Quantitatively Sensing Lipid Droplets Polarity in Cells and Atherosclerotic Model with a Novel Naphthalimide-Based Fluorescent Probe

Lipid droplets (LDs) are crucial organelles in lipid metabolism and energy storage, and their dysfunction is directly linked to metabolic diseases, such as atherosclerosis (AS). While many conventional LDs fluorescent probes are used for morphological visualization, several recently developed polarity-sensitive probes could be utilized to qualitatively monitor polarity changes. In this study, a novel polarity-ultrasensitive fluorescent probe namely Lipi-NIFD was developed to quantify LDs polarity, which was achieved by combined with hyperspectral fluorescence imaging (HSFI), a technique that provides spectral information beyond conventional fluorescence imaging and enables the quantitative detection of polarity changes. Based on the rational design of three naphthalimide derivatives (molecule 1, molecule 2, and Lipi-NIFD ), the π-bridge was identified as a critical structural determinant for polarity sensitivity. Lipi-NIFD, featuring a fluorene-based π-bridge, exhibited a high fluorescence quantum yield and superior polarity sensitivity, which is extremely useful for HSFI. Moreover, probe Lipi-NIFD has excellent LDs targeting specificity and remarkable photostability. Combined this novel probe with HSFI, we quantitatively mapped the LDs polarity distribution and changes in cells, aorta and liver of AS model mice. In particular, the LDs polarity in AS plaque ( E T (30) = 32.90 kcal mol –1 ) is substantially lower than that in the major region of the aorta ( E T (30) = 33.89 kcal mol –1 ). To our knowledge, this study represents the first quantitative analysis of LDs polarity in AS, offering a potential diagnostic tool for AS plaque.