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Polymer Surface Reorientation after Protein Adsorption Matthew L. Clarke and Zhan Chen* Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed March 21, 2006. In Final Form: August 17, 2006 Surface side-chain orientation changes of two polymers have been observed upon protein adsorption using sum frequency generation vibrational spectroscopy. Side-chain-deuterated poly(ethyl methacrylate) and poly(n-butyl methacrylate) were contacted with five protein solutions: albumin, fibrinogen, ubiquitin, cytochrome c, and lysozyme. The CD3/CD2 symmetric stretch ratios of the surface polymer side chains in contact with these different media were compared to each other and to that of the polymer contacting air or phosphate buffered saline. The adsorption of different proteins to the surfaces resulted in polymer side-chain orientations slightly different from each other, with orientations between the air and buffer cases.
Introduction The ability to tailor polymer bulk and surface properties has led to their use in biomedical applications.1-4 Much work has been devoted to understanding how polymer characteristics, such as the surface functional groups, hydrophobicity, flexibility, and additive concentration, affect the biocompatibility of materials.1-4 However, questions still remain as to how polymer and biological media interact, and knowledge of these interactions is vital for the improved development of biomedical materials. Proteins are the first biological material to adsorb onto implanted polymer materials, and the resulting protein conformations determine the overall host response.5 In studying the interactions between proteins and polymers, it is also important to consider how the polymer surface structure changes during the course of protein adsorption, since changes in the polymer surface structure affect its interactions with adsorbed molecules. As the reorientation of the polymer surfaces in response to a contacting environment change can be on the order of a few nanometers or even angstroms,6,7 it is important to employ highly surface-sensitive techniques. Obtaining molecular level information on the polymer surface functional groups is no simple task, and measuring changes in buried interfaces, such as the surface of polymer films with adsorbed proteins, is even more challenging. However, sum frequency generation (SFG) vibrational spectroscopy is aptly suited to monitor these interfaces. Because of its surface selectivity and its sensitivity to the orientation of the surface functional groups, SFG has been applied to the study of numerous polymer interfaces,8-19 showing polymer surface reorientation on the molecular level in various environments. * To whom all correspondence should be addressed. E-mail:
[email protected]. Fax: 734-647-4865. (1) Recum, A. F. Handbook of Biomaterials EValuation: Scientific, Technical, and Clinical Testing of Implant Materials; Macmillan Publishing Company: New York, 1986. (2) Klee, D.; Hocker, H. AdV. Polym. Sci. 1999, 149, 1-57. (3) Angelova, N.; Hunkeler, D. Trends Biotech. 1999, 17, 409-421. (4) Griffith, L. G. Acta Mater. 2000, 48, 263-277. (5) Horbett, T. A., Brash, J. L., Eds. Proteins at Interfaces II, Fundamentals and Applications; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (6) Lukas, J.; Sodhi, R. N. S.; Sefton, M. V. J. Colloid Interface Sci. 1995, 174, 421-427. (7) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 5897-5898. (8) Wang, J.; Woodcock, S. E.; Buck, S. M.; Chen, C.; Chen, Z. J. Am. Chem. Soc. 2001, 123, 9470-9471. (9) Wang, J.; Paszti, Z.; Even, M. A.; Chen, Z. J. Am. Chem. Soc. 2002, 124, 7016-7023. (10) Chen, Z.; Shen, Y. R.; Somorjai, G. A. Annu. ReV. Phys. Chem. 2002, 53, 437-465. (11) Chen, C.; Wang, J.; Woodcock, S. E.; Chen, Z. Langmuir 2002, 18, 1302-1309.
