CH719081A9 - Milk-derived extracellular vesicles and the process for isolating them. - Google Patents

Abstract

The present invention relates to the field of biotechnology and in particular milk-derived extracellular vesicles and provides a process for isolating such vesicles from milk and milk-related fluids. The present invention also relates to compositions containing said milk-derived extracellular vesicles, particularly suitable for use in pharmaceutical, veterinary, cosmetic and/or nutraceutical applications.

Description

Field of Invention

[0001] The present invention relates to the field of biotechnologies and, in particular, extracellular vesicles (EV, Extracellular Vesicles) 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 due to its scalability and its ability to maintain the structural integrity of the extracellular vesicles, as well as for the its ability to provide solutions of homogeneous purity, the obtained purity being higher than that of preparations previously known in the art. The invention further relates to milk-derived extracellular vesicle compositions by said process, which show surprisingly high recovery/extraction yields and provides EVs which have, 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 further relates to milk-derived extracellular vesicle compositions and methods for preparing the same, wherein the milk vesicles have been loaded, for example, with Amphotericin B or its derivatives, thereby providing potent antifungal, antiparasitic and antibacterial preparations. -infectious for oral use.

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

Technical background

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

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

[0005] In comparison with cell culture-derived exosomes, which are typically harvested from the supernatant of cultured human or higher organism cells, exosomes derived from natural sources used in the food industry (milk, fruit juice, etc. ) possess the distinct advantage of being available in relatively large quantities without the need to establish biotechnological production methods, cell bioreactors and processes for their production. However, the concentrations at which exosomes are present in different starting materials are far below levels useful for use directly as drug carriers or as therapeutic agents (such as drug carrier starting material or for therapeutic agents), and the presence of additional components (such as casein and other proteins, lipoproteins, sugars or fatty acids) limits applications in later stages.

[0006] Accordingly, there is a need for scalable methods for the isolation and purification of exosomes from these attractive natural sources.

[0007] In recent years, different processes have been described in the literature for the isolation of exosomes derived from cell cultures in the laboratory. Such 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 using heparin, membrane-based separations, microfluidics, or polyethylene glycol, as well as various commercial isolation kits, such as, for example, ExoQuick™ (Antes, 2013).

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

[0009] In contrast, the isolation of exosomes from natural sources poses additional challenges; typical natural sources such as milk contain a high degree of dry matter and/or large amounts 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). As a result, each matrix poses specific and unique challenges in isolating EVs or EV-enriched fractions that have defined characteristics and consistent, reproducible quality. This aspect is particularly important, since 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.

[0010] In the case of dairy EVs, ultracentrifugation to collect EVs in a pellet or in defined fractions on density gradients is still the method of choice used for their isolation. However, in addition to the need for specialized and expensive instrumentation, ultracentrifugation is technically difficult to scale industrially, labor intensive and time consuming. Furthermore, since ultracentrifugation separates molecular moieties based on their relative densities, 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 assessment is based on methods that determine the total protein content of a preparation. In addition, ultracentrifugation via pelletization itself promotes the formation of exosome clusters and EV aggregates, potentially leading to vesicle fusions, distortions of size distributions, and alterations of cellular uptake and other biological activities. Finally, the ultracentrifugation process produces variable yields and qualities, making process standardization difficult.

[0011] Alternative methods known in the art are based on commercial kits using affinity-based isolation strategies, which are not only severely limited with regards to the scale and yield of the preparations, but also require difficult conditions for the elution of vesicles bound to affinity resins with unknown consequences for EV integrity and function.

[0012] 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 EVs by size exclusion chromatography after pre-purification by ultracentrifugation or tangential flow filtration through a membrane with a molecular cut-off of 500 kDa. Therefore, both of these protocols require a chemical treatment of the milk using 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 column of Sepharose (SEC, Size Exclusion Chromatography). These two steps (ie the use of EDTA and the SEC), together with the other steps of the process (ultracentrifugation or filtration) are defined as mandatory, in order to obtain the desired quality of the final IV product. On the other hand, these steps by themselves pose an additional limitation to the process. First, the use of EDTA solubilizes casein aggregates, increasing the amount of freely diffusing low molecular weight impurities that must be removed by Tangential Flow Filtration (TFF). This leads, over time, to the formation of encrustation of the membrane thus limiting, as the authors themselves have commented, the purity that can be obtained with TFF. Second, the need to use SEC fractionation to further purify material after TFF leads to multiple different fractions of EV preparations having varying purities and compositions, with different levels of impurities, further limiting the possibility of scaling. of the process.

[0013] Consequently, in the field of milk-derived exosomes there is still a need to develop an isolation process that avoids the disadvantages of ultracentrifugation, that is suitable for handling large volumes of starting fluid (i.e., milk), that can work without specialized laboratory equipment, and that allows to obtain preparations of exosomes having a homogeneous, well-defined and reproducible quality.

[0014] With respect to the use of IV preparations for drug delivery, particular attention must be paid not only to the IVs and their preparation, but also to the types of cargoes with which the constituted delivery system is employed from EVs. Classic small molecules often show an oral bioavailability 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 by intravenous or subcutaneous injection or for local applications. However, there are also numerous natural products having molecular characteristics generally described in the field of pharmaceutical chemistry as failing the “rule of five” criteria (Lipinsky et al. 2001, Adv. Drug Deliv. Rev. 46) and similar assessments based on rule and/or that show no therapeutically useful oral bioavailability. Now, several limited attempts have been made to load milk EVs with cargo molecules.

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

[0016] US10420723B2 describes some preparations of milk EVs with a selection of natural products, the loading done by suspending the drug in PEG-400 or using ethanol as a co-solvent and, after co-incubation, separating the EVs from the 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 non-specific drug binding to non-vesicular proteins present per se in the EV samples, particularly when ultracentrifugation-based processes are used first for their isolation.

EP3620519A1 discloses the use of extrusion to perform loading of milk EVs with RNAs modified with a hydrophobic anchor.

[0018] Such overtly generalized procedures 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 parameters optimization without an a priori guarantee of success. An example demonstrating that this principle still holds true for EVs and EV-derived drug carrier systems is provided by Grossen et al. (Grossen et al., European Journal of Pharmaceutics and Biopharmaceutics, Volume 158, 2021), where no functional effect was observed after oral administration of RNA-loaded milk IVs, as described in EP 3 620 519 A1.

