Vascular Smooth Muscle Cells on Polyelectrolyte Multilayers

Nov 13, 2004 - Like smooth muscle cells in vivo, cultured A7r5 cells are capable of converting between a nonmotile “contractile” phenotype and a m...
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Biomacromolecules 2005, 6, 161-167

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Vascular Smooth Muscle Cells on Polyelectrolyte Multilayers: Hydrophobicity-Directed Adhesion and Growth David S. Salloum,†,§ Scott G. Olenych,‡,§ Thomas C. S. Keller,‡ and Joseph B. Schlenoff*,† Department of Chemistry and Biochemistry, Center for Materials Research and Technology (MARTECH), and Department of Biological Science, The Florida State University, Tallahassee, Florida 32306 Received May 21, 2004; Revised Manuscript Received September 10, 2004

Polyelectrolyte multilayer films were employed to support attachment of cultured rat aortic smooth muscle A7r5 cells. Like smooth muscle cells in vivo, cultured A7r5 cells are capable of converting between a nonmotile “contractile” phenotype and a motile “synthetic” phenotype. Polyelectrolyte films were designed to examine the effect of surface charge and hydrophobicity on cell adhesion, morphology, and motility. The hydrophobic nature and surface charge of different polyelectrolyte films significantly affected A7r5 cell attachment and spreading. In general, hydrophobic polyelectrolyte film surfaces, regardless of formal charge, were found to be more cytophilic than hydrophilic surfaces. On the most hydrophobic surfaces, the A7r5 cells adhered, spread, and exhibited little indication of motility, whereas on the most hydrophilic surfaces, the cells adhered poorly if at all and when present on the surface displayed characteristics of being highly motile. The two surfaces that minimized cell adhesion consisted of two varieties of a diblock copolymer containing hydrophilic poly(ethylene oxide) and a copolymer bearing a zwitterionic group AEDAPS, (3[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate). Increasing the proportion of AEDAPS in the copolymer decreased the adhesion of cells to the surface. Cells presented with micropatterns of cytophilic and cytophobic surfaces generated by polymer-on-polymer stamping displayed a surface-dependent cytoskeletal organization and a dramatic preference for adhesion to, and spreading on, the cytophilic surface, demonstrating the utility of polyelectrolyte films in manipulating smooth muscle cell adhesion and behavior. Introduction Surface modification using polyelectrolyte multilayers (PEMUs) to develop biocompatible materials has been attracting attention lately due to the ease of synthesis and cost-effectiveness of the layer-by-layer technique.1 Surface properties ranging from hydrophobic to hydrophilic, charged to uncharged, and smooth to rough can be generated using a variety of parameters including the chemical nature of the polyelectrolytes and the pH, ionic strength, and temperature used for multilayer synthesis. Because proteins play an important role in the adhesion, spreading, and growth of cells, considerable effort has been expended in developing polyelectrolyte thin films with properties that make the surface adhesive or resistant to protein adsorption.2-7 Although an understanding of PEMU-protein adsorption is necessary to intelligently engineer cell-biomaterial interaction, it is difficult to predict PEMU-cell biocompatibility from simple measurements of protein adsorption.8-10 Recent investigations using cultured cells revealed PEMU properties important for cell biocompatibility.11-17 These investigations have demonstrated that surfaces can be rendered cytophilic or cytophobic by embedding or attaching proteins or peptides to * To whom correspondence should be addressed. † Department of Chemistry and Biochemistry, The Florida State University. ‡ Department of Biological Science, The Florida State University. § These authors have equally contributed to this work.

the multilayer18,19 and by tuning the pH used for multilayer buildup.10 Other modifications such as chemical cross linking have improved the PEMU stability and cell adhesion.20 Certain surfaces such as polysaccharide films made by layerby-layer buildup have been investigated for use as antimicrobial coatings21 and bioactive endovascular stent coatings.22 In fact, polyelectrolyte complexes have a long history of use in preparing bioinert surfaces.23-25 The surface properties of endovascular stents may play an important role in the process of restenosis. During restenosis, vascular smooth muscle cells migrate to cover implanted stents, often building layers of tissue that cause occlusion of the blood flow.26 In evaluating biocompatibility, therefore, it is important to understand how smooth muscle cells interact with PEMU surfaces. Smooth muscle cells are capable of alternating between a “contractile” phenotype, characterized by a nonmotile cell type that possesses both a contractile smooth muscle cytoskeleton and a nonmuscle cytoskeleton for cell support, and a “synthetic” phenotype that is motile and possesses a nonmuscle cytoskeleton used for cell support and cell motility.27,28 The two cell phenotypes normally exhibit differences in cell shape, stability of adhesion, and organization of the underlying cytoskeleton structures. In this study, we have investigated the behavior of rat aortic smooth muscle A7r5 cells cultured on PEMUs. A7r5 cells, which have been used in numerous studies as a model

