EP4422597A1

EP4422597A1 - Method for obtaining concentrated populations of extracellular vesicles washed of the physiopathological load thereof

Info

Publication number: EP4422597A1
Application number: EP22809515.4A
Authority: EP
Inventors: Tania LIMONGI; Francesca SUSA; Roberto Pisano; Valentina CAUDA
Original assignee: Politecnico di Torino
Current assignee: Politecnico di Torino
Priority date: 2021-10-28
Filing date: 2022-10-27
Publication date: 2024-09-04
Also published as: IT202100027725A1; WO2023073609A1

Abstract

A method for obtaining a larger, more homogeneous, safer, more internalizable and engineered population of extracellular vesicles EVs, extracted from biological fluids following various isolation and/or conservation methods, where the isolation product consists of an amount of EVs, includes subjecting said isolation product to a lysis process by adding double-distilled water which causes an osmotic imbalance between the inner compartment of the vesicles and the surrounding hypotonic environment, resulting in the rupture of the lipid membranes of the treated EVs and the escape of the material contained in the vesicular lumen.

Description

METHOD FOR OBTAINING CONCENTRATED POPULATIONS OF EXTRACELLULAR VESICLES WASHED OF THE PHYSIOPATHOLOGICAL LOAD THEREOF

DESCRIPTION

Field of application

In the more general aspect thereof, the present invention relates to the field of biotechnology and nanomedicine based on extracellular vesicles (EVs). Specifically, the invention provides a new method for obtaining a larger, more homogeneous and intemalizable population of extracellular vesicles from products extracted with the most varied techniques available, such as ultracentrifugation, ultrafiltration, precipitation, etc.

The method consists in a simple and reproducible osmotic lysis of extracellular vesicles through the addition of a hypotonic solution, such as doubledistilled water, in a sufficient amount to cause the rupture of the lipid membranes, followed by a washing, again with double-distilled water, of the lysed sample, removing most of the components of the vesicular lumen which escaped following the lysis process.

According to a peculiar feature of the invention, all this occurs without the denaturation of the membrane proteins, maintaining the native state, i.e., the form which is operating and functional, since acidic, basic or surfactant solutions which could affect the morpho-functionality of the vesicles themselves are not used.

The removal of the components of the vesicular lumen is particularly indicated if the EVs are used in biomedical/clinical applications, as they could promote various types of immuno-stimulatory effects or induce undesired neoplastic transformations. At the same time, it could be used to screen the health of the donor/patient from whom they were isolated.

Therefore, both the method described below and the product obtained, i.e., the vesicles produced by post-lysis reassembly, and therefore, purified of the physiopathological load thereof, which makes the use thereof in biomedical and/or clinical applications particularly useful and advantageous, are the object of the present invention. Background art

Extracellular vesicles (EVs) are a very heterogeneous population of vesicles delimited by a double phospholipid layer, ranging in size from ten nanometers to a few microns and containing lipids, proteins and nucleic acids. The composition and pathophysiology of EVs significantly depend on the origin (parental) cell and on when 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 International Society of Cell Vesicles (ISEV) has provided a classification of EVs based on the chemical-physical features, size, microscopic appearance, setting, lipid composition, major protein markers, and subcellular origin thereof. EVs are classified into three main groups: exosomes, microvesicles, and apoptotic bodies. The exosomes originate by virtue of the invagination of the membrane of late endosomes, forming intraluminal vesicles (ILVs) within the multivesicular bodies (MVBs). The ILVs are released into the extracellular environment by fusing the MVBs with the plasma membrane, taking the name exosomes and having an average diameter 50-100 nm. Micro vesicles, also referred to as shedding vesicles or microparticles, of irregular shape having an average diameter of 100-1000 nm, are formed by direct budding of the cellular plasma membrane incorporating some cytoplasmic components therein. The apoptotic bodies, released by cells which undergo a programmed cell death process referred to as apoptosis, have average sizes between 1000 and 5000 nm. 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, add to these main types. [Yanez-Mo M., et al., Biological properties of extracellular vesicles and their physiological functions, J. Extracell Vesicles, 2015. 4: 27066].

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

The EVs taken from blood, urine and other biological fluids are very useful biomarkers. In particular, the proteins and lipids present on the membrane thereof, 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.