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

Purposes of the invention

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

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

Another object of the present invention is to provide IV preparations particularly suitable for further refinement and/or drug loading by methods described in the art.

Another object of the present invention is to also provide novel drug-loaded milk IV preparations based on the antifungal and antiparasitic drug Amphotericin B (AmB).

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

[0025] These objects, among others, will be dealt with in greater detail in the following paragraphs, together with experimental data and examples, in order to fully clarify the object of the present invention.

Summary of the Invention

The present invention addresses the foregoing and other needs, with reference to the prior art, by providing a novel process suitable for the industrial isolation of exosomes from different types of milk as starting material. The present invention also relates to compositions obtained with this isolation process, which have high purity with reference to the possible presence of contaminating proteins, protein aggregates, organic impurity aggregates 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, comprising an enzyme-based casein coagulation step, followed by ultrafiltration using membranes having specific pore sizes, and dialysis at controlled ionic strength, allows to obtain the isolation, purification and concentration of milk-derived exosomes (EV) with high efficiency.

The surprising effect of the discovery of the invention is related to the fact that starting materials rich in proteins and fats usually suffer from lack of possibility to be highly concentrated due to increasing aggregation, turbidity or precipitation. At the same time, dialysis with pure water can lead to depletion of stabilizing ions which are needed 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 characteristic surface-bound protein markers of exosomes. Said series of purification steps typically comprises a first step involving the coagulation of most of the protein components of the milk, followed by their removal by filtration or other separation methods, and a second step, comprising concentration and ionic strength dialysis controlled EV fraction by ultrafiltration using specifically selected pore size membranes.

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

[0031] Although the first step of the process, i.e. casein coagulation, can technically be carried out by acid-induced coagulation, this solution is less preferred than enzyme-based coagulation, due to the functional consequences observed in the preparations finally obtained when an acid is used. Indeed, EVs isolated using the latter, on average show larger particle sizes and a lower abundance of EV marker proteins, such as TSG101 or CD9 in Western blot analysis. Thus, acid-induced casein coagulation appears to provide EV isolates that do not meet all desired requirements.

[0032] Therefore, according to 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 (bovine and goat), loaded with Amphotericin B. When different loading conditions for Amphotericin B were compared, it was surprisingly found that co-incubation for a specified amount of time in a specified ratio of Amphotericin B to milk IV yielded functional preparations of Amphotericin B-loaded IVs, containing the highest Amphotericin B drug load with a stable drug-carrier association. TFF-based dialysis with 750 kDa membranes allowed ultracentrifugation-free re-isolation of biologically functional milk EVs loaded with Amphotericin B, suitable to treat conditions for which Amphotericin B is indicated in accordance with medical practice.

[0033] 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 trials have been performed with IV milk preparations in accordance with the present invention, it has been surprisingly found that the IV milk preparations of the invention can be used to effect highly efficient transport across the respiratory epithelium and are able to generate high levels of systemic exposure in many tissues, for example in the liver, kidney, heart or brain and potentially others.

Description of the Figures

Figure 1 : Characterization of different preparation steps of EVs from bovine milk by Nanoparticle Tracking Analysis (NTA). Particle size distribution histograms, mean and mode value were determined by NTA. Data are shown for representative samples of whey (Figure 1.1, a-c), preprocessed whey (Figure 1.2, d-f), and tangential flow filtration (TFF) IV preparations of preprocessed whey using two different membranes: with pores of 750 kDa or with pores of 100 kDa (Figure 1.3, g-i). Particulate concentrations are shown as yields, calculated backwards with reference to the volume of whey. For TFF-processed material, yields and particle sizes are shown for both whey and retentate (the fluid retained by the membrane filter) of pre-processed milk (graphs h and i). Samples were measured in triplicate; error bars represent standard deviations from the three technical replicates. Figure 2: Particle to protein ratios of preparation steps of different bovine milk EVs. Particle to protein ratios were determined by measuring particle numbers by NTA and total protein concentration was determined using Bradford's reagent. The results are expressed as the mean of n=3 experiments. (a) Three different samples (batch 1 - 3) of preprocessed whey, (b) two examples of TFF processing intermediates: The preprocessed whey was diluted 1:1 (bar „input“ ), concentrated three times and dialysed 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 pH 7.4 (Example 14). The 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 is shown as an overlay of the samples before (grey, Example 1) and after (black, Example 2) filtration. Panel (b) shows two independent batches of pre-processed whey. Panel (c) shows milk EV preparations after processing by TFF on membranes with pore sizes 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 EV preparations of milk following EV isolation 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 pre-processing (100 kDa molecular weight cutoff (MWCO)) of bovine milk EVs. The relative depletion of protein contaminants, as determined by size exclusion chromatography on a Superdex S200 column, was determined by comparing the area (black) and height (gray) of whey peaks 1-6 (Example 1) and of pre-processed whey (Example 4). Figure 4.1, panel (a) shows chromatograms recorded at 280 nm with peak assignment. Panel (b) shows peak areas and heights normalized to the EV peak (peak 1). Molecular weight was determined by an independent calibration run with a size exclusion standard. The % depletion of the main contaminants is shown as a function of the calculated molecular weight in Figure 4.2, panel (c). Figure 5: Characterization of different bovine milk EV preparations 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 by Ultracentrifugation (UC of purified EVs from milk, Example 12). Figure 6: Typical preparation of bovine EVs by 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 starting material for TFF of pre-processed whey: (a) chromatogram of SEC 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 whey (e) particle size measured by NTA, n=3. The lot shown was processed by 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS pH 7.4 (Example 5). Figure 7: Cellular uptake of EVs from bovine milk into human lung adenocarcinoma cells (A459). The TMR/Cy5-labeled EVs obtained in Example 17 were added to A459 cells and cell uptake was analyzed by fluorescence microscopy as in Example 17. The representative images obtained by large-field „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; gray-scale 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. Single curves represent independent biological replicates, while 5 single images were taken per well. Error bars illustrate standard deviations over individual images. Figure 8: Preparation of goat milk IVs. The characteristics of the following intermediates of the preparation of goat milk EVs are shown: Goat whey, pre-processed goat whey and purified EVs from goat milk on a TFF membrane having a pore size of 750 kDa, processed by 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS 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 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 milk using size exclusion analytical chromatography. SEC chromatograms recorded with an absorbance at 280 nm are shown for (a) goat whey, (b) preprocessed goat whey, and (c) membrane-processed goat EV for TFF 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 pH 7.4. Figure 10: Typical preparation of EVs from goat milk by the method of the present invention. Characteristics of EV from goat 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) chromatogram of SEC (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 whey (e) particle size measured by NTA, n=3. GW1PPEK1 was processed by 20 cycles of 4x concentration and dialysis by refilling to the original volume with PBS pH 7.4. Figure 11: Cellular uptake of goat milk EVs into human lung adenocarcinoma cells (A459). Goat milk EVs obtained in Example 11 were labeled with TMR and Cy5 as described for bovine milk EVs, added to A459 cells, and cell 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; gray-scale brightfield images. Quantification of dose-dependent EV uptake at 6 hours in panel (b) for TMR/Cy5-labelled goat milk EVs. Single curves represent independent biological replicates, while 5 single images were taken per well. Error bars illustrate standard deviations over individual images. Figure 12: Characteristics of EVs 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 whey (e) particle size measured by NTA, n=3. Figure 13: Characterization of bovine milk-derived EVs by TEM with negative staining. Bovine milk EVs of 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 µl of a 2.5 µM solution) with isocratic elution (Acetonitrile/20 mM EDTA (55% / 45% vol/vol, 1 ml/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 goat milk EVs. goat milk EVs at 1 nM were incubated with increasing concentrations of AmB and the loaded AmB concentration was quantified by RP-HPLC (a).AmB at 2 µM was incubated with increasing concentrations of goat milk EVs and the AmB concentration loaded was quantified by RP-HPLC (b).Figure 16: Co-fractionation of AmB with EVs from bovine and goat milk by size exclusion chromatography.The EVs from bovine milk of Figure 16.1(a) and goat of Figure 16.2(b) at 1 µM were loaded with Amphotericin B at 2 µM by co-incubation at room temperature for 6 hours and the samples were analyzed by size exclusion chromatography with detection by UV-VIS at 406 nm before and after washing by ultrafiltration. The chromatograms for free AmB and free milk EV are shown for comparison. Figure 17: Uptake of EVs from goat milk into 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. Uptake was then analyzed by wide-field fluorescence visualization and is shown for TMR/Cy5-labelled goat EVs loaded with AmB versus 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 the large-scale purification of dairy EVs, several aspects are important; among others, these are: type of starting material, concentration of EVs 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 is particularly important, given that contaminating factors may give rise to unwanted side effects and/or may interfere with further EV derivatization, such as loading with delivery molecules, etc. and/or may complicate interpretation of data that is generated with lower quality preparations.

