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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 3

EV INTERNALIZATION AND INTRACELLULAR TRAFFICKING - BREAKING THROUGH
MEMBRANE BARRIERS
EV-cell membrane interactions
The mechanism of internalization of EVs by recipient cells is the first essential step that defines their
intracellular route, intracellular fate and functionality. Besides fusion with the plasma membrane,
endocytosis is most often described as the mechanism of uptake of EVs [22,23] , including clathrin-mediated
endocytosis, phagocytosis, macropinocytosis and lipid raft-mediated endocytosis, including caveolae-
mediated endocytosis [6,24-33] . Interestingly, molecular determinants of other lipid raft-dependent endocytic
pathways, specifically flotillin, ARF6 and RhoA, were reported to play a role in EV biogenesis/release [11,34,35] .
Similar routes of internalization have been observed for a.o. viruses and synthetic nanoparticles and are
known to be cell type-dependent as well as dependent on nanoparticle characteristics [36-39] .

EV binding to recipient cells
How EVs interact with the cell surface and the route that EVs follow after subsequent internalization will
depend on properties and conditions such as size, composition, cell source, ligands, environmental pH,
presence of serum, and even isolation method [1,20,40-42] A size-dependent internalization of nanoparticles via
[43]
distinct endocytic routes was shown for nanoparticles with fixed sizes . Obviously, because of the
heterogeneity in the size of EV populations, such a relationship is difficult to detect for EVs, further
complicated by a probable heterogeneity in (surface) composition of EVs of different sizes. Using targeted
gold nanoparticles of different sizes, it was shown that small particles (15-30 nm) have a higher cell binding
[44]
probability than larger particles (90-150 nm), but result in a lower amount of mass bound per cell .
However, also here caution is needed, as the ligand density on the gold nanoparticles of different sizes may
differ. Overall, varying one parameter, e.g., size, while keeping all other parameters, including ligand
density, constant is a major challenge, also for synthetic nanoparticles.

The binding of EVs to recipient cells is a prerequisite for EV endocytosis. One class of proteins that
facilitates this interaction is tetraspanins, a class of membrane-spanning proteins. Tetraspanins are
commonly found in EVs and play a role in EV biogenesis, cargo sorting, cellular uptake, and functionality.
While tetraspanins are typically enriched within exosomes, other subpopulations of EVs may also contain
tetraspanins. CD9, CD63, and CD81 are examples of tetraspanins that are associated with EV-cell
interaction [45,46] . For example, researchers have shown that blocking CD9 can reduce the uptake of cancer
cell fibroblast (CAF)-derived EVs by pancreatic cancer cells, cancer cell migration, and epithelial-to-
mesenchymal transition (EMT). This finding reveals an important role for EVs in the interaction of the
[1]
tumor with the tumor microenvironment and its aggressiveness .

In contrast, a low CD9 expression on sEVs produced by colorectal cancer cells was shown to promote their
[47]
uptake , which could be explained by the inactivating effect of CD9 on ADAM17-mediated adhesion of
sEVs with α5β1 integrin on recipient cells. Tetraspanins are known to interact with lipids and other integral
membrane proteins, including integrins, forming so-called tetraspanin-enriched microdomains (TEMs).
TEMs play a role in the regulation of adhesion strength, and viruses have been shown to enter cells through
TEMs, with or without direct binding to tetraspanins . Interestingly, CD9 can dictate the mechanism of
[48]
coronavirus (CoV) entry. Specifically, CoV particles entered via fusion with the plasma membrane in the
presence of CD9, whereas in its absence, viral particles were taken up via endocytosis, followed by fusion
with the endosomal membrane . In a recent paper, CD63 and CD9 knockdown in producer cells and
[49]
recipient cells was used to indicate that these tetraspanins were not required for EV uptake and content
delivery . Unfortunately, the mechanism of uptake, i.e., whether this occurred via plasma membrane
[50]
fusion or endocytosis, was not evaluated. Moreover, downregulation of the expression of a specific protein