pH-/Viscosity-Activatable NIR Fluorescent Probes via Acceptor Engineering of Hemicyanine Dyes for High-Contrast Bioimaging

Real-time monitoring of the lysosomal microenvironment using small-molecule probes is critical for understanding the complex interactions between organelles and diseases associated with lysosomal dysfunction. Most traditional fluorescent-dye-based lysosome probes rely on the protonation of the nitrogen atom in the morpholine unit to visualize the lysosomal microenvironment by inhibiting the photoinduced electron transfer (PET) effect. However, these probes often face selectivity issues within the complex cellular microenvironment. For instance, classic hemicyanine dyes (HD) show nonspecific fluorescence responses in the liver due to inappropriate p<i>K</i>_a value, leading to low imaging contrast and the risk of false positives. Herein, a series of novel pH/viscosity-activatable, lysosomal-targeted near-infrared (NIR) fluorescent probes were developed by incorporating naphthalimide dye with hemicyanine dyes. These probes exhibit no fluorescence at physiological pH and weak fluorescence under acidic conditions (key 1), with substantial fluorescence activation triggered by abnormal viscosity in pathological tissues (key 2). Notably, <b>NpCy-4</b> demonstrated a superior signal-to-background ratio (SBR) for cellular imaging and proved effective for real-time in situ diagnosis of acute gastritis.