Langmuir 1995,11, 375-376 375

Blood Contact Properties of Surface Immobilized Cw

Lorraine M. Lander and William J. Brittain*

Department of Polymer Science, The University of Akron, Akron, Ohio 44325-3909

Erwin A. Vogler*

Becton Dickinson Research Center, 21 Davis Drive, P.O. Box 12016,

Research Triangle Park, North Carolina 27709

Received August 1, 1994. In Final Form: September 28, 1994

Several publications have recently discussed potential biological properties of fullerenes. Perhaps the most stimulating observation is that a water-soluble derivative of C60 can inhibit HIV-1 protease.' Fullerene derivatives have also exhibited photoinduced DNA scission properties2 and can operate as photoinduced electron transport centers in artificial lipid bilayer^.^ Tour and co-workers4 published data on the apparent uptake of unsubstituted fullerenes by human skin cells in culture. Parent c60 enriched in 14C was prepared and administered to the cells as a fine aqueous dispersion. It was found that the enriched c60 was rapidly taken up and remains cell-associated after washing with (260-free medium. The exact nature of this interaction with human cells is under continued inves- tigation.

As part of our larger program on the surface chemistry aspects of contact activation in blood coagulation, we have studied surface immobilized fullerene. Stimulated by the recent reports on the unique biological properties of fullerenes, we asked ourselves if surface-immobilized C ~ O confers any unexpected procoagulant or anticoagulant properties. It is known that pyrolytic carbon species, especially isotropic carbons, display excellent biocompat- ibility. Compared to other implant materials, low tem- perature isotropic carbons have demonstrated superior blood contact performance and corrosion re~istance.~

We have previously described the preparation of self- assembled monolayers (SAMs) with tethered fullerenes.6 Atomic force microscopy (AFM) demonstrated that these homogeneous, monomolecular films of ordered fullerenes were firmly attached to the surface in an ordered lattice Of C60 molecules corresponding to the expected molecular pattern for the (hOO) faces of a face centered cubic unit cell. AFM indicated a c60 film thickness of 1 nm which is consistent with a monomolecular layer. The water contact angle hysteresis was 12" (advancing contact angle = 76") which is typical of ordered self-assembled mono- layers; X-ray photoelectron spectroscopy and W-vis spectroscopy were also used to confirm the structure of the c 6 0 monolayer. The reader is referred to ref 6 for additional details. We have used a similar synthetic procedure to create well-ordered fullerene films on thin glass microdisks appropriate for use in blood coagulation studies. The research groups of Mirkin' and Smith8 have

(1) Sijbesma, R.; Sranov, G.; Wudl, F.; Castoro, J. A.; Wilkins, C.; Friedman, S. H.; Decamp, D. L.; Kenyon, G. L. J. Am. Chem. SOC. 1993, 115, 6510.

(2) Tokuyama, H.; Yamago, S.; Nakamura, E. J. Am. Chem. Soc. 1993,115, 7918. (3) Hwang, K. C.; Mauzerall, D. Nature 1993,361, 138. (4) Scrivens, W. A.; Tour, J. M.; Creek, K. E.; F'irisi, L. J. Am. Chem.

SOC. 1994,116, 4517. (5) Beavan, A. Mater. Eng. 1990,107,39. (6) Tsukruk, V. V.; Lander, L. M.; Brittain, W. J. Langmuir 1994,

10, 996.

also published methods for the preparation of monomo- lecular films of fullerenes.

The coagulation time assay employed herein measured the propensity of different surface chemistries to contact activate the intrinsic pathway of blood coagulation. By varying the number of identically prepared disks used in a coagulation assay, we can systematically vary the amount of surface area and thus study the dependence of coagulation time on both surface chemistry and surface area. The type of data that results is a plot of coagulation time versus surface area. Comparison of such raw data for different surface chemistry permits the development of structure-reactivity relationships that connect surface chemistry, energy, and the behavior ofblood at interfaces. In this communication, we compare results for c60 monolayers to methyl-terminated SAMs and clean glass.

Results and Discussion Voglel.9 has described a phenomenological model for

plasma coagulation in which the number of putative "activating" sites scales linearly with procoagulant surface area. According to this model, contact activation of the plasma coagulation cascade is catalytic in the sense that these activating sites are not consumed by irreversible or slowly reversible protein adsorption occurring during coagulation. This model was successfully applied to experimental measurements of porcine platelet poor plasma coagulation time in the presence of a known surface area oftest procoagulants spanning a wide range of surface energy (water wettability). These test procoagulants included a series of polystyrene samples with varying levels of surface oxidation and a series of SAMs with 12 different terminal functional groups.l0 An exponential- like trend of increasing coagulation activation with increasing wettability was observed for the oxidized polystyrene system. Results with SAM procoagulants followed this general trend with some interesting chemi- cally-specific interactions with proteins of the contact activation system, over-and-above the surface energy trend.

