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Page 16 Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26

To demonstrate the presence of EV cargo in its lumen and to eliminate the possibility of its association on
the EV surface, controls such as nuclease- (DNase, RNase), proteinase K-, and lipase-treated EVs,
depending on the cargo under investigation, should be included. When studying the natural mechanisms of
EV-mediated cell-cell communication, a physiologically relevant amount of EVs must be used, as excessive
amounts of EVs may give rise to artifacts and non-physiological outcomes, including toxicity. This may also
apply to EVs being used as drug carriers. It is still a matter of debate whether the 10 μg of EVs (equivalent to
10 particles) used in many in vitro experiments is a physiologically relevant amount. The yield of EVs from
11
raw materials (culture supernatant or body fluid) is often quite low (0.5 μg of protein/mL of supernatant),
and thus extensive enrichment of EVs is typically performed . Also, multiple producer-recipient cell
[88]
combinations could be tested to show the generality of the method as well as the mechanism being put
forward.

It is of utmost importance to analyze the stoichiometry of the whole process: number of EVs added, number
of cargo molecules per EV, % of uptake, % of EVs that release cargo, the (approximate) number of cargo
molecules released, functional effect, etc. This would greatly help to reach a comprehensive understanding
of EV-mediated cargo delivery. For some cargoes, it is relatively easy to obtain these data, for example, for
mRNAs (by RT-qPCR), while it is more challenging for others, such as for miRNA due to, e.g., possible
interference from endogenous miRNAs.

The investigation of EV-mediated mRNA delivery may suffer from undesired co-loading of translated
protein into EVs. Also, mRNA-protein aggregates can form and co-precipitate with EVs and overestimate
the outcomes. This can give rise to misleading results by giving false positives. To confirm that the outcome
is from mRNA and not from proteins, EVs loaded with mRNAs can be added to recipient cells transfected
with siRNA against the reporter mRNA. If the protein quantity is reduced in these conditions, it is a good
indication that the outcome results from mRNA and not the protein. Similarly, cycloheximide, which stops
the de novo synthesis of proteins, can also be used in recipient cells to analyze the contribution of newly
synthesized proteins resulting from mRNA delivery. Another way to investigate the functional delivery of
mRNA rather than protein is to treat EVs with heat shock and methylene blue [117,131,132] which penetrates the
lumen and interferes with the RNA. Additionally, EVs can be treated with propidium monoazide
(PMA) [117,133] , which is an impermeant nucleic acid intercalating photoreactive dye that blocks RT-qPCR. If it
affects mRNA amplification, this indicates that the mRNA is present on the surface of EVs rather than in
the lumen. For miRNAs, nonendogenous miRNAs (viral miRNAs) may be considered as cargo to allow
[119]
separation of its effects from endogenous miRNAs . To quantify EV RNA delivery, one can radioactively
label EV RNA by incubation of EV producer cells with 3H-labeled uracil .
[134]

COMPARISON OF EVS WITH OTHER NANOPARTICLES
Do EVs share similarities with other nanoparticles, such as viral particles and synthetic gene delivery
vectors, in their uptake and cargo release dynamics? In an interesting study, Murphy et al. tackled this
question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in
terms of uptake and cargo (specifically gRNA) delivery . With this study, they addressed the issue that
[135]
EVs are known to contain very low amounts of miRNA, i.e., only 1 copy per hundreds or thousands of
EVs [135,136] , making the scientific community question whether EVs can bring about a real change in cell
phenotype, despite several reports showing EV-mediated changes in the phenotype of recipient cells ,
[1-3]
hinting at high efficiency of cargo release by EVs. Indeed, Murphy et al. showed that when the particle-to-
gRNA ratio is kept the same, EVs are more efficient in imparting a functional effect, indicating successful
cargo delivery, than LNPs. In fact, LNPs containing less than 1 pM gRNA did not result in any functional
effect, while EVs containing 0.1-2.5 fM gRNA did. The authors concluded that EV-mediated gRNA delivery