Controlling Cell Attachment Selectively onto Biological Polymer

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Langmuir 2004, 20, 7215-7222

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Controlling Cell Attachment Selectively onto Biological Polymer-Colloid Templates Using Polymer-on-Polymer Stamping Haipeng Zheng,†,‡ Michael C. Berg,† Michael F. Rubner,*,§ and Paula T. Hammond*,† Departments of Chemical Engineering and Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received January 15, 2004. In Final Form: May 21, 2004 A new patterning approach using polymer-on-polymer stamping (POPS) has been developed to fabricate polymer-colloid templates for controlling selective cell attachment. In this paper, a polyamine surface patterned onto a poly(acrylic acid)/poly(allylamine hydrochloride) (PAA/PAH) cell resistant multilayer platform serves as a template for the deposition of close- or loose-packed colloidal particles. Peptides containing the RGD adhesion sequence were used to modify the PAH/colloid surface for specific cell attachment. Cell behavior was studied by varying colloidal packing array density, pattern geometry, and surface chemistry. It was found that loose-packed RGD-modified colloidal arrays enhance cell adhesion, as observed through the development of focal adhesion contacts and orientation of actin stress fibers, but close-packed colloidal arrays induce a rounded and nonadhesive cell morphology and yield a smaller number of attached cells. On loose-packed arrays, cells adjust their shapes to the pattern geometry when the stripe width is smaller than 50 µm and increase their extent of attachment when the concentration of surface RGD peptides is increased. This new biomaterials system allows the examination of cell behavior as a function of RGD surface distribution on the molecular to micrometer scale and reveals cellular response to different surface roughnesses.

Introduction In recent years, microfabrication and nanotechnology have played an important role in the examination of how chemical and topographical surface patterns influence cell biology. These techniques have resulted in many new fundamental studies and developments for biological and medical applications, including tissue engineering,1,2 medical implants,3 biosensors,4,5 DNA chips,6,7 and drugscreening devices.8 Various methods have been applied to pattern and modify surface functionality and topography for studying such cell functions as adhesion, morphology, proliferation, and migration. Among these techniques, both photolithography9-11 and soft lithography12-14 are * To whom correspondence may be addressed. E-mail: [email protected]. [email protected]. † Department of Chemical Engineering, MIT. ‡ Current address: Essilor of America, Inc., Saint Petersburg, FL 33709. § Department of Materials Science and Engineering, MIT. (1) Tan, W.; Desai, T. A. Tissue Eng. 2003, 9, 255-267. (2) Ang, T. H.; Sultana, F. S. A.; Hutmacher, D. W.; Wong, Y. S.; Fuh, J. Y. H.; Mo, X. M.; Loh, H. T.; Burdet, E.; Teoh, S. H. Mater. Sci. Eng., C 2002, 20, 35-42. (3) Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 561-567. (4) Park, T. H.; Shuler, M. L. Biotechnol. Prog. 2003, 19, 243-253. (5) Pancrazio, J. J.; Whelan, J. P.; Borkholder, D. A.; Ma, W.; Stenger, D. A. Ann. Biomed. Eng. 1999, 27, 697-711. (6) De Benedetti, V. M. G.; Biglia, N.; Sismondi, P.; De Bortoli, M. Int. J. Biol. Markers 2000, 15, 1-9. (7) Dimitrjevic, B. Jugosl. Med. Biohem. 2001, 20, 65-71. (8) Erickson, D.; Li, D. Q.; Krull, U. J. Anal. Biochem. 2003, 317, 186-200. (9) Scotchford, C. A.; Ball, M.; Winkelmann, M.; Voros, J.; Csucs, C.; Brunette, D. M.; Danuser, G.; Textor, M. Biomaterials 2003, 24, 11471158. (10) Clark, P. Biosens. Bioelectron. 1994, 9, 657-661. (11) Chehroudi, B.; Gould, T. R. L.; Brunette, D. M. J. Dent. Res. 1990, 69, 291-291. (12) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. Rev. Biomed. Eng. 2001, 3, 335-373.

