Serum Protein Adsorption and Platelet Adhesion to Polyurethane

Chapter 33

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Serum Protein Adsorption and Platelet Adhesion to Polyurethane Grafted with Methoxypoly(ethylene glycol) Methacrylate Polymers 1

2

Maria I. Ivanchenko , Eduard A. Kulik , and Yoshito Ikada Research Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan

The surface of a polyurethane film was subjected to UV-induced graft polymerization of methoxy-poly(ethylene glycol) (PEG) methacrylate monomers in the presence of L-cysteine as a chain transfer agent. The number of ethylene glycol (EG) units in the monomer side-chain was 4, 9, and 24. Adsorption of serum albumin and gamma-globulin as well as platelet adhesion to the grafted films were studied to evaluate the non-fouling property of the PEG enriched surface layer. It was found that the monomer with the smallest length of PEG chain of only 4 EG units was the most "inert" toward the blood components when the graft yield was largely reduced by the use of high concentrations of the chain transfer agent. Staining technique was employed to visualize the graft depth profile and protein penetration into the grafted films. It was concluded that extraordinarily high graft yields were not effective in preventing protein adsorption while very low graft yields were not sufficient to reduce interactions with proteins and platelets. For many years, segmented polyurethanes (PU) have been employed to fabricate medical devices because of easy molding processes and good mechanical properties. However, problems still remain in achieving good blood compatibility. Therefore, surface modification of PU has been a target of many research groups. Recently we have reported grafting of hydrophilic chains onto the surface of P U (7 ), where methoxy poly(ethylene glycol) (PEG) methacrylate (MnG) was used as the monomer having PEG units. PEG is reported to be relatively nontoxic (2) and to be capable of reducing the interactions between blood components and man-made materials (3,4). We found that cellulose surfaces grafted with PEG chains exhibited less complement activation as the length of the PEG chain was shorter (5). In contrast, other research groups reported that the optimum molecular weight (MW) of P E G for minimum Current address: Bakulev Institute of Cardiovascular Surgery, Leninsky Per. 9, Moscow, Russia Current address: Institute of Transplantology and Artificial Organs, Schukinskaya 1, Moscow, Russia

2

0097-6156/95/0602-0463$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


464

PROTEINS AT INTERFACES II

protein adsoiption was in a range from 2,000 to 20,000 (6-9). It was also shown that protein adsoiption decreased with increase in M W of PEG in spite of the decrease of total P E G content (70). Advantages of high molecular weight PEG in preventing protein adsoiption have been calculated also theoretically (11). However, there are several reports which show no significant effect of the P E G length on the nonfouling property of the modified surface (12-14). The puipose of this study is to gain a deeper insight into the structure of the grafted layer and its interaction with proteins and platelets. The grafted surface is obtained by photo-induced graft polymerization of MnG monomers onto a PU film. To investigate the interactions of the grafted surface with blood components, detailed surface analyses are essential, because graft polymerization occurs not only at the outermost surface but also in the bulk phase far from the surface (75), when polar polymers are used as the substrate of the graft polymerization. As a result, protein molecules may be not only adsorbed on the outermost surface but also can be sorbed into the inside of the grafted substrate polymer. A major difference between graft polymerization of monomers and chemical coupling of existing polymers for the preparation of grafted surfaces is the difficulty in controlling the molecular weight of graft chains obtained by the graft polymerization. M W of the graft chains may be a very important determinant of the microstructure, water content, and compositional depth profile of the grafted layer. In the present study, a chain transfer agent is added to the monomer solution for graft polymerization to vary the length of the graft chain and hence the graft yield, both of which should have a considerable effect on the interaction of the material with proteins and cells.