Recently, the interactions of proteins and polymers have been investigated by SFG.20-27 However, it is incorrect to assume that the polymer surface is static during protein adsorption, and elucidating the polymer surface changes may help us further understand the interactions between proteins and polymers. To our knowledge, the reorientation of polymer surfaces upon the adsorption of proteins has not been previously studied by SFG. Here, we investigate how protein adsorption affects the orientation of the polymer surface groups and the dependence of this reorientation on the particular protein and the length of polymer side chain. Experimental Section Reagents. Hydrogenated polymers were purchased from Scientific Polymer Products Inc. (Ontario, NY). Side-chain-deuterated poly(ethyl methacrylate) (d5-PEMA) was purchased from Polymer Source, Inc. (Dorval, QC, Canada). Side-chain-deuterated poly(n-butyl methacrylate) (d9-PBMA) was synthesized as detailed below. Tetrahydrofuran, toluene, phosphate buffered saline (PBS) (pH 7.4), methacrylic acid, and proteins were purchased from Sigma-Aldrich (Milwaukee, WI) and used as received. Figure 1 shows the molecular formulas of d5-PEMA and d9-PBMA. Fully deuterated ubiquitin was purchased from ASLA Biotech (Riga, Latvia). Partially Deuterated PBMA Synthesis. Approximately 1 g of methacrylic acid (Sigma-Aldrich), and 1 g of d10-n-butanol (CDN (12) Wilson, P. T.; Briggman, K. A.; Wallace, W. E.; Stephenson, J. C.; Richter, L. J. Appl. Phys. Lett. 2002, 80, 3084-3086. (13) Liu, Y.; Messmer, M. C. J. Phys. Chem. B 2003, 107, 9774-9779. (14) Hong, S.; Zhang, C.; Shen, Y. R. Appl. Phys. Lett. 2003, 82, 3068-3070. (15) Clarke, M. L.; Wang, J.; Chen, Z. Anal. Chem. 2003, 75, 3275-3280. (16) Rao, A.; Rangwalla, H.; Varshney, V.; Dhinojwala, A. Langmuir 2004, 20, 7183-7188. (17) McGall, S. J.; Davies, P. B.; Neivandt, D. J. J. Phys. Chem. B 2004, 108, 16030-16039. (18) Sung, J.; Kim, D.; Whang, C. N.; Ohe, M.; Yokoyama, H. J. Phys. Chem. B 2004, 108, 10991-10996. (19) Li, G.; Ye, S.; Morita, S.; Nishida, T.; Osawa, M. J. Am. Chem. Soc. 2004, 126, 12198-12199. (20) Wang, J.; Buck, S. M.; Even, M. A.; Chen, Z. J. Am. Chem. Soc. 2002, 124, 13302-13305. (21) Wang, J.; Buck, S. M.; Chen, Z. J. Phys. Chem. B 2002, 106, 1166611672. (22) Wang, J.; Clarke, M. L.; Zhang, Y.; Chen, X.; Chen, Z. Langmuir 2003, 19, 7862-7866. (23) Wang, J.; Even, M. A.; Chen, X.; Schmaier, A. H.; Waite, J. H.; Chen, Z. J. Am. Chem. Soc. 2003, 125, 9914-9915. (24) Kim, J.; Somorjai, G. A. J. Am. Chem. Soc. 2003, 125, 3150-3158. (25) Chen, X.; Clarke, M. L.; Wang, J.; Chen, Z. Int. J. Mod. Phys. B 2005, 19, 691-713. (26) Clarke, M. L.; Wang, J.; Chen, Z. J. Phys. Chem. B 2005, 109, 2202722035. (27) Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z. J. Phys. Chem. B 2006, 110, 5017-5024.
10.1021/la0607656 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/09/2006
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Figure 1. Molecular formulas of (a) d5-PEMA and (b) d9-PBMA. Isotopes, Pointe Claire, QC, Canada) were placed in a 10 mL flask with 3 drops of H2SO4. The solution was stirred and heated to 65 °C in a dry air environment for 2 days. The top organic layer was saved. Fourier transform infrared (FTIR) analysis of this layer shows the formation of an ester bond, indicating that butyl methacrylate (BMA) was synthesized. BMA was dissolved in hexane and washed with 1 M NaOH and then brine solution to remove any remaining methacrylic acid. Most hexane was removed by rotovap. Hydroquinone was removed by running the BMA through a column of inhibitor remover (Sigma-Aldrich). The remaining hexane was removed by rotovap. 2,2′-Azobis(2-methylpropionitrile) (1% molar, Sigma-Aldrich) was added to the BMA and heated to 60 °C for 5 days. The resulting d9-PBMA was dissolved in CH2Cl2, and methanol was added until a solid precipitated. The solid, containing the higher molecular weight polymer, was dried and stored. FTIR analysis indicated the product was d9-PBMA. Gel permeation chromatography showed Mn ) 170 kg/mol, Mw/Mn ) 1.8. Sample Preparation. Polymers were dissolved in toluene (2%, w/w). SFG substrates included fused-silica windows (for the C-H region) and right-angle CaF2 prisms (for the C-D region). The fused-silica windows were cleaned by a sulfuric acid bath saturated with potassium dichromate and thoroughly rinsed with water. The CaF2 prisms were cleaned using soap, water, toluene, and air plasma treatment. Polymer films were prepared by spin-coating the polymer solutions at 2000 rpm onto the windows or prisms. The film thickness was approximately 100 nm, measured by a Dektak profilometer. Prisms were coated on a leg