Mechanically tunable click-crosslinked alginate hydrogels for local cell-free protein synthesis and delivery

Hydrogels are widely used as drug-delivery platforms, yet conventional systems rely on the passive release of preloaded therapeutics, often resulting in limited control over dose and localization. In this work, we explore an alternative approach: transforming hydrogels into protein-producing materials by integrating cell-free protein synthesis (CFPS) within injectable click-crosslinked alginate hydrogels. By tuning alginate concentration and crosslinking density, we generated hydrogels with distinct mechanical properties and evaluated their impact on in situ protein production and diffusion. Softer hydrogels promoted higher protein synthesis, whereas stiffer networks restricted protein diffusion, demonstrating that hydrogel mechanics directly regulate both CFPS activity and protein transport. An intermediate formulation provided the best balance between injectability, protein production efficiency, and sustained transport. These findings demonstrate that hydrogel mechanics directly regulate both CFPS activity and protein transport, establishing click-crosslinked alginate hydrogels as promising platforms for localized therapeutic protein production and delivery.