Human Serum Albumin on Fluorinated Surfaces - American Chemical

Geiselbergstrasse 69, 14476 Golm, Germany. Received March 14, 2003. In Final Form: June 18, 2003. The aim of this work is to investigate the stabiliza...
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Human Serum Albumin on Fluorinated Surfaces Manuel A. N. Coelho,† Euridice P. Vieira,† Hubert Motschmann,‡ Helmuth Mo¨hwald,‡ and Andreas F. Thu¨nemann*,§ Deparamento de Engenharia Quimica, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal, Max Plank Institute of Colloids and Interfaces, Am Mu¨ hlenberg 1, 14424 Golm/Potsdam, Germany, and Fraunhofer Institute for Applied Polymer Research, Geiselbergstrasse 69, 14476 Golm, Germany Received March 14, 2003. In Final Form: June 18, 2003 The aim of this work is to investigate the stabilization of the conformation of adsorbed human serum albumin (HSA) on surfaces containing CF3 and/or OH groups. These groups are structural characteristics (mimics) of trifluoroethanol, which is known to stabilize the secondary structure of several proteins. We focused on three different types of organic surfaces: (i) self-assembled monolayers (SAMs) prepared from mixtures of thiols with CF3 and OH end groups, (ii) polymer surfaces of a poly(vinyl methyl ketone) (PVMKCF3/OH), which was modified with OH and CF3 groups, and (iii) surfaces of polymer fluorosurfactant complexes containing CF3 groups. It was revealed that the secondary structure of HSA in contact with the SAMs depends on the thiol composition. SAMs of mixed monolayers displayed the highest HSA recognition measured by antibody binding. CD measurements showed that PVMKCF3/OH surfaces retain the secondary structures of adsorbed HSA. Examples of polymer fluorosurfactant complexes are given that retain the secondary structure of adsorbed HSA and a complex that increase the content of R-helices. A combination of CF3 and OH groups is proposed as a new approach to prepare biocompatible surfaces.

1. Introduction In the medical field there is a growing demand for biocompatible materials used in short- or long-term applications within the human body. These materials fulfill quite diverse tasks ranging from low-end medical disposables such as catheters or lenses to high-end devices such as artificial joints or heart pacemakers. Each requires very specific material properties, and in addition their use should not cause thrombosis, tissue damage, inflammation, or toxic reactions.1 A biocompatible material has to provide two things: the desired bulk properties and compatibility with living tissue. Since all unwanted body reactions originate at the material-host interface, an efficient approach is the use of existing materials modified with a suitable surface coating. The first step in the interaction of a foreign material within the human body is exposure to blood and the subsequent adsorption of blood plasma proteins.2 The biological system analyzes this adsorption layer, and depending on the concentration and conformation of the blood proteins found, it can initiate a rather complex and not yet fully understood cascade of reactions.3,4 Ratner suggested in 1993 the idea of tailoring the surface to intentionally adsorb specific proteins in order to achieve a specific response of the body to a material.5 Pitt and Cooper attempted to promote the adsorption of a passi* To whom correspondence may be addressed. E-mail: [email protected]. Address: Fraunhofer Institute for Applied Polymer Research, Geiselbergstrasse 69, 14476 Golm, Germany, and also Bundesanstalt fu¨r Materialforschung und -pru¨fung, Richard-Wellsa¨tter-Strasse 11, 12489 Berlin, Germany. † Deparamento de Engenharia Quimica. ‡ Max Plank Institute of Colloids and Interfaces. § Fraunhofer Institute for Applied Polymer Research. (1) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837-850. (2) Coleman, R.W. J. Clin. Invest. 1984, 73, 12489. (3) Williams, D. Med. Device Techno. 1996, 7, 6-9. (4) Gobel, R. J.; Janatova, J.; Googe, J. M.; Apple, D. J. Biomaterials 1987, 8, 285-288. (5) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837-850.

vating layer of human serum albumin (HSA),6 as did Eberhart and co-workers.7 Understanding and controlling protein adsorption are therefore decisive for designing biocompatible materials. To address this problem, several strategies are currently being pursued and reviews can be found.8,9 In this paper we suggest a novel strategy. Instead of a minimization of the adsorbed amount as pursued by many workers in this field,10 we actively stabilize and preserve the native structure of the adsorbed proteins. The most important protein in this context is HSA.11 HSA is a globular protein of known crystal structure which is responsible for the transport of metabolites in the body. Its secondary structure is dominated by a high content of R-helices (67%).12 Albumin is the most abundant protein in the blood, and due to its high diffusion coefficient, it is the protein which is the first to be adsorbed to foreign surfaces. Adsorbed HSA may undergo conformational changes such as a loss in secondary structure as shown by CD spectroscopy in refs 13-15. The data provide evidence that the adsorbed HSA lost about 20-25% of their R-helicity. These distortions are responsible for the activation of the biological system, and for this reason HSA adsorption is an important event in the reaction cascade.16 (6) Pitt, W. G.; Cooper, S. L. J. Biomed. Mater. Res. 1988, 22, 359382. (7) Eberhart, R. C.; Munro, M. S.; Williams, G. B.; Kulkarni, P. V.; Shannon, W. A.; Brink, B. E.; Fry, W. J. Artif. Organs 1987, 11, 375382. (8) Ishihara, K. Trends Polym. Sci. 1997, 5, 401-407. (9) Hubbel, J. Trends Polym. Sci. 1994, 2, 20. (10) Ikada, Y. Adv. Polym. Sci. 1984, 57, 103. (11) Liebmann-Vinson, A.; Lander, L. M.; Foster, M. D.; Brittain, W. J.; Vogler, E. A.; Majkrzak, C. F.; Satija, S. Langmuir 1996, 12, 22562262. (12) Xiao M. H.; Carter, D. Nature 1992, 358, 209-215. (13) Kondo, A. J. Colloids Interface Sci. 1991, 143, 214. (14) Soderquist, M.; Walton A. G. J. Colloids Interface Sci. 1980, 75, 386. (15) Norde W.; Favier J. P Colloids Surf. 1992, 64, 87. (16) Vromann, L.; Adams, M. Fed. Proc. 1971, 30, 1494.