10.1021/bm0497015 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/13/2004

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smooth muscle cell line, exhibit characteristic smooth muscle cell phenotypic plasticity in converting between adherent contractile cells and highly motile proliferative cells and therefore serve as a useful model for investigating the migration and colonization of coated vascular stents by smooth muscle cells.29 The PEMUs tested here included common combinations of polyelectrolytes, as well as novel polyelectrolytes, yielding a range of surface charge and hydrophobicity properties. We demonstrate that surface charge and surface hydrophobicity alter the cell shape and attachment of cultured A7r5 cells and can be used to manipulate the cell adhesive properties of A7r5 cells. At the opposite ends of the cell adhesion spectrum, we show A7r5 cells on PEMUs made from Nafion and a perfluorinated polyelectrolyte, a hydrophobic surface, spread and adhere as nonmotile cells whereas cells on hydrophilic surfaces with a synthesized zwitterionic copolymer adhere poorly if at all. We further demonstrate the behavior of cells on micropatterned surfaces generated by polymer-on-polymer stamping (POPS). Materials and Methods Materials. 1,3-Propane sultone (PS), acrylic acid, poly(styrenesulfonic acid), PSS (molecular weight, MW 7.3 × 104, Mw/Mn ) 1.06), poly(diallyldimethylammonium chloride), PDADMAC (MW 3.7 × 105, Mw/Mn ) 2.1), poly(allylamine hydrochloride), PAH (MW ∼7 × 104), and poly(acrylic acid), PAA (MW ∼2.4 × 105) were used as received from Aldrich. 2-(Acrylamido)-ethyldimethylamine (AEDA) was obtained from Monomer-Polymer & Dajac Inc. Poly(N-methyl-2-vinyl pyridinium iodide-block-ethylene oxide), PM2VP-b-PEO (PM2VP block 86% quarternized, respective block molecular weights 56 500:5900 Mw/Mn ) 1.08), poly(methacrylic acid-block-ethylene oxide), PMAb-PEO (respective block molecular weights 41 000:30 700 Mw/Mn)1.5), and poly(4-vinyl pyridine), P4VP (MW ∼5 × 104) were from Polymer Source Inc. Nafion was purchased from Aldrich and used as 2.5 wt. % solution in ethanol: methanol 50:50 vol/vol for stamping. All polymer, monomer, and buffer solutions were prepared using 18 MΩ water (except fluorinated polymers). Polyelectrolyte Synthesis. 3-[2-(Acrylamido)-ethyl dimethylammonio] propane sulfonate AEDAPS.30 The zwitterionic monomer was made from 2-(acrylamido)-ethyldimethylamine (AEDA) and PS. Methyl ethyl hydroquinone inhibitor (200 ppm) was removed from AA and AEDA by passing the monomer through a silica column. AEDA (1.0 equiv; 1.42 g, 10 mmol) was dissolved in 28 mL of propylene carbonate. PS (1.1 equiv; 1.34 g, 10.1 mmol) was added to the reaction mixture. The reaction was stirred at 45°C for 1 h. The product, 3-[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate, AEDAPS, precipitated and was washed with petroleum ether with a product yield of 60%. 1H NMR (300 MHz, DMSO): δ 8.45 (bs, 1H), 6.15 (m, 2H), 5.55 (m, 1H), 3.21 (m, 2H), 3.32-3.59 (m, 6H), 3.06 (s, 6H), 2.47 (t, 2H). FTIR: N-H, 3270 cm-1 (m); CdC-H 3037 cm-1, aliphatic C-H, 2964 cm-1; amide C)O, 1668 cm-1(s); and 1545 cm-1 (s); CdC stretch 1628 cm-1; S-O, 1200 cm-1 (s).

