Rational Design of Fluoro-photoacoustic Probes for <i>In Situ</i> Imaging of Endogenous β-Galactosidase Activity and Autophagy

<i>In situ</i> tracking of lysosomal biomarkers has facilitated the unraveling of complex lysosome-associated biological processes and functions. Conventional lysosomal probes typically rely on amine-containing motifs for lysosomal accumulation; however, these motifs would impact lysosomal acidity and disrupt cellular homeostasis due to the proton buffering. To address these issues, this study reports a new strategy for constructing nonamino-dependent lysosomal tracking probes by incorporating an acid-responsive and noninvasive fluoro-photoacoustic scaffold HD-BTZ. Leveraging an ICT mechanism, the HD-BTZ scaffold could respond to the acid environment, enabling <i>in situ</i> visualization of lysosomal dynamics with minimal proton buffering effects. As a proof of concept, two activable molecular probes, <b>HD-BTZ-gal</b> and <b>HD-BTZ-photo</b>, were designed by integrating β-galactosidase's substrate or photolytic protecting groups into the HD-BTZ fluorophore scaffold for <i>in situ</i> tracking of β-galactosidase in lysosomes and lysosome-associated autophagy, respectively. The probes exhibit excellent dual-target activation properties, for which the <b>HD-BTZ-gal</b> probe enables <i>in situ</i> imaging of lysosomal β-galactosidase, while the <b>HD-BTZ-photo</b> probe could be employed for <i>in situ</i> tracking of autophagy by monitoring the dynamic changes in lysosomal pH in living cells. <i>In vivo</i> experiments showed that <b>HD-BTZ-gal</b> allowed for fluorescence/photoacoustic dual-mode imaging of chemotherapy-induced senescence in tumors, suggesting that this strategy could provide an effective approach for evaluating and monitoring age-related diseases.