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Page 12 Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26

producer cells. The tagging of GFP to CD63 made sure that the GFP signal would not be diluted in the
cytosol upon EV back fusion with the endosomal membrane. Further, recipient cells were genetically
engineered to express anti-GFP fluobody in the cytosol. Upon incubation of fluobody-expressing cells with
GFP-CD63 EVs, back fusion of EVs with the endosome led to GFP exposure to the cytosol, resulting in
anti-GFP fluobody binding at the endosomal membrane and formation of fluobody punctae. This assay
allowed for quantification of the number of EV-containing endosomes (GFP-positive) as well as the number
of endosomes exposing EV cargo to the cell cytosol (GFP-mCherry double positive). Subsequent
investigation of the fluobody punctae using CLEM revealed endosomes as the underlying cellular structures,
providing direct evidence for EV cargo release from endosomes. Cargo release from endosomal structures
was confirmed in another study by following quantum dot-labeled EVs carrying fluorescent miRNA in
[118]
recipient cells containing fluorescently labeled endosomes through single particle tracking .
Protease-based assays
Albanese et al. developed an assay to determine EV cargo delivery based on β-lactamase-mediated cleavage
of the FRET substrate CCF4 . EVs were generated by cells expressing CD63- β -lactamase and incubated
[119]
with recipient cells that were loaded with CCF4. Cleavage of CCF4 by β-lactamase was detected by a shift in
fluorescence emission from 520 nm to 447 nm, indicating the exposure of β-lactamase from EVs to the cell
cytosol. However, EV cargo release was not detected unless the EVs were surface functionalized with VSV-
G through co-expression of CD63- β-lactamase and VSV-G in EV producer cells.

A couple of reports [59,114] have shown the use of luciferase enzymes for the detection of EV cargo delivery,
[120]
especially NanoLuc , which gives a very high signal-to-noise ratio. The general idea is that a NanoLuc
protein is loaded in EVs by overexpression in donor cells, and its delivery in recipient cells is quantified as
luciferase activity upon the addition of a luciferase substrate. It can also be used in a converse strategy where
recipient cells express luciferase and functional delivery of anti-luciferase miRNAs via EVs can be detected
from a reduction in luciferase activity .
[119]
Somiya et al. developed the EV-mediated tetraspanin-tetracycline transactivator (tTA) delivery (ETTD)
assay [75,76,121] , in which a tetracycline-controlled transcription factor is delivered by EVs, which upregulates
the expression of a TRE (Tetracycline responsive element)-controlled reporter gene (luciferase or
fluorescence protein) in the recipient cells. Specifically, the tetraspanins CD9, CD63, or CD81 were fused at
the C-terminal with the TEVp cleavage site, followed by tetracycline transactivator (tTA). When the EV
membrane fuses with the endosome, the lumenal tTA is exposed to the cytosol, cleaved in the presence of
TEVp expressed in the cytosol, and releases the transcription activator tTA. The cytoplasmic release of tTA
leads to the tTA induction of the reporter gene expression under the TRE promoter.

Split protease-based assays
In the EV cargo delivery (EVCD) assay [75,76,121] , a split luciferase is used [120,122] . A small fragment of NanoLuc
(HiBiT) is fused to EV tetraspanins, while the large subunit of NanoLuc (LgBiT) is expressed in recipient
cells. When the HiBiT-tagged tetraspanins are exposed to the cytosol of recipient cells, the luciferase
fragments combine and emit luminescence signals when in the presence of a substrate. Because NanoLuc
produces much brighter luminescence than conventional luciferases such as firefly or Renilla luciferases,
NanoLuc-based assays are sensitive enough to detect the rare event of EV cargo delivery.

Perrin et al. used an interesting split protease-based method to shed light on the process of back fusion of
ILVs in which ILVs were shown to fuse back with the limiting membrane (LM) of the MVB . They
[87]
engineered an NLS-GFP-TCS-CD63 fusion protein consisting of CD63 tagged with GFP containing an N-