Self-Assembling Peptide Monolayers: Endothelial Cell Behavior on Functionalized Metal Substrates
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ABSTRACT Despite the high initial success rate with metallic stents for the treatment of a variety of vascular lesions, problems have included occlusion due to thrombus formation or intimal proliferation. Improving the biological behavior of these and other other implantable metallic devices may require the use of biomimetic peptide coatings which promote specific cellular responses at the biological-materials interface. Thiol-terminated peptides, without the addition of a cysteine residue, were synthesized by a modification of standard solid phase methodology. Gold/mica or gold/glass surfaces were exposed for 6 hours at 23 'C to one of three peptide solutions: GRGD(13A) 3YNH(CH 2)2 SH (RGD); (P3A) 6NH (CH2)2SH (bAla); or a 1:1 mix of both peptides. Peptide films were examined by external reflectance infrared (IR) spectroscopy and atomic force microscopy (AFM) which confirmed the presence of unique close-packed structures for bAla and the 1:1 mix. Endothelial cell proliferative, migratory, and adhesive behavior were evaluated using 3H-thymidine and 51Cr labeling techniques, respectively. Cell proliferation, migration, and adhesion were significantly higher on RGD containing peptide films. Well-ordered protein assemblies on metallic substrates can be produced with the proper choice of peptide chain structure and terminal residues. Biological activity is a function of film composition and oligopeptide pendant structure. INTRODUCTION Recent advances in surface engineering have been achieved by following the paradigm of the biological membrane which organizes highly ordered molecular assemblies via processes largely driven by van der Waals attraction forces [1]. Indeed, the stability and order of alkanethiolates on gold, as well as the kinetics of monolayer assembly are enhanced by increasing chain length and associated van der Waals intermolecular interactions. In protein assemblies, nature has provided another well-recognized model system for organizing molecules with intrinsic physiochemical stability which far exceeds that of the cell membrane. These differences are related, in part, to stronger non-covalent interactions mediated by hydrogen bonding, particularly in pleated secondary structures[2]. Here we describe the formation of highly ordered monolayer assemblies driven by hydrogen bond interactions between poly(J3alanine) thiolates. By choosing an appropriate peptide pendant or by creating a surface mixture of ligand and non-ligand containing species, biological responses can be controlled. EXPERIMENTAL METHODS Peptide Synthesis. Model peptides were synthesized and purified using a solid-phase strategy which has been described in detail elsewhere[3]. Briefly, the synthesis of thiol terminated peptides without the addition of a cysteine residue or the use of other post-synthesis derivatization steps was performed after first derivitizing chloromethylated polystyrene resin with N-t-butoxycarbonyl-2aminoethanethiol. In this case, after peptide synthesis a free thiol was generated by cleavage of the para-substitut
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