10.1021/la034445n CCC: $25.00 © 2003 American Chemical Society Published on Web 07/22/2003

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Table 1. Relative Percentages of Thiols with CF3 Groups in the SAMs

SAM

area at 1178 cm-1 (au)

CF3 thiol in SAMa (%)

area at 1141 cm-1 (au)

CF3 thiol in SAMb (%)

SAMCF3 SAM75%CF3 SAM50%CF3 SAM25%CF3

0.062 0.061 0.039 0.013

100 98 63 21

0.020 0.019 0.014

100 95 65

CF3 thiol in SAM calcd from eqs 2 and 3 (%) 100/100 87/91 62/69 19/24

a Relative amount of CF thiol in SAM calculated from IR band at 1178 cm-1. b Relative amount of CF thiol in SAM calculated from 3 3 IR band at 1141 cm-1.

Since the adsorption process is accompanied by a substantial loss in R-helicity, the coating of surfaces should actively stabilize this structural element. Efficient stabilizers of R-helices are fluorinated alcohols such as trifluoroethanol.17 Despite numerous investigations the mechanism of the molecular interaction remains obscure.18,19 The aim of our work is to produce surface coatings containing CF3 and/or OH groups and to study the adsorption behavior of HSA onto such surfaces. As a first approach SAMs prepared from mixtures of CF3- and OH-terminated thiols were used to study adsorption and conformational changes of proteins on surfaces with different properties. Thiols are suitable for the formation of model surfaces as it is easy to obtain well-defined monolayers just by controlling their chemistry in solution.20-24 When CF3 (hydrophobic)-terminated and OH (hydrophilic)-terminated thiols are mixed, it is possible to vary the wettability of surfaces and as a result control the protein adsorption processes. Nature itself makes use of the hydrophobic/hydrophilic character of complex systems. For example, vascular endothelium, known for its antithrombogenic properties, forms microphase separated structures consisting of hydrophobic and hydrophilic microdomains.25 As a second approach we use polymers in this work. These are either modified by OH and CF3 groups or complexed with CF3 group containing surfactants. Polymers offer strong advantages to self-assembled monolayers (SAMs), as it is possible to modulate their mechanical properties, which is absolutely necessary for different bioapplications; these include immobilization of enzymes and adsorbed layers of proteins. Polymer chemistry and biology come together through an artificial material, and it is here that extensive studies are being performed.26-29 It is necessary to study the functionality of the adsorbed protein layer to determine the body response to a foreign material and the activity of immobilized enzymes. HSA was used to perform the experiments in this study. This protein is known to have no cell receptors in the body, so when adsorbed on a surface it leads to passivation (17) Yang, J. J.; Pitkeathly, M.; Radford, S. E. Biochemistry 1994, 33, 7345-7353. (18) Rajan, R.; Balaram, P. Int. J. Peptide Protein Res. 1996, 48, 328-336. (19) Arunkumar, A. I.; Kumar, T. K. S.; Yu, C. Biochim. Biophys. Acta 1997, 1338, 69-76. (20) Tidwell, C. D.; Ertel, S. I.; Ratner, B. D.; Tarasevich, B. J.; Atre, S.; Allara, D. L. Langmuir 1997, 13, 3404-3413. (21) Bain, C. D.; Biebuyck, H. A.; Whitesides G. M. Langmuir 1989, 5, 723-727. (22) Sanassy, P.; Evans, S. D. Langmuir 1993, 9, 1024-1027. (23) Buijs, J.; Britt, D. W.; Hlady, V. Langmuir 1998, 14, 335-341. (24) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1165. (25) Fasman, G. D., Ed. Circular Dichroism and the Conformational Analysis of Biomolecules; Plenum Press: New York, 1996. (26) Cassie, A. B. D. Faraday Soc. 1948, 3, 11. (27) Kato, K.; Sano, S.; Ikada, Y. Colloids Surf., B 1995, 4, 221-230. (28) Courtney, J. M.; Lamba, N. M. K.; Gaylor, J. D. S.; Ryan, C. J.; Lowe, G. D. O.; Hubbell, J. A. Rev. Trends Polym. Sci. 1994, 2, 20. (29) Langer, R.; Cima, L. G.; Tamada, J. A.; Wintermantel E. Biomaterials 1990, 11, 738.