We have used this same experimental approach to test fullerene monolayers and have compared the results to a methyl-terminated SAM. The SAMs were prepared on small glass disks (0.5 cm diameter, Bellco Glass). The methyl-terminated SAMs were prepared via chemisorp- tion of hexadecyltrichlorosilane (Huls) using literature methods." The preparation of the c60 monolayers has been described? The monolayers were characterized using water contact angles and electron spectroscopy for chemi- cal analysis.

The coagulation time assay used was a modified recalcification time test in which the coagulation time of a fured volume of citrated platelet-poor porcine plasma was monitored as a function of added procoagulant surface area (which was controlled by the number of added glass disks). The recalcification time assay has been described in more detail elsewhereg but salient features are briefly recapitulated here. Approximately 0.5 mL of frozen

(7) Caldwell, W. B.; Chen, K; Mirkin, C. A.; Babinec, S. J. Langmuir 1993.9, 1946. Chen, K.; Caldwell, W. B.; Mirkin, C. A. J. Am. Chem. SOC. 1993,115, 1193.

(8) Chupa, J. A.; Xu, S.; Fischetti, R. F.; Strongin, R. M.; McCauley, J. P.. Jr.: Smith. A. B.. 111: Blaisie. J. K: Peticolas, L. J.: Bean, J. C. J . Am. Chem. Soc. 1993,115, 4383. (9) Vogler, E. A. Submitted for publication in J . Bwmed. Res. (10) Vogler, E. A.; Graper, J. C.; Sugg, H. W.; Harper, G. R.; Lander,

L. M.; Brittain, W. J. Submitted for publication in J . Biomed. Muter. Res. (11) Wassennan, S.R.;Tao,Y.-T.;Whitesides,G. M.Langmuir 1989,

5, 1074.

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0743-746319512411-0375$09.00/0 0 1995 American Chemical Society

376 Langmuir, Vol. 11, No. 1, 1995 Notes

Figure 1. Results of p4 coagulation with clean glass disks, disks with methyl-terminated SAMs, and disks with c 6 0 monolayers. The open circles correspond to cleaned glass disks, open triangles to disks with methyl-brminated SAMs, and open squares to disks with c60 monolayers.

platelet poor porcine plasma (P4) was aliquoted into polystyrene test tubes (Falcon, Becton Dickinson) con- taining a variable number of SAM-modified glass disks. A tube without disks served as a control that measured coagulation due to background activation. The plasma was recalcified by addition of 100 p L of 0.2 M CaC12 and a stopwatch started. The tubes were quickly sealed with Parafilm and the contents were mixed with an inverting hematology mixer (Fisher). Coagulation time was noted by a distinct change in a fluid-like rheology to gel formation; the end point was sharp and reproducible to 30 s. The coagulation time response ofglass was measured using commercial micronized glass (Min-u-cil, Pennsyl- vania Glass and Sand; 5.6 m2/gm) which served as a standard benchmark for different P4 lots.

Figure 1 shows the results of P4 coagulation with c60 monolayers. Coagulation time in minutes is plotted against the surface area of the glass disks. The curves do not coincide at the origin due to variations in porcine plasma lots. The 1-min variation in origins is comparable

to the deviation of experimental points from the statistical fits and does not affect conclusions based differences between the surfaces. Smooth curves through the data of Figure 1 result from the nonlinear statistical fit of the model described in ref 9. Interestingly the procoagulant properties of immobilized c60 were intermediate relative to the highly activating clean glass surface and the passivated silane surface. Purely on the basis of chemical structure, it might have been anticipated that the behavior of Cc0 would more closely resemble a methyl-terminated surface. Indeed the advancing water contact angles for the methyl-terminated and c60 SAMs were 111" and 76", respectively. A more detailed analysis of the dependence of blood plasma coagulation on procoagulant surface energy1° predicts that c60 monolayers should be nonac- tivating (like methyl-terminated SAMs). However, the difference in clot time behavior for c 6 0 versus methyl- terminated surfaces is modest. Perhaps, the more sig- nificant conclusion to draw from the data in Figure 1 is that there is nothing extraordinary about the procoagulant properties of surface immobilized c60.

The present study adds information to the growing library of biological effects of fullerenes. In this report, an immobilized monolayer of fullerenes has been shown to be slightly activating in plasma coagulation (relative to methyl-terminated SAMs). The more spectacular reports of high biological activity for derivatized fullerenes and cellular uptake by aqueous dispersions of the parent system may involve more specific interactions between Cm and the biomolecule mediating the measured response. We hope that our work brings to light another aspect of c 6 0 biological activity and fuels further investigations into potentially unique properties of c60.

Acknowledgment. The skilled technical assistance of Ms. Jane Graper is gratefully acknowledged. The financial support of the Army Research Office is also gratefully acknowledged. LA940603M