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Langmuir 1997, 13, 5900-5908
In Vitro Plasma Protein Adsorption on ω-Functionalized Alkanethiolate Self-Assembled Monolayers Magnus Lestelius,†,‡ Bo Liedberg,‡ and Pentti Tengvall*,† Biomaterials Consortium and Molecular Films and Surface Analysis Group, Laboratory of Applied Physics, Linko¨ ping University, S-581 83 Linko¨ ping, Sweden Received February 24, 1997. In Final Form: August 25, 1997X Functionalized self-assembled monolayers (SAMs) of alkanethiolates on gold were used to study citrated human plasma protein adsorption by ellipsometry and antibody techniques in vitro. The aspiration is to gain knowledge about how surface properties affect such events like coagulation and complement activation, which in turn influence cell activation at an implant site. Five functionalities, methyl (-CH3), trifluoromethyl ester (-O(CdO)CF3), sulfate (-OSO3H), carboxyl (-COOH), and hydroxyl (-OH), were subject to characterization by ellipsometry, contact angle measurements, scanning force microscopy (SFM), and Fourier transform infrared reflection-absorption spectroscopy (IRAS). The low-energy surfaces, with methyl and trifluoromethyl ester terminations, showed affinity for fibrinogen. The trifluoromethyl ester also deposited lipoprotein (LP). The sulfate and the carboxyl surfaces deposited the coagulation proteins high molecular weight kininogen (HMWK), factor XII (F XII), and prekallikrein (PK), indicating contact activation of coagulation. Hydroxyl-functionalized SAMs showed low deposition of plasma proteins in general, although a low binding of antibodies against the contact activation proteins, complement factor 3c (C3c), and lipoproteins was observed. The morphology of the formed plasma protein layers was studied using scanning force microscopy. On the low-energy surfaces the proteins tended to cluster into large formations. In the case of the methylated surface the formations had the appearance of dendrite-like networks. On the high-energy surfaces the proteins retained a more uniform spreading in small rounded clusters.
Introduction The chemical nature of a solid surface is a property believed to affect the biological processes that occur when foreign materials contact human tissues or blood.1 The manner in which the chemistry influences the outcome of implantations is still not understood, and the notions of “good” or “bad” for biomaterials chemistry is debated. A general perception is that different applications also require different properties with respect to morphology, mechanics, and wear. It is accepted that for titanium implants in bone, the surface morphology plays an important role.2 On the other hand, by immobilization of heparin on surfaces the clotting time of blood can be successfully prolonged.3 Thus the microscopic surface chemistry can have a great impact on the success in clinical practice. Proteins adsorb rapidly upon surface-blood contact when materials are implanted. It has been suggested that protein adsorption, as one of the first occurrences, may influence the final outcome by specific signals that are mediated for the recruitment of specific cells, which in turn affect further events at the implant site.1,4 Factors that influence protein adsorption are therefore of interest for biomaterials science. Several investigators have, during recent years, used various strategies for studying the influence of surface functionality on protein adsorption and cell adhesion.1,4 The use of self-assembled monolayers (SAMs) has improved upon the resolution of surface chemistry since they form densely packed monolayers with * Corresponding author. E-mail:
[email protected]. † Biomaterials Consortium. ‡ Molecular Films and Surface Analysis Group. X Abstract published in Advance ACS Abstracts, October 1, 1997. (1) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837-850. (2) Larsson, C.; Thomsen, P.; Lausmaa, J.; Rodahl, M.; Kasemo, B.; Ericson, L. E. Biomaterials 1994, 15, 1062-1074. (3) Larsson, R.; Larm, O.; Olsson, P. Ann. N. Y. Acad. Sci. 1987, 516, 102-115. (4) Hoffman, A. S. Ann. N. Y. Acad. Sci. 1987, 516, 96-101.