against high adsorption of other proteins such as fibrinogen, which is important for blood clotting.30 Passivation of a surface by HSA requires strong bonds between the surface and HSA but, most importantly, without inducing denaturation. In recent studies experimental results of induced desorption with trifluoroethanol (TFE) show that HSA binds more strongly to a hydrophobic surface (octadecyltriclorosilane) than to a hydrophilic one (quartz).31 Prime and Whitesides24 as well as of Wertz and Santore32 addressed the question of whether HSA binds more strongly to hydrophobic or hydrophilic surfaces. The remaining problem to be solved is that conformational changes (denaturation) of HSA have to be suppressed. 2. Materials and Methods Perfluorinated acids of different chain length (4, 7, 8, 10 and 12 carbon atoms), acetone, 1,1,1,3,3,3-hexafluoropropanol, methanol, hexane, poly(vinyl methyl ketone), HS(CH2)9OCH2CF3, and HS(CH2)11OH were supplied by Aldrich and used as received. Polyethyleneimine (PEI, Lupasol WF) was supplied by BASF. This polymer is highly branched with a molar ratio of primary to secondary to tertiary amino groups of 34:40:26 and a molecular weight of Mw ) 25000 g/mol.33 The polysiloxane (PDMS) contains an amount of 0.6 mol % aminoethyl aminopropyl functions (WR1100, Wacker Chemie, Germany). The kinematic viscosity was 5000 mm2 s-1, corresponding to a polymerization degree of about 1500. HSA, essentially free from fatty acids, was purchased from Sigma. HSA has a molecular weight of 69000 g/mol, a diffusion constant of (6-8) × 107 cm2/s, an isoelectric point of 5.4, and a globular shape. Its content in the blood is 50-60% of all proteins, and its secondary structure contains about 64-67% R-helix.34 2.1. SAMs. SAMs that contain a mixture of hydrophobic and hydrophilic groups on the surface were obtained by the competitive adsorption of thiols with different end groups. The thiol HS(CH2)9OCH2CF3 was mixed with HS(CH2)11OH in different percentages. The thiols were dissolved in ethanol with a concentration of 10-3 M, and the SAMs were prepared by adsorption on a gold surface for 12 h. For clarity, SAMCF3 was prepared from the OCH2CF3 terminated thiol. Amounts of 25, 50, and 75 mol % CF3-terminated thiol were used for the preparation of SAM25%CF3, SAM50%CF3, and SAM75%CF3, respectively. SAMOH was prepared from HS(CH2)11OH. Contact angle measurements and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used for the characterization of the SAMs (cf. the chapters wetting and structure). A summary is given in Table 1. 2.2. PVMKCH3/OH. The reaction of a commercial poly(vinyl methyl ketone) with (trifluoromethyl)trimethylsilane yields statistical copolymer poly(vinyl methyl ketone) containing CF3 and OH (PVMKCH3/OH) (cf. Figure 1). The polymer was characterized by 1H NMR, and elemental analysis revealed that 60% of the ketone groups reacted (containing CF3 and OH). Both modified (30) Rabinow, B. E.; Ding Y. S.; Qin C.; Mchalsky, M. L.; Schneider, J. H.; Ashline, K. A.; Shelbourn, T. L.; Albrecht, R. M. J. Biomater. Sci., Polym. Ed. 1994, 6, 91. (31) Coelho, M. A. N.; Vieira, E. P.; Motschmann, H.; Mo¨hwald, H. Biochim. Biophys. Acta 2003, 1645, 6-14. (32) Wertz, C. F.; Santore, M. M. Langmuir 2001, 17, 3006-3016. (33) Information, supplied by the manufacturer, BASF, specialty chemicals, Ludwigshafen, Germany. (34) Kragh-Hansen, U. Dan. Med. Bull. 1990, 37, 57-84.

