IT202100027167A1 - EXTRACELLULAR VESICULES DERIVED FROM MILK AND PROCESS TO ISOLATE THEM - Google Patents

Description

DESCRIPTION of the Invention entitled:

?Extracellular vesicles derived from milk and process for isolating them?

Field of Invention

The present invention concerns the field of biotechnology and, in particular, extracellular vesicles (EVs) derived from milk. The present invention provides a process for isolating such extracellular vesicles from milk and milk-related fluids particularly suitable for industrial production thanks to the possibility of of its scaling and its capacity? to maintain the integrity? structural structure of extracellular vesicles, so? as for its capacity? to provide solutions of homogeneous purity, the purity obtained being superior to that of preparations previously known in the field. The invention further concerns compositions of extracellular vesicles derived from milk by the said process, which show surprisingly high recovery/extraction yields and provides EVs which present, in particular, few contaminating proteins and other contaminants, making them suitable for chemical modification, for example by labeling with targeting molecules or loading of active ingredients for use in pharmaceutical, veterinary, cosmetic and nutraceutical applications. The invention also concerns compositions of milk-derived extracellular vesicles and methods for preparing the same, in which the milk vesicles have been loaded, for example, with Amphotericin B or its derivatives, thus providing potent antifungal, antiparasitic and -infectious for oral use.

The invention also provides a method for the systemic delivery of milk EVs and their cargoes, which results in surprisingly efficient systemic absorption and allows them to reach numerous organs through the respiratory tract.

Technical background

Extracellular Vesicles (EVs) are a known category of components derived from cell membranes including, among others, exosomes, ectosomes, apoptotic bodies and microvesicles. EVs, and in particular exosomes, have attracted enormous attention as part of a not yet fully understood long-distance cell-to-cell communication system that opens up entirely new avenues for clinical biomarkers and diagnostic products, cell-free cell therapy, vaccines, and potential drug carriers.

Exosomes are defined as particles enclosed in a double membrane, having a size of approximately 40-200 nm, derived from multivesicular bodies (MVBs) that are produced and released into the extracellular space continuously by virtually all cell types. eukaryotic. Furthermore, ? Exosomes have been shown to be present in a wide range of body fluids, such as blood, saliva, urine, etc. as in mammalian milk.

Compared to exosomes derived from cell cultures, which are typically collected from the supernatant of human cells or from cultured higher organisms, exosomes derived from natural sources used in the food industry (milk, fruit juices, etc.) possess the clear advantage of being available in quantity? relatively high without the need? to establish biotechnological production methods, cellular bioreactors and processes for their production. However, the concentrations at which exosomes are present in different starting materials are far below the levels useful for being used directly as drug carriers or as therapeutic agents (as well as drug carrier starting material or as a starting point for therapeutic agents), and the presence of additional components (such as casein and other proteins, lipoproteins, sugars or fatty acids) limits applications in subsequent phases.

Consequently, there is the necessity? of scalable methods for the isolation and purification of exosomes from these attractive natural sources.

In recent years, different processes have been described in the literature for the isolation of exosomes derived from cell cultures in the laboratory. These methods include many different procedures such as, for example, differential and high-speed ultracentrifugation, density gradient centrifugation, size exclusion chromatography, affinity chromatography, affinity-based adsorption and using heparin, membrane-based separations, microfluidics, or polyethylene glycol-based precipitation, so? such as various commercial isolation kits, such as, for example, ExoQuick? (Antes, 2013).

? It is important to note that the above isolation processes were reported primarily by researchers conducting laboratory-scale EV isolations for analytical, mechanistic, or cell-based investigations, with relatively low volumes to process. Furthermore, these methods have been mainly applied to the isolation from the supernatant of cell culture-derived exosomes, which contains a relatively low concentration of contaminating proteins and higher molecular weight impurities.

In contrast, isolation of exosomes from natural sources poses additional challenges; Do typical natural sources such as milk contain a high degree of dry matter and/or large quantities? of fatty acids, sugars, other emulsifiers and/or contaminating proteins. Furthermore, different starting materials represent different matrices with a wide range of potentially toxic, interacting, cross-reacting or interfering contaminants (e.g., other proteins, peptides, lipids, carbohydrates, oligonucleotides such as micro-RNA or other natural products and derivatives). Consequently, each matrix poses specific and unique challenges in the isolation of EVs or EV-enriched fractions, which have defined characteristics and quality. reproducible and constant. This aspect ? particularly important, given that only such reproducible preparations of high purity EVs are suitable for further functionalization, loading of drug molecules and/or modification for different targeting strategies commonly employed in future generation drug delivery systems.

In the case of milk-derived EVs, ultracentrifugation to collect the EVs into a pellet or defined fractions on density gradients? ? still the method of election used for their isolation. However, in addition to the need? of specialized and expensive equipment, ultracentrifugation is? difficult to scale industrially from a technical point of view, ? laborious and time consuming. Furthermore, given that ultracentrifugation separates molecular portions based on their densities? relative, it can? lead to the co-precipitation of aggregating proteins, lipoproteins and various oligonucleotides, such as small and micro RNAs. Such contaminants can lead to a significant overestimation of the actual yield of the preparation, especially when the evaluation is based on methods that determine the total protein content of a preparation. In addition, ultracentrifugation via pelletization itself promotes? the formation of clusters of exosomes and EV aggregates, potentially leading to vesicle fusions, distortions of size distributions and alterations of cellular uptake and other activities. biological. Finally, the ultracentrifugation process produces yields and quality? variables, making it difficult to standardize the process.

Alternative methods known in the art are based on commercial kits using affinity-based isolation strategies, which are not only significantly limited with respect to the scale and yield of the preparations, but also require harsh conditions for the elution of the vesicles linked to affinity resins? with unknown consequences for the integrity? and the function of EVs.

In a recent publication, Marsh et al. propose, in order to purify EVs from bovine milk, two slightly different protocols, both essentially based on the combined use of the complexing agent EDTA, to solubilize casein micelles and for the final fractionation of the EVs via size exclusion chromatography after prepurification via ultracentrifugation or cross-flow filtration through a membrane with a molecular cut-off of 500 kDa. Therefore, both of these protocols require a chemical treatment of milk that uses EDTA for the solubilization of casein aggregates and micelles. Furthermore, the EVs are recovered, at the end of the process, after a final passage through a Sepharose column (SEC, Size Exclusion Chromatography). These two phases (i.e. the use of EDTA and SEC), together with the other phases of the process (ultracentrifugation or filtration) are defined as mandatory, in order to obtain the quality desired of the final EV product. On the other hand, these phases pose in themselves an additional limitation to the process. First, the use of EDTA solubilizes casein aggregates, increasing the amount of of freely diffusing low molecular weight impurities that must be eliminated by tangential flow filtration (TFF). This leads, over time, to the formation of encrustations of the membrane, thus limiting, as the authors themselves have commented, the purity that can be achieved. be obtained through TFF. Secondly, the need? of using fractionation by SEC to further purify the material after TFF, leads to obtaining multiple different fractions of EV preparations having variable purities and compositions, with different levels of impurities, further limiting the possibility process scaling.

Consequently, in the field of milk-derived exosomes there is still the need? to develop an isolation process that avoids the disadvantages of ultracentrifugation, that is suitable for the manipulation of large volumes of starting fluid (i.e., milk), that can work without specialized laboratory instrumentation, and that allows to obtain preparations of exosomes having a quality? homogeneous, well defined and reproducible.

With reference to the use of EV preparations for drug delivery, particular attention must be paid not only to the EVs and their preparation, but also to the types of cargo with which they are used? the delivery system consisting of EVs was used. Classic small molecules often show bioavailability? oral in the order of 20-100%, while biological macromolecules and their derivatives, such as oligonucleotides and proteins, are not considered orally bioavailable. In general, these molecules, despite various attempts in the literature, are considered for use only for intravenous or subcutaneous injection or for local applications. However, there are also numerous natural products having molecular characteristics generally described in the field of medicinal chemistry as not meeting the "rule of five" criteria.

and similar assessments that are rule-based and/or show no bioavailability? oral therapeutically useful. Now, several limited attempts have been made to load milk EVs with cargo molecules.

WO2018102397A1 describes preparations of milk EVs loaded with biological macromolecules. However, the loading procedures shown are limited to biological macromolecules modified mainly with a hydrophobic anchor in order to allow a physical insertion or interaction of the macromolecular cargo with exosomes and/or by some kind of physico-mechanical destruction of the EV membranes , for example through sonication or freeze-thaw cycles.

