Extracellular vesicle compositions and preparation

Abstract

In various aspects and embodiments, compositions containing extracellular vesicles, including modified extracellular vesicles, methods of making formulations of extracellular vesicles, methods of modifying extracellular vesicles, and methods of using modified and / or unmodified extracellular vesicles are provided.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 465,073, filed May 9, 2023, U.S. Provisional Application Serial No. 63 / 624,637, filed January 24, 2024, and U.S. Provisional Application Serial No. 63 / 640,825, filed April 30, 2024, all of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0002] The present disclosure relates generally to extracellular vesicles (e.g., milk-derived vesicles or exosomes) and methods of making, modifying, and using such vesicles.

[0003] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant Numbers HL161237, HL141855, and HL056728 awarded by the National Institutes of Health and STTR #2203330 awarded by NSF. The government has certain rights in the invention. BACKGROUND

[0004] PCT Application Publication No. WO2022182782 discloses “Methods of isolating exosomes from biological fluids, such as those containing casein.”

[0005] PCT Application Publication No. WO2020010161 discloses “Milk vesicles as drug delivery vehicles, compositions comprising therapeutic agents encapsulated within or otherwise associated with milk vesicles, methods of producing such milk vesicles and compositions thereof, and methods of delivering such milk vesicles and compositions to specific patient tissues or organs.” SUMMARY

[0006] The present disclosure is based, at least in part, on compositions containing extracellular vesicles, including modified extracellular vesicles, methods of making preparations of isolated extracellular vesicles, methods of modifying extracellular vesicles, and methods of using modified and / or unmodified extracellular vesicles.

[0007] As used herein, the terms "extracellular vesicle" or "EV" or "vesicle" are used interchangeably and refer to small lipid bilayer membrane-bound structures that exist outside of cells. In many embodiments, extracellular vesicles are released from cells into the extracellular space. Extracellular vesicles have a lipid bilayer and are typically about 30 to 1,000 nm in diameter. In addition to the lipid bilayer and internal aqueous cavity, extracellular vesicles have additional components such as tetraspanins and IgG molecules; thus, detection of these components in isolated extracellular vesicles can be used to distinguish other (such as synthetic) particles that have a lipid bilayer. Extracellular vesicles often contain naturally bioactive agents in their interior and / or outer bilayer membrane. In addition, as discussed in more detail below, extracellular vesicles can be loaded with "cargo" such as therapeutic molecules, and when administered to a subject, the cargo-loaded vesicles can enhance delivery of the cargo; for example, under certain conditions, cargo-loaded vesicles can be used to effectively orally administer molecules that would otherwise not be bioavailable or poorly bioavailable when orally administered.

[0008] It has been discovered that milk is a rich source of extracellular vesicles, and recent studies have shown that isolated extracellular vesicle preparations can be made from milk, which can be loaded with "cargo" such as therapeutic molecules, and which can be used to successfully administer the cargo to a subject (see, e.g., WO2022182782).

[0009] Certain aspects and embodiments of the present disclosure are based on the discovery that isolated extracellular vesicles modified to have ATP and / or one or more ATP analogs can increase cellular uptake of the modified extracellular vesicles when administered to a subject, thereby increasing the administration effectiveness of a cargo if present in the vesicles. The term ATP means adenosine triphosphate. As used herein, the term "ATP-analog-vesicle" refers to an extracellular vesicle that has been modified to include ATP and / or one or more ATP analogs. In certain embodiments, the ATP-analog-vesicles of the present disclosure can be particularly useful for administering a cargo via a route other than oral administration, such as parenteral administration, intranasal administration, IV administration, injection, subcutaneous injection, or topical administration.

[0010] Thus, in one aspect, a composition comprising isolated ATP-analog vesicles is provided, wherein the vesicles comprise 0.01 to 1,000 mM ATP or ATP analog. In a similar aspect, a composition comprising isolated milk ATP-analog vesicles is provided, wherein the vesicles comprise 0.01 to 1,000 mM ATP or ATP analog. In yet another aspect, a composition is provided, wherein the composition comprises isolated bovine milk ATP-analog vesicles, wherein the vesicles comprise 0.01 to 1,000 mM ATP or ATP analog. In some embodiments, the ATP-analog vesicles comprise ATP or ATP analog at a concentration between: 0.01 to 1,000 mM; or 0.1 to 100 mM; or 0.25 to 175 mM; 0.5 to 50 mM; or 0.75 to 25 mM; or 0.8 to 20 mM; or 0.85 to 15 mM; or 0.9 to 10 mM; or 0.1 to 5 mM; or 0.5 to 5 mM; or 0.1 to 2 mM; or 0.5 to 1.5 mM; or 0.75 to 1.25 mM. In some embodiments, the ATP-analog vesicles comprise ATP or ATP analog at a concentration of: about 0.1 mM; 0.25 mM; 0.5 mM; 0.75 mM; 0.9 mM; 1 mM; 1.1 mM; 1.25 mM; 1.5 mM; 2 mM; 5 mM; 10 mM; 50 mM; or 100 mM. In this context, when a reagent in the vesicles, such as ATP, is described in molar concentration (e.g., as mM), the vesicles are exposed to a solution of the reagent at the indicated molar concentration until equilibrium is reached (e.g., using the methods described herein), such that the molar concentration within each vesicle is consistent with the molar concentration of the solution.

[0011] In another aspect, a composition comprising isolated ATP-analog vesicles is provided, wherein the vesicles comprise 1 ng to 1,000 μg ATP or ATP analogs per mg vesicle protein. In a similar aspect, a composition comprising isolated milk ATP-analog vesicles is provided, wherein the vesicles comprise 1 ng to 1,000 μg ATP or ATP analogs per mg milk vesicle protein. In yet another aspect, a composition comprising isolated bovine milk ATP-analog vesicles is provided, wherein the vesicles comprise 1 ng to 1,000 μg ATP or ATP analogs per mg bovine milk ATP-analog vesicle. In some embodiments, the ATP-analog vesicles comprise ATP or ATP analogs at a concentration between: 1 ng to 1,000 μg per mg vesicle protein; or 10 ng to 100 μg per mg vesicle protein; or 100 ng to 10 μg per mg vesicle protein; or 200 ng to 1 μg per mg vesicle protein; or 300 ng to 750 ng per mg vesicle protein; or 400 ng to 650 ng per mg vesicle protein; or 450 ng to 600 ng per mg vesicle protein; or 500 ng to 550 ng per mg vesicle protein. In some embodiments, the ATP-analog vesicles comprise ATP or ATP analogs at a concentration of: about 1 ng per mg vesicle protein; 10 ng per mg vesicle protein; 100 ng per mg vesicle protein; 200 ng per mg vesicle protein; 300 ng per mg vesicle protein; 400 ng per mg vesicle protein; 450 ng per mg vesicle protein; 500 ng per mg vesicle protein; 550 ng per mg vesicle protein; 600 ng per mg vesicle protein; 650 ng per mg vesicle protein; 750 ng per mg vesicle protein; 1 μg per mg vesicle protein; 10 μg per mg vesicle protein; 100 μg per mg vesicle protein; or 1,000 μg per mg vesicle protein.

[0012] In related aspects, a method is provided that includes obtaining a composition comprising isolated vesicles (e.g., any of the isolated vesicle compositions as described herein), contacting the vesicles with a solution comprising ATP and / or an ATP analog to obtain a composition comprising ATP-analog-vesicles. As used herein, the term “isolated” and the like refers to an agent (e.g., a vesicle) that has been separated from its original environment, i.e., the environment of the isolated agent (e.g., vesicle) is substantially free of at least one component found in the environment in which the “non-isolated” agent (e.g., vesicle) was present. The term includes an agent (e.g., a vesicle) removed from some or all of the components in which it is found in its natural environment, e.g., isolated from a tissue, biopsy fluid (such as milk) sample. The term also includes an agent (e.g., a vesicle) removed from at least one, some, or all components because the agent (e.g., vesicle) is present in a non-naturally occurring environment, e.g., isolated from a cell culture or cell suspension. Thus, an isolated agent (e.g., a vesicle) is partially or completely separated from at least one component (including other substances, cells, or cell populations) because it is found in nature or because it is grown, stored, or exists in a non-naturally occurring environment. In some embodiments, the isolated vesicles of the present disclosure are isolated extracellular vesicles that have been isolated from milk, e.g., vesicles that have been isolated from milk using one or more of the methods described herein and / or described in PCT WO2022182782. Vesicles isolated from milk are referred to herein as “milk vesicles.”

