Extracellular vesicles (EVs) are 50–1000 nm vesicles secreted by virtually any cell type in the body. They are expected to transfer information from one cell or tissue to another in a short- or long-distance way. RNA naturally present in EVs might be limited in a physiological context.
|Abels et al.
|Glioblastoma (GBM)||Short||Transfer of GBM EVs in 0.3% of microglial cells, presence of a GBM-miRNA in these cells||Partial (4/59) miRNA target induced silencing|
|Injection of GBM EVs induce partial siRNA target knockdown||Highly supra-physiologic EV dose|
|Lucero et al.
|Glioblastoma (GBM)||Short||GBM EVs induce angiogenesis in vitro and a transcriptomic fingerprint is described||Supra physiologic dose (105 EVs/cell)|
|The transcriptomic fingerprint is found is also found in patients||There is only a correlation between EV treated GBM cells and patient GBM, no demonstration of causality is proposed|
|Shen et al.
|Tumor derived EVs||Short||Tumor derived EVs induce stemness in vitro||Supra physiologic dose (25:1 producing to receptor ratio)|
|Limiting EV transfer in vivo diminish the effect on surrounding cells||KO of EV production in vivo is performed using a Rab-7 KO tumor model, this KO has a lot of other effects that may explain the difference observed|
|Ying et al.
|Glucose tolerance||Potentially both||miRNA is transferred from hematopoietic derived cells to liver cells in vivo||The transfer may be mediated by either EVs or Tunelling nanotubes (TNT) (and other?) mechanism, this miRNA being known to be transferred via TNT|
|Chen et al.
|Bone regeneration||Potentially both||MiR-375 is able to induce bone regeneration in vitro||No significant difference is observed compared to EVs not expressing miR-375 in vivo|
|Thomou et al.
|Transfer of miRNA from adipose tissue to liver||Potentially both||A serum-derived EV preparation transfers active miRNA to liver cells in vivo||The serum derived EV preparation purification protocol has a high chance to be comtaminated by extravesicular miRNA (up to 97.5% of miRNA purified)|
|CRE-mRNA transfer in vivo||Potentially both||CRE recombination is induced at long distance in the presence of EVs derived from cells expression CRE mRNA and protein||The CRE-Lox induced recombination may be mediated either by mRNA transfer via EVs but also or by transfer of mRNA or CRE protein by TNT, cell fusion, or extravesicular transfer|
|A particular phenotype is described in CRE-recombined cells compared to non recombined cells||The causality in not demonstrated as a cell with a particular phenotype may be more prone to be transfected by CRE, in particular a more mobile and phagocytic cell.
The sole endocytosis of nano-objects like EVs is also impacting the cell phenotype, even in the absence of cargo.
Sverdlov claimed that it is very unlikely that naturally circulating EVs transfer a significant part of information through RNA in vivo at long distances in physiological states . He argued that the best candidates for information transfer would be self-amplifying (e.g., mRNA) and/or have a regulatory function (e.g., a transcription factor, a miRNA). At the time, he made the hypothesis that RNA inside EVs was not subject to strong selection. Baglio et al.  and other groups found that most RNA in various types of EVs (from tumor, MSCs, immune cells and serum, isolated by various methods (ultra)-centrifugation or affinity column) were small <400 nucleotides (nt) long RNA . Among them, most are tRNAs (that can hardly be expected to have an effect) and miRNA only constituted ∼0.9%  of RNA reads. Although the miRNA are relatively enriched (∼10 fold compared to cell RNA4), enrichment may largely be due to the nonspecific size selection biased to the smaller sizes such as tRNAs. As an example, 16 S RNA (1,6 kB), a typical medium-size RNA has a hydrodynamic diameter of ∼30 nm , whereas miRNA (20–83 nts) have a cylinder shape with a 2 nm diameter and a 7–20 nm length. mRNA encapsulation inside EVs also depends on their local concentration around EV formation sites, as well as mRNA interaction with membrane lipids and proteins . Before being functional, miRNA are getting through the pri- and pre-miRNA state. To be potentially active if they get to the target cell cytosol, miRNA needs either (i) to be not yet associated with Ago2 to form the RISC complex but still able to bind to it (i.e., being pri- or pre-miRNA) and therefore they would be able to bind it later on in the recipient cell cytosol or (ii) to already be associated with the RISC complex as a miRNA, a state in which they can exert their silencing activity directly. Importantly, association of miRNA to the RISC complex allows them to be much more stable than if left alone where it can be rapidly degraded by nuclease, in particular in the context of EV travel through endosomes (containing nucleases) in the target cell.
