New Lipophilic Fluorescent Dyes for Labeling Extracellular Vesicles: Characterization and Monitoring of Cellular Uptake.

PKH dyes, which are currently the most widely used fluorescent probes for extracellular vesicle (EV) labeling, have some limitations. For example, these dyes tend to aggregate, leading to formation of EV-like nanoparticles that can be taken up by cells. Moreover, it has been suggested that PKH dyes trigger an enlargement of EVs because of membrane fusion or intercalation. To overcome these limitations, we developed three novel extracellular vesicular-membrane-binding fluorescent probes-Mem dye-Green, Mem dye-Red, and Mem dye-Deep Red-for monitoring EV uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold and amphiphilic moieties on the cyanine. The three dyes have different photophysical characteristics. To investigate the characteristics of the Mem dyes for EV labeling, we performed nanoparticle tracking, zeta potential measurements, and confocal microscopy. The dyes enable highly sensitive fluorescence imaging of EVs. They can also be used to observe EV dynamics in live cells. The Mem dyes show excellent EV labeling with no aggregation and less particle enlargement.