[0013] In certain aspects and embodiments, the vesicles herein are provided in a liquid solution. As used herein, vesicles in a “liquid solution” means that the vesicles are present in a liquid, e.g., the liquid can be one or more solvents such as water, vegetable oil, terpene, aldehyde, ketone, diacetone alcohol, glycol ether, methanol, ethanol, isopropyl alcohol. In certain embodiments, the liquid is an aqueous liquid. In certain embodiments, the liquid is a physiological solution with electrolytes and / or buffers. In certain embodiments, the liquid can also include one or more solutes including sodium chloride, potassium chloride and other salts, magnesium, calcium, disaccharides, amino acids, nucleic acids, and other solute solutions known to one of skill in the art.

[0014] In certain aspects and embodiments, the extracellular vesicle compositions are provided in a dry or lyophilized state. As used herein, the terms “dry” and “lyophilized” mean a product, such as a cake or powder, that has been removed of water by freeze-drying or sublimation, wherein the final product has a lower concentration of water than the original liquid starting material. In certain embodiments, other forms of drying or “lyophilization” as described herein can include spray drying, pharmaceutical drying, crystallization, extrusion, and other forms of drying known to one of skill in the art.

[0015] In certain aspects and embodiments, the dried product has a moisture content (defined by [mass of water / mass of sample] * 100) of less than about 10 wt%, or less than about 9 wt%, or less than about 8 wt%, or less than about 7 wt%, or less than about 6 wt%, or less than about 5 wt%, or less than about 4 wt%, or less than about 3 wt%, or less than about 2 wt%, or less than about 1 wt%, or less than about 0.5 wt%, or less than about 0.1 wt% of moisture.

[0016] As used herein, the term “analog” means a compound that is similar in structure and / or function to another compound but differs in structure from that compound. This difference can be one single element or group, or more than one group (e.g., 1, 2, 3, 4, or more groups), provided that it retains the same chemical scaffold and preferably functions as the parent chemical. Such modifications are routine to one of skill in the art and include, for example, additional or substituted chemical moieties. In some embodiments, an analog as used herein can have a different or very different chemical structure from the parent compound, but have similarity in a relevant functional or biological activity. In some embodiments, an analog as used herein can have a different or very different chemical structure from the parent compound, but have at least one relevant functional or biological activity that is increased, decreased, or absent.

[0017] As used herein, the term “ATP analog” refers to an analog of ATP. In certain embodiments, the ATP analog of the present disclosure is a non-hydrolysable analog of ATP. In some embodiments, the ATP analog of the present disclosure is one or more selected from the group consisting of: a, b-methylene-ATP (a, b mATP); b, g-methylene-ATP (b, g mATP); 2-thio-ATP (2-SH-ATP); 2-methylthio-ATP (2-MeS-ATP); 2',3'-O-2,4,6,-trinitrophenyl-ATP (TNP-ATP); 2',3'-9-(4-benzoyl)-ATP (BzATP); N-alkyl-2 ATP; adenosine 5'-(b, g-imido)triphosphate (AMP-PNP); ATP-MgCl2; 5-aminoimidazole-4-carboxamide riboside, disoproxil fumarate, ribavirin, azidothymidine, fludarabine, efavirenz, and oxidized ATP (oATP), adenosine, ADP, AMP, and other nucleotides (e.g., GTP, CTP, UTP, TTP).

[0018] As used herein, the term "tryptophan analog" refers to tryptophan itself or an analog of tryptophan. In certain embodiments, the tryptophan analog of the present disclosure is tryptophan. In some embodiments, the tryptophan analog of the present disclosure is one or more selected from the group consisting of L-tryptophan, D-tryptophan, a tryptophan dimer, a tryptophan trimer, a tryptophan peptide, histidine, L-histidine, D-histidine, a histidine dimer, a histidine trimer, a histidine peptide, tyrosine, L-tyrosine, D-tyrosine, a tyrosine dimer, a tyrosine trimer, a tyrosine peptide, arginine, L-arginine, D-arginine, an arginine dimer, an arginine trimer, an arginine peptide, cysteine, L-cysteine, D-cysteine, a cysteine dimer, a cysteine trimer, a cysteine peptide, lysine, L-lysine, D-lysine, a lysine dimer, a lysine trimer, a lysine peptide, phenylalanine, L-phenylalanine, D-phenylalanine, a phenylalanine dimer, a phenylalanine trimer, a phenylalanine peptide, D-carnitine, L-carnitine, a carnitine dimer, a carnitine trimer, a carnitine peptide, acetyl L-carnitine, L-carnitine L-tartrate, propionyl L-carnitine, D-taurine, L-taurine, a taurine dimer, a taurine trimer, a taurine peptide.

[0019] Other aspects and embodiments of the present disclosure are based on the discovery that certain molecules, such as tryptophan and / or tryptophan analogs, can be used to disrupt the association of extracellular vesicles with caseins and other milk protein molecules that can aggregate and complicate the isolation of extracellular vesicles from milk; and related methods of isolating extracellular vesicles from milk, wherein the methods comprise contacting extracellular vesicles-containing milk with tryptophan and / or a tryptophan analog.

[0020] Accordingly, in one aspect, there is provided a method of isolating extracellular vesicles from a milk sample, wherein the method involves contacting (or incubating) the milk sample with tryptophan and / or one or more tryptophan analogues, and subsequently isolating the extracellular vesicles from one or more milk components, such as, for example, casein-containing aggregates. In some embodiments, the method comprises contacting (incubating) the vesicle-containing sample with tryptophan and / or one or more tryptophan analogues at a concentration of between 10 pM to 1 mM. In some embodiments, the method comprises contacting (incubating) the vesicle-containing sample with tryptophan and / or one or more tryptophan analogues at a temperature of between 4 to 50 degrees Celsius for at least 10 minutes. In some embodiments, the method comprises contacting (incubating) the vesicle-containing sample with tryptophan and / or one or more tryptophan analogues at a concentration of between 10 pM to 1 mM at a temperature of between 10 to 50 degrees Celsius for at least 10 minutes. In some embodiments, the method comprises contacting (incubating) the vesicle-containing sample with tryptophan and / or one or more tryptophan analogues at a concentration of between 10 pM to 1 mM at a temperature of between 10 to 50 degrees Celsius for 10 to 60 minutes.

