Article pubs.acs.org/Langmuir

Patterned Friction and Cell Attachment on Schizophobic Polyelectrolyte Surfaces Rana M. Jisr,† Thomas C. S. Keller, III,‡ and Joseph B. Schlenoff*,§ †

Department of Physical Sciences, West Virginia University Institute of Technology, Montgomery, West Virginia, 25136, United States ‡ Department of Biological Science, §Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306, United States S Supporting Information *

ABSTRACT: A series of copolyelectrolytes with randomly positioned fluorinated (hydrophobic) and zwitterionic (hydrophilic) repeat units was synthesized and used to assemble multilayers. Regular layer-by-layer growth was observed for polymers with a charge density as low as 6%. The hydrophobicity of these “schizophobic” surfaces increased with increasing fluorine content. Polymer-on-polymer stamping was used to create patterned areas of low and high friction, probed by lateral force microscopy using a modified hydrophobic tip. “Contractile” A7r5 smooth muscle cells adhered to the fluorinated surfaces, but the introduction of zwitterion functionality induced a motile, less firmly attached morphology consistent with the “synthetic” motile phenotype of this cell line. In contrast with cells well adhered (on fluorinated) or completely nonadhering (on zwitterionic) films, incorporation of closely spaced repeat units with strongly contrasting hydrophobicity appears to generate intermediate cell adhesion behavior.

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INTRODUCTION Modification of surfaces with thin films is a widespread method of controlling adhesion at the biological interface. Adhesion/ adsorption can be desired, for example, in directed cell patterning, growth, and differentiation, or undesired, in which case the adhesion falls under the broad heading of “biofouling.”1 Recent research on biofouling has focused on manipulating the chemical and physical properties of surfaces such as morphology,1−3 thickness, roughness,4 surface charge, and surface free energy5 to prevent or reduce biofilm formation. Controlled surface hydration is among various approaches used to improve antifouling performance.6,7 For example, surfaces modified with oligo- or poly(ethylene glycol) (PEG), well hydrated, have a long history of demonstrated antifouling properties.7−9 More recently, adsorption and grafting of certain zwitterionic copolymer brushes provides an effective hydration layer, which acts as a barrier for protein adhesion.10−12 Neutral, hydrophilic polymer coatings contrast with more traditional low surface energy fluorinated “nonstick” coatings, sometimes termed “foul-release” surfaces, from which adherent films may be easily removed.13,14 Mixed hydrophilic/hydrophobic films effective in this respect were reported by Wooley and co-workers, who used hyperbranched fluoropolymers and linear poly(ethylene glycol) chains cross-linked to obtain amphiphilic surfaces with complex morphology and composition.15 The antifouling and fouling-release behavior of amphiphilic surfaces was attributed to nanoscale variations in surface morphology and composition.16,17 Amphiphilic charac© 2013 American Chemical Society

ter is usually built in via block architecture. For example, Ober and co-workers18−20 reported bilayer coatings based on mixed semifluorinated surface active block copolymers with grafted PEG side chains. It was found that surfaces with higher proportions of hydrophobic fluoroalkyl side chains failed to resist the attachment of cells whereas surfaces with PEG side chains showed no settlement of spores. High advancing contact angle (CA) measurements were reported for these surfaces.19,20 Another path to rugged surface modification is via the assembly of ultrathin films of polyelectrolyte complex formed by the multilayer method, which employs alternating deposition of oppositely charged polyelectrolytes.21 Various multilayer surfaces have been shown to discourage cell and protein adsorption and fouling by bacteria.22−29 Zwitterions incorporated into the multilayer surface strongly inhibited cell and protein adsorption, whereas fluorinated multilayers encouraged cell adhesion.29 The components in these polyelectrolyte multilayers (PEMUs) are known to be amorphous and well blended on the molecular level.21 This level of mixing counteracts phase segregation likely to occur when mixed hydrophobic/hydrophilic surfaces are immersed in water. We were interested in the bioadhesive properties of multilayer surfaces incorporating units of high hydrophilicity “contrast.” The present work is Received: October 7, 2013 Revised: November 14, 2013 Published: December 5, 2013 15579

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concerned with the synthesis of a series of random copolyelectrolytes with both fluorinated and zwitterionic functionalities on the same chain and the wetting response of such a “schizophobic” surface. These copolymers were assembled with common polyelectrolytes to yield a range of surface hydrophobicities. Polymer-on-polymer stamping30−32 (POPS) was used to pattern these fluorinated copolymers and AFM and LFM were performed to assess the topology and friction contrast which resulted from differences in surface composition. The various technical challenges included a nontrivial surface modification of PDMS-based stamps so that they could be wetted by a fluorinated solvent.33,34 The response of cells to the biointerface was assessed by culturing A7r5 aortic vascular smooth muscle cells on these surfaces. A7r5 smooth muscle cells are known for their phenotypic plasticity, by which cells can adopt either a “contractile” (differentiated) state in which cells organized to produce contractile force spread and adhere to the surface or the “synthetic” state in which the cells are proliferative and highly motile.

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Figure 1. Structures of polyelectrolytes used in this study.

