Langmuir 2002, 18, 2975-2979
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Micropatterns of a Cell-Adhesive Peptide on an Amphiphilic Comb Polymer Film Jinho Hyun,† Hongwei Ma,† Pallab Banerjee,‡ Janet Cole,‡ Kenneth Gonsalves,‡ and Ashutosh Chilkoti*,† Department of Biomedical Engineering, Campus Box 90281, Duke University, Durham, North Carolina 27708-0281, and Department of Chemistry & C.C. Cameron Applied Research Center, University of North Carolina, Charlotte, 9201 University City Boulevard, Charlotte, North Carolina 28223-0001 Received December 5, 2001. In Final Form: January 30, 2002 We report in this paper a generic method to modify the surfaces of common polymeric biomaterials that enables spatially resolved attachment and growth of mammalian cells in a biologically relevant milieu. We demonstrate that an amphiphilic comb polymer presenting short oligoethylene glycol side chains can be coated onto a number of different polymeric biomaterials, namely polystyrene, poly(methyl methacrylate), and poly(ethylene terephthalate) from a methanol/water mixture. The comb polymer film is stable in water and presents reactive COOH groups at the oligoethylene glycol chain ends, thereby permitting the surface of the comb polymer to be patterned with a cell adhesive, arg-gly-asp peptide. The micropatterned surfaces spatially confine the attachment and growth of fibroblasts for ∼24 h in 10% serum to the patterned regions.
Introduction The ability to control the placement of cells in an organized pattern on a substrate is important for the development of cellular biosensors, biomaterials, and highthroughput drug screening assays.1-4 The critical problem in spatially directing cellular interactions at a biomaterial * To whom correspondence should be addressed. Telephone: (919) 660-5373. FAX: (919) 660-5362. E-mail:
[email protected]. † Duke University. ‡ University of North Carolina. (1) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M. Science 1994, 264, 696-698. Singhvi, R.; Stephanopoulos, G.; Wang, D. I. C. Biotechnol. Bioeng. 1994, 43, 764771. Kapur, R.; Giuliano, K. A.; Campana, M.; Adams, T.; Olson, K.; Jung, D.; Mrksich, M., Vasudevan, C.; Tayor, D. L. Biomed. Microdev. 1999, 2, 99-109. Voldman, J.; Gray, M. L.; Schmidt, M. A. Annu. Rev. Biomed. Eng. 1999, 1, 401-425. Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. Biotechnol. Prog. 1998, 14, 378-387. Borkholder, D. A.; Bao, J.; Maluf, N. I.; Perl, E. R.; Kovacs, G. T. A. J. Neurosci. Methods 1997, 77, 61-66. Dodd, S. J.; Wiliams, M.; Suhan, J. P.; Wiliams, D. S.; Koretsky, A. P.; Ho, C.; Biophys. J. 1999, 76, 103-109. Fromherz, P. Phys. Rev. Lett. 1997, 78, 4131-4134. Humes, H. D.; Buffington, D. A.; MacKay, S. M.; Funke, A. J.; Weitzel, W. F. Nat. Biotechnol. 1999, 17, 451-455. Huynh, T.; Abraham, G.; Murray, J.; Brockbank, K.; Hagen, P. O.; Sullivan, S. Nat. Biotechnol. 1999, 17, 1083-1086. Kapur, R.; Calvert, J. M.; Rudolph, A. S. J. Biomech. Eng.-Trans. ASME 1999, 121, 65-72. Pancrazio, J. J.; Bey, P. P.; Cuttino, D. S.; Kusel, J. K.; Borkholder, D. A.; Shaffer, K. M.; Kovacs, G. T. A.; Stenger, D. A. Sens. Actuators, B 1998, 53, 179-185. St. John, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1998, 70, 11081111. Hammarback, J. A.; Palm, S. L.; Furcht, L. T.; Letourneau, P. C. J. Neurosci. Res. 1985, 13, 213-220. Healy, K. E.; Lom, B.; Hockberger, P. E. Biotechnol. Bioeng. 1994, 43, 792-800. (2) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356363. Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305-313. Lopez, G. P.; Biebuyck, H. A.; Harter, A. R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1077410781. (3) Patel, N.; Padera, R.; Sanders, G. H. W.; Cannizzaro, S. M.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff, K. M. FASEB J. 1998, 12, 1447-1454. (4) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992-5996. Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811-7819. Mkohliso, S. A.; Giovangrandi, L.; Leonard, D.; Mathieu, H. J.; Ilegems, M.; Aebischer, P. Biosens. Bioelectron. 1998, 13, 1227-1235. Groves, J. T.; Mahal, L. K.; Bertozzi, C. R. Langmuir 2001, 17, 5129-5133. Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 561-567. Bhatia, S. N.; Balis, U. J.; Yarmush, M. l.; Toner, M. FASEB J. 1999 13, 1883-1900.
