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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 13

terminal nuclear localization signal (NLS) and a C-terminal tobacco etch virus (TEV) protease-specific
cleavage site (TCS). In cells that co-expressed NLS-GFP-TCS-CD63 and split TEV protease, ILVs were
formed that contained NLS-GFP-TCS in their lumen, which upon back fusion, would become exposed to
the cytosol. Upon the addition of a protease dimerizer, TEV protease in the cytosol was activated, resulting
in the cleavage and release of NLS-GFP from cytosolically exposed NLS-GFP-TCS-CD63. Subsequent
nuclear accumulation of NLS-GFP served as a measure for ILV back fusion with the MVB limiting
membrane. In this way, the authors showed that ~30% of ILVs underwent back fusion. Interestingly, Joshi et
[22]
al. showed a similar extent of back fusion (~24%) for sEVs internalized by recipient cells . How back
fusion of ILVs and sEVs is controlled is a central question that remains to be answered, as was discussed
above.

Interestingly, it was reported that a maximum of a third of sEVs are generated from dynamic ILVs (i.e.,
backfused and then regenerated into ILVs) while the majority come from inert ILVs (i.e., that have not
[87]
undergone back-fusion and new formation in MVBs) . Do these dynamic ILVs, after their secretion and
subsequent uptake by recipient cells, represent the population of EVs that undergoes back-fusion?
Alternatively, there may exist MVBs that permit back-fusion, while other MVBs are non-permissive.
Currently, we do not know if inert ILVs and dynamic ILVs arise from the same MVB or distinct MVBs. It is
not unimaginable to think that there is a decision-making process in MVBs directing different populations
over different trajectories. It is important to find the answers to these questions in order to elucidate the
mechanisms behind ILV (re)formation and ILV/EV back-fusion to improve our understanding and the
translation of EV-mediated cargo delivery.

Techniques to study endosomal permeabilization
Galectin-based assay
Galectins, including galectin (GAL) 1, GAL3, GAL4, GAL8 and GAL9, are beta-galactoside-binding
proteins that are employed as markers for endosomal and lysosomal damage. Because beta-galactosides are
exclusively present within the endo/lysosomal lumen, the expression of fluorescent fusion proteins of
galectins in the cell cytosol will result in the formation of fluorescent punctae upon galectin accumulation in
permeabilized endosomes . GAL8 and GAL9 were shown as the more sensitive markers for endosomal
[123]
[123]
permeabilization compared to GAL1, GAL3 and GAL4 for lipoplexes , lipid nanoparticles [123,124] and
cholesterol-conjugated siRNA .
[125]
In HEK293T cells genetically engineered to express monomeric azami green-tagged GAL3 (mAG-GAL3),
endosomal permeabilization was studied upon incubation with sEVs, but no endosomal permeabilization
was detected . Immuno-labeling against GAL3 in HeLa cells exposed to sEVs similarly did not reveal
[22]
GAL3 accumulation in EV-containing endosomes . These results suggest that EVs release their cargo from
[59]
endosomes in a non-destructive manner, which fits with the involvement of back fusion in EV cargo
release.

All methods discussed above mainly focus on the direct visualization of membrane fusion or cargo release
[Table 2], which is undoubtedly important to understand the underlying compartments and processes in
EV cargo release. However, verifying the functionality of these cargoes is indispensable as that is the final
outcome of interest. De Jong et al. developed the CROSS-FIRE system to detect EV-mediated RNA
delivery . Using the CROSS-FIRE system, they revealed important molecular determinants of the process
[126]
of EV-mediated RNA delivery. Rho GTPases Rac1 and RhoA, PAK1, Cav1, ITGB1, Rab5 and Rab7, and
ROCK1 were found to be important for functional RNA delivery. Unfortunately, the cellular uptake nor
endosomal escape was measured, making it impossible to decide at which step in the delivery process the