Effect of Flow on Human Serum Albumin Adsorption to Self

The variation in the adsorption tenacity on the SAMs with an alkyl chain packing ...... University of Texas Health Science Center at Dallas, Dallas, T...
0 downloads 0 Views 144KB Size


Langmuir 2003, 19, 5464-5474

Effect of Flow on Human Serum Albumin Adsorption to Self-Assembled Monolayers of Varying Packing Density Eugene J. Choi and Mark D. Foster* Maurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio 44325

Susan Daly, Robert Tilton, and Todd Przybycien Department of Chemical Engineering, Center for Complex Fluids Engineering, Polymers and Surfaces Program, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

Charles F. Majkrzak National Institute of Standards and Technology (NIST) Center for Neutron Research, Gaithersburg, Maryland 20899

Peter Witte and Henning Menzel Macromolecular Materials, Institute for Technical Chemistry, Technical University of Brunswick, Hans-Sommer-Strasse 10, D-38106 Brunswick, Germany Received November 6, 2002. In Final Form: April 25, 2003 Adsorption of human serum albumin (HSA) to self-assembled monolayers (SAMs) of different packing densities under dynamic conditions was investigated in situ using optical reflectometry and neutron reflectometry. When optical reflectometry was used, it was observed that HSA has a greater apparent steady-state surface concentration and higher initial rate of adsorption on a less densely packed SAM than on a densely packed SAM. These results are consistent with the contention that HSA specifically binds to the loosely packed chains. Changes in the morphology of an adsorbed protein layer with the imposition of continuing flow during adsorption were studied for the first time using neutron reflectometry. HSA interpenetrates the less densely packed SAM but sits on top of the densely packed SAM when adsorbing under persistent flow or without persistent flow. However, the orientation of the adsorbed molecules changes with the details of the flow. In the absence of persistent flow, a side-on orientation prevails. The adsorbed layers are thicker when adsorbed under persistent flow, consistent with a reasonably uniform tilting of the molecules up away from the surface. The variation in the adsorption tenacity on the SAMs with an alkyl chain packing density is greater when the adsorbed layer structure is equilibrated under quiescent conditions than when the adsorbed layer is subjected to continuing flow.

Introduction Protein adsorption at surfaces and interfaces is one of the most important events that occurs when blood contacts a foreign surface. Studying the interactions between proteins and surfaces is also important in designing biocompatible devices and surfaces because the formation of a thrombus can inhibit the function of such a biomedical device.1,2 One way to control thrombus formation is to design a surface that is resistant to protein adsorption, and numerous studies have been conducted in this area using poly(ethylene oxide) surfaces.3-13 However, the

problem with designing such surfaces is that although protein adsorption is reduced, protein-resistant surfaces do not entirely prevent protein adsorption. Another way to control thrombogenesis is to adsorb a protective layer of protein that will prevent other blood proteins from adsorbing to the surface and, thus, prevent the formation of a thrombus. One candidate protein for this passivating layer is human serum albumin (HSA). HSA is the most abundant protein in the human blood and acts as a fatty acid transporter.14 Crystallographic studies15,16 of the structure of HSA show that there are

* To whom correspondence should be addressed. E-mail: [email protected] Phone: (330) 972-5323. Fax: (330) 972-5290.

(8) Lee, J. H.; Kopecek, J.; Andrade, J. D. Proc. Polym. Mater. Sci. Eng. Div. ACS 1987, 57, 613. (9) Andrade, J. D.; Nagaoka, S.; Cooper, S.; Okano, T.; Kim, S. W. ASAIO J. 1987, 10, 75. (10) Wang, R. L. C.; Kreuzer, H. J.; Grunze, M. J. Phys. Chem. B 1997, 101, 9767. (11) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426. (12) Pertsin, A. J.; Grunze, M.; Garbuzova, I. A. J. Phys. Chem. B 1998, 102, 4918. (13) Feldman, K.; Ha¨hner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134. (14) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (15) Curry, S.; Brick, P.; Franks, N. P. Biochim. Biophys. Acta 1999, 1441, 131. (16) Bhattacharya, A. A.; Gru¨ne, T.; Curry, S. J. Mol. Biol. 2000, 303, 721.