Experimental Materials. A PU film from Pellethane (Dow Chemical, 2363-90AE) with a thickness of about 0.2 mm was used after purification by Soxhlet extraction with methanol for 24 h. M n G monomers having 4 (M4G), 9 (M9G), and 23 (M23G) ethylene glycol units were donated by Shin-Nakamura Chemical Co., Ltd., Wakayama, Japan. The chemical structure of the monomers is shown in Fig. 1. The monomers were dissolved in benzene and washed with saturated sodium chloride aqueous solution. Benzene was then evaporated to obtain the pure monomers. L Cysteine monohydrochloride of extra pure grade and riboflavin (vitamin B ) were purchased from Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and used as received. Human serum albumin (HSA, crystallized) and human gamma-globulin (IgG, crystallized) were purchased from Sigma Co., Ltd., U S A , and used without further purification. Na I for protein labeling was purchased from Dai-ichi Pure Chemical Co., Ltd., Tokyo, Japan. 2

1 2 5

Graft polymerization. U V irradiation of the PU film was performed with a highpressure mercury lamp (75W, Toshiba SHL-100 U V type, X> 254 nm). The density of peroxide formed on the PU film by U V irradiation was determined with the iodide method as described previously (Kulik E . A . , Ivanchenko M . I., Kato K . , Sano S., and Ikada Y . J. Polym. ScL, Polym. Chem, Ed., in press). The aqueous monomer solution containing riboflavin, L-cysteine, and PU film in a quartz glass tube was irradiated for 2 h at 40 °C. Unless otherwise noted, the monomer concentration was kept at 4 wt% for M4G and M9G and 10 wt% for M23G. To remove homopolymer from the grafted films, they were first washed with running tap water and then in double-distilled water at 75 ° C for 24 h under stirring. The amount of grafted MnG polymers was determined by measuring the weight increase of grafted films using an electronic balance with an accuracy of 10" g. The M W of homopolymers was determined by high performance liquid chromatography 5

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

33. IVANCHENKO ET AL.

Platelet Adhesion to Polyurethane

465


with the T O S O H apparatus, equipped with an RI detector and two gel permeation columns (TOSOH PW 3.000 and PW 6.000). Polymers were eluted at a flow rate of 0.9 ml/min using double-distilled water. A set of PEG standards were used for the calibration. Surface analysis of grafted films. The stationary contact angle of films using a water droplet of about 7 pm diameter was measured using a telescopic goniometer (M 2010-6G II type, Elma Inc., Tokyo, Japan). The measurement was done on at least five different parts of film and averaged. Dynamic contact angle analysis of hydrated films was performed at 25 ° C with the Wilhelmy plate technique using equipment manufactured by Shimadzu Inc., Kyoto, Japan (16). Five hysteresis loops were collected to give an average value. FT-IR analysis was done using a spectrophotometer manufactured by Shimadzu Inc. (type 8100) in the ATR mode with a KRS-5 crystal. Spectra were collected as 800 scans at 4 wave number resolution. XPS spectra were obtained with a spectrometer using a MgK a X-ray source (ESCA 750, manufactured by Shimadzu Inc.). Emitted photoelectrons were detected at an angle of 9 0 ° with respect to the sample surface. For determination of O/C stoichiometry, a collecting factor of 2.9 was used for Ols. The XPS data were integrated with an E S C A PAC 760 analyzer. Cross-sections of grafted films were stained with 1 wt% aqueous solution of Sky Blue 6B dye at room temperature for 2 days, followed by observation under a light microscope. 1 2 5

Protein adsorption. Labeling of proteins with I was performed with the chloramine-T method(77). The protein concentration was adjusted with phosphate buffered saline solution (PBS, 0.1M, pH 7.4) to 0.15 and 0.2 g/L for HSA and IgG solutions, respectively. Prior to contact with the protein solution, all samples were hydrated in PBS solution for 2 h at room temperature. Protein adsorption was carried out at 37 ° C for lh. After protein adsorption, the films (1cm xlcm) were rinsed six times with 2 ml of PBS. The washing was performed for a total of about 2 h. Five species of each sample were counted and averaged. Grafted films were subjected to adsorption of H S A and IgG labeled with fluorescein isothiocyanate (FITC). The protein labeling was made in accordance with the procedures described by Goldman(7#). After liquid gel chromatography the labeled protein solutions were dialyzed against PBS for 8 h at room temperature. The level of labeling, determined from the adsorption spectra of the conjugates, was about one FITC molecule per molecule of HSA and two FITC molecules per IgG molecule. Protein adsorption (staining) was carried out at 37 ° C for 24 h from 20 and 6 g/L HSA and IgG solutions in PBS, respectively. After staining, the films were blotted with tissue paper and the cross-section of films with a thickness of 10 pm was observed with a confocal microscope using an argon laser as a source of light. Repeated gel chromatography of protein solutions showed that less than 2 % of the bound FITC became free after the staining procedure. Platelet adhesion. Venous blood from male rabbits was used to prepare washed platelet suspensions of 1.5xl0 cells/mL in PBS (1 and Tamada Y . , Kulik E . , and Ikada Y . Biomaterials, in press). One ml of the platelet suspension was added to a PU film of 15 mm diameter in multidish 24 wells made of polystyrene (Corning, USA) and kept for 30 min or 1 h at 37 ° C under static conditions. After incubation the film was taken out and dip-rinsed twice with PBS in order to remove the platelets which were not attached to the film surface. After washing, the lysis buffer was added to the film for the subsequent determination of adhered platelets on the same 8


