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Page 6 Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26

thus, the relation between electrical stimulation of cells and EV uptake remains to be investigated.
Importantly, EV size distribution and zeta potential were unaffected by electrical stimulation of the
producer cells, as was the expression level of CD9, HSP70 and CD81, which may explain the unaltered
uptake of the EVs. The production of EVs is a process of relatively low yield and efforts to increase the yield
are fundamental to translating EVs to the clinic, e.g., through the use of bioreactors and stimuli that boost
[66]
release while maintaining consistent production of EVs with defined content .
Employing high-speed atomic force microscopy (HS-AFM), Sajidah et al. observed nanotopological
[67]
changes in sEVs caused by stress conditions . Low (4.0) and high (10.0) pH were compared to pH 7.5
(near physiological conditions), temperature was changed from 4 to 100 °C and compared to 37 °C, and
physiological buffer condition (50 mM Tris HCl with 150 mM NaCl) was compared to hypotonic and
hypertonic environments, i.e., 0.0 M NaCl and 1.8 M NaCl, respectively. Low pH did not affect the sEVs
morphology, which may have been expected because EVs originate from acidic MVBs. Hypotonic
conditions also showed preservation of the spherical morphology of the sEVs, whereas high temperature (60
and 100 °C), high pH (pH 10) and hypertonic conditions (1.8 M NaCl) were damaging to the structure of
the EVs. This may set limitations to EV isolation and sterilization procedures.

Intracellular trafficking of EVs
The mode of internalization of EVs has a great impact on their subsequent intracellular trafficking and fate.
EVs that are internalized via endocytosis may follow the canonical pathway through early endosomes, late
endosomes to (degradative) lysosomes. Phenotypic changes in recipient cells can be triggered through the
activation of cell signaling pathways upon binding of EVs to cells and/or the release of functional cargo into
the cell cytosol. Membrane fusion enables cargo release from EVs into the cytosol. This fusion process can
[58]
occur at the plasma membrane level and the endosome level, the latter being known as back
fusion [22,59,68-70] . Membrane fusion can be stimulated by the presence of fusogenic proteins in EVs. Syncytin-1,
syncytin-2, VSV-G and Hur proteins are known for their fusogenic activity [71-73] . Overexpression of Syn 1 in
EVs was shown to boost the uptake and cargo delivery of EVs in acceptor cells . Even at endogenous levels
[72]
of Syn 1, Uygur et al. showed that GFP gene transfer occurred between GFP-transduced and non-
transduced cells by means of Syn 1-mediated fusion of extracellular membrane vesicles (EMVs, i.e., EVs
plus retroviral particles) with cells . In contrast, Somiya et al reported that EV-mediated cargo delivery did
[74]
not occur unless the EVs were engineered to express the VSV-G fusion protein [75,76] . However, the EVs were
isolated by PEG precipitation and the presence of residual PEG in the EV formulation could have
influenced the fusion capacity of the EVs . When Zhang and Schekman investigated the intercellular
[77]
transfer mechanism of Cas9 protein and split GFP fragments, they showed that direct cell-cell contact was a
requirement for effective protein transfer between donor and acceptor cells. Specifically, syncytin-2-
dependent membrane fusion at the contact point between a microtube (2-4 µm diameter) on one cell and
the plasma membrane of another cell was necessary for the formation of an open-ended tubular connection
[73]
between the cells . Conversely, accumulating evidence supports protein-independent fusion of EVs with
biological membranes.

It has been suggested that proteins play a minor role in the fusion process, as paraformaldehyde treatment
of EVs (crosslinking of EV proteins) did not alter their fusogenic properties [22,78] . Still, membrane proteins
seem to be needed as structural elements contributing to membrane fusion, because lipid vesicles
mimicking cell membrane lipid composition or EVs solubilized with octylglucoside (removing proteins
from the membrane) abrogated fusion activities [58,79] . In addition, EV fusion increased for EVs that were
generated by cells grown under acidic conditions, which correlated with a decrease in membrane fluidity
due to a change in lipid composition. Specifically, an increase in sphingomyelin (SM), cholesterol, and GM3