tokyo, Japan) at 280 nm to determine the surface concentration of adsorbed protein after being rinsed 5 times. The mean molar residual ellipticity [u] at a specific wavelength was calculated from the results of CD and UV spectroscopic measurements. The secondary structure of the adsorbed protein was estimated from the [u] value. According to Greenfield and Fasman, the polypeptide, poly(L-lysine) chains having 100% a-helix content had a [u] value at 222 nm of −4.0 × 104.16 The a-helix content of the adsorbed protein was estimated using the following equation: a-helix (%) = [[u]222 * 100] / −4.0 × 104
(2)
Figure 2. Procedure for measurement of amount and conformation of protein adsorbed on polymer surface by UV–CD method.
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After these spectroscopic measurements for determination of the secondary structure of adsorbed protein were taken, the sample plate was immersed and shaken mildly 100 times in 500 mL of PBS for detachment of the loosely bound protein to determine the equilibrium surface concentration of adsorbed protein. The experiments were carried out three times for each polymer, and the mean value (± standard deviation) of each polymer is indicated. Comparative analyses were determined using analysis of variance (ANOVA) and the Student’s t test.
necessary to obtain almost the same degree of hydration. Figure 3 demonstrates DSC curves of the hydrated polymer membranes. In the case of pure water, the shape of the exothermic peak around 0°C, corresponding to the freezing of water, was observed (data not shown). The heat capacity during freezing was 332.2 mJ/mg. The DSC curve of hydrated poly(HEMA) had a main peak around 0°C (free water region), and a shoulder peak around −20°C also was observed. A small and broad peak in the temperature range between −20°C and −40°C (intermediate water region) was observed in PAB70 and PVB90. Each MPC polymer had only one peak near 0°C. The exothermic peak around 0°C was assumed to be due to free water (bulk water), and the free water fraction in the hydrated polymer membrane was calculated from the Heq and heat capacity during freezing. As indicated in Table II, free water fractions in PMB30 and PMD30 at the equilibrium hydration state were extremely large compared with those of the other hydrated polymer membranes tested. In Table II, the free water fraction in the hydrated membrane, the hydration degree of which was adjusted to 0.36, also is summarized for comparison of the free water fraction under the same degree of hydration. The free water fractions in the PMB30 and PMD30 polymers were 0.69 and 0.62, respectively. On the other hand, the free water fractions in poly(HEMA), PAB70, and PVB90 were less than 0.42. Thus it appears that the hydrated MPC polymer had a large amount of free water.
RESULTS Characterization of polymer samples Table I summarizes the results of polymer synthesis. The MPC unit mole fraction in the polymer was the same as that in the monomer feed. Moreover, the molecular weight of every MPC polymer exceeded 105. The prepared MPC polymer could be dissolved in ethanol, and PMD, having a longer alkyl methacrylate unit, also dissolved in chloroform. However, each MPC polymer was insoluble but became swollen and assumed a hydrogel state in water. The PAB70 and PVB90 also adsorbed water and assumed a hydrogel state.
Water state in hydrated polymer membrane Protein adsorption
Table II indicates the Heq and the fraction of the free water in the various polymer membranes. The Heq increased with increases in the MPC mole fraction. Compared to the MPC polymers, for both PAB70 and PVB90 a higher hydrophilic unit in the polymer was
The equilibrium amount of proteins adsorbed on the polymer surface is summarized in Table II. The amounts of both proteins adsorbed on poly(HEMA),
TABLE II Characteristics of Hydration State of Polymers and Protein Adsorption on Polymer Surfaces PMB
Heq* Free water fraction at Heq at H = 0.36 Equilibrium amount of adsorbed protein (mg/cm2)† BSA BPF
PMD
Poly(HEMA)
10
30
10
30
0.40 0.39‡
0.23
0.84
0.19
0.34 (0.34)‡ 0.28
0.25
0.84
—
0.69
1.7 ± 0.7 3.4 ± 0.1
0.50 ± 0.2§ 2.0 ± 0.5§
0.22 ± 0.1§,\ 1.1 ± 0.2§,\
PAB70
PVB90
0.70
0.36
0.41
0.22
0.70
0.42
0.39
—
0.62
0.42
0.31
0.51 ± 0.2§ 2.0 ± 0.4§
0.35 ± 0.1§,\ 1.2 ± 0.3§,\
1.3 ± 0.2\ 2.3 ± 0.4§
1.8 ± 0.3\ 2.6 ± 0.3§
*Heq = (weight of water in the polymer membrane)/(weight of polymer membrane saturated with water) at 25°C; †initial concentration of proteins in PBS: [BSA] = 0.45 g/dL, [BPF] = 0.03 g/dL; ‡The values in literature (Ref. 34); The values indicated here are mean ± SD for three experiments; §p < 0.01 vs. poly(HEMA); \p < 0.01 vs. PMB10.
