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Fibronectin and Cell Attachment to Cell and Protein Resistant Polyelectrolyte Surfaces Scott G. Olenych,† Maroun D. Moussallem,‡ David S. Salloum,‡,§ Joseph B. Schlenoff,‡ and Thomas C. S. Keller*,† Department of Biological Science, The Florida State University, Tallahassee, Florida 32306, and Department of Chemistry and Biochemistry, Center for Materials Research and Technology, The Florida State University, Tallahassee, Florida 32306 Received April 28, 2005; Revised Manuscript Received August 17, 2005
Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces. Introduction Cells secrete proteins, including fibronectin (FN), laminin, vitronectin, and collagen, which form an extracellular matrix (ECM). The secreted ECM can vary in composition depending on a number of factors including the cell type and extracellular signals controlling cell behavior. In vivo, the ECM can form a three-dimensional scaffold to which cells bind. In culture, ECM proteins can interact with the substrate surface. Cells attach to the ECM through the binding of plasma membrane receptor proteins known as integrins to specific ECM proteins.1 Integrins act as contact points connecting cytoskeletal structures such as stress fibers inside cells to the surrounding ECM.2 Consequently, the number and quality of integrin contacts with the ECM plays an important role in determining cytoskeletal organization and cell behavior.3,4 The quality of cell-ECM contact depends on a number of factors such as the stability, organization, and composition of the ECM. In cell culture, different surfaces can affect the properties of bound ECM and the behavior of cells. Layer-by-layer deposition provides a means to generate polyelectrolyte multilayer film (PEMU) surfaces with a wide variety of different biofunctional properties.5,6 Multiple controllable variables (polymer pairs, number of layers, chemical cross-linking, pH, ionic strength, and temperature dependence) make it possible to “tune” PEMU properties * To whom correspondence may be addressed. E-mail:
[email protected]. † Department of Biological Science. ‡ Department of Chemistry and Biochemistry. § Current Address: The Procter and Gamble Company, Miami Valley Innovation Center, Cincinnati, OH 45252.
that are important for manipulating protein adsorption and cell behavior. Properties such as the rigidity, energy (hydrophobicity7,8 or hydrophilicity7-9), temperature sensitivity,10,11 charge density (strong or weak charge),7-9,12-14 porosity, and topology of PEMU surfaces have been used to manipulate protein and cell adhesion. Understanding interaction of certain proteins with PEMU surfaces and the affects of protein binding on cell behavior is central to improving the design of surfaces for better biofunctionality. Most studies of PEMU biofunctionality have focused exclusively on either protein adsorption or cell adhesion.7,14-17 Along these lines, we recently completed an investigation examining the effect of PEMU surface charge and hydrophobicity on the attachment and spreading of A7r5 rat aortic smooth muscle cells.8 Like many other types of cells, A7r5 cells can interact with ECM proteins such as FN and that interaction can influence cell behavior.18 On the other hand, it has been demonstrated that protein adsorption to a surface does not necessarily correlate with cell adhesion to that surface.19 In an effort to better understand effects ECM proteins may have on smooth muscle cell interaction with PEMUs, we have explored the relationship between adsorption of FN, a model ECM protein, and smooth muscle cell spreading and motility on a variety of PEMU cell culture surfaces, including micropatterned surfaces incorporating a newly developed cell-protein-resistant PEMU. Understanding the interaction of ECM proteins and smooth muscle cells with PEMUs, which may be useful in coating implantable devices, is important because of the roles smooth muscle cells and ECM proteins play in wound healing.
