Extracellular vesicle mechanism of action

A wide range of effects

The field of extracellular vesicles has also to face regulator questions, in particular a tricky one: “what is the mechanism of action?” This question can be addressed saying that extracellular vesicles in regenerative medicine are inducing an anti-apoptotic, anti-oxidative, pro-angiogenic, immunomodulatory effect, promote inflammation resolution and change the extracellular matrix remodeling process as well as their parental cells [35]. It raises a very interesting question “can’t you just purify the molecule of interest?”. This question is still difficult to address with the current knowledge in the field, the effect of extracellular vesicles seem to be dependent on many proteins and RNAs, with no clear major actor among others. Therefore, the current paradigm is that the complexity of extracellular vesicles allows it to play on many pathways at the time, explaining the pleiotropic effect. From a pharma and a regulator perspective, this kind of response is unusual, and will probably be complex to justify. 

From a research perspective, the problem of “how extracellular vesicles function” would probably need to be simplified, and we consider that the investigation of whether the effect is mainly mediated through a receptor ligand interaction or a cargo delivery effect would be a major first step. Another simple and still not really investigated effect could be the simple use of extracellular vesicles as nutrients for cells in a starving stressful situation. 

From a physicist point of view, the most likely mechanism of action would of course be the receptor ligand interaction, based on simple approximations on surface to volume ratios: taking a cell radius of 7.5 µm, a vesicle radius of 75 nm and assuming they are perfect sphere, there would be a need for 10 000 extracellular vesicles to obtain the same surface than the parental cell, and 1 000 000 EVs (100 fold more) to obtain the same volume than the parental cell. It means that receptor ligand interaction would be much more efficient than cargo delivery as a way to transfer information. Extracellular vesicles are like classical nanoparticles, where the idea is to use small objects to obtain a potent surface-surface interaction and a homogenous cell to cell distribution, but in large amounts to ensure that a sufficient quantity/volume is delivered. 

Moreover, the actual proportion of cargo delivered without being degraded is also an open question, but is probably very low [98]. Finally, this is supported by the fact that most extracellular vesicle preparations used in the field were frozen before use, a method that can lead to the loss of a part of the inner cargo. In general, the small size of nanoparticles gives them high surface/volume ratio, enabling potent interaction with cells. Moreover, this small size allows a good diffusion inside tissues. 

The effect of lipids in the extracellular vesicles have not been investigated up to now, and they may be key actors in the regenerative effect of stem cell derived extracellular vesicles, in particular the role of bioactive lipids like Pro Resolving Lipids [99]. 

Finally, in vitro experiments are often carried out using highly overestimated concentrations compared to extracellular vesicle concentration in vivo, and therefore need to be considered with caution [100]. 

Once again, to get a glimpse of the actual numbers of extracellular vesicles present in plasma, the seminal paper published by Brisson et al estimates its concentration to 30 000 EVs/µL [101]. If we take a half-life of roughly 5 min, a 3L amount of plasma, then the amount of EVs produced every 5 min is 4.5 x 10¹⁰, 6.9 x 10¹¹ per hour and 1.3 x 10¹³ per day, corresponding to roughly a total hydrated weight of 0.21 mg, 2.6 mg and 62 mg respectively with radius = 75 nm and EV density = 1,15. Compared to the total amount of cells in our body, which is estimated to be around 10¹⁴, the EV/Cell ratio is much lower than the one used in vitro in the context of a whole body IV injection. The actual concentration of EVs/cell can anyway be much higher in case of local delivery methods, for example in the eye, or on the skin. 

Delivering a functional cargo?

Albeit the fact that extracellular vesicles are able to enter cells efficiently is confirmed by various experiments, their ability to deliver their cargo efficiently inside the cell to the cytoplasm, i.e. escaping the endosome, is still under debate, with a huge bias toward published positive results. Once discovered inside extracellular vesicles in 2007 [7], miRNAs were considered as the major actor of extracellular vesicle effect. Later, stoichiometry analysis revealed that their concentration was finally not so high, with roughly 1 out of 100 EVs loaded with one copy of a specific common miRNA [102]. Moreover, extracellular vesicles were initially thought to deliver their cargo through fusion with the plasma membrane but were rather found to be internalized and get through the classical endosome lysosome pathway that is usually associated with degradation [103]. Another possibility to enter the cell cytoplasm, the place where miRNA, siRNA or various proteins cargoes would be able to act is to fuse with the plasma membrane inside endosomes, a mechanism called “back fusion” [104]. The exosomes, with an intra endosomal origin (ILVs) are expected to be resistant to acidic pH and proteases inside the endo-lysosome pathway and be able to fuse with endosomes. This potential fusion process would be enhanced by acidic pH that induces in many proteins and lipids a change in charge [105], leading to a change of surface zeta potential and a change in the proteins structure. This zeta potential change and protein conformation change [106] could be an enhancer of fusion [107].