surface is the rapid adsorption of a complex layer of proteins within minutes of contact with serum in cell culture or upon implantation in vivo.5 The adsorbed layer of proteins can physically obscure the micropatterned celladhesive ligand, or present a multitude of alternative cellular signals, which can prevent the formation of cellular patterns, mediated by the micropatterned cell-adhesive ligand. One approach to solving this problem involves the presentation of a biochemical ligand of interest against a protein-resistant, nonfouling surface. The most common method to prevent nonspecific protein adsorption involves the incorporation of poly(ethylene glycol) (PEG) at the surface. A number of methods have been developed to incorporate PEG at surfaces, including physisorption,6 chemisorption,7 chemical grafting,8 and plasma-initiated grafting9 and deposition.10 Most of these methods, however, either require multiple processing steps that must be optimized for the substrate of interest (e.g., grafting of PEG) or are restricted to specific substrates (e.g., chemisorption of oligoethylene glycol-functionalized alkanethiols on gold) and, hence, do not offer a generic route for the surface modification of diverse materials. In contrast, amphiphilic comb polymers that present short oligoethylene glycol side chains11,12 are an attractive alternative because they offer a simple and generic, one(5) Horbett, T. A.; Brash, J. L. ACS Symp. Ser. 1987, No. 343, 1-33. Andrade, J. D.; Hlady, V.; Jeon, S. I. In Hydrophilic polymers; Glass, J. E., Eds.; Advances in Chemistry Series No. 248; American Chemical Society: Washington, DC, 1996; p 51. (6) Lee, J. H.; Kopeckova, P.; Kopecek, J.; Andrade, J. D. Biomaterials 1990, 11, 455-464. Neff, J. A.; Caldwell, K. D.; Tresco, P. A. J. Biomed. Mater. Res. 1998, 40, 511-519. (7) Prime, K. L.; Whitesides, G. M. Science 1991, 25215, 1164-1167. Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1071410721. (8) Harris, J. M. Poly(ethylene glycol) Chmistry: Biotechnical and Biomedical Applications; Plenum Press: New York, 1992. Park, K. D.; Kim, W. G.; Jacobs, H.; Okano, T.; Kim, S. W. J. Biomed. Mater. Res. 1992, 26, 739-756. Tseng, Y. C.; Park, K. J. Biomed. Mater. Res. 1992, 26, 373-391. Amiji, M.; Park, K. J. Biomater. Sci., Polym. Ed. 1993, 4, 217-234. Bearinger, J. P.; Castner, D. G.; Golledge, S. L.; Rezania, A.; Hubchak, S.; Healy, K. E. Langmuir 1997, 13, 5175-5183, (9) Sheu, M. S.; Hoffman, A. S.; Terlingen, J. G. A.; Feijen, J. Clin. Mater. 1993, 13, 41-45. (10) Lopez, G. P.; Ratner, B. D.; Tidwell, C. D.; Haycox, C. L.; Rapoza, R. J.; Horbett, T. A. J. Biomed. Mater. Res. 1992, 26, 415-439.
10.1021/la015712x CCC: $22.00 © 2002 American Chemical Society Published on Web 03/01/2002
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step coating procedure from solution to modify the surface of existing biomaterials to simultaneously attain two important objectives: first, the oligoethylene glycol side chains in the comb polymer prevent the nonspecific adsorption of proteins, and second, the comb polymers can be synthesized to incorporate chemically reactive groups at the termini of the oligoethylene glycol moieties to enable spatially resolved presentation of cell-adhesive ligands at the surface of the comb polymer.12 In essence, this approach mimics the presentation of biological ligands on the termini of oligoethylene glycol-functionalized SAMs of alkanethiols on gold that are the preferred model system to control cellular interactions,2 without the limitation of gold as the substrate. A complementary approach that involves incorporation of PEG into the polymer itself to render the surface nonfouling followed by micropatterning the polymer with a cell-adhesive ligand has also been previously demonstrated.3 We demonstrate in this paper that an amphiphilic comb polymer with a methacrylate backbone and which presents short oligoethylene glycol side chains can be coated onto a number of different polymeric biomaterials, namely, polystyrene (PS), poly(methyl methacrylate) (PMMA), and poly(ethylene terephthalate) (PET) from a methanol/water mixture. The comb polymer film is stable in water and presents reactive COOH groups at the oligoethylene glycol chain ends, which are used to pattern a cell adhesive, arg-gly-asp (RGD) peptide, onto the surface of the comb polymer by microstamping on an activated polymer surface (MAPS),13,14 a soft lithography technique derived from microcontact printing. The micropatterned surfaces spatially confine the attachment