EXPERIMENTAL SECTION

Materials and Reagents. 1,1,1,2,2,3,3,4,4,5,5,6,6-Tridecafluoro-8iodooctane, (TDFI, C8H4F13I), poly(4-vinylpyridine), P4VP (Mw ∼ 300 000), 1,3-propanesultone (99%), poly(4-styrenesulfonate sodium salt) (PSS; Mw = 64 000, M̅ w/M̅ n = 1.4, 18 wt % in water), and poly(diallyldimethylammonium chloride), (PDADMA; Mw = (4−5) × 105, 20 wt % in water) were used as received from Sigma Aldrich. Sodium chloride (NaCl) and H2SO4 (conc.) were from Fisher. Heptadecafluoro-1,1,2,2-tetrahydrodecyl-triethoxysilane (HFS) was obtained from Gelest. Deionized water (Barnstead, E-pure, Milli-Q) was used to prepare all aqueous solutions. 2,2,2-Trifluoroethanol (TFE, Sigma) was used to prepare all organic solutions. Poly(dimethylsiloxane) (PDMS; Sylgard 184A) prepolymer and curing agent (Sylgard 184 B) were from Dow Corning. Synthesis of Series of Poly(4-vinyltridecafluorooctylpyridinium iodide-co-4-vinylpyridiniumpropanesulfonate), PVFOPco-VPPS. P4VP (1 g, 9.5 mmol) was dried under vac at 110 °C for 4 h, then dissolved in a 100 mL 1:1 v/v mixture of dimethylformamide (DMF) and nitromethane at 50 °C. Under Ar, TDFI (4.875 g, 0.01 mol) was injected into the solution which was heated at 80 °C for 48 h. The product was divided into two portions. The first (PVFOP, Figure 1) was precipitated in ethyl acetate and used as a reference with 38% fluorination as determined by elemental analysis (E.A., Atlantic Microlab Inc.) shown in Table 1. The remaining portion was treated with 20% excess 1,3-propanesultone (PS) (0.348 g, 2.85 mmol) for 24 h at 60 °C. The resulting copolymer was precipitated out of solution using both methanol and ethyl acetate, filtered and dried under vac for 24 h at 60 °C (see Scheme S1, Supporting Information). The copolymer obtained was only soluble in TFE. Elemental analysis showed 62 mol % of the product to be sulfobetaine units (Table 1). Following the same steps, a series of random PVFOP-co-VPPS copolymers were synthesized: (6:94 mol %), (10:90 mol %), (18:82 mol %), (21:79 mol %), (28:72 mol %), (32:68 mol %), and (38:62 mol %). Propanesultone used for all series was 50% in excess. Synthesis was carried in 40 mL 1:1 v/v mixture of DMF/ nitromethane. For PVFOP-co-VPPS (6:94 mol %), P4VP (1 g, 9.5 mmol), TDFI (0.936 g, 1.97 mmol), and PS (1.39 g, 11.4 mmol) were used. For PVFOP-co-VPPS (10:90 mol %), P4VP (0.5, 4.76 mmol), TDFI (0.936 g, 1.97 mmol), and PS (0.78 g, 6.38 mmol) were used. For PVFOP-co-VPPS (18:82 mol %), P4VP (0.5 g, 4.76 mmol), TDFI (1.443 g, 3.04 mmol), and PS (1.58 g, 12.9 mmol) were used. For PVFOP-co-VPPS (21:79 mol %), P4VP (0.5 g, 4.76 mmol), TDFI (1.872 g, 3.95 mmol), and PS (0.697 g, 5.71 mmol) were used. For PVFOP-co-VPPS (28:72 mol %), P4VP (0.5 g, 4.76 mmol), TDFI (2.145 g, 4.53 mmol), and PS (1 g, 8.18 mmol) were used. For PVFOP-co-VPPS (32:68 mol %), P4VP (0.5 g, 4.76 mmol), TDFI

(2.35 g, 4.95 mmol), and PS (0.654 g, 5.35 mmol) were used. Elemental analyses for the PVFOP-co-VPPS copolymers are listed in Table 1. The copolymers were characterized by FTIR spectroscopy and were identifiable by the distinctive C−F stretch in the 1200 cm−1 region of the spectrum. See Figure S1 in the Supporting Information. The sulfonate stretch appeared at ∼1035 cm−1. The N−H peak at 1414 cm−1 disappeared, and a peak at 1640 cm−1 appeared indicating high percent quaternization (Figure S1, Supporting Information). Synthesis of Poly(4-vinylpyridiniumpropanesulfonate)-co(vinylpyridine), PVPPS-co-VP. Using modified literature techniques,35,36 P4VP was alkylated with PS. P4VP (1 g, 10 mmol) was dried under vacuum and dissolved in a 50 mL of 1:1 v/v mixture of DMF and nitromethane. At 50 °C under argon, 1,3-propanesultone (1.74 g, 14.25 mmol) was injected into the reaction flask dropwise for 30 min and the temperature was raised to 80 °C. The mixture was stirred under argon for 36 h. The heterogeneous product was precipitated in ethyl acetate and dried under vac for 24 h at 60 °C (Scheme S1B, Supporting Information). Elemental analysis showed that PVPPS-co-VP was ∼80% quaternized. Elemental analysis for (C10 H13NSO3)0.80-co-(C7H7N)0.20· H2O calcd (found): C, 51.1% (50.12%); H, 6.26% (6.36%); N, 6.34% (6.49%); S, 11.60% (11.59%). FTIR showed two characteristic sulfonate peaks at ∼1035 and ∼1200 cm−1. The IR peak at 1414 cm−1 was still present, indicating that the copolymer was not 100% alkylated (Figure S2, Supporting Information). Multilayer Buildup and Thickness Measurements. Sequential adsorption of PVFOP-co-VPPS (1 mM) and PSS (1 mM 0.5 M NaCl) polyelectrolytes on polished Si[100] wafers was performed by hand dipping where the exposure time for the two polymers was 10 min with three 1 min rinses with TFE and water in the following, cyclical, order: PVFOP-co-VPPS, TFE, TFE, water, PSS, water, water, TFE, and so forth. For cell culture, glass coverslips (cover glass, no. 11/2, 22 mm sq., Corning) were cleaned sequentially in ethanol, then in 70% H2SO4 (conc.)/30% H2O2(aq) for 20 min, and then in hot H2O2/ammonia/ water, 1:1:7 v/v, rinsed in water, and dried under a stream of nitrogen. Fluorinated zwitterionic copolymers were assembled with PAA (at pH= 7.4). Polymer solution concentrations were 1 mM (with respect to the monomer repeat unit) with no salt added to the polymer solutions. Sequential adsorption of polyelectrolytes on coverslips was performed by hand dipping, where the exposure time for the two polymers was 10 min with three rinses of fresh distilled water, 1 min each between. 15580

