Bioluminescence Resonance\nEnergy Transfer (BRET)-Mediated\nProtein Release from Self-Illuminating Photoresponsive Biomaterials

Phototriggered release of various\ncargos, including soluble\nprotein\nfactors and small molecules, has the potential to correct aberrant\nbiological events by offering spatiotemporal control over local therapeutic\nlevels. However, the poor penetration depth of light historically\nlimits implementation to subdermal regions, necessitating alternative\nmethods of light delivery to achieve the full potential of photodynamic\ntherapeutic release. Here, we introduce a strategy exploiting bioluminescence\nresonance energy transfer (BRET)–an energy transfer process\nbetween light-emitting Nanoluciferase (NLuc) and a photosensitive\nacceptor molecule–to drive biomolecule release from hydrogel\nbiomaterials. Through a facile, one-pot, and high-yielding synthesis\n(60–70%), we synthesized a heterobifunctional ruthenium cross-linker\nbearing an aldehyde and an azide (CHO-Ru-N<sub>3</sub>), a compound\nthat we demonstrate undergoes predictable exchange of the azide-bearing\nligand under blue-green light irradiation (>550 nm). Following\nsite-specific\nconjugation to NLuc via sortase-tag enhanced protein ligation (STEPL),\nthe modified protein was covalently attached to a poly(ethylene glycol)\n(PEG)-based hydrogel via strain-promoted azide–alkyne cycloaddition\n(SPAAC). Leveraging the high photosensitivity of Ru compounds, we\ndemonstrate rapid and equivalent release of epidermal growth factor\n(EGF) via either direct illumination or via BRET-based bioluminolysis.\nAs NLuc-originated luminescence can be controlled equivalently throughout\nthe body, we anticipate that this unique protein release strategy\nwill find use for locally triggered drug delivery following systemic\nadministration of a small molecule.