[0036] As indicated above, the purification method commonly used in the current state of the art to obtain dairy EV preparations is essentially based on numerous centrifugation steps, of which at least one, but commonly two, are ultracentrifugation (i.e. > 50,000 x g), this causes the EVs to pellet and separate from the liquid and low/lower molecular weight contaminant components.

[0037] The technical disadvantages of this ultracentrifugation process are numerous: – ultracentrifugation is in itself difficult to scale up and the plants are expensive; – ultracentrifugation leads to co-pelletization of cellular debris, as well as protein and oligonucleotide aggregates, such non-EV components often leading to gross overestimation of EV content; – ultracentrifugation leads to distortion/modification of the EVs (change in size, fusion of vesicles) potentially reducing the biological activity of the preparations containing them.

[0038] 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 specific density (molecular weight), even when low in , and simultaneously also remove a large excess of impurities having lower and/or higher density (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 step, on pelletizing whole cells, lipid aggregates of organelles and larger structures using high-speed centrifugation. inferior. This pre-purification step, removing larger impurities, is followed by an ultracentrifugation step, e.g. 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.

[0039] Such a pre-purification step is described, for example by (Wolf et al., 2015), to be 13,200 x g for 30 minutes, starting with skim milk, while Izumi et al use 2x 1,200 x g for 10 min ( Izumi et al., 2015), starting from raw milk. Other largely similar values can be found in this area. For example, Gao et al. describe a two-step pre-purification process when EVs are purified from yak milk, first using 8,000 x g for 30 minutes, followed by 13,000 x g in order to remove heavier structures (Gao et al., 2019; Gao et al. al., 2021a; Gao et al., 2021b).

[0040] In general, such removal of the largest impurities is followed by an ultracentrifugation step which, according to (Wolf et al., 2015) comprises 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., (Izumi et al., 2015)) employ e.g. 100,000 x g for 90 min and discard the resulting pellet in order to then obtain material from the remaining supernatant again using a greater g-force of 120,000 x g for 90 min, which it is therefore considered of sufficient quality to be used directly without further washing.

[0041] To further assist in the isolation of EVs by ultracentrifugation, Gao et al. (Gao et al., 2019; Gao et al., 2021a; Gao et al., 2021b) also describe a process involving the coagulation of milk proteins by rennet treatment, before subsequently isolating the EVs using ultracentrifugation with the parameters commonly known (initial pelletizing at 120,000 x g for 90 minutes and washing followed by re-pelletizing using the same parameters).

When considering alternative isolation methods, other than ultracentrifugation, a candidate methodology is size-based separation based on size exclusion chromatography. Blans et al., (Blans et al., 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 only shown at small scales (processing volumes of ml), and the authors themselves acknowledge that the size exclusion step is used to separate the remaining casein and whey proteins from the vesicular material after the steps of centrifugation at 340,000 g or 20,000 g. Therefore, a specific challenge of using milk as starting material is the high abundance of lipids, fatty acids and lipoproteins which, in the current state of the art, is usually previously removed by centrifugation, as otherwise it would lead to scale formation. size exclusion resin or separation membranes. For this reason, in the present art, these methods have rather been used as additional purification methods for the separation of EVs after initial (ultra)centrifugation.

[0043] 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 modified physicochemical and chemical properties, such as displacement towards smaller size, changed surface composition, lack of biological activity, etc. Unfortunately, it is still unclear to which size exclusion resin parameters the adverse effects are related and, for this reason, size exclusion based separation processes are therefore less preferred.

