Controlling cell adhesion to surfaces via associating bioactive triblock proteins

A surface functionalization strategy that produces substrates with well-controlled ligand density is critical to investigating the role of cell–substrate interactions in regulating cell adhesion, viability, migration, proliferation and differentiation. Towards this end, we have designed and synthesized a triblock protein, CRC, comprising a polyelectrolyte domain flanked by two amphiphilic leucine zipper domains. The amphiphilic end domains of CRC adsorb onto surfaces and preferentially associate into trimeric aggregates, forming a hydrogel coating layer. Under serum-free conditions, the CRC coating was found to render both 2D substrates and 3D scaffolds non-adhesive to cells. A RGDS sequence was inserted in the middle domain of CRC (generating the protein CRC-RGDS) and found to introduce cell-binding activity. Incorporation of the RGDS sequence did not significantly impact the surface activity of CRC, allowing us to titrate the RGDS surface density simply by adjusting the relative ratios of the two proteins. Ligand density dependent cell–substrate interactions were demonstrated in human foreskin fibroblasts, human umbilical vein endothelial cells, and rat neural stem cells. The versatility to functionalize a range of different substrate surfaces, combined with the ease of controlling surface ligand density, makes these triblock proteins an attractive tool for developing cell-specific surface coatings with tailored biofunctional attributes.