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Figure 1. Structures of the polymer and the polymer fluorosurfactant complexes which were used for the preparation of surfaces for adsorption of HSA: (a) PMVKCF3/OH with 60% of CF3 and OH containing monomers; (b) complexes of PEI and perfluorinated carboxylic acids with 4 (PEI-C4), 7 (PEI-C7), and 10 carbon atoms (PEI-C10); (c) complexes of a polysiloxane functionalized with amino groups and perfluorinated acids with 8 (PDMS-C8) and 12 carbon atoms (PDMS-C12). The amount of complexed amino groups of the polysiloxane was 50% (PDMS50-C8, PDMS50-C12) and 100% (PDMS100-C8, PDMS100C12). The degree of amino group functionalized monomers is 6%. and unmodified PVMK were processed as thin films on quartz and silicon wafers by dip coating, using acetone as a solvent. Homogeneous smooth films, as observed by atomic force microscopy (AFM) and ellipsometry, of defined thickness were prepared. The films did not show any signs of degradation in a phosphate buffer with time. 2.3. Preparation of Polymer Fluorosurfactant Complexes. 2.3.1. Poly(ethyleneimine) (PEI) Complexes. For complexation, 1 mmol of perfluorinated acid (chain length 4, 7, and 10) was dissolved in 100 mL of water (Millipore). At 90 °C the solution was stirred, and a solution of 1.0 equiv of PEI in 50 mL of water was added. The stoichiometry was calculated with respect to the charges. A solid complex was obtained as a white precipitate, separated, and dried for 12 h at a reduced pressure of 0.1 mbar. The yields for the complexes PEI-C4, PEI-C7, and PEI-C10 were in the range of 90-95%. Details of the physicochemical properties of the complexes are described elsewhere,35 and the chemical structures are shown in Figure 1. The complexes PEI-C4, PEI-C7, and PEI-C10 were dissolved in 1,1,1,3,3,3hexafluoroisopropyl alcohol (IsoF). These complexes were bound to quartz surfaces by dip coating (∼2 min). 2.3.2. Polysiloxane Complexes. Four complexes of the PDMS and perfluorinated acids with 8 (C8) and 12 carbon atoms (C12) were prepared (cf. Figure 1). The amounts of complexed amino groups of the PDMS in the complexes were 50% (PDMS50-C8, PDMS50-C12) and 100% (PDMS100-C8, PDMS100-C12). For complexation formation of complex PDMS100-C12, 1 equiv of PDMS (20.83 g) was dissolved in 100 mL of hexane. An 1.0 equiv of C12 (2.0 g) was dissolved in 50 mL of 2-butanol and 0.5 mL of methanol. While the mixture was stirred, the surfactant solution was added in drops to the PDMS solution resulting in a transparent complex solution. Films were prepared by solvent casting as described earlier,36 and cross-linking on quartz slides was performed in air for 12 h at a temperature of 125 °C. The same conditions (with adjusted amounts of perfluorinated acids) were used for the synthesis of the other complexes. Details on (35) Thu¨nemann, A. F. Langmuir 2000, 16, 824-828. (36) Antonietti, M.; Conrad, J.; Thu¨nemann, A. F. Macromolecules 1994, 27, 6007-6011.

Coelho et al. the wetting behavior of the complexes PDMS50-C12 and PDMS100-C12 are given elsewhere.37 The complex formation is a self-assembly reaction as described in several reviews (cf. for example G. Wegner and C. Ober38). 2.4. Measurements. For each polymer and complex, stacks of three dip-coated quartz slides were placed in a flow cuvette. All quartz slides coated with polymers and complexes formed stable thin films in buffer solution and were optically inactive and transparent in the range 180-300 nm. Solutions of HSA proteins were prepared on a phosphate buffer at pH 7.4 with concentrations of 0.068 g/L (10-6 mol/L) and allowed to adsorb. After equilibrium the protein solution was replaced by a pure buffer. Circular dichroism (CD) measurements were then performed on a Jasco J710 spectropolarimeter at 23 °C in a 0.1 cm path length cell over the wavelength range 195-260 nm. The scan speed was 20 nm/min with a 0.2 nm resolution. Every spectrum obtained was the average of four measurements. Several mathematical methods have been proposed for the calculation of the secondary structure of proteins based on the linear dependence between structure fractions and spectra. The methods used in this work are CONTIN,39 and the self-consistent method SELCOM.40 These methods were chosen to quantify the variations in secondary structure deduced from the CD spectra measured. Fourier transformed infrared spectroscopy (FTIR)41 measurements were performed in transmission geometry on an Equinox 55s with a resolution of 4 cm-1 and 200 accumulations. An attenuated total reflection (ATR) was used for the investigation of adsorbed proteins at the interface between a solid substrate and the liquid protein solution. With ATR the light does not pass the liquid and thereby avoids any disturbing light absorption from the liquid. An enzyme-linked immuno sorbent assay (ELISA) was used to detect conformational changes of the HSA on the SAMs. In the total internal reflection fluorescence experiments (TIRF), the proteins were labeled with FITC as fluorescence dye so that they could be excited by the beam of an argon laser at λ ) 488 nm as a light source. The protein and buffer solutions flowed in the system in the laminar regime. The fluorescence signal was detected via a CCD camera (details of this setup are describe elsewhere42) and with a photomultiplier tube (details can be found elsewhere43-45). A flow cell was designed to perform the TIRF measurements with such dimensions (64 × 12 × 2 mm) that a laminar flow could be obtained in the cell.46 Thereby well-defined conditions were achieved to control the protein and TFE diffusion onto the surface. A laminar flow is provided for a Reynolds number inferior to 2100. The Reynolds number is defined by

Re ) Fvd/µ

(1)

where F is the density of the liquid, v is the velocity in the direction of the flow, d is the thickness of the rectangular system, and µ is the dynamic viscosity of the liquid. The flow rate of the solvent varied between 1 and 2 mL/min. Thus Re was in the range of 10-20 for the pure buffer system and from 7 to 14 for the TFE buffer system. Such low Re numbers indicate that the shear stress applied to HSA was low. The contact angle measurements were performed on a G10 contact angle goniometer (Kru¨ss, Germany); the angles reported here are the average of five measurements. The advancing contact (37) Thu¨nemann, A. F.; Kublickas, R. H. J. Mater. Chem. 2001, 11, 381-384. (38) Wegner, G.; Ober, C. Adv. Mater. 1997, 9, 17 ff. (39) Luo, P.; Baldwin, R. L. Biochemistry 1997, 36, 8413-8421. (40) Myers, J. K.; Pace, C. N.; Scholtz J. M. Protein Sci. 1998, 7, 383-388. (41) Joseph, D.; Andrade, K. Surface and Interfacial aspects of Biomedical Polymers; Plenum Press 1985; Vol. 2. (42) Auch, M. Thesis, Max Planck Institute of Colloid and Interfaces, 1999. (43) Watkins, R. W.; Robertson, C. J. Biomed. Mater. Res. 1977, 11, 915. (44) Lok, B. K.; Channing R. J. Colloid Interface Sci. 1983, 91, 0000. (45) Axelrod, D.; Burghart, T. P. Annu. Rev. Biophys. Bioeng. 1984, 13, 247. (46) Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. Transport Phenomena; John Wiley & Sons: New York, 1960.