10.1021/bm050298r CCC: $30.25 © 2005 American Chemical Society Published on Web 09/24/2005
Cell and Protein Resistant Polyelectrolyte Surfaces
Materials and Methods Materials. 1,3-Propane sulfone (PS), acrylic acid (AA), poly(allylamine hydrochloride) (PAH; MW ≈ 7 × 104), and poly(acrylic acid) (PAA; MW ≈ 2.4 × 105) were used as received from Aldrich. 2-(Acrylamido)-ethyl dimethylamine (AEDA) was obtained from Monomer-Polymer & Dajac Inc. Poly(4-vinyl pyridine) (P4VP; MW ≈ 5 × 104) was obtained from Polymer Source Inc. Nafion, a perfluoronated sulfonated polymer, was purchased from Aldrich and used as 2.5 wt % solution in ethanol:methanol 50:50 (vol/vol) for stamping. All polymer (except fluorinated polymers), monomer, and buffer solutions were prepared using 18 MΩ water. Polyelectrolyte Synthesis. PAA-co-PAEDAPS copolymer was made from 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (AEDAPS)8 and AA via free radical polymerization, as described by McCormick and Salazar.20 The mole ratio for AA and AEDAPS was varied to obtain different percentages of zwitterionic units in the copolymer (90:10, 75:25, or 50:50 AA:AEDAPS molar ratios).8 The polymers were characterized using Fourier transform infrared (FTIR) and attenuated total reflection FTIR, which confirmed the presence of both carbonyl bond stretch CdO (1725 cm-1, AA; 1670 cm-1, AEDAPS) and of sulfonate bond (νSO3-) stretch at ∼1200 cm-1. Poly(4-vinyl-trideca-fluoro-octyl pyridinium iodide)-co-poly(4-vinyl pyridine) (PFPVP)8 was synthesized to obtain a positively charged perfluoronated polyelectrolyte for layering with the negatively charged polyelectrolyte Nafion. The polymer exhibited the distinctive C-F bond stretch in the 1200-cm-1 region of the IR spectrum. Polyelectrolyte Multilayers on Glass Cover Slips. Glass coverslips (cover glass, No. 1, 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 a closed container) and then in hot H2O2/ ammonia/water (1:1:7 vol/vol). The cleaned coverslips were rinsed in water and blown dry with a stream of nitrogen. Polymer solution concentrations were made 10 mM (with respect to the monomer repeat unit) in 0.25 M sodium chloride salt, except for Nafion, which was made as a 0.25 wt % solution in ethanol:methanol 50:50 vol/vol and PFPVP, which was made 2 mM. Sequential adsorption of polyelectrolytes on coverslips was performed by hand dipping for 10 min in each polymer solution and followed by three rinses with water for 1 min each. PEMU Nomenclature. For clarity, we employ the following shorthand for multilayers: (A/B)x where A is the starting polyelectrolyte contacting the substrate (coverslip), B is the terminating polyelectrolyte layer, and x is the number of layer pairs. In odd numbers of layers, (A/B)xA, A is the terminating polyelectrolyte layer. Optical Waveguide Lightmode Spectroscopy (OWLS). OWLS, which measures the effective refractive index of a thin layer adsorbed on a silicon titanium oxide waveguide film, was used to monitor PEMU build up and FN adsorption onto the PEMUs. The OWLS data were acquired with a Bios-1 instrument (Artificial Sensing Instruments, Zu¨rich, ASI) and an optical waveguide grating coupler sensor chip
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composed of a 200 nm thick silicon titanium oxide film SixTi(1-x)O2 (x ) 0.25 ( 0.05) with a refractive index nf ) 1.77 ( 0.03 spin coated onto a glass substrate (ASI). The sensor chip had a 20-nm diffraction grating depth and a periodicity of 2400 lines/nm. Polyelectrolyte solutions (1 mM with respect to the monomer unit) and the FN solution (50 µg mL-1) were made in 10 mM Tris (150 mM Ionic strength, pH 7.4). The polyelectrolyte solutions, except for PFPVP and Nafion, were pumped through the OWLS cell at a flow rate of 8.3 µL s-1 at 25 ( 0.5 °C for 4 min for each layer followed by a 4 min rinse with buffer. The PFPVP/Nafion PEMU was made with a multilayering robot (nanoStrata, Inc.) due to the limitation of solvent use in the OWLS instrument. The terminating layer was rinsed with buffer in the instrument until a stable baseline was established before exposure to FN. The quantity of adsorbed material was calculated to have an average value of dn/dc ) 0.188 mL g-1 using the following equation14,21 Γ)
tA(nA - nC) dn/dc