EVs are increasingly used in the native state thereof or, after engineering, for various types of therapeutic applications. Generally biocompatible and with low immunogenicity, EVs have a high capacity of loading substances such as pharmaceutical molecules, proteins, nucleic acids or nanomaterials. Characterized by a long half-life in 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.

EVs show apparent specificity and tropism towards tumor cells since they are about ten times more internalized as compared to similarly sized liposomes [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 by virtue of 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 certain types of tumor, acting as mediators in processes such as the invasion of healthy tissues, drug resistance, angiogenesis, and possible escape mechanisms from immune system control. Indeed, tumor cells not only communicate with one another through EVs, but also with healthy cells, in some cases modifying the phenotype thereof or differentiating them. For example, it has been shown that TDEVs are capable of directing the differentiation of normal fibroblasts in the tumor microenvironment into cancer-addicted fibroblasts (CAFs), in turn capable of secreting factors and EVs capable of promoting cancer proliferation, invasion and metastasization [Liu Han, Eric W. F. Lam, and Yu Sun, Extracellular vesicles in the tumor microenvironment: old stories, but new tales. Mol Cancer. 2019; 18: 59]. From this it is easy to understand how the use of EVs secreted from tumor cells as devices for dispensing drugs should be carefully evaluated, however 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 the cells in culture taken from the same patients to be treated, in a mere context of personalized medicine, greatly reduces the events described above.

Many cell lines such as immature dendritic cells, mesenchymal stems (MSC) and lymphocytes are excellent candidates for EV isolation for drug delivery applications, combining the effects of the original load thereof with those of a possible post-isolation load.

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

For these reasons, the use of EVs for therapeutic applications is still far from becoming a well-established reality in clinical practice.

The vesicles can be isolated from any type of biological fluid, such as cell culture supernatants, blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, ascitic fluid, broncho-alveolar lavage fluid, synovial fluid, breast milk, sweat, tears, joint fluid. Precisely because of the variability and biological complexity of biological fluids, the process of isolating the vesicles is very delicate and critical. Various processes are known in the prior art by 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. As a rule, the isolation product consists of a certain amount of EVs resuspended in aqueous saline solutions such as PBS (Phosphate buffered saline) or saline solution.

Patent literature

Patent application WO 2017/161010 describes a method for producing a membrane vesicle from an extracellular vesicle. Such a method includes obtaining open membrane vesicles, removing the content thereof so as to eliminate unwanted molecules, and reducing the possible risk of side effects, capable of reassembling to form new ones. The opening of the membrane of the extracellular vesicle is obtained by treating the vesicles with an aqueous solution having a pH between 9 and 14. Reassembly is achieved through sonication techniques, mechanical vibrations, extrusion through porous membranes, electric current and combinations thereof. Concurrent with the first reassembly step of the emptied vesicles, the newly formed membrane vesicles can be loaded with specific loads.

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

WO 2020/060479 describes extracellular vesicles containing an exogenous load, observing that it could be loaded into the vesicles by means of hypotonic dialysis without other indications. However, extracellular vesicles obtained from red blood cells are discussed and not from biological fluids as in the present invention, which are "liquids expelled and secreted from the human body".

In the article by Nasiri Kenari Amirmohammad et al. entitled: “Methods for loading therapeutic vesicles into extracellular vesicles and generating mimetic-nanovesicle extracellular vesicles”, in METHODS, ACADEMIC PRESS NL (XP086150058), various methods for the exogenous loading of therapeutic vesicles in extracellular vesicles are discussed. It says that "In general, the molecules can be loaded in the extracellular vesicles by mechanical methods such as sonication and extrusion or chemical methods such as the use of surfactants, hypotonic saponin, CACI2 and lipofectamine reagent". In the case of hypotonic dialysis, it is stated that by mixing the EVs and the therapeutic vesicles with a hypotonic solution, due to the osmolarity, the EV membranes permeabilize, allowing the therapeutic vesicles to pass through the membrane. No other indication is provided, nor are the different steps of the method itself clearly defined.

In the article by Gregor Fuhrmann et al., in the JOURNAL OF CONTROLLED RELEASE, XP055385619, a hypotonic dialysis method is reported which includes transferring the extracellular vesicles and drug in dialysis membranes.

It results that the methods mentioned above, using the reagents and procedures described for the lysis and subsequent reassembly of the vesicles, do not fully meet the requirements of the market in terms of clarity, biocompatibility, safety and efficiency. In particular, from the aforementioned prior art, it arises that there is a great need to have standardized and reproducible extracellular vesicle production methods capable of providing biocompatible products with a high targeting and/or loading capacity, maintaining the native state of the proteins, i.e., the form in which the proteins naturally replicate and which is responsible for the function thereof.