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Figure 2. cos θ of water contact angle measurements of SAM surfaces that were prepared of mixtures of CF3- and OHterminated thiols. The amount of CF3 thiol refers to its concentration in solution, which was used for SAM preparation. angle was measured by injecting a 5 µL liquid drop on the surface. Hexadecane and water were used as test liquids.

3. Results and Discussion 3.1. SAMs. 3.1.1. Wetting. CF3 and OH end groups display a large difference in the wetting properties of the resulting SAMs (SAMCF3 and SAMOH, respectively). SAMOH displayed contact angle values with water of about 15°, which is in accordance with the literature.47 For OCH2CF3-terminated chains a contact angle of 89° was found which is smaller than that for a CH2CH2CF3-terminated thiol, which gives a contact angle of about 100°. It has been shown that water influences atomic layers at a distance of approximately 5 Å of the uppermost region of the thiol surfaces and penetrates this by the formation of hydrogen bonds.48 In the OCH2CF3 thiol containing SAM the ether group lies at a distance of ∼3 Å from the surface hence in the region where water can interact via hydrogen bonding. This is an explanation for the lower contact angle of SAMs of OCH2CF3 than that of CH2CH2CF3-terminated thiols. The cos θ values (see Figure 2) of the SAMs show no linear decrease with the CF3 to OH ratio used for SAM preparation. The contact angles are higher than expected. Calculating the fraction of each thiol on the surface via Cassie’s26 and Israelachvili49 law (see eqs 2 and 3, respectively) a preferential adsorption of the OCH2CF3 thiol for mixtures above 25% CF3 was derived (cf. Table 1). Cassie’s and Israelachvili’s laws are given by

cos θ )

∑ xi cos θi

(2)

∑i xi(1 + cos θi)2

(3)

and

(1 + cos θ)2 )

where xi is the molar fraction of compound i at the surface and θi is the corresponding contact angle of a surface made only by compound i. These two laws can only be used accurately in cases of a molecular homogeneous surface. So the calculated values are only rough approximations. On the other hand the applicability of these equations to surfaces where hydrogen (47) Whitesides, G. M.; Laibnis P. E. Langmuir 1990, 6, 87-96. (48) Wagner, R.; Richter, L.; Wu, Y.; Weissmuller, J.; Kleewein, A.; Hengge, E. Appl. Organomet. Chem. 1998, 12, 265-276. (49) Israelachvili, J. N.; Gee, M. L. Langmuir 1989, 5, 288-289.

Figure 3. IR spectra of SAMs. The right-hand figure shows the CH2 symmetric and asymmetric vibration bands of SAMs made of solutions with different amounts of CF3 thiol. The lefthand figure shows the region with the absorption bands typical for CF3 and OH groups.

bonding and dispersion forces are mixed has as not yet been extensively studied.50 3.1.2. Structure. The ratio of the integrated areas of the infrared bands of the SAMs was analyzed (Figure 3), and a preferential adsorption of CF3-terminated thiol relative to the OH thiol was obtained (cf. Table 1). It is important to note that calculating adsorbed amounts via integrated areas of infrared curves is only an approximate measure. Nevertheless these results indicate that the SAMs contain a higher amount of CF3 than was expected from the stoichiometry of the thiols used for their preparation. The CH2 stretching region (3000-2800 cm-1) has been thoroughly studied in the literature. It provides information on the structural organization of the alkyl chains.51,52 The CH2 stretches occur at 2850 cm-1 (symmetric) and 2918 cm-1 (asymmetric). Closely ordered monolayers were assumed for SAM25%CF3 by surface plasmon resonance measurements and the peak positions of the OH thiol (Figure 3). The spectrum of SAMOH shows an additional peak at 2878 cm-1. This peak has been assigned by Nuzzo et al.53 to the terminal CH2 bonded to OH.28 It can be seen from the diagrams that this peak vanishes for SAM50%CF3, SAM75%CF3, and SAMCF3. The variations in contact angle (Figure 2) confirmed the OH thiol presence on the surface. The 50% and 75% mixtures of the CF3/OH thiols behaved “nonideally” as the CH2 stretching bands shifted with the increase of CF3 on the surface, toward 2853 and 2925 cm-1, for pure CF3 (Figure 3b). This indicates a less densely packed structure for SAM50%CF3, SAM75%CF3, and SAMCF3 rather than a densely packed structure of the SAMOH and SAM25%CF3. The disorder in the SAMs is a result of the strong dipole produced by the CF3CH2O end groups. This dipole is not compensated along the hydrocarbon chain which leads to local repulsion between side groups. For SAM25%CF3 mixtures, the monolayer structure is not affected by the introduction of OCH2CF3. If this thiol is “diluted” on the (50) Atre, S. V.; Liedberg, B.; Allara D. L. Langmuir 1995, 11, 38823893. (51) Sydner, R. G.; Hsu, S. L.; Krimm S. Spectrochim Acta 1978, 34, 395. (52) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (53) Nuzzo, R. G.; Dubois, L. H.; Allara, D. I. J. Am Chem Soc. 1989, 111, 321-335.

