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Peptide Attachment to Vapor Deposited Polymeric Thin Films Shashi K. Murthy,† Bradley D. Olsen,‡ and Karen K. Gleason*,‡ Departments of Materials Science and Engineering and Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received November 7, 2003. In Final Form: March 25, 2004
Introduction Chemical vapor deposition (CVD) is a technique by which polymeric thin films can be synthesized and deposited onto a variety of substrates in a single step. An important advantage of CVD is the ability to coat substrates that have complex, three-dimensional topographies and small overall dimensions, such as micrometer-scale neural prostheses.1-3 Applying conformal and reproducible coatings onto such substrates by conventional solution-based techniques is difficult because of surface tension and capillary effects. The CVD technique is, however, limited to materials whose precursors can be easily volatilized. Biologically active coatings, such as films capable of immobilizing biomolecules like fibronectin,4,5 laminin,5 or DNA,6 are extremely difficult to synthesize using CVD alone because of the low volatility of the complex bioactive functional groups. Such coatings are of interest because they can enhance the biocompatibility of medical implants and improve their performance.4,5,7 In neurological implants, for example, surfaces that promote neuron adhesion and proliferation can mitigate the effects of tissue damage caused during implantation and allow better electrical contacts to be made between the implants and surrounding cells.4,5 There are two approaches available for the synthesis of bioactive coatings on substrates with small dimensions and three-dimensional topographies, both of which involve CVD. In most instances, the technique of plasma-enhanced CVD (PECVD, also referred to as gas-discharge treatment) is used to modify existing surfaces by introducing functional groups (such as amine and carboxyl groups) that are capable of directly bonding with biological molecules.7-12 In the second approach, CVD is used to create a thin film that is subsequently modified by solution chemistry to attach molecules capable of binding with biological molecules or cells.6,13 The preliminary step in * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (617) 253-5066. † Department of Materials Science and Engineering. ‡ Department of Chemical Engineering. (1) Murthy, S. K.; Edell, D. J.; Gleason, K. K. In Neuroprosthetics: Theory and Practice; Horch, K. W., Dhillon, G., Eds.; World Scientific: Singapore, 2004; Vol. 2. (2) Limb, S. J.; Gleason, K. K.; Edell, D. J.; Gleason, E. F. J. Vac. Sci. Technol., A 1997, 15, 1814. (3) Pryce Lewis, H. G.; Edell, D. J.; Gleason, K. K. Chem. Mater. 2000, 12, 3488. (4) Cui, X. Y.; Lee, V. A.; Raphael, Y.; Wiler, J. A.; Hetke, J. F.; Anderson, D. J.; Martin, D. C. J. Biomed. Mater. Res. 2001, 56, 261272. (5) Ignatius, M. J.; Sawhney, N.; Gupta, A.; Thibadeau, B. M.; Monteiro, O. R.; Brown, I. G. J. Biomed. Mater. Res. 1998, 40, 264-274. (6) Yang, W.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253. (7) Uyama, Y.; Kato, K.; Ikada, Y. Adv. Polym. Sci. 1998, 137, 1-39.