S0743-7463(97)00187-X CCC: $14.00
a well-defined interface exposed to the environment.5,6 SAMs have been prepared on different substrates by the use of alkyltrichlorosilanes on titanium,7 silicon,8 glass,8 polystyrene,9 and polydimethyl siloxane10 or by alkanethiols on gold11-13 for studies on surface biological events in vitro and ex vivo. The question of steric repulsion has been addressed with alkanethiolate monolayers on gold by anchorage of flexible ethylene glycol chains in order to minimize protein adsorption.14 Only recently have experiments with alkanethiolate monolayers in vivo been performed.15 From a biomaterials and interface biology point of view it is interesting to study the protein deposition on model surfaces from, for instance, human blood plasma to determine the presence of proteins such as albumin (Alb), immunoglobulins (Ig), complement factors, fibrinogen (FG), fibronectin (FN), coagulation factors (activators and down regulators), and lipoproteins (LP). These proteins all have various biological roles: Alb is considered a biological passivator;4 immunoglobulin G (IgG) activates the complement system and, for example, binds (5) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Israelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932-950. (6) Ulman, A. An Introduction to ULTRATHIN ORGANIC FILMS. From Langmuir-Blodgett to Self-Assembly; Academic Press Inc.: San Diego, 1991. (7) Sukenik, C. N.; Balachander, N.; Culp, L. A.; Lewandowska, K.; Merritt, K. J. Biomed. Mater. Res. 1990, 24, 1307-1323. (8) Margel, S.; Vogler, E. A.; Firment, L.; Watt, T.; Haynie, S.; Sogah, D. Y. J. Biomed. Mater. Res. 1993, 27, 1463-1476. (9) Vogler, E. A.; Graper, J. C.; Harper, G. R.; Sugg, H. W.; Lander, L. M.; Brittain, W. J. J. Biomed. Mater. Res. 1995, 29, 1005-1016. (10) Silver, J. H.; Hergenrother, R. W.; Lin, J.-C.; Lim, F.; Lin, H.-B.; Okada, T.; Chaudhury, M. K.; Cooper, S. L. J. Biomed. Mater. Res. 1995, 29, 535-548. (11) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (12) Lo´pez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877-5878. (13) Deng, L.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 5136-5137. (14) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (15) Lindblad, M.; Lestelius, M.; Johansson, A.; Tengvall, P.; Thomsen, P. Biomaterials 1997, 18 (15), 1059-1068.
© 1997 American Chemical Society
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Scheme 1. Simplified Overview of Sequential Events in Two Protein-Mediated Defense Systemsa
a Rounded boxes represent some health status intervention, square boxes with solid lines represent the plasma protein systems, and square boxes with dashed lines represent some key proteins or protein complexes involved.
lymphocytes;16-18 the complement factors (including C3) are a part of the immune defense16-18 ending in the lysis of cells with the membrane attack complex rupturing the cell membrane; FG binds platelets;19,20 FN can bind a number of cell types and bacteria have receptors for FN;16,21,22 R2-macroglobulin (R2M) is a down regulator of the coagulation cascade20,23 that ends in the formation of a blood clot in which factors like high molecular weight kininogen (HMWK), factor XII (F XII), factor VIII (F VIII) and prekallikrein (PK) are components;20,24,25 anti thrombin III (ATh III) is another potent down regulator of coagulation, as it binds thrombin;20, 23 LP can act as transport protein of such agents as cholesterol.26 In Scheme 1, where some protein key factors have been marked out, possible events upon injury or infection that involve the protein-mediated defense systems intrinsic coagulation and complement can be seen. The dissolution of a blood clot by the fibrinolysis,27 and the kinin system, coupled to coagulation brings about effects like vasodi(16) Austyn, J. M.; Wood, K. J. Principles of Cellular and Molecular Immunology; Oxford University Press: Oxford, UK, 1994. (17) Mu¨ller-Eberhard, H. J. Annu. Rev. Biochem. 1988, 57, 321347. (18) Walport, M. In Immunology; Roitt, I., Brostoff, J., Male, D., Eds.; Grower Medical Publishing: London, 1989. (19) Bennet, J. S.; Shattil, S. J. In Hematology; Williams, W. J., Beutler, E., Erslev, A. J., Lichtman, M. A., Eds.; McGraw-Hill, Inc.: New York, 1990. (20) Kozin, F.; Cochrane, C. G. In Inflammation: Basic Principles and Clinical Correlates; Gallin, J. I., Goldstein, I. M., Snyderman, R., Eds.; Raven Press, Ltd.: New York, 1988. (21) Holmsen, H. In Hematology; Williams, W. J., Beutler, E., Erslev, A. J., Lichtman, M. A., Eds.; McGraw-Hill, Inc.: New York, 1990. (22) Gristina, A. G. Science 1987, 237, 1588-1595. (23) Comp, P. C. In Hematology; Williams, W. J., Beutler, E., Erslev, A. J., Lichtman, M. A., Eds.; McGraw-Hill, Inc.: New York, 1990. (24) Colman, R. W.; Scott, C. F.; Schmaier, A. H.; Wachtfogel, Y. T.; Pixley, R. A.; Edmunds, L. H., Jr. Ann. N. Y. Acad. Sci. 1987, 516, 253-267. (25) Nemerson, Y. In Hematology; Williams, W. J., Beutler, E., Erslev, A. J., Lichtman, M. A., Eds.; McGraw-Hill, Inc.: New York, 1990. (26) Evans, W. H.; Graham, J. M. Membrane Structure and Function; IRL Press Ltd.: Oxford, UK, 1989. (27) Francis, C. W.; Marder, V. J. In Hematology; Williams, W. J., Beutler, E., Erslev, A. J., Lichtman, M. A., Eds.; McGraw-Hill, Inc.: New York, 1990.