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Figure 4. Molecular structure of a SAM which is prepared from mixtures of thiols containing OH and CF3 end groups. The CH2CF3 group remains above the OH group being the differences in thickness between both groups of around 3 Å. The highest order was found for SAM25%CF3. If the amount of CF3 thiol is higher than 25%, the monolayer tends to disorganize.

surface, the side chains are separated far enough to avoid local repulsion and the monolayer formed is organized with the closely packed trans configuration. As soon as we increase the percentage of OCH2CF3 on the surface, the proximity of the chains and the formation of small clusters of molecules lead to side chain repulsion. Other possible causes of disorder of SAM50%CF3, SAM75%CF3, and SAMCF3 may be steric effects due to the large diameter of CF3 (5.6 Å) compared to CH2 (4.7 Å). This has an effect on a SAM rich in OCH2CF3. For SAMs of mixtures with more than 30% OH thiol, possible steric effects (resulting from near fluorinated groups) are expected to be small. The OH thiol is approximately 3 Å shorter than the CF3 thiol, so when adsorbed together with CF3, the OH groups stay below the CF3 groups (Figure 4). C-F and C-O-C stretches were assigned based on infrared spectra of 2,2,2-trifluoroethanol54 and 2,2,2trifluoroethyl methyl ether.55 In the bulk material the molecules show a peak at 1278 cm-1 and at 1315 cm-1 due to asymmetric stretches of CF3, which shifted to 1282 and 1317 cm-1 for the monolayer. The symmetric stretches appear at 1159 and 1178 cm-1 for bulk material and surface, respectively. The bulk C-O-C stretches are at 1141 cm-1 (asymmetric) and 979 cm-1 (symmetric). These stretches are present in our SAMs at almost the same wavenumbers as in solution, 1141 and 978 cm-1 (Figure 3). The relative intensities of the 1178 cm-1 (symmetric) and 1282 cm-1 (asymmetric) stretch of CF3 are roughly the same for all concentrations except for mixtures of 25% where the 1282 cm-1 stretches are hardly seen. These results suggest that the orientation of the CF3 groups on the monolayer was similar for all experiments except for 25% mixtures, which form well-packed monolayers. It is possible to consider the orientation of the CF3 groups almost perpendicular to the surface due to the high intensity of the symmetric stretch band when compared to the asymmetric stretch band. HSA Adsorption Adsorption of fluorophore labeled HSA on the SAMs was followed by TIRF42,43 which was normalized by surface plasmon spectroscopy.56 The results are shown in Figure (54) Kalasinsky, V. F.; Anjaria, H. V. J. Phys. Chem. 1980, 84, 19401944. (55) Li, Ying-Sing; Cox, F. O.; During, J. R. J. Phys. Chem. 1987, 91, 1334-1334.

Coelho et al.

Figure 5. HSA adsorption onto SAM surfaces followed by TIRF: SAMOH (circles); SAM50%CF3 (squares); SAMCF3 (triangles). The decrease after the maximum levels is due to rinsing with water (solid lines are to guide the eye).

Figure 6. Results from antibody binding to HSA adsorbed on the SAMs. The SAMs were prepared of thiols with OH and CF3 end groups mixed with different ratios in solution (0, 25, 50, 75, and 100 mol % CF3 thiol). The optical density values were normalized by the adsorbed amount of HSA.

5. It can be seen that the adsorption plateaus followed the order SAMOH > SAMCF3 > SAM50%CF3. But a different order retained in the adsorbed amount of HSA after rinsing the adsorbed layer with water, which is SAM50CF3 > SAMCF3 > SAMOH. The irreversibly adsorbed HSA layer retained an adsorbed amount, which is not directly correlated to the contact angles (Figure 2), which are a measure of the surface energy. The SAM surface structure may play a more important role than the surface energy in HSA adsorption. One cause for this effect can be related to the fact that for the SAM50%CF3 the protruding thiols give rise to a steric instability for HSA adsorption. The gaps between the thiol molecules have a depths of the order of 3 Å (Figure 4). Another cause could be the presence of “arrested” water molecules in gaps formed between the thiols. Proteins would not easily displace this water. Thus these water molecules would mask the surface for adsorption of proteins functioning like a “cushion” against protein adsorption. 3.1.3. Conformation of HSA. ELISA was used as a screening test to indicate conformational changes of adsorbed proteins. The antibodies recognize the assessable active epitopes of the protein. The SAMs of the pure components (SAMCF3 and SAMOH) provoked the lowest adsorbed amount of antibodies when comparing SAMs of mixtures of these two thiols (Figure 6). The introduction of hydrophilicity was important for the protein stability and caused a strong increase in antibody binding. (56) Laschitsch, A.; Menges, B.; Johannsmann, D. 2000, 77, 22522254.