466


sample by the lactate dehydrogenase (LDH) method (Tamada Y . , Kulik E . , and Ikada Y . Biomaterials, in press).


Results The density of peroxides generated by U V irradiation on the PU film increased linearly with irradiation time as shown in Fig. 2. This indicates that U V is capable of generating the active groups in PU which are necessary for the subsequent graft polymerization. To avoid the time-consuming procedure of excluding free oxygen which inhibits graft polymerization, riboflavin was added to the monomer solution. Riboflavin functions not only as a photoinitiator, but also consumes oxygen dissolved in the aqueous medium in the course of U V irradiation to form lumichrome (79). In the present study, graft polymerization was carried out at a riboflavin concentration of 2.5xl0 g/L because an excess of riboflavin reduced the effective U V dose on the film surface, and the maximum graft yield and the minimum static contact angle of grafted PU were observed at this initial concentration, as shown in Fig. 3. A certain weight increase was found even without the use of riboflavin, but graft polymerization did not take place unless the monomer solution containing the PU film was U V irradiated. Effects of L-cysteine addition to the monomer mixture on the graft polymerization are shown in Figs. 4-6. L-cysteine added as a chain transfer agent for radical polymerization (20) effectively reduced the M W of homopolymer, as seen in Fig. 4. As shown in Fig. 5, the effect of L-cysteine addition on the weight increase of the grafted PU film was much less significant than on the M W of the homopolymer. It is evident from Fig. 5 that the monomer polymerizability decreased with an increase in PEG length in the side-chain. The grafted PU surface became more hydrophobic with increasing chain transfer agent concentration, as seen in Fig.6. Protein adsorption to the grafted surfaces is shown in Figs. 7 and 8 for serum albumin and gamma-globulin, respectively. The adsorption dependence on the L-cysteine concentration was similar for both proteins but completely different for the monomers used for graft polymerization. When grafted with M 9 G and M23G polymers without L-cysteine, PU showed 5 times lower protein adsorption than the virgin PU. This low adsorption was observed for L-cysteine concentrations up to 4 x l 0 wt %. Adsorption was enhanced by a factor of two at 4 wt% L-cysteine concentration. Protein adsoiption to the PU grafted with M 4 G polymer was considerably reduced with an increase in L-cysteine concentration, approaching one tenth of the adsorption compared to the untreated PU. It follows that die best grafting conditions for the minimization of protein adsorption is to graft polymerize the monomer with the shortest PEG side chain (M4G) at the highest concentration of chain transfer agent. It is interesting to note that M4G was essentially ineffective in reducing albumin adsorption unless L-cysteine was used, as seen in Fig. 7. The lowest platelet adhesion was observed when the PU films were subjected to polymerization of M4G, regardless of the L-cysteine concentration. The results are given in Fig. 9. It is interesting that the PU film grafted with M4G polymer in the absence of L-cysteine adsorbed a relatively high amount of protein, but adhered a very small number of platelets. In the case of M4G-grafted PU the amount of adsorbed proteins markedly decreased with a decrease in the graft yield; in other words, with an increase in L cysteine concentration. To characterize the grafted surface, analysis was done for PU grafted with M 4 G polymer using XPS, ATR-FTIR, and dynamic contact angle measurements. The results are given in Table I. IR spectra of the M 4 G homopolymer, the PU film grafted with M4G polymer without L-cysteine, and die virgin PU film are shown in Fig. 10. The absorbance ratio of 11460/(Il530+11460) -5

- 2


IVANCHENKO ET AL.