Figure 1. Schematic picture of the terminal functionalities of the self-assembled monolayers at pH 7.4. The inset in the upper left corner is a schematic cartoon of the structure of a alkanethiolate SAM on gold. Key: -CH3 (I); -OC(O)CF3 (II); -OSO3- (III); -COO- (IV); -OH (V).
lation16 are also part of the inflammatory system but are not discussed in this paper. In a previous work, we evaluated the plasma protein adsorption pattern, the kallikrein formation (as an indicator of contact activation of the coagulation cascade), and to some extent complement activation in vitro on three different modifications containing carboxyl and amine functionalities in different combinations.28 It was then observed that the carboxylated surfaces bound contact activation proteins and promoted kallikrein formation. The combination of amine and carboxylate functionalities seemed to activate complement, and glutathione (with two carboxylate groups and one amine, as well as a polypeptide-like backbone) showed low protein deposition in the phosphate-buffered system but large amounts deposited when calcium and magnesium were added. No activation of either the intrinsic pathway of coagulation28 or complement28 could be detected at short incubation times (10 min). In our present study we have chosen to use five different functionalizations (see Figure 1), HS(CH2)15CH3 (I), HS(CH2)16OCOCF3 (II), HS(CH2)16OSO3H (III), HS(CH2)15COOH (IV), and HS(CH2)16OH (V) on gold. The SAMs were characterized with contact angle measurements, ellipsometry, and IRAS to confirm high-quality ordered monolayers and successful functionalizations. The plasma protein adsorption pattern with antibodies and ellipsometry was investigated similarly to previous studies.28,29 In addition, we imaged the formed plasma protein layers with SFM. All the functionalized surfaces then exhibited individual morphological features. This is in accordance with previous observations of protein layers on various substrates.30-34 (28) Lestelius, M.; Liedberg, B.; Lundstro¨m, I.; Tengvall, P. J. Biomed. Mater. Res. 1994, 28, 871-880. (29) Lestelius, M.; Tengvall, P.; Lundstro¨m, I. J. Colloid Interface Sci. 1995, 171, 533-535. (30) Schakenraad, J. M.; Stokroos, I.; Busscher, H. J. Biofouling 1991, 4, 61-70.
