IT202100027725A1 - METHOD FOR OBTAINING CONCENTRATED POPULATIONS OF EXTRACELLULAR VESICULES WASHED OF THEIR PHYSIO-PATHOLOGICAL LOAD - Google Patents

Description

Patent application for industrial invention entitled: "METHOD FOR OBTAINING CONCENTRATED POPULATIONS OF EXTRACELLULAR VESICULES WASHED OF THEIR PHYSIO-PATHOLOGICAL LOAD"

DESCRIPTION

Field of application

In its most beautiful aspect In general, the present invention concerns the sector of biotechnology and nanomedicine based on extracellular vesicles (EVs). Specifically, the invention provides a new method for obtaining greater numerous, homogeneous, and internalizable, population of extracellular vesicles starting from products extracted with the most various techniques available, such as ultracentrifugation, ultrafiltration, precipitation, etc...

The method consists of a simple and reproducible osmotic lysis of extracellular vesicles through the addition of a hypotonic solution, such as double-distilled water, followed by washing, again with double-distilled water, of the lysed sample, removing most of the components of the vesicular lumen leaked following the lysis process.

The removal of these components is particularly indicated when EVs are used in biomedical/clinical applications as they could promote immunostimulatory effects of various types or induce unwanted neoplastic transformations.

The method described below and their product, i.e. the vesicles produced by post-lysis reassembly, and therefore purified of their pathophysiological load, both constitute the object of the present invention, which is equipped with particular characteristics which make its use particularly useful and advantageous in biomedical and/or clinical applications.

State of art

Extracellular vesicles (EVs) are a very heterogeneous population of vesicles delimited by a phospholipid bilayer, have dimensions ranging from tens of nanometers to a few microns and contain lipids, proteins and nucleic acids within them. The composition and pathophysiology of EVs significantly depend on the cell of origin (parental) and the moment in which they are produced. To date, several subgroups of EVs have been defined such as apoptotic bodies, microparticles, microvesicles, shedding vesicles, ectosomes, exosomes and exosome-like vesicles.

The company? International Cellular Vesicle (ISEV) classification provided a classification of EVs based on their chemical-physical characteristics, size, appearance under microscopy, sedimentation, lipid composition, main protein markers and subcellular origin. EVs are classified into three main groups: exosomes, microvesicles and apoptotic bodies. Exosomes originate thanks to the invagination of the membrane of late endosomes, forming intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs). ILVs are released into the extracellular environment through the fusion of MVBs with the plasma membrane, taking the name of exosomes and have an average diameter of 50-100 nm. Microvesicles, also called shedding vesicles or microparticles, of irregular shape with an average diameter of 100-1000 nm, are formed by direct budding of the cellular plasma membrane, incorporating some cytoplasmic components within them. The apoptotic bodies, released by cells that undergo a process of programmed cell death, called apoptosis, have an average size between 1000 and 5000 nm. To these main types are added ectosomes, round-shaped membrane particles with an average diameter of 50-80 nm and ectosome-like vesicles, with an average diameter of 20-50 nm. [

Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles, 2015.4: p.27066].

EVs are produced by both prokaryotic and eukaryotic cells, and are universally recognized as fundamental elements of intercellular communication due to their ability to to transfer proteins, lipids and nucleic acids, influencing physiological and pathological functions in both parental and target cells.

EVs taken from samples of blood, urine and other biological fluids are very useful biomarkers. In particular, the proteins and lipids present on their membrane, reflecting the protein and lipid content of the parental cells, together with the various molecules contained in the lumen are used for diagnostic and prognostic screening.

Are EVs used more and more? frequently in their native state or, after engineering, for therapeutic applications of various types.

EVs, generally biocompatible and with low immunogenicity, have a high capacity loading of substances such as pharmaceutical molecules, proteins, nucleic acids or nanomaterials. Characterized by a long half-life in the blood circulation and, depending on the case, capable of crossing biological barriers, such as cytoplasmic membranes and the blood-brain barrier, they are excellent candidates for drug delivery applications.

Do the EVs show clear specificity? and tropism towards tumor cells since they are internalized about ten times more? compared to what liposomes of similar size do [Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro, Smyth, 2014]. Furthermore, nanometric vesicles tend to accumulate in tumor tissue rather than in healthy tissue thanks to the increased permeability. and retention of the former (enhanced permeability and retention (EPR) effect).