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decrease in MPC composition, then became almost zero in the case of BSA adsorbed on poly(HEMA). In the case of BPF adsorbed on the polymers, the same tendency was observed (data not shown). The secondary structure of the adsorbed proteins was determined to calculate the a-helix content. Figure 5 demonstrates the a-helix content of protein adsorbed on the polymer surface. The a-helix contents of BSA and BPF in PBS, which are assumed to be the ‘‘native’’ secondary structure, were 54% and 19%, respectively. When these proteins adsorbed on the polymer surface, changes in the a-helix content were observed. On the surface of PMB30 the a-helix content of both adsorbed BSA and BPF was almost the same as that of native proteins (p > 0.01). The proteins adsorbed on the MPC polymers could maintain their original higher a-helix level compared with those on poly(HEMA) (p < 0.01). Figure 3. DSC curves of hydrated polymer.
DISCUSSION PAB70, and PVB90 were larger than those on the MPC polymers (p < 0.01). The increase in MPC mole fraction reduced the amount of protein adsorption (p < 0.01). Figure 4 shows the CD spectra of BSA in PBS and that adsorbed on the polymer surface. In the case of BSA in PBS, the mean molar residual ellipticity, [u], was negative between 205 nm and 250 nm, and a large negative peak at 222 nm was observed. The CD spectrum of BSA adsorbed on PMB30 was almost the same as that in PBS. The negative ellipticity at 222 nm of BSA adsorbed on the MPC polymers increased with a
Figure 4. CD spectra of BSA adsorbed on polymer surface and in PBS.
Protein adsorption is one of the most important phenomena in determination of the biocompatibility of materials.17,18 In general, proteins adsorb onto a surface within a few minutes when the material contacts body fluids such as blood, plasma, and tears. Many investigations concerned with protein adsorption have been reported.17,18 Some methods to reduce protein adsorption also have been proposed. Among them, construction of a hydrophilic surface has been believed to be effective.19–21 Therefore, surface modification of materials with hydrophilic or a watersoluble polymer chain was investigated. Whether some modifications of hydrophilic polymers would be effective not only in obtaining blood compatibility but also in reducing protein adsorption was considered.22–25 The environment surrounding the biomolecules and polymeric materials used in our bodies is an aqueous medium. The characteristics of the water in the material or on the surface of the material are important in recognizing the interactions.26,27 Particularly, it is considered that the structure of water surrounding proteins and polymer surfaces influences protein adsorption behavior.28,29 We have investigated the blood compatibility of various phospholipid polymers.1–5 Polymers having a phosphorylcholine group, MPC polymers, suppress blood cell adhesion even when the polymers contact human whole blood without an anticoagulant.3 We hypothesized that the main reason for this phenomenon was less protein adsorption. In fact, MPC polymers could reduce protein adsorption from plasma or buffered solutions containing plasma.2,3,30,31 These protein adsorption-resistant properties were confirmed by other groups.6,7
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Figure 5. a-Helix content of BSA and BPF adsorbed on polymer surface and in PBS. * represents no significant difference versus the value for native protein in PBS (p > 0.01) and ** represents significant difference versus poly(HEMA) case (p < 0.01).