[0044] Membrane-based separation has been successfully applied to obtain IV preparations derived from less complex sources than milk. In fact, in the case of the use of milk as a 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 the EVs, such as milk fat globules (range size range: 0.1-15 µm) 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 coagulate at concentrations above the range of 10 mg/mL. Due to these complicating factors, it is commonly known that the use of membrane filtrations is inconvenient and can lead to the occurrence of artifacts and membrane fouling, starting with milk as the source of EVs.

[0045] However, and contrary to the teaching deriving from the prior art it has been surprisingly found that the process according to the present invention, based on membrane filtration, can be successfully employed for the efficient isolation of high purity EVs from milk or milk derivatives, when preceded by both a pre-purification phase of intentional protein coagulation and a clarification phase on an inert filtration bed.

[0046] Based on these premises, the present invention relates to a process for the isolation of milk-derived EVs and their use as a carrier of drugs or preparations having intrinsic biological activity, useful for a range of applications including, but not limited to limited to, pharmaceutical, veterinary, cosmetic and/or nutraceutical applications or as an additive to human or animal diets.

[0047] The general term "milk", unless otherwise specified, is used to define the product supplied by all mammalian species, as commonly dealt with in the dairy industry. Examples of types of milk 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.

[0048] In accordance with the present invention, a process is provided for the isolation of purified EVs from milk, said process being characterized in that it comprises at least one milk protein coagulation step and at least one ultrafiltration and concentration step. membrane-based, providing at least a single homogeneous fraction having uniform characteristics, e.g. in terms of bioanalytical parameters, particle concentration, protein/particle ratio, chromatographic purity, particle size distribution, etc. In particular, according to the present invention, the process for the isolation of purified EVs from milk of the present invention provides a single homogeneous fraction of purified EVs from milk having uniform characteristics.

[0049] The process for the isolation of purified EVs according to the present invention comprises the following steps of: a) providing a milk sample; b) optionally clarifying said sample by means of traditionally known processes of filtration or decantation to obtain clarified milk; c) subjecting said milk or clarified milk to a treatment with a mixture of enzymes suitable for the coagulation of milk proteins at a pre-defined 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 clear solution to at least one ultrafiltration and concentration step using membranes with defined pore sizes and dialysis medium, water or buffer of adjusted 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 EV concentrated preparation; g) subjecting said concentrated EV preparation, or said stabilized EV concentrated preparation, to a second filtration/clarification step in order to obtain an essentially pure clarified isolate of dairy EV; h) optionally subjecting said essentially pure clarified isolate of dairy EVs to sterilizing filtration using standard sterile filters of typical pore size less than 1 µm, preferably between 0.45 µm and 0.2 µm, in order to obtain a clarified isolate essentially pure and sterile of dairy IV.

Optionally, said essentially pure clarified dairy EV isolate obtained from step (g) or said essentially pure and sterile clarified dairy EV isolate 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 still, 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 limit yourself to, mannitol, sucrose, trehalose, or proteins such as BSA or casein, to make a frozen or freeze-dried IV preparation.

In another embodiment of the present invention it has been found that the EV isolate obtained from the process of the invention is suitable for being preserved by freezing with minimal losses of EV particles after thawing. This procedure requires freezing substantially pure EV preparations containing as numerous particulate concentrations as possible in the order of 10^12 particles per mL, more preferably 10^13 and higher. In general, it has been found that EV preparations of a certain purity, as well as a requirement for precise chemical modification and controlled loading, tend to exhibit large particle losses after freeze/thaw. Therefore, it is an intrinsic advantage of the process of the invention that it leads to obtaining EV preparations which consist of a single fraction having controllable and uniformly high particulate concentrations, suitable for freezing and storage.

[0052] 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 material filter media such as a cardboard filter or other filter cloth or lining, or by or otherwise using suitable clarification and filtration methods known in the art.

[0053] In the following description, different embodiments of the present invention will be referred to as part of the present invention. The information provided herein, along with the specific details of the examples, are set forth herein for purposes of clarity and should not be construed as limiting the invention nor should unnecessary limiting conclusions be drawn from these exemplary embodiments and the details contained in the examples.

It should be noted that, in one embodiment of the present invention, various pre-processed forms of milk and milk products may be used as starting materials in place of the pure milk sample. For example, these may be, but are not limited to, dry milk, skim milk, heat-treated milk, pasteurized milk, or colostrum, provided that these derivatives contain "intact" EVs, i.e. EVs that have the potential to exert their effects. biological functions.

[0055] When powdered milk or powdered colostrum is supplied, the process of the present invention further comprises a re-hydration step, in which said powders and granulates are dispersed in water to give a uniform suspension. This is usually achieved by combining the powders or granules with water and agitating the mixture to produce a homogeneous suspension. All known rehydration and suspension methods are suitable for said additional step, provided they do not damage the EV particles.

In another embodiment of the present invention, step (c) is performed using an enzyme mixture containing pepsin and chymosin, which is suitable for casein-based coagulation of milk proteins. Enzyme mixtures suitable for achieving this coagulation consist, for example, of pepsin (in the range of 0 to 90%) and chymosin (in the range of 100 to 10%). Preferred mixtures are composed of 5% pepsin and 95% chymosin, even more preferably 20% pepsin and 80% chymosin. The enzyme mixture can be used in an amount and for times well known by the person skilled in the art, for example about 40 mg of enzyme mixture or about 50 International Milk Clotting Units (IMCU) per liter of milk . The reaction time will depend on the amount of enzyme mixture and can range 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 is 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.

[0057] The most preferred enzyme mixture to be used in accordance with the invention is rennet of bovine origin, containing 5% pepsin and 95% chymosin. The possibility of using this enzymatic mixture can be considered one of the many advantages of the present invention with respect to the prior art which, for example, describes the contrary, the use of acid solutions or complexing agents such as EDTA. Rennet, in fact, is routinely 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 administrations.

[0058] In a preferred embodiment of the invention, after said phase (c) a further phase (c') can be provided in order to improve the microbiological characteristics of the product, carrying out a short pasteurization. Preferably, the liquid containing the coagulated proteins obtained in step (c) is heated to 45-65°C, preferably between 53-56°C for at least tens of minutes.

