Rational design of a lipophilic β-galactosidase-responsive near-infrared probe for in vivo imaging of cellular senescence.

Senescence-associated β-galactosidase (SA-β-gal) is a key biomarker of cellular senescence and has been demonstrated to be a major driver of various age-related diseases and tumor resistance. However, noninvasive in vivo imaging of SA-β-gal remains challenging due to the poor membrane permeability of existing probes, their reliance on intratumoral injection, and limited blood-brain barrier (BBB) permeability. This study reports a novel, rationally designed near-infrared (NIR) fluorescent probe, DCIP-AcGal, which involves a synergistic lipophilicity-oriented design strategy by leveraging a BBB-penetrating fluorophore with acetylated β-galactose. Its SA-β-gal-sensitive NIR emission property enables deep-tissue imaging of senescent cells with low background and high signal-to-noise ratio. The probe's favorable lipophilicity facilitates passive diffusion through the cell membranes, ensuring efficient activation by SA-β-gal within the lysosomes. Its specificity for imaging senescent cells has been validated through GLB1 knockdown, and its fluorescence intensity and distribution correlate positively with p21 expression. Following intravenous injection, DCIP-AcGal enables noninvasive imaging of palbociclib-induced tumor senescence in living mice. Furthermore, due to its favorable lipophilicity, the probe crosses the BBB, allowing for the assessment of senescence accumulation in the brains of aged mice. These findings demonstrate that probe DCIP-AcGal provides a valuable tool for investigating senescence biology and monitoring the efficacy of senotherapeutic interventions.