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Langmuir 2004, 20, 348-356
Evaluation of the Stability of Nonfouling Ultrathin Poly(ethylene glycol) Films for Silicon-Based Microdevices Sadhana Sharma,† Robert W. Johnson,‡ and Tejal A. Desai*,§ Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, Structural Chemistry, Abbott Laboratories, Abbott Park, Illinois 60064, and Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215 Received May 2, 2003. In Final Form: October 31, 2003 The creation of nonfouling surfaces is one of the major prerequisites for microdevices for biomedical and analytical applications. Poly(ethylene glycol) (PEG), a water soluble, nontoxic, and nonimmunogenic polymer has the unique ability of reducing nonspecific protein adsorption and cell adhesion and, therefore, is generally coupled with a wide variety of surfaces to improve their biocompatibility. The performance of these modified surfaces for long-term biomedical applications largely depends on the stability of these PEG films. To this end, we have investigated the stability of covalently coupled ultrathin PEG films on silicon in aqueous in vivo like conditions for a period of 4 weeks. The PEG-modified silicon substrates were incubated in PBS (37 °C, pH 7.4, 5% CO2) for different periods of time and then characterized using the techniques of ellipsometry, contact angle measurement, X-ray photoelectron spectroscopy, and atomic force microscopy. The ability of the PEG-modified surfaces to control protein fouling was examined by protein adsorption studies using fluorescein isothiocyanate labeled bovine serum albumin and ellipsometry. Furthermore, the ability of these films to control fibroblast adhesion was examined. Studies suggest that the PEG-modified surfaces retain their protein and cell repulsive nature even though the PEG film thickness decreases for the period of investigation.
* To whom correspondence may be addressed. Tel: 617 3583054. Fax: 617-353-6766. E-mail:
[email protected]. † The Ohio State University. ‡ Abbott Laboratories. § Boston University.
both to the presumed biological inertness of the polymer backbone and also to its solvated configuration. However, in more than 80% of the cases, steric stabilization force and chain mobility effect have been sufficient enough to explain the protein-resistant behavior.5 The two main contributions to this repulsive force are excluded volume component and mixing interaction component. When protein molecules approach PEG-coupled surfaces, the available volume for each polymer segment is reduced, and consequently a repulsive force develops due to the loss of conformational entropy of the PEG chains. Also, the number of available conformations of the PEG segments is reduced owing to their compression or interpenetration of the protein chains generating an osmotic repulsive force. Besseling6 suggested that the chemical properties of surfaces might affect their states of hydration and the repulsive or attractive forces that result from the interactions of two such surfaces as they are allowed to interact. Theoretical analysis indicated that the interaction between two surfaces that causes changes in the orientation of water molecules (compared to bulk water) is repulsive; such surfaces were identified as having an excess of either proton donors or acceptors. Grunze and co-workers have also proposed that the interaction of water with the surface of SAMs is more important than steric stabilization of the terminal (EG)nOH chains. Theoretical and experimental work from Grunze’s group indicated that the conformation and packing of the chains in SAMs affect the penetration of water in the ethylene glycol layer and the inertness of the surface.7,8 In essence, despite all these investigations, the mechanism of PEG’s resistance to protein adsorption is still a mystery and will continue to attract the attention
(1) Turner, J. N.; Shain, W. Exp. Neurol. 1999, 156, 33. (2) Edell, D. J.; Toi, V. V.; McNeil, V. M.; Clark, L. D. IEEE Trans. Biomed. Eng. 1992, 39 (6), 635. (3) Schmidt, S.; Horch, K.; Normann, R. J. Biomed. Mater. Res. 1993, 27 (11), 1393. (4) Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum: New York, 1992.
(5) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 1043. (6) Besseling, N. A. M. Langmuir 1997, 13, 2113. (7) Pertsin, A. J.; Grunze, M. Langmuir 2000, 16, 8829. (8) Zolk, M.; Eisert, F.; Pipper, J.; Herrwerth, S.; Eck, W.; Buck, M.; Grunze, M. Langmuir 2000, 16, 5849.
Introduction In recent years, silicon-based materials (e.g., silicon, glass, silicon dioxide, quartz) have been extensively employed for the development of microdevices for analytical, separation, and biomedical technologies. However, silicon surfaces exposed to air or water develop a native oxide layer with surface silanol groups. These silanol groups are ionizable in water and make silicon surfaces negatively charged at neutral pH. A charged surface creates a streaming potential in the fluid flow, promotes biofouling, and in turn limits the long-term functioning of these microdevices.1-3 This necessitates the creation of nonfouling interfaces that can be achieved by the immobilization of certain neutral and hydrophilic macromolecules, such as poly(hydroxyethyl methacrylate), poly(acrylamide), poly(N,N-dimethyl acrylamide), dextran, and poly(ethylene glycol). Among them, poly(ethylene glycol) (PEG)/poly(ethylene oxide) (PEO, MW >10 000), a water-soluble, nontoxic, and nonimmunogenic polymer, has been found to be the most effective compared to other polymers.4 Actually the protein-repelling behavior of PEG is far from being simple to comprehend. It is a field in itself and an active area of research. Several theories have been proposed by physicist and chemists, but none of them is adequate to explain its protein-resistant behavior under all the conditions. The unusual efficacy of PEG as an apparently biologically passivating surface film is linked
10.1021/la034753l CCC: $27.50 © 2004 American Chemical Society Published on Web 12/20/2003
Stability of Ultrathin Films
of the researchers in future; what is clear and well established is the proven ability of PEG to control biofouling. PEG-coupled silicon surfaces can be created either by physical adsorption9-15 or by covalent immobilization such as grafting and chemical coupling.16-25 PEG thin films prepared by simple adsorption techniques are likely to elute off the surface due to weak forces of adhesion.26 Theoretically, covalently coupled PEGs are considered to be more stable.4,5 It has been determined that PEG does not degrade in a vacuum for up to 1000 h, suggesting that it should be minimally exposed to oxygen as well as light during long-term storage.26 Nevertheless, PEGs are susceptible to oxidative degradation and chain cleavage like other polyethers and many other hydrophilic polymers when exposed to aqueous environments. This may lead to loss of PEG film thickness and ultimately result in a loss of coating functionality. Quite a few studies have addressed the stability of PEG in storage conditions as well as in extreme conditions of pH and temperature.27-29 Nevertheless, the issue of stability in in vivo like environments remains rather unexplored.30 As the maintenance of PEG film functionality is crucial for limiting biofouling, it is essential to develop insights into the causes and consequences of PEG film functionality over prolonged exposures to aqueous environments. In the present research effort, we have focused on the detailed investigation of the stability of ultrathin PEG films on silicon in vivo like environments (phosphatebuffered saline (PBS), 37 °C, pH 7.4, 5% CO2). PEGmodified surfaces were developed using the coupling conditions optimized previously.31,32 The immobilization scheme used in this investigation involves a one-step coupling procedure (i.e., coupling the silicon surface with PEG-silane for the formation of PEG thin films) as opposed to the other common methods that involve two or more steps to create PEG films. This makes the development of ultrathin PEG films (