Therefore, the present invention aims to overcome the problems and inconveniences described above related to the use of extracellular vesicles with particular application to nanomedicine and the clinic by providing a new simple, safe and economical method to purify, increase the yield, biocompatibility and internalization of extracellular vesicles from products extracted with the most varied techniques available to date.

Summary of the invention

It is the object of the present invention to overcome the limits of the currently known prior art, linked to low yield and heterogeneity of EV isolates, immunogenicity due to the origin of the samples, and inefficiency of loading molecules into the EVs, providing a novel treatment method of EVs post-isolation and/or conservation.

The lysis of the EVs is performed by resuspending the vesicles in double- distilled water: this process causes the rupture of the lipid membranes of the vesicles, favoring the escape of the substances contained therein.

During the experiment performed, the inventors verified that subsequent washing steps in double-distilled water favor the removal of the intravesicular substances released after lysis, allowing the removal of the content of the vesicular lumen, before the reassembly of the membranes of the vesicles which, once purified, are larger in number in terms of particles per unit volume (ml).

A loading process can be coupled to the lysis process. In more detail, molecules and materials such as drugs, nanoparticles, nucleic acids, peptides or biological derivatives can be dispersed in the hypotonic solution in the presence of extracellular vesicles. Thereby, following lysis, the new vesicles will be reassembled, encapsulating the molecules and/or nano-materials previously dispersed in solution.

Brief description of the drawings

The present invention will be described below by means of some preferred embodiments, provided by way of non-limiting example, with reference to the accompanying drawings, in which:

Figure 1 is a graph of the concentration of extracellular vesicles measured by Nanoparticle Tracking Analysis (NTA);

Figure 2 is a graph of the distribution of frequency classes of extracellular vesicle sizes;

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

Figure 4 is a graph of the internalization of extracellular vesicles detected by cytofluorometry, expressed as a percentage of positive events;

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

Figure 6 is a graph depicting the load of fluorescent gold nanoparticles in lysed vesicles, detected by cytofluorometry and expressed as average fluorescence intensity.

Detailed description of the invention

The first part of the method shown in the present invention includes diluting extracellular vesicles in a hypotonic solution with respect to that of the starting sample, such as double-distilled water, capable of triggering a lysis and reassembly process of the vesicles, the purpose of which is to obtain a larger population of EVs than the starting one and the purification of the EVs from the physiopathological load thereof.

It is a further object of the present invention to provide a method for obtaining new vesicles having specific and advantageous features which can be used for different types of treatments and/or for the targeted release of active ingredients.

In the following description, the use of "e.g.", "etc.", "or" indicates nonexclusive alternatives without limitation, unless otherwise indicated; the use of "also" means "including, but not limited to” unless otherwise indicated; the use of "includes / comprises" means "includes / comprises, but not limited to” unless otherwise indicated.

As used herein, the term "extracellular vesicle" comprises exosomes, shedding vesicles, microparticles, ectosomes, ectosome-like vesicles, membrane vesicle, and refers to a vesicle derived from any cell, comprising a membrane delimiting an inner space, typically of average diameter between 20 nm and 400 nm, better 40 nm to 300 nm, even better 50 to 250 nm, containing various endogenous macromolecular entities therein, 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 outer coating of cells and organelles with the typical structure thereof consisting of a double lipid layer forming a semipermeable barrier which separates an inner compartment from the outer environment, allows the passage of a particular compound and is capable of merging with other membranous systems which have the same or similar structure, then exposing the content of the extracellular vesicle or dissociating the content from the membrane once open.

The method for treating extracellular vesicles described in the invention can be applied either immediately after the isolation process, or after any time on isolated and suitably conserved vesicles.