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PAA-co-PAEDAPS Copolymer. The polymer was made from AEDAPS and AA via free radical polymerization as described by McCormick and Salazar.30 The monomers were dissolved in aqueous 0.5 M NaCl to assist homogeneity of the copolymer. PAA-co-PAEDAPS (90:10 mol %) was made by copolymerizing 2.4 g (33 mmol) of AA with 3.2 g (12 mmol) of AEDAPS. Similarly, for PAA-co-PAEDAPS (75: 25 mol %), 0.77 g (11 mmol) of AA and 4.27 g (16 mmol) of AEDAPS were used. Total monomer concentration was 0.9 M in 30 mL of distilled water. Potassium persulfate, 7.2 mg (0.1 mol %), was then added to the mixture which was heated at 50 °C under argon and stirring for 120 h. The product was dialyzed against distilled water using 14 000 molecular-weight-cutoff dialysis tubing. Elemental analysis, E.A., (Atlantic Microlab Inc.) for PAA-co-PAEDAPS (nominal 90:10 mol %) (C3H4O2)0.9-co-(C10H20N2O4S)0.1, theory (found): C, 47.98% (46.75%), H 6.14% (6.04%), N, 3.10% (2.30%), S, 3.51% (2.65%). E. A. for PAA-co-PAEDAPS (75:25 mol %), (C3H4O2)0.75-co-(C10H20N2O4S)0.25, theory (found): C, 47.50% (45.41%); H, 6.66% (6.93%); N, 5.83% (5.38%); S, 6.66% (6.60%). The polymers were characterized using FTIR which confirmed the presence of both carbonyl stretch CdO (1725 cm-1, AA and 1670 cm-1, AEDAPS) and the appearance of sulfonate stretch SO3- at ∼1200 cm-1. P4VTDFOP-co-P4VP Copolymer, “PFPVP”. 1,1,1,2,2,3,3,4,4,5,5,6,6 -Tridecafluoro-8-iodooctane (TDFI, C8H4F13I) was reacted with P4VP to give poly(4-vinyl-trideca-fluorooctyl pyridinium iodide)-co-poly(4-vinyl pyridine). DMF and nitromethane, previously dried using molecular sieves, were used to dissolve the reactants. P4VP (1.0 equiv; 1.05 g, 10 mmol) was dried at 110 °C for 4 h then dissolved in a 50 mL of a 1:1 v/v dry mixture of DMF and nitromethane. Under stirring and argon, 1.2 equivalents (5.7 g, 12 mmol) of TDFI was then added to the reaction mixture and left for 48 h at 50 °C. The product was precipitated using ethyl acetate then washed with petroleum ether and dried under vacuum for 24 h at 60°C. E.A. for PFPVP, (C15H11NF13I)0.45co-(C7H7N)0.55, theory (found): C, 40.90% (40.95%); H, 3.39% (3.34%); N, 5.06% (5.11%); F, 29.46% (29.54%); I, 17.58% (17.48%). The elemental analysis results showed the polymer to be 45 ( 3% quaternized with the fluorinating reagent. Experimental reaction yield was 90%. The polymer was characterized by FTIR spectroscopy and exhibited the distinctive C-F stretch in the 1200 cm-1 region of the spectrum. Polyelectrolyte Multilayers on Glass Cover Slips. Glass cover slips (cover glass, No. 1 1/2, 22 mm sq., Corning) were cleaned in 70% H2SO4 (concentrated)/30% H2O2(aq) (“piranha:” caution, piranha is a strong oxidizer and should not be stored in closed containers) then in hot H2O2/ammonia/ water, 1:1:7 vol/vol, rinsed in water and blown dry with a stream of nitrogen. Polymer solution concentrations were 10 mM (with respect to the monomer repeat unit) in 0.25 M sodium chloride except for Nafion which was 0.25 wt % solution in ethanol:methanol 50:50 vol/vol and PFPVP which was 2 mM. Sequential adsorption of polyelectrolytes on cover slips was performed by hand dipping, where the exposure time for the two polymers was 10 min with three rinses of pure solvent, 1 min each, between.