important tools to make high-resolution micrometer and sub-micrometer scale patterns. Furthermore, nanotopographical surfaces and protein functionalized templates have been fabricated and patterned for cell recognition, through techniques such as interference lithography,15 dip-pen nanolithography,16 polymer demixing,17 and phase separation of block copolymers.18,19 The length scales of such patterns can go down to 1-200 nm. Recently, colloidal particles have also been used as templates to direct cell attachment and spreading for biological applications.20-23 Such techniques provide a convenient strategy to tune surface chemistry and topography over large areas by simply changing particle functionality, size, packing density, and pattern geometry. New approaches have been developed to selectively direct colloidal particle arrays onto patterned polyelectrolyte multilayer films, as reported in previous work.24-26 By changing particle size and pattern dimension, we have (13) Zhang, S. G.; Yan, L.; Altman, M.; Lassle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biomaterials 1999, 20, 1213-1220. (14) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305-313. (15) Fan, Y. W.; Cui, F. Z.; Hou, S. P.; Xu, Q. Y.; Chen, L. N.; Lee, I.-S. J. Neurosci. Methods 2002, 120, 17-23. (16) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S. H.; Mirkin, C. A. Science 1999, 283, 661-663. (17) Dalby, M. J.; Riehle, M. O.; Johnstone, H.; Affrossman, S.; Curtis, A. S. G. Biomaterials 2002, 23, 2945-2954. (18) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2000, 113, 1677-1686. (19) Irvine, D. J.; Ruzette, A. V. G.; Mayes, A. M.; Griffith, L. G. Biomacromolecules 2001, 2, 545-556. (20) Gleason, N. J.; Nodes, C. J.; Higham, E. M.; Guckert, N.; Aksay, I. A.; Schwarzbauer, J. E.; Carbeck, J. D. Langmuir 2003, 19, 513-518. (21) Banerjee, P.; Irvine, D. J.; Mayes, A. M.; Griffith, L. G. J. Biomed. Mater. Res. 2000, 50, 331-339. (22) Miyaki, M.; Fujimoto, K.; Kawaguchi, H. Colloids Surf., A 1999, 153, 603-608. (23) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428.

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fabricated patterned particle monolayers and single particle chains with high selectivity.26 We have systematically controlled the size of these particle clusters by tuning the diameter of patterned polymer templates,27 and further developed an approach to assemble twocomponent colloidal arrays side-by-side on the patterned templates.25 Besides many promising applications in microphotonics and chemical or molecular based sensors, these colloidal arrays can be functionalized with biological ligands for the creation of highly controlled cellular templates for use in biosensors, tissue engineering, and drug-screening assays. Here we apply one of the above-mentioned approaches, polymer-on-polymer stamping (POPS),28 to direct biocompatible micrometer-sized particles into two-dimensional colloidal arrays on polyelectrolyte multilayer platforms, followed by selective peptide functionalization and cell recognition.29 These particles are patterned onto a unique bioinert polyelectrolyte multilayer film formed from the alternating deposition of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). It has been shown that this polyelectrolyte multilayer platform exhibits long-term cell-resistant properties,30 outperforming the more commonly used ethylene glycol self-assembled monolayers (SAMs)13,31-34 or poly(ethylene glycol) (PEG)10,35 and its oligomers.36,37 The POPS process provides the opportunity to directly pattern these multilayers with a functionalizable surface; furthermore, multilayers can be deposited on a wide variety of surfaces, ranging from glass to plastic and flexible polymer surfaces. We have recently reported the use of such patterned surfaces for RGD peptide functionalization and subsequent directed cell attachment via manipulation of RGD density on a molecular length scale.38 It is possible to manipulate RGD surface density on a micrometer scale using patterned colloidal particles on a multilayer template.29 This approach possesses many advantages to systems which utilize gold as a substrate,20 for example, the use of a variety of rigid or flexible substrates and the enhanced adhesion gained by the use of a multilayer film as a conformal and tunable platform. The physical interactions between gold and colloidal particles are much weaker than electrostatic interactions between multilayer films and functionalized colloidal particles in our system. The (24) Zheng, H.; Rubner, M. F.; Hammond, P. T. Langmuir 2002, 18, 4505-4510. (25) Zheng, H.; Lee, I.; Rubner, M. F.; Hammond, P. T. Adv. Mater. 2002, 13, 569-572. (26) Chen, K. M.; Jiang, X.; Kimerling, L. C.; Hammond, P. T. Langmuir 2000, 16, 7825-7834. (27) Lee, I.; Zheng, H.; Rubner, M. F.; Hammond, P. T. Adv. Mater. 2002, 13, 572-576. (28) Jiang, X.; Zheng, H.; Gourdin, S.; Hammond, P. T. Langmuir 2002, 18, 2607-2611. (29) Zheng, H. P.; Berg, M.; Kim, H. J.; Cohen, R.; Rubner, M. F.; Hammond, P. T. Abstr. Pap. Am. Chem. Soc. 2002, 223, 453-COLL. (30) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96-106. (31) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336-6343. (32) McClary, K. B.; Ugarova, T.; Grainger, D. W. J. Biomed. Mater. Res. 2000, 50, 428-439. (33) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (34) Mrksich, M.; Chen, C. S.; Xia, Y. N.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10775-10778. (35) Kim, M. K.; Park, I. S.; Dal Park, H.; Wee, W. R.; Lee, J. H.; Park, K. D.; Kim, S. H.; Kim, Y. H. J. Cataract Refractive Surg. 2001, 27, 766-774. (36) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56, 406-416. (37) Jo, S.; Shin, H.; Mikos, A. G. Biomacromolecules 2001, 2, 255261. (38) Berg, M. C.; Yang, S. Y.; Hammond, P. T.; Rubner, M. F. Langmuir, in press.