US10420723B2 describes some preparations of milk EVs with a selection of natural products, loading carried out by suspending the drug in PEG-400 or using ethanol as a co-solvent and, after co-incubation, separating the EVs from excess free drug using centrifugation and ultracentrifugation methods, such as those used for EV isolation. Such a procedure suffers from the same limitations (scalability, yield, purity, etc.) as the procedures commented above for the isolation of milk EVs, and has the additional disadvantage of the non-specific binding of the drug to the non-vesicular proteins present per se ? in EV samples, particularly when ultracentrifugation-based processes are first used for their isolation.

EP3620519A1 exposes the use of extrusion to load milk EVs with RNAs modified with a hydrophobic anchor.

Such procedures openly so? generalized tests do not automatically lead to obtaining biologically active drug carriers but require laborious and case-by-case experimentation, and are subjected to the identification of a feasible combination by testing an unlimited list of additives, variables and optimization parameters without an a priori guarantee successfull. An example that demonstrates that this principle is still valid for EVs and EV-derived drug carrier systems? provided by European Journal of Pharmaceutics and Biopharmaceutics, Volume 158, 2021), in which it is not? No functional effect was observed after oral administration of RNA-loaded milk EVs, as described in EP 3620 519 A1.

Therefore, new and improved preparation methods for loading and applying milk EVs loaded with specific molecules are needed.

Purpose of the invention

A first aim of the present invention? is to provide a method to isolate EVs from the milk of different mammalian species, as commonly produced in the dairy industry. These isolated EVs have high purity and reproducible characteristics.

A further purpose of the invention? is to provide a method to isolate EVs from the colostrum of different species, including human colostrum, so? as from milk already? processed of different species, such as milk powders (produced by freeze-drying or spray-drying) or pasteurized, skimmed or otherwise processed milk and powders derived from such processed milk, which still contain at least partially intact EVs.

Another purpose of the present invention? that of providing EV preparations particularly suitable for further refinement and/or drug loading via methods described in the art.

Another purpose of the present invention? to also provide new drug-loaded EV milk preparations based on the antifungal and antiparasitic drug Amphotericin B (AmB).

Yet another purpose of the present invention? is to provide effective methods for the systemic delivery of EV preparations resulting in drug exposure in different mammalian organs.

These purposes, among others, will be addressed? in more detail in the following paragraphs, together with experimental data and examples, in order to fully clarify the subject matter of the present invention.

Summary of the invention

Does the present invention address the needs? previous and other types, with reference to the prior art, providing a new process suitable for the industrial isolation of exosomes from different types of milk as starting material. The present invention also concerns compositions obtained with this isolation process, which have high purity with reference to the possible presence of contaminating proteins, protein aggregates, aggregates of organic impurities and lipids, and their use as nano-carriers for the release of drugs .

The isolation method of the present invention is ? based on the surprising discovery that a process consisting of a series of distinct steps, including an enzyme-based casein coagulation step, followed by ultrafiltration using membranes having specific pore sizes and controlled ionic strength dialysis, achieves the isolation, the purification and concentration of milk-derived exosomes (EVs) with high efficiency.

The surprising effect of the discovery of the invention? related to the fact that starting materials rich in proteins and fats usually suffer from the lack of possibility? to be highly concentrated due to aggregation, turbidity? or increasing precipitation. At the same time, dialysis with pure water can? lead to the depletion of stabilizing ions that are necessary to prevent protein aggregation.

Furthermore, the process of the invention comprises a pre-determined series of purification steps, leading to the isolation of EV-rich extracts having average size ranges of 30-200 nm and bearing the surface-bound protein markers characteristic of exosomes. This series of purification steps typically includes a first step involving coagulation of most of the protein components of milk, followed by their removal through filtration or other separation methods, and a second step, including concentration and ionic strength dialysis. controlled EV fraction via ultrafiltration using specifically selected pore size membranes.

Therefore, the invention allows the isolation of large quantities? of EVs, maintaining their intrinsic biological function to a degree comparable with, or superior to, that of EVs isolated by methods currently employed in accordance with the prior art, which are typically based on differential ultracentrifugation or gradient ultracentrifugation of density? which are characterized, as already? said, by numerous disadvantages, i.e. they take time, they are difficult to standardize, they are laborious and limit the possibility? of scale-ups.

Although the first step of the process, i.e. casein coagulation, can technically be carried out by means of acid-induced coagulation, this solution is less preferred than enzyme-based coagulation, due to the functional consequences observed in the preparations finally obtained when ? used an acid. Indeed, EVs isolated using the latter show on average larger particle sizes and a lower abundance of EV marker proteins, such as TSG101 or CD9 in Western blot analyses. Thus, acid-induced casein coagulation appears to provide EV isolates that do not meet all the desired requirements.

Therefore, in accordance with the present invention, the procedures for obtaining milk EV preparations largely free of non-vesicular protein contaminants have been applied to obtain, for example, milk EVs of two different species (bovines and goats), loaded with Amphotericin B. When different loading conditions for Amphotericin B were compared, ? It was surprisingly found that co-incubation for a quantity of time in a specified ratio of Amphotericin B and milk EV, provided functional Amphotericin B-loaded EV preparations, containing the highest drug load? high in Amphotericin B with a stable association between drug and carrier. TFF-based dialysis with 750 kDa membranes enabled ultracentrifugation-free re-isolation of biologically functional milk EVs loaded with Amphotericin B, suitable for treating conditions for which ? indicated Amphotericin B in accordance with medical practice.

In the scientific literature, milk EVs have generally been considered as drug carriers for local applications or to obtain products previously found unsuitable for oral drug delivery. When experiments with EV milk preparations have been carried out in accordance with the present invention,? It was surprisingly found that the EV milk preparations of the invention can be used to effect extremely efficient transport across the respiratory epithelium and are capable of generating high levels of systemic exposure in numerous tissues, for example in the liver, kidney, heart or brain and potentially in others.

Description of the Figures

Figure 1: Characterization of different stages of preparation of EV from bovine milk using Nanoparticle Tracking Analysis (NTA). Particle size distribution histograms, mean value and mode were determined using NTA. Data are shown for representative samples of whey (Figure 1.1, a-c), pre-processed whey (Figure 1.2, d-f), and EV preparations after tangential flow filtration (TFF) of pre-processed whey using two different membranes: with 750 kDa pores or with 100 kDa pores (Figure 1.3, g-i). Particle concentrations are shown as yields, calculated backwards with reference to the volume of whey. For material processed by TFF, yields and particle sizes are shown for both the whey and the retentate (the fluid retained by the filter membrane) of preprocessed milk (graphs h and i). Samples were measured in triplicate; error bars represent standard deviations over the three technical replicates.

Figure 2: Ratios between particles and proteins of preparation phases of different EVs from bovine milk. Particle to protein ratios were determined by measuring particle numbers via NTA and total protein concentration ? was determined using Bradford reagent. The results are expressed as the average of n=3 experiments. (a) Three different samples (batch 1 - 3) of pre-processed whey, (b) two examples of TFF processing intermediates: The pre-processed whey ? was diluted 1:1 (bar ?input?), concentrated three times, and dialyzed three times (bar ?retentate?) using a membrane having a pore size of 750 kDa or 100 kDa.

Figure 3: Characterization by size exclusion chromatography of preparation steps of different EVs from bovine milk. Samples were injected into a Superdex S200 column and eluted at 0.8 ml/min at 4?C in PBS at pH 7.4 (Example 14). Chromatograms recorded at 280 nm are shown in Figure 3.1 for whey (a), pre-processed whey (b) and EV preparation after processing by TFF (c). The whey chromatogram? shown as an overlay of the samples before (grey, Example 1) and after (black, Example 2) filtration. Panel (b) shows two independent batches of preprocessed whey. Panel (c) shows milk EV preparations after processing by TFF on membranes of pore size of 750 kDa (left) or 100 kDa (right). The black rectangle highlights the EV peak. Figure 3.2, panel (d) shows two representative chromatograms of final milk EV preparations following isolation of EVs according to the method of the present invention using TFF through a membrane having a pore size of 750 kDa (Example 5) .

Figure 4: Relative depletion of protein contaminants during preprocessing (100 kDa molecular weight cutoff (MWCO)) of EV from bovine milk. The relative depletion of protein contaminants, as determined by size exclusion chromatography on a Superdex S200 column, is ? was determined by comparing the area (black) and height (gray) of peaks 1-6 of whey (Example 1) and pre-processed whey (Example 4). Figure 4.1, panel (a) shows chromatograms recorded at 280 nm with peak assignment. Panel (b) shows the peak areas and heights normalized to the EV peak (peak 1). The molecular weight? was determined using an independent calibration cycle with a size exclusion standard. The depletion in % of the main contaminants? shown as a function of calculated molecular weight in Figure 4.2, panel (c).

