A dual-responsive NIR fluorescent probe for GSH and viscosity: applications in models of cellular inflammation and apoptosis

Cancer remains a leading cause of global death. Because high levels of glutathione (GSH) and increased intracellular viscosity are known as key biomarkers linked to cancer cells, we developed a dual-responsive near-infrared (NIR) fluorescent probe, NIR-NOS, for simultaneous detection of mitochondrial GSH and viscosity. NIR-NOS uses NOS-Br as the GSH-responsive component and a C[double bond, length as m-dash]C bond within the NIR-1 part as the viscosity-sensitive element, with its fluorescence enhanced through the restriction of intramolecular rotation. When exposed to GSH, breaking the ether bond causes a significant fluorescence turn-on. At the same time, increased viscosity limits the rotation of the C[double bond, length as m-dash]C bond, boosting the fluorescence at 640 nm by about 50 times. Spectroscopic tests showed excellent selectivity for GSH over other substances, with a detection limit of 0.44 µM. Importantly, NIR-NOS shows low toxicity and effectively targets mitochondria. This probe allows for visualization of both natural and induced changes in mitochondrial GSH, as well as real-time observation of intracellular viscosity shifts, demonstrated in studies using nystatin (Nys) and lipopolysaccharide (LPS) to alter these factors. Additionally, because of its mitochondrial targeting and dual-response function, NIR-NOS enables real-time tracking of key mitochondrial activities, such as vesicle formation and programmed cell death triggered by Nys. This offers deeper understanding of mitochondrial redox regulation and environmental changes. Overall, this study introduces NIR-NOS, a thoughtfully designed, multifunctional NIR fluorescent probe with great potential for advanced cancer imaging and live, <i>in situ</i> tracking of interconnected mitochondrial markers.