The application of EVs as drug delivery devices requires from time to time a careful evaluation of the cellular source from which they come. For example, ? It has been observed that tumor cell-derived EVs (TDEVs) can play a key role in the progression and spread of some types of tumors, acting as mediators in processes such as invasion of healthy tissues, drug resistance, angiogenesis and possible escape mechanisms from the control of the immune system. In fact, through EVs, tumor cells communicate not only with each other, but also with healthy cells, in some cases modifying their phenotype or differentiating them. ? For example, it has been demonstrated that TDEVs are able to direct the differentiation of normal fibroblasts in the tumor microenvironment into tumor fibroblasts (cancer-addicted fibroblasts CAF) which are in turn capable of secreting factors and EVs capable of promoting cancer proliferation, l? invasion and metastasis [ Extracellular vesicles in the tumor microenvironment: old stories, but new tales. Mol Cancer. 2019; 18:59].

From there? ? It is easy to understand how the use of EVs secreted by tumor cells as devices for drug dispensing must be carefully evaluated while enhancing the innate tumor tropism of these vesicles.

Certainly the use of EVs isolated from healthy cells significantly reduces the risks described above, although the in vivo use of non-autologous materials does not exclude possible side effects and adverse immune responses after administration in the recipient organism.

The use of EVs isolated from body fluids or from the supernatants of cultured cells taken from the same patients to be treated, in a mere context of personalized medicine, significantly reduces the eventualities described above.

Many cell lines such as immature dendritic cells, mesenchymal stem cells (MSCs) and lymphocytes are excellent candidates for the isolation of EVs for drug delivery applications by combining the effects of their original cargo with those of a possible post-isolation load.

Nanomedicine based on the use of EVs therefore presents many advantages including strong biomimicry and high specificity. of targeting. However, this approach still has some critical issues. such as, for example, the lack of high-efficiency protocols for isolations, immunogenicity problems? and low efficiency in loading therapeutic content (e.g. nanoparticles, drugs or nucleic acids).

For these reasons, the use of IV for therapeutic applications is ? still far from becoming a reality? consolidated in clinical practice.

Vesicles can be isolated from any type of biological fluid, such as, for example, cell culture supernatants, blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, ascitic fluid, bronchus lavage fluid -alveolar, synovial fluid, breast milk, sweat, tears, joint fluid. Precisely because of the variability? and the complexity? biology of biological fluids, the vesicle isolation procedure is very delicate and critical. In the state of the art, various procedures are known through which extracellular vesicles can be isolated, such as ultracentrifugation, ultrafiltration or with the aid of commercial kits such as optiprep, exoquick, exo-spin or others. The product of isolation? consisting, as a rule, of a certain quantity? of EVs resuspended in aqueous saline solutions such as PBS (Phosphate buffered saline) or physiological solution.

Patent documents

Patent application WO 2017/161010 describes a method of producing a membrane vesicle starting from an extracellular vesicle. This method involves obtaining open membrane vesicles, removing their contents in order to eliminate unwanted molecules, and reduce the possible risk of side effects, capable of reassembling to form new ones. The opening of the extracellular vesicle membrane? obtained by treating the vesicles with an aqueous solution having a pH between 9 and 14. Reassembly is obtained through sonication techniques, mechanical vibrations, extrusion through porous membranes, electric current and their combinations. Concomitant with the first phase of reassembly of emptied vesicles, newly formed membrane vesicles can be loaded with specific cargoes.

Patent application KR20180122433 describes a method for producing a membrane vesicle derived from an extracellular vesicle. This method involves obtaining an open membrane vesicle and removing the contents of the vesicle in order to eliminate unwanted molecules and reduce the possible risk of side effects. The opening of the membrane of the extracellular vesicle is obtained by treating the vesicles with an aqueous solution of sodium carbonate (200 mM, pH 1) for approximately 2 hours.

However, the two methods mentioned above using the reagents and procedures described for the lysis and subsequent reassembly of the vesicles do not fully respond to market demands in terms of biocompatibility, safety and efficiency. In particular, from the state of the art mentioned above, it emerges that there is a great need to have standardized and reproducible extracellular vesicle production methods to provide biocompatible products with a high capacity? targeting and/or loading.