In particular, according to the preferred form of the invention, the method for treating post-isolation extracellular vesicles comprises the following steps: a) providing an amount of extracellular vesicles; preferably suspended in an aqueous saline solution (e.g., PBS, saline, culture medium); b) treating the solution containing the extracellular vesicles preferably with double-distilled water, or in any case with hypotonic solution with respect to the sample, to obtain the opening of the lipid membranes of the EVs. The formation of an osmotic gradient between the inner compartment of the same vesicles, rich in salts, miRNA, DNA, proteins and enzymes, and the hypotonic compartment of double-distilled water causes the rupture of the membrane of the treated EVs and the escape of the material contained therein. The open or fragmented vesicles are reorganized in a more energetically favorable state, i.e., the double lipid layers are re-associated, including therein part of the liquid in which they are dispersed; c) subjecting the lysed sample to washing (preferably with doubledistilled water) for the removal of the material contained in the vesicular lumen. This operation is particularly important for biomedical and/or therapeutic applications both for the possibility of recovering and analyzing the content of the lysed vesicles, and to avoid immuno-stimulatory and/or inflammatory effects in the treatment recipients; d) optionally, the lysed EVs can be engineered and loaded for nanotechnology and drug delivery applications by adding nanomaterials, drugs, nucleic acids, fluorescent molecules in the aqueous solution during the lysis step, so that they are loaded in the vesicles during their reassembly.

The lysis and purification process of EVs from the pathophysiological load is particularly effective, fast and easy to perform and allows obtaining neo- assembled vesicles with improved capacities as compared to the starting population, such as the internalization efficacy in tumor cells. Such an aspect makes the use thereof particularly interesting and efficient in the therapeutic and personalized medicine fields.

Once opened and purified of most of the molecules contained therein, in a hypotonic environment, extracellular vesicles tend to re-associate, selfassembling 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, if 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 physiopathological content, it is possible to proceed with a loading step (step d), to be carried out during the first or a subsequent lysis operation, adding the component to be loaded into the EVs in 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 the lysis, the newly-formed vesicles encompass them therein.

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 volume (ml) as compared to the control sample (Figure 1), with a size distribution the higher frequency classes of which are concentrated between 100 and 220 nm (Figure 2) and an ability to be significantly more internalized in both healthy and tumor cells. More specifically, again referring to internalization in cells, an increase in internalization in tumor cells approximately 5 times greater than that in healthy cells was observed (Figure 4). Furthermore, the lysed vesicles are capable, following a short incubation, of loading molecules or nanoparticles, such as gold nanoparticles (Figure 5) with significantly higher efficiency (p<0.001) than the control sample (Figure 6). For such features, the vesicles obtained by the method according to the present invention lend themselves to a wide range of applications, including 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 such a method have been tested under different operating conditions, as amply described in the examples of the experimental section below.

However, it is to be understood that there is no intention to limit the invention to the specific embodiments discloses, but on the contrary, the invention is intended to cover all modifications, equivalent forms and variants falling within the scope of the invention as defined in the claims.

EXAMPLES

Example 1 - Osmotic lysis of vesicles

The EVs used by the inventors to evaluate the goodness and efficiency of the method of the invention were isolated from B lymphocytes by differential ultracentrifugation and conserved at -80°C for various times in saline solution. For the test, an amount of 30 pl of EVs was diluted 1:50 in double-distilled water so as to perform the osmotic lysis; for the control sample, 30 pl of EVs were diluted again with a 1:50 ratio in saline (hypotonic solution with the vesicular lumen).

Both test and control samples were resuspended with “vortex” stirrer for 3 minutes and incubated at 37°C for 6 minutes, then further stirred with vortex for 1 minute.

The samples were then subjected to ultrafiltration with Amicon Ultra-0.5 ml 50 kDa (Merck Millipore). A volume of 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 ultrafiltration product was then resuspended in double-distilled water, saline, or in medium, in a variable volume depending on the application.

Example 2 - Analysis of the concentration and size distribution of extracellular vesicles purified of the physiopathological load thereof

The extracellular vesicles of the test sample, resuspended in 150 pl double- distilled water (1:5 with respect to the starting concentration), or those of the control, in 150 pl of saline solution (1:5 with respect to the starting concentration), treated as described in example 1, were subjected to concentration and size distribution analysis using the Nanoparticle Tracking Analysis (NTA) technique, using a Nanosight NS300 (Malvern Panalytical) provided with a laser source with

1 = 505 nm, a syringe pump and NTA software vers. 3.4.

The measurements, with the samples diluted so as to ensure an adequate particle/frame, were performed by acquiring three 60-second videos each with a pump infusion rate 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.

Each analysis was performed on each sample five times (n=5) and expressed as the average value of the results obtained. The measurement data obtained on the extracellular vesicles after osmotic lysis (EVsl) and the respective controls after lysis in saline (EVsl CTRL) after a first lysis cycle are reported in the following Table 1 and shown in Figure 2.