The quantification of these phenomena is a trend in the field, but they currently need complicated methods to do so. As an example, methods of choice are the expression of palmitoylated fluorescent proteins that label cell membranes, the use of fluorescent tagged RNAs, the detection of fluorescent proteins in the recipient cell transferred either directly or transferred through a mRNA that is later expressed in the recipient cell. Finally, a very sensitive ON/OFF method to detect the efficient delivery of proteins or RNA was described recently. CRE recombinase is a very interesting protein tool that once delivered is able to go in the nucleus (efficiency highly depends on the localization signal) and cut a specific section of the DNA (between two loxP sequences), an irreversible event that can be linked with the permanent expression of a fluorescent protein as a simple readout. As an example, it is possible to insert a stop codon between two loxP sequences, that once removed allow the expression of a fluorescent protein or any protein of interest. The fact that there is a need for such very sensitive methods (roughly 0,1 % of the white blood cells are recombined in an in vivo tumor model where CRE is expressed by the tumor [98]) to detect an efficient protein cytosolic delivery is interesting on its own, meaning that the delivery efficiency is at least not highly effective. 

[35] J.L. Spees, R.H. Lee, C.A. Gregory, Mechanisms of mesenchymal stem/stromal cell function, Stem Cell Research & Therapy, 7 (2016) 125.

[98] A. Zomer, C. Maynard, Frederik J. Verweij, A. Kamermans, R. Schäfer, E. Beerling, Raymond M. Schiffelers, E. de Wit, J. Berenguer, Saskia Inge J. Ellenbroek, T. Wurdinger, Dirk M. Pegtel, J. van Rheenen, In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior, Cell, 161 (2015) 1046-1057.

[99] J. Dalli, C.N. Serhan, Specific lipid mediator signatures of human phagocytes: microparticles stimulate macrophage efferocytosis and pro-resolving mediators, Blood, 120 (2012) e60-e72.

[100] S.E. D., Amedeo Avogadro’s cry: What is 1 µg of exosomes?, BioEssays, 34 (2012) 873-875.

[101] A. N., L. R., T. S., G. C., P. J.-M., M. S., B.A. R., Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration, Journal of Thrombosis and Haemostasis, 12 (2014) 614-627.

[102] J.R. Chevillet, Q. Kang, I.K. Ruf, H.A. Briggs, L.N. Vojtech, S.M. Hughes, H.H. Cheng, J.D. Arroyo, E.K. Meredith, E.N. Gallichotte, E.L. Pogosova-Agadjanyan, C. Morrissey, D.L. Stirewalt, F. Hladik, E.Y. Yu, C.S. Higano, M. Tewari, Quantitative and stoichiometric analysis of the microRNA content of exosomes, Proceedings of the National Academy of Sciences, 111 (2014) 14888-14893.

[103] K.J. McKelvey, K.L. Powell, A.W. Ashton, J.M. Morris, S.A. McCracken, Exosomes: Mechanisms of Uptake, Journal of Circulating Biomarkers, 4 (2015) 7.

[104] T. Falguières, P.-P. Luyet, J. Gruenberg, Molecular assemblies and membrane domains in multivesicular endosome dynamics, Experimental Cell Research, 315 (2009) 1567-1573.

[105] S.A. Akimov, M.A. Polynkin, I. Jiménez-Munguía, K.V. Pavlov, O.V. Batishchev, Phosphatidylcholine Membrane Fusion Is pH-Dependent, International Journal of Molecular Sciences, 19 (2018) 1358.

[106] N.V. Di Russo, D.A. Estrin, M.A. Martí, A.E. Roitberg, pH-Dependent Conformational Changes in Proteins and Their Effect on Experimental pK(a)s: The Case of Nitrophorin 4, PLoS Computational Biology, 8 (2012) e1002761.