(1) Schultz, J. S.; Lindenauer, S. M.; Penner, J. A. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S. L., Peppas, N. A., Eds.; American Chemical Society: Washington, DC, 1982; p 43. (2) Hanson, S.; Ratner, B. D. In Biomaterials Science; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, CA, 1996; p 228. (3) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159. (4) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (5) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. J. Biomed. Mater. Res. 1998, 42, 165. (6) Gregonis, D. E.; Buerger, D. E.; Van Wagenen, R. A.; Hunter, S. K.; Andrade, J. D. Trans. Soc. Biomater. 1984, 7, 766. (7) Kjellander, R.; Florin, E. J. Chem. Soc., Faraday Trans. 1 1981, 77, 2023.

10.1021/la026811t CCC: $25.00 © 2003 American Chemical Society Published on Web 05/24/2003

Human Serum Albumin

six large binding sites and at least one smaller binding site for stearic acid, a common fatty acid in the human body. There have been attempts to use the presence of these binding sites to promote the tenacious adsorption of HSA to polymer surfaces by grafting alkyl chains to the surface.17-21 Eberhart et al.21 studied the in vivo adsorption of HSA to polyurethane grafted with (CH2)18 and observed no thrombus formation. Another strategy suggested for improving the biocompatibility has been the treatment of a surface with dextran functionalized with Cibracron blue, “blue dextran”, to provide specific, reversible HSA binding.22,23 Thrombus formation may be affected by the adsorption of HSA because HSA is the most abundant protein in the blood and it adsorbs to the surface first. Because it has an affinity for the surface, it stays adsorbed. The formation of a preadsorbed layer of HSA hinders the subsequent adsorption of other blood proteins. A difficulty with grafting to a polymer surface is that the polymer surface can rearrange from a hydrophobic to a hydrophilic character when the environment changes, disrupting the structure of the surface.24,25 Self-assembled monolayers (SAMs) provide well-defined surfaces for studying protein adsorption26 and also a potential means of creating monomolecular coatings for surface modification. SAMs provide chemical homogeneity, robustness, and variable surface functionality. Hydrophobic SAMs composed of CH3-terminated alkyl chains have been of great interest in studying surface interactions.26,27 An important property that can be varied to control adsorption is the packing density of the SAM alkyl chains. The surfaces of densely packed SAMs cannot appreciably rearrange when placed in an aqueous environment, and even the chains of less densely packed SAMs are limited in their ability to rearrange because the alkyl chains are chemically bonded to the substrate. In this study, the dependence of HSA adsorption under persistent flow on the SAM chain packing was investigated in situ. Earlier studies by some of the authors28-31 have already demonstrated a dependence of the adsorption tenacity on the SAM chain packing when the initial contact between the protein solution and the SAM occurs under flow but the flow is not continued while the structure is probed. Comparison between the behavior of lipidized and and that of delipidized HSA28 under these adsorption conditions (denoted hereafter as “nonpersistent flow”) is (17) Pitt, W. G.; Cooper, S. L. J. Biomed. Mater. Res. 1988, 22, 359. (18) Plate´, N. A.; Matrosovich, M. N. Dokl. Akad. Nauk SSSR 1976, 229, 496. (19) Munro, M. S. Ph.D. Thesis, University of Texas Health Science Center at Dallas, Dallas, TX, 1986. (20) Eberhart, R. C.; Munro, M. S.; Frautschi, J. R.; Lubin, M.; Clubb, F. J.; Miller, C. W.; Sevastianov, V. I. Ann. N.Y. Acad. Sci. 1987, 516, 78. (21) Eberhart, R. C.; Munro, M. S.; Frautschi, J. R.; Sevastianov, V. I. In Proteins at Interfaces; Brash, J., Horbett, T., Eds.; ACS Symposium Series 343; American Chemical Society: Washington, DC, 1987; Chapter 24. (22) Keogh, J. R.; Eaton, J. W. J. Lab. Clin. Med. 1994, 124, 537. (23) Keogh, J. R.; Velander, F. F.; Eaton, J. W. J. Biomed. Mater. Res. 1992, 26, 441. (24) Ruckenstein, E.; Gourisankar, G. V. J. Colloid Interface Sci. 1985, 107, 488. (25) Ruckenstein, E.; Gourisankar, G. V. J. Colloid Interface Sci. 1986, 109, 557. (26) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 11644. (27) Lu, J. R.; Su, T. J.; Thirtle, P. N.; Thomas, R. K.; Rennie, A. R.; Cubitt, R. J. Colloid Interface Sci. 1998, 206, 212. (28) Petrash, S.; Sheller, N. B.; Dando, W.; Foster, M. D. Langmuir 1997, 13, 1881. (29) Sheller, N. B.; Petrash, S.; Foster, M. D. Langmuir 1998, 14, 4535. (30) Petrash, S.; Cregger, T.; Zhao, B.; Pokidysheva, E.; Foster, M. D.; Brittain, W. J.; Sevastianov, V.; Majkrzak, C. F. Langmuir 2001, 17, 7645. (31) Choi, E. J.; Foster, M. D. Langmuir 2002, 18, 557.