[0059] Subsequently, the mixture is cooled and subjected to one or more filtrations, in accordance with step (d). In a preferred embodiment of the present invention, step (d) involves a first coarse filtration to remove most of the coagulated protein, followed by deep filtration using a filter aid such as diatomaceous earth or similar filter aids. , as well as commercially available and routinely used as filter sheets in deep filtration. The clarified material obtained at the end of step (d) has a refractive index of 3-5°Brix.

[0060] The clarified and de-caseinized liquid obtained at the end of step (d) is then subjected to a series of at least one concentration and dialysis step in accordance with step (e). In a preferred embodiment, a first pre-concentration step is performed, followed by dialysis to efficiently remove the remaining contaminating proteins and other, smaller components, before performing a final concentration step in order to reach the final concentration. desired EV of milk.

Membranes having different pore sizes were tested for the concentration and dialysis steps. In general, the majority of contaminating proteins will be removed by membranes having a size cut-off limit greater than 70 kDa, more appropriately having a cut-off close to or greater than 100 kDa, even more appropriately 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 SEC chromatography). analytical, for example, on a Sephadex S-200 30/10 column). The choice of such a membrane is not a priori obvious to those skilled in the art since on the one hand contaminating proteins typically have molecular weights much lower than 300 kDa and on the other hand EVs have been shown to pass substantially unimpeded through membranes having pore sizes of 0.22 µm or inert filter aids, capable of removing soluble aggregates, thus suggesting that larger membrane sizes may lead to significant EV losses, not improving protein removal. Again, for non-dairy EVs, the use of ultrafiltration membranes with molecular weight cutoffs significantly greater than 100kDa, such as 300 kDa and higher, have been associated with EV losses. Furthermore, membranes with a molecular weight cut-off of 750 kDa are not usually commonly available.

[0062] It should be noted that for the isolation method of the invention, the fraction to be recovered (that is, the one containing the EVs) is that which remains above the filter, not the fraction which passes through it.

[0063] In a preferred embodiment of the present invention, in order to obtain the desired concentration and purity, a first pre-concentration is performed, during which the material is concentrated numerous times, typically 2-6 times with a membrane appropriate. This first pre-concentration is followed by another filtration, in dialysis mode, 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 has been found that the inclusion of sodium chloride in concentrations in the range of 0.5-1.5%, more preferably 0.8-1% is sufficient for this purpose. To those skilled in the art, other solutions will be apparent, such as a change of the specific salt form or a change of one of several buffer systems used in biochemical preparations, such as, but not limited to, buffers based on phosphate, citrate, carbonate, Tris , HEPES and others.

Upon completion of dialysis, the EV preparation may be further concentrated to the desired final EV concentration. This final concentration can be determined by methods well established in the art, such as Nano-Particle Tracking analysis. Generally, the values for the 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.

[0065] As a final step of the preparation method, the material obtained in step (e) is further clarified in step (g), using for example a diatomaceous earth as in step (d).

[0066] Optionally, such a filtration step may also be necessary after the first ultrafiltration and concentration steps of the pre-processing or during ultrafiltration if, for example, the material to be processed is particularly rich in fats, depending on its exact characteristics and of the species from which the starting material is derived. These filtration steps also serve to remove soluble protein aggregates early, derived for example from shear stress to which the material is 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 may be subjected to further filtration, which reduces microbial contamination, or to effective sterile filtration (h). The skilled in the art is perfectly capable of selecting a suitable method for this step, for example, the use of standard sterile filters having pore sizes of less than 1 µm.

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

[0070] The present invention therefore also relates to preparations of EVs, obtained by means of the above process, which satisfy numerous criteria of exceptionally high quality. Such stringent quality standards are important for the use of natural isolates, as IVs in pharmaceutical preparations or finished pharmaceutical products. EV preparations according to 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 of protein or more preferably at least 10^13 particles/mg of protein and an EV peak purity not less than 75% or more preferably at least 80% and more preferably still having an EV peak purity greater than 85% or most 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.

[0071] The present invention also relates to the use of the EV product, isolated from milk or milk derivatives following the method according to 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.

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

[0073] 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, compounds with cosmetic and/or nutraceutical value can also be considered an "active ingredient" in the present invention. The person skilled in the art is perfectly capable of selecting the most suitable compound and the correct way to load it into the EVs in relation to the purpose for which the preparation of EVs is intended.

In a specific embodiment of the present invention, the 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 activity.

A further aspect of the present invention relates to preparations containing the 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.

[0076] In particular, the EVs optionally 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, the IVs 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 on the mucosa.

[0077] 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 the bioavailability of the loaded drug whenever a physical barrier is present. By way of example, the use of IVs as a delivery system can enhance the bioavailability of 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, IV milk preparations have been found to be particularly suitable for inclusion in an inhalation formulation.

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

It is therefore surprising and notable that for the efficient systemic delivery mechanism via the respiratory epithelium in accordance with the present invention, no further modification of the milk EVs is required and it has been found that such systemic exposure following upon contact with the respiratory epithelium is several orders of magnitude greater than the systemic exposure obtained with equivalent dosages of oral milk IVs. After application of small volumes of IV milk preparations via the nose, systemic exposure in numerous tissues, including brain tissue and liver, has been detected in adult mammals.

All the discussed advantages presented by the invention will be further proved in the following experimental part which is not intended to be limiting with reference to the complete scope of the invention reported above.

Experimental section

Example 1:

[0082] Fresh cow's milk (2.5 L) was collected and left to rest in a graduated cylinder at 10°C for 22 hours, during which time natural skimming took place. The upper milk layer (ca. 150 mL) was removed by decanting.

[0083] The pH of the skim milk was adjusted to pH = 6.4 using citric acid. Then, the skimmed milk (2 L) was preheated to 35°C for 15 minutes with a heat exchange unit connected to a jacketed tank. To this heated solution was added rennet of bovine origin containing 20% pepsin and 80% chymosin, at a dosage of 50 IMCU (international milk coagulation units) per L. The coagulation temperature was maintained at 35°C for 25 minutes. After which the mixture was heated, as quickly as possible, to 56°C and was maintained at this temperature for 10 minutes. The mixture was then rapidly cooled below 10°C and the liquid whey was separated from the coagulated casein protein solid mass 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.

