Enzymatically Triggered Deprotection and Cross-Linking of Thiolated Alginate-Based Bioinks

Thiolated polymers are widely used in hydrogels for drug delivery, tissue engineering, and biofabrication. The oxidation of thiols is spontaneous, resulting in the formation of disulfide bridges and cross-linking of polymers. The cross-linking process is, however, difficult to control and is initiated directly when the thiolated components are exposed to ambient conditions, which significantly complicates handling of the materials. Here, we show a fully bioorthogonal enzyme-mediated thiol-based chemistry for dynamic covalent cross-linking of carbohydrate-based hydrogels that circumvents the problems with uncontrolled thiol oxidation. Alginate was modified with cysteine residues, protected by an enzyme-labile thiol-protecting group (Phacm). Releasing the Phacm group by penicillin G acylase generates free thiols that oxidize under physiological conditions, resulting in a reversible cross-linking and formation of hydrogels with tunable stiffness. Prior to deprotection, the components can be exposed to ambient conditions. The enzyme-triggered deprotection and subsequent gelation allows for encapsulation of cells and 3D bioprinting of cell-laden hydrogel structures. Remaining deprotected thiols enabled postprinting modifications and hydrogel self-healing. The proposed hydrogel synthesis strategy significantly increases the versatility of thiol-based cross-linking chemistries and provides new possibilities to generate dynamic covalent hydrogels for a broad range of biomedical applications.