Langmuir, Vol. 19, No. 13, 2003 5465

consistent with the contention that specific binding to the alkyl chains plays a role in the greater tenacity of adsorption on loosely packed SAMs. Here, the effect on the adsorbed layer structure of continuing flow after initial adsorption is considered. We also seek evidence that the difference in the adsorption tenacity with SAM packing is peculiar to HSA by considering a few measurements with human gamma globulin (HGG). HGG is a mixture of antibody proteins without any known binding sites specific for fatty acids. It has a larger average molecular weight than does HSA (150 000 vs 66 439 g/mol).32,33 Optical reflectometry (OR)34-37 was used to study the adsorption kinetics. In principle, total internal reflection fluorescence (TIRF)30,38-41 could also have been used for this purpose. However, the alteration of the structure of the protein by the attachment of fluorescein labels and concentration quenching due to high labeling ratios30,42 can make the interpretation of TIRF results problematic. OR does not require the use of labels. Butler et al.34 have used OR to study the initial rates of adsorption of bovine serum albumin (BSA) to poly(lactide-co-glycolide), while Robeson and Tilton37 have studied the kinetics of adsorption of lysozyme on silica using the same method. In this work, OR was used to obtain both the adsorbed amount after a finite adsorption time and the initial rates for adsorption of HSA and HGG to SAMs of different packing densities. A primary objective of the study was to characterize the effect of persistent flow on the structure of the adsorbed layer by means of neutron reflectometry (NR). NR measurements complement those made with OR by providing information on the thickness and roughness of the adsorbed layer by way of its sensitivity to variations in the neutron scattering length density (SLD). The study of adsorption under flow is made possible by the ease with which neutrons penetrate many solids. The use of deuterated SAMs and D2O in NR measurements provides an excellent contrast between the protein and the SAM, allowing for the resolution of subtle differences in the adsorbed layer structure. Even though NR is not directly sensitive to the orientations of the adsorbed molecules, bounds on the possibilities for molecular orientations can be inferred from the adsorbed HSA layer thickness derived from the NR data. Experimental Section Reagents and Solutions. Hydrogenous hexadecyltrichlorosilane (HTS) (Geleste, Inc.) was used as was received. Deuterated HTS (hexadecyltrichlorosilane-d33, d-HTS) was synthesized by the hydrosilation of 1-bromohexadecane-d33 (CDN Isotopes).43 (32) Peters, T. All about Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996; p 9. (33) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Co.: New York, 1995. (34) Butler, S. M.; Tracy, M. A.; Tilton, R. D. J. Controlled Release 1999, 58, 335. (35) Charron, J. R.; Tilton, R. D. J. Phys. Chem. 1996, 100, 3179. (36) Tilton, R. D. In Colloid-Polymer Interactions, From Fundamentals to Practice; Farinato, R. S., Dubin, P. L., Eds.; John Wiley & Sons: New York, 1999; Chapter 13. (37) Robeson, J. L.; Tilton, R. D. Langmuir 1996, 12, 6104. (38) Lassen, B.; Malmsten, M. J. Colloid Interface Sci. 1996, 179, 470. (39) Tremsina, Y. S.; Sevastianov, V. I.; Petrash, S.; Dando, W.; Foster, M. D. J. Biomater. Sci., Polym. Ed. 1998, 9, 151. (40) Ball, V.; Schaaf, P.; Voegel, J.-C. In Biopolymers at Interface, Surfactant Science Series; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; Vol. 75, Chapter 13. (41) Santore, M. M. In Colloid-Polymer Interactions, From Fundamentals to Practice; Farinato, R. S., Dubin, P. L., Eds.; John Wiley & Sons: New York, 1999; Chapter 14. (42) Robeson, J. L.; Tilton, R. D. Biophys. J. 1995, 68, 2145.


Langmuir, Vol. 19, No. 13, 2003

Solutions of 0.1 mg/mL delipidized HSA (Sigma, 99%, Fraction V, essentially fatty acid- and globulin-free) and 0.1 mg/mL solutions of HGG (Sigma, 99%, γ-globulins prepared from Cohn Fraction II and III,