Methoxy-PEG Methacrylates (MnG)

CH = C P

d

I


C—(OCH^-CH

II

0

monomer code

number of EG groups PEG in monomer, wt % n

M4G

4

60.3

M9G

9

77.3

23

89.7

M23G

Figure 1. The chemical structure and E G content of monomers used.


467



468

Figure 4. Molecular weight (MW) of homopolymers formed at different concentrations of L-cysteine. M4G ( • ), M9G (O ), and M23G ( • ).





[ L-cysteine ], wt % Figure 5. Weight increase of the PU film after graft polymerization. M4G ( • ), M 9 G (O ), and M23G ( • ) .

[ L-cysteine ], wt % Figure 6. Contact angle of the PU film grafted at different concentrations of L cysteine. M4G ( • ), M9G (O ), and M23G ( • ) .


469


470


[ L-cysteine ], wt % Figure 7. Albumin (HSA) adsorption onto the PU films grafted at different concentrations of chain transfer agent. Monomer: M4G ( • ), M9G (O ), and M23G ( • ) .

0

//

10

-3

10

-

2

-

10

1

0

10

10

1

[ L-cysteine ], wt % Figure 8. Gamma-globulin (IgG) adsorption onto the PU films grafted at different concentrations of chain transfer agent. Monomer: M4G ( • ), M9G (O ), and M23G ( • ).





[ L-cysteine ], wt % Figure 9. Platelet adhesion to the PU films grafted at different concentrations of chain transfer agent. Monomer: M4G ( • ), M9G (O ), and M23G ( • ).

"i

r

I

i

i

i

1700

1600

1500

1400

1300

Wavenumber, cm

Figure 10. ATR-FTIR spectra of M4G homopolymer (a), virgin P U (b), PU grafted with M 4 G at 10 wt % monomer concentration in the absence of L cysteine (c).


471


472

was used to compare the graft yield of the modified films. The absorbances at 1460 cnv and 1530 c n r correspond to methoxy groups of homopolymers and aromatic groups of the PU, respectively. As the absorbance ratio did not decrease with an increase in the depth of ATR-FTIR analysis, as shown in Table I, it is likely that the grafted polymer was distributed in the bulk phase of PU over a depth of about 100 nm. The data from XPS analysis, which are represented in Table I in terms of the atomic ratio 0/(C+0), suggest that even in a very thin surface layer of the film, probably 3-5 nm depth, the content of grafted MnG polymers was likely to be less than 50 % . Both the XPS and ATR-FTIR data confirmed that the surface content of P E G decreased with an increase in L-cysteine concentration. The equilibrium water content, given in Table I, was measured after 24 h incubation in water at 25 °C. It is obvious that the water content of PU films grafted with the M4G polymer decreased with an increase in L-cysteine concentration. However, the significant weight increase seen for the film grafted with the M4G polymer was not observed for those grafted with the M9G and M23G polymers after hydration. The dynamic contact angles of hydrated films revealed that the increase in L-cysteine concentration resulted in decreased contact angle hysteresis.


1

1

Table I. Effect of L-cysteine addition to the monomer solution for graft polymerization on the surface properties of PU films modified by graft polymerization of M4G at 10 wt % monomer concentration

Sample Parameter

PU

L-cystein, wt %

-

Graft yield, wt %

-

ATR-FTIR, 11460/(11530+11460) Incident angle: 60 : 45 0 0

XPS, 0/(C+0) Water content, wt%

M4G (homopolymer) -

PU-M4G

PU-M4G -Cysteine 4.0

0.0

-

34.9

.

29.6

0.04 0.04

1.00 1.00

0.32 0.32

0.14 0.13

0.181

0.352

0.263

0.232

-

20.1

9.3

0.0

a

Contact angle ):

a

advancing, 0a

78

-

45

67

receding, 0r

48

-

8

41

hysteresis,0a- 0 r

30

-

37

26

Determined after hydration of samples

Fig. 11 gives the microphotographs of grafted films after staining with Sky Blue dye. Obviously, the staining is limited to the surface region for the film grafted with M 9 G , while the films grafted with M4G in the presence and absence of L cysteine are stained deep into the subsurface region.