[0021] In various embodiments, the extracellular vesicles of the compositions and methods of the present disclosure have a diameter (or, in the case of a composition comprising a plurality of vesicles, an average (mean) diameter of the particles is between) of between 1 to 2,000 nm; or 10 to 1,500 nm; or 20 to 1,000 nm; or 1 to 1,000 nm; or 1 to 500 nm; or 1 to 300 nm; or 1 to 200 nm; or 1 to 185 nm; or 1 to 175 nm; or 1 to 170 nm; or 1 to 165 nm; or 1 to 160 nm; or 1 to 155 nm; or 1 to 150 nm; or 1 to 145 nm; or 1 to 140 nm; or 1 to 135 nm; or 1 to 130 nm; or 10 to 1,000 nm; or 10 to 500 nm; or 10 to 300 nm; or 10 to 200 nm; or 10 to 185 nm; or 10 to 175 nm; or 10 to 170 nm; or 10 to 165 nm; or 10 to 160 nm; or 10 to 155 nm; or 10 to 150 nm; or 10 to 145 nm; or 10 to 140 nm; or 10 to 135 nm; or 10 to 130 nm; or 15 to 1,000 nm; or 15 to 500 nm; or 15 to 300 nm; or 15 to 200 nm; or 15 to 185 nm; or 15 to 175 nm; or 15 to 170 nm; or 15 to 165 nm; or 15 to 160 nm; or 15 to 155 nm; or 15 to 150 nm; or 15 to 145 nm; or 15 to 140 nm; or 15 to 135 nm; or 15 to 130 nm; or 20 to 1,000 nm; or 20 to 500 nm; or 20 to 300 nm; or 20 to 200 nm; or 20 to 185 nm; or 20 to 175 nm; or 20 to 170 nm; or 20 to 165 nm; or 20 to 160 nm; or 20 to 155 nm; or 20 to 150 nm; or 20 to 145 nm; or 20 to 140 nm; or 20 to 135 nm; or 20 to 130 nm; or 25 to 1,000 nm; or 25 to 500 nm; or 25 to 300 nm; or 25 to 200 nm; or 25 to 185 nm; or 25 to 175 nm; or 25 to 170 nm; or 25 to 165 nm; or 25 to 160 nm; or 25 to 155 nm; or 25 to 150 nm; or 25 to 145 nm; or 25 to 140 nm; or 25 to 135 nm; or 25 to 130 nm; or 30 to 1,000 nm; or 30 to 500 nm; or 30 to 300 nm; or 30 to 200 nm; or 30 to 185 nm; or 30 to 175 nm; or 30 to 170 nm;or 30 to 165 nm; or 30 to 160 nm; or 30 to 155 nm; or 30 to 150 nm; or 30 to 145 nm; or 30 to 140 nm; or 30 to 135 nm; or 30 to 130 nm; or 35 to 1,000 nm; or 35 to 500 nm; or 35 to 300 nm; or 35 to 200 nm; or 35 to 185 nm; or 35 to 175 nm; or 35 to 170 nm; or 35 to 165 nm; or 35 to 160 nm; or 35 to 155 nm; or 35 to 150 nm; or 35 to 145 nm; or 35 to 140 nm; or 35 to 135 nm; or 35 to 130 nm; or 40 to 1,000 nm; or 40 to 500 nm; or 40 to 300 nm; or 40 to 200 nm; or 40 to 185 nm; or 40 to 175 nm; or 40 to 170 nm; or 40 to 165 nm; or 40 to 160 nm; or 40 to 155 nm; or 40 to 150 nm; or 40 to 145 nm; or 40 to 140 nm; or 40 to 135 nm; or 40 to 130 nm; or 45 to 1,000 nm; or 45 to 500 nm; or 45 to 300 nm; or 45 to 200 nm; or 45 to 185 nm; or 45 to 175 nm; or 45 to 170 nm; or 45 to 165 nm; or 45 to 160 nm; or 45 to 155 nm; or 45 to 150 nm; or 45 to 145 nm; or 45 to 140 nm; or 45 to 135 nm; or 45 to 130 nm; or 50 to 1,000 nm; or 50 to 500 nm; or 50 to 300 nm; or 50 to 200 nm; or 50 to 185 nm; or 50 to 175 nm; or 50 to 170 nm; or 50 to 165 nm; or 50 to 160 nm; or 50 to 155 nm; or 50 to 150 nm; or 50 to 145 nm; or 50 to 140 nm; or 50 to 135 nm; or 50 to 130 nm; or 55 to 1,000 nm; or 55 to 500 nm; or 55 to 300 nm; or 55 to 200 nm; or 55 to 185 nm; or 55 to 175 nm; or 55 to 170 nm; or 55 to 165 nm; or 55 to 160 nm; or 55 to 155 nm; or 55 to 150 nm; or 55 to 145 nm; or 55 to 140 nm; or 55 to 135 nm; or 55 to 130 nm; or 60 to 1,000 nm; or 60 to 500 nm; or 60 to 300 nm; or 60 to 200 nm; or 60 to 185 nm; or 60 to 175 nm; or 60 to 170 nm; or 60 to 165 nm; or 60 to 160 nm; or 60 to 155 nm; or 60 to 150 nm; or 60 to 145 nm; or 60 to 140 nm; or 60 to 135 nm; or 60 to 130 nm; or 65 to 1,000 nm; or 65 to 500 nm; or 65 to 300 nm; or 65 to 200 nm; or 65 to 185 nm; or 65 to 175 nm; or 65 to 170 nm; or 65 to 165 nm; or 65 to 160 nm; or 65 to 155 nm; or 65 to 150 nm; or 65 to 145 nm; or 65 to 140 nm; or 65 to 135 nm; or 65 to 130 nm; or 70 to 1,000 nm; or 70 to 500 nm; or 70 to 300 nm; or 70 to 200 nm; or 70 to 185 nm; or 70 to 175 nm; or 70 to 170 nm; or 70 to 165 nm; or 70 to 160 nm; or 70 to 155 nm; or 70 to 150 nm; or 70 to 145 nm; or 70 to 140 nm; or 70 to 135 nm; or 70 to 130 nm; or 75 to 1,000 nm; or 75 to 500 nm; or 75 to 300 nm; or 75 to 200 nm; or 75 to 185 nm; or 75 to 175 nm; or 75 to 170 nm; or 75 to 165 nm; or 75 to 160 nm; or 75 to 155 nm; or 75 to 150 nm; or 75 to 145 nm; or 75 to 140 nm; or 75 to 135 nm; or 75 to 130 nm; or 80 to 1,000 nm; or 80 to 500 nm; or 80 to 300 nm; or 80 to 200 nm; or 80 to 185 nm; or 80 to 175 nm; or 80 to 170 nm; or 80 to 165 nm; or 80 to 160 nm; or 80 to 155 nm; or 80 to 150 nm; or 80 to 145 nm; or 80 to 140 nm; or 80 to 135 nm; or 80 to 130 nm; or 85 to 1,000 nm; or 85 to 500 nm; or 85 to 300 nm; or 85 to 200 nm; or 85 to 185 nm; or 85 to 175 nm; or 85 to 170 nm; or 85 to 165 nm; or 85 to 160 nm; or 85 to 155 nm; or 85 to 150 nm; or 85 to 145 nm; or 85 to 140 nm; or 85 to 135 nm; or 85 to 130 nm; or 90 to 1,000 nm; or 90 to 500 nm; or 90 to 300 nm; or 90 to 200 nm; or 90 to 185 nm; or 90 to 175 nm; or 90 to 170 nm; or 90 to 165 nm; or 90 to 160 nm; or 90 to 155 nm; or 90 to 150 nm; or 90 to 145 nm; or 90 to 140 nm; or 90 to 135 nm; or 90 to 130 nm; or 95 to 1,000 nm; or 95 to 500 nm; or 95 to 300 nm; or 95 to 200 nm; or 95 to 185 nm; or 95 to 175 nm; or 95 to 170 nm; or 95 to 165 nm; or 95 to 160 nm; or 95 to 155 nm; or 95 to 150 nm; or 95 to 145 nm; or 95 to 140 nm; or 95 to 135 nm; or 95 to 130 nm; or 100 to 1,000 nm; or 100 to 500 nm; or 100 to 300 nm; or 100 to 200 nm; or 100 to 185 nm; or 100 to 175 nm; or 100 to 170 nm; or 100 to 165 nm; or 100 to 160 nm; or 100 to 155 nm; or 100 to 150 nm; or 100 to 145 nm; or 100 to 140 nm; or 100 to 135 nm; or 100 to 130 nm.Or 60 to 165 nm; or 60 to 160 nm; or 60 to 155 nm; or 60 to 150 nm; or 60 to 145 nm; or 60 to 140 nm; or 60 to 135 nm; or 60 to 130 nm; or 65 to 1,000 nm; or 65 to 500 nm; or 65 to 300 nm; or 65 to 200 nm; or 65 to 185 nm; or 65 to 175 nm; or 65 to 170 nm; or 65 to 165 nm; or 65 to 160 nm; or 65 to 155 nm; or 65 to 150 nm; or 65 to 145 nm; or 65 to 140 nm; or 65 to 135 nm; or 65 to 130 nm; or 70 to 1,000 nm; or 70 to 500 nm; or 70 to 300 nm; or 70 to 200 nm nm; or 70 to 185 nm; or 70 to 175 nm; or 70 to 170 nm; or 70 to 165 nm; or 70 to 160 nm; or 70 to 155 nm; or 70 to 150 nm; or 70 to 145 nm; or 70 to 140 nm; or 70 to 135 nm; or 70 to 130 nm; or 75 to 1,000 nm; or 75 to 500 nm; or 75 to 300 nm; or 75 to 200 nm; or 75 to 185 nm; or 75 to 175 nm; or 75 to 170 nm; or 75 to 165 nm; or 75 to 160 nm; or 75 to 155 nm; or 75 to 150 nm; or 75 to 145 nm; or 75 to 140 nm; or 75 to 135 nm; or 75 to 130 nm; nm; or 90 to 1,000 nm; or 90 to 500 nm; or 90 to 300 nm; or 90 to 200 nm; or 90 to 185 nm; or 90 to 175 nm; or 90 to 170 nm; or 90 to 165 nm; or 90 to 160 nm; or 90 to 155 nm; or 90 to 150 nm; or 90 to 145 nm; or 90 to 140 nm; or 90 to 135 nm; or 90 to 130 nm; or 95 to 1,000 nm; or 95 to 500 nm; or 95 to 300 nm; or 95 to 200 nm; or 95 to 185 nm; or 95 to 175 nm; or 95 to 170 nm; or 95 to 165 nm; or 95 to 160 nm; or 95 to 155 nm; or 95 to 150 nm nm; or 95 to 145 nm; or 95 to 140 nm; or 95 to 135 nm; or 95 to 130 nm; or 100 to 1,000 nm; or 100 to 500 nm; or 100 to 300 nm; or 100 to 200 nm; or 100 to 185 nm; or 100 to 175 nm;Or 100 to 170 nm; or 100 to 165 nm; or 100 to 150 nm; or 100 to 155 nm; or 100 to 150 nm; or 100 to 145 nm; or 100 to 100 nm; or 100 to 135 nm; or 100 to 130 nm; or 105 to 1,000 nm; or 105 to 500 nm; or 105 to 300 nm; or 105 to 200 nm; or 105 to 185 nm; or 105 to 175 nm; or 105 to 170 nm; or 105 to 165 nm; or 105 to 160 nm; or 105 to 155 nm; or 105 to 150 nm; or 105 to 145 nm; or 105 to 140 nm; or 105 to 135 nm; or 105 to 130 nm; nm; or 110 to 1,000 nm; or 110 to 500 nm; or 110 to 300 nm; or 110 to 200 nm; or 110 to 185 nm; or 110 to 175 nm; or 110 to 170 nm; or 110 to 165 nm; or 110 to 150 nm; or 110 to 155 nm; or 110 to 150 nm; or 110 to 145 nm; or 110 to 100 nm; or 110 to 135 nm; or 110 to 130 nm; or 115 to 1,000 nm; or 115 to 500 nm; or 115 to 300 nm; or 115 to 200 nm; or 115 to 185 nm; or 115 to 175 nm; or 115 to 170 nm; or 115 to 165 nm; or 115 to 160 nm nm; or 115 to 155 nm; or 115 to 150 nm; or 115 to 145 nm; or 115 to 140 nm; or 115 to 135 nm; or 115 to 130 nm; or 120 to 1,000 nm; or 120 to 500 nm; or 120 to 300 nm; or 120 to 200 nm; or 120 to 185 nm; or 120 to 175 nm; or 120 to 170 nm; or 120 to 165 nm; or 120 to 150 nm; or 120 to 155 nm; or 120 to 150 nm; or 120 to 145 nm; or 120 to 100 nm; or 120 to 135 nm; or 120 to 130 nm.

