Langmuir 1999, 15, 8405-8411
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Protein Interactions with Poly(ethylene glycol) Self-Assembled Monolayers on Glass Substrates: Diffusion and Adsorption Zhihao Yang,† Jeffrey A. Galloway,‡ and Hyuk Yu* Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706 Received March 4, 1999. In Final Form: July 20, 1999 Hydrophilic polymer chains, poly(ethylene glycol) (PEG), are attached to glass surfaces by silylation of the silanol groups on glass surfaces with (ω-methoxy-terminated PEG)trimethoxysilanes. We consider these tethered polymer chains to resemble self-assembled monolayers (SAMs) of PEG since the grafting process is entirely spontaneous. They are shown to exhibit excellent biocompatibility and represent a model system for studying the interactions of proteins with polymer surfaces. The PEG SAMs are prepared with two different molecular weight polymers (MW ) 750 and 5000) and characterized with the techniques of angular-dependent X-ray photoelectron spectroscopy and atomic force microscopy. For the low molecular weight sample, the polymer chains tend to extend, forming a brush-like monolayer, whereas for the large molecular weight sample, the longer polymer chains tend to interpenetrate each other, forming a mushroomlike PEG monolayer on the surface. Interactions between a plasma protein, bovine serum albumin, and the PEG SAMs are investigated in terms of protein adsorption and diffusion on the surfaces by the technique of fluorescence recovery after photobleaching. The diffusion and aggregation behaviors of the protein on the two monolayers are found to be quite different despite the similarities in adsorption and desorption behaviors. The results are analyzed with a hypothesis of the hydrated surface dynamics.
Introduction Most conventional materials for construction of endovascular medical devices often fail to be compatible with blood and plasma proteins since they are not compatible with plasma components. The clinical applications of these endovascular interventional materials invariably experience, in the long term, problems from adverse reactions of blood such as thrombosis1 and complement activation.2 Attempts to synthesize special biocompatible materials have been made in many ways; however, few of them are successful due to problems from poor mechanical properties, difficult processing, and toxicity of the compounds. It is generally believed that blood clotting is caused by the conformation change of the plasma proteins upon adsorption to the material surface, initiating a sequence of reactions leading to thrombus formation.3 Thus, the surface modification of conventional materials leading to surface biocompatibility is considered as a more practical and potentially successful approach to fabricating endovascular devices. Far beyond this narrow applications area, the interactions of proteins or other biomacromolecules with polymeric solid substrates are of importance in many other areas such as protein chromatography,4 biological sample supporting, enzyme immobilization,5 and DNA surface computing.6 The most widely used method for making surfaces biocompatible in clinical practice is to treat the surfaces † Current address: Research Laboratories, Eastman Kodak Co., Rochester, NY 14560-2121. ‡ Current address: Department of Chemical Engineering & Materials Science, University of Minnesota, Minneapolis, MN 55455.
(1) Bayer, R. E. Artif. Organs 1978, 2, 422. (2) Chenoweth, D. E.; Cheung, A. K.; Henderson, L. W. Kidney Int. 1983, 24, 764. (3) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1. (4) Sevastianov, H. P. Ber. Bunsen-Ges. Phys. Chem. 1989, 93, 948. (5) Stark, M. B.; Hobuberg, K. Biotechnol. Bioeng. 1989, 34, 942. (6) Smith, L. M.; Corn, R. M.; Condon, A. E.; Lagally, M. G.; Frutos, A. G.; Liu, Q.; Thiel, A. J. J. Comput. Biol. 1998, 5, 255.