where Γ is the amount of adsorbed material in the layer in mg m-2, tA is the thickness of adsorbed layer in nm, nA is the refractive index of the adsorbed layer, nC is the refractive index of the solution, dn/dc is the change of refractive index with the change of concentration in mL g-1. Polymer Stamping. Polymer-on-polymer stamping (POPS),22-24 was done with a poly(dimethylsiloxane) stamp (PDMS) that had defined channels. In this technique, the surface of the patterned PDMS stamp was inked by applying 2.5 wt % Nafion using a cotton swap and dried with a nitrogen stream. The Nafion-patterned side of the stamp was brought into contact with the PAA-co-PAEDAPS surface for 10 min and then gently removed. The topology of the stamped surface was characterized with a KLA-Tencor P15 Surface Profiler (KLA-Tencor). Cell Culture. The A7r5 rat aortic smooth muscle cells (American Type Culture Collection) were cultured in high glucose Dulbecco’s modified Eagle medium 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% confluence. Cells were released from culture flasks using a trypsin and ethylenediamine tetraacetic acid (EDTA) solution in Hanks buffered salt solution. Microscopy. Fibronectin-Coated Coverslips. PEMUcoated coverslips were incubated overnight with 50 µg mL-1 FN in phosphate-buffered saline (PBS) at 4 °C. These coverslips were used for the cell adhesion and FN adsorption studies. To detect FN binding, the treated coverslips were gently rinsed twice with PBS prior to fixing with 3.7% paraformaldehyde in PBS. Following fixation, the coverslips were washed twice with PBS and blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. The coverslips were incubated with antifibronectin antibody (1: 1000 dilution) for 45 min, washed twice with PBS, and incubated with Alexa 488-goat antirabbit secondary antibody
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(1:200 dilution) for 45 min. The coverslips were then washed three times in PBS and mounted in Vectashield. The coverslips were imaged with the same exposure setting for each coverslip using a Zeiss color Axiocam on a Nikon Microphot-FX microscope. Densitometry of the original images was performed using Quantity One software (Bio Rad Laboratories, Inc.). Cells on Coverslips. Equal numbers (∼1 × 104 cell mL-1) of A7r5 cells were plated onto PEMU-coated glass coverslips with or without adsorbed FN in six-well dishes and grown for 30-48 h to allow cell attachment before imaging. Live cell phase contrast images were obtained using a Zeiss Axiovert-35 microscope equipped with a NEC Ti-24A charge-coupled device camera and Metamorph Imaging Software. For immunofluorescent localization of actin and FN, cells were washed twice with PBS, pH 7.4, fixed with freshly prepared 3.7% paraformaldehyde in PBS for 10 min, followed with a 3 min incubation in 0.1% Triton X-100 in PBS. Following three washes with PBS, the cells were blocked with 3% BSA in PBS for 30 min. The cells were incubated for 20 min at room temperature with one unit of Alexa Fluor 488 phalloidin (Molecular Probes, Inc.) or a 1:75 dilution of phalloidin-TRITC (Sigma-Aldrich Co.) in PBS. Cells also were incubated for 45 min with a 1:400 dilution of anti-R-actin antibody, clone 1A4 (Sigma-Aldrich, Co), or a 1:1000 dilution of antifibronectin rabbit polyclonal (SigmaAldrich, Co). The coverslips were washed three times with PBS and then incubated with Alexa 546-goat antimouse or 488-goat antirabbit secondary antibodies (1:200 dilutions) for 45 min. The cells were washed three 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 Zeiss laser confocal microscope (LSM-510). Cell migration, FN tracks, was measured using Metamorph imaging software. Results and Discussion We previously investigated the adhesion and spreading of A7r5 vascular smooth muscle cells on uncoated PEMU surfaces.8 We found that varying the PEMU surface charge and energy (hydrophobicity) altered A7r5 cell shape, attachment, and cytoskeletal arrangement. In addition, we developed zwitterion-containing surfaces that effectively prevented cell attachment. The investigation described here specifically addresses the role the ECM may play in cell behavior on PEMUs. Interactions between a cell and the ECM are critical in determining cell shape, motility, and cytoskeletal arrangement in vivo.25-27 The framework of the ECM consists of several different proteins including FN, vitronectin, collagen, and thrombospondin. FN deposition regulates and supports other proteins in the ECM,28-30 and therefore is a useful model ECM protein to examine when investigating cellular attachment to substrates. Fibronectin interacts with integrins in the cell membrane and has a well-established effect of promoting cellular spreading and motility.18,31-34 OWLS and immunofluorescence microscopy were used to examine attachment of FN to a sampling of PEMU
Olenych et al.