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multilayer systems used as a platform for cell attachment can have a variety of functions, such as electronic or ionic conductance,39-43 which might be useful in the monitoring of cellular behavior, and it has been demonstrated that drugs or other small molecules can be imbedded in multilayer systems for gradual release, an aspect which is interesting for a variety of tissue engineering and cellular study applications. Finally the patterning methods demonstrated here can be used to gain features down to the micrometer to sub-micrometer scale, a range significantly smaller than that which has been thus far presented;24,28 we have also demonstrated the ability to pattern bicomponent arrays of such particle systems using the methods described here, thus introducing the possibility of directed bicellular systems.44 The most important advantage to this approach is the flexibility to precisely control the ligand patterns and density by changing the transferred pattern area, as well as the functionality and surface charge density of particles, particle size, and particle packing density. Variation of these parameters allows us to tune the chemical distribution of peptides on the surface, as well as the surface roughness, or topography. This work demonstrates the use of multilayer chemistry and controlled colloid deposition to elucidate the roles of different topographies and distributions of surface functional groups in cellular responses for biomedical applications. Experimental Methods Materials. Poly(allylamine hydrochloride) (PAH) (Mw ) 70 000) and methylene blue were purchased from Sigma. Poly(acrylic acid) (PAA) (Mw ) 90 000) was purchased from Polysciences as a 25% aqueous solution. Poly(dimethylsiloxane) (PDMS) was purchased from Dow Corning. Aliphatic amine polystyrene (CAPS) latex particles (1.0 µm), which include carboxyl and amine groups, were purchased from Interfacial Dynamics Inc. Sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido] hexanoate (Sulfo-LC-SPDP) was purchased from Pierce. RGD peptides and dansyl chloride labeled peptides were prepared by MIT biopolymers laboratory. Preparation of Patterned Polyelectrolyte Multilayer Templates. As demonstrated in previous papers,30,45 PAH/PAA multilayer thin films were assembled using an HMS programmable slide stainer (Zeiss, Inc.). Both the PAH and PAA solutions (0.01 M based on repeat unit) were adjusted to pH 2.0 with HCl. The layer-by-layer process was repeated until 20 layers were constructed, leaving PAA as the outermost layer. A 0.01 M PAH aqueous solution with pH 6.0 was used as polymer ink. PDMS stamps were inked by spin coating a 0.01 M PAH aqueous solution at pH 6 atop patterned features and then dried with N2. The stamps were then brought into contact with a PAH/PAA multilayer platform for 5 min. The stamped templates were then rinsed with agitated water for 2 min to remove additional layers. Assembly and Modification of Colloidal Arrays. The CAPS particle suspension was diluted to 0.1 g/100 mL with pHadjusted deionized (DI) water (pH ) 4.6) for the selective deposition of CAPS particles on the patterned PAH surface. Varying deposition time from 10 min to 2 h controlled the packing density of colloidal arrays. The samples were then immersed in 1 mM Sulfo-LC-SPDP solution for 30 min, rinsed, and then (39) Fou, A. C.; Rubner, M. F. Macromolecules 1995, 28, 7115-7120. (40) Fou, A. C.; Onitsuka, O.; Ferreira, M.; Rubner, M. F.; Hsieh, B. R. J. Appl. Phys. 1996, 79, 7501-7509. (41) DeLongchamp, D. M.; Hammond, P. T. Chem. Mater. 2003, 15, 1165-1173. (42) DeLongchamp, D. M.; Kastantin, M.; Hammond, P. T. Chem. Mater. 2003, 15, 1575-1586. (43) DeLongchamp, D. M.; Hammond, P. T. Adv. Funct. Mater. 2004, 14, 224-232. (44) Zheng, H. P.; Rubner, M. F.; Hammond, P. T. In preparation. (45) Berg, M. C.; Choi, J.; Hammond, P. T.; Rubner, M. F. Langmuir 2003, 19, 2231-2237.

