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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 17

was at least 2-fold more efficient than delivery mediated by LNPs. Put the other way around, the
development of a synthetic gene delivery vector, i.e., LNPs, with an endosomal escape capacity almost equal
to that of a natural delivery vector, i.e., EVs, can be considered a big achievement.

Studies suggested that EVs or EV-like liposomes are more efficiently taken up by cells than LNPs [137,138] .
About 10% of EVs were taken up in 2 h and 26%-32% in 24 h; in contrast, 0.6% of LNPs were taken up in 2 h
and 1% in 24 h. Thus, it appears that the uptake, as well as the efficiency, of cargo delivery of EVs, is
generally higher than that of artificial delivery vehicles such as LNPs. About only 1%-2% of endocytosed
[139]
LNPs undergo endosomal escape to release their cargo , and this is the rate-limiting step for their efficacy.
EVs similarly use endocytosis for uptake and cargo release efficiency turns out to be ~25% , an order of
[22]
magnitude higher than with LNPs.

An important point for consideration is the amount of EVs that is added to recipient cells in order to induce
a phenotypic effect. For example, multiple doses of EVs were required in the CROSS-FIRE system to bring
about the shift from RFP to GFP expression [126,140] . And even then, only a small percentage of cells showed
reporter gene (GFP) expression. Do these large amounts of EVs represent the physiological situation and is
this question even relevant? We believe the question is relevant if the physiological roles of EVs, e.g., their
role in metastasis , are under investigation, but may be less relevant when EVs are exploited for drug
[52]
delivery purposes, provided that the large amounts of EVs do not induce toxic side effects.

Overall, the downside of using assays that depend on the downstream effects of the cargo is the fact that the
measured functional effect does not necessarily reflect the efficiency of cargo delivery, but may be
influenced by other factors. This can be illustrated by an example from the gene delivery field: endosomal
escape of pDNA into the cytosol does not necessarily result in the expression of the DNA if it is not
successfully transferred to the cell nucleus, for transcription to take place. Therefore, the transfection
efficiency does not necessarily reflect the efficiency of DNA delivery into the cytosol. Likewise, high cellular
uptake of gene delivery vectors does not necessarily correlate with high transfection efficiency, because the
endosomal escape of the genetic cargo also needs to be efficient . Therefore, it is important to analyze the
[109]
cellular uptake of nanoparticles, the intracellular release of the cargo, and the instigation of a functional
effect altogether. The combined quantitative assessment of the different steps in EV cargo trafficking and
[118]
functional effects is key in the elucidation of the spatiotemporal dynamics of EVs in cells .

Various factors can impact the EV uptake and cargo delivery process. Upstream (donor/producer cell type,
cargo expression and loading efficiency, isolation procedure, etc.) as well as downstream factors (acceptor/
recipient cell type, microenvironment, etc.) play a role in deciding the fate of EVs in recipient cells. Thus,
there is a growing need for a golden standard assay to analyze and quantify EV-mediated cargo delivery and
its functional outcome. Ideally, such an assay should be simple, sensitive, quantitative, and scalable.

SUMMARY AND OUTLOOK
EVs display a natural homing/targeting capacity together with the potential to deliver cargo molecules into
cells, thereby providing a unique platform for the delivery of therapeutic agents to even challenging-to-
reach organs like the brain . However, the composition of EVs is heterogeneous, which can impact their
[141]
overall functionality as well as trigger undesired side effects. Additionally, although progress has been made,
a significant obstacle to clinical applications of EVs is the challenge of obtaining high quantities of EVs at a
reasonable cost and with consistency between batches. The development of universal or patient-matched
EV donor cell lines could be helpful, considering the presence of Human Leukocyte Antigens (HLAs) on
[142]
EVs . A thorough and standardized isolation and characterization procedure is essential to the