[0084] The resulting material was characterized by the number of particles and their size distribution, as well as the total protein content, as described in Examples 13 and 15. 1.14E+12 1.29E+11 186.5 1.6 154.8 5.8 1.55 0.04 7.32E+11 1.14E+12

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 µm) using a standard sintered glass filtration funnel of 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 was first concentrated four times to reach a total volume of approx. 370 mL. Then four volumes (1.5 L) of a 1% sodium chloride solution were added and the material was concentrated again to give the previous volume of 370 mL. The relative depletion of protein contaminants with different molecular weights during this pre-processing step was determined by Size Exclusion Chromatography (SEC), as described in Example 14 and is shown in Example 4.

Example 4:

[0087] The product of Example 3 was clarified following the procedure described in Example 2. After clarification, the sample was sterile filtered on a disposable 0.45 µm filtration unit to obtain approx. 350 mL of a clear opaque solution having a refractive index of 2. The product was then divided into different aliquots of 50 mL each and some aliquots were flash frozen for long-term storage.

[0088] The resulting material was characterized by the number of particles and their size distribution, as well as the total protein content, as described in Examples 13 and 15. 1.80E+12 4.97E+11 161.1 2 ,3 147.0 9.6 1.14 0.14 1.57E+12 4.49E+11

Example 5:

The frozen product of Example 4 (4x 50 mL) was slowly thawed overnight and clarified again by the method described in Example 2. The material was then subjected to a second round of concentration/dialysis 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 were exchanged by dialysis by repeatedly adding 100 mL of PBS pH 7.4. Finally, the sample was concentrated to obtain approx. 40 mL of purified and concentrated dairy EVs. The material was sterile filtered using a 0.2 µM PES syringe filter. The resulting material was characterized by the number of particles and their size distribution, as well as the total protein content, as described in Examples 13 and 15. 9.26E+12 5.90E+11 165.33 0.6 146 .17 7.87 1.67 0.016 5.55E+12 2.01E+11

Example 6:

[0090] Fresh cow's milk (1,500 L) was collected and partially skimmed leaving it to settle in a tank for 24 hours.

[0091] The skimmed milk (1,300 L) was then pre-heated at 35°C for 15 min. Rennet of bovine origin (containing 5% pepsin-95% chymosin) was added to this heated solution at a dosage of 50 IMCU/L. The coagulation temperature was maintained at 32°C for 60 minutes. After which the mixture was heated, as quickly as possible, to 56°C and was maintained at this temperature for 15 minutes. The mixture was then allowed to cool and the liquid whey was separated from the solid mass of coagulated casein protein using a metal mesh filter.

[0092] In total, approximately 1,000 L of slightly cloudy whey were obtained.

Example 7:

[0093] The whey (600 L) obtained in Example 6 was clarified using a plate and frame filtration device. The filter press 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 was passed over the filter. After filtration, 500 L of clear serum were obtained. The clear serum thus 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% NaCl solution in distilled water was added to the product and the solution was again concentrated to a final volume of 100 L. This material was then clarified once more using the deep filtration setup and immediately thereafter the filter was passed through a sterile filtration cartridge (Filtrox, 0 .5 µm).80 L of EV from clarified pre-concentrated milk was then obtained and frozen. 57E+12 7.36E+11

Example 8:

A 6 L sample obtained from Example 7 was concentrated six times (to give IL) and subjected to six cycles of dialysis/concentration 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 IL of EVs from sterile filtered and purified milk. 2.41E+13 4.40E+11 142.4 1.7 126.0 5.3 1.0 0.021 2.41E+13 5.13E+11

Example 9:

The whey (2,000 L) obtained as described in Example 6 was clarified using a plate and frame filtration device. The filter press 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 was passed over the filter. After filtration, 1800 L of transparent serum were obtained. The clear serum thus 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% NaCl solution in distilled water was added to the product and the solution was concentrated again to give 100 L. At this point, another 500 L of PBS (pH 7.4) was added to the product and the solution was concentrated to approximately 60 L. This material was then clarified another time using the deep filtration set-up and immediately thereafter the filter was passed through a sterile filtration cartridge (Filtrox, 0.5 µm).Therefore, 50 L of purified dairy EVs were obtained.

Example 11:

[0096] This example proceeds as Examples 1 to 5, starting with fresh goat milk (2.5 L) to obtain 20 mL of EV from purified goat milk.

[0097] The goat milk was pre-processed according to the steps described in Examples 1-4. 150 mL of pre-processed goat whey was slowly thawed overnight. The material was then subjected to a second round of concentration/dialysis 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 were exchanged by dialysis by repeatedly adding 112 mL of PBS pH 7.4. Finally, the sample was concentrated to obtain approx. 20 mL of purified and concentrated goat's milk IV. The material was sterile filtered using a 0.2 µM PES syringe filter. The resulting material was characterized by the number of particles and their size distribution, as well as the total protein content, as described in Examples 13 and 15. 1.18E+13 1.12E+12 137.5 1.5 103.0 6.4 1.44 0.13 8.19E+12 4.33E+11

Example 12:

[0098] As a comparison, a protocol from the literature for the isolation of EVs from milk based on ultracentrifugation was performed. 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.

[0099] Fresh whole milk was defatted by swirling for 30 min at 4°C at 13,000 g. The supernatant was passed through a Whatman filter paper. The defatted milk was centrifuged for 60 min at 4°C at 100,000 g (corresponding to rcf max 31,400 rpm, Sorvall Discovery Ultracentrifuge, with fixed angle rotor T865). The supernatant (not exceeding 70% of the total volume) was collected carefully with a glass pipette without disturbing the bottom slush layer. The EVs were pelletized by vortexing the supernatant for 90 min at 4°C at 135,000 g, corresponding to an rcf max = 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 syringe with a 0.2 µM PES filter. 1.14E+11 0.20E+11 142.8 1.6 119.0 3.0 0.287 n a 2.28E+11 2.13E+09

Example 13: Nanoparticle Tracking (NTA) analysis of EV preparations

The 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.

