Fluorescent probes based on aza-Nile Red for visualizing mitochondrial polarity fluctuation

Abstract

Nile Red is intensely fluorescent in a lipid-rich environment while it has minimal fluorescence in aqueous media. Whereupon, three neutral red/near-infrared fluorescent dyes 1a-1c with good water solubility were synthesized by introducing nitrogen atoms into the 2, 3 and 4 positions of Nile Red in order to improve the biocompatibility in this paper. Optical testing showed that the maximum absorption wavelengths of 1a-1c were in the range of 525–617 nm, while the maximum emission ones located at 584–668 nm in different solvents. When the solvent polarity (Δf) was reduced from 0.316 to 0.020, the maximum absorption wavelengths of 1a-1c were blue-shifted by 73–97 nm, and the fluorescence intensities were enhanced by 32–44 folds. Meanwhile, excellent linear relationships between the fluorescence lifetimes, absolute fluorescence quantum yields of 1a-1c and Δf (0.229–0.307) were established with high correlation coefficients (R² = 0.98–0.99). In addition to excellent low toxicity and mitochondrial targeting properties, 1a-1c were able to detect polarity changes in models of inflammation induced by Lipopolysaccharide at 50 nM. Furthermore, based on the significant mitochondrial polarity differences between cancer and normal cells, 1a-1c had the ability to differentiate them. And 1a-1c also were capable of real-time monitoring mitochondrial polarity during starvation or rapamycin-induced autophagy. Overall, the excellent optical properties and biocompatibility of aza-Nile Red (1a-1c) suggest that they have great potential in studying mitochondrial physiology and pathological biological processes.