the second approach can be carried out by nonplasma CVD techniques, such as hot-filament CVD (HFCVD, also known as hot-wire CVD). A significant limitation of the first approach is the potential for substrate damage due to UV irradiation, ion bombardment, and electron-impact fragmentation, all of which occur in any plasma-enhanced process.14-16 The resulting changes in the chemical and physical properties of the substrate, which are often difficult to characterize, could affect the biological function of the coated substrates.17 This limitation can be overcome by following the second approach using a nonplasma CVD technique. Furthermore, since solution chemistry techniques can be used to synthesize a wide variety of biological conjugates (molecules capable of binding to biomolecules or cells), the second approach is considerably more versatile compared to the first. This paper describes the attachment of two different peptides, poly-L-lysine (PLL) and the arginine-lysineaspartic acid (RGD) tripeptide, to fluorocarbon-organosilicon copolymer thin films prepared by HFCVD. Fluorocarbon-organosilicon copolymers synthesized by HFCVD are being investigated as potential biopassivation coatings on neurological implants1 because they combine the desirable characteristics of fluorocarbon polymers (low surface energy and low dielectric constant) and organosilicon polymers (good adhesion to silicon substrates18 and low surface roughness19). Following the HFCVD synthesis, the copolymer films are functionalized by grafting chains of poly(acrylamide) (PAM) onto the surface. The PAM chains are then hydrolyzed to poly(acrylic acid) (PAA) to facilitate peptide attachment. PLL and RGD were chosen because of their known ability to promote the adhesion and proliferation of cells on surfaces.20-25 The methodology described here can be easily extended to other peptides (8) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677-710. (9) Foerch, R.; McIntyre, N. S.; Sodhi, R. N. S.; Hunter, D. H. J. Appl. Polym. Sci. 1990, 40, 1903-1915. (10) Hoffman, A. S. J. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 341. (11) Hayat, U.; Tinsley, A. M.; Calder, M. R.; Clarke, D. J. Biomaterials 1992, 13, 801-806. (12) Terlingen, J. G. A.; Brenneisen, L. M.; Super, H. T. J.; Pijpers, A. P.; Hoffman, A. S.; Feijen, J. J. Biomater. Sci., Polym. Ed. 1993, 4, 165-181. (13) Lahann, J.; Balcells, M.; Rodon, T.; Lee, J.; Choi, I. S.; Jensen, K. F.; Langer, R. Langmuir 2002, 18, 3632-3638. (14) Yasuda, H. Plasma Polymerization; Academic Press: Orlando, FL, 1985. (15) Wrobel, A. M.; Czeremuszkin, G. Thin Solid Films 1992, 216, 203-210. (16) Yasuda, H.; Hsu, T. J. Polym. Sci., Polym. Chem. Ed. 1977, 15, 81-97. (17) Martinez, A. J.; Manolache, S.; Gonzalez, V.; Young, R. A.; Denes, F. J. Biomater. Sci., Polym. Ed. 2000, 11, 415-438. (18) Wrobel, A. M.; Wertheimer, M. R. In Plasma Deposition, Treatment, and Etching of Polymers; d’Agostino, R., Ed.; Academic Press: San Diego, 1990; p 163. (19) Pryce Lewis, H. G.; Casserly, T. B.; Gleason, K. K. J. Electrochem. Soc. 2001, 148 (12), F212-F220. (20) Griscom, L.; Degenaar, P.; LePioufle, B.; Tamiya, E.; Fujita, H. Sens. Actuators, B 2002, 83, 15-21. (21) James, C. D.; Davis, R.; Meyer, M.; Turner, A.; Turner, S.; Withers, G.; Kam, L.; Banker, G.; Craighead, H.; Isaacson, M.; Turner, J.; Shain, W. IEEE Trans. Biomed. Eng. 2000, 47, 17-21. (22) Reed, J.; Hull, W. E.; Vonderlieth, C. W.; Kubler, D.; Suhai, S.; Kinzel, V. Eur. J. Biochem. 1988, 178, 141-154. (23) Drumheller, P. D.; Hubbell, J. A. Anal. Biochem. 1994, 222, 380-388. (24) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548-6555. (25) Tirrell, M.; Kokkoli, E.; Biesalski, M. Surf. Sci. 2002, 500, 6183.