[0022] In some embodiments, it may be preferred that all or most of the extracellular vesicle particles in the composition have a size of less than about 600 nm, or in some embodiments less than about 500 nm. For example, in some cases, extracellular vesicles used to deliver therapeutic payloads can elicit an immune response in subjects when the particle size exceeds 500 nm or 600 nm. In some embodiments, extracellular vesicles with a particle size exceeding about 600 nm, or exceeding about 500 nm, or exceeding about 250 nm can be removed by filtration, for example using a 0.2 µm (or 0.22 µm) filter. Thus, in some embodiments, at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the extracellular vesicles in the isolated vesicle composition of this disclosure have a particle size of less than about 600 nm, or less than about 500 nm, or less than about 250 nm.

[0023] In some embodiments, it may be preferred that all or most of the extracellular vesicle particles in the composition have a size greater than about 20 nm. For example, in some cases, extracellular vesicles for delivering therapeutic payloads may be less effective when a large number of particles smaller than about 20 nm are present. In some embodiments, extracellular vesicles with a particle size smaller than about 20 nm can be removed by filtration, such as using a 500 kDa filter. Therefore, in some embodiments, at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the vesicles in the isolated extracellular vesicle composition of this disclosure have a particle size greater than about 20 nm.

[0024] In some embodiments, it may be preferred that all or most of the extracellular vesicle particles in the composition have a size greater than about 20 nm, and also have vesicles with a size less than about 600 nm (or less than about 500 nm in some embodiments). Therefore, in some embodiments, at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the isolated vesicle compositions of this disclosure have a particle size less than about 600 nm, or less than about 500 nm; or less than about 250 nm; and at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the isolated vesicle compositions of this disclosure have a particle size greater than about 20 nm.

[0025] In some embodiments, the extracellular vesicles of this disclosure (such as isolated milk extracellular vesicles; and / or ATP-analog-vesicles) include one or more carrier molecules. As used herein, the term "carrier" refers to a molecule (e.g., a biomolecule) added to a vesicle, such as a molecule intended to be administered to a subject using the vesicle as a delivery medium. In some embodiments, the carrier molecule is a peptide, protein, nucleic acid, polysaccharide, small molecule, etc. In many embodiments, the carrier molecule is non-bioavailable or has poor bioavailability when administered via a particular route, but has increased or improved bioavailability when administered via the vesicle composition of this disclosure. In some embodiments, the carrier molecule has a molecular weight greater than 20 Daltons and less than 500,000 Daltons, between 2 and 1,000 amino acids, or a size between 1 and 200 nanometers. In some embodiments, the carrier is a molecule that is not naturally present in vesicles in its natural source or is not endogenous to vesicles. In some embodiments, the carrier molecule may be endogenous in its natural source within the milk vesicle, but exogenously added, for example, to increase the amount of molecule in the vesicle. Non-limiting exemplary molecules that can be used as carriers include antibodies, hormones, growth factors, enzymes, cytokines, chemokines, toxins, antitoxins, coagulation factors, interfering RNA (iRNA), microRNA (miRNA), antisense RNA, messenger RNA (mRNA), non-coding RNA, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), long non-coding RNA, and iRNA (such as, for example, siRNA or shRNA). Any nucleotide molecule or fragment thereof disclosed herein may contain a naturally occurring nucleotide sequence. Alternatively, the nucleotide molecule may be synthetic (not naturally occurring). Detailed Implementation

[0026] Vesicle size, type and determination thereof As used herein, the terms “size” and “diameter” are used interchangeably. In embodiments relating to compositions comprising multiple vesicles (such as milk vesicles, exogenous bodies, milk exogenous bodies, milk extracellular vesicles, etc.), the average (average) size or diameter of the vesicles in the composition may be any size indicated herein for a single vesicle (e.g., where a size range is indicated, the average size of the particles falls within the specified range). The vesicles of this disclosure may be any size specified herein. In some embodiments, and where specifically indicated, when the size (diameter) is expressed as a range and in the case of a composition comprising multiple vesicles, the average size of the particles falls within the stated range and a specific percentage (e.g., more than 60%, or more than 65%, or more than 70%, or more than 75%, or more than 80%, or more than 85%, or more than 90%, or more than 95%) of the vesicle particles fall within the indicated range.

[0027] Particle size can be determined by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), or microfluidic resistance pulse sensing.