face. The polymer films made by these methods were optically flat. Protein solutions of 1 mg/mL were prepared by dissolving the protein in PBS and were used within 15 min of preparation. Instrumentation. Detailed explanations of the SFG technique have been published previously.28-34 SFG spectra were collected using an EKSPLA system (Vilnius, Lithuania). Specifics about our system have been described in our previous publications.8,35 A near total reflection geometry was employed.23 SFG spectra were collected using the ssp (s - polarized SFG output, s - polarized visible input, and p - polarized IR input) and sps polarization combinations of the input and output laser beams. Although sps spectra were collected and fitted, the spectral differences between the different interfaces were small. Therefore, the discussion will focus on the differences in the ssp spectra. The intensity of the SFG signal at a given IR wavelength (ω) can be fitted by Iijk(ω) ∝ |χijk,nr +
Aijk,q
∑ω - ω q
|2
q + iΓq
(1)
where χijk,nr is the nonresonant background contribution, and Aijk,q, ωq, and Γq are the strength, resonant frequency, and damping coefficient of the vibrational mode q, respectively. Spectra were normalized for variations in the visible and IR beam powers. For (28) Shen, Y. R. The Principles of Nonlinear Optics; John Wiley & Sons: New York, 1984. (29) Shen, Y. R. Annu. ReV. Phys. Chem. 1989, 40, 327-350. (30) Bain, C. D. J. Chem. Soc., Faraday Trans. 1995, 91, 1281-1296. (31) Chen, Z.; Ward, R.; Tian, Y.; Malizia, F.; Gracias, D. H.; Shen, Y. R.; Somorjai, G. A. J. Biomed. Mater. Res. 2002, 62, 254-264. (32) Buck, M.; Himmelhaus, M. J. Vac. Sci. Technol., A 2001, 19, 27172736. (33) Richmond, G. L. Chem. ReV. 2002, 102, 2693-2724. (34) Voges, A. B.; Al-Abadleh, H. A.; Geiger, F. M. EnViron. Catal. 2005, 83-128. (35) Wang, J.; Chen, C.; Buck, S. M.; Chen, Z. J. Phys. Chem. B 2001, 105, 12118-12125.
Figure 2. SFG ssp spectra of (a) d5-PEMA and (b) d9-PBMA in air and in PBS buffer. Dots are experimental data, lines are fitting results. Table 1. Spectral Assignment of d-PAMA C-D Stretching Modes d5-PEMA peaks (cm-1)
d9-PBMA peaks (cm-1)
stretching mode assignment
2068 2098 2131 2141 2172 2221 2241
2067 2107 2139 2152 2173 2194 2216
CD3 symmetric CD2 symmetric CD3 symmetric Fermi resonance CD2 symmetric Fermi resonance CD2 symmetric Fermi resonance CD2 asymmetric CD3 asymmetric
each type of sample, spectra from three consecutive scans were averaged, and two or three of these trials were performed.
Results and Discussion The reorientations of the surface methyl groups of PEMA and PBMA have been previously reported.9,36 It is important to first confirm that d9-PBMA reorients in a manner similar to that of PBMA. The SFG spectra of d9-PBMA in air and in PBS buffer are shown in Figure 2. In fact, it is easily observed that significant reorientation of the surface methyl groups has occurred. Yang et al. previously assigned the C-D stretching modes from selfassembled monolayers (SAMs) of dioctadecyl disulfide.37 Although much shorter, the chemical structures of the side chains of the poly(alkyl methacrylate)s (PAMAs) are similar to those of the SAM surfaces, and similar assignments will be used. The observed peaks for both d9-PBMA and d5-PEMA with assignments of the vibrational modes are shown in Table 1. It is deduced from Figure 2 that polymer surface reorientation occurs for both PAMAs when the contacting medium is changed from air to PBS buffer. Additionally, the reorientation of the surface methyl groups is consistent with previously published results: the methyl groups align more along the surface normal in air, and lie down more in an aqueous environment. To study polymer surface reorientation in response to protein adsorption, several proteins were chosen on the basis of structure and size. Since this work is focused on discerning the differences in polymer surface responses to the adsorption of different proteins, the biological function of the proteins will be ignored. A relatively high protein concentration (1 mg/mL) was chosen to ensure that adsorption was close to or above monolayer adsorption. SFG ssp spectra of d9-PBMA and d5-PEMA in contact with protein solutions of albumin, fibrinogen, lysozyme, cytochrome c, and ubiquitin are shown in Figures 3 and 4, respectively. (36) Chen, C. Y.; Clarke, M. L.; Wang, J.; Chen, Z. Phys. Chem. Chem. Phys. 2005, 7, 2357-2363. (37) Yang, C. S. C.; Richter, L. J.; Stephenson, J. C.; Briggman, K. A. Langmuir 2002, 18, 7549-7556.
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Figure 3. SFG ssp spectra of the d9-PBMA/protein solution interface. Dots are experimental data, lines are fitting results.