Figure 5: Characterization of different EV preparations from bovine milk by Western blot. Western blot analysis of (a) CD9, (b) tsg101, and (c) MFGE8. HEK cell lysate (CL) ? served as a positive control. Bovine milk EVs prepared by different methods are shown: By ultrafiltration using a membrane having a pore size of 100 kDa followed by Size Exclusion Chromatography (UF SEC), by Tangential Flow Filtration using a membrane having a pore size of 750 kDa ( TFF, Example 5) and via Ultracentrifugation (UC of EVs purified from milk, Example 12).

Figure 6: Typical preparation of bovine EVs with the method of the present invention. The characteristics of EV from bovine milk processed by Tangential Flow Filtration (TFF) using a membrane having a pore size of 750 kDa and the corresponding pre-processed whey TFF starting material: (a) SEC chromatogram recorded with absorbance at 280 nm (b) particle size distribution (measured by NTA, histogram represents one of three measurements), (c) particle to protein ratio based on particle numbers measured by NTA and total protein measurements by Bradford , n=3, (d) particles per ml of whey (e) particle size measured by NTA, n=3. The lot shown? was processed through 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS at pH 7.4 (Example 5).

Figure 7: Cellular uptake of bovine milk EVs in human lung adenocarcinoma cells (A459). The TMR/Cy5-labeled EVs obtained in Example 17 were added to A459 cells and cellular uptake ? was analyzed by fluorescence microscopy as in Example 17. Representative images obtained by wide-field viewing (?imaging?) with a 0.95 NA 40x objective are shown in panel (a). BW images: DAPI channel for nucleus staining, TMR channel for EV detection, Cy5 channel for EV detection, and TMR-Cy5 FRET channel; grayscale brightfield images. Quantification of dose-dependent EV uptake at 6 hours in panel (b) for TMR/Cy5-labeled bovine milk EVs stored at 4?C or -80?C. Individual curves represent independent biological replicates, while 5 individual images were taken per well. Error bars illustrate standard deviations over individual images.

Figure 8: Preparation of EV from goat's milk. The characteristics of the following intermediates of EV preparation from goat milk are shown: Goat milk whey, pre-processed goat milk whey and purified EV from goat milk on TFF membrane having pore size of 750 kDa, processed by 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS at pH 7.4). Samples were characterized in terms of (a) particle size distribution (measured by NTA, histogram shown for one of three measurements), (b,c) particles per ml of whey, and particle size, both measured by NTA ( n=3) and particle to protein ratios based on particle numbers measured by NTA and total protein measurements by Bradford (n=3).

Figure 9: Characterization of EVs from goat's milk using analytical size exclusion chromatography. SEC chromatograms recorded with an absorbance at 280 nm are shown for (a) goat's milk whey, (b) pre-processed goat's milk whey, and (c) EV from goat's milk processed on a TFF membrane having pore size of 750 kDa. Purification by TFF? was performed by 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS at pH 7.4.

Figure 10: Typical preparation of EV from goat's milk with the method of the present invention. Characteristics of EV from goat's milk processed by Tangential Flow Filtration using a membrane having a pore size of 750 kDa and the corresponding pre-processed whey as starting material: (a) SEC chromatogram (recorded with absorbance at 280 nm ) (b) particle size distribution (measured by NTA, histogram represents one of three measurements), (c) particle to protein ratio based on particle numbers measured by NTA and total protein measurements by Bradford, n= 3, (d) particles per ml of whey (e) particle size measured by NTA, n=3. GW1PPEK1 ? was processed through 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS at pH 7.4.

Figure 11: Cellular uptake of goat milk EVs in human lung adenocarcinoma cells (A459). The goat milk EVs obtained in Example 11 were labeled with TMR and Cy5 as described for bovine milk EVs, added to A459 cells, and cellular uptake ? was analyzed by fluorescence microscopy as in Example 17. Representative images obtained by wide-field viewing with a 0.95 NA 40x objective are shown in panel (a). BW images: DAPI channel for nucleus staining, TMR channel for EV detection, Cy5 channel for EV detection, and TMR-Cy5 FRET channel; grayscale brightfield images. Quantification of dose-dependent EV uptake at 6 hours in panel (b) for TMR/Cy5-labeled goat milk EVs. Individual curves represent independent biological replicates, while 5 individual images were taken per well. Error bars illustrate standard deviations over individual images.

Figure 12: Characteristics of EV from bovine milk processed by Ultracentrifugation. (a) chromatogram of SEC with absorbance at 280 nm (b) particle size distribution (measured by NTA, histogram shown for one of three measurements), (c) particle-to-protein ratio based on particle numbers measured by NTA, and total protein measurements by Bradford, n=3, (d) particles per ml of whey (e) particle size measured by NTA, n=3.

Figure 13: Characterization of bovine milk-derived EVs by negative-stained TEM. EVs from bovine milk from Example 5 were analyzed by TEM with negative staining and are shown at 40,000x (a) and 80,000x (b) magnification.

Figure 14: Quantification of Amphotericin B using high performance liquid chromatography. RP-HPLC chromatogram of Amphotericin B (50 ?l of a 2.5 ?M solution) with isocratic elution (Acetonitrile/20 mM EDTA (55% / 45% vol/vol, 1ml/min) on a C18 column and detection at 405 nm (a) and standard curve for different concentrations of Amphotericin B (AmB) derived from the peak area (b).

Figure 15: Concentration dependence of AmB loading on EV from goat milk. Goat milk EVs at 1 nM were incubated with increasing concentrations of AmB and the concentration of AmB loaded ? was quantified by RP-HPLC (a). AmB at 2 ?M ? was incubated with increasing concentrations of EV from goat's milk and the concentration of AmB loaded ? was quantified by RP-HPLC (b).

Figure 16: Co-fractionation of AmB with EV from bovine and goat milk using size exclusion chromatography. The EVs from bovine milk in Figure 16.1 (a) and goat milk in Figure 16.2 (b) at 1 ?M were loaded with Amphotericin B at 2 ?M by coincubation at room temperature for 6 hours and the samples were analyzed by high temperature chromatography size exclusion with detection by UV-VIS at 406 nm before and after washing by ultrafiltration. Chromatograms for free AmB and free milk EV are shown for comparison.

Figure 17: Goat milk EV uptake in A549 cells with and without AmB loading. Goat milk EVs were labeled with TMR-NHS and Cy5 NHS, loaded with AmB, and incubated with A549 cells for 6 hours. The uptake? was then analyzed by large-field fluorescence visualization and was shown for AmB-loaded TMR/Cy5-labeled goat EVs compared to untreated ones as the number of positive vesicles per 1000 pixels of cell area. Error bars represent standard deviations over 25 fields of view, data for three independent wells are shown. Figure 17.1 (a). Example images are shown in Figure 17.2(b).

Detailed description of the invention

When considering large-scale purification of dairy-derived EVs, several aspects are important; among others, these are: type of starting material, concentration of EV that can? be obtained (enrichment) and purity of the final product (i.e., how many other components other than EVs are present in a typical preparation). The latter? particularly important, given that contaminating factors can give rise to undesirable side effects and/or can interfere with further EV derivatization, such as loading with molecules to be delivered, etc. and/or can complicate the interpretation of data that are generated with quality preparations? inferior.

As indicated previously, the purification method commonly used in the current state of the art to obtain EV preparations from milk is ? essentially based on numerous centrifugation phases, of which at least one, but commonly two, are ultracentrifugation (i.e. > 50,000 x g), this is does it? that the EVs form pellets and separate from the liquid and contaminating components having a reduced/lower molecular weight.

The technical disadvantages of this ultracentrifugation process are numerous:

- ultracentrifugation? in itself? difficult to scale and the facilities are expensive; - ultracentrifugation leads to co-pelletization of cellular debris, so? as of protein and oligonucleotide aggregates, such non-EV components often lead to gross overestimation of EV content;

- ultracentrifugation leads to a distortion/modification of the EVs (change in size, fusion of vesicles) potentially reducing the activity? biological nature of the preparations that contain them.

On the other hand, ultracentrifugation provides a very efficient small-scale methodology, commonly employed in biochemistry to "condense" and extract a material from a liquid based on its density. specific (molecular weight), even when poorly present, and at the same time also remove a large excess of impurities having density? lower and/or higher (with lower/higher molecular weight). Applied to EV isolation, the currently generally accepted method for EV preparation in accordance with the prior art is ? based, as a first phase, on the pelletization of whole cells, lipid aggregates of organelles and more complex structures? large using high-speed centrifugation? inferior. This pre-purification phase, removing the most impurities? big, ? followed by an ultracentrifugation step, for example 100,000 x g for 90 minutes, to obtain the desired EVs which can also be resuspended and ultracentrifuged again to obtain higher purity. Some researchers also describe a coagulation step involving milk proteins, which must be performed before ultracentrifugation.