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Table 1. Elemental Analysis for the Series of Random Copolymers, PVFOP-co-VPPS and for PVPPS-co-VP Polyzwitteriona calcd % (found %) C (C15H11INF13)0.06(C10H13NSO3)0.94·H2O (C15H11INF13)0.10 (C10H13NSO3)0.90·H2O (C15H11INF13)0.18(C10H13NSO3)0.82·H2O (C15H11INF13)0.21(C10H13NSO3)0.79·H2O (C15H11INF13)0.28(C10H13NSO3)0.72·H2O (C15H11INF13)0.32(C10H13NSO3)0.68·H2O (C15H11INF13)0.38(C10H13NSO3)0.62·H2O (C15H11INF13)0.38(C7H7N)0.62·H2O a

46.4 45.0 42.4 41.6 39.8 38.9 37.7 39.8

(43.1) (44.2) (42.2) (40.7) (38.5) (36.7) (37.1) (39.8)

H 5.4 5.3 4.7 4.6 4.2 4.0 3.8 3.5

(4.9) (5.5) (4.8) (4.9) (4.1) (4.0) (3.7) (3.1)

N 5.3 5.0 4.5 4.4 4.1 3.9 3.7 4.6

(5.0) (4.9) (4.8) (5.1) (4.0) (3.7) (4.1) (4.7)

I 2.9 4.5 7.4 8.4 10.3 11.4 12.7 15.9

(7.8) (4.9) (5.4) (9.1) (10.1) (13.2) (12.0) (18.6)

F 5.6 8.8 14.4 16.3 20.1 22.1 24.8 31.0

(5.7) (8.9) (14.2) (16.2) (20.4) (21.6) (25.1) (31.7)

S 11.3 10.3 8.5 7.9 6.7 6.1 5.3

(9.5) (9.6) (8.6) (7.2) (7.0) (6.0) (5.1)

F/S 0.50 0.86 1.69 2.05 3.00 3.62 4.70

(0.60) (0.93) (1.65) (2.25) (2.91) (3.60) (4.92)

All samples contained small amounts of water (approximately 1 H2O per repeat unit). Quasielastic Light Scattering (QELS). The hydrodynamic radius (Rh) of the fluorinated zwitterionic copolymers was measured in solution using a QELS unit from Wyatt Technologies. Measurements were performed in scintillation vials filled with 0.2 wt % PVFOP-coVPPS in TFE. The pure solvent was filtered through 0.02 μm filters (Whatman, ANOTOP), and the polymer solutions were filtered again through 0.2 μm filters.

For POPS, PEI and PSS were deposited on Si wafers with PSS as the terminating layer. Both solutions were 10 mM with respect to monomer repeat unit. A robotic platform exposed the silicon wafers alternately to the two polymer solutions for 5 min each, with three intermediate rinses in water for 30 s each. Thickness was measured for dry surfaces using a Gaertner Scientific L116B autogain ellipsometer with a 632.8 nm radiation at 70° incidence angle. A refractive index (RI) of 1.6 and 1.7 was employed for high % fluorinated multilayers, and a RI of 2 was used for low % fluorinated multilayers. The thickness of multilayers used in cell cultures was 360 ± 15 Å. Contact Angle Measurements. Static contact angles of 10 μL water droplets were measured using a Rame-Hart NRL goniometer model 100. Contact angles were averaged over seven measurements on different areas of sample surface (RSD 10%). Dynamic contact angles (both advancing (Θa) and receding (Θr)) were measured using the Wilhelmy plate technique (Cahn DCA 300 Analyzer) for surfaces deposited on Si[100] (2.5 cm × 2 cm) with an immersion rate in water of 100 μm s−1. Cell Culture and Microscopy. A7r5 rat aortic smooth muscle cells were grown for 24−36 h on PEMU coated coverslips in Dulbecco’s modified Eagle’s high glucose medium supplemented with 10% fetal bovine serum (FBS) at 37 °C. Cells were washed twice with phosphate buffered saline (PBS) pH 7.4 at 37 °C and fixed in PBS with 3.7% paraformaldehyde (without Mg2+ or Ca2+). Cells were then washed twice in PBS and then permeabilized in 0.2% Triton X-100/ PBS. After washing twice in 0.05% Triton X-100/PBS, cells were blocked for 30 min in 1% bovine serum albumin, BSA/0.05% Triton X-100/PBS. Cellular actin was stained using 1 unit of AlexaFluor 568 phalloidin (Molecular Probes, Inc.) in PBS for 30 min at room temperature, washed three times in 0.05% Triton X-100/PBS, and mounted in Cytoseal 30 medium. Stained cells were imaged using a 40× objective on an Olympus IX71 microscope with a Q-imaging Retiga EXi camera. For live cell imaging, coverslips were transferred to a Bioptechs Delta T controlled temperature dish in culture media at 37 °C in a CO2/air environment. Differential interference contrast (DIC) images were collected using a 20× objective on a Nikon TE-2000 inverted microscope with Nikon Elements software and a Qimaging Retiga EXi camera. Images were collected every 5 s, and images were later compiled into .avi files at 30 frames per second. Lateral Force Microscopy (LFM). Contact mode topographic and friction images were collected using a Dimension 3001 unit (Digital Instruments, Inc.) in the constant-deflection mode using oxidesharpened V-shaped Si3N4 cantilevers (Park Scientific Instruments) with typical radius of curvature of 20 nm and a nominal spring constant of 0.06 N m−1. The normal force between tip and sample (FN) was estimated from a force curve plotted against z-displacement of the cantilever. The normal sensitivity (SN) was obtained from the slope of the linear portion of the force−distance curve. The estimated normal force for all the samples was 45 ± 18 nN. Root-mean-square (RMS) roughness was measured over an area of 5 μm × 5 μm. For LFM measurements, probes were coated with PVOP by dipping them in a solution of PVOP (10 mM in TFE) for 1 h followed by three rinses, 1 min each, in TFE.