Human Serum Albumin of Fluorinated Surfaces

Figure 7. CD spectra of HSA adsorbed to the PVMK (dashed line) and to PMVKCF3/OH (solid line).

The water structure around mixtures of hydrophilic and hydrophobic thiols can be decisive as the only parameter that was varied was the chemical distribution of CF3 and OH groups on the surface. Water layers separate the protein from the surface. The structure of these layers determines the kind of interaction as proteins themselves are surrounded by several layers of water. If the SAM surfaces are able to retain water, for instance in the gaps between the molecular chains of the SAMs (Figure 4), disturbances of the water structure around the proteins are minimized and an unfolding of the protein does not occur. This could explain the increase in functionality of HSA when adsorbed on SAMs consisting of mixtures of thiols. Another reason for this increased stability could be the R-helix retention in the adsorbed HSA molecules via the presence of CF3 and OH units on the surface of these mixtures. 3.2. PVMKCF3/OH. In the previous section it was shown that HSA adsorbed onto SAMs maintains its structure better when thiols with hydrophobic (CF3) and hydrophilic (OH) end groups are mixed. SAMs of thiols are interesting model surfaces for studying the behavior of adsorbed proteins, but for medical applications they underlie many restrictions. They need a metal surface for chemical adsorption, which can be hard to manipulate. Therefore polymer surfaces offer an alternative because of their great variety and possible modifications. In this section HSA was adsorbed onto surfaces of PVMK and PVMKCF3/OH (CF3 and OH groups are bound to 60% of its monomers). HSA was adsorbed for 2 h on PMVK and PMVKCH3/OH. Then the CD spectra of the protein layers, which were irreversibly adsorbed (not sensitive against solvent exchange from HSA solution to HSA free solution), were recorded. Figure 7 shows the difference of HSA adsorbed to PVMKCH3/OH and PVMK. We found that HSA maintains its native state when adsorbed to PVMKCH3/OH (the CD spectrum is identical to that in solution). By contrast, a significant amount of random coil structures are present in the HSA adsorbed on PMVK as indicated by the CD band at 195. In addition we have shown earlier31 that 2 h of adsorption induces strong conformational changes in HSA when adsorbed onto quartz. Therefore we conclude that the combination of CF3/OH groups of the modified polymer maintain a high content of R-helical structure in HSA. This is a similar result as obtained for HSA adsorbed on SAMs consisting of mixtures of thiols. 3.3. Polymer Fluorinated Surfactant Complexes. It was seen in the previous sections that the combination of CF3 and OH groups has an effect in preserving the structure of adsorbed HSA. This is similar to the effect of TFE, which recovers the natural secondary structure of

Langmuir, Vol. 19, No. 18, 2003 7549

Figure 8. CD spectra of HSA adsorbed on surfaces of poly(ethyleneimine) fluorosurfactant complexes. The numbers of carbon atoms of the surfactants are 4 (PEI-C4, dash-dotted line), 7 (PEI-C7, dashed line), and 10 (PEI-C10, dotted line). A CD spectrum of HSA in its native state in solution is shown for comparison (solid line). Table 2. Adsorbed Amount of HSA and the Surface Energy of the Poly(ethyleneimine) Perfluorocarboxylate Complexes PEI-C4, PEI-C7, and PEI-C10 PEI-C4 PEI-C7 PEI-C10 (mg/m2)

amt of adsorbed HSA 5.9 contact angle with n-hexadecanea 50 (deg) surface energy of the complexa 19.0 (mN/m)

4.1 70

1.6 79

13.1

10.5

a The contact angles (advancing) and the surface energy values where taken from ref.35

denaturated HSA when used in suitable concentrations.57 The addition of too much TFE and also IsoF to HSA and other protein solutions has an adverse effect as it increases the amount of helicity to values above native. We assumed that polymer fluorosurfactant complexes may have a similar R-helix inducing influence on the structure of proteins as that of TFE. Therefore the adsorption of HSA to surfaces of fluorinated polymer complexes are studied in this section. 3.3.1. Poly(ethyleneimine) Complexes. HSA was adsorbed for 2 h on films of the complexes of poly(ethyleneimine) and perfluorinated carboxylic acids with 4, 7, and 10 carbon atoms (PEI-C4, PEI-C7, PEI-C10). These polymer complexes form thin films on quartz and silicon wafer surfaces deposited by the self-assembly process.35 The CD spectra of HSA adsorped onto the surfaces of PEI-C4, PEI-C7, and PEI-C10 are shown in Figure 8. It can be seen that the conformation of adsorbed HSA was maintained in contact with PEI-C4 and PEIC7. By contrast, the HSA on a PEI-C10 surface showed a clear decrease in its content of R-helix. The total adsorbed amounts of HSA (determined by surface plasmon resonance spectroscopy,56 cf. Table 2) were 5.9 mg/m2 (PEIC4), 4.1 mg/m2 (PEI-C7), and 1.6 mg/m2 (PEI-C10). They seem to be correlated to the surface energies, which decreases in the line PEI-C4, PEI-C7, PEI-C10 (see Table 3). The higher the surface energy of the poly(ethylene imine) complex, the higher the adsorbed amount of HSA. As an approximation we assume that the amount of adsorbed HSA has a minor effect on its structure. A factor that influences the structure of HSA can be the roughness presented by these surfaces resulting from their deposition onto quartz. This can cause nucleation of the proteins around defects of the surface layer. Indeed surfaces of PEI-C4 and PEI-C7 on quartz were smooth (57) Vieira, E. P.; Motschmann, H.; Mo¨hwald, H.; Coelho, M. A. N. Submitted to Biomacromolecules.