[0028] As used herein, the term "exosome" refers to a subset of extracellular vesicles that are generally characterized by a specific size, but may also be characterized by other unique features, such as relatively increased amounts of protein markers, such as CD81, CD9, and syntenin. Exosomes are characterized by a size less than 200 nanometers, containing tetraspan membrane proteins including CD81, CD9, and CD63, lumen proteins including syntenin and TSG-101, being composed of a lipid bilayer membrane, and stably transferring carriers between cells. In some embodiments, the exosomes of this disclosure (such as lacteal exosomes) have a diameter between 10 and 200 nm (or, in the case of compositions comprising multiple vesicles, an average (mean) diameter of the particles within this range). In some embodiments, the diameter of the exosomes (such as lactal exosomes) disclosed herein is between the following (or, in the case of a composition comprising multiple vesicles, the average (mean) diameter of the particles is between the following): 10 to 200 nm; or 10 to 185 nm; or 10 to 175 nm; or 10 to 170 nm; or 10 to 165 nm; or 10 to 160 nm; or 10 to 155 nm; or 10 to 150 nm; or 10 to 145 nm; or 10 to 140 nm; or 10 to 135 nm; or 10 to 130 nm; or 15 to 200 nm; or 15 to 185 nm; or 15 to 175 nm; or 15 to 170 nm; or 15 to 165 nm; or 15 to 160 nm; or 15 to 155 nm; or 15 to 150 nm; or 15 to 145 nm; or 15 to 140 nm; or 15 to 135 nm. nm; or 15 to 130 nm; or 20 to 200 nm; or 20 to 185 nm; or 20 to 175 nm; or 20 to 170 nm; or 20 to 165 nm; or 20 to 160 nm; or 20 to 155 nm; or 20 to 150 nm; or 20 to 145 nm; or 20 to 140 nm; or 20 to 135 nm; or 20 to 130 nm; or 25 to 200 nm; or 25 to 185 nm; or 25 to 175 nm; or 25 to 170 nm; or 25 to 165 nm; or 25 to 160 nm; or 25 to 155 nm; or 25 to 150 nm; or 25 to 145 nm; or 25 to 140 nm; or 25 to 135 nm; or 25 to 130 nm; or 30 to 200 nm; or 30 to 185 nm nm; or 30 to 175 nm; or 30 to 170 nm; or 30 to 165 nm; or 30 to 160 nm; or 30 to 155 nm; or 30 to 150 nm; or 30 to 145 nm; or 30 to 140 nm; or 30 to 135 nm; or 30 to 130 nm; or 35 to 200 nm; or 35 to 185 nm;Or 35 to 175 nm; or 35 to 170 nm; or 35 to 165 nm; or 35 to 160 nm; or 35 to 155 nm; or 35 to 150 nm; or 35 to 145 nm; or 35 to 140 nm; or 35 to 135 nm; or 35 to 130 nm; or 40 to 200 nm; or 40 to 185 nm; or 40 to 175 nm; or 40 to 170 nm; or 40 to 165 nm; or 40 to 160 nm; or 40 to 155 nm; or 40 to 150 nm; or 40 to 145 nm; or 40 to 140 nm; or 40 to 135 nm; or 40 to 130 nm; or 45 to 200 nm; or 45 to 185 nm; or 45 to 175 nm; or 45 to 170 nm; or 45 to 165 nm nm; or 45 to 160 nm; or 45 to 155 nm; or 45 to 150 nm; or 45 to 145 nm; or 45 to 140 nm; or 45 to 135 nm; or 45 to 130 nm; or 50 to 200 nm; or 50 to 185 nm; or 50 to 175 nm; or 50 to 170 nm; or 50 to 165 nm; or 50 to 160 nm; or 50 to 155 nm; or 50 to 150 nm; or 50 to 145 nm; or 50 to 140 nm; or 50 to 135 nm; or 50 to 130 nm; or 55 to 200 nm; or 55 to 185 nm; or 55 to 175 nm; or 55 to 170 nm; or 55 to 165 nm; or 55 to 160 nm; or 55 to 155 nm; or 55 to 150 nm; or 55 to 150 nm; nm; or 55 to 145 nm; or 55 to 140 nm; or 55 to 135 nm; or 55 to 130 nm; or 60 to 200 nm; or 60 to 185 nm; or 60 to 175 nm; or 60 to 170 nm; or 60 to 165 nm; or 60 to 160 nm; or 60 to 155 nm; or 60 to 150 nm; or 60 to 145 nm; or 60 to 140 nm; or 60 to 135 nm; or 60 to 130 nm; or 65 to 200 nm; or 65 to 185 nm; or 65 to 175 nm; or 65 to 170 nm; or 65 to 165 nm; or 65 to 160 nm; or 65 to 155 nm; or 65 to 150 nm; or 65 to 145 nm; or 65 to 140 nm; or 65 to 135 nm nm; or 65 to 130 nm; or 70 to 200 nm; or 70 to 185 nm; or 70 to 175 nm; or 70 to 170 nm; or 70 to 165 nm; or 70 to 160 nm; or 70 to 155 nm; or 70 to 150 nm; or 70 to 145 nm; or 70 to 140 nm;Or 70 to 135 nm; or 70 to 130 nm; or 75 to 200 nm; or 75 to 185 nm; or 75 to 175 nm; or 75 to 170 nm; or 75 to 165 nm; or 75 to 160 nm; or 75 to 155 nm; or 75 to 150 nm; or 75 to 145 nm; or 75 to 140 nm; or 75 to 135 nm; or 75 to 130 nm; or 80 to 200 nm; or 80 to 185 nm; or 80 to 175 nm; or 80 to 170 nm; or 80 to 165 nm; or 80 to 160 nm; or 80 to 155 nm; or 80 to 150 nm; or 80 to 145 nm; or 80 to 140 nm; or 80 to 135 nm; or 80 to 130 nm; or 85 to 200 nm nm; or 85 to 185 nm; or 85 to 175 nm; or 85 to 170 nm; or 85 to 165 nm; or 85 to 160 nm; or 85 to 155 nm; or 85 to 150 nm; or 85 to 145 nm; or 85 to 140 nm; or 85 to 135 nm; or 85 to 130 nm; or 90 to 200 nm; or 90 to 185 nm; or 90 to 175 nm; or 90 to 170 nm; or 90 to 165 nm; or 90 to 160 nm; or 90 to 155 nm; or 90 to 150 nm; or 90 to 145 nm; or 90 to 140 nm; or 90 to 135 nm; or 90 to 130 nm; or 95 to 200 nm; or 95 to 185 nm; or 95 to 175 nm; or 95 to 170 nm; nm; or 95 to 165 nm; or 95 to 160 nm; or 95 to 155 nm; or 95 to 150 nm; or 95 to 145 nm; or 95 to 140 nm; or 95 to 135 nm; or 95 to 130 nm; or 100 to 200 nm; or 100 to 185 nm; or 100 to 175 nm; or 100 to 170 nm; or 100 to 165 nm; or 100 to 150 nm; or 100 to 155 nm; or 100 to 150 nm; or 100 to 145 nm; or 100 to 100 nm; or 100 to 135 nm; or 100 to 130 nm; or 105 to 200 nm; or 105 to 185 nm; or 105 to 175 nm; or 105 to 170 nm; or 105 to 165 nm nm; or 105 to 160 nm; or 105 to 155 nm; or 105 to 150 nm; or 105 to 145 nm; or 105 to 140 nm; or 105 to 135 nm; or 105 to 130 nm; or 110 to 200 nm; or 110 to 185 nm; or 110 to 175 nm;Or 110 to 170 nm; or 110 to 165 nm; or 110 to 150 nm; or 110 to 155 nm; or 110 to 150 nm; or 110 to 145 nm; or 110 to 100 nm; or 110 to 135 nm; or 110 to 130 nm; or 115 to 200 nm; or 115 to 185 nm; or 115 to 175 nm; or 115 to 170 nm; or 115 to 165 nm; or 115 to 160 nm; or 115 to 155 nm; or 115 to 150 nm; or 115 to 145 nm; or 115 to 140 nm; or 115 to 135 nm; or 115 to 130 nm; or 120 to 200 nm; or 120 to 185 nm; or 120 to 175 nm nm; or 120 to 170 nm; or 120 to 165 nm; or 120 to 150 nm; or 120 to 155 nm; or 120 to 150 nm; or 120 to 145 nm; or 120 to 140 nm; or 120 to 135 nm; or 120 to 130 nm.

[0029] Vesicles carry payloads.

[0030] In some embodiments, the method further includes loading vesicles of the compositions and methods described herein onto one or more carriers. Vesicles can be loaded by any suitable method. Exemplary methods for loading vesicles (such as those prepared by the methods described herein) are any of those set forth in International Patent Application Publication WO2020 / 028439 (particularly pages 83-87).

[0031] Breast vesicles can carry any suitable or desired delivery vehicle. In some embodiments, the delivery vehicle is a therapeutic compound or molecule. Exemplary delivery vehicles include, but are not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory agents, antihistamines, anti-infectives, radiation sensitizers, chemotherapeutic agents, imaging agents, immunogens, anticancer drugs, any combination thereof, and / or similar substances.

[0032] In various embodiments, the delivery vehicle may be a peptide, such as the ACT-11 peptide. In some exemplary embodiments, the delivery vehicle may be the ACT-11-minus I peptide. Other peptide delivery vehicles include those set forth in International Patent Application Publication WO2020 / 028439 (particularly pages 67-82 and 85, and 106-111). In some embodiments, the delivery vehicle compound is esterified, as described on pages 81-86 of International Patent Application Publication WO2020 / 028439. In some embodiments, the delivery vehicle compound has multiple esterifications, as described on pages 81-86 of International Patent Application Publication WO2020 / 028439.