Hydrophobicity-Directed Adhesion and Growth

Film Thickness. The film thickness was determined on Si wafer using a Gaertner Scientific L116S autogain ellipsometer with 632.8 nm radiation at 70° incident angle. A refractive index of 1.54 was employed for the multilayer film. For fluorinated polymers, a refractive index of 1.35 was used. Thicknesses are quoted as “dry” thickness. Contact Angle Measurements. Water contact angles measurements were recorded using a contact angle goniometer (Rame´-Hart, Inc.) with the sessile drop technique. Measurements were done at five different locations of the sample and averaged. The volume of the drop was maintained at 10 µL. Polymer Stamping. Polymer-on-polymer stamping (POPS)31,32 was done with a poly(dimethylsiloxane) stamp (PDMS) that had defined channels. In this technique, the surface of a patterned PDMS was inked by applying 2.5 wt % Nafion using a cotton swap and drying with a nitrogen stream. The patterned side of the stamp was brought in contact with the multilayer surface for 10 min. The surface was characterized with a KLA-Tencor P15 Surface Profiler. Cell Culture. The A7r5 rat aortic smooth muscle cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle medium high glucose supplemented with 10% fetal bovine serum, 100 units mL-1 penicillin G, 100 µg mL-1 streptomycin, and 10 µg mL-1 gentamicin. Cells were plated onto 75 mm2 culture flasks and maintained at 37 °C in a humidified atmosphere with 5% CO2 in air. Cells were fed once per week and passaged when they reached 85% confluency. Cells were released from culture flasks using a trypsin and ethylenediamine tetraacetic acid solution in Hanks buffered salt solution. Microscopy. Equal numbers (∼1 × 104 cell mL-1) of A7r5 cells were plated onto bare or polyelectrolyte coated glass cover slips in 6-well dishes and grown for 30-48 h to allow cell attachment before imaging. Live cell phase images were obtained using a Zeiss Axiovert-35 microscope equipped with a NEC Ti-24A CCD camera and Metamorph Imaging Software. For staining, cells were washed once with cold phosphate buffered saline pH 7.4 (PBS) and then fixed with ice cold acetone for 1 min. Following three washes with PBS, the cells were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. The cells were stained using 1 unit of Alexa Fluor 488 phalloidin (Molecular Probes, Inc.) in PBS for 20 min at room temperature. The cells were washed 3 times in PBS and the cover slips were mounted in Vectashield (Vector Laboratories Inc.) mounting medium containing 1.5 µg mL-1 4′,6-diamidino-2-phenylindole (DAPI). Stained cells were observed with a Nikon Microphot-FX microscope and imaged using a Zeiss color Axiocam. Cell surface area measurements on images of cells for each surface were performed using the area measurement function of ImageJ software ver.1.32J (Wayne Rasband, National Institutes of Health, USA). Statistical analysis was performed using SigmaPlot (Systat Software, Inc.). PEMU Nomenclature. For clarity, we employ the following shorthand for multilayers: (A/B)x where A is the starting polyelectrolyte contacting the substrate, B is the terminating polyelectrolyte in contact with subsequent cell solutions and x is the number of layer pairs. In (A/B)xA, A

