A “turn-on” intracellular pH probe for the quantitative monitoring of lysosomal alkalization in living cells

Abstract

A slight elevation in lysosomal pH can lead to indigestion or nonspecific hydrolysis, thereby increasing the risk of various neurodegenerative diseases and cancer. Therefore, accurate monitoring of lysosomal pH changes in living cells is essential for the diagnosis and treatment of such diseases, despite the significant challenges involved. In this study, we synthesized a pH-dependent fluorescent probe, B26, which comprises 1,8-naphthalimide as the fluorescent chromophore, an N-(2-hydroxyethyl) piperazine group for lysosome targeting, and a hydroxyethyl group to increase solubility and regulate pKa. B26 demonstrated high sensitivity, selectivity, and reversibility in response to H⁺, and exhibited a remarkable 98-fold increase in fluorescence intensity between pH 2.0 and pH 11.0, with a pKa value of 7.0, highlighting its “turn-on” fluorescence property. Density functional theory calculations and ¹H NMR titration revealed that the pH-sensing mechanism of B26 relies on the inhibition of photoinduced electron transfer from the N-(2-hydroxyethyl) piperazine group to the naphthalimide moiety under acidic conditions. Importantly, B26 effectively labeled lysosomes and displayed significant sensitivity to pH changes, facilitating the quantitative detection of pH shifts during lysosomal alkalization in living cells due to its elevated pKa. These findings suggest that B26 successfully addresses the limitations of existing lysosomal pH probes, particularly in detecting pH changes within the near-neutral range. Furthermore, both the zebrafish model and subcutaneous imaging support the application of B26 in in vivo settings. Given its exceptional properties, B26 holds enormous potential for the research and diagnosis of pH-related diseases.