Real-Time Luminescence Assay for Cytoplasmic Cargo Delivery of Extracellular Vesicles

Extracellular vesicles (EVs) have
been considered to deliver biological
cargos between cells and mediate intercellular communication and potential
drug delivery carriers. However, the mechanisms that underlie the
biological process of EV uptake and cytoplasmic cargo release in recipient
cells are largely unknown. Quantitative and real-time assays for the
assessment of cargo delivery efficiency inside recipient cells have
not been feasible. In this study, we developed an EV cargo delivery
(EVCD) assay using a split luciferase called a NanoBiT system. Recipient
cells expressing LgBiT, a large subunit of luciferase, emit luminescence
when EV cargo proteins fused with a small luminescence tag (HiBiT
tag) that can complement LgBiT are delivered to the cytoplasm of recipient
cells. Using the EVCD assay, the cargo delivery efficiency of EVs
could be quantitatively measured in real time. This assay was highly
sensitive in detecting a single event of cargo delivery per cell.
We found that modification of EVs with a virus-derived fusogenic protein
significantly enhanced the cytoplasmic cargo delivery; however, in
the absence of a fusogenic protein, the cargo delivery efficiency
of EVs was below the threshold of the assay. The EVCD assay could
assess the effect of entry inhibitors on EV cargo delivery. Furthermore,
using a luminescence microscope, the cytoplasmic cargo delivery of
EVs was directly visualized in living cells. This assay could reveal
the biological mechanism of the cargo delivery processes of EVs.