[0101] The measurements were performed in triplicate at a controlled temperature of 25°C. Each sample was diluted 1 to 100 in sterile filtered PBS pH 7.4 to 1 mL. Samples were further diluted in order to perform measurements in the range of 80-120 particles/frame. The camera level was held at 12 throughout all measurements and five consecutive 60 second recordings were made for each sample. Samples were tested with a detection threshold of 5. Characterization of dairy EV preparations by methods of the present invention using NTA is illustrated in Figure 1, Figures 2, 6(b-e), 8, 10(b-e) and 12 (b-e).

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

[0102] 50 µL of the samples were subjected to size exclusion chromatography using a Superdex 200 10/300 column (Cytiva) on a Shimadzu LC-20Ai system equipped with an SPD-M20A PDA Diode Array Detector and a Fluorescence Detector RF-20A. Size exclusion was performed in PBS 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 ). Characterization of milk EV preparations by the methods of the present invention using SEC is illustrated in Figures 3, 4, 6(a), 9, 10(a) and 12(a).

Example 15: Protein Analysis of EV Preparation

[0103] 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 instructions of the manufacturer using serial dilutions of BSA as a standard. 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 onto a 0.45 µM nitrocellulose membrane by semi-dry blot at 20 Volts for 45 min. Blocking was performed using TBS + 0.2% Tween-20 + 5% BSA (for markers MFGE8, CD9) or TBS + 0.2% Tween-20 + 2% nonfat dry milk ( tsg101) for 1 hour at room temperature with stirring. After blocking, the membranes were incubated at 4°C o/n with the following primary antibodies: MFGE8 (HPA002807, Sigma, dilution 1:1000), tsg101 (ab83, Abcam dilution 1:1000) or CD9 (AHS0902, Invitrogen , dilution 1:2000) diluted in TBS + 0.2% Tween-20 + 1% BSA (MFGE8, CD9) or TBS + 0.2% Tween-20 + 2% nonfat dry milk (tsg101 ) with shaking. After incubation, the membrane 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). The membranes were washed 5 times for 5 minutes with TBS + 0.2% Tween-20 before analysis in a LI-COR visualization instrument. Characterization of milk EV preparations by the methods of the present invention using Western Blot is illustrated in Figure 5.

Example 17: Cellular uptake after EV labelling

[0105] The milk EVs obtained in Example 8, as well as in Example 11, were each diluted to 10 µM in PBS pH 7.4 and reacted with 25 µM of (5',6'-)TMR-NHS and 50 µM of Cy5-NHS for 1 hour at 37°C. Unreacted dye was removed by six rounds of concentration/dialysis each time with six volumes of PBS 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 confluency (5% CO2 , 95% humidity, 37°C). Double Cy5/TMR-labelled 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) enriched with Hoechst 33258 at 0.5 µM.Cells were visualized by fluorescence microscopy at wide field (Olympus IX83) using an ApoPlan 40x objective with a numerical aperture of 0.95 Over 10 images were taken per well, where each image represented a maximum intensity projection of 7 individual Z images and 5 different images were displayed Channels: Brightfield, Hoechst (ex385nm, em417-477nm), TMR (ex545nm, em605/70nm), CY5 (ex640nm, em700/75nm) and TMR- CY5 FRET (ex545, em700/75).The detection of cells and vesicles it was done with a self-programmed ImageJ Fiji plugin. Briefly, cells were detected by angle detection of the channel in bright field followed by smoothing and thresholding of the image. EVs were detected using auto Li threshold (similar thresholds are used at all concentrations) on the CY5 channel. The EVs in the cell area have been counted and are shown as cell area „EV/1,000 pixel“.

Example 18: Transmission Electron Microscopy of negatively stained EVs

The milk EVs obtained in Example 8 were diluted to a concentration of 1 to 5x10^10 particles per mL in PBS and pipetted onto grids coated with Formvar and carbon. After air-drying for 30-45 min at room temperature, excess IV solution was removed by pressing grids on filter paper at an angle of approx. 45th. Grids were then washed 3x with distilled water by placing them in 100 µL drops on parafilm, followed by fixation with 1% glutaraldehyde in PBS for 5 min on 40 µL drops, washing 8x with water in 40 µL drops and negative staining with 2% aqueous uranyl acetate for 5 minutes. The 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 (Tröndle camera). Typical images of milk EV preparations from Example 8 are shown in Figure 13.

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

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 µL samples and were injected and separated on a C18 column (3.0 × 150 mm with 5 µm particle size) by isocratic elution with a mobile phase of Acetonitrile/20 mM EDTA (55 % / 45% vol/vol) with a flow rate of 1 mL/min. EDTA has been used in the mobile phase, as it improves the chromatographic behavior of AmB by direct competition with Amphotericin B for metal ion chelation. AmB was detected by measuring the absorbance of UV at 406 nm. Calibration standard solutions were prepared at concentrations ranging from 0.0125 - 10 µg/mL. Representative chromatograms and calibration curves of AmB are shown in Figure 14 a and b, respectively.

Example 20: Amphotericin B loading on bovine milk exosomes

For loading AmB onto bovine milk exosomes, a number of methods have been tested, including room temperature incubation, 37°C incubation, hypotonic condition, sonication, extrusion, saponin incubation, and freeze-thaw. AmB was added from 1 mM stock solution in DMSO to milk EV samples in PBS to give final concentrations of 1 nM MEV, 2 µM AmB and 0.5 % DMSO and were treated under different conditions , as follows: – Incubation: The formulations were incubated at room temperature (21°C), 37°C or 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: formulations were sonicated using a probe sonicator (80 % power with 30 second pulse / 30 second pause). – Hypotonic condition: the formulations were diluted with H2O up to a final concentration of PBS 0.5x. – Freeze-thaw: the formulations were flash frozen, kept at -80°C for 0.5 hours and thawed at room temperature for 3 cycles. – Extrusion: formulations were extruded (x10-fold and x20-fold) using a Avanti Lipids extruder having a pore diameter of 200 nm.

[0109] To remove free AmB from the formulation, the mixture was ultrafiltered six times with PBS by centrifugation in a Vivaspin ultrafiltration unit (MWCO of 10 kDa) at 10,000 x g for 15 minutes. The final washing step led to obtaining 10 times concentrated samples. The AmB was then extracted by adding acetonitrile to the original volume, resulting in a final ACN:H2O 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 AmB concentrations retained for the different conditions are listed in Table 1, revealing that loading by incubation at room temperature for ≥ 6 hours resulted in the highest loading densities.