Cell Attachment Selectivity immersed in 1 mM RGD solution overnight, both followed by drying with a stream of N2. Cell Culture and Immunostaining. All cell culture materials were purchased from Gibco/Invitrogen unless otherwise stated. Wild type (WT) NR6 cells were obtained from the Griffith lab at MIT and cultured in MEM-R supplemented with 7.5% (v/v) FBS, 1% (v/v) penicillin (10 000 U/mL, Sigma), 1% (v/v) streptomycin (10 mg/mL, Sigma), 1% (v/v) nonessential amino acids (10 mM), 1% (v/v) sodium pyruvate (100 mM), 1% (v/v) L-glutamine (200 mM), and 1% (v/v) Geneticin G418 antibiotic (350 µg/10 mL of PBS). Cells were passaged at subconfluence by trypsinization (0.25%, 1 mM EDTA, Sigma), incubated at 37 °C, 90% humidity, and 5% CO2, and seeded at 10 000 cells/cm2 on polymer-colloid templates in cell media. After being washed with PBS, cells were fixed with 3.7% formaldehyde solution (Sigma) for 15 min at room temperature. Cells were then rinsed three times with PBS and permeabilized with 0.1% Triton X-100 (Sigma) for 3 min and rinsed twice with PBS. Then, samples were blocked with PBS + 2% BSA (Sigma) for 20 min and incubated with diluted monoclonal anti-vinculin antibody (50:1, Sigma) solution in a humidified chamber at room temperature for 45 min. After being washed with 1% BSA in PBS three times, the samples were incubated with diluted Alexa Fluor 488 goat anti-mouse IgG (H+L) (50:1, Molecular Probes) and rhodaminephalloidin solution (75:1, Molecular Probes) in a dark humidified chamber at room temperature for 45 min. Finally, samples were coated with ProLong Antifade solution after three washes with PBS. Radiolabeling and Measurement. The Sulfo-LC-SPDP modified surfaces were radiolabeled with the peptide, YGRGDSPC, with 125I using a technique developed in the Griffith lab at MIT. The surface radioactivity was then measured to quantify ligand surface density. The Iodobead method was applied to iodinate the tyrosine in the sequence. A solution of 1 mg/mL peptide in 2-[N-morpholinoethane sulfonic acid] buffer (Sigma) was mixed 1 to 4 with a solution of sodium iodide-125 (PerkinElmer) in PBS in the presence of an Iodo-Bead (Pierce Biotechnology). Sodium metabisulfate (12 mg/mL, Sigma) and potassium iodide (Sigma) in PBS were used to quench and chase the reaction, respectively. The radiolabeled peptide was separated from unreacted 125I by fractionating the reaction mixture in a C18 Sep-Pak reverse phase cartridge (Waters) with solutions of water, methanol, and trifluoroacetic acid. The fractions of radiolabeled peptides were diluted with unlabeled peptides (200:1) and coupled to the surfaces activated with Sulfo-LC-SPDP. A Packard Cobra II Auto-Gamma counter was used to measure the activities of samples and standards of known peptide concentrations. An average of six samples subtracted from the background was calculated in each case. Characterization. An optical microscope (Kramer Scientific Corp., Valley Cottage, NY) equipped with a CCD camera (TK-1280U) was used to confirm methylene blue stained polymer patterns and patterned colloidal arrays. A Nikon TE300 epifluorescence microscope (Nikon, Inc., Instrument Group, Melville, NY) equipped with a CCD camera (ORCA-ER) and Openlab system (Improvision Inc., Boston MA) was used to observe colloidal arrays with the modification of dansyl chloride labeled RGD peptides and cell behavior on colloidal arrays. The surface coverage and cell spreading were determined using NIH Image software. For visualization of the rhodamine-phalloidin and antivinculin labeled cells, a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss Inc., Thornwood, NY) was utilized.

Results and Discussion In this study, a colloidal template was created for specific cell interactions by forming a pattern of peptide functionalized colloidal particles atop a cytophobic polyelectrolyte multilayer. In previous papers,46-48 it has been demonstrated that the morphology and degree of swelling (46) Mendelsohn, J. D.; Barrett, C. J.; Chan, V. V.; Pal, A. J.; Mayes, A. M.; Rubner, M. F. Langmuir 2000, 16, 5017-5023. (47) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 42134219. (48) Yoo, D.; Shiratori, S. S.; Rubner, M. F. Macromolecules 1998, 31, 4309-4318.

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Figure 1. Schematic illustration of patterning approach for selective cell attachment.

and hydration of PAH/PAA multilayer films can be tuned by changing the deposition conditions; thick and loopy adsorbed polymer layers are assembled from pH 2.0/2.0 solutions due to the high ionization of PAH and low ionization of PAA. These films have been proven to resist cell attachment for cultures maintained over 1 month because of their high degree of hydration.30 Here we use a (PAH2.0/PAA2.0)10 multilayer film system as a cell-resist platform to direct cell adhesion on a patterned biological colloidal surface. Polymer-on-polymer stamping24,28,45 is used as a tool to create a template of spatially controlled surface charge for the deposition of colloidal particles on the surface. Figure 1 outlines the procedure for this approach. First, 10 bilayers of alternating PAH/PAA were assembled on clean glass, with each polyion adsorbed from pH 2.0 solutions. A patterned PAH monolayer was then transferred by stamping PAH on the PAA top surface of the multilayer. The PAH layer was transferred based on electrostatic interactions between PAH and PAA, leaving a surface of spatially defined positive and negative charge. In previous work,30 we achieved a maximal surface density of transferred amine functional groups on the PAA surface with the PAH ink at pH 11.0; however, in this case, the PAH ink solution was optimized to ensure strong adhesion between the particles and the surface. At a PAH ink solution of pH 6.0, CAPS particles were stable on the patterned PAH/PAA templates even after rinsing with water. The lowered stability observed with more basic ink solutions, and thus denser amine functional surfaces may be due to partial repulsive interactions between charged amine groups on the surface and the CAPS particles. To confirm the presence of a patterned PAH monolayer on the PAA surface of the (PAH/PAA)10 multilayer platform, methylene blue dye solution was used

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Figure 2. Time-dependent average surface coverage of colloidal particles on multilayer polyelectrolyte substrates, colloidal suspension [c] ) 0.1 g/100 mL.

