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Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26 Page 11

recipient cells by EVs provides indirect evidence for the functional delivery of their cargo, evading
degradation within lysosomes. Multiple studies have shown that EVs are able to escape endosomes by
membrane fusion with the endosomes and this seems to be the primary pathway of EV cargo release [68,75,76] .
Contrastingly, some studies point to a lack of cargo release due to the failure of EVs to undergo endosomal
escape in their native form, which can be overcome through EV surface functionalization with a fusogen
such as VSV-G [75,76] . In addition, alternate pathways have been suggested, including cargo release through
endosomal permeabilization and nuclear import of EV cargo from Rab7+ endosomes through nuclear
pores [46,115] . Taken together, EV uptake, recycling, and degradation determine the extent of intracellular EV
accumulation, while EV fusion with the plasma membrane or endosomal membrane, or endosomal rupture
determines functional cargo delivery into the cell cytosol, of which back fusion with the endosomal
membrane has been most widely described [Figure 1].

In the following section, we have listed the hitherto reported techniques to shed light on EV cargo release
pathways. Thereafter, we discuss in detail various factors impacting the cargo delivery process. As the pool
of methods for cargo delivery identification is still growing, we also lay out controls to be used in cargo
delivery studies for reducing false positives and attaining valid conclusions.

Techniques to study EV-membrane fusion and cargo delivery
Lipid mixing-based assays
The introduction of fluorescent probes, e.g., octadecyl Rhodamine B chloride (R18), in (artificial)
[116]
membranes at self-quenching concentrations can be used to measure lipid mixing/membrane fusion .
Costrafeda et al. used liposomes containing equal amounts of phosphatidylserine (PS), phosphatidylcholine
[117]
(PC), and cholesterol labeled with R18 (Chol-R18) to interact with EVs . Chol-R18 dequenching upon its
dilution as caused by the fusion of the PS:PC:Chol-R18 liposomes with EV membranes was visible as an
increase in fluorescence. Similarly, Parolini et al. incubated cells with R18-labeled EVs to monitor an
[58]
increase in fluorescence intensity as a measure for the fusion of the EVs with cellular membranes .

Morandi et al. used a FRET-based lipid mixing assay together with cryogenic transmission electron
microscopy (cryo-TEM) and electron cryotomography (cryo-ET) to provide important insights into the
membrane fusion process between EVs and artificial membranes . They probed the interaction of EVs that
[68]
were labeled with the FRET pair DiI and DiD, with unlabeled liposomes. In intact EVs, DiI excitation
results in DiD fluorescence emission, because of the close apposition of both fluorophores and the overlap
between DiI emission wavelength and DiD excitation wavelength. In the case of fusion between EVs and
liposomes, the distance between the FRET pair increases, resulting in an increase in the donor fluorescence
intensity (and a concomitant decrease in acceptor fluorescence intensity). Cryo-TEM and cryo-ET revealed
EV hemifusion intermediates and internal content mixing, respectively, which correlated with the
membrane mixing data obtained with FRET.

However, the use of liposomes as cell mimics precludes the identification of the cellular location of cargo
release, and in case cells are used, live cell imaging is required to visualize the site of membrane fusion,
because of the temporary nature of a change in fluorescence intensity.

Nanobody-based assay
A pioneering technique to quantify EV-mediated cargo delivery that also pinpointed the subcellular site of
EV cargo release was introduced by Joshi et al. . Their assay used a combination of GFP-loaded EVs,
[22]
mCherry-labeled anti-GFP nanobody-expressing recipient cells, and correlative light and electron
microscopy (CLEM). Specifically, GFP was loaded inside EVs through the expression of GFP-CD63 in EV