Rational Design of Enzyme-Responsive Fluorescent Organoruthenium Prodrug for Real-Time Tracking of Cargo Release and Cytotoxicity

The precise design strategies that achieve structural optimization of both metallodrugs and self-reported metallodrug carriers can overcome the major limitations of metallodrug development. Herein, we structurally modified a conventional half-sandwich Ru(arene)(bipyridine) complex into a fluorogenic and esterase-responsive prodrug nanosystem via functionalizing the <i>N</i>,<i>N</i>'-bidentate bipyridine ligand with 10,12-pentacosadiynoic acid derivative, thereby designing an amphiphilic complex with the ability for coassembly. Prior to obtaining the nanoassembly, in an attempt to infer the benefits of functionalizing the ligand, a series of four organoruthenium complexes, including the amphiphilic complex, was prepared and characterized. The nanoassembly is found to release the Ru complex in the presence of porcine liver esterase concomitantly with a 4-fold increase in its fluorescence intensity. Such a stimuli-responsive behavior is exploited for real-time release monitoring of Ru complex and understanding its functionality to induce cell death in HeLa and THP-1 cancer cells. Compared to the free Ru complex, this nanosystem exhibits better cellular uptake and 100-fold higher cytotoxicity in HeLa cancer cells while being less toxic in noncancerous GM5756 cells. <i>N</i>-Acetylcysteine-induced GSH augmentation studies revealed that the nanoassembly exerts antiproliferative activity through oxidative stress. Toxicity analysis in zebrafish embryos confirmed the biocompatibility of the nanoformulations, suggesting a promising future <i>in vivo</i> studies.