4396
Langmuir 2001, 17, 4396-4404
Surface Properties, Fibrinogen Adsorption, and Cellular Interactions of a Novel Phosphorylcholine-Containing Self-Assembled Monolayer on Gold Vassiliki A. Tegoulia† and Weisun Rao‡ Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716
Anand T. Kalambur and John F. Rabolt Department of Materials Science and Engineering, University of Delaware, Newark, Delaware 19716
Stuart L. Cooper* Illinois Institute of Technology, 10 W. 33rd Street, Chicago, Illinois 60616 Received December 26, 2000. In Final Form: April 17, 2001
A novel synthetic route for the preparation of a phosphorylcholine terminated thiol is presented. The new molecule was characterized by FTIR and NMR and was used to prepare one- and two-component monolayers rich in phosphorylcholine. The new surfaces had an intermediate hydrophilicity based on their sessile contact angles, but dynamic contact angle measurements showed that the surfaces became completely wettable when in contact with water. Ellipsometric measurements revealed that the molecules extended toward the monolayer-air interface similar to well-studied monolayers prepared from methyl- or hydroxylterminated thiols. Angle-resolved XPS survey measurements verified that phosphorus, nitrogen, and oxygen were situated closer to the monolayer-air interface, a conclusion also supported by angle-resolved C 1s measurements. Grazing-angle FTIR data indicated a less ordered structure for all the PC-containing monolayers than that of a methyl-terminated SAM. Fibrinogen adsorption and neutrophil adhesion on the new surfaces were significantly lower than on gold and on CH3-terminated SAMs. Bacterial adhesion was not found to vary significantly between the surfaces examined.
Introduction The clinical application of devices or biomaterials that come in contact with blood is of major importance in modern medicine. Adverse reactions between foreign prosthetic surfaces and blood components such as proteins or cells are the preeminent factors restricting the extended use of certain biomaterials. Typical examples are the inability to have biosensors that operate efficiently for any length of time due to protein accumulation and the need for anticoagulants to be used during extracorporeal procedures such as dialysis and heart-lung bypass operations to alleviate complications associated mainly with thrombosis. Finally, there are serious problems arising from device-centered infections.1,2 One of the most intriguing developments in the past decade has been the recognition that cell membranemimetic systems based on phosphatidylcholine or its major component, phosphorylcholine, exhibit very low protein adsorption and cell adhesion when in contact with blood and as a result limit the induction of surface associated blood clot formation. This concept evolved from the study of cell membranes. Cell walls are composed of various * To whom correspondence should be addressed. † Current address: Genentech, Inc., South San Francisco, CA 94080. ‡ Current address: Fish & Richardson P. C., Boston, MA 02110. (1) Brash, J. L.; Horbett, T. A. In Proteins at interfaces: Physicochemical and Biochemical Studies; ACS Symposium Series; American Chemical Society: Washington, DC, 1987; p 343. (2) Mittelman, M. W. Bacterial Adhesion. Molecular and Ecological Diversity; Wiley-Liss, Inc.: New York, 1996.
amphiphilic molecules.3 Proteins and cells in the bloodstream do not foul onto the surface of other cells, suggesting that their outer surface is biocompatible. However, the mechanism for this behavior is not fully understood. Initially, it was thought that biocompatibility was provided by the whole phospholipid molecule at the external side of the cell membrane. As a result, a great deal of effort was made to immobilize phospholipid molecules onto solid substrates.4-6 Hayward et al. later suggested that it was the phosphorylcholine headgroup instead of the whole molecule that was responsible for the good biocompatibility.7 The most prominent hypothesis to explain these observations is the suggestion that the phosphorylcholine zwitterion binds significant amounts of water, creating a hydration layer that does not allow proteins and cells to adhere on the surface.3,8,9 The strong hydration of the PC groups has been supported by simulation work that has shown a “heavy”, nonrandom association of water molecules with the phosphorylcholine moiety.10 It has also (3) Murphy, E. F.; Lu, J. R.; Brewer, J.; Russell, J.; Penfold, J. Langmuir 1999, 15, 1313. (4) Malmsten, M. J. Colloid Interface Sci. 1995, 172, 106. (5) Ong, S.; Cal, S.-J.; Bernal, C.; Rhee, D.; Qiu, X.; Pidgeon, C. Anal. Chem. 1994, 66, 782. (6) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126. (7) Hayward, J. A.; Chapman, D. Biomaterials 1984, 5, 135. (8) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323. (9) Yianni, Y. P. Structural and Dynamic Properties of Lipids and Membranes; Portland Press Ltd: London, 1992. (10) Sheng, Q.; Schulten, K.; Pidgeon, C. J. Phys. Chem. 1995, 99, 11018.
