Bio-Orthogonal Cross-Linking Chemistry Enables In Situ Protein Encapsulation and Provides Sustained Release from Hyaluronic Acid Based Hydrogels

Chemically cross-linked hydrogels are promising systems for protein delivery applications, but their utility may be limited due to the possibility of protein reaction with hydrogel precursors. Herein, a catalyst-free inverse-demand Diels-Alder reaction between tetrazine and norbornene groups was used to demonstrate the bio-orthogonal nature of cross-linking chemistry that is chemically inert to proteins. Tetrazine-modified hyaluronic acid and norbornene-modified polyethylene glycol were used as hydrogel precursors for in situ encapsulation of a model protein, Fab1. Measurement of gelation kinetics demonstrates that network formation and gel stiffness are temperature-dependent but independent of Fab1 concentration. In vitro release testing shows that Fab1 is completely released from the hydrogel matrix over a period of several weeks. Analytical characterization suggests that Fab1 is released without any physical or chemical modifications and retains its antigen binding capacity. Thus, the bio-orthogonal and catalyst-free aqueous phase chemistry enables efficient in situ protein encapsulation in a single step and provides sustained protein release.