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Well-defined substrates, like SAMs on gold or similar systems, can be conveniently employed to investigate protein adsorption and short-time implantation phenomena and other aspects of biomaterials science. This is made possible by the versatility and the rich flora of synthetic protocols available that allows a rigorous control of the SAM/ambient interface. These types of surfaces have also been successfully used to monitor interface properties and events in wetting,35,36 lubrication,37 tribology,38 corrosion inhibition,39 and biosensing.40,41 Materials and Methods Gold Substrates. Two types of gold surfaces were used. First, a 200 nm thick gold (99.99% purity, Nordiska Affineriet, Helsingborg, Sweden) film was electron beam evaporated onto Si[100] surfaces primed with an adhesion layer of 1 nm Ti. Prior to evaporation, the silicon was cut to 20 × 40 mm pieces, cleaned in a solution of five parts deionized and filtered water (Millipore, 18 MΩ cm, low organic content), one part ammonium hydroxide (25%, Merck, Darmstadt, Germany), and one part hydrogen peroxide (30%, Merck) at 80 °C for 10 min (TL1) and in another solution consisting of six parts deionized water, one part hydrogen chloride (37%, Merck), and one part hydrogen peroxide at 80 °C for 10 min (TL2), and finally rinsed in copious amounts of deionized water, and the samples were mounted in the evaporation chamber.42,43 A pressure of (2-7) × 10-8 mbar was maintained, and evaporation proceeded at a rate of 0.5 nm/s. The so-prepared films were used for infrared characterization, and sometimes also contact angle and ellipsometric measurements. Sputter-deposited gold substrates were obtained from Biacore AB, Uppsala, Sweden, and employed in the protein adsorption experiments. These were prepared via sputter deposition of a 200 nm thick layer of gold on glass substrates with an adhesion layer of 1 nm of Cr at a background pressure of 10-6 mbar. Samples sizes of 20 × 40 mm and 9 × 9 mm were used for IRAS and protein experiments, respectively. The two procedures yielded gold films with equivalent properties, as evidenced by SFM and ellipsometry of the bare gold and IRAS, ellipsometry, and contact angle measurements of the formed monolayer films. Monolayer Preparation. Alkanethiolate SAMs were prepared by immersion of the gold substrates, previously cleaned according to the TL1 procedure, in ethanolic (99.5%, Kemetyl, Stockholm) solutions of the desired thiol species, normally by overnight incubations, and never less than 18 h. The various modifications were achieved as follows. A 2 mM ethanolic solution of HS(CH2)15CH3 (I) (90-95%, Fluka, Buchs) was prepared, and the freshly cleaned gold surfaces were immersed. After incubation, the surfaces were ultrasonicated in ethanol for 10 min. For HS(CH2)16OCOCF3 (II), first a monolayer of 16-mercaptohexadecanol, HS(CH2)16OH (>99.5%, Biacore AB, Uppsala, Sweden), was formed in a 2 mM (ethanol) solution overnight and subsequently ultrasonicated in the solvent. The trifluoromethyl (31) Warkentin, P.; Wa¨livaara, B.; Lundstro¨m, I.; Tengvall, P. Biomaterials 1994, 15, 786-795. (32) Wa¨livaara, B.; Warkentin, P.; Lundstro¨m, I.; Tengvall, P. J. Colloid Interface Sci. 1995, 174, 53-60. (33) Taborelli, M.; Eng, L.; Descouts, P.; Ranieri, J. P.; Bellamkonda, R.; Aebischer, P. J. Biomed. Mater. Res. 1995, 29, 707-714. (34) Takahara, A.; Kojio, K.; Ge, S.-R.; Kajiyama, T. J. Vac. Sci. Technol. A 1996, 14, 1747-1754. (35) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164. (36) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (37) Salmeron, M.; Neubauer, G.; Folch, A.; Tomitori, M.; Ogletree, D. F.; Sautet, P. Langmuir 1993, 9, 3600-3611. (38) Overney, R.; Meyer, E. Mat. Res. Soc. Bull. 1993, 18, 26-34. (39) To¨rnkvist, C.; Thierry, D.; Bergman, J.; Liedberg, B.; Leygraf, C. J. Electrochem. Soc. 1989, 136, 58-64. (40) Lo¨fås, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1990, 1256-1258. (41) Schierbaum, K. D.; Weiss, T.; Thoden van Velzen, E. U.; Engbersen, J. F. J.; Reinhoudt, D. N.; Go¨pel, W. Science 1994, 265, 1413-1415. (42) Kern, W.; Puotinen, D. A. RCA Rev. 1970, 31, 187-206. (43) Kern, W. J. Electrochem. Soc. 1990, 137, 1887-1892.