10.1021/la001790t CCC: $20.00 © 2001 American Chemical Society Published on Web 06/07/2001
Phosphorylcholine-Containing SAM on Gold
been suggested that specific plasma proteins that inhibit the blood clotting are selectively adsorbed on this headgroup.11,12 Recently, a number of researchers have focused on mimicking biologically inert surfaces using the phospholipid approach in the search for a nonthrombogenic antifouling substrate. Membrane-mimetic systems for blood contacting applications have been designed as copolymers containing the phosphorylcholine functional groups in either the side chain or on the polymer backbone using a variety of polymers such as polymethacrylates, polyurethanes, poly(dimethylsiloxane)s, or polysulfones.13-17 Literature also exists on the two-dimensional polymerization of lipid monomers self-assembled in the form of vesicles,18 direct adsorption of unilamellar lipid vesicles to hydrophobic substrates,19 and vesicle fusion on surfaces.20-22 At the same time, Langmuir-Blodgett techniques have been used to construct supported bilayers via a process of controlled dipping of an appropriate substrate through an organic amphiphilic monolayer.4,23 The overall significance of these design strategies lies in the ability to engineer surfaces in which the constituent members can be controlled, modified, and easily assembled with a high level of control over both order and chemistry. The incorporated phospholipid/phosphorylcholine deposited by the cited methods has been proven quite successful in minimizing protein adsorption and cell adhesion. However, in the case of the polymeric materials, it is not possible to accurately control the position of the phosphorylcholine moieties. Also, the thickness of the polymers or the fused vesicles may limit their use in applications where nanometer-scale dimensional requirements must be enforced as in the case for biomedical microdevices (BioMEMs).24 An alternative approach to the coating of PC polymers would be to covalently bind an organic monolayer bearing terminal PC groups onto a solid substrate. An approach like this would have the following advantages: one could create monolayers whose thickness can be accurately controlled at the angstrom level, the PC coverage could be directly manipulated, and since the monolayers are covalently bound they would be able to withstand relatively harsh conditions. Hayward has demonstrated the potential of the formation of PC monolayers on silicon wafers, but in his case, the attached alkyl chain that contained the PC group was not very stable because of the presence of an oxygen atom between carbon and silicon.7,25 As a result, the resulting alkyl silicate was readily (11) Iwasaki, Y.; Nakabayashi, N.; Nakatani, M.; Mihara, T.; Kurita, K.; Ishihara, K. J. Biomater. Sci. Polym. Ed. 1999, 10, 513. (12) Chapman, D. Langmuir 1993, 9, 39. (13) Campbell, E. J.; O’Byrne, V.; Stratford, P. W.; Quirk, I.; Vick, T. A.; Wiles, M. C.; Yianni, Y. P. ASAIO J. 1994, 40, M853. (14) Chen, T.-M.; Wang, Y.-F.; Li, Y.-J.; Nakaya, T.; Sakurai, I. J. Appl. Polym. Sci. 1996, 60, 455. (15) Ueda, T.; Oshida, H.; Kurita, K.; Ishihara, K.; Nakabayashi, N. Polym. J. 1992, 24, 1259. (16) Kohler, A. S.; Parks, P. J.; Mooradian, D. L.; Rao, G. H. R.; Furcht, L. T. J. Biomed. Mater. Res. 1996, 32, 237. (17) Baumgartner, J. N.; Yang, C. Z.; Cooper, S. L. Biomaterials 1997, 18, 831. (18) Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L. Macromolecules 1997, 30, 6483. (19) Salamon, Z.; Wang, Y.; Tollin, G.; MacLeod, H. A. Biochim. Biophys. Acta 1994, 1195, 267. (20) Williams, L. M.; Evans, S. D.; Flyn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751. (21) Winger, T. M.; Chaikof, E. L. Langmuir 1998, 14, 4148. (22) Winger, T. M.; Ludovice, P. J.; Chaikof, E. L. Langmuir 1999, 15, 3866. (23) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, 1991. (24) Desai, N. P.; Hossainy, S. F. A.; Hubbell, J. A. Biomaterials 1992, 13, 417.
