ESIPT Nano‐Emitter to Probe Lysosome Biogenesis in Live Cells

Endosome-lysosome fusion and endo-lysosome fission-mediated lysosome biogenesis are crucial in regulating cellular health, and their dysregulation signifies disease. Tracking such intricate events with minimal disturbance remains elusive due to the scarcity of single-component synthetic probes capable of distinctly and simultaneously labeling both endosomes and lysosomes. Here, an amphiphilic π-conjugated imine probe is designed that forms micellar self-assemblies in water, called Nano-emitter, which distinctly and simultaneously labels endosomes and lysosomes upon monochromatic-wavelength excitation. ESIPT (Excited State Intramolecular Proton Transfer) active Nano-emitter shows red fluorescence at endosomal pH. Its hydrolysis to fluorescent amine, PEG-Naph at lysosomal pH illuminated lysosomes fluorescent green, with both imine and amine forms excitable using a 405 nm confocal laser. The two-color labeling of endosomes and lysosomes enabled tracking of their fusion and lysosome-biogenesis processes in living cells. Using multiplexed time-lapse imaging with Nano-emitter and anti-cancer drug doxorubicin, the role of these processes is investigated in lysosome-mediated doxorubicin sequestration in MCF-7 cells. The results show that endosomes as well as endo-lysosomes also sequestered doxorubicin apart from lysosomes. Interestingly, doxorubicin-sequestered endo-lysosomes underwent fission and generated more doxorubicin-sequestered lysosomes, preventing the drug's nuclear localization. Such versatile probes can enhance the understanding of drug sequestration and foster therapeutic strategies.