This pre-purification phase? described, for example by, to be 13,200 x g for 30 minutes, starting from skimmed milk, while they use 2x 1,200 xg for 10 min, 2015), starting from raw milk. Other largely similar values can be found in this area. For example, they describe a two-step pre-purification process when EVs are purified from Yak milk, first employing 8,000 x g for 30 minutes, followed by 13,000 x g in order to remove the smallest structures. heavy (

In general, this removal of impurities is more big? followed by an ultracentrifugation phase which, according to (2015) includes 100,000 x g for 90 minutes to obtain a pellet containing the desired EVs, which is then resuspended and the material pelletized again under the same conditions. Others (e.g., (2015)) employ for example 100,000 x g for 90 min and discard the resulting pellet in order to then obtain material from the remaining supernatant using again a greater g-force of 120,000 x g for 90 min, which is ? therefore considered of quality? enough to be used directly without further washing.

To further assist EV isolation by ultracentrifugation,

also describe a process involving the coagulation of milk proteins by treatment with rennet, before subsequently isolating the EVs using ultracentrifugation with commonly known parameters (initial pelleting at 120,000 x g for 90 minutes and washing followed by re-pelletisation using the same parameters ).

When considering alternative isolation methods, other than ultracentrifugation, as a candidate methodology? size separation based on size exclusion chromatography. 2017) describe such a methodology in detail. Their methodology uses a column packed with Sephacryl S 500 size exclusion resin to obtain EV fractions, however only after prior centrifugation at 20,000 g. Furthermore, the methodology described by the authors is ? shown only on a small scale (ml processing volumes), and the authors themselves recognize that the size exclusion phase is used to separate casein and whey proteins remaining from vesicular material after centrifugation steps at 340,000 g or 20,000 g. Therefore, a specific challenge of using milk as a starting material? the high abundance of lipids, fatty acids and lipoproteins which, in the current state of the art, is usually removed previously by centrifugation, as this would otherwise lead to the formation of encrustations of the size exclusion resin or separation membranes. For this reason, in the current technique, these methods have been used rather as additional purification methods for the separation of EVs after initial (ultra)centrifugation.

? It is also important to mention that size exclusion processes with some types of resins (for example, Sepharose CL-2B), have sometimes led to obtaining EV preparations having properties modified physico-chemical and chemical properties, such as a shift towards smaller sized particles, modified surface composition, lack of activity? biological, etc. Unfortunately, ? It is still unclear to which parameters of the size exclusion resin the negative effects are connected and, for this reason, separation processes based on size exclusion are therefore less preferred.

Membrane-based separation? has been successfully applied to obtain EV preparations derived from less complex sources than milk. In fact, in the case of using milk as raw material for their isolation, specific attention must be paid to the other colloidal structures that are present in this natural fluid and are found in the same size range as EVs, such as milk fat globules (range size: 0.1-15 ?m) and casein micelles (size range: 100-200 nm). In addition, milk contains a large excess of non-EV-associated proteins having a broad size distribution and a tendency to clot at concentrations above the 10 mg/ml range. Because of these complicating factors, ? It is commonly known that the use of membrane filtration is inconvenient and can? lead to the occurrence of artifacts and the formation of membrane encrustations, starting from milk as a source of EV.

However, and contrary to the teaching deriving from the known art? It was surprisingly found that the process according to the present invention, based on membrane filtration, can be successfully used for the efficient isolation of high purity EVs from milk or milk derivatives, when preceded by both an intentional protein coagulation prepurification phase and a clarification phase on an inert filtration bed.

On the basis of these premises, the present invention concerns a process for the isolation of EVs derived from milk and their use as carriers of drugs or preparations with active activity. intrinsic biological properties, useful for a range of applications including, but not limited to, pharmaceutical, veterinary, cosmetic and/or nutraceutical applications or as additives to human or animal diets.

The general term "milk", unless otherwise specified, is used to define the product supplied by all mammalian species, as commonly covered in the dairy industry. Examples of milk types include, but are not limited to, bovine milk, bovine colostrum, human milk, human colostrum, sheep's milk, goat's milk, buffalo's milk, donkey's milk, or camel's milk, including their respective forms of colostrum.

In accordance with the present invention, ? provided a process for the isolation of EVs purified from milk, said process being characterized by comprising at least one milk protein coagulation step and at least one membrane-based ultrafiltration and concentration step, providing at least a single homogeneous fraction having uniform characteristics, for example in terms of bioanalytical parameters, particle concentration, protein/particle ratio, chromatographic purity, particle size distribution, etc. In particular, in accordance with the present invention, the process for the isolation of EVs purified from milk of the present invention provides a single homogeneous fraction of EVs purified from milk having uniform characteristics.

The process for the isolation of purified EVs according to the present invention includes the following steps:

a) provide a milk sample;

b) optionally clarifying said sample by means of traditionally known filtration or decantation processes to obtain clarified milk; c) subjecting said milk or said clarified milk to a treatment with a mixture of enzymes suitable for the coagulation of milk proteins at a predefined temperature and time to obtain a liquid containing coagulated proteins;

d) separating said coagulated protein from their supernatant by carrying out one or more? filtrations of said liquid with an inert layer of a filter medium, to obtain a transparent solution;

e) subjecting said transparent solution to at least one ultrafiltration and concentration step using membranes with defined pore sizes and dialysis medium, water or buffer of regulated ionic strength, in order to obtain a concentrated EV preparation;

f) optionally treating said concentrated EV preparation with a stabilizing agent, such as potassium sorbate and, simultaneously, adjusting the pH to obtain a stabilized concentrated EV preparation;

g) subjecting said concentrated preparation of EV, or said concentrated preparation of stabilized EV, to a second filtration/clarification phase in order to obtain an essentially pure clarified isolate of EV from milk;

h) optionally subjecting said essentially pure clarified isolate of EV from milk to sterilizing filtration using standard sterile filters of typical pore size less than 1 ?m, preferably between 0.45 ?m and 0.2 ?m, in order to obtain a Essentially pure and sterile clarified isolate of EV from milk.

Optionally, said essentially pure clarified isolate of EV from milk obtained from step (g) or said clarified essentially pure and sterile isolate of EV from milk obtained from said step (h) may be stored at 4?C until further use or frozen at temperatures between 1?C and -80?C or more? preferably again, flash frozen in liquid nitrogen and stored at temperatures between -20 and -80 ?C or freeze-dried in the absence or presence of common cryopreservatives such as, but not limited to, mannitol, sucrose, trehalose or proteins such as BSA or casein, for obtain a frozen or freeze-dried IV preparation.

In another embodiment of the present invention? It was found that the EV isolate obtained from the process of the invention was suitable for storage by freezing with minimal losses of EV particles after thawing. This procedure requires the freezing of substantially pure EV preparations containing the highest particle concentrations. numerous possible in the order of 10^12 particles per ml, more? preferably 10^13 and above. In general, ? EV preparations have been found to have a certain purity, so as required for precise chemical modifications and controlled loading, they tend to show large particle losses after freezing/thawing. Therefore, ? An intrinsic advantage of the process of the invention is the fact that it leads to obtaining EV preparations which consist of a single fraction having controllable and uniformly high particle concentrations, suitable for freezing and preservation.

The filtrations according to steps (d) and (g) of the process described above, in accordance with the present invention, can be performed using any method known in the art, for example, using disposable or reusable physical filters comprising a filter material such as a cardboard filter or other filter coatings or fabrics, or using or otherwise using suitable clarification and filtration methods known in the art.

In the following description, different embodiments of the present invention will be referred to as part of the present invention. The information provided herein, together with specific details of the examples, is set forth herein for purposes of clarity and should not be construed as limiting the invention n? unnecessary limiting conclusions should be drawn from these exemplary embodiments and the details contained in the examples.

? Of note, in one embodiment of the present invention, various pre-processed forms of milk and milk derivatives may be used as starting materials in place of the pure milk sample. For example, these may be, but are not limited to, powdered milk, skimmed milk, heat-treated milk, pasteurized milk or colostrum, provided that these derivatives contain "intact" EVs, i.e. EVs having the potential to exert their biological functions.

When powdered milk or powdered colostrum are supplied, the process of the present invention further comprises a re-hydration step, in which said powders and granules are dispersed in water to give a uniform suspension. This ? usually obtained by combining powders or granules with water and stirring the mixture to produce a homogeneous suspension. All known rehydration and suspension methods are suitable for this additional step, provided that they do not damage the EV particles.