Langmuir, Vol. 17, No. 14, 2001 4397
hydrolyzed. Ong et al. have also immobilized single-chain phospholipids containing ω-carboxyl groups in the alkyl chain on silica propylamine forming immobilized artificial membranes (IAM) that could be used as chromatography packing material.5,26,27 Recently, Wang et al. reported a new synthetic route by reacting a hydroxyl-terminated silane monolayer with phosphorus oxychloride (POCl3) and HOCH2CH2N+Me3OAc-.28 Previously, our group has also prepared PC-rich surfaces by reacting a hydroxylterminated thiol monolayer on gold with 2-chloro-1,3,2dioxaphospholane and then with trimethylamine.29 However, in all these efforts, the incorporation of the phosphorylcholine was performed after a monolayer terminated in a reactive end group had been deposited on the silicon or gold substrate, and most of the times the yield of the modification reaction was low. Also, it was not possible to control the positioning of the PC moieties or to vary the amount of the PC functionality on the surface. This would be possible if we could prepare a PC-terminated thiol that would enable us to prepare pure PC or mixed SAMs where the ratio of the two functionalities could be controlled by the thiol ratios in the binary solution. This is why a novel method for the covalent binding of phosphorylcholine moieties to a gold-coated surface is presented in this paper. To our knowledge, there has been only one other attempt to synthesize a thiol-terminated double-chain phospholipid (1,2-bis[16-mercaptohexadecanoyl)-sn-glycero-3-phosphocholine) that was used to prepare chemisorbed monolayers.30,31 This new surface was found to be hydrophilic, and the molecules were oriented such that the polar headgroups were extended outward from the surface. The behavior of this new surface toward cell adhesion and protein adsorption was not evaluated. In our work, a new phosphorylcholine (PC) end-functionalized thiol containing 11 methylene groups was prepared, characterized, and used for preparation of self-assembled monolayers on gold. Single- and twocomponent monolayers of pure PC and mixed monolayers with PC thiol and methyl- or hydroxyl-terminated thiols have been prepared. Surfaces were characterized using sessile and dynamic contact angle measurements, ellipsometry, X-ray photoelectron spectroscopy (XPS), and grazing-angle Fourier transform infrared spectroscopy (FTIR-GA) to acquire information on the surface properties as well as on the structural order and composition of the monolayers. Adsorption and elution of fibrinogen along with neutrophil and bacterial adhesion under flow were studied. The performance of the new surfaces was compared to the gold surface and two well-studied SAMs terminated in methyl and hydroxyl groups. Experimental Methods Materials. N-Undecylmercaptan (HS(CH2)10CH3) and 10undecylenyl alcohol (CH2dCH(CH2)9OH) were purchased from Pfaltz and Bauer (Waterbury, CT). The 11-mercapto-1-undecanol (HS(CH2)11OH) was bought from Aldrich Chemical Co. (Milwaukee, WI). All reagents used for the synthesis of 11-mercaptoundecylphosphorylcholinethiol were bought from Aldrich Chemical Co. (Milwaukee, WI) and were used without further purification unless otherwise specified. (25) Hayward, J. A.; Durrani, A. A.; Shlton, C. J.; Lee, D. C.; Chapman, D. Biomaterials 1986, 7, 252. (26) Pidgeon, C.; Venkataram, U. V. Anal. Biochem. 1989, 28, 36. (27) Pidgeon, C.; Stevens, J.; Otto, S.; Jefcoate, C.; Marcus, C. Anal. Biochem. 1991, 30, 163. (28) Wang, Y.; Su, T. J.; Green, R.; Tang, Y.; Styrkas, D.; Danks, T. N.; Bolton, R.; Lu, J. R. Chem. Commun. 2000, 587. (29) Tegoulia, V. A.; Cooper, S. L. J. Biomed. Mater. Res. 2000, 50, 291. (30) Diem, T.; Czajka, B.; Weber, B.; Regen, S. L. J. Am. Chem. Soc. 1986, 108, 6094. (31) Fabianowski, W.; Coyle, L. C.; Weber, B. A.; Granata, R. D.; Castner, D. G.; Sadownik, A.; Regen, S. L. Langmuir 1989, 5, 35.
4398
Langmuir, Vol. 17, No. 14, 2001
Tegoulia et al.