MPC polymers having less than 0.35 mole fraction of an MPC unit were insoluble in water but assumed a hydration state when the polymer was immersed in aqueous medium.8,13 However, the hydration state of the MPC polymer was quite different from that of other amphiphilic polymers such as poly(HEMA), poly(vinyl alcohol), and polymers having AAm units or 2-acrylamide-2-methyl propane sulfonate units.8 The temperature dependence of the degree of hydration of the MPC polymer is positive whereas that of the other amphiphilic polymers is negative. This phenomenon suggests that there is little interaction between the MPC polymer and water molecules. As shown in Table II, the Heq of PMB30 and PMD30 was higher than that of poly(HEMA). This also was indicated by the DSC result, as shown in Figure 3. Water molecules interacting with the polymer chain were frozen at a lower temperature compared with free water molecules. In the case of poly(HEMA), an exothermic reaction based on the freezing of water began at a lower temperature while PMB30 showed a sharp DSC peak around 0°C, which was close to that of pure water. This result indicated that PMB30 had a large amount of free water in the hydrated state. On MPC polymer surfaces, as indicated in Table II, protein adsorption was significantly reduced, in particular for PMB30. It was reported that theoretical amounts of BSA and BPF adsorbed on the surface in a monolayered state are 0.9 mg/cm2 and 1.7 mg/cm2, respectively.32 On the surface of PMB30, the amount of adsorbed proteins was less than these theoretical values. Thus it is possible that the phosphorylcholine
group can reduce protein adsorption effectively. We already have reported that poly(BMA) or poly(HEMA) surfaces, treated with phosphatidylcholine liposomal solution reduced protein adsorption compared to the original polymers.30 Water molecules bind at the hydrophobic part of the polymer through van der Waals force, which is the so-called ‘‘hydrophobic hydration.’’33,34 These bound water molecules cause protein adsorption by hydrophobic interaction. When a protein molecule is adsorbed on a polymer surface, water molecules between the protein and polymer need to be replaced.35 Protein adsorbed on the surface lost bound water at the surface-contacting portion. This phenomenon induces conformational change in the proteins, that is, the hydrophobic part of the protein is exposed and contacts the polymer surface directly. If the water state at the surface is similar to an aqueous solution, protein does not need to release bound water molecules even if protein molecules contact the surface. This means that the hydrophobic interaction does not occur between proteins and the polymer surface. Moreover, conformational change during protein adsorption on or contact with the surface also is suppressed. Recently, Tsuruta considered the importance of water structure on biomedical polymers. He described in his review article that polymers having a hydroxyl group such as poly(HEMA) can incorporate water molecules at the surface and form a network structure of water molecules.36 Protein adsorption starts with protein trapping by a network structure of water molecules on the surface. The longer the contact of a pro-
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tein on the surface, the greater is the chance of the protein’s interacting with the surface, undergoing a conformational change, and inducing irreversible adsorption. This is a highly probable explanation for the difference in protein adsorption behavior between MPC polymers and other amphiphilic polymers, including poly(HEMA). The CD spectrum of a protein adsorbed onto a polymer surface showed useful information about the conformational change during adsorption.15,37 The CD spectra of the BSA adsorbed on the surface indicated that the secondary structure did not change on PMB30 and that the a-helix content was at the same level as in the native state; however, it decreased by contact with poly(HEMA). We concluded that when the free water fraction on the polymer surface is kept at a higher level, proteins can contact the surface reversibly, without significant conformational change. The free water fraction must be one of the more important factors to consider in the blood compatibility of polymeric materials. Thus, phospholipid polymers having a phosphorylcholine group, such as MPC polymers, are effective biomedical materials and could be used to develop new bloodcontacting artificial organs.
