AFFINITY PATTERNING OF BIOMATERIALS USING PLASMA GAS DISCHARGE

A. Goessl, L. Jung, D. Bowen-Pope, A.S. Hoffman Department of Bioengineering,

University of Washington, Seattle, Washington, United States of America

Abstract. Patterned surfaces were prepared by combination of gas discharge and photolitographic processes. First a protein-repellant surface was prepared by glow discharge deposition of tri- and tetraglyme vapor on poly (ethylene terephtalate) surface, then on top of it fluorocarbon was deposited on selected domains by photolithography. Cell attachment was shown to be dependent of the surface hydrophilicity.

1. INTRODUCTION

One of the most frequent modes of failure of vascular implants (grafts, stents, etc.) is the occlusion of the lumen due to anastomotic hyperplasia, where an excessive proliferative response of the medial smooth muscle cells (SMC) leads to a thickening of the tissue at the joint of the vessel and a synthetic implant. The activation of SMC by the events occurring at the wound site, the reversion from the contractile to the proliferative phenotype, is hypothesized to result from changes in the chemical and mechanical environment of the cells.

In this paper we present a method to manufacture cell culture substrates for the patterned immobilization of cell receptor ligands on a non-fouling background.

We have developed a novel technique that allows the patterned immobilization of peptide or protein ligands on polymeric cell culture substrates. We first deposit a thin, protein and cell resistant coating from a triglyme or tetraglyme vapor onto a clean PET (poly[ethylene terephthalate]) cell culture substrate. The gas discharge source is mostly made of high energy excited molecules and free radicals and short wavelength UV. The deposited polyether polymer film resists the adsorption of proteins or cells. Then, using a photolithographic process we deposit on the nonfouling surface small domains of a thin, hydrophobic fluorocarbon plasma polymer. The domain sizes range from 5–100 microns.

These patterned surfaces were characterized using AFM (atomic force microscopy) to assess topographical properties and pattern fidelity. We also used ToF-SIMS (time of flight secondary ion mass spectroscopy) to assay the chemical composition of the different domains on the surface. Both methods show excellent pattern fidelity and the desired chemical compositions in the different regions. We can adsorb various cell adhesion proteins of interest (eg, laminin, fibronectin, or gelatin) onto the fluorocarbon domains. We demonstrated that by adsorbing fluorescently labeled BSA (bovine serum albumin) to these surfaces; they were then imaged using epifluorescent microscopy.

2. MATERIALS AND METHODS

2.1. Glow discharge and lithography process

Cleaned PET substrates (Lux Thermanox coverslips) were treated with an Ar plasma, then a thin polymer film of tetraglyme (tetra(ethylene oxide)-dimethyl ether) was deposited by plasma polymerization. The thickness was measured by ellipsometry (Rudolph EL, nf=1.5) the

films were chemically characterized by ESCA. To assess the non-fouling properties of the glyme film, SMC (Fisher Rat 344) were seeded onto the films in the presence of 10% calf serum and incubated for 24h. Onto this non-fouling polymer a positive photoresist (Hoechst AZ 1512) was applied and exposed to UV light through a photomask. The exposed areas of the resist were washed away with an aqueous developer (Hoechst AZ 3510), exposing these areas of the tetraglyme layer for the subsequent plasma deposition of a thin fluorocarbon polymer film (process gas tetrafluoroethylene). Again, the thickness was measured by ellipsometry (nf=1.395). Subsequently the remaining photoresist was removed by treatment with EtOH, aqueous developer (2x) and water (2x), leaving behind the pattern of a fluorocarbon polymer on a non-fouling background. These substrates were chemically characterized using ToF-SIMS imaging.

