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

may result in compensatory expression of other proteins, which may take over (part of) its function.

Besides tetraspanins, also integrins are membrane-bound proteins found in EVs that mediate their
interaction with cells. Integrins are adhesion molecules that mediate cell-cell adhesion and cell adhesion to
[51]
the extracellular matrix . Likewise, in EVs, the integrins support adhesion to the extracellular matrix and
recipient cells, providing the means for targeting. Vice versa, inhibition of integrin activity can be used to
[52]
reduce the uptake of EVs by recipient cells . For example, disintegrin inhibitor (DisBa-01) that inhibits αvß
3 integrins, YIGSR peptide that inhibits β1-containing integrins, and RGD peptides that inhibit RGD-
binding integrins such as αvβ3, αvβ5, αvβ6, αvβ1, αvβ8, α5β1, αIIbβ3, and α8β1, have been shown to reduce
the uptake of EVs by specific cell types [53-56] .

Thirdly, the glycan composition of EVs mediates cell interaction and may confer cell specificity. sEVs
isolated from adipose-derived stem cells (ADSC-sEVs) were deglycosylated by means of α2-3,6,8
neuraminidase, ß1-4 galactosidase, ß-N-acetylglucosaminidase or a combination of those enzymes .
[42]
Cellular uptake of the ADSC-EVs by HeLa cancer cells was reduced for deglycosylated EVs, but when the
EVs were exposed to non-malignant human lung fibroblasts, deglycosylated EVs showed higher uptake
than the untreated control. Glycan engineering of EVs with sialyl Lewis X and Lewis X ligands increased
cellular specificity for endothelial and dendritic cells, respectively . These results reinforce that EV glycan
[57]
profile, including glycoproteins and glycolipids, has an impact on the interaction of EVs with cells.

It is important to notice that an increase in the intracellular accumulation of EVs is generally referred to as
an increase in "uptake". However, enhanced accumulation of EVs does not necessarily mean that there was
an increase in internalization of EVs. Equally possible is their entrapment within the cell due to enhanced
evasion of degradative and/or recycling pathways [Figure 1].

Environmental factors influencing the cellular secretion and uptake of EVs
Environmental factors can affect EV biogenesis, release, and cellular uptake. Cancer cells are shown to
secrete large amounts of EVs that reflect characteristic stress-related phenotypic changes in tumor cells
induced by, e.g., acidic pH and hypoxia, i.e., hallmarks of the tumor microenvironment (TME).
Interestingly, the number of secreted EVs at acidic pH was shown to be reduced even though the protein
concentration was increased compared to pH 7.4 conditions [41,58] . Therefore, when determining EV
amounts, it is important to report not only EV protein content, but also EV numbers. Environmental pH
influences not only the production of EVs, but also EV uptake. Intriguingly, EVs secreted at low pH (pH 5)
were more efficiently internalized than EVs secreted at pH 7 (both at environmental pH 5 and 7) when in
the absence of serum, but the opposite behavior occurred in the presence of serum . Overall, spontaneous
[41]
uptake of EVs occurs at a low rate of about ~1% at 1 h . However, EVs intrinsic targeting to specific cell
[59]
types offers an advantage that can be exploited for the delivery of therapeutics . Natural or engineered
[60]
surface ligands are crucial determinants of the uptake and uptake rate of EVs. Under hypoxic conditions,
endothelial cells exhibited a higher uptake of EVs compared to normoxic conditions. Additionally, changes
were observed in both the quantity and composition of EVs that were secreted under hypoxic
conditions [61-64] . Interestingly, Cerezo-Magaña et al. observed a correlation between hypoxia-stimulated EV
uptake and increased heparan sulfate proteoglycan (HSPG) endocytosis via lipid raft-mediated
endocytosis . The examples given above illustrate the interplay between environmental factors (e.g.,
[64]
presence of serum, pH, hypoxia), EV release and EV uptake.

In addition, non-physiological stimuli can be used to enhance EV secretion. Low electricity levels applied to
murine melanoma B16F1 and murine fibroblast 3T3 Swiss Albino cells increased EV secretion, while it did