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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.

The lack of techniques to trace brain cell behavior in vivo hampers the ability to monitor status of cells in a living brain. Extracellular vesicles (EVs), nanosized membrane-surrounded vesicles, released by virtually all brain cells might be able to report their status in easily accessible biofluids, such as blood. EVs communicate among tissues using lipids, saccharides, proteins, and nucleic acid cargo that reflect the state and composition of their source cells. Currently, identifying the origin of brain-derived EVs has been challenging, as they consist of a rare population diluted in an overwhelming number of blood and peripheral tissue-derived EVs. Here, we developed a sensitive platform to select out pre-labelled brain-derived EVs in blood as a platform to study the molecular fingerprints of brain cells. This proof-of-principle study used a transducible construct tagging tetraspanin (TSN) CD63, a membrane-spanning hallmark of EVs equipped with affinity, bioluminescent, and fluorescent tags to increase detection sensitivity and robustness in capture of EVs secreted from pre-labelled cells into biofluids. Our platform enables unprecedented efficient isolation of neural EVs from the blood. These EVs derived from pre-labelled mouse brain cells or engrafted human neuronal progenitor cells (hNPCs) were submitted to multiplex analyses, including transcript and protein levels, in compliance with the multibiomolecule EV carriers. Overall, our novel strategy to track brain-derived EVs in a complex biofluid opens up new avenues to study EVs released from pre-labelled cells in near and distal compartments into the biofluid source.