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RESULTS AND DISCUSSION Copolymers and Multilayer Buildup. Copolymers were made with two alkylation steps, summarized in Scheme S1, Supporting Information. In the first step, up to 38% of the starting PVP was alkylated with a heavily fluorinated chain. We have previously used this fluorinated PVP (PVFOP) to make superhydrophobic multilayers.37 In the second step, the remaining pyridine units in the PVFOP polyelectrolyte were subjected to exhaustive alkylation with propane sultone. Elemental analysis supported essentially complete quaternization of PVP in the PVFOP-co-VPPS product, with 6−38 mol % fluorinated repeat units. In addition, FTIR spectra (Figure S1, Supporting Information) show the disappearance of the 1414 cm−1 band characteristic of unalkylated PVP. In contrast, zwitterion derivatization of PVP itself by propane sultone was incomplete (see also FTIR spectra in Figure S2, Supporting Information), probably because the reaction became heterogeneous as the sulfonated PVP precipitated out of solution. All PVFOP-co-VPPS copolymers were soluble in TFE. Despite the very different natures of the fluorinated and the zwitterion functionality, there was no evidence of aggregation as seen in QUELS, which provided a hydrodynamic radius of 13 ± 1 nm for all the copolymers in TFE. The hydrid aqueous/fluorous solvent conditions for assembling the two multilayer components, PSS and PVFOPco-VPPS, were somewhat unusual. However, the layer-by-layer buildup depicted in Figure 2 showed typical linear behavior for the 38:62 mol % fluorinated zwitterionic copolymer. It has been shown that purely zwitterionic polyelectrolytes do not build up in multilayers and that a charged comonomer is needed to promote multilayering.29,38 The lower charge density copolymers also provided multilayer buildup but at a rate that scaled with the % fluorinated content. The 6% net charge density is lower than “critical” minimum charge densities, between 50% and 75%, below which multilayer growth for some systems is not possible.39−41 On the other hand, charge densities as low as 10 mol %42,43 have been used for multilayering when polymer− polymer interactions are aided by hydrophobic or H-bonding interactions. The fluorinated copolymers here probably rely on a strong hydrophobic component to their ion pairing to allow them to build up as multilayers. 15581

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conventional, fully charged polyelectrolytes.45 In the PEMU surface terminated with 6:94 copolymer, zwitterionic groups outnumber fluorinated ones but the CA was still about 40°. A silicon surface terminated with a monolayer of sulfobetaine units gave contact angles approaching zero.46 A PEMU terminated with a copolymer of acrylic acid and sulfobetaine yielded a contact angle of about 10°.29 CA hysteresis (CAH) was present in all cases, becoming more pronounced with greater %F. CAH governs the sliding angle of water on the solid surface. Usually, CAH arises from three sources: chemical heterogeneity, surface roughness, and surface reorganization during the CA measurement.47,48 The roughness of these surfaces was low, about 5 nm, and did not vary much between surfaces. Chemical heterogeneity is also intrinsically built into the system, but the scale of this heterogeneity is smaller than the size (about 10 nm) of a polymer chain since repeat units on the polymer are constrained to be in close proximity. For small molecules self-assembled on a surface, larger hydrophilic/hydrophobic domains are possible, through surface phase separations. Surface reorganization is a strong possibility, expected for this schizophobic composition. In air, the fluorinated groups are expected to present themselves at the interface, whereas in water they might be buried as the zwitterionic groups orient toward the surface. It should be noted that there was no unusual or contradictory wetting behavior such as the interesting “contraphilic” behavior reported by Makal and Wynne, where exposure to water makes a surface hydrophobic,49 or a “zwitter-wettable” surface, which stays hydrophobic as it adsorbs water.50 Our films are rather glassy, stitched together via multiple ion pairing which does not allow large scale reorganization of materials at the surface.37 PDMS Surface Modification. The use of soft elastomeric materials, such as PDMS, offers numerous attractive properties in several lithographic techniques.51 However, poor wettability of PDMS surfaces is a significant drawback. The chemical structure of the repeating −OSi(CH3)2− unit yields hydrophobic PDMS surfaces with a contact angle of approximately 108°.52 It is also “fluorophobic” where most fluorinated solvents do not wet the PDMS surface. Several approaches have used traditional wet chemical methods to modify the surface of the PDMS in order to make it more wettable for patterning aqueous-based inks.53 Increased PDMS hydrophilicity can quickly, but temporarily, be achieved by plasma treatment which introduces silanol (Si−OH) and removes methyl (Si−CH3) groups from the surface.54

Figure 2. Layer-by-layer buildup of different percentages of PVFOPco-VPPS with PSS (in 0.5 M NaCl) on Si wafer monitored by ellipsometry for 6:94 mol % (●); 10:90 mol % (▲); 18:82 mol % (○); 21:79 mol % (■); 28:72 mol % (◆); and 38:62 mol % (△). Solvents used in the multilayer buildup were 2,2,2-trifluorethanol and water. The thickness includes a ca. 20 Å native oxide layer on the Si wafer.