[0033] In some exemplary embodiments, the exogenous delivery vehicle is a biomolecule. In some exemplary embodiments, the exogenous delivery vehicle is a lipid, polypeptide, peptide, nucleic acid, or cellular metabolite. In some exemplary embodiments, the exogenous delivery vehicle is a Cx43-terminal peptide, ACT1 peptide, ACT11 peptide, ACT minus I peptide, a selectable peptide, or any combination thereof. In some exemplary embodiments, the exogenous delivery vehicle is any of those delivery vehicles set forth in WO 2020 / 028439, ACT1 or ACT11 peptide, ACT minus peptide (see, for example, the ACT minus peptide set forth in WO 2022 / 076932), Gap19, JM2 (see, for example, US Patent No. 9,345,744 B2), a selectable peptide (see, for example, WO 2022 / 076932), or any combination thereof. In some exemplary embodiments, the exogenous delivery vehicle is a peptide, including but not limited to the ACT-11 peptide. In some exemplary embodiments, the exogenous delivery vehicle is a peptide, including but not limited to the ACT-11-minus I peptide. Other peptide delivery systems include those described in International Patent Application Publication WO2020 / 028439 (particularly pages 67-82, 85, and 106-111). In some embodiments, the delivery compound is esterified, as described on pages 81-86 of International Patent Application Publication WO2020 / 028439. In some embodiments, the delivery compound has multiple esterifications, as described on pages 81-86 of International Patent Application Publication WO2020 / 028439. Other exemplary peptide delivery systems include: Corticorelin, Cosyntropin, Seracide, Sincalide, Protectilin, Sermorelin, Somatorelin, Tesamorelin, Secretin, Secretin (human), Secretin (pig), Thymosin, thyroid hormones, Thymalfasin, and thymosin. Thymopentin, Calcitonin, Salmon Calcitonin, Elcatonin, Human Calcitonin, Teriparatide, Atosiban, Carbetocin, Oxytocin, Buserelin, Ozempic, Gonadorelin, Goserelin, Histrelin, LeuprolideNafarelin, Triptorelin, Abarelix, Cetrorelix, Degarelix, Ganirelix, Depreotide, Edotreotide, Lanreotide, Octreotide, Pentetreotide, Somatostatin, Vapreotide, Arginine vasopressin rgipressin, desmopressin, lysine vasopressin, phenyl lysine vasopressin, terlipressin, enfuvirtide, ziconotide, saralasin, bivalirudin, eptifibatide, βadp1, carperitide, nesiritide, aCT1, aCT11 -I, aCT1-I, angiotensin, ANP, BMP, liraglutide, ghrelin, ANP, Ac2-26, LL-37, FF / CAP18, VIP, PIPS, AcF, FeG, CNP, BNP, MOTS-c, Elampretide, SBS272, SBT-550, Icatibant, Exenatide, liraglutide, Lixisenatide, Albiglu Tide, Dulaglutide, Semaglutide, Pramlintide, Linasclotide, Sinaspultide, Pasireotide, Teduglutide, Peginesatide, Pentagastralin, Cecropin, Moricin, Insect Defensins, Drosomicin Diptericins, Metchnikowin, Ponericins, Jelleines, Apisimin, PyrrhocoricinPersulcatusin, melittin, apidaecin, drosocin, lebocin, attacin, and gloverin. Esterified and natural forms of Gap19, rotigaptide, peptif, L2, JM, ACT017, CB1, AXT107, and other therapeutic or bioactive delivery systems as described in WIPO patent application WO / 2022 / 182782, which are incorporated herein by reference. Exemplary carrier nutrients may include, but are not limited to, minerals (e.g., potassium, sodium, chloride, magnesium, manganese, cobalt, molybdenum, calcium, copper, zinc, iodine, iron, chromium, fluoride, selenium, etc.), vitamins (vitamins A, E, D, C, K, etc.), creatine, ATP, ADP, AMP, adenosine, sugars (e.g., glucose, fructose, mannose, galactose, lactose, etc.), hyaluronic acid, forms of vitamin A (retinol and other forms), vitamin C (L-ascorbic acid), biotin, niacin, pantothenic acid, omega-3 fatty acids, fats, and fatty acids.

[0034] Exemplary hormones that can be used as carriers include amino acid-derived hormones (e.g., melatonin and thyroxine), small peptide hormones and protein hormones (e.g., thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), arachidonic acid (e.g., arachidonic acid, lipoxygenase, and prostaglandins), purines (e.g., ATP), pyrimidines (e.g., thymine), enzymes (e.g., creatine), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone, and cortisol).

[0035] Exemplary immunomodulators that can be used as delivery vehicles include prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN-a, IFN-b, IFN-e, IFN-K, IFN-co, and IFN-g), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), cytosine-guanosine monophosphate, oligodeoxynucleotides, dextran, antibodies, and aptamers).

[0036] Exemplary antipyretics include, but are not limited to, nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., salicylcholine, magnesium salicylate, and sodium salicylate), paracetamol / acetaminophen, metamizole, nabumetone, phenazone, and quinine.

[0037] Exemplary anxiolytics include benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clonazepam, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisop). serotonergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), tetramethylglucuril, fabomotizole, selank, bromantane, emoxypine, azapirones, barbiturate, hydroxyzine, pregabalin, isovaleric acid, and beta-blockers.

[0038] Exemplary antipsychotic agents that can be used as carriers include benperidol, bromperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, and desildenafil. Dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thiodiazine hioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, morinone Indone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapinePaliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, bifeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetadmethionil, vabicaserin, xanomeline, and zicronapine.

[0039] Exemplary analgesics that can be used as delivery vehicles include acetaminophen / paracetamol, nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, and fluoride. Flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritriamide, and aspirin, as well as related salicylates (e.g., salicylcholine, magnesium salicylate, and sodium salicylate).

[0040] Exemplary antispasmodics that can be used as carriers include mebeverine, papaverine, cyclobenzalin, carisoprodol, olphenadrine, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, succinylcholine, and dantrolene. Suitable anti-inflammatory agents include, but are not limited to, prednisone, nonsteroidal anti-inflammatory agents (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immunoselective anti-inflammatory derivatives (e.g., submandibular peptide-T and its derivatives).

[0041] Exemplary antihistamines that can be used as delivery vehicles include H1-receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromdiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine). iramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine Fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, bisacodyl H2 receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and p2-adrenergic agonists.

[0042] Exemplary anti-infective agents that can be used as carriers include amoebic insecticides (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin B, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin...). (namycin and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), and antifungals (e.g., azole antifungals (e.g., itraconazole, fluconazole, posaconazoline)). Le), ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin and amphotericin B) Antimalarial agents (e.g., pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g., aminosalicylates, etc.)Aminosalicylic acid), isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antiviral agents (e.g.,Amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / lopinavir / ritonavir Tenofovir, interferon alpha-2v / ribavirin, pegylated interferon alpha-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, sodium foscamet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delaviridine dine), nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, doxorinosine, tenofovir, abacavir, zidovudine, stavudine, trientabine, zalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir / ritonavir, fosanavir (samprenavir), darunavir, ritonavir, tipranavir, atazanavir, nelfmavir, amprenavir, indinavir, saquinavir, ribavirin, valacyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g.,Doripenem, meropenem, ertapenem, and cilastatin / imipenem), cephalosporins (e.g., cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefclor, ceftibuten, ceftriaxone) Cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefzoxime, and ceftazidime; glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin); glycylcyclines (e.g., tigecycline); antileprosy agents (e.g., clofazimine and thalidomide); lincomycin and its derivatives (e.g., clindamycin and lincomycin); macrolides and their derivatives (e.g.,Telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, linezolid, sulfamethoxazole / trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole dazole), aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, clavulanate / ticarcillin, penicillin, procaine penicillin) Penicillin, oxacillin, dicloxacillin, and nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g.,Doxycycline, demeclocycline, minocycline, doxycycline / salicylic acid, doxycycline / omega-3 polyunsaturated fatty acid and tetracycline) and urinary tract anti-infectives (e.g., nitrofurantoin, methemine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim and methylene blue).

