Switchable Fluorescence of Doxorubicin for Label‐Free Imaging of Bioorthogonal Drug Release

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans‐cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond‐cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA‐MB‐231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems. A little light release: The release of doxorubicin from an iron oxide nanodrug formulation through bioorthogonal cleavage is accompanied by an increase in fluorescence. This behavior can validate drug release upon its uncagingreaction with the highly specific chemical partner tetrazine. The increase in doxorubicin fluorescence was exploited to study its release profile from the nanoparticle surface upon chemical activation.