ABSTRACT: EVs are a heterogeneous population of nano-sized lipid-bilayer enclosed particles ubiquitously secreted by eukaryotic and prokaryotic cells in physiological and pathological conditions and they can be found in all human body fluids. EVs are involved in intercellular communications and they play a key role both in physiological conditions, regulating homeostasis, guiding the immune response, mediating intercellular communication, and in pathological conditions, such as in the spread of metastases, in the progression of neurodegenerative, metabolic and cardiovascular diseases. One of the basic mechanisms through which EVs exert their influence is interacting with recipient cells binding to specific receptors, like tetraspanin proteins, integrins, immunoglobulins, and proteoglycans.In the following study we introduce an innovative, fast, reproducible and sustainable method to engineer EVs that can overcome many issues associated with the therapeutic and the diagnostic use of EVsand to favour their translation from research to clinical use. This technique is based on osmotic lysis, carried out with a sudden dilution of EVs with a hypotonic aqueous solution.We here show that applying this hypo-osmotic shock to EVs allows to obtain a more enriched population of vesicles in terms of concentration and purity, starting from EVs isolated from different sources and with different purification approaches, overcoming the low yields and the presence of impurities that characterized many isolation methods and opening the way to a possible scaling up of production.This osmotic lysis method enables the separation of EVs inner cargoes from their membranes, with a triple result: (I) the removal of inner RNAs and proteins circumvent the tumorigenic risk associated with tumor-derived EVs, whose targeting properties could be, in this way, exploited for targeted drug delivery; (II) the interactions between exogenous and endogenous cargoes could be prevented in case of use of these EVs as drug delivery vehicles; (III) finally, the inner content of vesicles could be collected for further analysis to make diagnostic a prognostic evaluations. In addition to these advantages, results showed that the rearrangements of lipid and protein composition of the EVs lipid bilayers upon the lysis process, surprisingly, favor the internalization inside cells and demonstrate also a targeting ability towards diseased cells, in the case of this study, cancerous cells.
Marcello Manfredi