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and could potentially serve as a useful approach in the synthesis of bioactive coatings on substrates with small sizes and complex topographies. Experimental Section Fluorocarbon-organosilicon copolymer and homopolymeric organosilicon thin films were deposited onto silicon wafers by HFCVD, as described previously.26,27 For copolymer synthesis, the precursors used were 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane (obtained from Gelest) and perfluorooctane sulfonyl fluoride (obtained from Aldrich). For homopolymer synthesis, the precursor used was hexamethylcyclotrisiloxane, also obtained from Gelest. Reagents for all further steps were obtained from Aldrich. For the solution polymerization of PAM, a 16 wt/vol % solution of acrylamide (solution 1) was first prepared in distilled water. A second solution (solution 2) containing 0.05 M sodium nitrate and 0.02 wt/vol % sodium azide was prepared in distilled water (these compounds prevent the growth of bacteria or algae in the PAM solution). To prepare a solution of 8 wt % PAM, 100 mL of solution 1 and 100 mL of solution 2 were combined in a flask and mixed well. Approximately half of the mixture was then poured into a second flask. In the next step, 0.20 g of ammonium persulfate was added to one flask, and 0.10 mL of tetramethylethylenediamine was added to the other (these compounds are a redox initiator pair). Each flask was then sparged for 10 min using dry nitrogen. The contents of both flasks were then combined, mixed, and poured into a reaction vessel containing the silicon wafer coated with HFCVD film. The PAM polymerization reaction was then allowed to proceed for 30 min. The silicon wafer was then removed from the reaction vessel and washed thoroughly with water and acetone before drying in air. This procedure was followed with the fluorocarbon-organosilicon copolymer film as well as the homopolymeric organosilicon film. Prior to peptide attachment, the grafted PAM chains were hydrolyzed using 1.5 M hydrochloric acid. For this step, coated silicon wafers were immersed in the acid (along with a stirrer), and the container was then closed and placed in a boiling water bath for 2 h. Retention of the underlying siloxane functional groups was verified by Fourier transform infrared (FTIR) spectroscopy using a using a Nicolet Nexus 870 spectrometer in transmission mode. Poly-L-lysine (FITC labeled, molecular weight 15000-30000) and the RGD tripeptide, both obtained from Aldrich, were attached to the hydrolyzed films using a carbodiimide kit (obtained from Polysciences). The procedure followed using this kit is given in detail as Supporting Information. X-ray photoelectron spectroscopy (XPS) was performed on all samples using a Kratos Axis Ultra spectrometer using a monochromatized aluminum KR source operating at 150 W.
Results and Discussion Figure 1 shows carbon (1s) (C(1s)) high-resolution scans performed before and after the grafting of PAM onto the fluorocarbon-organosilicon copolymer films. The carbonyl group peak is absent prior to grafting and appears after grafting. In addition, the reduction in height of the CF2 and CF3 peaks is consistent with the HFCVD film surface being covered by a thin layer of a different material. The nitrogen and carbonyl content of the PAM-grafted films was unaffected by repeated rinsing with distilled water and prolonged immersion (for periods of 30 min duration followed by rinsing and immersion in distilled water). XPS survey and C(1s) scans were performed after each cycle, and no reduction was observed in the concentration of nitrogen atoms and carbonyl groups. XPS analysis was also performed on fluorocarbon-organosilicon films that were immersed in aqueous solutions of PAM. In this case, the survey and C(1s) indicated that there was no adsorption of PAM onto the HFCVD film surface. (26) Murthy, S. K.; Olsen, B. D.; Gleason, K. K. Langmuir 2002, 18, 6424-6428. (27) Murthy, S. K.; Gleason, K. K. Macromolecules 2002, 35, 19671972.
Figure 1. Carbon (1s) high-resolution scans of the fluorocarbon-organosilicon copolymer film before and after PAM grafting. Peak assignments are taken from refs 25 and 26. Chart 1
These results suggest that the PAM layer is indeed covalently bonded to the fluorocarbon-organosilicon copolymer. The fluorocarbon-organosilicon copolymer consists of a hydrocarbon backbone with siloxane rings as pendant groups and fluorocarbon or sulfonyl fluoride chain ends (Chart 1).26 The siloxane rings originate from the cyclic vinylmethylsiloxane precursor (1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, V3D3), and not all of the vinyl groups in the rings are converted into the hydrocarbon backbone units. In the free radical polymerization of acrylamide, chain propagation occurs along the vinyl bonds of the monomer. It is therefore postulated that bonding between growing PAM chains and the HFCVD fluorocarbon-organosilicon copolymer occurs when the radical chain ends of the former react with vinyl groups present in the latter. The critical role of vinyl groups in the grafting of PAM was confirmed by carrying out the same grafting procedure with homopolymeric organosilicon films deposited by HFCVD film hexamethylcyclotrisiloxane (D3). This precursor is similar to V3D3 but has no vinyl groups. The composition of the film (as determined by XPS) was the same before and after the grafting procedure indicating that PAM chains were not attached to the organosilicon film. The attachment of PAM to the fluorocarbon-organosilicon copolymer film transforms a hydrophobic surface into a hydrophilic surface, which can be functionalized further. One such step is the hydrolysis of the amide groups in PAM into carboxylic acid groups followed by protein attachment via the carbodiimide method. Figure 2 shows C(1s) high-resolution scans performed at various stages of this process. Hydrolysis of the PAM using hydrochloric
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Figure 2. Carbon (1s) high-resolution scans of the PAM-grafted HFCVD films (a) after hydrolysis, (b) after PLL attachment, and (c) after PLL attachment followed by rinsing and immersion in water.