473

Figure 11. Optical photographs of cross-section of the PU film grafted with M4G using 10 wt % monomer concentration without L-cysteine (a), M 4 G at 10 wt % monomer concentration and at 4 wt % L-cysteine concentration (b), and M 9 G at 4 wt % monomer concentration without L-cysteine (c) after staining with Sky Blue. ( i — i : area stained by Sky Blue)


474


The fluorescent microphotographs of the Films after (ad)sorption of FITCH S A and FITC-IgG are shown in Fig. 12. It is evident from Fig. 12 that the depth of protein penetration decreases in the following order: HSA/PU-M4G(b) > HSA/PUM4G-Cystein(c) > IgG/PU-M4G(d) > HSA/PU(a) > HSA/PU-M9G (not shown). Interference from free FITC molecules was unlikely to occur, as seen from comparison of Fig. 12(b) and 12(d).


Discussion The following important findings of this work are helpful in clarifying the mechanism of protein interaction with PEG enriched surface layers: 1. The MnG monomer with 9 ethylene glycol units is very effective in repulsion of proteins and cells from the grafted PU surface unless the chain transfer agent is added. 2. Protein adsoiption onto the PU surface is drastically decreased, when it is grafted with M 4 G polymer in the presence of high concentrations of L-cysteine. 3. The amount of proteins adsorbed to the grafted PU films generally does not correlate well with the number of adhered platelets. A large amount of adsorbed HSA and a small amount of adhered platelets are found for the M4G-grafted PU. To explain the above findings we assume that the distribution profile of graft chains in the cross-sectional direction of the PU film is considerably different among the monomers used. Recently we have found that only 3 % of peroxide groups generated in the PU film by U V irradiation are located in the vicinity of the film surface and can be digested by peroxidase in aqueous solution (Kulik E . A . , Ivanchenko M . I., Kato K., Sano S., and Ikada Y. J. Polym. ScL, Polym. Chem, Ed., in press). Thus one can expect that the graft polymerization has proceeded also into the bulk of the PU. Indeed, as shown in Fig. 11, the M4G polymer chains are present in the deep subsurface of the PU film. The addition of L-cysteine hampered this penetration to some extent, whereas the penetration of M9G polymer into the PU film is limited to a very thin layer from the outermost surface as the stained cross-section indicates. It is likely that penetration of proteins into the grafted samples is possible , at least when M4G is used as the monomer for graft polymerization. Staining of the cross-section of grafted films by labeled proteins, shown in Fig. 12, confirmed this assumption. Thus, it is conceivable that the main difference among the grafted samples is the variation in depth of protein penetration. From this point of view the lack of correlation between protein adsoiption and platelet adhesion to the PU film grafted with the M4G monomer may be explained by the fact that the small-sized protein can penetrate into the bulk of the hydrated sample in marked contrast to the large platelet. The apparently complicated effects of L-cysteine addition on the protein adsoiption may be explained in terms of the microstructure of the graft layer which should be closely related to the surface PEG content, the graft yield, and the distribution of graft chains. The chain transfer agent effects appear particularly remarkable for the PU film grafted with the shortest side-chain monomer (M4G). When the graft profile depth is much larger than the protein size, as shown in Fig. 13, the nature of the upper polymer layer is of limited importance. The protein molecules are able to pass through the upper polymer layer as easily as they penetrate into the highly swollen hydrogels which have been used for protein separation and analysis. Whether or not the protein is finally adsorbed may depend on the structure of the semipermeable layer. It is conceivable that penetration and sorption of protein often occurs when hydrophobic polymers are grafted, coupled or blended extensively with a hydrophilic polymer, because the produced hydrophilic layer will attract protein molecules less extensively, at least, at the outermost surface, but allow them to enter into the water-swollen bulk phase. Therefore, extraordinarily high graft yields are not effective in preventing protein adsorption while very low graft yields are not sufficient to reduce the interactions with proteins and platelets.





