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Page 8 Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26

However, as EVs (ILVs) are generated in an acidic milieu already, there must be a mechanism to activate
the EV fusogen after the EVs are released and taken back into cells. Intriguingly, when EVs are treated with
acidic pH, neutralized with buffer, and allowed to fuse with endosomes at acidic pH, they readily fuse with
the endosomal membranes with comparable efficiency as non-treated EVs. This suggests that these putative
EV fusogens indeed can get reversibly activated [22,87] . Thus, EVs present a paradoxical scenario: ILVs are
formed in an acidic environment, i.e., in MVBs, without undergoing massive back-fusion, whereas
following the endocytosis of EVs, cargo release by means of back-fusion occurs upon endosomal
acidification. This raises the question of how fusion activation at low pH is compatible with ILV biogenesis
in MVBs [68,93,94] . Moreover, the occurrence of back fusion of ILVs has been reported , suggesting that a
[87]
general block of ILV back fusion does not exist. We hypothesize that the fusion propensity of ILVs/EVs
with endosomal membranes is controlled by physicochemical properties, e.g., macromolecular crowding or
liquid-liquid phase separation [94-97] . Alternatively, the non-fusogenic ILVs in MVBs may represent the
population of ILVs that was generated in early endosomes and lacks LBPA. Similarly, the absence of LBPA
in early endosomes was held responsible for the absence of release of genetic cargo from early endosomes,
[98]
as observed in non-viral gene delivery using antisense oligonucleotides and polyplexes [99,100] .

Likely, fusogenicity is context-dependent and will vary among and within EV subpopulations, while being
affected by the source cells and their state. This may partially explain the differential impacts of EVs from
different sources under different conditions. For example, there are differences in EV behavior between
different donor-recipient cell combinations, underscoring the possibility that different cells may use
different pathways for EV endocytosis and may differently regulate EV cargo release.

Next to inducing escape from endosomes, EVs can avoid lysosomal degradation through recycling
pathways. Rab11 is a marker of recycling endosomes and has been implicated in the tethering and
homotypic fusion of vesicles [101,102] . Together with other Rab GTPases, e.g., Rab27 and Rab35, Rab11
promotes EV sorting and recycling [103,104] .

Assays for detection and quantification of EV internalization and intracellular trafficking
Analytical methods to determine the internalization mechanism of EVs, their subsequent intracellular
trafficking, as well as cargo release are essential to further improve our understanding of how EVs interact
with in vitro and in vivo systems [Table 1]. Fluorescently labeled EVs can be directly visualized in cells,
while enzyme labeling allows for their detection through substrate conversion.

Detection of EVs can be obtained by labeling the EVs employing fluorescent dyes such as DiI, DiO, PHK26
and PHK76 membrane dyes, and CFDA and calcein AM membrane-permeable dyes [25,56,61,105-107] . Although
easy to use, labeling of EVs with fluorescent dyes has its downsides. EV membrane labeling with lipophilic
dyes may change EV characteristics, influencing their behavior, and may suffer from exchanges with other
biological membranes, causing faulty identification of EV localization in cells . To ensure that the
[108]
fluorescence signal accurately reflects the localization of EVs, a non-exchangeable dye should be used and
free dye should be used as a control in the experiments. Finally, it should be verified if labeled and unlabeled
EVs show the same functional effect in recipient cells to exclude a possible effect of EV labeling on EV
function.

Alternatively, reporter proteins, for example, GFP, YFP, mCherry fluorescent protein, or nanoluciferase and
Gaussia luciferase bioluminescent proteins, are used for EV labeling [22,25,59,72] . Fluorescence or
bioluminescence detection using microscopy, flow cytometry and spectroscopy are then employed to
determine EV uptake by cells, although early stages in EV uptake, i.e., binding versus internalization, are