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

[0110] Ambient temperature 1 hour 0 nM Ambient temperature 2 hours 21 nM Ambient temperature 4 hours 179 nM Ambient temperature 6 hours 227 nM Ambient temperature 24 hours 252 nM 37°C for 6 hours 171 nM 37°C for 24 hours 128 nM 50 °C for 6 hours 87 nM 50°C for 24 hours 52 nM Hypotonic condition 189 nM Sonication for 2.5 min (ice bath) 294 nM Sonication for 5 min (ice bath) 162 nM Sonication for 10 min (ice bath) ice) 81 nM Sonication for 2.5 min (without ice bath) 44 nM Sonication for 5 min (without ice bath) 24 nM Extrude 10 times 184 nM Extrude 20 times 172 nM Room temperature for 24 hours + 0.2% of Saponin 254 nM 37°C for 24 hours + 0.2% of Saponin 132 nM 50°C for 24 hours + 0.2% of Saponin 54 nM 3 Freeze-thaw cycles 108 nM

Example 21: Amphotericin B loading on goat milk exosomes

Loading of AmB onto goat milk exosomes was tested using the procedures as described in Example 20. Retained AmB concentration was performed using RP-HPLC as described in Example 19.

Table 2: Retained AmB concentration in goat milk IV (1 nM) under different conditions

[0112] Ambient temperature 2 hours 82 nM Ambient temperature 4 hours 271 nM Ambient temperature 6 hours 392 nM Ambient temperature 24 hours 388 nM 37°C for 6 hours 199 nM 37°C for 24 hours 112 nM 50°C for 6 hours 91 nM 50°C for 24 hours 48 nM Sonication for 2.5 min (ice bath) 209 nM Sonication for 5 min (ice bath) 103 nM Sonication for 10 min (ice bath) 73 nM

Example 22: Saturation of AmB loading on goat milk IV

[0113] To additionally test the saturation of EV loading as a function of AmB concentration, EVs from 1 nM goat milk were incubated for 6 hours at room temperature with increasing AmB concentrations ranging in the range from 1 µM to 10 µM (DMSO at 1% v/v in all samples). Furthermore, we tested the formulation of AmB-loaded exosomes with increasing EV concentration while keeping the ratio of EV:AmB constant. MEV:AmB concentrations were at 1 nM: 2 µM; 2 nM: 4 µM; 3 nM: 6 µM; 4 nM: 8 µM and 5 nM: 10 µM. All formulations contained 1% DMSO. The retained AmB concentration 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

[0114] To test whether AmB retained with the EVs after loading is actually physically associated with the vesicles, we used size exclusion analytic chromatography under native conditions to verify co-fractionation. 10 µL of the samples as prepared in Example 20 (1 nM of bovine or goat milk EVs loaded with 2 µM AmB by incubation for 6 hours at room temperature) before and after elimination of free AmB by ultrafiltration were injected on a Sephadex 200 30/10 column and separated by isocratic elution with PBS pH 7.4 at a flow rate of 0.8 mL/min in a Shimadzu LC-20Ai instrument equipped with a UV/Vis and fluorescence diode array detector. 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 analysed. As shown in Figure 16, AmB loaded on both bovine milk EVs (Figure 16a) and goat milk EVs (Figure 16b) is 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 AmB-loaded goat milk EVs

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 EV loaded with 2 µM AmB by incubation for 6 hours at room temperature). Cell 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 uptake by the cells identically to dairy EVs from unloaded goats, confirming that AmB loading does not influence the functionality of milk EVs in cell uptake.

Example 25: Systemic IV 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/B16 mice (female 8 weeks). After aspiration of 5 µL of 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 pH 7.4) and analyzed by epifluorescence visualization in an IVIS Spectrum visualization instrument. The intensities of the fluorescence signals relating to organs from PBS-treated animals are shown in Table 3.

Table 3: Intensities of fluorescence signals of different organs from IV-administered mice compared to the corresponding organs of PBS-treated control animals:

[0117] Brain 1.2 GI tract 44.2 Heart 7.7 Left kidney 32.4 Right kidney 29.5 Liver 18.5 Lymph node 5.7 Left lung 386.0 Right lung 412.7 Spleen 7.0

Claims (10)

1. A process for the isolation of purified EVs from milk, characterized in that it comprises at least one milk protein coagulation step and at least one membrane-based ultrafiltration and concentration step. 2. A process for the isolation of purified EVs from milk according to claim 1, characterized in that it comprises the following steps: a) provide a sample of milk; b) optionally clarifying said sample by means of traditionally known processes of filtration or decantation to obtain clarified milk; c) subjecting said milk or 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 filtering medium, to obtain a transparent solution; e) subjecting said clear solution to at least one step of ultrafiltration and concentration using membranes with defined pore sizes and water or buffer of adjusted 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 EV concentrated preparation; g) subjecting said concentrated EV preparation or said stabilized EV concentrated preparation to a second filtration/clarification step in order to obtain an essentially pure clarified isolate of dairy EV; h) optionally subjecting said essentially pure clarified isolate of dairy EVs to sterilizing filtration using standard sterile filters of typical pore size less than 1 µm, preferably between 0.45 µm and 0.2 µm, in order to obtain a clarified isolate essentially pure sterile IV dairy. 3. The process according to claim 1 or claim 2 characterized in that said milk protein coagulation step or enzyme mixture of step (c) contain pepsin and chymosin, preferably obtained by fermentation or rennet, preferably containing 5% of pepsin and 95% of chymosin. 4. The process according to claim 1, 2 or 3 characterized in that said at least one ultrafiltration and concentration step (e) is carried out through a membrane having a molecular cut-off of 750 kDa and is preferably preceded by a pre -concentration and ultrafiltration 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 carrier for active ingredients in the pharmaceutical, veterinary, nutraceutical and/or cosmetic fields. The composition comprising the essentially pure clarified isolate of dairy EV according to claims 1-4 and pharmaceutically acceptable excipients. 7. A composition according to claim 6, characterized in that it further comprises Amphotericin B loaded into said essentially pure clarified isolate of dairy EV. 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 antiparasitic.