Stepwise Design of a High Signal-to-Background Ratio Dual-Emission Probe for Imaging Mitochondrial HClO and Microenvironmental Changes in Diabetic Liver and Kidney Injury

Abstract Diabetic hepatorenal injury is linked to oxidative stress, mitochondrial dysfunction, and ferroptosis. Elevated hypochlorous acid (HClO) levels, together with alterations in mitochondrial polarity and viscosity, are key indicators of diabetic hepatorenal injury. Early, in situ, real-time monitoring of these signals is essential for disease assessment and therapeutic intervention. However, most existing fluorescent probes target only single analytes or organs, restricting multi-signal and dual-organ monitoring, while their imaging signal-to-background ratios (SBRs) remain suboptimal. To overcome these limitations, we developed a series of dual-emission fluorescent probes, PVPH1–PVPH3, through a stepwise molecular engineering strategy. Extension of the π-conjugation system induced a red-shift in PVPH2 relative to PVPH1, whereas further incorporation of a dicyanovinyl electron-withdrawing unit into PVPH3 enhanced intramolecular charge transfer and molecular rotor characteristics, improving the probe SBR. PVPH3 enables dual-emission monitoring of HClO and viscosity/polarity-related mitochondrial microenvironmental changes. Spectroscopic studies demonstrated fluorescence enhancement factors of 201.83-fold, 85.75-fold, and 20.67-fold for viscosity, polarity, and HClO, respectively, together with excellent selectivity, stability, and low cytotoxicity. Cellular imaging further revealed that PVPH3 predominantly localizes in mitochondria and effectively visualizes HClO elevation and microenvironmental disruption during apoptosis, ferroptosis, and hyperglycemia-induced injury. In the streptozotocin (STZ)-induced diabetic mouse model, PVPH3 achieved noninvasive dual-channel in vivo imaging of the liver and kidneys and enabled monitoring of Met- and Dhq-induced intervention responses. Overall, PVPH3, with a high SBR achieved through layered molecular optimization, provides a visualization tool for early assessment of diabetic liver and kidney injury, investigation of ferroptosis-related mechanisms, and monitoring of drug-intervention responses.