Lestelius et al. ester was then introduced on the surface by exposure of the hydroxyl groups to 0.3 mL of trifluoroacetic anhydride F3CC(O)OC(O)CCF3 (>99%, Fluka) in 30 mL of tetrahydrofuran (THF, >99.5%, Fluka) in the presence of 0.3 mL of triethylamine (Et3N, >99%, Kodak, Rochester, NY) for 1 h under a N2 purge.44 This was followed by ultrasonication in THF, ethanol, and finally deionized water in order to remove physisorbed species. The HS(CH2)16OSO3H (III) modification was produced in a similar way. The hydroxyl monolayer on gold was treated with 45 mL of chlorosulfonic acid, ClSO2OH (>98%, Fluka), and 150 mL of pyridine (>99.8%, Fluka) in 30-40 mL of diethyl ether (>99.5%, Fluka) for 1 h under a N2 purge.44 Successive ultrasonication in diethyl ether, ethanol, and deionized water was employed. Caution is advised, chlorosulfonic acid reacts violently with water residues in solvents. HS(CH2)15COOH (IV) (>99%, Biacore AB) and HS(CH2)16OH (IV) were introduced on the gold films by overnight incubations in 2 mM ethanolic solution and subsequent ultrasonication in ethanol (99.5%). All surfaces were stored in ethanol until use during the same day. At each preparation occasion, the set of prepared surface samples were always accompanied by samples for characterization with contact angle measurements, ellipsometry, and IRAS (see below). Contact Angle Measurements. The advancing and receding contact angles were measured in a laboratory atmosphere with a Rame´-Hart NRL model 100 goniometer. Both water (Millipore MilliQ 185 purification system, low organic content, ∼3 ppb, and high resistivity, ∼18 MΩ cm) and hexadecane (>98%, Fluka, chromatographed over aluminum oxide, Brockman I, Merck before use) were used to probe the surfaces by the sessile drop technique. Ellipsometry. SAMs thicknesses and thicknesses of the adsorbed protein layers were determined with a Rudolph Research AutoEl III ellipsometer, equipped with a HeNe laser (λ ) 632.8 nm) as the light source. The light was incident at an angle of 70° relative to the surface normal. For the monolayer characterization, a three-phase (ambient/ organic film/gold substrate) model45-47 was used to evaluate subsequent changes in the measured ellipsometric angles, ∆ and ψ. These were recorded for the TL1-cleaned gold, for the intermediate synthesis steps, and for the formed monolayers. The organic phase was assumed to be isotropic with a refractive index N ) 1.50 + i0.48-50 For protein adsorption experiments, the three-phase model was expanded to a four-phase one (ambient/protein film/SAM/ gold) since the monolayers were even and reproducible. ∆ and ψ were measured and thicknesses evaluated with an assumed refractive index of the transparent adsorbed protein film Nnative protein ) 1.465 + i0. The obtained thicknesses were subsequently converted to surface concentrations (Γ) according to the method of Stenberg and Nygren, which accounts for the dehydration of the adsorbed protein layer.51 2 1 - 1/Nnative protein Γ ) ddry proteinF0 ) dnative protein F0 ≈ 2 1 - 1/Ndry protein
Kdnative protein (ng/mm2) (1) The density (F0) of a dry protein layer is approximately ∼1.37 g/cm3 and the refractive index for the same dry layers is Ndry protein ∼ 1.55, which gives the constant (K) the value of ∼1.2 × 106 ng/mm3. For example, a measured thickness of 10 nm results (44) Bertilsson, L.; Liedberg, B. Langmuir 1993, 9, 141-149. (45) McCrackin, F. L.; Passaglia, E.; Stromberg, R. R.; Steinberg, H. L. J. Res. Natl. Bur. Stand. A 1963, 67, 363-377. (46) Azzam, R. M. A.; Rigby, P. G.; Kreuger, J. A. Phys. Med. Biol. 1977, 22, 422-430. (47) Azzam, R. M. A.; Rigby, P. G.; Krueger, J. A. Optik 1978, 50, 249-250. (48) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (49) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (50) Shi, J.; Hong, B.; Parikh, A. N.; Collins, R. W.; Allara, D. L. Chem. Phys. Lett. 1995, 246, 90-94. (51) Stenberg, M.; Nygren, H. J. Phys. 1983, 44, 83-86.
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Table 1. Advancing (θa) and Receding (θr) Contact Angles of Water and Hexadecane and the Ellipsometric Thickness (d) of the Alkanethiolate Monolayers on Golda monolayer compound
θa(H2O) (deg)
θr(H2O) (deg)
θa(HD) (deg)
θr(HD) (deg)
d (nm)
HS(CH2)15CH3 (I) HS(CH2)16OCOCF3 (II) HS(CH2)16OSO3H (III) HS(CH2)15COOH (IV) HS(CH2)16OH (V)
110 ( 1 92 ( 3 18 ( 3 12 ( 1 e10
102 ( 1 77 ( 3