Figure 4. SFG ssp spectra of the d5-PEMA/protein solution interface. Dots are experimental data, lines are fitting results. Table 2. CD3/CD2 Symmetric Stretch Ratios for d-PAMAs in Contact with Different Environmentsa environment
d5-PEMA CD3/CD2 symmetric stretch ratio
d9-PBMA CD3/CD2 symmetric stretch ratio
air PBS buffer ubiquitin albumin fibrinogen cytochrome c lysozyme
1.10 0.37 0.45 0.52 0.47 0.42 0.47
1.05 0.61 0.96 0.95 0.92 0.78 0.83
a All protein solutions are 1 mg/mL in PBS buffer. Standard deviation is (0.05 for the d5-PEMA ratios and (0.10 for the d9-PBMA ratios.
It is clear that these spectra are more similar to each other than either the polymer/air or polymer/PBS spectra. This indicates that the adsorption of different proteins to the surface elicits a similar response. However, the response is not identical, and it will be fruitful to investigate the differences in these spectra. The ratio of the ssp CD3/CD2 symmetric stretching modes, including Fermi resonances, can be used as an order parameter of the polymer side chains to characterize the surface hydrophobicity. The spectra of d5-PEMA and d9-PBMA in all environments have been fitted, and the ratios of the ssp CD3/CD2 symmetric stretching modes are shown in Table 2. In air, the ratio is large, which we believe is indicative of the polymer in contact with a hydrophobic medium. When in contact with buffer, the ratio is much smaller, indicating contact with a hydrophilic medium. When in contact with the protein solutions, the ratio is between these values. As the proteins adsorb on the surface, they expose hydrophobic side chains to the surface for more favorable interactions with the polymer.20,21 Additionally, water molecules will be displaced from the surface, creating an environment at the interface that is more hydrophobic. However, since the proteins also contain polar side chains, and some water will still be present in the protein film, the environment still
exhibits hydrophilic aspects. Therefore, as the proteins adsorb, the polymer side chains simultaneously orient intermediate to the two extreme cases (air and buffer) as a result of the new environment that is between the conditions experienced by the polymer in air and in buffer. When examining the adsorption of the proteins to d9-PBMA, it is observed that lysozyme and cytochrome c induce lower CD3/CD2 ratios than the other proteins. If the structures of these five proteins are classified by the ratio of the hydrophobic/polar amino acids, lysozyme and cytochrome c would be the most polar. Therefore, the d9-PBMA surface will be exposed to a more “water-like” environment when lysozyme or cytochrome c are adsorbed compared to the other proteins. Naturally, proteins cannot be solely classified by the ratio of hydrophobic and polar groups, and detailed analysis of the protein-surface interactions should consider the domains of the proteins interacting with the surface and protein conformational changes or denaturation. The CD3/CD2 ratios for d5-PEMA and d9-PBMA are not directly comparable with each other because of the different chemical structures of the side chains. However, examining the differences in reorientation may be possible. It is observed that a greater change in the CD3/CD2 ratios of the side chains occurs when the two most polar proteins adsorb to d9-PBMA, compared to d5PEMA, as evidenced by the larger difference in the CD3/CD2 ratios between lysozyme or cytochrome c and the other proteins. This is attributed to the longer side-chain length of d9-PBMA, which gives the chains a higher degree of flexibility. To further confirm that polymer surface reorientation occurs upon the adsorption of proteins, hydrogenated PBMA was contacted with fully deuterated ubiquitin solution, and SFG C-H stretching signals were collected from the PBMA/deuterated ubiquitin solution interface (data not shown). The reorientation behavior observed was similar to that of the deuterated polymer analogues.
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Conclusion Polymer surface reorientation was observed upon protein adsorption. Additionally, previous SFG studies have observed protein structural changes after adsorption onto various polymer surfaces.26,27 This indicates that the polymer and protein interactions can result in conformation and orientation changes in both the polymer and protein at the interface. Therefore, the polymer surface onto which a protein adsorbs cannot be considered a static surface having the same properties as that in contact with air or buffer. Additionally, we expect that, after adsorption, subsequent protein conformation change or protein exchange will also occur with simultaneous changes in the polymer surface group orientation. Here the polymer orientation change is likely to be similar to the differences observed previously for the adsorption of different proteins. The ability of these
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polymer groups to respond to the proteins may have important roles in determining which proteins adsorb to the surface and in which conformations. Future studies will correlate polymer surface structures to protein structures at the polymer/protein solution interface to understand molecular interactions between polymer surfaces and proteins. Such interactions play important roles in biocompatibility, marine biofouling, food processing, and protein separations. Acknowledgment. The authors thank Christopher Price and Adam Matzger for their help with the polymer synthesis. M.C. thanks the University of Michigan for a Rackham Predoctoral Fellowship. This research is supported by the National Science Foundation (CHE-0315857 and CHE-0449469). LA0607656
