Design and Optimization of Lysosome-Targeted β‑Galactoside Senolytic Prodrugs: Harnessing the Aromatic Ring of Self-Immolative Linkers

To improve senolytic efficacy and selectivity, we designed gemcitabine (Gem)-based galactoside prodrugs activated by senescence-associated β-galactosidase (SA-β-gal), bearing lysosome-targeting groups at the 3- and 5-positions of the self-immolative linker aromatic ring. This strategy avoids stereocenter formation and promotes faster, electron-donating-effect-driven drug release. Gal-dMor-Gem, with two morpholine groups, showed the strongest activity. Its senolytic index reached 16.1–56.7 across six senescent cell (SnC) models, a 2.8- to 3.7-fold improvement over the nontargeted SSK1. Gal-dMor-Gem released Gem faster and preferentially induced SnC apoptosis, as validated in a coculture model. Biodistribution studies confirmed its preferential accumulation and activation in senescent tissues. In senescent mice, Gal-dMor-Gem (0.5 mg/kg) surpassed SSK1 in restoring body weight, improving biochemical parameters, and reducing SA-β-gal, IL-6, and lamin B1 abnormalities in multiple organs. At 1.0 mg/kg, most markers returned to healthy levels. This work identifies Gal-dMor-Gem as a potent senolytic and highlights a generalizable strategy for developing targeted SA-β-gal-responsive prodrugs.