Contact Angles. The multilayers are terminated with a copolymer bearing well integrated groups of very different hydrophilicities. On the one hand, the fluorinated repeat unit has been used to make hydrophobic and even ultrahydrophobic (defined as high contact angle with low CAH) surfaces.37 In contrast, the sulfobetaine zwitterionic group is hydrophilic, noninteracting (in water) and is often used to prevent fouling of surfaces;10,29 hence, the term “schizophobic” is used to describe our mixed surfaces. Because the hydrophilic/phobic units are neighbors on a polymer chain, they are forced to be in close proximity and any phase segregation is likely to be highly local, perhaps as aggregates of fluorophilic randomly neighboring -C6F13 chains. The term “schizophrenic” has been used by the Armes group44 to describe block polymers made from responsive, self-assembling hydrophobic and hydrophilic blocks. Static and dynamic contact angles were measured on (PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS PEMUs (i.e., 21layer films terminated with PVFOP-co-VPPS). Both surface hydrophobicity and hysteresis increased with increasing fluorination (Table 2). The fact that CA increases steadily with percent of fluorination suggests the contradictory effects of the zwitterion and fluorinated groups average. However, even a small percentage of fluorination yields contact angles that are higher than those for multilayers terminated, for example, with

Table 2. Water Contact Angles (both static and dynamic) for (PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS Multilayers (PVFOPco-VPPS outermost layer)a dynamic contact angle (±3°)

a

PEMU system

static contact angle (±4°)

advancing, Θa°

receding, Θr°

hysteresis, ΔΘ°

blank Si (6%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS (10%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS (18%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS (21%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS (28%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS (32%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS (38%PVFOP-co-VPPS/PSS)10PVFOP-co-VPPS

8 42 45 50 63 66 85 93

21 41 57 48 62 69 80 82

19 18 23 20 30 33 35 31

2 23 34 28 32 36 45 51

Ambient conditions. 15582

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Figure 3. LFM images and section analysis of surface patterned with 38:62 PVFOP-VPPS and PDADMA stripes using an uncoated (left) versus PVOP-coated (right) probe. Fluorinated zwitteronic stripes were darker (lower friction) compared to PDADMA stripes (higher friction). Little contrast in the LFM images was observed between PVFOP-VPPS and PDADMA stripes when the surface was scanned with an uncoated tip. Image scan direction was from left to right.

TFE showed poor wetting behavior on the surface of the PDMS. Therefore, surface modification of the PDMS was required in order to stamp the fluorinated zwitterionic copolymers. Ö ner and McCarthy34 described fluorination of PDMS by fluorosilanes using a vapor-phase reaction for 3 days at ∼70 °C. We modified their approach somewhat. The surface modification of our PDMS stamp was done with a two step processes depicted in Figure S4 in the Supporting Information. First, the PDMS stamp was rendered hydrophilic by creating −OH groups on the surface with an air plasma. The PDMS− OH stamp showed a low water contact angle (7°) and was stored in deionized water prior to further modification in order to preserve its hydrophilic character. A flat PDMS stamp was inked with 100 mM (heptadecafluoro-1,1,2,2,-tetrahydrodecyl)triethoxysilane (HFS) solution in ethanol, dried under nitrogen, and put in conformal contact with the patterned PDMS−OH substrate and heated under vacuum at 90 °C for 2 h. After heating, the two stamps were separated, wherein the fluorinated silane was transferred to the treated PDMS−OH to yield a fluorosilane-modified patterned PDMS with a water contact angle of 114°. After treatment, good wetting behavior of TFE (static contact angle 32 ± 4°) was observed on the fluorinated stamp surface (Figure S4, Supporting Information). Surface Patterning: Polymer on Polymer Stamping (POPS). Microcontact printing has been extended to create polymer patterned surfaces on multilayers.31 In POPS, the surface of a stamp is inked with polymer, and, after drying, the stamp is pressed on the multilayer. Usually, a polyelectrolyte of opposite charge is used to facilitate adhesion. In the present study, well-defined areas of fluorinated and nonfluorinated material were required. Since PVFOP-co-VPPS is positively charged, a method was needed to ensure the entire substrate surface was positively charged. For this purpose, the areas of the substrate not modified with PVFOP-co-VPPS were “backfilled” with PDADMA as depicted in Figure S5, Supporting