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would be the terminating polymer. Salt, MY (cation M and anion Y), has an important role in the buildup process and is represented by (A/B)x @c MY, where c is the molarity of the salt (MY) in the polymer solution. The pH can be included in the nomenclature esp. when using pH dependent PEMUs. Example, (PAH/PAA)2PAH @0.25 M NaCl @pH 7.4, represent 2.5 pairs of PAH/PAA built at 0.25 M NaCl and a pH of 7.4. Results and Discussion Polyelectrolyte multilayers were fabricated to test the effect of both surface charge and surface hydrophobicity on smooth muscle cell attachment and spreading. Using a panel of negatively and positively charged surfaces with varying hydrophobicities, we developed surfaces that encourage, or discourage, the adhesion and proliferation of cells. Surfaces that lead to both cell adhesion and cell spreading are termed “adhesive” in the context of the present work. On some surfaces, cells were observed to adhere loosely, if at all, but did not spread; these we define as nonadhesive. Both types of surfaces may be useful for different types of biological applications. To minimize surface roughness, the number of polyelectrolyte layers was limited to 4 or 5, sufficient to cover the surface, as shown by reduced transport of ions through multilayers of this thickness.33 The A7r5 cells are vascular smooth muscle cells originally derived from rat aortic tissue. Smooth muscle cells were chosen considering the potential application of polyelectrolyte multilayers as coatings for stents, where the problem of restenosis is at least partially caused by the invasion of vascular smooth muscle cells onto implanted stents. A highly cell resistant surface would be of use as a coating for implantable devices. We found that the nature of the polyelectrolyte surface controls whether the cells become highly spread, nonmotile, and contractile with prominent stress fiberlike actin structures or more rounded and highly motile with actin filament-rich lamellipodia and filopodia. These different morphology-motility states are consistent with the cells being either contractile or synthetic, but confirmation of whether the nature of the polyelectrolyte surface can control smooth muscle phenotypic plasticity requires further investigation. A sampling of polyelectrolyte surfaces was chosen to explore the effect of surface charge and hydrophobicity on smooth muscle cell morphology and motility (motility of cell extensions and cell locomotion was confirmed by video microscopy but data is not shown). Table 1 shows the measured contact angle (a measure of hydrophobicity) and thickness of the surfaces tested. A7r5 cells cultured on these layers exhibited graded adhesion and spreading responses with respect to hydrophobicity on both negatively and positively charged polyelectrolyte surfaces, with the negatively charged surfaces being more cell adhesive than the positively charged surfaces of comparable or greater hydrophobicity (Figure 2). A7r5 cells cultured on Nafion and PFPVP, which differ in their surface charges but are similar in hydrophobicity with contact angles of ca. 100°, exhibit the flat and spread appearance of the smooth muscle cell “contractile” phenotype. The effect of

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Table 1. Thickness, Surface Charge, and Contact Angles of Polyelectrolytes Used in This Study multilayer

thickness, Å

surface charge

contact angle

(PAH/PAA)2 (PAH/PAA)2 PAH (PAH/PAA-co-PAEDAPS)2 (PAH/PAA-co-PAEDAPS)2 PAH (PM2VP-b-PEO/PMA-b-PEO)2 (PM2VP-b-PEO/PMA-b-PEO)2 PM2VP-b-PEO (PDADMA/PSS)2 (PDADMA/PSS)2 PDADMA (PFPVP/Nafion)2 (PFPVP/Nafion)2 PFPVP

125 ( 6 181 ( 13 131 ( 3 175 ( 11 22 ( 4 37 ( 2 38 ( 2 55 ( 4 116 ( 6 186 ( 11

+ + + + +

5 ( 2° 9 ( 2° 10 ( 2° 12 ( 2° 15 ( 2° 20 ( 2° 30 ( 3° 55 ( 5° 100 ( 5° 100 ( 5°

Figure 1. Structures of polyelectrolytes used in this study. The fraction alkylated in PFPVP is defined as y.

the charge difference between Nafion and PFPVP appears to be minimal, as the cell shapes appear nearly identical on both surfaces (Figure 2A, 2D). Although well spread, the

cells cultured on PSS (Figure 2B) display more edge “ruffling” than those on Nafion, suggesting that the PSS cells have a more dynamic intracellular organization than the Nafion cells. The PDADMA surface (Figure 2E) although more hydrophobic than the oppositely charged PSS (Figure 2B) appears to be less cell adhesive. Many of the cells plated on the PDADMA surface failed to attach at all and those that did appear to be very active with cellular processes extending out in all directions. A7r5 cells cultured on the most hydrophilic surfaces, PAA (5 ( 2°, Figure 2C) and PAH (9 ( 2°, Figure 2F), appear to be motile, with little spreading and multiple filopodia. PAH, the most hydrophilic positively charged surface tested, appears to be less cell adhesive than PAA. The morphology and motility (not shown) of these cells is similar to that of “synthetic” smooth muscle cells. Table 2 shows the mean cell surface area obtained by measuring images of A7r5 cells cultured on polyelectrolyte multilayers. The utility of fluorinated polyelectrolytes, which are distinctly nonbiological materials, in promoting adhesion and growth of cells was unexpected. In particular, it could be reasoned that the positive polyelectrolyte might mimic the behavior of positive surfactant-type molecules by disrupting the cell membrane and compromising cell integrity. No evidence for dead cells on the perfluorinated surfaces could be found. It is possible that cells respond as they do to the hydrophobic nature of the perfluorinated surface, while the “fluorophobic” qualities of the cell membranes prevent them

Figure 2. Phase images of live A7r5 cells cultured on (A) (PFPVP/Nafion)2, (B) (PDADMA/PSS)2, (C) (PAH/PAA)2, (D) (PFPVP/Nafion)2PFPVP, (E) (PDADMA/PSS)2PDADMA, (F) (PAH/PAA)2PAH. Top panel are negatively charged surfaces (A-C). Bottom panel are positively charged surfaces (D-F). Hydrophobicity decreases from the left (A, D) panel to the right panel (C, F). Right bottom tags represent polymer on outermost surface (scale bar ) 20 µm).