Table 1 - Frequency classes of lysis product size averages in descending order (n=5). EVsl CTRL is the control sample, EVsl is the test sample.

The results obtained show that the distribution order 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 includes the 5 osmotic lysis step is much more concentrated (3xlOⁿ±7xlO¹⁰ vesicles/mL) than the control group in which the lysis process is replicated but in an isotonic solution (7.4x10⁹± 1.7x10⁹ vesicles/mL), as shown in Figure 1, although in both cases the dimensions of the vesicles produced are well distributed with a peak around 100 nm (Figure 2). 0 The phenomenon could be explained by highlighting how with the lysis of the starting vesicles, many new vesicles could have been formed from the postlysis reassembled lipid bilayer patches. The internalization of part of the lysis and/or washing liquid does not lead to an apparent reduction in the size of the new population. Example 3 - Determination of the extracellular vesicles protein content

The protein content of the newly formed vesicles after a first (EVsl) and a second cycle (EVs2) of osmotic lysis was determined by Bradford assay. The analyses conducted on the samples three times showed that the vesicles, obtained by the lysis process of the invention, are significantly richer than the control, both after a single lysis cycle (EVsl) and after a second lysis cycle (EVs2). The results are presented in Figure 3 which shows the graph of the averages and standard errors of the measurements obtained. Statistical comparisons made by 2-way ANOVA. *: p<0.05.

Example 4 - Internalization capacity of extracellular vesicles obtained by osmotic lysis

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

5 pg/ml EVs per sample (test and control), per line (lymphocytes and Daudi) and per timeframe (24 and 48 hours), were labeled with Wheat Germ Agglutinin conjugated with Alexa Fluor 647 dye. The marked vesicles were treated as described in Example 1. For each line and timeframe, 500 pl of treatment was plated with 200000 cells/ml for both the control and for the test.

After 24 or 48 hours of incubation the cells were recovered, centrifuged and resuspended in 500 pl PBS for internalization analysis in cytofluorometry. The analyses were conducted with the Guava Easycyte 6-2L flow cytometer (Merck Millipore) with a flow rate of 0.59 pL/s, using the red laser (Xe_X =642 nm) on IxlO⁴ events. Data from untreated cells (0 pg/ml) are used as a reference. The results are represented in terms of positive events, characterized by a shift in the fluorescence intensity of Red-R (661/15 filter emission) and the percentages of positive events were compared with untreated cells. The results obtained are shown in Figure 4 and show that the neo-vesicles obtained after the lysis process are internalized to a significantly higher extent 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 significantly more internalized by the tumor cell line as compared to the healthy one, and this difference is already detected after 24 hours of treatment. Statistical comparisons made by 3-way ANOVA three times. *:p<0.05, **:p<0.001.

Example 5 - Loading gold nanoparticles into 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 pg of lymphocyte-derived EVs and 10 pg of commercial gold nanoparticles (AuNPs) with a diameter of 5 nm were used. For the test, the EV and AuNP solution was diluted 1:50 in double-distilled water to perform the osmotic lysis and loading, while the control sample was diluted again with a 1:50 ratio in saline.

Both test and control samples were resuspended with “vortex” stirrer for 3 minutes and incubated at 37°C for 30 minutes under stirring at 180 rpm, then further stirred with vortex for 1 minute.

Example 6 - Load capacity of extracellular vesicles obtained by osmotic lysis

The load capacity of extracellular vesicles, produced by the method of the invention, was evaluated by transmission electron microscopy (TEM). In detail, 5 pg of EVs and 10 pg of AuNPs were treated as described in Example 5. The sample was deposited on a TEM analysis screen and contrasted with uranyl acetate. As can be noted from Figure 5, the AuNPs, opaque to the electrons, were successfully loaded in the clearest, because less electron dense, extracellular vesicles.

Example 7 - Load capacity of extracellular vesicles obtained by osmotic lysis

The load capacity of the extracellular vesicles, produced by the method of the invention, was evaluated by adapting the analysis protocol of the 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 pg of EVs and 5 pg of Cy5 -labeled AuNPs per sample (test and control) were treated as described in Example 5.

