Exploring ratiometric endolysosomal pH nanosensors with hydrophobic indicators responding at the nanoscale interface and multiple fluorescence resonance energy transfers

Compared with conventional water-soluble fluorescence probes, pH-sensitive fluorescent nanosensors based on hydrophobic indicators remain largely unexplored. We report here the unique pH response of the nanosensors with a hydrophobic indicator (Ch3, a Nile Blue derivative) in polymeric nanoparticles (NPs). At the aqueous-organic interface of the NPs, spectral overlap and dye accumulation caused significant Forster resonance energy transfer (FRET) not only between the protonated and deprotonated Ch3 (hetero-FRET), but also between the protonated and deprotonated Ch3 themselves (homo-FRET). The pH response was simulated according to an interfacial response mechanism and the dynamic range was found to depend on the size of the NPs and dye distribution (Kp). Therefore, adjusting the size of the NPs and the local dye concentration gave rise to a series of dynamic sensing ranges with apparent pKa values from 2.7 to 9.6 based on a single indicator. The nanosensors were successfully delivered to HeLa cells to monitor subcellular pH values in the endosomes and lysosomes. Based on cellular calibrations, the average pH in the organelles were determined to be ca. 4.7. Moreover, the pH neutralization process during lysosome membrane permeabilization (LMP) induced by hydrogen peroxide stimulation was also successfully visualized with the nanosensors.