Information. A positively charged copolymer (0.5 wt % in TFE, either fluorinated zwitterionic copolymer or PVFOP) was applied to the surface of the stamp with a cotton swab. The stamp was then pressed for 7 s on top of the two layer PEI/PSS multilayer surface deposited on Si wafer and left for 15 min in contact with the polymer surface. Patterns obtained were 10 μm in width, 15−17 nm in depth with a 5 μm gap. The patterned surface was dipped in PDADMA (10 mM in DI water) for 1 h and then rinsed in deionized water. This approach provided stamped fluorinated lines of width 10 μm separated by 5 μm lines of nonfluorinated polymer. Unlike our previous polymer stamping methods,37 the height difference between fluorinated and nonfluorinated areas was minimal, on the order of 10 nm. Surface roughness was therefore not significant. Stamping with PDADMA (0.5 wt % in methanol) was done with the same procedure except the patterned surface was dipped in PVOP (10 mM in CH2Cl2) for 1 h then rinsed in CH2Cl2. TFE was not used for dissolving PVOP because it was also a good solvent for PDADMA, which resulted in dissolution of the pattern. Methylene chloride was the alternative solvent for PVOP. Friction Measurements. In addition to height images, used to correlate friction to position, friction images were collected for the patterned surfaces using LFM in the contact mode. In LFM, the sample is scanned perpendicular to the long axis of the cantilever causing the cantilever to deflect (torque) due to intermolecular forces between the tip and the surface.55 The torque of the tip, recorded as a voltage, is a measure of relative friction of different areas on a sample. LFM is best applied to samples with small topographic features, as in our case, because changes in topography can also cause changes in the cantilever torsion. LFM probe tips were coated with poly(vinyloctylpyridinium iodide), PVOP, a positively charged polyelectrolyte, to avoid 15583

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Figure 4. AFM (left) versus LFM (right) images of surface patterned with 38:62 mol % PVFOP-VPPS and PDADMA stripes, with their section analysis, using a modified PVOP probe tip. Fluorinated zwitterion stripes were darker (lower friction) compared to the PDADMA stripes (higher friction). The difference in height between the two different components was 12 nm.

Figure 5. AFM (left) versus LFM (right) images of a surface patterned with 38:62 mol % PVFOP and PDADMA stripes, with their section analysis, using a modified PVOP probe tip. Fluorinated region showed darker areas (lower friction) compared to the PDADMA region (higher friction). The difference in height between the two different components was 13 nm.

attractive interactions with, and possible adhesion to, the positively charged surface and the probe tip. With an

unmodified Si3N4 tip (which bears a negative charge), the friction contrast in LFM image was weak. The uncoated tip also 15584

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adequately predict the behavior of live cells on biological interfaces. Live cells possess extraordinarily complex machinery with which to probe various physical properties of surfaces, such as hydration level, charge, and modulus. In the absence of specifically recognized sites, it is a combination of these gross physical properties that cue cell adhesion, survival, and proliferation. Here, rat aortic A7r5 smooth muscle cells were used to probe cell behavior on the schizophobic multilayer surfaces. This particular cell line was chosen because of our past experience investigating their behavior on both zwitterionic and fluorinated multilayers. For example, a PEMU terminated with an acrylic acid copolymer containing about 25% zwitterion defeated both protein adsorption56 and A7r5 cell adhesion.29 In contrast, when a multilayer was capped with the same PVFOP polymer used here, or Nafion, the A7r5 cells spread and attached with great efficiency,29 although robust cell adhesion to a fluorinated surface is counterintuitive. The polyelectrolyte surface charge and hydrophobicity were found to control whether the cells became highly motile, with a more rounded morphology and multiple lamellipodia and filopodia, or highly spread and contractile, with numerous well-developed actin stress fibers. These different morphology-motility states reflect those of smooth muscle cell “synthetic” and “contractile” phenotypes. A7r5 cells cultured on (PVFOP/PAA)4 surfaces with PVFOP as the terminating layer showed behavior consistent with a “contractile” phenotype, with well spread and firmly attached cells containing robust stress fibers (Figure 6). Here, the cell morphology, adhesion, and phenotypic state depended more on the hydrophobicity and the charge of the top polyelectrolyte layer than on the thickness of layers, since films were all about the same thickness. A study by Salloum et al.29 showed cells cultured on Nafion and PVFOP, which differ in their surface charges but are similar in hydrophobicity (Table 3), have the same “contractile” phenotype behavior, with cell shapes nearly identical on both surfaces. The cytotoxic and bacteriocidal properties of tetraalkylammonium polymer repeat units at surfaces appear to contraindicate their use in cell culture. However, we have shown that the cytotoxic nature of a polycation in solution is neutralized by complexation with polyanions during multilayer formation.57 We have reported robust cell adhesion and proliferation on fluorinated anions and cations atop multilayers.29 While the C8 hydrocarbon alkylammonium (alkylpyridinium) cation is strongly toxic,58 the fluorous nature of the side chain in

showed spikes on transitioning between lines, possibly due to adhesive interactions. However, LFM images with the PVOP coated tip exhibited enhanced friction contrast. An example is shown in shown in Figure 3, which shows a comparison of LFM on a 38:62 PVFOP-co-VPPS (10 μm width) PDADMA (5 μm width) pattern using either uncoated or PVOP-coated tips. The coated tip showed a clear contrast between fluorinated (lower friction, dark) areas and nonfluorinated (higher friction, light) areas. As a cross-check, PDADMA was stamped on the surface of the multilayer and this pattern was backfilled by immersing in PVOP. LFM scans (Figure S6, Supporting Information) show that the PDADMA surface has higher friction than the PVOP surface. Areas of friction pattern correlate with areas of height pattern, as seen in Figure 4 for contrast between 38:62 PVFOPco-VPPS and PDADMA stripes. Patterns with 21:79 copolymer (Figure S7, Supporting Information) show somewhat lower contrast. Compositions of 6:94 and 28:72 PVFOP-co-VPPS showed similar contrast to 21:79 despite the difference in fluorination levels. The contrast between stamped PVFOP and PDADMA was the greatest (Figure 5), consistent with the higher water contact angle on the PVFOP surface. Effect of Surface Hydrophobicity on Cell Adhesion. Table 3 shows the static contact angle and dry thickness of Table 3. Thickness and Advancing Contact Angles of Polyelectrolyte Surfaces PEMU system (6%PVFOP-co-VPPS/PAA)75PVFOP-co-VPPS (21%(PVFOP-co-VPPS/PAA)75PVFOP-co-VPPS (38%PVFOP-co-VPPS/PAA)75PVFOP-co-VPPS (PVFOP/PAA)4PVFOP (PVFOP/PAA)50PVFOP (PVFOP/Nafion)7Nafion (PVFOP/Nafion)7PVFOP