In another embodiment of the present invention, step (c)? performed using an enzyme mixture containing pepsin and chymosin, which is suitable for casein-based coagulation of milk proteins. Enzyme mixtures suitable for obtaining this coagulation are composed, for example, of pepsin (in a range from 0 to 90%) and chymosin (in a range from 100 to 10%). Preferred mixtures are composed of 5% pepsin and 95% chymosin, even more? preferably 20% pepsin and 80% chymosin. Can the enzyme mixture? be used in a quantity? and for times well known to those skilled in the art, for example about 40 mg of enzyme mixture or about 50 units? international milk coagulation units (IMCU, International Milk Clotting Units) per liter of milk. The reaction time will depend on? from the quantity? of enzymatic mixture and can? vary from a few minutes to several days. In a preferred embodiment of the invention, temperatures in the range of 25-37°C are used to carry out the reaction. The pH? adjusted to a weakly acidic value, between 5 and 7, using standard methods and ingredients known in the art, or left unadjusted to the initial value of the starting material.

The most preferred enzyme mixture to use in accordance with the invention is? rennet of bovine origin, containing 5% pepsin and 95% chymosin. The possibility? to use this enzymatic mixture can? be considered one of the many advantages of the present invention compared to the prior art which for example, to the contrary, describes the use of acid solutions or complexing agents such as EDTA. The rennet, in fact, is usually used for the production of different types of cheeses and, therefore, can? be considered completely safe for its use in the EV isolation process to be further used for human or animal administration.

In a preferred embodiment of the invention, after said step (c) can? a further phase (c?) should be envisaged in order to improve the microbiological characteristics of the product, carrying out a short pasteurisation. Preferably, the liquid containing the coagulated proteins obtained in step (c)? heated to 45-65?C, preferably between 53-56?C for at least tens of minutes.

Afterwards, the mixture? cooled and subjected to one or more filtrations, in accordance with phase (d). In a preferred embodiment of the present invention, step (d) considers an initial coarse filtration to remove the majority of the coagulated protein, followed by a deep filtration using a filter aid such as diatomaceous earth or similar filter aid , what? as commercially available and routinely used as filter layers in deep filtration. The clarified material obtained at the end of phase (d) has a refractive index of 3-5?Brix.

The clarified and de-caseinized liquid obtained at the end of phase (d)? then subjected to a series of at least one concentration and dialysis phase in accordance with phase (e). In a preferred embodiment? an initial pre-concentration phase was performed, followed by dialysis to efficiently remove the remaining contaminating proteins and other components, plus? small, before carrying out a final concentration phase in order to reach the desired final concentration of milk EVs.

For the concentration and dialysis phases, membranes with different pore sizes were tested. In general, most contaminating proteins will be removed using membranes having a dimensional cut-off limit greater than 70 kDa, in a more appropriate having a cut-off close to or greater than 100 kDa, even more so? appropriate having a cut-off close to or greater than 300 kDa, and more? preferably a membrane having a cut-off of 750 kDa. In particular, the use of a membrane having a molecular weight cut-off of or near 750 kDa in at least one ultrafiltration step following casein coagulation allows virtually all contaminating vesicular proteins to be eliminated (as measured by chromatography analytical SEC, for example, on a Sephadex S-200 30/10 column). The choice of such a membrane is not ? obvious a priori to those skilled in the art given that on the one hand the contaminating proteins typically have molecular weights much lower than 300 kDa and on the other hand? EVs have been shown to pass essentially unimpeded through membranes with pore sizes of 0.22 ?m or inert filter aids capable of removing soluble aggregates, thus suggesting that larger membrane sizes may lead to significant EV losses, not improving protein elimination. Again, for non-dairy EVs, the use of ultrafiltration membranes with molecular weight cutoffs significantly greater than 100kDa, such as 300kDa and higher, is ? been associated with EV leaks. Furthermore, membranes with molecular weight cutoffs of 750 kDa are not routinely commonly available.

? it should be noted that for the isolation method of the invention, the fraction to be recovered (i.e., the one containing the EVs) is that which remains above the filter, not the fraction that passes through it.

In a preferred embodiment of the present invention, in order to achieve the desired concentration and purity, it is an initial pre-concentration took place, during which the material was concentrated numerous times, typically 2-6 times with an appropriate membrane. This first pre-concentration? followed by another filtration, in mode? dialysis, and at least 5-10 volumes are exchanged. ? It has been found that for this dialysis the ionic strength of the water or aqueous buffer must be controlled in order to prevent aggregation of the remaining macromolecules and the solutions becoming cloudy. ? It was found that the inclusion of sodium chloride in concentrations in the range between 0.5 and 1.5%, more? preferably between 0.8-1%? sufficient for this purpose. To those skilled in the art, other solutions will be apparent, such as a change of the specific salified form or a change of one of the numerous buffer systems used in biochemical preparations, such as, but not limited to, buffers based on phosphate, citrate, carbonate, Tris , HEPES and others.

At the end of dialysis, the IV preparation can be further concentrated to the desired final EV concentration. This final concentration can? be determined with methods consolidated in the art, such as Nano-Particle Tracking analysis. Generally, values for particle numbers achieved with the method according to the invention are in the range of 10^12-10^13 particles/ml, with typical values of particles/mg of total protein also in the range of 10^12-10^14 particles/mg of protein.

As the final step of the preparation method, the material obtained in step (e) ? further clarified in step (g), using for example diatomaceous earth as in step (d).

Optionally, such a filtration step can? be necessary even after the first phases of ultrafiltration and concentration of pre-processing or during ultrafiltration if, for example, the material to be processed is ? particularly rich in fat, depending on its exact characteristics and the species from which it is derived. derived the starting material. These filtration phases also serve to early remove aggregates of soluble proteins, derived for example from shear stress to which the material is exposed. subjected during dialysis, and which could otherwise catalyze or induce further protein aggregation. Such aggregates may further lead to membrane fouling during subsequent dialysis and concentration steps, or give rise to precipitation or loss of EV material after prolonged storage in liquid or frozen form or after reconstitution following lyophilization or spray drying.

These filtration steps for clarification can be useful in obtaining an aggregate-free, well-soluble, and stable EV preparation.

Optionally, in a further embodiment of the invention, the preparation can be subjected to further filtration, which reduces microbial contamination, or to true sterile filtration (h). The art expert? perfectly capable of choosing a suitable method for this stage, for example, the use of standard sterile filters having pore sizes less than 1 ?m.

Furthermore, in another embodiment, different methods can be applied to the preparation in order to improve its stability. and shelf life. For example, stabilizers can be added to the final product and/or EV extracts can be frozen with or without different cryoprotectants, freeze-dried and/or subjected to spray drying.

The present invention therefore also concerns EV preparations, obtained through the above process, which satisfy numerous quality criteria. exceptionally high. Such stringent quality standards are important for the use of natural isolates, as EVs in pharmaceutical preparations or in finished pharmaceutical products. The EV preparations in accordance with the present invention are substantially pure EV preparations and show the following analytical characteristics: high particle concentrations of at least 10^12 particles/ml, even more? preferably at least 10^13 particles per ml, or more? preferably at least 10^14 particles as determined by NTA analysis and typically a high particle to protein ratio on the order of 5x10^12 particles/mg protein or more? preferably at least 10^13 particles/mg of protein and an EV peak purity of no less than 75% or more? preferably at least 80% and more preferably still having an EV peak purity greater than 85% or more preferably having an EV peak purity greater than 90%, as measured by FPLC using a Sephadex-S200 30/10 analytical column with a detection at 280 nm or equivalent chromatographic setup.

The present invention also concerns the use of the EV product, isolated from milk or milk derivatives following the method in accordance with the invention, in the pharmaceutical, veterinary, nutraceutical and/or cosmetic fields. In particular, the EVs obtained can be refined using the methods known in the art in order to enhance their uptake and/or their delivery towards specific organs or tissues and/or they can be loaded with active ingredients and, consequently, be used as drug carriers and delivery systems.

In fact, the EV preparations obtained in accordance with the present invention are particularly suitable for use as drug carriers given that they are characterized by a low degree of contaminating proteins, typically otherwise derived from co-precipitation with the EVs during ultracentrifugation using milk as starting material. ? It is known that such contaminants and protein co-precipitates interfere with the subsequent modification and loading steps of exosomes and/or can give rise to false positives or unwanted side reactions, for example, during bioconjugation, lipid insertion or other chemical methods or physical for loading EVs with active drug molecules or with pro-drugs.

The terms "active ingredient" and "drug" can be considered synonymous and they can indicate different types of molecules or compounds capable of having an effect on the human or animal body in terms of improving health or treating or preventing syndromes or diseases . In addition to the known pharmaceutically active compounds, in the present invention compounds with cosmetic and/or nutraceutical value can also be considered an "active ingredient". The person expert in art? perfectly capable of selecting the most suitable compound? suitable and the correct way to load it into the EVs in relation to the purpose for which it is? IV preparation is expected.

