Bioavailability of immobilized epidermal growth factor: Covalent versus noncovalent grafting

Abstract

In an effort to rationalize and optimize an antiapoptotic coating combining chondroitin sulfate (CS) and epidermal growth factor (EGF) for vascular applications, the authors here report the comparison of two grafting strategies aiming to display EGF in an oriented fashion on CS. For that purpose, the authors produced, purified, and characterized a chimeric protein corresponding to EGF that was N-terminally fused to a cysteine and a coil peptide. The chimera was covalently immobilized via its free thiol group or captured via coiled-coil interactions at the surface of a biosensor or on a chondroitin sulfate coating in multiwell plates, mimicking the coating that was previously developed by them for stent-graft surfaces. The interactions of grafted EGF with the soluble domain of its receptor or the impact of grafted EGF upon vascular smooth muscle survival in proapoptotic conditions indicated that the coiled-coil based tethering was the best approach to display EGF. These results, combined to direct enzyme-linked immunosorbent assay measurements, indicated that the coiled-coil tethering approach allowed increasing the amount of bioavailable EGF when compared to covalent coupling, rather than the total amount of grafted EGF, while using much lower concentrations of tagged EGF during incubation.

Immobilization of Growth Factors for Cell Therapy Manufacturing

“…In general, this technique involves the reaction of free thiols in the GF with thiol-reactive groups in the substrate surface. In GFs, free thiol groups exist in cysteine residues, but may also be introduced chemically or with recombinant technology (Place et al, 2012;Chen et al, 2014;Riahi et al, 2017). Functionalized GFs react with free thiols on the substrate surface, leading to their immobilization.…”

“…Due to their highly specific and stable interaction, coiledcoil structures have been used in various biomedical and biotechnological applications, including GF immobilization (Boucher et al, 2008(Boucher et al, , 2010Apostolovic et al, 2010;Murschel et al, 2013;Assal et al, 2015;Noel et al, 2016;Riahi et al, 2017). The target GF is expressed as a fusion protein with one coiled-coil strand, while the substrate surface is functionalized with the complementary coiled-coil strand.…”

Advances in Growth Factor Delivery for Bone Tissue Engineering

“…Moreover, the HAp-1:1 group showed high cysteine-knot stability through adsorption/desorption processes, indicating that nano-textured HAp surfaces can better incorporate BMP-2 molecules through adsorption and can aid in BMP-2 biological activity. Alginate microbeads were surface condensed with heparin through polyelectrolyte complexes (diethylaminoethyldextran (DEAE-D), poly-l-ornithine, and poly-l-arginine) to provide a delivery system for BMP-2 [128]. The authors observed distinct release profiles for each of the systems designed.…”

Growth Factor Engineering for Biomaterials

“…To realize and optimize an antiapoptotic coating for vascular applications, EGF was combined with chondroitin sulfate. Riahi and Murschel designed and prepared a chimeric EGF in which the N-terminal was fused with a cysteine or coil-forming peptide. In the former case, the chimeric EGF was covalently tethered through the thiol group.…”