Sverdlov proposed a rough approximation of the maximal amount of RNA per EV if they are densely packed in EV of 100 nm diameter: ∼1600 RNA/EV for 1000-nt RNA and ∼6700 RNAs/EVs for 200 nts RNA. However, when measured by total RNA quantification , the number of RNA per EV was less than one in serum-derived EVs. Another team reported the presence of ∼7 µg of RNA per 1010 EVs dosed by bulk representing ∼6500 RNA molecules per EV , but the presence, as discussed by the authors, of contaminating surrounding extra-vesicular RNA may artificially enhance this number. As an example, once extra-vesicular RNA is removed from serum-derived EV preparations (using differential centrifugation and size-exclusion chromatography) only ∼2.5% of total miRNA remains in the serum-derived EV fraction . Most of the time, purification strategies used are not allowing complete extra-vesicular RNA removal (in particular in serum where it represents a large fraction of RNA), therefore attribution of a particular effect to intra-vesicular EVs may be difficult. Quantitative results on the amount of miRNA per EVs estimates that most represented miRNA can hardly be found in 1 out of 100 exosomes (the range varies for each miRNA from one copy per 9 exosomes to one copy per 47,162 exosomes, mean of 1 copy per 121 exosomes using digital PCR, a reliable and sensitive quantitative method) . Knowing that they detected 131 miRNA in total, the estimated miRNA per EV should be considered to be ∼1 per EV.
|Ctot (EV)||1012 EV/L|||
|f (EV subtype)||All EVs||100%|||
|All non hematopoietic tissue EVs||0.2%|
|Other non hematopoietic tissue||0.04%|
|Half life (τ½)|
|7 min (mice)|||
|f (target tissue)||All tissues||100%|||
|Nb Cell (tissue)||All tissues||3.72 × 1013|||
|Liver||2.41 × 1011|
|Spleen||2 × 1011|
|Brain||3 × 1012|
The RNA-based mechanism of action (MOA) for effects mediated by non-modified native EVs in therapeutic conditions has previously been challenged by comparing it to data obtained from siRNA experiments. In most preclinical studies, EV doses usually range from ∼1 to 200 µg per mouse , corresponding to about 1010 to 1012 EV/mouse depending on EV preparation and dosage methods. If we consider ∼1 miRNA per EV, this dose represents ∼1010 to 1012 miRNA per dose, corresponding to about ∼0.2–20 ng of miRNA/mouse or ∼0.016–1.6 pmol/mouse. siRNA doses reported to be efficient in vivo in systemic injections are rather in the microgram range (27 to 750 µg/mouse ). One explanation is that the observed therapeutic effect of native EVs is not mediated by their naturally loaded (mi)RNAs. Indeed, this ∼103–104 fold difference was though too big to be explained by a very high difference in delivery efficacy . However, this may be now discussed in view of recent results comparing engineered EVs to synthetic RNA nanovectors.
Indeed, recently reported delivery efficacy of EVs obtained in vivo show a ∼10–300 fold improvement in favor of EVs  compared to lipid nanoparticles (although the authors discuss the estimation of miRNA concentration with their method may favor EV reported efficacy by ∼10 fold ). The authors used the natural ability of pre-miR-451 to be enriched preferentially in EVs and used it as a backbone to couple with an siRNA of interest in order to target it inside EVs . They then used these engineered EVs to target the liver, intestine or kidney glomeruli and achieve various target knockdown. Interestingly, this ∼10 to-300 fold improvement in terms of RNA cytosolic delivery in favor of EV in vivo is fully consistent with independent data on delivery efficacies reported for synthetic vectors: EVs reach a ∼20% endosomal escape rate  compared to 0.1 to 2% for synthetic vectors , which leads to a ∼50 fold increased cytosolic delivery. Even higher differences (up to 104) were reported in the delivery efficacy in favor of EVs in vitro . Importantly, such a fold change also takes into account the very different endocytosis rate that favors EVs compared and synthetic vectors in vitro but not in vivo .
Altogether, these quantitative estimates show (Figure 1) that distant communication by EVs via RNAs probably has limited efficacy in physiological conditions, although it may be a bit different in pathological conditions and in the therapeutic use of EVs that are engineered to load large amounts of specific RNA.