The present invention therefore aims to overcome the problems and drawbacks described above related to the use of extracellular vesicles with particular application to nanomedicine and clinics by providing a new simple, safe and economical method for purifying, increasing yield, biocompatibility. ? and the internalization of extracellular vesicles starting from products extracted with the most various techniques currently available.

Summary of the invention

The aim of the present invention is to overcome the limitations of the currently known state of the art, linked to low yield and heterogeneity. of EV isolations, to immunogenicity? due to the origin of the samples and the inefficiency of loading of molecules within the EVs, providing an innovative method of processing EVs post-isolation and/or conservation.

EV lysis is performed by resuspending the vesicles in double-distilled water: this process causes the rupture of the lipid membranes of the vesicles, encouraging the release of the substances contained within them.

During the experimentation carried out, the inventors verified that subsequent washing phases in double-distilled water favor the removal of the intravesicular substances released after lysis, allowing the removal of the contents of the vesicular lumen, before the reassembly of the membranes of the vesicles which, once purified, turn out to be more numerous in terms of particles per unit? of volume (ml).

To the lysis process can? be coupled to a load process. More? in detail, molecules and materials such as, for example, drugs, nanoparticles, nucleic acids, peptides or biological derivatives can be dispersed in the hypotonic solution in the presence of extracellular vesicles. In this way, following lysis, the new vesicles will reassemble, encapsulating the molecules and/or nanomaterials previously dispersed in solution.

Brief description of the figures

Will this invention come? described below through some preferred embodiments, provided by way of example and not by way of limitation, with reference to the attached drawings in which:

Figure 1 ? a plot of extracellular vesicle concentration measured by Nanoparticle Tracking Analysis (NTA);

Figure 2? a plot of the frequency class distribution of extracellular vesicle sizes;

Figure 3? a graph of the protein content of the extracellular vesicles after a first and second cycle of osmotic lysis;

Figure 4? a graph of the internalization of extracellular vesicles detected by flow cytometry, expressed as a percentage of positive events;

Figure 5? a transmission electron microscopy image contrasted with Uranyl Acetate of the load of gold nanoparticles in the lysed vesicles obtained with the method covered by the patent;

Figure 6? a graph representing the load of fluorescent gold nanoparticles in the lysed vesicles, detected by flow cytometry and expressed as intensity? of average fluorescence.

Detailed description of the invention

The first part of the method illustrated in the present invention involves the dilution of the extracellular vesicles in a hypotonic solution compared to that of the starting sample, such as double-distilled water, capable of triggering a process of lysis and reassembly of the vesicles which has as its aim obtaining a larger population? number of EVs than the starting one and the purification of the EVs from their physio-pathological cargo.

Object of the present invention? also to provide a method for obtaining new vesicles endowed with specific and advantageous characteristics that can be used for different types of treatments and/or for the targeted release of active ingredients.

In the following description, the use of ?for example?, ?etc.?, ?or / or? means non-exclusive alternatives without limitation, unless otherwise indicated; the use of ?also? means ?including, but not limited to? unless otherwise indicated; the use of ?include / comprises? means ?includes / includes, but not limited to? unless otherwise indicated.

As used herein, the term "extracellular vesicle" includes exosomes, shedding vesicles, microparticles, ectosomes, ectosomelike vesicles, membrane vesicles, and refers to a vesicle derived from any cell, comprising a membrane that delimits an internal space, typically, of average diameter between 20 nm and 400 nm, better from 40 nm to 300 nm, even better from 50 to 250 nm, containing various entities within it? endogenous macromolecular molecules, such as nucleic acids, proteins, carbohydrates, lipids, small molecules and/or combinations thereof, or can? be functionalized on the outer membrane or loaded with various exogenous compounds such as therapeutic, diagnostic contrast, and/or targeting agents.

As used herein, the term "membrane" refers to a biological membrane, i.e. an external covering of cells and organelles with its typical structure formed by a double lipid layer that forms a semi-permeable barrier that separates an internal compartment from the external environment, allows the passage of a particular compound and is capable of fusing with other membranous systems that have the same or similar structure, subsequently exposing the contents of the extracellular vesicle or dissociating the contents from the membrane once opened.

