Chemically Orthogonal Protein Ligation Domains for Independent Control of Hydrogel Modification with Adhesive Ligands and Growth Factors

The twin, chemically orthogonal protein ligation domains, SpyCatcher and SnoopCatcher, were used to link two engineered proteins into poly(ethylene glycol) (PEG) hydrogels in order to control both endothelial cell adhesion and material-mediated pro-mitotic stimulation. SpyCatcher was appended with an N-terminal adhesion ligand RGDS to form RGDS-SC, and SnoopCatcher was appended with the vascular endothelial growth factor (VEGF)-mimetic peptide QK to form QK-SnpC. QK-SnpC formed a spontaneous covalent bond with SnoopTag peptide with 40% reaction efficiency, both in solution, in a PEG gel containing SnoopTag peptide, and in a PEG gel with both SnoopTag and SpyTag sites. QK-SnpC added to cell culture media enhanced endothelial cell proliferation compared to a negative control, and was statistically indistinguishable from the positive control of 130 pM VEGF165. Endothelial cells seeded onto PEG gels presenting both RGDS-SC and QK-SnpC showed ∼50% of cells actively proliferating (defined as Ki67+), compared to ∼31% of cells seeded on gels presenting RGDS-SC alone. These results show that complementary nondiffusing biochemical signals can be linked into PEG-DA hydrogels simultaneously using 'Catcher-based ligation strategies, thereby inducing more nuanced cell–material interactions.