Tethered protein/peptide-surface-modified hydrogels

Abstract

We investigated a wet chemistry method to covalently bond polyethylene glycol (PEG)-tethered extracellular matrix (ECM) proteins (laminin and fibronectin) or peptide (fibronectin-adhesion-peptide sequence) onto the surface of a poly(2-hydroxylethyl methacrylate-co-methylacrylic acid) (PHEMA/MAA) hydrogel that could potentially be used as a replacement for corneal tissue in the eye. An essential requirement for the success of such a surface in the biological environment is its ability to support the growth and attachment of corneal epithelial cells; ECM proteins are known to promote cellular attachment and growth. We hypothesized that the use of tethers or long hydrophilic chains would allow the attached ECM protein/peptide molecules to move two-dimensionally in space, thereby increasing their ability to bind with epithelial cell membranes. Additionally, the tethers would prevent the specifically added growth-enhancing factors from being obscured by any non-specific protein binding occurring on the hydrogel surface. In this surface-modification study, carbodiimidazole (CDI) was used to activate the carboxylic groups (-COOH) on the hydrogel surface in anhydrous dimethylsulfoxide (DMSO) before addition of the PEGylated proteins/peptide. The resulting tethered protein/peptide surface-modified hydrogels were analyzed in terms of percent grafting efficiency, biological activity and mechanical properties. X-ray photoelectron spectroscopy (XPS) and 125I radioactive labeling demonstrated the successful covalent bonding between the moieties and the hydrogel surface. Radiolabeling and enzyme-linked immunosorbent assay (ELISA) studies indicated that the tethered proteins attached to the hydrogel surface at a concentration of approx. 0.1 microg/cm2. ELISA testing further showed that tethered proteins remained biologically active. However, mechanical tensile testing indicated that the mechanical properties of these chemically modified hydrogels were somewhat altered in comparison with the unmodified hydrogel. Future studies will evaluate the cellular response to these surface-modified materials in vitro and in vivo.