The method of processing extracellular vesicles described in the invention can? be applied both immediately after the isolation process and after any time on isolated and appropriately preserved vesicles.

In particular, according to the preferred form of the invention, the method for treating post-isolation extracellular vesicles includes the following steps:

a) provide a quantity? of extracellular vesicles; preferably suspended in an aqueous saline solution (e.g., PBS, saline, culture medium);

b) treat the solution containing the extracellular vesicles preferably with double-distilled water, or in any case with a hypotonic solution compared to the sample, to obtain the opening of the lipid membranes of the EVs. The formation of an osmotic gradient between the internal compartment of the vesicles themselves, rich in salts, miRNA, DNA, proteins and enzymes, and the hypotonic one of the double-distilled water determines the rupture of the membrane of the treated EVs and the leakage of the material contained within them . The open or fragmented vesicles reorganize themselves into an energetically more stable state. favorable, i.e. the lipid bilayers re-associate, including part of the liquid in which they are dispersed within them;

c) subject the lysed sample to washing (preferably with double-distilled water) to remove the material contained in the vesicular lumen. This operation is particularly important for biomedical and/or therapeutic applications both for the possibility to recover and analyze the contents of the lysed vesicles, both to avoid immunostimulatory and/or inflammatory effects in recipients of the treatments;

d) optionally, the lysed EVs can be engineered and loaded for nanotechnological and drug delivery applications by adding nanomaterials, drugs, nucleic acids, fluorescent molecules into the aqueous solution during the lysis phase, so that they are loaded into the vesicles during their reassembly.

The process of lysis and purification of EVs from the pathophysiological burden? particularly effective, fast and simple to perform and allows to obtain newly assembled vesicles with improved compared to the starting population such as the efficacy of internalization into tumor cells. This aspect makes its use in the therapeutic and personalized medicine fields particularly interesting and efficient.

The extracellular vesicles, once opened and purified of most of the molecules contained within them, in a hypotonic environment, tend to re-associate, self-assembling into new vesicles, which, in turn, can be subjected to a further series of lysis and/or washing, depending on the type of application.

According to the invention, in the case in which the extracellular vesicles are used for nanotechnological applications for diagnostic and/or therapeutic purposes, in conjunction with the lysis and the consequent removal of the physio-pathological contents,? It is possible to proceed with a loading phase (phase d), to be carried out during the first or subsequent lysis operation, adding the component to be loaded into the EVs into the lysis solution.

In general, materials such as drugs, therapeutic molecules, nanoparticles, nucleic acids, peptides or biological derivatives, etc. are dispersed in the lysis solution containing the extracellular vesicles, so that, following lysis, the newly formed vesicles incorporate them into their internal.

The inventors have observed that the population of vesicles obtained with the method described above is ? up to 100 times more? concentrated in terms of particles per unit? of volume (ml) compared to the control sample (Figure 1), with a size distribution whose classes of greatest frequency are concentrated between 100 and 220 nm (Figure 2) and a capacity to be significantly more? internalized in cells, both healthy and tumorous. More? in detail, always referring to internalization in the cell? An increase in internalization was observed in tumor cells approximately 5 times greater than that in healthy cells (Figure 4). Furthermore, the lysed vesicles are able, following a short incubation, to load molecules or nanoparticles, such as gold nanoparticles (Figure 5) with a significantly higher efficiency (p<0.001) compared to the sample control (Figure 6).

Due to these characteristics, the vesicles obtained with the method according to the present invention are suitable for a wide range of applications, including those of regenerative medicine, therapeutic and diagnostic ones.

The effectiveness of the process according to the invention and the clear advantage deriving from the use of the vesicles obtainable with this method were tested in different operating conditions, as fully described in the examples of the experimental section reported below.

It must be understood, however, that there is no no intention to limit the invention to the specific embodiments illustrated, but, on the contrary, the invention is intended to cover all modifications, equivalent forms and variations that fall within the scope of the invention as defined in the claims.

EXAMPLES

Example 1 - Osmotic lysis of vesicles

The EVs used by inventors to evaluate goodness? and the efficiency of the method object of the invention were isolated from B lymphocytes via differential ultracentrifugation and stored at -80 ?C for various times in physiological solution. For the test, a quantity? equal to 30 ?l of EV ? was diluted 1:50 in double-distilled water in order to carry out osmotic lysis; for the control sample, 30 ?l of EV were always diluted with a 1:50 ratio in physiological solution (hypotonic solution with the vesicular lumen).