to stain the unstamped PAA region. A clear contrast between the deep blue PAA regions and the PAH regions was observed in optical microscopy in reflection mode. The positively charged PAH surface was then used to direct the deposition of net negatively charged carboxylamine functionalized polystyrene (CAPS) particles containing both carboxyl and primary amine groups. The pH of the colloidal suspension was maintained at pH 4.6, well above the isoelectric point of the particles (3.2), as determined using ζ potential measurements, thus ensuring a negative charge during deposition. The particle density can be controlled on the PAH template by changing the pH and concentration of the colloidal suspension, as well as the deposition time of the colloidal particles. It was previously29 found that the CAPS particles yield the most selective deposition on the patterned PAH surface when the pH is adjusted to approximately 4.6, due to strong charge attractions between the PAH surface and particles. In these experiments, the concentration of the colloidal suspension was fixed at 0.1 g/ 100 mL. After the CAPS particles were selectively deposited on the PAH surface, GRGDSPC peptides, which contain the cell adhesive RGD sequence, were used to further modify the surfaces of the CAPS particles and the patterned PAH layer. The surface of the CAPS particles and the patterned PAH layers both present primary amine groups at pH 7.4, which can be used for the attachment of the peptides. A cross-linker, sulfo-LC-SPDP, consisting of two terminal functional groups, was used to link the RGD peptides to the patterned CAPS-PAH surface. The terminal Nhydroxysuccinimide group reacts with the primary amine groups, and the second terminal group, 2-pyridine disulfide, reacts with the sulfhydryl group of the cysteine at the end of the peptide sequence. Finally, mouse fibroblast NR6 cells were selectively attached to the desired RGD functionalized colloidal region. Time was the primary variable for control of particle packing density in this work, with pH maintained at 4.6 and particle suspension concentrations held at 0.1 g/100 mL. Figure 2 shows that at a particle deposition time of 10 min, the adsorbed particles were loosely packed on the patterned PAH monolayer, with 10% surface coverage. From the micrograph shown in Figure 3a, individual particles confined within 20 µm stripes can be clearly observed. When the adsorption time was increased to 30 min, the surface coverage increased to 35%, as shown in Figure 3b. After a 2 h deposition time, the resulting particles were closely packed with ∼95% coverage of the PAH region, as shown in panels c and d of Figure 3. Figure 4 shows fluorescent micrographs of patterned CAPS particles that have been modified with dansyl

Figure 3. arrays on coverage: picture of

Optical micrographs of packed 1.0 µm CAPS colloidal the patterned PAH surfaces with different surface (a) 10%; (b) 35%; (c) 95%; and (d) a low magnification c. The scale bars are 5 µm, but 100 µm in panel d.

chloride labeled GRGDSPC peptides as described above. For loose-packed colloidal arrays, the CAPS particles exhibited strong fluorescence signals, so that individual CAPS particles and particle clusters were observed on the patterned PAH surface. Close-packed colloidal arrays, on the other hand, presented intense uniform green stripes. Given the same degree of peptide functionalization for each CAPS particle on the surface, it is expected that the RGD surface density should increase with particle packing density. The relative RGD density on the patterned CAPS-PAH surface regions at varying particle packing densities was obtained by determining sulfur atomic ratios in X-ray photoelectron spectroscopy (XPS). Assuming that the sulfo-LC-SPDP molecules completely react with the RGD peptides, there are two sulfur atoms left in each linked RGD peptide atop the CAPS-PAH surface. Therefore, half of the atomic concentration of sulfur from a total atomic concentration of surface elements including C, O, N, and S can be regarded as the relative RGD density on the patterned surface. XPS data (see Table 1) shows that there was 0.10%, 0.16%, and 0.21% average atomic concentration of sulfur on the surfaces (based on the average of four samples in each case) for a PAH top layer only, a loose-packed CAPS colloidal (PAH) surface with 40-50% coverage, and a closepacked CAPS surface with about 95% coverage, respectively. This corresponds to 0.05%, 0.08%, and 0.11% relative RGD density on these surfaces, respectively. The fact that more RGD peptide exists on the modified colloidal particles than on the bare PAH surface results from the different surface amine group densities available on the PAH platform and the colloidal surface and the increased surface area of the three-dimensional colloidal spheres. The number of primary amine groups of the CAPS particles able to react with the cross-linker molecules and RGD peptides is related to the number of unprotonated reactive amine groups available on the surface (pKa ) ∼9.0) of the CAPS particles at pH 7.4, whereas the number of primary amine groups available on the PAH surface is strongly dependent on the pH of the ink solution. As observed in previous work,38 a lower density of amine groups is available when the PAH ink pH is 6.0. Data obtained using XPS represent only relative values. Peptides radiolabeled with 125I were therefore employed to more quantitatively determine the ligand density of samples as a function of particle density. The radioactivities of surfaces patterned with radiolabeled ligands

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Figure 4. Fluorescent micrographs of colloidal arrays modified with dansyl chloride labeled GRGDSPC peptides: (a) loose packed; (b) close packed. The scale bars are 50 µm. Table 1. Surface XPS Analysis of RGD-Modified Polymer (PAH)-Colloid (CAPS) Templates avg atomic concn (%)

