Uptake, functionality, and re-release of extracellular vesicle-encapsulated cargo

Extracellular vesicles (EVs) are membrane-encapsulated particles that carry genetically active and protein/lipid cargo that can affect the function of the recipient cell. A number of studies have described the effect of these vesicles on recipient cells and demonstrated their promise as therapeutic delivery vectors. Here we demonstrate functional delivery of EV-encapsulated RNA and protein cargo through use of luminescence and fluorescence reporters by combining organelle-targeted nanoluciferase with fluorescent proteins. We highlight a mechanism by which cells retain internalized cargo in the endosomal compartment for days, usually leading to content degradation. We also identify a mode through which recipient cells re-release internalized EVs intact after uptake. Highlighting these different fates of EVs in recipient cells sheds light on critical factors in steering functional cargo delivery and will ultimately allow more efficient use of EVs for therapeutic purposes.