In vivo quantitative visualization of hypochlorous acid in the liver using a novel selective two-photon fluorescent probe

摘要

Hypochlorous acid (HOC1) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOC1 is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOC1, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOC1, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygenitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOC1 concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 uM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOC1 molecules. The cellular concentration of HOC1 was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOC1, and rapid "off-on" fluorescence. It is suitable for dynamic quantitatively monitoring HOC1 generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.