Ratiometric near-infrared fluorescence probes for quantitative monitoring of mitochondrial pH dynamics in Drosophila melanogaster and HeLa cells during metformin treatment

Abstract

This study introduces two new ratiometric probes, AH⁺ and BH⁺, designed to monitor mitochondrial pH dynamics. Derived from IR-780 iodide, these probes incorporate imidazole, hydroxy-xanthene, and hemicyanine groups. Owing to their positively charged hemicyanine groups, both probes selectively accumulate in mitochondria through electrostatic interactions. Their sensing mechanism is governed by deprotonation-induced π-conjugation rearrangements, enabling dual-emission fluorescence for real-time visualization of mitochondrial pH fluctuations. Monitoring of various cellular processes, including mitophagy, hypoxia, oxidative stress, and mitochondrial dysfunction induced by metformin, is reported. In their protonated states (AH⁺ and BH⁺), the probes exhibit large Stokes shifts (>90 nm) due to excited state intramolecular proton transfer (ESIPT). In contrast, the unprotonated forms (A and B) show anti-Stokes shifts. While theoretical calculations using the APFD/6-311+g(d,p) functional/basis set were conducted to simulate these effects, the results were inconsistent with experimental findings. The probes' suitability for measuring mitochondrial pH in HeLa cells was confirmed through a series of tests for photostability, selectivity, reversibility, and cytotoxicity. Colocalization experiments yielded high Pearson Correlation Coefficients (PCC) of 0.96 for AH⁺ and 0.93 for BH⁺, affirming their specific mitochondrial targeting. The probes successfully measured pH changes in HeLa cells under various conditions, including changes in buffer pH, starvation, oxygen deprivation, and chemical treatments with NAC, FCCP, and H₂O₂. Their ability to sense pH was also validated in vivo using Drosophila melanogaster larvae. Significantly, treatment with metformin was shown to alter mitochondrial pH levels, demonstrating the probes' utility in studying drug effects. Collectively, these findings establish AH⁺ and BH⁺ as powerful tools for probing mitochondrial function and cellular stress responses, offering new opportunities to explore fundamental mechanisms underlying health and disease.