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

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

The analyses were conducted with the Guava Easycyte 6-2L flow cytometer (Merck Millipore) with a flow rate of 0.12 pL/s, using the red laser (Xe_X Q

=642 nm) on 5x10 events. The results are depicted in Figure 6 in terms of mean fluorescence intensity, and show how EVs treated with the proposed method, when loaded with labeled AuNPs, have a statistically higher fluorescence than the control ones kept in PBS and therefore not lysed. Statistical comparisons performed by t-test on ten replicates, **:p<0.001.

All the experimental data have confirmed that the present invention has the following advantages with respect to the current solutions: reduction of the costs of nanotechnological processes related to the use of EVs; increased reproducibility in the preparation of EV samples; increased reliability in terms of reducing the possibility of generating possible rejects or inductions of unwanted differentiations /transformations; and compatibility with industrial-scale production.

Claims

1) A method for obtaining a larger, more homogeneous, safer, more internalizable and engineerable population of extracellular vesicles EVs, extracted from biological fluids following various isolation and/or conservation methods, where the isolation product consists of an amount of EVs, characterized in that it includes subjecting said isolation product to a lysis process by adding doubledistilled water in a sufficient amount to cause, by osmotic imbalance between the inner compartment of the vesicles and the surrounding hypotonic environment, the rupture of the lipid membranes of the treated EVs and the escape of the material contained in the vesicular lumen, without denaturing the membrane proteins.

2) A method according to the preceding claim, characterized in that, after lysis, the open or fragmented vesicles are reassembled including therein part of the liquid in which they are immersed.

3) A method according to the preceding claim, characterized in that the lysed sample is subjected to washing to remove the content of the vesicular lumen.

4) A method according to any one of the preceding claims, wherein the isolation product is obtained with any one of the available techniques including ultracentrifugation, ultrafiltration and the use of various commercial kits such as optiprep, exoquick, exo-spin.

5) A method according to claim 1, characterized in that the purified vesicles obtained from the lysis step are subjected to further lysis steps with double-distilled water performed in conjunction with the addition of components to be loaded in the lysis solution, thus obtaining that following the lysis the new vesicles are reorganized in an aqueous environment by re-association and selfassembly, so as to encapsulate the molecules and/or nanomaterials previously dispersed in solution.

6) A method according to claim 5 which is usable for the production of EVs for applications of nanomedicine, regenerative medicine and drug delivery capable of combining the effects of the natural content of the EVs themselves with a new one engineered a posteriori. 7) A method according to the preceding claims which allows improving the loading and internalization efficiency of nanoparticles or molecules of therapeutic or diagnostic interest in newly formed vesicles in conjunction with the lysis process.

8) A method according to any one of the preceding claims, characterized in that it optimizes the yields, thus reducing the costs thereof, of EV production processes as it is capable of increasing the concentration of EVs obtained post-isolation up to two orders of magnitude.

9) A method for treating post-isolation extracellular membrane vesicles comprising the following steps: a) providing an amount of extracellular vesicles obtained following any isolation and/or conservation process; b) treating the solution containing the extracellular vesicles by diluting it with a hypotonic solution, such as double-distilled water, with a dilution factor from 1:25 to 1:80, vortexing it for about 3 minutes, incubating it at 37 °C for 5-10 minutes, and then vortexing it again for 1 minute, to obtain the opening of the lipid membranes of the EVs present, due to the osmotic gradient between the inner compartment of the same vesicles and the surrounding hypotonic environment, resulting in the escape of the material contained therein; c) washing the lysed sample to remove the material which was contained inside the vesicles; d) reassembling said membranes with the formation of new vesicles purified of the original physiopathological load thereof.

10) A method for treating post-isolation extracellular membrane vesicles according to claim 9, characterized in that it further includes the step of loading components for applications of nanomedicine, regenerative medicine and drug delivery in the lysed and reassembled vesicles.

11) A method according to claim 9, characterized by the use of a dilution factor adapted to cause the lysis of the starting material, adjustable depending on the process. 19

12) A method according to claim 10, characterized in 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. 13) Extracellular vesicles comprising vesicles consisting of lipid membranes derived from extracellular vesicles isolated, purified and reassembled in a hypotonic environment according to the method of claim 9 without denaturing the membrane proteins.

14) Extracellular vesicles to be used as diagnostic and therapeutic solutions in nanomedicine and regenerative medicine: comprising vesicles consisting of lipid membranes derived from extracellular vesicles isolated, lysed, purified and reassembled in a hypotonic environment, according to the method of claim 9.