Figure 1. Synthesis scheme of the phosphorylcholine-terminated thiol (PC). Synthesis and Characterization of the 11-Mercaptoundecylphosphorylcholinethiol. The reaction route for the synthesis of 11-mercaptoundecylphosphorylcholinethiol (PC) is presented in Figure 1.32 A benzene solution (70 mL) containing a mixture of 10-undecylenyl alcohol (2.43 g) (1) and 2-chloro1,3,2-dioxaphospholane (2.33 g) was cooled to 0 °C in an ice bath. 2.96 mL of triethylamine ((CH3CH2)3N, TEA) was added slowly into the stirred solution. The solution was kept at 0 °C for 15 min before increasing the temperature to ambient where it was then kept under stirring for 4 h. The white precipitate, (CH3CH2)3N‚ HCl, was removed by filtering, and the solvent was evaporated using a rotary evaporator. A clear and colorless oily substance (4.40 g) (2) was obtained at the yield of 92%. 1H NMR (250 MHz, CDCl3): δ 5.77 (dCH-, 1H), 4.93 (dCH2, 2H), 4.22 (-OCH2CH2O-, 4H), 4.04 (-CH2-, 2H), 1.99 (-CH2O-, 2H), 1.24 (-C(CH2)7-C-, 14H). Compound 2 was immediately dissolved into 80 mL of anhydrous acetonitrile in a pressure bottle, and the solution was cooled in a dry ice/acetone bath. 20 mL of trimethylamine (TMA) was added. The reaction was left to proceed in a water bath (65-70 °C) for 48 h. After removing the white precipitate (0.83 g) by filtering and the solvent by rotary evaporation, a yellow viscous compound (3) of 4.51 g was obtained. Characterization demonstrated that the white solid precipitate and the yellow viscous compound had the same composition, giving a total yield of 89%. 1H NMR (250 MHz, CDCl3): δ 5.78 (dCH-, 1H), 4.93 (dCH2, 2H), 4.27 (-OCH2-, 2H), 3.80 (-CH2-, 2H), 3.41 (-CH2N-(CH3) 3, 11H), 1.99 (-CH2O-, 2H), 1.24 (-C-(CH2)7-C-, 14H). The yellow compound 3 was subsequently dissolved in 35 mL of anhydrous acetonitrile (CH3CN), 2.4 mL of thiolacetic acid, and 0.16 g of recrystallized 2,2′-azobis(isobutyronitrile) (AIBN). The solution was heated under reflux in an inert atmosphere (argon) in a 55 °C water bath for 18 h under UV irradiation. The solvent was then removed by rotary evaporation. A yellow oily compound with strong odor (4) of 4.68 g was obtained at the yield of 79%. 1H NMR (250 MHz, CDCl3): δ 4.29 (-OCH2C-, 2H), 3.81 (-SCH2-, 2H), 3.62 (-C-CH2-C-, 2H), 3.35 (-CH2-N(CH3) 3, 11H), 2.29 (H3C, 3H), 1.96 (-C-CH2O-, 2H), 1.23 (C(CH2)8-C, 16H). The odorous oil compound 4 was dissolved into 40 mL of methanol and 1.2 mL of 1.0 M HCl. The solution was heated in a water bath at 55 °C under nitrogen for 12 h. The methanol solvent was then removed by rotary evaporation in a vacuum, and a yellow oily compound with odor (5) of 2.63 g was obtained at the yield of 63%. FT-IR (cm-1): 3365 (OH, water), 2934, 2853 (-CH2-, -CH3), 1467 (-OCH2-), 1649, 1256 (-PdO), 1091 (-PO-CH2-), 984 (N-(CH3) 3), 766 (-CH2-)n. 1H NMR (250 MHz, CDCl3): δ 4.51 (-OCH2C-, 2H), 3.40 (-CH2-N-(CH3) 3, 11H), 2.60 (-C-CH2-C-, 2H), 2.49 (-C-CH2O-, 2H), 1.42 (-SCH2C-, 2H), 1.25 (C-(CH2)8-C, 16H). (32) Tegoulia, V. A., Ph.D. Thesis, 2000.
Preparation of Glass Substrates. 3 in. diameter glass disks of BK7 optical quality (Paragon Opticals, Reading, PA), 25 × 75 mm glass slides (Corning, Corning, NY), and 24 × 50 mm glass coverslips (Fisher Scientific, Pittsburgh, PA) were used in this work. The surfaces were cleaned by heating at 80 °C for 10 min in a mixture of H2O:H2O2:NH4OH (ratio 5:1:1 v/v) (procedure TL1) followed by a similar heating in a second mixture of H2O: H2O2:HCl (ratio 6:1:1 v/v) (procedure TL2). The water used in this procedure was deionized and filtered (Nanopure, 18 MΩ‚ cm, low organic content). This treatment removes the ionic contaminants and increases the number of silanol groups on the surface, resulting in a hydrophilic surface with an advancing contact angle for water