and growth of fibroblasts to the patterned regions for up to 24 h in 10% serum. Experimental Section PS, PMMA, and PET were purchased from GoodFellow Corp. and were washed with ethanol prior to use. A comb polymer was synthesized, as described previously, by free radical polymerization of methyl methacrylate (MMA), poly(ethylene glycol) methacrylate (referred to herein as hydroxy-poly(oxyethylene) methacrylate, HPOEM, Mn ∼ 526 g/mol, corresponding to m ∼ 10 in Figure 1), and poly(ethylene glycol) methyl ether methacrylate (referred to herein as poly(oxyethylene) methacrylate, POEM, Mn ∼ 475 g/mol, n ∼ 8.5).12 The comb polymer was characterized by 1H NMR in CDCl3: 4.12 ppm (-OCH3), 3.63.65 ppm (CH2-CH2-O-), 0.5-2 ppm (CH2-C-(CH3)-), and 3.39 ppm (-OH). The composition of the terpolymer was 61 wt % MMA, 21 wt % HPOEM, and 18 wt % POEM using the peaks at 4.12 and 3.39 ppm for quantification. The number average molecular weight (MW) of the comb polymer (Mn) was ∼25 000 with a polydispersity of ∼2.7, as measured by gel permeation chromatography using PS calibration standards. After synthesis, the hydroxyl-functionalized comb polymer was carboxylated by reaction with succinic anhydride in solution, as described elsewhere.15 Thin films of the carboxylated poly(MMA/ HPOEM/POEM) comb polymer were prepared by spin casting a 1% (w/v) water/methanol (20/80 v/v) solution of the comb polymer on different substrates at 2000 rpm and then drying the films at room temperature for 24 h. The COOH groups in spin-cast films of the carboxylated comb polymer were activated by immersion in an aqueous solution of 1-ethyl-3-(dimethylamino)propylcarbodiimide (EDAC, 0.1 M) and N-hydroxysuccinimide (NHS, 0.2 M) for 30 min. The samples were then rinsed with (11) Banerjee, P.; Irvine, D. J.; Mayes, A. M.; Griffith, L. G. J. Biomed. Mater. Res. 2000, 50, 331-339. (12) Irvine, D. J.; Mayes, A. M.; Griffith, L. G. Biomacromolecules 2001, 2, 85-94. (13) Yang, Z.-P.; Chilkoti, A. Adv. Mater. 2000, 12, 413-417. (14) Yang, Z.-P.; Belu, A. M.; Liebmann-Vinson, A.; Sugg, H.; Chilkoti, A. Langmuir 2000, 16, 7482-7492. (15) Storey, R. F.; Hickey, T. P. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 1825-1838.
Letters deionized water, dried under a stream of nitrogen, and used immediately thereafter. The thickness of the comb polymer film, spin-cast onto a silicon wafer was measured on a home-built single-wavelength ellipsometer.16 Contact mode atomic force microscopy (AFM) imaging was performed on a MultiMode SPM (Digital Instruments) in air using a standard silicon nitride tip (Nanoprobe SPM, Digital Instruments) with a spring constant of 0.12 N/m at a scan rate of 3.05 Hz. The sessile water contact angles of the comb polymer films on the different polymer substrates were measured on a Ra´me-Hart goniometer (100-00, Mountain Lakes, NJ) using deionized water. X-ray photoelectron spectroscopy was performed on an SSX-100 instrument (Surface Science Labs, Mountain View, CA) at a takeoff angle of 35°, as described elsewhere.13,14 The fabrication of elastomeric stamps with micrometer-size relief features has been described previously,14 as has the use of MAPS to µCP EZ-Link biotin-PEO-LC-amine ((+)-biotinyl-3,6,9trioxaundecanediamine) (biotin-amine) onto polymers.14 After biotin-amine micropatterning, the micropatterned surfaces were incubated with 0.1 µM Alexa488-labeled streptavidin or with unlabeled streptavidin in HEPES buffered saline (HBS, pH 7.4) containing 0.02% (v/v) Tween 20 for 1 h. The streptavidin patterns were subsequently incubated with 0.1 µM biotin-gly-arg-gly-aspser-pro-lys (biotin-GRGDSPK) in the same buffer for 1 h or with biotin-GRGDSP(K-TMR). NIH 3T3 cells were grown in DMEM (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL), 100 units/mL penicillin, 100 mg/mL streptomycin, and 7.5 mM HEPES at 37 °C in 5% CO2. Cells were plated on the peptide micropatterned slides or controls at a density of 1 × 106 cells/mL in DMEM supplemented with 10% serum. Cells were incubated at 37 °C for either 3 or 24 h, gently rinsed with culture media to remove loosely adherent cells, and imaged under phase contrast optics.
Results and Discussion We synthesized a random terpolymer of MMA, HPOEM, and POEM by free radical copolymerization of the three monomers (Figure 1A).12 NMR showed that the comb polymer contained 61 wt % MMA, 21 wt % HPOEM, and 18 wt % POEM, corresponding to a molar ratio of 16:1:1 in Figure 1A. After derivatization of the terminal OH groups in HPOEM with succinic anhydride,15 a small fraction (