475

Figure 12. Fluorescent photographs of cross-section of virgin PU film (a, HSA/PU), PU film grafted with M4G at 10 wt % monomer concentration without L-cysteine (b, HSA/PU-M4G), M 4 G at 10 wt % monomer concentration and at 4 wt % L-cysteine concentration (c, H S A / P U - M 4 G Cysteine) after staining with FITC-HSA, and PU film grafted with M4G at 10 wt % monomer concentration without L-cysteine (d, IgG/PU-M4G) after staining with FITC-IgG. ( i — i : area having fluoresence)



476


Figure 13. Schematic representation of protein and cell interaction with a modified surface having a graft depth profile much larger than the protein sizes.




477

Acknowledgments One of the authors (M.I.I.) thanks the Japan Society for the Promotion of Science for financial support of this study. We would like to acknowledge Dr. E . Uchida, Kacho Junior College, Kyoto, for her help in the grafting experiments.


Literature Cited 1. Fujimoto,K.;Inoue, H.; Ikada, Y. J. Biomed. Mater.Res. 1993, 27, 347-355. 2. Chaikof,E.L.; Merrill,E.W.; Callow,A.D.; Connolly, R.J.; Verdon, S.L.; Ramberg, K. J. Biomed. Mater.Res. 1992, 2, 1163-1168. 3. Merrill, E.W.; Pekala, R.W.; Mahmud, N.A. V.3. In Hydrogels in Medicine ; Peppas, N.A. Ed.:New York, NY, 1987, V.3, pp..1-16. 4. Sevastianov,V.I.;Kulik, E.A.; Kim, S.W.; Eberhart, R.C. Biomaterial-Living System Interaction 1993, 1, 3-11. 5. Akizawa,T.; Kino,K.; Koshikawa, S.; Ikada,Y.; Kishida,A.;Yamashita, M.; Imamura, K. Trans.Am.Soc.Artificial Internal Organs 1989, 35, 333-335. 6. Nagaoka, S.; Mori, Y.; Takiuchi, H.; Yokota,K.;Tanzawa, H.; Nishiiumi, S. Polym. Preprints 1982, 24, 67-68. 7. Desai, N.P.; Hubbell, J.A. Biomaterials 1991, 12, 144-153. 8. Sheu, M.S.; Hoffman,A.S.;Feijen, J. J.Adhesion Sci.Technol. 1992, 6, 9951009. 9. Park, K.D.; Okano, T.; Nojiri,C.;Kim, S.W. J. Biomed. Mater.Res. 1988, 22, 977-992. 10. Gombotz, W.R.; Guanghui, W.; Horbett T.A.; Hoffman, A.S. J. Biomed. Mater. Res. 1991, 25, 1547-1562. 11. Jeon, S.L.; Lee, J.H.; Andrade, J.D.; De Gennes, P.G. J. Colloid Inter. Sci. 1989, 142, 149-158. 12. Pekala, R.W.; Merrill, E.W.; Lindon,J.; Kushner, L.; Salzman, E.W. Biomaterials 1986, 7 , 379-385. 13. Amiji, M.; Park, K. Biomaterials 1992, 13, 682-692. 14. Llanos, G.R.; .Sefton, M.V.; J. Biomater.Sci. Polym.Ed. 1993, 4, 381-400. 15. Uchida, E.; Uyama, Y.; Ikada,Y. J.Polym..Sci. Polym.Chem. 1990, 28 , 28372844. 16. Uyama, Y.; Inoue,H.; Ito,K.; Kishida,A.; Ikada,Y. J.Colloid Interface Sci. 1991, 141, 275-279. 17. Methods in Immunology: a laboratory text for instruction and research. Garvey J.S., Cremer N.E., and Sussdorf D.H. Eds., Benjamin Inc., London, 1977. 18. Fluorescent Antibody Techniques; Goldman, M.,Ed.; Academic Press, New York, NY,1968. 19. Song, P.S.; Metzler, D.E.; Photochem. and Photobiology 1967, 6, 691-698 . 20. Kulik, E.A.; Kato, K.; Ivanchenko, M.I.; Ikada, Y. Biomaterials 1993, 14, 763769. RECEIVED February 10, 1995


Serum Protein Adsorption and Platelet Adhesion to Polyurethane

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