Both test and control samples were resuspended with a ?vortex? for 3 minutes and incubated at 37?C for 6 minutes, then further mixed by vortexing for 1 minute.

The samples were then subjected to ultrafiltration with Amicon Ultra-0.5 ml 50 kDa (Merck Millipore). A volume equal to 0.5 ml of each sample? was loaded for each filter, centrifuged at 14000 g for 10 minutes and eluted at 1000 g for 2 minutes at room temperature.

The product of ultrafiltration? It was then resuspended in double-distilled water, saline solution, or in medium, in a variable volume depending on the application.

Example 2 - Analysis of the concentration and size distribution of purified extracellular vesicles of their pathophysiological cargo

The extracellular vesicles of the test sample, resuspended in 150 ?l of double-distilled water (1:5 compared to the starting concentration), or those of the control, in 150 ?l of physiological solution (1:5 compared to the starting concentration), treated as described in example 1, they were subjected to concentration and size distribution analysis using the Nanoparticle Tracking Analysis (NTA) technique, using a Nanosight NS300 (Malvern Panalytical) equipped with a laser source with ? = 505 nm, a syringe pump and the NTA vers.3.4 software.

The measurements, with the samples diluted in such a way as to ensure an adequate particle/frame, were carried out by acquiring three videos of 60 seconds each with a speed of of pump infusion of 50 A.U., the screen gain set to 1 and the camera level between 15 and 16. The videos were then analyzed via software with a detection threshold of 5.

Any analysis? was carried out on each sample in quintuplica (n=5) and expressed as the average value of the results obtained. The measurement data obtained on extracellular vesicles after osmotic lysis (EVs1) and the respective controls after lysis in physiological solution (EVs1 CTRL) after a first cycle of lysis are reported in the following Table 1 and shown in Figure 2.

Table 1 - Frequency classes of the average sizes of the lysis products in descending order (n=5). EVs1 CTRL refers to the control sample, EVs1 refers to the test sample.

The results obtained show that the order of distribution of the frequency classes for the two types of vesicles are comparable, while the population of vesicles obtained with the process according to the invention which involves the osmotic lysis phase? much more? concentrated (3x10<11>?7x10<10 >vesicles/mL) compared to the control group in which the lysis procedure is replicated but in an isotonic solution (7.4x10<9>?1.7x10<9 >vesicles/mL), as shown in Figure 1, although in both cases the sizes of the vesicles produced are well distributed with a peak around 100 nm (Figure 2).

The phenomenon could be explained by highlighting how with the lysis of the initial vesicles many vesicles could be formed de novo from the lipid bilayer patches reassembled after lysis. The internalization of part of the lysis and/or washing liquid does not lead to a clear reduction in the size of the new population.

Example 3 - Determination of the protein content of extracellular vesicles

The protein content of the newly formed vesicles after a first (EVs1) and a second cycle (EVs2) of osmotic lysis? was determined by Bradford assay. The analyzes conducted on triplicate samples showed how the vesicles, obtained through the lysis process of the invention, are significantly more richer than the control, both after a single lysis cycle (EVs1) and after a second lysis cycle (EVs2). The results are presented in Figure 3 which shows the graph of the means and standard errors of the measurements obtained. Statistical comparisons performed using 2-way ANOVA. *: p<0.05.

Example 4 - Capacity? of internalization of extracellular vesicles obtained by osmotic lysis

The capacity? of internalization of extracellular vesicles, produced with the method of the invention? was evaluated in healthy cells (B lymphocytes), and in a Burkitt lymphoma line (Daudi).

5 ?g/ml of EV per sample (test and control), per line (lymphocytes and Daudi) and per time frame (24 and 48 hours), were labeled with Wheat Germ Agglutinin conjugated with the dye Alexa Fluor 647. The labeled vesicles were treated as described in Example 1. For each line and each timing, 500 ?l of treatment were plated with 200,000 cells/ml for both the control and the test.

