Spatiotemporal Immune Modulation Using Light‐Responsive Nanoparticles With Confinement‐Enhanced Photocleavage

ABSTRACT Spatiotemporal control over immune agonist delivery remains a critical unmet need in immuno‐oncology, where systemic administration risks off‐target immune activation. Here, we present a self‐reporting, light‐responsive polymeric nanoparticle (NP) system for triggered drug release via one‐photon (1P) UV or two‐photon (2P) near‐infrared (NIR) excitation. The system employs a photo‐uncaging prodrug strategy, in which drug molecules are covalently linked to a hydrophobic, light‐responsive o ‐hydroxycinnamoyl ( o ‐HC) platform, enabling NP encapsulation via Flash NanoPrecipitation. Upon photorelease, the o ‐HC platform stoichiometrically generates a coumarin‐based fluorescent reporter, enabling reaction tracking. Using a series of model prodrugs bearing functional groups common to therapeutics, we demonstrate efficient photo‐uncaging in both solution and NP core. Notably, photo‐uncaging reaction rates and action cross sections are at least one order of magnitude larger within NPs than in solution, suggesting a nanoconfinement‐enhanced reaction mechanism accompanied by a confinement‐driven shift in reaction equilibrium. We further apply this platform to deliver a TLR 7/8 immune agonist to RAW 264.7 macrophages, achieving light‐controlled, tunable pro‐inflammatory cytokine secretion. In addition, we report the first application of label‐free optical metabolic imaging for non‐destructive multiphoton laser cell phototoxicity assessment. These results establish o ‐HC prodrug‐loaded NPs as a promising platform for spatiotemporally precise immune agonist delivery in immuno‐oncology.