[0043] Exemplary chemotherapeutic agents that can be used as delivery vehicles include paclitaxel, brentuximab vedotin, doxorubicin, and 5-FU. (Fluorouracil), everolimus, pemetrexed, melphalan, pamidronate disodium, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, bebnostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine Mustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, vidarabine, cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprobde, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, recombinant chrysanthemum erwin's asparaginase Erwinia chrysanthemin), amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarebx, pralatrexate, methotrexate, fluxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatemesylate, carmustine, eribubn, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alpha-2a, gefitinib, romidepsin, ixabepilone, ruxobtinib, cabazitaxel, ado-trastuzumab Emtansine, Carfilzomib, Chlorambucil, Sargramostim, Cladribine, Mitotane, Vincristine, Procarbazine, Megestrol, Trametinib, Mesna, Strontium-89 Chloride, Mechlorethamine, Mitomycin, Busulfan, Gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukinDiftitox, abtretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib histrebn, sunitinib, siltuximab, omacetaxine, thioguanine / tioguanine, dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorebn, arsenic trioxideTrioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine Enzalutamide, ipilimumab, goserebn, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid.

[0105] Suitable radiation sensitizers include, but are not limited to, 5-fluorouracil, platinum analogs (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, DNA topoisomerase I targeting agents (e.g., camptothecin derivatives (e.g., topotecan and irinotecan)), epidermal growth factor receptor blocking agents (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors (e.g., L-778-123), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), bFGF and VEGF targeting agents (e.g., bevacizumab and thalidomide), NBTXR3, Nimoral, trans-saffron sodium NVX-108, and combinations thereof. See also, for example, Kvols, LK., J Nucl Med 2005; 46:187S-190S.

[0044] Exemplary immunogens that can be carried or attached to the outer surface of isolated vesicles as a carrier may include keyhole hemocyanin (KLH), shellfish hemocyanin (CCH) (also a blue carrier immunogenic protein), bovine serum albumin (BSA), ovalbumin (OVA), and antigens for generating an immune response against pathogens that cause disease, including diphtheria, tetanus, pertussis, measles, mumps, rubella, hepatitis A, hepatitis B, meningococcal disease (e.g., meningitis), human papillomavirus varicella, rabies, influenza, rotavirus, HIV, malaria, and coronavirus disease.

[0045] Exemplary polynucleotide modification systems that can be used as carriers or attached to the outer surface of isolated vesicles may include, but are not limited to, CRISPR-Cas systems, OMEGA systems, PRIME editing systems, base editors, a wide range of nucleases, zinc finger nucleases, recombinases, TALE nucleases, CAST systems, non-LTR retrotransposon systems, transposons, RNAi, antisense nucleic acids, etc. See, for example, Crooke ST, Liang XH, Baker BF, Crooke RM. Antisense technology: Areview. J Biol Chem. 2021;296:100416. doi:10.1016 / j.jbc.2021.100416; Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, DOI: dx.doi.org / 10.1016 / j.molcel.2015.10.008; Peters et al., PNAS 114 (35) (2017); DOI: 10.1073 / pnas.1709035114; Makarova et al., 2018. The CRISPR Journal, v. 1, n5, Fig. 5; Kim, YG et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. USA 91, 883-887; Kim, YG et al., 1996, Hybrid restriction enzymes: zincfinger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156-1160; Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); Zhang et al., Nature Biotechnology 29:149-153 (2011); Rees and Liu. 2018. Nat. Rev. Gent. 19(12):770-788; Komor et al., 2016. Nature. 533:420-424; Nishida et al., 2016. Science.353; Gaudeli et al., 2017. Nature. 551:464-471; Cox et al., 2017. Science 358: 1019-1027; Levy et al., Nature Biomedical Engineering doi.org / 10.1038 / s41441-019-0505-5 (2019); Gorsuch et al. (2022). Targeting the hepatitis B cccdna with a sequence-specific arcus nuclease to eliminate hepatitis B virus in vivo. Molecular Therapy, 30(9), 2909–2922. doi.org / 10.1016 / j.ymthe.2022.05.013; Anzalone et al., 2019. Nature. 576: 149-157, especially Figures 1b and 1c, related discussion and supplementary discussion; Anzalone AV, Gao XD, Podracky CJ et al., Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol . 2022;40(5):731-740; Yarnall et al., Nat Biotechnol(2022). doi.org / 10.1038 / s41587-022-01527-4;Groth, AC and Calos, MP (2004) J. Mol. Biol. 335, 667-678;Lei et al., FEBS Lett. April 2018;592(8):1389-1399;Singh et al., Attachment Site Selection and Identity in Bxb1 SerineIntegrase-Mediated Site-Specific Recombination, PLoS Genet. May 2013;9(5):e1003490;and Gupta et al., Nucleic Acids Res. May 2007;35(10):3407–3419;Tou et al., bioRxiv 2022.01.07.475005, doi.org / 10.1101 / 2022.01.07.475005;Klompe et al. Nature, doi:10.1038 / s41586-019-1323;Strecker et al. Science. 10 / 1126 / science.aax9181 (2019);Christensen SM et al., RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA targetsite, Proc Natl Acad Sci US A. 21 Nov 2006;103(47):17602-7;Eickbush TH et al., Integration, Regulation, and Long-Term Stability of R2 Retrotransposons, Microbiol Spectr. 2015 Apr;3(2):MDNA3-0011-2014. doi: 10.1128 / microbiolspec.MDNA3-0011-2014; Han JS, Non-long terminal repeat (non-LTR)retrotransposons: mechanisms, recent developments, and unanswered questions, Mob DNA.May 12, 2010; 1(1):15. doi: 10.1186 / 1759-8753-1-15;Malik HS et al., Theage and evolution of non-LTR retrotransposable elements, Mol Biol Evol. June 1999; 16(6):793-805;US Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,58 5,849, 7,595,376, 6,903,185, 6,479,626, 8,163,514, 8,133,697, 8,021,867, 8,119,361, 8,119,381, 8,124,369, 8,129,134, 10,851,358; U.S. Patent Application Publication Nos. 2020 / 0239544, 2018 / 0346934; International Patent Application Publication Nos. WO 2018 / 213708, WO 2018 / 213726, WO2014 / 093622, WO 2019 / 005884, WO 2019 / 005886, WO 2019 / 071048, WO 2016 / 106236, WO2020 / 206231, WO 2021 / 138469, WO 2020 / 131862, WO 2021 / 257997, WO 2021 / 087394, WO2022 / 147321, WO 2022 / 07682, WO 2022 / 150651, WO 2021 / 102042, WO 2022 / 173830...

[0046] In some implementations, the exogenous transport vehicle is in the range of 0.1 to / or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 49 0, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pg, ng, μg / mg vesicle protein, or any non-zero amount of any value or subrange within any of these ranges, are present in breast vesicles.

[0047] In other embodiments, therapeutic delivery vehicles, such as those listed in any of the embodiments herein, can facilitate delivery loading, for example, by esterification of endogenous vesicle enzymes as described in WIPO patent application WO / 2022 / 182782, which is incorporated herein by reference.

[0048] Vesicles as medicines, therapeutic and nutritional preparations and uses.

[0049] Extracellular vesicles can be a powerful tool for administering therapeutic delivery vehicles to subjects due to their ability to transport and protect biological signaling molecules to and from subjects, as well as the unique ability of certain extracellular vesicle populations to cross tissue boundaries such as the skin barrier and blood-brain barrier. Extracellular vesicles can also evade immune surveillance in certain situations and exhibit good immunological tolerance, even during autotransmission between individuals and species. The use of membrane cloaking and surface display (JNanobiotechnology) to target extracellular vesicles to injured tissue has further increased interest in their potential to translate into clinical applications as a novel means of improving drug delivery safety. Therefore, this description also includes pharmaceutical formulations that may contain an amount, an effective amount, and / or a minimum effective amount and / or a therapeutically effective amount of one or more vesicles as described herein (such as carrier-loaded mammary vesicles) and a pharmaceutically acceptable carrier or excipient. Formulations comprising extracellular vesicles are described in certain exemplary embodiments herein, wherein the formulation is produced at least in part by any of the methods described in any of the preceding paragraphs and / or elsewhere herein, such as in the working examples below. Certain exemplary embodiments described herein include methods for administering formulations, as described in any of the preceding paragraphs and / or elsewhere herein (such as in the working examples below), to a subject. In some embodiments, the formulation administered to the subject comprises a breast vesicle, such as those described elsewhere herein and / or prepared by methods described elsewhere herein. In some embodiments, the breast vesicle is a breast vesicle carrying a delivery vehicle.

