Protein-polyelectrolyte-PGE1 modified biomer platelet adhesion

LETTERS TO THE EDITORS Protein-Polyelectrolyte-PGE1 Modified Biomer Platelet Adhesion In our previous work we have described (1) about the radiation-induced bonding of albumin onto polyetherurethane surfaces. It is also discussed that polyelectrolyte can be trapped within the albumin network via glutaraldehyde. Here a similar procedure is being used to modify the biomer surface, but with a closer outlook toward a natural vessel. It is known (2) that the presence of a carbohydrate-rich outer coat, which is hydrophilic, gelatinous, and of very high water content, contributes to the low interfacial tension at the interface between cells and the extracellular environment. It is also known (3) that arterial wall releases prostacyclin PGI: which is extremely antithrombogenic in character. Further it is observed (4) that albuminated surfaces showed minimal platelet adhesion as compared to fibrinogen and 3'-globulin adsorbed surfaces. Now considering these aspects we have modified the biomer surface and investigated the platelet adhesion on these surfaces as described earlier (1) in different batches. We fabricated the samples in the form of vascular grafts from 15% biomer solution in dimethylacetamide on clean dried glass rods as described elsewhere (5). The grafts were cleaned (4) with 0.1% soap solution (Teepol), distilled water, rinsed finally with acetone, and were dried in a vacuum oven. The clean grafts were immersed in the phosphate bUffer ofpH 7.4 and shaken. Then the mixture of proteins and polyelectrolyte solution in buffer was added to make the final concentration as 100 rag% albumin (human, essentially fatty acid-free, Fraction V, Sigma Co., -tool wt 69,000), 150 rag% polyelectrolyte (Synthesized (6) from natural rubber--poly(cis-l,4)-isoprene, source Havea Brasiliensis "Para Rubber"), and 100 rag% gelatin (S. D. Fine Chem. Pvt. Ltd., India) to reduce air/water interface. The adsorption was done under vacuum for 24 hr at room temperature. After the exposure, the samples were rinsed and various batches were prepared for platelet adhesion: I. Cleaned bare samples. II. Samples after the above exposure. III. The exposed samples were irradiated using 6°Co source with a total dose of 0.275 mrad (0.11 mrad/hr) under N2 atmosphere. IV, Some of the irradiated samples were again exposed to a freshly prepared solution of albumin, gelatin, and polyelectrolyte as mentioned above, the concentrations under same conditions. Then they were crosslinked with 1.0% glutaraldehyde solution (7) in buffer for 1 hr and rinsed. V. Some samples from set IV were exposed to albumin (100 rag%) for 1 hr and rinsed.

VI. Some samples from batch IV were exposed to PGEI (Sigma Co.), 400 #g% at 4°C overnight, rinsed, and stored. Platelet adhesion studies were performed using washed calf platelets. Fresh citrated call's blood was centrifuged to get platelet-rich plasma (PRP). The platelet separated from that, washed and suspended in Tyrode solution as described elsewhere (4). The samples were exposed to the platelet suspension for 20 min under static condition and rinsed with buffer under controlled flow. The platelets were fixed with 2.5% glutaraldehyde, stained with Coomassie blue G, and rinsed with buffer. The platelet density was estimated using an optical microscope. Different fields were read randomly and then averaged. The surface energy analysis (8) has indicated that PGE,immobilized surfaces are relatively hydrophobic in character compared to proteinated surfaces (e.g., albumin). However, from present studies it seems that biological activity plays an important role besides the hydrophobic/ hydrophilic nature of the surface done. Table I shows a reduction of platelet adhesion in all cases compared to the bare one, which may be due to the albumin, gelatin, the sulfamate and carboxylate groups of polyelectrolyte and PGE,. Although the surfaces IV, V, and VI seem to be similar from the platetet-adherence point of view, we believe such modifications may be helpful for varied applications based on short- or long-term needs. Further biological evaluations for the feasibility of the above surfaces toward biomedical applications are in progress.

TABLE I

Surface

III IV V

VI

Bare 1st exposed sample 1st exposed and irradiated 2nd time exposed and glutaraldehyde-treated Albumin-treated after glutaraldehyde treatment Exposed in PGEj after glutaraldehyde treatment

Meariplateletswithin the microscopicvision field_+SD

3.27 + 1.16 1.73 ± 1.03 1.47 __+0.69 1.33 + 0.82 1.25 + 0.77

1.33 +__0.67

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Journal of Colloid and InterfaceScience, Vol. 101,No. 2. October1984

0021-9797/84 $3.00 Copyright© 1984by AcademicPress.Inc. All rightsof reproductionin any formreserved.

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LETTERS TO THE EDITORS ACKNOWLEDGMENTS

We appreciate the help received from Mr. Bhas for the irradiation of samples; Dr. A. V. Lal for providing calf blood; and Mr. Thomas Chandy, Mr. George Joseph, and Miss Ajanta Nair for their suggestions and help. REFERENCES 1. Sharma, C. P., and Geetha Kurian, J. Colloidlnterface Sci. 97, 38 (1984). 2. Rigby, B. J., Biochem. Biophys. Acta 133, 472 (1967). 3. Moncada, S., and Vane, J. R., Fed, Proc. Fed. Amer. Soc. Exp. Biol. 38, 66 (1979). 4. Sharma, C. P., and Chandy, T., J. Colloid Interface Sci. 89, 479 (1982). 5. Sharma, C. P., Curr. Sci. 52, 1057 (1983). 6. Van Der Does, L., Beugeling, T., Frochling, P. E.,

Journal of Colloidand InterfaceScience.Vol. 101,No. 2, October1984

and Bantjes, A., J. Polym. Sci. Polym. Symp. 66, 337 (1979). 7. Cambic, H., Picha, G., Kiraly, R. J., Koshino, J., and Nose, Y., Trans. Amer. Soc. Artif. Intern. Organs 22, 664 (1976). 8. Chandy, T., and Sharma, C. P., J. Biomed. Mater. Res., in press; Sharma, C. P., and George, J., "Abstracts, 1st International Symposia-cum-workshop on Bioengineering, Jadavpur University, India," p. 43, 29th Dec. 1983. CHANDRA P, SHARMA N. V. NIRMALA Biosurface Technology Division SCTIMST (BMT WING) Poojapura, Trivandrum-695012 India Received November 15, 1983