C 1s

N 1s

O 1s

S 2p

PAH surfaces loose-packed CAPS-PAH surfaces (40-50% coverage) close-packed CAPS-PAH surfaces (95% coverage)

68.05 76.41 84.40

6.28 4.10 3.71

25.57 19.33 11.68

0.10 0.16 0.21

Figure 5. Ligand surface density as a function of packed particles atop PAH surfaces calculated from radiolabeling data: (1) PAH surface without particles; (2) 2-5%; (3) 3040%; (4) about 95% particle coverage.

were compared to known peptide concentrations to calculate the ligand surface density. For samples stamped with PAH, and samples with 2-5% coverage of colloidal particles packed on the PAH surfaces, approximately 80 000 molecules/µm2 of YGRGDSPC were attached to the surfaces. However, samples packed with 30-40% colloidal particles had much higher peptide surface densities of 1.5 × 106 molecules/µm2. When particle density increased to 95%, the ligand density further increased to 5.8 × 106 molecules/µm2. The data trends shown in Figure 5 confirm that more closely packed particle densities lead to higher surface concentrations of RGD. XPS provides a relative RGD density with respect to surfaces incident to the beam (beam direction was fixed), but the radiolabeling method counts RGD molecules attached to all surface regions, including the areas between individual particles and the PAH surfaces; it should be noted that these densities are expected to be very high due to the large surface area of the colloids. In cell culture experiments, cells selectively attached onto the patterned RGD functionalized colloidal array/ PAH surfaces, avoiding the PAA areas of the cytophobic multilayer surface. Fibroblasts were chosen because they are known for their highly adhesive nature and the large number of RGD receptors which these cells exhibit. A great number of studies by Griffith and co-workers have established the behavior of mouse fibroblast cells on RGD functionalized solid surfaces with molecular level varia-

tions19,21,49 and more recently on patterned multilayers.30,38 The ability to demonstrate directed adhesion with fibroblast cells suggests that directed adhesion should be fairly achievable in a broad range of tissue-forming cells. The specificity of the cell-substrate interactions was confirmed using soluble GRGDS peptides to detach the adherent cells from the GRGDSPC patterns, as previously demonstrated.38 Cells on patterned colloidal particles without RGD modification exhibited small rounded or elliptical shapes (∼300 µm2), indicating their inability to further spread; the rounded cell morphology indicates a lack of cell attachment and proliferation on the surface. Cell attachment results were obtained on RGD-modified patterned colloidal arrays, in which the colloid packing density and patterned stripe width were varied systematically. Examples representative of the effects of these parameters are shown in Figure 6. For loose-packed colloidal arrays, cells attached and spread over large areas on the CAPSPAH surface with high selectivity. In this case, cell morphology strongly depended on particle density and pattern geometry. When the stripe width was about 20 µm, confinement appears to result in cells primarily extended along the stripe direction, forming long narrow shapes directed by the underlying particles. Figure 6a shows a cell that has reached its maximum spread area (∼3500 µm2) when the particle surface coverage is limited to 1-2%. The cell exhibits extended branched psuedopod arrangements at either end around the particles on the surface. When the stripe width is increased to 40 µm or larger, most cells spread into an elliptical or rectangular shape along the stripe direction and vertical direction. Figure 6b shows a cell spread 3000 µm2 when the particle surface coverage was 35%. Elongation of the cells on the surface is further enhanced by a sparse distribution of RGD functionalized particles. When the stripe width was larger than 50 µm, however, as shown in Figure 6c, cells spread out randomly into spindle, oblong, or narrow line shapes along the stripe direction on the patterned CAPSPAH surface, which has 10% particle surface coverage. After modification with GRGDSPC peptides, the closepacked colloidal arrays did not promote cell spreading at all, although a few cells selectively attached onto these particles, as shown in panels d and e of Figure 6. To make the experiments simpler and more comparable, we studied the effect of colloidal density or surface coverage on cell spreading by investigating approximately 50 cells (49) Maheshwari1, G.; Brown, G., Lauffenburger, D. A., Wells, A.; Griffith L. G. J. Cell Sci. 2000, 113, 1677-1686.

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Figure 6. Cell attachment results on RGD-modified patterned colloidal arrays with different colloidal surface coverage and stripe dimension: (a) 1-2% coverage/20 µm stripes; (b) 35% coverage/40 µm stripes; (c) 10% coverage/50 µm stripes; (d) and (e) 95% coverage/50 µm stripes. The scale bars are 50 µm.