Figure 1. Layer-by-layer buildup of (PAH/PAA)2 monitored in situ with OWLS. Arrows represent the designated injection of polyelectrolyte. Fibronectin (FN, 50 µg mL-1) was introduced after multilayer buildup and is indicated by the arrow in the figure prior to the last buffer rinse. Buffer rinses were also used between layer buildup (arrows not shown).
Figure 2. OWLS measurement of surface thickness change for fibronectin (50 µg mL-1) adsorption to PEMU surfaces terminated with: (A) PAH, (B) Nafion, (C) PAA, (D) PAA-co-PAEDAPS 75:25 mol %, and (E) PAA-co-PAEDAPS 90:10 mol %. Arrows represent the time each buffer rinse was initiated.
surfaces characterized in our previous study of cell attachment. Figure 1 shows a typical OWLS experiment tracking (PAH/PAA)2 layer buildup followed by FN adsorption. The rapid buildup of each layer to a saturated value strongly suggests that each successive layer fully coats the surface. This conclusion is supported by atomic force microscopy (AFM) analysis of a similar PEMU for which the surface roughness parameter was found to be on the order of 1 nm for a 10 nm thick surface (data not shown). The AFM image also showed that the features on the surface attributed to the PEMU had an average diameter of 10 nm, which is extremely small relative to the size of A7r5 cells exposed to the surfaces. The other PEMUs examined with OWLS in this study were layered with the same procedure, with similar coverage, except for (PFPVP/Nafion)2, which was layered on the OWLS chip ex situ and then put into the OWLS instrument to measure fibronectin adsorption. Figure 2 shows a representative comparison of FN adsorption to each of the surfaces tested after PEMU buildup. Table 1 summarizes the mean multilayer thickness, FN thickness, and calculated FN protein coverage on the surface of each PEMU tested. FN caused an increase of almost 20 nm in thickness on the PAH surface. This thickness and
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Cell and Protein Resistant Polyelectrolyte Surfaces
Table 1. Multilayer Thickness, Protein Layer Thickness, and Protein Coverage on Different Polyelectrolytes Studied with OWLS PEMU (PAH/PAA)1PAH (PFPVP/Nafion)2 (PAH/PAA)2 (PAH/PAA-co-PAEDAPS 90:10)2 (PAH/PAA-co-PAEDAPS 75:25)2 (PAH/PAA-co-PAEDAPS 50:50)2
PEMU thickness (nm)a protein layer thickness (nm)a protein coverage (mg m-2) FN-antibody bindingc 4.7 ( 1.3 15.0 ( 0.4b 14.5 ( 3.0 7.8 ( 0.5 11.2 ( 0.3 ND
19.4 ( 5.1 5.4 ( 2.3 0.6 ( 0.3
