Development of a semi-synthetic cyanin-based mitochondria-targeting viscosity-responsive fluorescent probe: application in fluorescent imaging of cells and zebrafish

The precise monitoring of viscosity in the mitochondrial microenvironment holds significant theoretical value and practical importance for the early prevention of diseases, precise diagnosis, and the optimization of treatment strategies. We have therefore embarked upon a programme targeting probes that are capable of the fluorescent visualization of changes in viscosity. In this study, 4-(4-methylpiperazin-1-yl)benzaldehyde and 4-morpholinobenzaldehyde were subjected to Knoevenagel condensation reactions with 3-(2,3,3-trimethylindol-1-ium-1-yl)propane-1-sulfonate and 1,2,3,3-tetramethyl-3H-indolium iodide, respectively, resulting in the one-step synthesis of electron-donor (D)-electron-acceptor (A) fluorescent probes PpIp, MpIp, PpIn, and MpIn, Experimental results show that the fluorescence intensity ratio (log(I/I₀)) of this series of probes exhibits a good linear relationship (R2 > 0.98) with the viscosity (logη) of the system over a range of 2.24–827.5 cP. Combining DFT calculations, solvent polarity analysis, and fluorescence lifetime experiments, we demonstrate that the viscosity response of this series of probes primarily stems from the restriction of intramolecular rotation in high-viscosity environments and the suppression of TICT-related nonradiative decay. This series of probes exhibits low cytotoxicity. Cell fluorescence imaging experiments demonstrated that probes MpIn and PpIn specifically target mitochondria, with Pearson correlation coefficients exceeding 0.85 for colocalization with the commercial mitochondrial probe Mito-Tracker Green. Furthermore, this series of probes has successfully enabled the fluorescent visualization of viscosity changes in HepG2 cells and in vivo in zebrafish.