Endogenous Fluorescence Labeling of Extracellular Vesicles with a Hydrophilic Cyanine Dye via Caveolin-1 Dependent Endocytosis.

Fluorescent labeling of extracellular vesicles (EVs) can provide an understanding of their biological properties from biogenesis to uptake and function. Since EVs have been found to play a crucial role in diseases, including cancers, the fluorescent EVs can be used in diagnostic or therapeutic applications. Previously, several methods using fluorescent imaging agents are reported to label and characterize the spatiotemporal properties of EVs. Among them, lipophilic membrane dyes such as PKH26/PKH67, DiR/DiD, and MemGlow are typically used for exogenous EV labeling, however, the application of lipid dyes is complicated by unbound dye, aggregate formation, and non-EV labeling. To overcome these limitations, an endogenous labeling method is reported for the first time to visualize EVs using a hydrophilic cyanine dye, named CA800-EV, without the need for membrane labeling or immunolabeling. Importantly, this simple and efficient method of endogenous fluorescence labeling the CA800-EV dye to multivesicular bodies is successfully used for monitoring the biogenesis and secretion of EVs. It is demonstrated that CA800-EV enables unambiguous and accurate detection of EVs through the time-lapsed tracking of EV release from MDA-MB-231 cells. Therefore, the newly developed CA800-EV dye provides a general and practical labeling strategy for EV-specific targeting and imaging in real-time.