thickness (Å) 356 362 375 55 350 344 365

± ± ± ± ± ± ±

3 4 10 11 11 11 6

contact angle (Θa, deg) 36 45 60 100 100 104 114

± ± ± ± ± ± ±

3 3 4 3 3 3 3

various polyelectrolyte multilayers used here. All PEMUs (except one) were built to about the same thickness to remove thickness as a variable. The effects of surface modifications for the biological interface are often probed via the adsorption of individual or mixtures of proteins (e.g., serum). Whereas protein adsorption is an important property, it fails to

Figure 6. A7r5 smooth muscle cells “contractile” behavior on (PVFOP/PAA)4PVFOP PEMUs (terminated with a PVFOP layer). (Left image) Two uncontracted cells. (Right image) Cell in which contraction was stimulated by treatment with the Ca2+ ionophore A23187. Actin filaments (mostly in contractile stress fibers) were labeled with rhodamine-phalloidin. Scale bar = 20 μm. 15585

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PVFOP may prevent interaction with the cell membrane. The films used here were stable to cell growth conditions for the time period (up to about a week) for which they were employed. In this study, the effect of a fluorinated zwitterionic copolymer surface on cell attachment was tested. Cells were grown on multilayers, all between 350 and 375 Å in thickness, with 6:94, 21:79, and 38:62 mol % PVFOP-co-VPPS terminated surfaces. Cell mobility and morphology depended on the hydrophobicity of the surface. In contrast to the contractile phenotype of the cells on PVFOP-terminated PEMUs, the cells on PVFOP-co-VPPS were more loosely attached and had morphologies indicative of the synthetic phenotype, with more active actin-containing filopodia in the cells on the PEMUs with the most plentiful zwitterion (Figures 7 and 8). Interestingly,

Figure 8. Differential interference contrast images of live A7r5 cells cultured on (A) 6:94 mol % PVFOP:VPPS fluorinated zwitterionic terminated surface and (B) (PVFOP-PAA)50 with a PVFOP terminating layer (scale bar = 100 μm).

to removal of adhering or fouling components by a gentle fluid shearing force.

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CONCLUSIONS The synthesis of a series of novel random fluorinated/ zwitterionic copolymers with different surface wetting properties is reported. Polymer-on-polymer stamping using a fluorinated PDMS stamp was used to pattern these fluorinated copolymers. AFM and LFM showed well-resolved friction contrast between fluorinated and nonfluorinated areas. The best contrast was obtained with a probe tip modified with a positively charged, hydrophobic polyelectrolyte, which presumably avoided electrostatic attractive interactions between the tip and the sample surface. Image contrast in LFM showed all the fluorinated zwitterionic areas had lower friction compared to the hydrophilic (nonfluorinated) PDADMA surface. Maximum friction contrast was observed for micropatterns of fluorinated (with no zwitterion) polymer and PDADMA. The effect of a fluorinated/zwitterion “schizophobic” copolymer surface on the attachment of smooth muscle cells was studied. The plastic nature of smooth muscle cell cytoskeleton allows muscle cells to convert from a “contractile” nonmotile to a “synthetic” motile state. Depending on the surface properties, cells appeared to exhibit either contractile behavior, as seen on PVFOP hydrophobic surfaces, or motile behavior with multiple filopodia, as seen on fluorinated/zwitterionic surfaces. The

Figure 7. A7r5 smooth muscle cells “synthetic” behavior on (A, B) 6:94 mol % PVFOP:VPPS, (C, D) 21:79 mol % PVFOP:VPPS, and (E, F) 38:62 mol % PVFOP:VPPS surfaces. Cells were all motile and loosely attached to the surface. Cells on the highest zwitterionic content surface (E, F) adopted a spiky appearance with many filopodia. Actin filaments were labeled with rhodamine-phalloidin. Scale bar = 20 μm.

the surface with the highest zwitterionic content failed to prevent cell attachment due to the presence of fluorine. Conversely, the highest fluorinated content failed to promote good adhesion. The cells thus appear to be in a state of adhesion that reflects the contrast/competition in hydrophobicity of the zwitterionic and fluorinated components. Even the lowest content of fluorinated repeat unit (6%) was enough to allow loose attachment of the highly adherent A7r5 cells. Such a schizophobic surface might therefore be well suited 15586