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Hydrophobicity-Directed Adhesion and Growth Table 2. Cell Spreading on Polyelectrolyte Multilayers Tested in This Studya multilayer (PFPVP/Nafion)2 (PFPVP/Nafion)2 PFPVP (PAH/PAA)2 (PAH/PAA)2 PAH (PAH/PAA-co-PAEDAPS)2 (90:10 copolymer) (PDADMA/PSS)2 (PDADMA/PSS)2 PDADMA (P2VMP-b-PEO/PMA-b-PEO)2 (P2VMP-b-PEO/PMA-b-PEO)2 P2VMP-b-PEO (PAH/PAA-co-PAEDAPS)2 (75:25 copolymer)

mean cell area,b µm2 241 ( 17 239 ( 21 157 ( 9 132 ( 10 27 ( 2 191 ( 16 108 ( 9 37 ( 3 29 ( 2 ND

a Cell surface areas covered by individual cells were measured using images of cells cultured on each of the surfaces tested. b Mean surface area ( SEM (n ) 50 cells).

Figure 3. Phase images of live A7r5 cells cultured on diblock polymers of (A) (PM2VP-b-PEO/PMA-b-PEO)2PM2VP-b-PEO and (B) (PM2VP-b-PEO/PMA-b-PEO)2. Right bottom tags represent outermost surface (scale bar ) 20 µm).

from mixing with the multilayer components. Indeed, phase separations of perfluorinated hydrocarbons from other (nonfluorinated) hydrophobic molecules are common. It is known that PEMUs swell when wet, and thick layers may become spongy (except for fluorinated polymers which are very hydrophobic).34 Other studies have shown that the thickness and rigidity of a substrate can affect cell attachment and alter cell shape.35,36 To assess the effect of layer thickness on cell behavior in these studies, we compared cells cultured on 2 and 2.5 layer pairs with those cultured on 4.5 and 5 layer pairs of PSS/PDADMA and PAA/PAH. The cell morphologies were identical on the same upper layer regardless of multilayer thickness (data not shown). To minimize the effects of surface thickness, subsequent experiments were done with 2 and 2.5 layer pairs for which we determined dry thickness (Table 1). Across the range of substrate thicknesses for the 2 and 2.5 layer pairs of surfaces tested here, cell morphology and motility depended more on the hydrophobicity and charge of the top polyelectrolyte layer than on the thickness of the layers (Figure 2, A and C, B and C, and D and F).