acryloyloxyethyl phosphorylcholine with various vinyl monomers,’’ Polym. J., 26, 561–569 (1994). K. Ishihara, T. Ueda, and N. Nakabayashi, ‘‘Preparation of phospholipid polymers and their properties as hydrogel membrane,’’ Polym. J., 22, 355–360 (1990). K. Ishihara, T. Ueda, and N. Nakabayashi, ‘‘Drug release from hydrated membrane having phospholipid structure,’’ Kobunshi Ronbunshu, 46, 591–595 (1989). T. Ueda, K. Ishihara, and N. Nakabayashi, ‘‘Thermally responsive release of 5-fluorouracil from biocompatible hydrogel membrane with phospholipid structure,’’ Makromol. Chem.: Rapid Commun., 11, 345–348 (1990). T. Ueda, K. Ishihara, and N. Nakabayashi, ‘‘Temperature effect on drug release from poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) membrane,’’ Membrane, 17, 101–106 (1992). M. N. Sarbolouki, ‘‘Probing the state of adsorbed water by diffusion technique,’’ J. Appl. Polym. Sci., 17, 2407 (1973). T. Ueda, H. Oshida, K. Kurita, K. Ishihara, and N. Nakabayashi, ‘‘Preparation of 2-methacryloyloxyethyl phosphorylcholine copolymers with alkyl methacrylates and their blood compatibility,’’ Polym. J., 24, 1259–1269 (1992). K. Ishihara, N. Muramoto, and I. Shinohara, ‘‘Controlled release of organic substrates using polymer membrane with responsive function for amino compounds,’’ J. Appl. Polym. Sci., 29, 211–217 (1984). T. Akaike, Y. Sakurai, K. Kosuge, Y. Senba, K. Kuwana, S. Miyata, K. Kataoka, and T. Tsuruta, ‘‘Study on the interaction between plasma proteins and polyion complex by circular dichroism and ultraviolet spectroscopy,’’ Kobunshi Ronbunshu, 36, 217–222 (1979). N. Greenfield and G. D. Fasman, ‘‘Computed circular dichroism spectra for the evaluation of protein conformation,’’ Biochemistry, 8, 4108–4115 (1969). J. L. Brash and T. A. Horbett, eds., Proteins at Interfaces: Physicochemical and Biochemical Studies, in ACS Symposium Series, Vol. 343, American Chemical Society, Washington, D.C., 1987. T. A. Horbett and J. L. Brash, eds., Proteins at Interfaces II— Fundamentals and Applications, in ACS Symposium Series, Vol. 602, American Chemical Society, Washington D.C., 1995. T. Tsuruta, T. Hayashi, K. Kataoka, K. Ishihara, and Y. Kimura, eds., Biomedical Applications of Polymeric Materials, CRC Press, Boca Raton, Florida, 1993. B. D. Ratner, A. S. Hoffman, S. R. Hanson, L. A. Harker, and J. D. Whiffen, ‘‘Blood-compatibility-water-content relationships for radiation-grafted hydrogels,’’ in Medical Polymers: Chemical Problems, B. Sedlacek, C. G. Overberger, and H. F. Mark (eds.), J. Polym. Sci. Polym. Symp., 66, 1979, pp. 363–376. E. W. Merrille and E. W. Salzman, ‘‘Polyethylene oxide as a biomaterial,’’ Am. Soc. Artif. Intern. Org. J., 6, 60–64 (1983). G. R. Llanos and M. V. Sefton, ‘‘Does polyethylene oxide possess a low thrombogenicity?,’’ J. Biomater. Sci. Polym. Edn., 4, 381–400 (1993). E. L. Chaikof, E. W. Merril, A. D. Callow, and R. J. Connolly, ‘‘PEO enhancement of platelet deposition, fibrinogen deposition, and complement C3 activation,’’ J. Biomed. Mater. Res., 26, 1163–1168 (1992). K. Bergstro¨m, K. Holmberg, A. Safranj, A. S. Hoffman, M. J. Edgell, A. Kozlowski, B. A. Hovanes, and J. M. Harris, ‘‘Reduction of fibrinogen adsorption on PEG-coated polystyrene surface,’’ J. Biomed. Mater. Res., 26, 779–790 (1992). K. Bergstro¨m, E. Osterberg, K. Holmberg, A. S. Hoffman, T. P. Schuman, A. Kozlowski, and J. M. Harris, ‘‘Effect of branching and molecular weight of surface-bond poly(ethylene oxide) on protein rejection,’’ J. Biomater. Sci. Polym. Edn., 6, 123–132 (1994). J. D. Andrade, H. B. Lee, M. S. Jhon, S. W. Kim, and J. B. Hibbs,
The authors appreciate the helpful comments of Dr. Tomoko Ueda-Yukoshi.
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