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

In situ 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 in situ visualization of lysosomal dynamics with minimal proton buffering effects. As a proof of concept, two activable molecular probes, HD-BTZ-gal and HD-BTZ-photo, were designed by integrating β-galactosidase's substrate or photolytic protecting groups into the HD-BTZ fluorophore scaffold for in situ tracking of β-galactosidase in lysosomes and lysosome-associated autophagy, respectively. The probes exhibit excellent dual-target activation properties, for which the HD-BTZ-gal probe enables in situ imaging of lysosomal β-galactosidase, while the HD-BTZ-photo probe could be employed for in situ tracking of autophagy by monitoring the dynamic changes in lysosomal pH in living cells. In vivo experiments showed that HD-BTZ-gal 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.