Bioorthogonal Engineering of Cellular Microenvironments Using Isonitrile Ligations

Hydrogels are routinely used as scaffolds to mimic the extracellular matrix for tissue engineering. However, common strategies to covalently crosslink hydrogels employ reaction conditions with potential off-target biological reactivity. The limited number of suitable bioorthogonal chemistries for hydrogel crosslinking restricts how many material properties can be independently addressed to control cell fate. To expand the bioorthogonal toolkit available for hydrogel crosslinking, we identify isonitrile ligations as a promising class of reactions. Isonitriles are compact, stable, selective, and biocompatible moieties that react with chlorooxime (ChO), tetrazine (Tz), and azomethine imine (AMI) functional groups under physiological conditions. We demonstrate that all three ligation reactions can form hydrogels, with isonitrile-ChO ligation exhibiting optimal gelation properties. Synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked by isonitrile-ChO ligation exhibit rapid gelation kinetics, elastic mechanical properties, stability under physiological conditions, and high biocompatibility. By combining ChO-functionalized multi-arm PEGs with isonitrile-functionalized engineered elastin-like proteins (ELPs), we demonstrate simultaneous control over network connectivity and adhesive ligand presentation, which in turn regulate cell spreading. These hydrogels enable the long-term culture of numerous human cell types relevant to regenerative medicine. Furthermore, we demonstrate that isonitrile-ChO ligation is orthogonal to common azide-alkyne cycloaddition, enabling independent, bioorthogonal functionalization of hydrogels containing live cells.