In a specific embodiment of the present invention, EVs derived from milk or dairy products following the isolation process of the invention are loaded with Amphotericin B, a well-known compound having antifungal and antiparasitic.

A further aspect of the present invention? related to preparations containing EVs isolated following the process of the invention, used to be administered, and if present, with their loaded drug, to a human or animal subject.

In particular, EVs possibly functionalized and/or loaded with one or more? active ingredients can be formulated in different preparations for systemic and/or topical administration. For example, but not limited to, EVs can be mixed with appropriate excipients in order to obtain formulations suitable for oral and/or parenteral administration, for inhalation, for topical administration on the skin or mucosa.

Thanks to their intrinsic characteristics, in fact, EV particles are particularly biocompatible with the biological barriers of human and animal bodies. Their structure and composition can facilitate the absorption of EVs at the cell membrane level, thus enhancing bioavailability as a consequence. of the drug loaded every time? There is a physical barrier. For example, the use of EV as a delivery system can? enhance bioavailability? of the drug transported along the gastrointestinal tract, bronchial mucosa and/or skin, leading to an enhanced concentration of the active ingredient in the bloodstream and at the target site.

In a preferred embodiment of the present invention, ? EV milk preparations have been found to be particularly suitable for inclusion in an inhalation formulation.

? It was surprisingly found that the administration of EV milk preparations, in accordance with the present invention, effects a very efficient systemic exposure after bringing them into contact with the respiratory epithelium of individuals of all ages, including adults.

? therefore surprising and noteworthy that due to the efficient systemic release mechanism via the respiratory epithelium in accordance with the present invention, it is not? No further modification of the milk EVs is necessary and is it necessary? It has been found that such systemic exposure following contact with the respiratory epithelium is greater by several orders of magnitude than the systemic exposure obtained with equivalent dosages of oral milk EVs. After application of small volumes of IV milk preparations via the nasal route, systemic exposure in numerous tissues, including brain tissue and liver, is ? been detected in adult mammals.

All the discussed advantages presented by the invention will be further proven in the following experimental part which is not intended to be restrictive with reference to the full scope of the invention reported previously.

Experimental section

Example 1:

Fresh cow's milk (2.5 L) ? was collected and left to rest in a graduated cylinder at 10?C for 22 hours, during this time ? natural skimming occurred. The top layer of milk (approx. 150 mL) is ? was removed by decanting.

The pH of skimmed milk? was adjusted to pH = 6.4 using citric acid. Then, the skimmed milk (2 L)? been pre-heated to 35?C for 15 minutes with a unit? of heat exchange connected to a jacketed tank. To this heated solution? Bovine rennet containing 20% pepsin and 80% chymosin was added, at a dosage of 50 IMCU (international milk coagulation units) per liter. The coagulation temperature is ? was held at 35?C for 25 minutes. After that the mixture? been heated, how much more? quickly as possible, at 56?C and ? was kept at this temperature for 10 minutes. The mixture ? was then rapidly cooled below 10?C and the liquid whey was was separated from the solid mass of coagulated casein protein using a sintered glass funnel having porosity? 1 with a lined filtration inlet. In total, approximately 1.7 L of slightly cloudy whey was obtained.

The resulting material? been characterized by the number of particles and their size distribution, so? as the total protein content, as described in Examples 13 and 15.

Example 2:

The cloudy whey (1.5 L) obtained in Example 1 ? was subjected to clarification by filtration on a filter aid (Diacel CF/S with 0.1 -0.2 Darcy units, average particle size of 13 ?m) using a standard sintered glass filtration funnel having porosity 3 to obtain a transparent yellowish solution with a refractive index of approximately 6.

Example 3:

The product of Example 2 (1.5 L) ? was then processed using a Vivaflow 200 TFF system (Sartorius) with disposable membrane cartridges having a MW cutoff of 100 kDa. The material ? It was first concentrated four times to reach a total volume of approximately 370 mL. Then four volumes (1.5 L) of a 1% sodium chloride solution were added and the material was added. was concentrated again to give the previous volume of 370 mL. The relative depletion of protein contaminants with different molecular weights during this preprocessing step? was determined by Size Exclusion Chromatography (SEC), as described in Example 14 and ? shown in Example 4.

Example 4:

The product of Example 3 ? was clarified following the procedure described in Example 2. After clarification, the sample was clarified. Was it filtered to sterility? on a unit? of disposable filtration from 0.45 ?m to obtain approx. 350 mL of a clear opaque solution having a refractive index of 2. The product is ? was then divided into different aliquots of 50 mL each and some aliquots were quickly frozen for long-term storage.

The resulting material? been characterized by the number of particles and their size distribution, so? as the total protein content, as described in Examples 13 and 15.

Example 5:

The frozen product of Example 4 (4x 50 mL) is ? It was slowly thawed overnight and clarified again using the method described in Example 2. The material was thawed. was then subjected to a second concentration/dialysis cycle using a hollow fiber membrane having a molecular weight cutoff of 750 kDa (MWCO) (D02-E750-10-N mPES). The material ? was concentrated four times to reach a volume of 50 mL. Then, a total of 20 volumes? was exchanged by dialysis by repeatedly adding 100 mL of PBS pH 7.4. Finally, the champion? concentrated to obtain approximately 40 mL of purified and concentrated milk EV. The material ? Was it filtered to sterility? using a 0.2 ?M PES syringe filter. The resulting material? been characterized by the number of particles and their size distribution, so? as the total protein content, as described in Examples 13 and 15.

Example 6:

Fresh cow's milk (1,500 L) ? was collected and partially skimmed, leaving it to settle in a tank for 24 hours.

Skimmed milk (1,300 L) ? was then pre-heated at 35?C for 15 min. To this heated solution? Bovine rennet was added (containing 5% pepsin-95% chymosin) at a dosage of 50 IMCU/L. The coagulation temperature? was maintained at 32?C for 60 minutes. After that the mixture? been heated, how much more? quickly as possible, at 56?C and ? was kept at this temperature for 15 minutes. The mixture ? was then left to cool and the whey was liquid? separated from the solid mass of coagulated casein protein using a wire mesh filter.

In total, approximately 1,000 L of slightly cloudy whey was obtained.

Example 7:

The whey (600 L) obtained in Example 6? clarified using a plate and frame filtration device. The filter press? It was first assembled with filter sheets (Pall EK filters) and pre-coated by passing a solution of filter aid (Diacel CF/S, 10 kg) in water. Then, another 5 kg of this filter aid was added to the whey and the whey ? passed over the filter. After filtration, 500 L of transparent serum were obtained. Transparent serum like this? obtained ? was then concentrated to 100 L using a 5?? hollow fiber cartridge. with a MWCO of 100 kDa. Then, 500 L of a 1% solution of NaCl in distilled water was added to the product and the solution was added. was concentrated again to a final volume of 100 L. This material is was then clarified once again using the deep filtration setup and immediately after the filter ? was passed through a sterile filtration cartridge (Filtrox, 0.5 ?m). 80 L of EV from clarified pre-concentrated milk was then obtained and frozen.

Example 8:

A 6 L sample obtained from Example 7? concentrated six times (to give 1L) and subjected to six dialysis/concentration cycles, as described in Example 5 using a hollow fiber membrane with a MWCO of 750 kDa (D02-E750-10-N mPES) by addition of 6 volumes (6L) of PBS (pH 7.4) to obtain 1L of milk EV filtered to sterility? and purified.

Example 9:

The whey (2,000 L) obtained as described in Example 6? clarified using a plate and frame filtration device. The filter press? It was first assembled with filter sheets (Pall EK filters) and pre-coated by passing a solution of filter aid (Diacel CF/S, 10 kg) in water. Then, another 5 kg of this filter aid was added to the whey and the whey ? passed over the filter. After filtration, 1800 L of transparent serum were obtained. Transparent serum like this? obtained ? was initially concentrated to 400 L using a 5?? hollow fiber cartridge. with a MWCO of 500 kDa. Then, 2,000 L of a 1% solution of NaCl in distilled water was added to the product and the solution was added. was concentrated again to obtain 100 L. At this point, another 500 L of PBS (pH 7.4) was added to the product and the solution was was concentrated at approx. 60 L. This material? was then clarified once again using the deep filtration setup and immediately after the filter ? was passed through a sterile filtration cartridge (Filtrox, 0.5 ?m). 50 L of purified milk EVs were then obtained.

Example 11:

This example proceeds as Examples 1 through 5, starting with fresh goat's milk (2.5 L) to obtain 20 mL of purified goat's milk EV.