with albumin solutions, since albumins adsorb irreversibly on most solid surfaces, presumably by means of hydrophobic and electrostatic interactions. It has been demonstrated that the preadsorbed proteins on the surfaces significantly reduce the adhesion of fibrinogen and platelets. However, the gradual exchange between the preadsorbed albumin and the proteins in solutions always results in short-lived surface passivation.7 The grafting of biocompatible polymer chains on solid surfaces has been shown to be the most effective method to prevent adsorption and denaturation of proteins in contact with the surfaces.8 Among various polymers, poly(ethylene glycol), PEG, or poly(ethylene oxide), PEO, appears to be the best one for providing “protein friendly” surfaces,9-11 due to the hydrated, neutral, highly mobile, and flexible chains.12 A number of experimental reports and theoretical considerations suggest that the brushlike PEG monolayers are the best for preventing protein adsorption, since they provide maximum entropic repulsion between the proteins and surfaces.13,14 An alternative model for the cause of protein-surface repulsion is to attribute it to the rapid movement of the hydrated chains.15 However, systematic experimental tests for the mechanism of the repulsive interactions with grafted PEG (7) Vroman, L.; Adams, A. L. J. Colloid Interface Sci. 1986, 111, 391. (8) Hoffman, A. S. Macromol. Symp. 1996, 101, 443. (9) Andrade, J. D.; Hlady, V.; Jeon, S. I. In Hydrophilic Polymers; Glass J. E., Ed.; American Chemical Society: Washigton DC, 1996. (10) Yang, Z.; Yu, H. Adv. Mater. 1997, 9, 426. (11) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (12) Harris, J. M. Poly(ethylene glycol) Chemistry; Plenum Press: New York, 1992. (13) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (14) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. Jeon, S. I.; Andrade J. D. J. Colloid Interface Sci. 1991, 142, 159. (15) Nogaoka, S.; Mori, Y.; Takiuchi, H.; Yokota, K.; Tanizawa, H.; Nishiumi, S. In Polymers as Biomaterials; Shalaby, S. W., Hoffman, A. S., Ratner, B. D., Horbett, T. A., Eds.; Plenum Press: New York, 1984; p 361.
10.1021/la990260y CCC: $18.00 © 1999 American Chemical Society Published on Web 09/18/1999
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Figure 1. Schematic representation of the end-grafted PEG self-assembled monolayers (SAMs) on glass substrates.
monolayers are yet to be understood in molecular terms and fully documented in the literature. To date, most investigations of protein interactions with polymeric solid surfaces are performed by studying protein adsorption on and desorption from the surfaces. Little attention has been given to the dynamic behavior and conformation of interacting proteins on the surfaces,16 which are also important issues directly relevant to protein functionality on the surfaces. In our perspective, the mobility of proteins on surfaces should be very sensitive to interactions of proteins with the surfaces, especially to the dynamic interactions between adsorbed proteins and grafted polymer chains. For our experimental tests, we have used (ω-methoxyterminated PEG)trimethoxysilanes to form grafted PEG monolayers on glass surfaces, and in a broader sense, we consider them to resemble self-assembled monolayers, SAMs. Two different molecular weight PEG derivatives result in two different chain conformations of the grafted PEG on the surfaces. The interactions between the PEG SAMs and a plasma protein, bovine serum albumin (BSA), have been investigated by monitoring adsorption and diffusion of the protein on the surfaces, and the aggregation behavior and conformational change of the protein upon adsorption have been probed by atomic force microscopy. Materials and Methods Materials. (ω-Methoxy terminated PEG)trimethoxysilanes (1), drawn in Figure 1 with PEG molecular weights of 750 and 5000, were custom synthesized by Shearwater Polymers, Inc. Fluorescein isothiocyanate (FITC) and anhydrous toluene were purchased from Aldrich Chemical Co. The microscope glass slides were from Clay Adams, and BSA was a commercial product from ICN Biomedical, Inc. The water used throughout the experiments was house-deionized water further purified by a Milli-Q system (Millipore) with a specific resistivity of 17 MΩ/cm at the withdrawal outlet. Preparation of Substrates. A cleaning solution of alcoholic sodium hydroxide was prepared as follows: 60 g of NaOH was dissolved in 60 mL of H2O, and then the solution was diluted to 500 mL with ethanol. After the microscope glass slides were soaked in this solution for 30-60 min, they were thoroughly rinsed with water, then placed in an aluminum-foil-covered container, and annealed at 585 °C for 20 min. This procedure is known to produce a maximum hydroxyl group density on the (16) Gasper, P. B.; Robertson, C. R.; Gast, A. P. Langmuir 1994, 10, 2699
Yang et al. glass surface.17 The glass slides thus prepared are highly hydrophilic;18 i.e., the contact angles with water are nearly zero (
