Quinoxaline-derived aza-BODIPY AIEE fluorescent probes for lysosomal viscosity imaging

In this study, two novel quinoxaline-derived aza-BODIPY fluorescent probes (1 and 2) responsive to lysosomal viscosity were synthesized via a one-pot Schiff base reaction. Both probes feature a rotatable benzothiazole group linked via a CN bond, while probe 2 incorporates a morpholine moiety to enhance lysosomal targeting. Single-crystal X-ray diffraction confirmed that the morpholine group increases π-electron delocalization and affects molecular stacking. Both probes exhibited characteristic UV–visible absorption and emission with large Stokes shifts. Probe 2 showed a red-shift due to the electron-donating effect of morpholine. Aggregation-induced emission enhancement (AIEE) was observed in both probes. Both displayed strong linear fluorescence responses to viscosity (2–610 cP), with up to 24-fold enhancement and low detection limits (0.27 and 0.26 cP). They demonstrated excellent selectivity over biological interferents and negligible cytotoxicity in MTT assays. Co-localization experiments revealed better lysosomal targeting for probe 2 (R = 0.86) compared to probe 1 (R = 0.83), attributed to the dual-targeting effect of the benzothiazole and morpholine groups. Both probes successfully monitored dexamethasone-induced lysosomal viscosity changes (10–40 μM) in live cells, enabling real-time tracking of the lysosomal microenvironment. Probe 2 consistently showed stronger fluorescence intensity than probe 1, highlighting its superior sensitivity and detection performance. These findings suggest the potential of these aza-BODIPY-based probes as effective tools for real-time intracellular viscosity imaging and lysosome-related disease research.