Goat's milk? been pre-processed according to the steps described in Examples 1-4. 150 mL of pre-processed goat's milk whey was slowly thawed overnight. The material ? was then subjected to a second concentration/dialysis cycle using a hollow fiber membrane having a MWCO of 750 kDa (D02-E750-10-N mPES). The material ? was concentrated four times to reach a volume of 38 mL. Then, a total of 20 volumes? was exchanged by dialysis by repeatedly adding 112 mL of PBS pH 7.4. Finally, the champion? been concentrated to obtain approx. 20 mL of purified and concentrated goat's milk IV. The material ? Was it filtered to sterility? using a 0.2 ?M PES syringe filter. The resulting material? been characterized by the number of particles and their size distribution, so? as the total protein content, as described in Examples 13 and 15.

Example 12:

For comparison, ? A protocol taken from the literature for the isolation of EVs from milk based on ultracentrifugation was carried out. The protocol was based on experimental procedures reported in the materials and methods sections of Betker et al., 2019; Agrawal et al., 2017; Munagala et al., 2017.

Fresh whole milk? was degreased by swirling for 30 min at 4?C at 13,000 g. The supernatant? was passed through a Whatman filter paper. Defatted milk? was centrifuged for 60 min at 4?C at 100,000 g (corresponding to a max rcf 31,400 rpm, Sorvall Discovery Ultracentrifuge, with T865 fixed angle rotor). The supernatant (not exceeding 70% of the total volume) is was carefully collected with a glass pipette without disturbing the lower mush layer. The EVs were pelleted by vortexing the supernatant for 90 min at 4?C at 135,000 g, corresponding to a max rcf = 36,400 rpm (Figure 12). The pellets were washed 3 times with PBS pH 7.4, dissolved in PBS pH 7.4 and filtered to sterility. using a 0.2 ?M PES filter syringe.

Example 13: Nanoparticle Tracking Analysis (NTA) of EV preparations Particle numbers and size distribution were analyzed using a NanoSight LM10 instrument (Malvern), configured with a 488 nm laser. Videos were collected and analyzed using NTA 3.1 software.

Measurements were performed in triplicate at a controlled temperature of 25?C. Each sample? was diluted from 1 to 100 in PBS filtered to sterility? pH 7.4 up to 1 mL. The samples were further diluted in order to carry out measurements in a range of 80-120 particles/frame. The camera level? was maintained at 12 during all measurements and five consecutive 60-second recordings were made for each sample. The samples were analyzed with a detection threshold of 5. The characterization of dairy EV preparations by the methods of the present invention using NTA ? illustrated in Figure 1, in Figures 2, 6 (b-e), 8, 10 (b-e) and 12 (b-e).

Example 14: Analysis by Size Exclusion Chromatography (SEC) of the EV Preparation

50 ?L of the samples were subjected to size exclusion chromatography using a Superdex 20010/300 column (Cytiva) on a Shimadzu LC-20Ai system equipped with an SPD-M20A PDA Diode Array Detector and an RF-20A Fluorescence Detector . Dimensional exclusion? was performed in PBS at pH 7.4 at a flow rate of 0.8 mL/min. The size of the eluted peaks? was determined using a gel filtration standard (BioRad #5119011, Figure 4). The characterization of milk EV preparations by the methods of the present invention using SEC? illustrated in Figures 3, 4, 6 (a), 9, 10 (a) and 12 (a).

Example 15: Protein Analysis of EV Preparation

Protein concentrations of EV samples were determined with Bradford (Pierce Detergent Compatible Bradford Assay Kit, Thermo Fisher Scientific 23246) or BCA (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific 23225) reagents according to the manufacturer's instructions using serial dilutions of BSA as standards. Protein concentrations were determined in triplicate.

Example 16: Western Blot Analysis of EV Preparations

EV samples were analyzed by SDS-PAGE using 4-20% TGX Gel (BioRad) under reducing (for markers MFGE8, tsg101) or non-reducing (CD9) conditions at 150 Volts for approximately 50 min. The separated proteins were transferred to a 0.45 ?M nitrocellulose membrane by semi-dry blotting at 20 Volts for 45 min. The blocking? was performed using TBS 0.2% Tween-20 5% BSA (for MFGE8, CD9 markers) or TBS 0.2% Tween-20 2% nonfat dry milk (tsg101) for 1 hour at room temperature agitated environment. After blocking, membranes were incubated at 4?C o/n with the following primary antibodies: MFGE8 (HPA002807, Sigma, 1:1000 dilution), tsg101 (ab83, Abcam 1:1000 dilution) or CD9 (AHS0902, Invitrogen , 1:2000 dilution) diluted in TBS 0.2% Tween-20 1% BSA (MFGE8, CD9) or TBS 0.2% Tween-20 2% nonfat dry milk (tsg101) with stirring. After incubation, the membrane is was washed 5 times with TBS 0.2% Tween-20 for 5 min each time and subsequently incubated with the corresponding secondary antibody (LI-COR) for 1 hour at room temperature with shaking (1:15000 of IRDye 680RD anti-mouse to detect tsg101 and CD9 or 1:15000 of IRDye 800 CW anti-rabbit to detect MFGE8). Membranes were washed 5 times for 5 min with TBS 0.2% Tween-20 before analysis in a LI-COR visualization instrument. The characterization of milk EV preparations by the methods of the present invention using Western Blot? illustrated in Figure 5.

Example 17: Cellular uptake after EV labeling

The milk EVs obtained in Example 8, so? as in Example 11, they were each diluted to 10 ?M in PBS at pH 7.4 and reacted with 25 ?M of (5?,6?-)TMR-NHS and 50 ?M of Cy5-NHS for 1 hour at 37?C. The unreacted dye? was removed by six concentration/dialysis cycles each time with six volumes of PBS at pH 7.4 on a 750kDa hollow fiber mPES membrane as described in Example 5. EV concentrations were determined by NTA as described in Example 13. A549 (human lung adenocarcinoma) cells were plated in 96-well plates pre-coated with collagen and grown under standard cell culture conditions to 50-60% cell confluence (5% CO2, 95% humidity). %, 37?C). Cy5/TMR double-labeled extracellular vesicles (bovine whey-derived EVs stored at 4?C or -80?C (Figure 7) or goat whey-derived EVs stored at 4?C (Figure 11) were added at increasing concentrations with three independent biological replicates per condition. After 6 hours of incubation, cells were fixed with PenFix (Richard Allen Scientific) supplemented with Hoechst 33258 at 0.5 ?M. Cells were visualized by fluorescence microscopy wide field (Olympus IX83) using a 40x ApoPlan objective with a numerical aperture of 0.95. Over 10 images were taken per well, with each image representing a maximum intensity projection of 7 individual Z images and displayed 5 different channels: Brightfield, Hoechst (ex385nm, em417-477nm), TMR (ex545nm, em605/70nm), CY5 (ex640nm, em700/75nm) and TMR-CY5 FRET (ex545, em700/75).Cell detection and vesicles was performed with a self-programmed ImageJ Fiji plugin. Briefly, cells were detected by brightfield channel angular detection followed by smoothing and thresholding the image. EVs were detected using the Li self-threshold (similar thresholds are used in all concentrations) on the CY5 channel. EVs in the cell area were counted and are shown as cell area ?EVs/1,000 pixels?.

Example 18: Transmission Electron Microscopy of Negatively Staining EVs The milk EVs obtained in Example 8 were diluted to a concentration of 1 to 5x10^10 particles per mL in PBS and were pipetted onto grids coated with Formvar and carbon. After air drying for 30-45 min at room temperature, the excess of the EV solution is was removed by pressing the grids onto filter paper at an angle of approx.45?. The grids were then washed 3x with distilled water by placing them in 100 ?L drops on parafilm, followed by fixation with 1% glutaraldehyde in PBS for 5 minutes on 40 ?L drops, washing 8x with water in 40? Negative staining with 2% aqueous uranyl acetate for 5 minutes. Samples were air dried for 1 hour and viewed on a Zeiss EM 910 Transmission Electron Microscope at 80,000 kV and 40,000 x magnification (Tr?ndle camera). Typical images of the milk EV preparations of Example 8 are shown in Figure 13.

Example 19: Quantification of Amphotericin B (loaded on milk EV) using high performance liquid chromatography

The quantification of Amphotericin B (AmB)? was performed using Reversed Phase High-Performance Liquid Chromatography (RP-HPLC) in a Shimadzu Prominence instrument. The loaded MEV samples were 50 ?L samples and were injected and separated on a C18 column (3.0 ? 150 mm with 5 ?m particle size) by isocratic elution with an Acetonitrile/20 mM EDTA mobile phase (55% / 45% vol/vol) with a flow rate of 1 mL/min. EDTA? was used in the mobile phase, given that it improves the chromatographic behavior of AmB by direct competition with Amphotericin B for chelation with metal ions. AmB ? was detected by measuring UV absorbance at 406 nm. Calibration standard solutions were prepared at concentrations varying in the range of 0.0125 ? 10 ?g/mL. Representative chromatograms and calibration curves of AmB are shown in Figure 14 a and b, respectively.