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Coelho et al.

Table 3. Adsorbed Amount of HSA, Secondary Structure Motifs of HSA Adsorbed on Surfaces of Polysiloxane Fluorosurfactant Complexes, and the Surface Energies of the Complexes adsorbed amt (mg/m2) R-helix (%) β-sheet (%) random coil (%) deg of complexation advancing contact angle of n-hexadecane surface energy (mN/m)

PDMS100-C12

PDMS50-C12

PDMS100-C8

PDMS50-C8

4.2 40 21 39 100 85a 8.9a

1.3 35 24 42 50 84a 9.2a

1.4 76 0 24 100 50 19.0b

1.5 64 4 32 50 26 25.0b

a The contact angles and surface energy values were taken from ref 37. b The surface energy values were calculated by the approached derived by Neumann and Li: Li, D.; Neuman, A. J. Colloid Interface Sci. 1992, 148, 190.

while those of PEI-C10 had larger amounts of defects as proved by AFM measurements (pictures not shown). The nucleation process of HSA would then probably induce a higher amount of conformational changes on a surface of PEI-C10 due to local concentration effects. The surface structure influences the conformation of the HSA adsorbed on PEI complexes, as the chemical composition of the outermost angstrom of the surface is the same for all surfaces, CF2CF3. Instead of inducing new R-helices, PEI-C4 and PEI-C7 surfaces were able to maintain the conformation of HSA. 3.3.2. Polysiloxane Complexes. Four surfaces of polysiloxane-perfluorinated acid complexes were used to measure the conformation behavior of HSA. These complexes were made of amino group functionalized polysiloxanes and perfluorinated acids.37 They differ from the PEI-perfluorinated acid complexes due to the change in the polymer backbone. Polysiloxane itself is hydrophobic while PEI is hydrophilic. Results of CD measurements are shown in Table 3. HSA on surfaces of the complexes with perfluorododecanoic acid (PDMS100-C12 and PDMS50-C12) show a lower content of R-helix (40 and 35%, respectively) than those with perfluorooctanoic acid (PDMS100-C12 and PDMS50C12). The R-helix content on PDMS50-C8 is significantly higher than that of the native (76%) while that on PDMS100-C8 is identical to the native (64%). It can be seen further that the R-helix content is higher for a degree of complexation of 100% than for 50%. For the surfaces of PDMS50-C12, PDMS50-C8, and PDMS100-C8, the adsorbed amount of HSA was similar (1.3-1.5 mg/m2). For PDMS100-C12 it was slightly higher (4.2 mg/m2). We assume again that the differences in adsorbed HSA are not decisive for the protein conformation on the complexes. When polysiloxane complexes are considered, perfluorooctanoic acid seems to be more suitable for the preparation of R-helix inducing surfaces than perfluorododecanoic acid. PDMS50-C8 is a good candidate as an R-helix inducing agent similar to TFE and will be investigated in more detail in the future.

4. Conclusions The object of this work has been to investigate the combined influence of CF3 and OH groups (similar to TFE) on the conformation of adsorbed HSA. A long-term intention is to look for possibilities in designing biocompatible surfaces. The results of the present study can be summarized as follows: 1. HSA adsorbed on mixed SAMs retains its native secondary structure, while it denaturates on SAMs made only of the OH or CF3 thiol. 2. CD measurements of HSA that were adsorbed on polymer surfaces of PVMKCF3/OH showed that this polymer retains the secondary structure of HSA while it denaturates significantly on a surface of PVMK. 3. Effects of CF3 groups on the HSA secondary structure were measured in contact with surfaces of polymerer fluorosurfactant complexes. In general the conformation of HSA was maintained. For one complex it was possible to achieve 76% helix for adsorbed HSA, a value ∼10% above the native one. In summary the surfaces investigated in this study could be interesting for the passivation of materials with HSA: the 2 h exposure time does not denaturate the protein. This is in contrast to quartz, octadecyltrichlorosilane, and other typical hydrophobic surfaces on which HSA denaturates quickly. Surfaces enriched in CF3 and OH groups in general maintain the conformation of adsorbed proteins, but the final conformation is strongly dependent on the structure of the substrates where the proteins are going to be adsorbed. Acknowledgment. We thank Andreas Pawlik who was responsible for polymer synthesis and financed together with the help of Hu¨ls. This work was supported by the EC project Amphiphile Monolayers at Fluid Interfaces and on Solid Support (Contract No. ERBCHRXCT930128). Manuel Coelho acknowledges Fundac¸ a˜o Luso Americana para o Desenvolvimento (FLAD) for financial assistance. The Max Planck Society and Fraunhofer Society are gratefully acknowledged. LA034445N