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Benign Ultralow Fouling Marine Coatings. Langmuir 2009, 25 (23), 13516−13521. (13) Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Hexemer, A.; Sohn, K. E.; Kramer, E. J.; Fischer, D. A. Comparison of the Fouling Release Properties of Hydrophobic Fluorinated and Hydrophilic PEGylated Block Copolymer Surfaces: Attachment Strength of the Diatom Navicula and the Green Alga Ulva. Biomacromolecules 2006, 7 (5), 1449−1462. (14) Lejars, M.; Margaillan, A.; Bressy, C. Fouling Release Coatings: A Nontoxic Alternative to Biocidal Antifouling Coatings. Chem. Rev. 2012, 112 (8), 4347−4390. (15) Gan, D.; Mueller, A.; Wooley, K. L. Amphiphilic and Hydrophobic Surface Patterns Generated from Hyperbranched Fluoropolymer(HBFP)Linear Polymer Networks: Minimally-adhesive coatings via crosslinking of hyperbranched fluoropolymers. J. Polym. Sci., Part A: Polym. Chem. 2003, 41 (22), 3531−3540. (16) Weinman, C. J.; Gunari, N.; Krishnan, S.; Dong, R.; Paik, M. Y.; Sohn, K. E.; Walker, G. C.; Kramer, E. J.; Fischer, D. A.; Ober, C. K. Protein Adsorption Resistance of Anti-biofouling Block Copolymers Containing Amphiphilic Side Chains. Soft Matter 2010, 6 (14), 3237− 3243. (17) Wang, Y.; Finlay, J. A.; Betts, D. E.; Merkel, T. J.; Luft, J. C.; Callow, M. E.; Callow, J. A.; DeSimone, J. M. Amphiphilic Conetworks with Moisture-Induced Surface Segregation for HighPerformance Nonfouling Coatings. Langmuir 2011, 27 (17), 10365− 10369. (18) Weinman, C. J.; Finlay, J. A.; Park, D.; Paik, M. Y.; Krishnan, S.; Sundaram, H. S.; Dimitriou, M.; Sohn, K. E.; Callow, M. E.; Callow, J. A.; Handlin, D. L.; Willis, C. L.; Kramer, E. J.; Ober, C. K. ABC Triblock Surface Active Block Copolymer with Grafted Ethoxylated Fluoroalkyl Amphiphilic Side Chains for Marine Antifouling/FoulingRelease Applications. Langmuir 2009, 25 (20), 12266−12274. (19) Park, D.; Weinman, C. J.; Finlay, J. A.; Fletcher, B. R.; Paik, M. Y.; Sundaram, H. S.; Dimitriou, M. D.; Sohn, K. E.; Callow, M. E.; Callow, J. A.; Handlin, D. L.; Willis, C. L.; Fischer, D. A.; Kramer, E. J.; Ober, C. K. Amphiphilic Surface Active Triblock Copolymers with Mixed Hydrophobic and Hydrophilic Side Chains for Tuned Marine Fouling-Release Properties. Langmuir 2010, 26 (12), 9772−9781. (20) Dimitriou, M. D.; Zhou, Z.; Yoo, H.-S.; Killops, K. L.; Finlay, J. A.; Cone, G.; Sundaram, H. S.; Lynd, N. A.; Barteau, K. P.; Campos, L. M.; Fischer, D. A.; Callow, M. E.; Callow, J. A.; Ober, C. K.; Hawker, C. J.; Kramer, E. J. A General Approach to Controlling the Surface Composition of Poly(ethylene oxide)-Based Block Copolymers for Antifouling Coatings. Langmuir 2011, 27 (22), 13762−13772. (21) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277 (5330), 1232−1237. (22) Rubner, M. F.; Cohen, R. E. Layer-by-Layer Processed Multilayers: Challenges and Opportunities. Decher, G., Schlenhoff, J. B., Eds.; In Multilayer Thin Films; Wiley-VCH: Weinheim, 2012; pp 23−41. (23) Lichter, J. A.; Van Vliet, K. J.; Rubner, M. F. Design of Antibacterial Surfaces and Interfaces: Polyelectrolyte Multilayers as a Multifunctional Platform. Macromolecules 2009, 42 (22), 8573−8586. (24) Gribova, V.; Auzely-Velty, R.; Picart, C. Polyelectrolyte Multilayer Assemblies on Materials Surfaces: From Cell Adhesion to Tissue Engineering. Chem. Mater. 2011, 24 (5), 854−869. (25) Wong, S. Y.; Han, L.; Timachova, K.; Veselinovic, J.; Hyder, M. N.; Ortiz, C.; Klibanov, A. M.; Hammond, P. T. Drastically Lowered Protein Adsorption on Microbicidal Hydrophobic/Hydrophilic Polyelectrolyte Multilayers. Biomacromolecules 2012, 13 (3), 719−726. (26) Lichter, J. A.; Thompson, M. T.; Delgadillo, M.; Nishikawa, T.; Rubner, M. F.; Van Vliet, K. J. Substrata Mechanical Stiffness Can Regulate Adhesion of Viable Bacteria. Biomacromolecules 2008, 9 (6), 1571−1578. (27) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Layer by Layer Buildup of Polysaccharide Films: Physical Chemistry and Cellular Adhesion Aspects. Langmuir 2003, 20 (2), 448−458.

response of cells to the schizophobic surface showed intermediate behavior between purely hydrophilic (zwitterionic) and hydrophobic (fluorinated) surfaces. It was found that even 6 mol % fluorinated content in the copolymer was enough to prevent the formation of completely cell resistant zwitterionic surfaces.

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ASSOCIATED CONTENT

S Supporting Information *

Procedure and FTIR spectra of quaternized polymers, PDMS silanization and POPS procedure. LFM of PDADMA/PVOP surface, and LFM of PVFOP-co-VPPS/PDADMA surface. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Science Foundation. REFERENCES

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