Example 20: Loading of Amphotericin B onto bovine milk exosomes

For loading AmB onto bovine milk exosomes, numerous methods were tested, including incubation at room temperature, incubation at 37 ?C, hypotonic condition, sonication, extrusion, incubation with saponin, and freeze-thaw. AmB ? was added from a 1 mM stock solution in DMSO into milk EV samples in PBS to give final concentrations of MEV at 1 nM, AmB at 2 ?M and DMSO at 0.5 % and were treated under different conditions, as follows:

? Incubation: The formulations were incubated at room temperature (21 ?C), at 37 ?C or at 50 ?C for different times (as specified in Table 1) in PBS.

? Saponin: saponin? was added to a final concentration of 2% w/v and the formulations were incubated under different conditions (as specified in Table 1).

? Sonication: The formulations were sonicated using a probe sonicator (80% power with 30 second pulse/30 second pause).

? Hypotonic condition: the formulations were diluted with H2O to a final concentration of 0.5x PBS.

? Freeze-thaw: Formulations were flash frozen, held at -80?C for 0.5 hours, and thawed at room temperature for 3 cycles.

? Extrusion: The formulations were extruded (x10 times and x20 times) using an Avanti Lipids extruder having a pore diameter of 200 nm.

To remove free AmB from the formulation, the mixture is was ultrafiltered six times with PBS by centrifugation in units? of Vivaspin ultrafiltration (MWCO of 10 kDa) at 10,000 x g for 15 minutes. The final washing phase resulted in samples concentrated 10 times. AmB? was then extracted by adding acetonitrile to the original volume, resulting in a final ACN:H<2>O concentration of 90:10. The mixture ? was vortexed at room temperature at 1,000 rpm for 30 minutes and centrifuged at 1,000 x g for 15 minutes before injection to determine the AmB concentration using RP-HPLC as described in Example 19. The retained AmB concentrations for the different conditions are listed in Table 1, revealing that loading by incubation at room temperature for ? 6 hours allowed to obtain the densities? greater loading times.

Table 1. Concentration of AmB retained in bovine milk exosomes under different loading conditions

Example 21: Loading of Amphotericin B onto goat milk exosomes

AmB loading onto goat milk exosomes? was tested using the procedures as described in Example 20. The concentration of AmB retained was ? was performed using RP-HPLC as described in Example 19.

Table 2: Concentration of AmB retained in EV from goat's milk (1 nM) under different conditions

Example 22: Saturation of AmB loading on EV from goat's milk

To additionally test the saturation of EV loading as a function of AmB concentration, EVs from 1 nM goat's milk were incubated for 6 hours at room temperature with increasing AmB concentrations varying in the range of 1 ?M to 10 ?M (DMSO at 1% v/v in all samples). Furthermore, we tested the formulation of AmB-loaded exosomes with increasing concentration of EV while keeping the ratio of EV:AmB constant. MEV:AmB concentrations were at 1nM:2 ?M; 2 nM: 4 ?M; 3 nM: 6 ?M; 4 nM: 8 ?M and 5 nM: 10 ?M. All formulations contained 1% DMSO. The concentration of AmB retained? was performed using RP-HPLC as described in Example 19. The results are shown in Figure 15.

Example 23. Co-fractionation of AmB with milk EV using size exclusion chromatography

To test whether AmB retained with EVs after loading? actually physically associated with the vesicles, we used analytical size exclusion chromatography under native conditions to verify co-fractionation. 10 ?L of the samples as prepared in Example 20 (1 nM EV from bovine or goat milk loaded with 2 ?M AmB by incubation for 6 hours at room temperature) before and after removal of free AmB by ultrafiltration are were injected onto a Sephadex 200 30/10 column and separated by isocratic elution with PBS at pH 7.4 at a flow rate of 0.8 mL/min in a Shimadzu LC-20Ai instrument equipped with a UV/Vis diode array detector and fluorescence. AmB ? was detected with an absorbance at 406 nm, milk EVs were detected by autofluorescence at 488nm/510 nm (ex/em). In addition to the formulations, free AmB and MEV were also analyzed. As shown in Figure 16, AmB loaded on both bovine milk EVs (Figure 16a) and goat milk EVs (Figure 16b) ? also co-fractionated with the MEV peak as analyzed by native size-exclusion chromatography, demonstrating the physical association of AmB with vesicles.

Example 24: Cellular uptake of goat milk EVs loaded with AmB

Goat milk EVs were labeled with Cy5 NHS and Tamra NHS as described in Example 17 and loaded with AmB as described in Example 21 (1 nM goat milk EVs loaded with 2 ?M AmB by incubation for 6 hours at room temperature). Cellular uptake in A549 cells? was then quantified by fluorescence microscopy, as described in Example 17. As shown in Figure 17, AmB-loaded goat milk exosomes were taken up by cells identically to non-loaded goat milk EVs, confirming that the loading AmB does not affect the functionality? of milk EVs in cellular uptake.

Example 25: Systemic EV exposure of the milk of the present invention after nasal delivery via the respiratory tract.

The TMR- and Cy5-labeled bovine milk EVs obtained in Example 17 were used for intranasal administration in C57/Bl6 mice (8-week-old female). After aspiration of 5 ?L of the labeled EVs at 3.4x10^13 particles/mL in PBS through the nose, the mice were sacrificed after 6 hours, the organs were fixed with 4% PFA, transferred to a Cryobuffer (30 % w/v sucrose, 1 % w/v polyvinylpyrrolidone-40, 30 % v/v ethylene glycol in 100 mM PBS at pH 7.4) and analyzed by epifluorescence visualization in an IVIS Spectrum visualization instrument. The intensities? Fluorescence signals for organs from PBS-treated animals are shown in Table 3.

Table 3: Intensity? of the fluorescence signals of different organs from EV-administered mice compared with the corresponding organs of PBS-treated control animals:

Claims (10)

1. A process for the isolation of purified EVs from milk, characterized by comprising at least one milk protein coagulation step and at least one membrane-based ultrafiltration and concentration step. 2. A process for the isolation of EVs purified from milk according to claim 1, characterized by comprising the following steps: a) provide a milk sample; b) optionally clarifying said sample by means of traditionally known filtration or decantation processes to obtain clarified milk; c) subjecting said milk or said clarified milk to a treatment with a mixture of enzymes suitable for coagulating milk proteins at a pre-defined temperature and time, to obtain a liquid containing coagulated proteins; d) separating said coagulated proteins from their supernatant by carrying out at least one filtration of said liquid, with an inert layer of a filter medium, to obtain a transparent solution; e) subjecting said transparent solution to at least one ultrafiltration and concentration step using membranes with defined pore sizes and water or buffer of regulated ionic strength as dialysis medium in order to obtain a concentrated EV preparation; f) optionally treating said concentrated EV preparation with a stabilizing agent, such as potassium sorbate and, simultaneously, adjusting the pH to obtain a stabilized concentrated EV preparation; g) subjecting said concentrated preparation of EV or said concentrated preparation of stabilized EV to a second filtration/clarification step in order to obtain an essentially pure clarified isolate of EV from milk; h) optionally subjecting said essentially pure clarified isolate of EV from milk to sterilizing filtration using standard sterile filters of typical pore size less than 1 ?m, preferably between 0.45 ?m and 0.2 ?m, in order to obtain a essentially pure sterile clarified isolate of EV from milk. 3. The process according to claim 1 or claim 2 characterized in that said milk protein coagulation phase or enzyme mixture of phase (c) contains pepsin and chymosin, preferably obtained by fermentation or ? rennet, preferably containing 5% pepsin and 95% chymosin. 4. The process according to claim 1, 2 or 3 characterized by the fact that it dictates at least one phase (e) of ultrafiltration and concentration? carried out through a membrane having a molecular cut-off of 750 kDa and? preferably preceded by a preconcentration and ultrafiltration phase through a membrane having a molecular weight cut-off of 100 ? 500 kDa. 5. Essentially pure clarified isolate of dairy EV according to any of claims 1-4 for use as a carrier for active ingredients in the pharmaceutical, veterinary, nutraceutical and/or cosmetic fields. 6. Composition comprising the essentially pure clarified isolate of dairy EV according to claims 1-4 and pharmaceutically acceptable excipients. 7. Composition according to claim 6, characterized in that it further comprises Amphotericin B loaded in said essentially pure clarified isolate of EV from milk. 8. Composition according to claim 6 for use as a carrier for active ingredients in the pharmaceutical, veterinary, nutraceutical and/or cosmetic fields. 9. Composition according to claim 8 for inhalation. 10. Composition according to claim 7 for use as an antifungal and/or pesticide.