Figure 7. Effect of colloidal surface coverage on spreading area of a cell on nonpatterned colloidal/PAH surfaces.

on nonpatterned colloidal PAH surfaces as shown in Figure 7. The average area of a spread cell on a continuous, unpatterned RGD modified PAH surface is 600-800 µm2 but increased almost three times when packed colloidal density atop the PAH surface was 10-30%. If particle surface coverage continued to increase to 45%, the cell spreading area became smaller, around 920-1100 µm2. The average size of cells spread on the 60% particle surface coverage system was about 600 µm2, similar to those on RGD modified PAH surface. But the cells presented the smallest size (300-400 µm2) on close-packed colloidal arrays with 95% of surface coverage. In this case, we believe that surface topography plays an important role in limiting cell spreading and attachment. The increased surface roughness, and the specific topography presented by the spherical array, appears to discourage cell attachment, despite the fact that much higher RGD densities exist on the surface due to the higher density of peptide functionalized particles. Studies of astroglial cells performed by Craighead and co-workers50,51 on etched silicon pillars or wells revealed that the topography presented to a cell can have a large (50) Turner, A. M. P.; Dowell, N.; Turner, S. W. P.; Kam, L.; Isaacson, M.; Turner, J. N.; Craighead, H. G.; Shain, W. J. Biomed. Mater. Res. 2000, 51, 430-441.

impact on its ability to attach and spread. In those studies, pillars of 0.5-2 µm that were well-spaced (1-5 µms) were preferred regions of attachment over smooth etched silicon surfaces. On the other hand, wells of width 0.5 µm and depth of 1 µm set in a continuous etched silicon surface at 0.5-2 µm interwell distances were less favorable surfaces for cellular attachment and yielded rounded cell shapes and minimal spreading. The close-packed particle arrays are somewhat similar to the wells in the Craighead study;50,51 the packing is such that isolated “wells” or pockets can form between the particles, resulting in similar surface features. Turner et al.50 suggested that for such surfaces, the free circulation of culture media in the well structures may be compromised. Green et al. observed similar results in studies of fibroblast cells, in which isolated posts fared well in cell attachment and isolated wells fared poorly at length scales of 2 and 5 µm.52 An alternative explanation is that the RGD functional surface area accessible to cells on the close-packed particle surface, though as yet undetermined, is likely to be considerably lower than that available on intermediate and sparse distributions of particles. This is due to the fact that closepacked particles will expose less surface area to the cell due to close packing with neighboring particles and the inability of the cell walls to conform to such a nonuniform surface. Finally, the actual tortuosity and surface roughness of the close-packed particle surface in general may be too high for favored cell attachment.53 To determine the effect of increasing the GRGDSPC functionality on the colloid surface, the relative RGD density (atomic % from XPS results) of loose-packed particle templates with intermediate (40-50%) coverage was increased to 0.18% (compared to 0.08%), by doubling the concentration of the sulfo-LC-SPDP and RGD solutions and the immersion time. In this case, more cells attached onto the patterned colloidal regions, forming a densely packed layer of cells. For patterned PAH-CAPS tem(51) Craighead, H. G.; James, C. D.; Turner, A. M. P. Curr. Opin. Solid State Mater. Sci. 2001, 5, 177-184. (52) Green, A. M.; Jansen, J. A.; Vanderwaeraden, J. P. C. M.; Vonrecum, A. F. J. Biomed. Mater. Res. 1994, 28 (5), 647-653. (53) Ponsonnet L, Comte V, Othmane A, Lagneau C, Charbonnier M, Lissac M, Jaffrezic N, Mater. Sci. Eng. 2002, C 21 (1-2) (Sp. Iss. SI SEP 1), 157-165.

Cell Attachment Selectivity

Figure 8. Optical micrographs of cell attachment onto patterned colloidal arrays atop PAH surfaces with different surface coverage: (a) 0%; (b) 2%; (c) 30%; (d) 95%. The scale bars are 20 µm.

plates, an area of 1000 × 1000 µm2 includes 10 alternating stripes of PAH-colloid (50 µm) and PAA (50 µm) regions, and the effective cell-attachment area is about 500 000 µm2. When the relative RGD density was 0.08%, as the previously described experiments, an average of approximately 68 cells attached and spread on the colloidal arrays within the 1000 × 1000 µm2 area, with a typical surface coverage of about 1000 µm2 per cell. When the RGD density on the colloids was increased to 0.18%, 117 cells on average were present in the unit area, with a surface coverage of 1500 µm2 per cell, indicating that both cell attachment and spreading were improved by increasing the density of RGD peptides on the colloid surface. Cell morphology can also be studied on randomly arranged colloidal particles atop a cytophobic multilayer, for which PAH (assembled from pH 2.0 solution) was the topmost polyelectrolyte layer. In this study, we wanted to examine the effect of surface topography due to the presence of the colloidal particles, while holding the averaged RGD density of the surface constant. The relative RGD density was maintained fixed at the high relative value of 0.18% (as determined by XPS) through variation of the SPDP linker concentration and reaction times, and different morphologies were observed as the density or surface coverage of particles was increased from 0% coverage to densely packed arrays. In Figure 8a, cells on an RGD-modified PAH surface appear spindle-shaped. On loosely packed CAPS particles (2% coverage), cells began to show a more fully branched shape with several psuedopods directed in various directions, as observed in Figure 8b. This behavior is very similar to the cell behavior atop nanometric topographical templates with approximately 100 nm islands,17 on which cells move along the tops of the islands using “pseudopodial-like” processes, forming a branched morphology. Figure 8b also indicates that stronger adhesion exists between the particles and the polymer platform than between the cells and the particles, such that the particles act as adhesive sites directing cells to spread. When the particles are packed closer (30% coverage), this branched morphology becomes more obvious, as shown in Figure 8c. However, Figure 8d shows that cells maintain a rounded, unspread shape on