Figure 3. Carbon (1s) high-resolution scans of PAM-grafted HFCVD films (a) after hydrolysis, (b) after RGD attachment, and (c) after RGD attachment and washing with water.
acid results in a slight shift in position of the carbonyl peak relative to its position in PAM (Figure 2a). This reflects the transformation of amide groups into carboxylic acid groups. Note that this transformation is not intended to apply to just the terminal amide groups of the PAM chain; any or all of these amide groups can be transformed to carboxylic acid groups. Preservation of the underlying siloxane functional groups was verified by FTIR spectroscopy. The position and shape of the asymmetric siloxane stretch between 1018 and 1104 cm-1 26 were unchanged by the hydrolysis step. Following the attachment of PLL onto these carboxylic acid groups by the carbodiimide reaction, the C(1s) scan shows three peaks: a carbonyl peak at 286.5 eV; a peak at 284.6 eV corresponding to carbon atoms adjacent to the amino groups in PLL ([-C*H2-NH2] and [-NH-C*H-CdO]);28 and a peak at 283.4 eV corresponding to methylene groups. After the sample was rinsed with water and immersed for 30 min, the peak at 284.6 eV is retained although its intensity is reduced. This decrease in intensity indicates that some PLL molecules are washed off the surface. However, the peak does not undergo any further reduction in intensity when the rinsing and immersion processes are repeated, indicating that the PLL molecules remaining after the first rinsing and immersion are covalently bound to the underlying film. The retention of PLL on the surface was also verified by fluorescence microscopy. The increase in intensity of the 283.4 eV peak is most likely due to exposure of the underlying PAA backbone. Similar results were obtained when the carbodiimide reaction was carried out with a different peptide, the
arginine-glycine-aspartic acid (RGD) sequence. Figure 3 shows C(1s) high-resolution scans obtained before and after RGD attachment and after treatment of the sample with water. The trends seen in Figure 2 are observed in Figure 3 as well. Specifically, the peak at 284.6 eV remains after rinsing and immersion in water, although its intensity is reduced. The retention of this peak indicates that the RGD molecules remaining after the rinsing and immersion process are indeed covalently bonded to the underlying film. Last, the peak at 283.4 eV increases in intensity following rinsing and immersion, again due to the PAA backbone. The attachment of peptides to fluorocarbon-organosilicon copolymer thin films presents an opportunity to modify a material with biopassivation characteristics to make it bioactive. This type of transformation could be useful in the fabrication of implantable integrated circuit devices where the protection of the electronic components is critical and enhancement of the interaction between the device and surrounding cells is desirable.
(28) Huang, N. P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17, 489-498.
Acknowledgment. We acknowledge the support of the National Institutes of Health under Contract NO1NS2-2347 and the U.S. Army (through the Institute of Soldier Nanotechnologies) under Contract DAAD-19-020002 with the U.S. Army Research Office. This work also made use of MRSEC Shared Facilities supported by the National Science Foundation under Award DMR-0213282. Supporting Information Available: Synthetic procedure describing the use of the carbodiimide kit for the peptide attachment step. This material is available free of charge via the Internet at http://pubs.acs.org. LA036102V