Multilayers containing diblock polyelectrolyte polymers or zwitterionic copolymers produced some of the more pronounced and interesting effects on cell attachment (Figure 3). A previous study using self-assembled monolayers of oligo(ethylene glycol) showed them to be cell resistant.37 Similarly, we found that PEMUs generated using two diblock polymers, each containing poly(ethylene oxide), PEO, with one containing PM2VP (poly(N-methyl-2-vinyl pyridinium iodide)), Figure 3A, and the other containing PMA (poly(methacrylic acid)), Figure 3B, also produced nonadhesive hydrophilic surfaces. The cells cultured on these diblock polyelectrolytes were highly rounded, were loosely attached, and had many filopodia. The positively charged diblock polymer (Figure 3A) appeared to be less cell adhesive than the negatively charged PMA surface (Figure 3B). Polymer surfaces with exposed zwitterion groups may mimic certain biological surfaces better than uniformly charged surfaces, because the headgroups of three of the four major membrane phospholipids are zwitterions. The effect of a zwitterionic polymer surface on cell attachment was evaluated using copolymers containing AEDAPS, a zwitterion synthesized for this purpose, and acrylic acid, AA. As shown above (Figure 2C), cells grown on the experimental control PAA surface were less spread than the more hydrophobic negatively charged surfaces, but cells adhered reasonably well and many displayed the prominent leading edge filopodia characteristic of motile cells (Figure 4A). Our PAA-terminated (PAH/PAA)x multilayers appear to be more cell adhesive than those of Mendelson et al.,10 possibly due to differences in the deposition conditions (pH), which maximized the proportion of PAA in their multilayers. Inclusion of AEDAPS in the copolymer made the surfaces even less cell adhesive. On a 90:10 mol % PAA:PAEDAPS copolymer, the cells appeared to be much more loosely attached and have adopted a spiky appearance associated with active filopodia (Figure 4B). Increasing the amount of the zwitterion in the copolymer to 75:25 mol % PAA:PAEDAPS yielded an extremely cell resistant surface. Indeed, the plated cells failed to attach and instead clumped together in nonadherent clusters that floated in the culture medium above the surface. Micropatterning of cultured cells has been used to investigate the effect of cell shape on various cell functions.19,38-40 Frequently, micropatterning of cells is accomplished using complex microfabrication techniques often requiring masking of patterned areas. The technique of polymer-on-polymer stamping31,32 makes the task of micropatterning polyelectrolyte multilayers a simpler process. For POPS, a polyelectrolyte applied to a patterned stamp can be transferred to a

Figure 4. Phase images of live A7r5 cells cultured on (A) (PAH/PAA)2 (B) a 90:10 mol % copolymer of PAA:PAEDAPS and (C) a 75:25 mol % copolymer of PAA:PAEDAPS. Right bottom tags represent outermost surface (scale bar ) 20 µm).

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Figure 5. Micropatterning of A7r5 cells grown on 20 µm wide ridges of Nafion stamped on 80 µm wide troughs of 75:25 mol % PAA:PAEDAPS copolymer. (A) Fluorescently labeled phalloidin staining actin (B) Cell nuclei fluorescently labeled with DAPI and (C) a phase image of the same micropatterned area (scale bar ) 80 µm).

Figure 6. (A) A7r5 cells growing on Nafion (right) stamped onto PAA:PAEDAPS (75:25 mol %) copolymer (left) showing the interface where the cell adhesive (Nafion) stamped surface meets the cell repulsive background surface (scale bar ) 20 µm). (B) A7r5 cells growing on Nafion (right) stamped onto (PAH/PAA)2PAH (left) (scale bar ) 20 µm). (C) Fluorescently labeled phalloidin staining actin in A7r5 cells growing at the interface between the Nafion stamp (right) and PAH background (left) (scale bar ) 10 µm).

polyelectrolyte multilayer surface of the same or opposite charge. In this investigation, we used a PDMS stamp with 20 µm wide parallel ridges separated by 80 µm wide grooves. These stamps were used to create cell-adhesive 20 µm wide lines of Nafion on a cell resistant surface of 75:25 mol % PAA:PAEDAPS. When presented with this micropatterned surface, the A7r5 cells adhered only to the lines of Nafion. Nuclear staining of cells on these surfaces revealed a regular distribution of elongated cells along the Nafion lines (Figure 5B). Phalloidin staining of the actin filaments in the cells revealed a distinct orientation along the Nafion stripe, demonstrating that not only the cell shape can be guided by the stamped surface but also the underlying organization of the cell cytoskeleton (Figure 5A). The peripheral regions of the PDMS stamp created a continuous Nafion surface for a direct side-by-side comparison of cells growing on two different continuous layers of polyelectrolytes (Figure 6). Analysis of cell behavior on these contrasting layers confirmed two important properties of the PEMUs: a single layer is sufficient to create a distinct cell behavior, regardless of the composition of the underlying surface. Cells adhered and spread to the same degree on Nafion stamped as a single layer on underlying surfaces with different properties of cell resistance, PAA:PAEDAPS (Figure 6A) or (PAH/PAA)2PAH (Figure 6B), or on a continuous layer of Nafion overlying PFPVP (Figure 2A). This side-by-side analysis of cells on contrasting surfaces also revealed that the nature of the PEMU surface has a dramatic affect on the organization of the cell actin cytoskeleton. The cells associated with Nafion stamped on PAH adopted very different cytoskeletal arrangements (Figure 6C). Actin filaments in the cells on the PAH surface were located primarily in the spiky filopodia that are mediating the cellsurface interaction. This arrangement of actin filaments is consistent with the cells being primarily the nonmuscle motile “synthetic” phenotype.

