Rational design of a ratiometric fluorescent probe for imaging lysosomal nitroreductase activity

Overexpressed nitroreductase (NTR) is often utilized to evaluate the hypoxic degree in tumor tissues, thus it is of great importance to develop high selective and efficient optical method to detect NTR. The dynamic fusion and function of lysosome promoted us to explore the possible appearance of NTR inside this organelle and to probe its behavior in a cellular context. In this work, a ratiometric fluorescent probe based on an extended 7C-7C conjugation of a triphenylamine unit was designed for NTR detection and lysosomes imaging. The dual-emission mechanism of the probe in the presence of catalytic NTR was confirmed by theoretical study. The structure-function relationship between probe and NTR was revealed by docking calculations, suggesting a suitable structural and spatial match of them. The photophysical studies showed the probe had high selectivity, rapid response and a wide pH range towards NTR. MTT assay indicated the probe had low cytotoxicity in both normal (HUVEC) and tumor (MCF-7) cells. Furthermore, the inverse fluorescent imaging results confirmed the probe was NTR-active and exhibited time-and concentration-dependent fluorescence signals. In addition, the relatively high Pearson's correlation coefficient (0.99 in HepG2 and 0.97 in MCF-7 cells, compared to Lyso-Tracker Red) demonstrated the probe had excellent lysosomes colocalization. This study illustrates a ratiometric detection of NTR agent for lysosomes fluorescent imaging, which may provide a novel insight in molecular design.

Automated summary

In this study, a ratiometric fluorescent probe based on extended 7C-7C conjugation of a triphenylamine unit was designed for the detection of NTR and lysosomes imaging. The probe showed high selectivity, rapid response, and a wide pH range towards NTR. It also exhibited low cytotoxicity in both normal and tumor cells, as well as excellent lysosomes colocalization.