After 24 or 48 hours of incubation, the cells were recovered, centrifuged and resuspended in 500 liters of PBS for flow cytometric analysis of internalization. The analyzes were conducted with the Guava Easycyte 6-2L flow cytometer (Merck Millipore) with a speed of flow of 0.59 ?L/s, using the red laser (?ex =642 nm) on 1x10<4 >events. Data from untreated cells (0 ?g/ml) are used as a reference. The results are represented in terms of positive events, characterized by a shift in intensity? of Red-R fluorescence (661/15 filter emission) and the percentages of positive events were compared to untreated cells. The results obtained are represented in Figure 4 and show that the neovesicles obtained after the lysis process are internalized to a significantly greater extent? higher than the respective controls, both after 24 and 48 hours of treatment, for both cell lines. It also appears that the newly produced vesicles are internalized significantly more by the tumor cell line than by the healthy one, and this difference is already? detected after 24 hours of treatment. Statistical comparisons performed using 3-way ANOVA on triplicates. *:p<0.05, **:p<0.001.

Example 5 - Loading of gold nanoparticles inside extracellular vesicles obtained by osmotic lysis

The gold particles were loaded into the EVs by adapting the osmotic lysis protocol described in example 1. For both EV samples (control and test), 5?g of EVs derived from lymphocytes and 10?g were used. g of commercial gold nanoparticles (AuNPs) with a diameter of 5 nm. For the test, the solution of EV and AuNPs ? was diluted 1:50 in double-distilled water to carry out osmotic lysis and loading, while the control sample was diluted 1:50. always diluted with a 1:50 ratio in physiological solution.

Both test and control samples were resuspended with a ?vortex? for 3 minutes and incubated at 37?C for 30 minutes with stirring at 180 rpm, then further mixed with vortex for 1 minute.

The samples were then subjected to ultrafiltration with Amicon Ultra-0.5 ml 50 kDa (Merck Millipore). A volume equal to 0.5 ml of each sample? was loaded for each filter, centrifuged at 14000 g for 10 minutes and eluted at 1000 g for 2 minutes at room temperature.

Example 6 - Capacity? loading of extracellular vesicles obtained by osmotic lysis

The capacity? of loading of the extracellular vesicles, produced with the method of the invention? was evaluated by transmission electron microscopy (TEM). In detail, 5 ?g of EV and 10 ?g of AuNPs were treated as described in Example 5. The sample? was deposited on a screen for TEM analysis and counterstained with uranyl acetate. How can you? Notice from Figure 5, the electron-opaque AuNPs were successfully loaded into the pi? clear, why? less electron-dense, extracellular vesicles.

Example 7 - Capacity? loading of extracellular vesicles obtained by osmotic lysis

The capacity? of loading of the extracellular vesicles, produced with the method of the invention? was evaluated by adapting the analysis protocol of EVs adsorbed on latex aldehyde/sulfate beads described by Thery et al. (Isolation and characterization of exosomes from cell culture supernatants and biological fluids, Thery, 2006).

2.5 ?g of EV and 5 ?g of Cy5-labeled AuNPs per sample (test and control) were treated as described in Example 5.

The product eluted from ultrafiltration is incubated for 15 minutes at room temperature with 5 ?l of beads, then it is brought to a volume of 500 ?l with PBS and incubated for 2 hours on a sample wheel for mixing.

At the end of the two hours, three washes are performed in PBS to eliminate particles not bound to the beads and resuspended in a volume of 250 ?l. The sample is then prepared for analysis by diluting 5 ?l of the bead solution in 200 ?l of PBS.

The analyzes were conducted with the flow cytometer Guava Easycyte 62L flow cytometer (Merck Millipore) with a speed of flow of 0.12 ?L/s, using the red laser (?ex =642 nm) on 5x10<3 >events. The results are represented in Figure 6 in terms of intensity? of average fluorescence, and show how the EVs treated with the proposed method, if loaded with labeled AuNPs, have statistically more fluorescence? higher than control ones kept in PBS and, therefore, not lysed. Statistical comparisons carried out using t-tests on ten replicates, **:p<0.001.

