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Page 10 Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26

glioma

difficult to discern due to the resolution limit of conventional light microscopes. Toribio et al. described a luciferase/GFP-based assay that is sensitive enough

[70]
to quantify and trace EV uptake at early stages employing a pair of chimeric reporter proteins, Dual Split 1 (DSP1) and Dual Split 2 (DSP2) . DSP1 was fused
to the N-terminus of CD9 and co-expressed with DSP2 in EV producer cells. Recipient cells were preloaded with luciferase substrate and incubated with
DSP1-CD9/DSP2 EVs, i.e., carrying luciferase activity. Upon their internalization, EVs get exposed to the luciferase substrate, which will be converted,

resulting in a luminescent signal. The subcellular localization of EVs was detected by the GFP fluorescence signal. Finally, labeling the content of the EVs, e.g.,
[25]
mRNA, is also an option .

Inhibition or competition assays can be employed to characterize specific uptake routes or (receptor-mediated) endocytosis pathways [Table 1]. Examples of
classical pharmacological inhibitors of endocytosis are dynasore (dynamin-dependent endocytosis), amiloride (macropinocytosis); genistein (caveolae-

dependent/clathrin-independent endocytosis); chlorpromazine and Pitstop 2 (clathrin-mediated endocytosis); cytochalasin D (actin-dependent endocytosis)
and bafilomycin A1 (endosomal acidification) [24,25,39,59] . Importantly, pharmacological inhibitors of endocytosis are non-specific and warrant the use of

alternative approaches to study nanoparticle/EV endocytosis. Moreover, investigation of the mechanisms of nanoparticle internalization has revealed that
inhibition of specific endocytic pathways may result in the upregulation of other pathways, and that not all uptake pathways result in functional cargo
delivery [100,109-112] . Therefore, adding a functional experiment to confirm that a proposed mechanism is involved in enhancing or abrogating the functional

outcome is highly recommended.

The cellular uptake of EVs can be deduced from observing a functional effect, i.e., a specific response of cells upon their exposure to EVs, which is associated
with the EVs content. For example, EVs loaded with siRNA may trigger the degradation of a specific mRNA which can be examined by quantifying the mRNA
or protein level . Recipient cells that are transfected with reporter genes coding for fluorescent or bioluminescent proteins can be used to quantify EV-
[113]
mediated siRNA silencing efficacy by means of fluorescence detection.

INTRACELLULAR DELIVERY OF EV CARGO

Functional delivery by EVs is determined by their ability to release the cargo contained in them into the recipient cell. Various aspects need to come together
to achieve functional EV-mediated delivery: cellular uptake; intracellular trafficking; cargo release; functional outcome. While the uptake of EVs is heavily
researched, we are just beginning to understand the molecular basis of intracellular trafficking and cargo release by EVs.

After uptake, EVs generally end up in endosomes [22,64] , although uptake via plasma membrane fusion is also a possibility, which will result in immediate
[58]
release of EV content into the cell cytosol. Early endosomes are known to mature into late endosomes that ultimately fuse with lysosomes, although endosomal
sorting can also result in recycling of endocytic cargo. Such recycling or re-release has been described for EVs . The induction of phenotypic changes in
[114]