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close-packed colloidal arrays (95% coverage). These results are consistent with the above patterned colloidal systems, demonstrating that not only the relative density of RGD peptides in the colloid-PAH regions but also particlepacking density or surface topography is important in directing cell spreading. Topography and RGD density were also found to affect the formation of stress fibers and focal adhesion contacts in the cell studies. Figure 9 contains micrographs of cells stained for actin (top) and vinculin (bottom). Three samples presenting different surface topographies and different amounts of RGD peptides were used, including a PAH platform, a loose-packed colloidal template (5-10% surface coverage), and a close-packed colloidal array (95%). The fluorescent micrographs in panels a and b of Figure 9 show spindle-shaped cells on the PAH platform. The fibroblast cells had moderately developed actin stress fibers in this morphology and a few spikes of vinculin at the cell periphery. Cells on loose-packed colloidal templates (Figure 9c-f) had well-defined stress fibers; the pattern of stress fibers appeared to correlate with cell spreading. The merged reflectance and fluorescence images shown in panels d and f of Figure 9 clearly demonstrated differences in cellular behavior on colloidal templates. The focal adhesion contacts formed sharp spikes of vinculin (Figure 9d,f) at the cell periphery, which have obvious correlation with the particles on the surface. Particles in these systems were located proximate to the vinculin focal adhesions; these results are consistent with observations in panels b and c of Figure 8, in which particles act as adhesive sites that direct cells to spread into different arrangements. Focal contacts also occurred on the PAH surface between particles at the cell periphery, as well as on the colloidal surfaces. However, it is apparent that loose-packed particles promoted cell spreading, with well-defined stress fibers and focal contacts. Panels g and h of Figure 9 show the distribution of actin fibers and vinculin in small rounded or elliptical cells attached to close-packed particle arrays. These cells exhibited limited spreading with very few actin stress fibers and focal adhesion contacts. This phenomenon is similar to results from another cell adhesion colloidal system reported by Carbeck.20 Furthermore, focal contact assembly usually depends on the degree of matrix “morphology”, “deformation”, or “rigidity”.50-54 Our systems provide a unique advantage for the comparison of cell spreading and migration on surfaces of varying rigidity, chemical composition, surface topography, and peptide distribution atop polymer-colloid templates by controlling the swellability of the underlying polymer multilayers and tuning particle size and surface functionality, which will be reported in a future paper. Conclusions This work demonstrates a simple means of tuning surface chemistry and topography to direct cell attachment and spreading through the creation of colloidal arrays deposited on patterned polymer multilayer templates and the control of particle packing density on the template surface. In particular, cells readily attached and spread onto surfaces containing loosely packed particle arrangements on the patterned surface; however, when the RGDfunctionalized particles were arranged in a close-packed manner, the surface became an undesirable region for cell attachment, with fewer cells adhered to the surface with smaller areas and rounded morphologies. It was (54) Katz, B.-Z.; Zamir, E.; Bershadsky, B.; Kam, Z.; Yamada, K. M.; Geiger, B. Mol. Biol. Cell 2000, 11, 1047-1060.

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Figure 9. Fluorescent micrographs showing actin stress fibers and vinculin stains of attached cells onto colloidal arrays atop PAH surfaces with different surface coverage: (a, b) 0%; (c-f) 5-10%; (g and h) 95%. The red color pictures present actin stains, and the green color pictures are with vinculin stains.

observed that fibroblasts can tune their spreading morphology to the pattern geometry when the stripe width is smaller than 50 µm and increase their attachment when the surface RGD density of the particles is increased on the RGD peptide modified loose-packed colloidal arrays atop the PAH platform. Such templates can further promote cell spreading, as evidenced by well-defined stress fibers and focal adhesion contacts. To further explore these colloid template systems, we have also systematically controlled the size of RGD-modified particle clusters, cluster spacing, and particle size and designed twocomponent colloidal arrays on which cells were directed to selectively attach to desired colloidal arrays and spread into different morphologies; these results will be reported separately. We believe the approach used here to pattern and control chemistry and topography in biological col-

loidal arrays can be a very powerful and flexible tool to develop new biomaterial systems for cell recognition, tissue engineering, and the fabrication of cell-based biosensors. Acknowledgment. This work was supported by the DuPont-MIT Alliance. This work utilized shared experimental facilities, which are supported by the MRSEC Program of the National Science Foundation under award number DMR 02-13282. The authors thank Dr. Carlos E. Semino and Dr. Linda Griffith for their helpful suggestions in cell culture experiments. In addition, the authors thank Dr. Shuguang Zhang for the use of his cell laboratory in the Center for Biomedical Engineering, MIT. LA049856Y