Claims (18)

1) Method to obtain a pi? numerous, homogeneous, safe, internalizable and engineerable population of EV extracellular vesicles, extracted from biological fluids following various isolation and/or conservation methods, where the product of isolation is? consisting of a quantity? of EV, characterized by the fact that it involves subjecting said isolation product to a lysis process by adding double-distilled water which causes an osmotic imbalance between the internal compartment of the vesicles and the surrounding hypotonic environment, with the consequent rupture of the lipid membranes of the IV treated and the leakage of the material contained in the vesicular lumen. 2) The method as per the previous claim characterized by the fact that, after lysis, the open or fragmented vesicles are reassembled by including part of the liquid in which they are immersed inside them. 3) The method as per the previous claim characterized by the fact that the lysed sample is subjected to washing to remove the contents of the vesicular lumen. 4) The method as in any of the previous claims in which the product of the insulation is? obtained with any of the available techniques including ultracentrifugation, ultrafiltration and the use of various commercial kits such as optiprep, exoquick, exo-spin. 5) The method as in claim 1 characterized by the fact that the purified vesicles, obtained from the lysis step, are subjected to further lysis steps with double-distilled water carried out in conjunction with the addition of components to be loaded into the lysis solution, obtaining ? that following lysis the new vesicles are reorganized in an aqueous environment by reassociation and self-assembly, so as to encapsulate the molecules and/or nanomaterials previously dispersed in solution. 6) The method as in claim 5 can be used for the production of EVs for nanomedicine, regenerative medicine and drug delivery applications capable of combining the effects of the natural content of the EVs themselves with a new one engineered a posteriori. 7) The method as per the previous claims which allows the improvement of the loading efficiency and internalization of nanoparticles or molecules of therapeutic or diagnostic interest in the newly formed vesicles in conjunction with the lysis process. 8) The method as any of the previous claims, characterized by the fact that it optimizes the yields, consequently reducing the costs, of EV production processes as it is? capable of increasing the concentration of EVs obtained post isolation by up to two orders of magnitude. 9) A method for processing post-isolation extracellular membrane vesicles comprising the following steps: a) provide a quantity? of extracellular vesicles obtained following any isolation and/or conservation process; b) treat the solution containing the extracellular vesicles with a hypotonic solution to obtain the opening of the lipid membranes of the EVs present, due to the osmotic gradient between the internal compartment of the vesicles themselves and the surrounding hypotonic environment, with the consequent leakage of the material contained in the their interior; c) subject the lysed sample to washing to remove the material contained within the vesicles; d) reassemble said membranes with the formation of new vesicles purified of their original physio-pathological load. 10) The method for treating post-isolation extracellular membrane vesicles as per claim 9, characterized by the fact that it further involves the loading phase of components for nanomedicine, regenerative medicine and drug delivery applications into the lysed and reassembled vesicles. 11) The method for treating post-isolation extracellular vesicles as per claim 9 characterized by the fact that the lysis phase is carried out in a hypotonic environment by diluting the extracellular vesicles, extracted by ultracentrifugation, in double-distilled water with a dilution factor of 1:25 to 1 :80, vortex for approximately 3 minutes, incubate at 37 ?C for 5-10 minutes, and then vortex again for 1 minute. 12) The method as per claim. 11 characterized by the use of a dilution factor capable of causing the lysis of the starting material which can be adjusted depending on the process. 13) The method as in claim 10 characterized by the fact that said materials are drugs, therapeutic molecules, nanoparticles, nucleic acids, peptides or biological derivatives, which are dispersed in the hypotonic solution in the presence of the lysed extracellular vesicles. 14) Extracellular vesicles comprising vesicles formed by lipid membranes derived from extracellular vesicles isolated, purified and reassembled in a hypotonic environment according to the method of claim 9. 15) Extracellular vesicles to be used as diagnostic and therapeutic solutions in nanomedicine and regenerative medicine: comprising vesicles formed by lipid membranes derived from extracellular vesicles isolated, lysed, purified and reassembled in a hypotonic environment, according to the method of claim 9. 16) Extracellular vesicles as in claim 15, derived from biological liquids, healthy and/or tumor eukaryotic cells and from plant cells loaded with a therapeutic load according to the method of claim 10. 17) Use of extracellular vesicles according to claim 16 as a means of administering drugs and active ingredients into tumor tissue. 18) Use of extracellular vesicles according to claim 15 as a means of administering drugs and active ingredients in regenerative medicine applications.