In contrast, the actin filaments in the cells growing on the Nafion surface are associated primarily with long stress fiberlike structures (Figure 6C). It is possible that the cells growing on the Nafion surface are in the “contractile” phenotype and the ordered actin bundles are a part of the smooth muscle contractile apparatus. Clearly, the polyelectrolyte surface is capable of modulating the appearance of not only the cell adherence and spreading but also the underlying arrangement of the cellular cytoskeleton. Further investigation is required to elucidate PEMU affects on other phenotypic characteristics of these cells. Conclusion We tested a panel of polyelectrolyte multilayer surfaces designed to examine the effects of substrate surface charge and surface hydrophobicity on attachment of smooth muscle cells. The plastic nature of the smooth muscle cell cytoskeleton allows smooth muscle cells to convert from a contractile nonmotile state to a cytoskeleton arranged for cell locomotion. Alterations of the surface charge and hydrophobicity produced cells that appeared to possess the typical smooth muscle contractile cytoskeleton on hydrophobic surfaces and cells with a cytoskeleton organized for locomotion on more hydrophilic surfaces. Thus, altering cell surface hydrophobicity is a useful approach to manipulate smooth muscle cell cytoskeletal rearrangements. Manipulations of surface charge on hydrophilic surfaces produced the widest spectrum of cell attachment behaviors, which may partially reflect the “spongy” nature of hydrophilic surfaces due to retained water. Hydrophilic charged surfaces of PAA, as documented in previous studies, produced attached motile fibroblast like cells. In contrast, novel diblock polymers with a partially neutral character produced minimally adhesive surfaces. Interestingly, a polyelectrolyte copolymer specifically engineered to mimic the

Hydrophobicity-Directed Adhesion and Growth

zwitterionic nature of cell membranes produces a surface that can be tuned from slightly cell adhesive to completely noncell adhesive. The zwitterionic copolymer, the most important new polymer tested here, provides interesting possibilities for directing cell growth. Using smooth muscle cells, we demonstrate the concept of controlling and directing cell behavior by micropatterning a cell non adhesive zwitterionic copolymer with a cell adhesive Nafion surface. With the appropriate ratio of hydrophobic to hydrophilic lines widths, we were able to induce parallel, single-file, end-toend growth of muscle cells - one of many potential applications for such high “hydrophobicity contrast” patterned surfaces. Acknowledgment. This work was supported by grants from the National Science Foundation (DMR-0309441) and the Florida State University. We are grateful to Rana Jisr and Hassan Rmaile for help in synthesis, Joan Hare for help with the tissue culture, and Tarek Zeidan for NMR analysis. Note Added after ASAP Publication. There were errors in the data reported in Table 2 in the version published ASAP November 13, 2004; the corrected version was published ASAP December 20, 2004. References and Notes (1) Decher, G., Schlenoff, J. B. Multilayer Thin Films - Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2003. (2) Mu¨ller, M.; Rieser, T.; Ko¨the, M.; Kessler, B.; Brissova, M.; Lunkwitz, K. Macromol. Symp. 1999, 145, 149-159. (3) Mu¨ller, M.; Brissova, M.; Rieser, T.; Powers, A. C.; Lunkwitz, K. Mater. Sci. Eng. C 1999, 8-9, 163-169. (4) Mu¨ller, M.; Rieser, T.; Lunkwitz, K.; Meier-Haack, J. Macromol. Rapid Commun. 1999, 20, 607-611. (5) Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G. Biomacromolecules 2000, 1, 674-687. (6) Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J. C. Langmuir 2001, 17, 878-882. (7) Salloum, D. S.; Schlenoff, J. B. Biomacromolecules 2004, 5, 10891096. (8) Han, D. K.; Ryu, G. H.; Park, K. D.; Jeong, S. Y.; Kim, Y. H.; Min, B. G. J. Biomater. Sci.-Polym. Ed. 1993, 4, 401-413. (9) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336-6343. (10) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96-106. (11) Ito, Y.; Chen, G. P.; Imanishi, Y. Bioconjugate Chem. 1998, 9, 277282.

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