2.2. Surfactant adsorption

A fluorocarbon-PEG-peptide amphiphile has been synthesized; the surfactant will be used to bind peptide ligands to the hydrophobic flurocarbon pattern on the substrate. Here we used surface plasmon resonance (SPR, custom buit instrument) to study the adsorption of a FC(9)-PEG(11) surfactant (no peptide attached) to homogeneous FC films deposited on SPR substrates (gold deposited on glass slides). The critical micell concentration (CMC) was measured using the surface tension method. An adsorption isotherm was recorded by measuring the adsorption from different concentrations under stop-flow conditions. SMC were seeded on substrates perpared in the same way to assess the effect of varying surface densities of PEG on attachment and spreading of cells in serum-free media.

3. RESULTS AND DISCUSSION

As written above, the PET substrates were first cleaned with an Ar plasma, then a thin polymer film of tetraglyme was deposited by glow discharge polymerization. On Fig. 1. SEM photomicrographs of pristine PET control surface and a tetraglyme-deposited surface is shown.

The thickness of the tetraglyme plasma films was linearly dependent on the treatment time, a film thickness of 115nm was used for further processing. Elemental analysis by ESCA showed the expected C:O ratio of ~2:1. The high resolution C1s spectrum shows the C-O bond (286.5eV) as the predominant bond. SMC seeded onto these substrates were not able to attach or spread even in the presence of 10% calf serum.

FIG. 1. Photomicrographs of: (a) untreated PET, and (b) tetraglyme-deposited PET surface.

The spatial resolution of the photolithography was found to be almost exclusively dependent on the quality of the photomask; with an appropriate mask feature sizes of <5µm can easily be obtained. The thickness of the FC plasma film, which also increases linearly with treatment time, was found to be critical for the clean removal of the photoresist. Films with a thickness of 10–30nm allow a quick removal of the underlying photoresist; thicker films significantly reduce the edge definition of the pattern. Given a homogeneous tetraglyme film, the photoresist can be completely removed using the above washing protocol. The complete removal was monitored using ToF-SIMS by the absence of resist-typical peaks from the cresol novolak (+121Da, -107Da). The glyme/FC patterned surfaces were analysed using ToF-SIMS in the imaging mode. On Fig. 2 we show the ion maps of a: CF3+(typical for the FC pattern) and of b: (C3H8O⁺), characteristic of the glyme background. The line width is 20µm.

FIG. 2. Ion maps of: (a) CF₃⁺(typical for the FC pattern); (b) (C3H8O⁺), characteristic of the glyme background.

On Fig. 3. we show the adsorption isoterm of the FC-PEG onto the gold-covered glass, as well as the cell attachment on surfaces treated with various amounts of FC-PEG surfactant.

Cells seeded onto a sub-monolayer of the FC-PEG surfactant were severely impaired in attachment and spreading due to the "non-fouling" properties of even relatively short-chained, surface-bound PEG. On substrates covered with a dense monolayer cell attachment was completely inhibited.

We have also synthesized fluorocarbon-PEG-peptide surfactant conjugates that can be used to anchor peptide ligands to the fluorocarbon domains through the hydrophobic interactions between the fluorocarbon tail of the surfactant and the patterned FC domains.

These maps, showing the spatial distribution of the typical ionic fragments, demonstrate clearly that the FC polymer is exclusively found on the 20 microns wide line, whereas the tetraglyme polymer is masked underneath the line and found only on the background. A surfactant concentration of >200 microgram/ml results in the formation of a monolayer within minutes of the exposure to the surface. The adsorption kinetic is dependent on the solution concentration. The monolayer surface concentration was calculated to be

~400 nanograms/cm², which corresponds to a specific area of 38Ų/molecule. By controlling the ligand pattern and the density within the pattern, we can control the size and the shape of cells and study the influence of these parameters on SMC physiology [1].

FIG. 3. Photomicrographs of cell attachment onto bare, and FC-PEG covered surfaces superimposed on the FC-PEG adsorption isoterm to the SPR surface.

ACKNOWLEDGEMENTS

NSF provided the funding for this research through the University of Washington Engineered Biomaterials (UWEB) Engineering Research Center Grant ERC 9529161.

REFERENCE [1] INGBER EL AL, Science 276 (1997) 1425.

CMC: 390 µg/ml