[0050] As used herein, the term “treating” can generally refer to achieving a desired pharmacological and / or physiological effect. An effect may be, but does not necessarily have to be, preventative in relation to the prevention or partial prevention of a disease, its symptoms or conditions (such as cancer, inflammatory diseases or conditions, and / or mechanical or non-mechanical injuries). An effect may be therapeutic in relation to the partial or complete cure of a disease, condition, symptom, or side effects attributable to that disease, condition, or condition. In some embodiments, an effect may be the relief or anticipated prevention of undesirable symptoms. As used herein, the term “treatment” covers any treatment of a subject, particularly a human or non-human animal (such as a non-human mammal), of cancer, inflammatory diseases or conditions, and / or mechanical or non-mechanical injuries, and may include any and more of the following: (a) prevention of disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppression of the disease, i.e., prevention of its development; and (c) relief of the disease, i.e., reduction or improvement of the disease and / or its symptoms or conditions. As used herein, the term "treatment" can refer to therapeutic treatment alone, preventive treatment alone, or both. Those who require treatment (subjects in need) may include those who already have a condition and / or those for which the condition needs to be prevented. As used herein, the term "treatment" may include suppressing a disease, symptom, or illness, such as preventing its progression; and alleviating a disease, symptom, or illness, such as causing its resolution. Treating a disease, symptom, or illness may include improving at least one symptom of a particular disease, symptom, or illness, even if the underlying pathophysiology is not affected, such as treating a subject's pain by administering an analgesic, even if such an agent does not treat the cause of the pain.

[0051] In some implementations, the subject administering the vesicles or formulations thereof suffers from a disease or condition. Exemplary diseases or conditions include, but are not limited to, any cancer, viral infection, bacterial infection, parasitic infection, external and internal wounds and tissue damage, cancer, ischemic and / or hypoxic injury (e.g., myocardial infarction, ischemic wound and / or stroke), multiple sclerosis, psoriasis, scleroderma, acne, eczema or skin and / or connective tissue disease, heart disease or condition, neurodegenerative disease or condition, neurological condition, atherosclerosis, pathology involving epithelial permeability and / or neovascularization (e.g., angiogenesis or vascularization), respiratory distress syndrome (RDS), reperfusion injury, skin vascular defects or malformations, macular degeneration, etc. Neovascularization of the choroidal capillaries through the Bruch membrane, diabetic retinopathy (inflammatory and inflammatory-related diseases and conditions), radiation damage, acute radiation syndrome, side effects of radiotherapy, radiation dermatitis, and diseases caused by high-dose radiation, including acute radiation syndrome, chronic radiation syndrome, cholangiocarcinoma, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gallbladder cancer, liver cancer, lung cancer, pancreatic cancer, pharyngeal cancer, ovarian cancer, salivary gland cancer, intestinal cancer, stomach cancer, thyroid cancer, urinary tract cancer, leukemia, lymphoma, multiple myeloma or any other cancer, non-malignant thyroid nodular disease, parathyroid adenoma, or posterior subcapsular cataract.

[0052] Wounds can be chronic or appear not to have fully healed. For example, a wound that has not healed within three months is considered a chronic wound. Chronic wounds include diabetic foot ulcers, ischemic ulcers, venous ulcers, venous leg ulcers, venous stasis, arterial ulcers, pressure ulcers, vasculitis ulcers, infected ulcers, pressure ulcers, traumatic ulcers, gangrenous ulcers, and mixed ulcers. Chronic wounds include those characterized by and / or chronic inflammation, insufficient granulation tissue differentiation and excessive fusion, epithelial reformation and failed wound closure, and prolonged repair time. Chronic wounds can include ocular ulcers, including corneal ulcers. The use of the disclosed invention in wound healing and tissue regeneration can be applied in humans and agricultural animals, sporting animals, and pet animals.

[0053] Tissue damage can be caused by, for example, cuts, scrapes, pressure wounds, stretching wounds, lacerations, squeezing wounds, bite wounds, abrasions, gunshot wounds, blast wounds, puncture wounds, surgical wounds, surgical interventions, medical interventions, host rejection after cell, tissue, or organ transplantation, drug effects, drug side effects, bedsores, radiation damage, radiation sickness, cosmetic skin wounds, internal organ damage, disease processes (e.g., asthma, cancer), infections, infectious agents, developmental processes, maturation processes (e.g., acne), genetic abnormalities, developmental abnormalities, environmental toxins, allergens, scalp injuries, facial injuries, jaw injuries, sexual organ injuries, joint injuries, excretory organ injuries, foot injuries, finger injuries, toe injuries, bone injuries, eye injuries, corneal injuries, muscle injuries, adipose tissue injuries, lung injuries, airway injuries, hernias, anal injuries, hemorrhoids, ear injuries, skin injuries, abdominal injuries, etc. Head injuries, retinal injuries, eye injuries, corneal injuries, arm injuries, leg injuries, sports injuries, back injuries, birth injuries, premature birth injuries, toxic bites, stings, barrier function impairment, endothelial barrier function impairment, epithelial barrier function impairment, tendon injuries, ligament injuries, heart injuries, heart valve injuries, vascular system injuries, cartilage injuries, lymphatic system injuries, head injuries, dislocations, esophageal perforation, fistulas, nail injuries, foreign bodies, fractures, frostbite, hand injuries, heat stress disorders, lacerations, neck injuries, self-harm, shock, traumatic soft tissue injuries, spinal cord injuries, spinal cord injuries, sprains, strains, tendon injuries, ligament injuries, cartilage injuries, chest injuries, dental injuries, trauma, nervous system injuries, burns, burn wounds, wind burns, sunburns, chemical burns, aging, aneurysms, strokes, radiation injuries, surgical radiation injuries, gastrointestinal injuries, infarctions or ischemic injuries.

[0054] Cardiac diseases and conditions that can be treated with the compositions and methods described herein include, but are not limited to, myocardial infarction, cardiomyopathy (e.g., hypertrophic cardiomyopathy), arrhythmias, and congestive heart failure. The regenerative effects of the provided compositions can lead to beneficial changes in cardiac membrane excitability and ion transients. Many different types of arrhythmias cause abnormalities in human cardiac function. Cardiac arrhythmias include, but are not limited to, bradycardia, tachycardia, alteman's alternation, automaticity defects, reentrant arrhythmias, fibrillation, atrioventricular nodal arrhythmias, atrial arrhythmias and triggered beats, long QT syndrome, short QT syndrome, Brugada syndrome, premature atrial contractions (PVCs), wandering atrial pacemakers, multifocal atrial tachycardia, atrial flutter, atrial fibrillation (AV nodal reentrant tachycardia is the most common cause of paroxysmal supraventricular tachycardia), junctional rhythms, junctional tachycardia, atrioventricular junctional PVCs, Wolff-Parkinson-White syndrome, Lown-Ganong-Levine syndrome, and premature ventricular contractions (PVCs). (Sometimes called premature ventricular contractions), electrical alternation and inconsistent electrical alternation, accelerated ventricular voluntary rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, ventricular fibrillation, first-degree heart block (which manifests as prolonged PR), second-degree heart block, type I second-degree heart block, type II second-degree heart block, third-degree heart block, and several accessory pathway conditions (e.g., Woj-Patwee syndrome (WPW)).

[0055] Neurodegenerative and neurological disorders include, but are not limited to, dementia, Alzheimer's disease, Parkinson's disease and related PD diseases, amyotrophic lateral sclerosis (ALS), motor neuron disease, schizophrenia, spinocerebellar ataxia, prions, spinal muscular atrophy (SMA), multiple sclerosis, epilepsy and other seizure disorders, and Huntington's disease.

[0056] Inflammatory diseases and inflammation-related diseases and conditions can include asthma, eczema, sinusitis, atherosclerosis, arthritis (including but not limited to rheumatoid arthritis), inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis. As used herein, the term "inflammatory condition" can include diseases or conditions that are at least partially caused or exacerbated by inflammation, typically characterized by increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), fever, redness, swelling, pain, and / or loss of function of the affected tissue or organ. Causes of inflammation can include physical injury, chemicals, microorganisms, tissue necrosis, cancer, or other agents or conditions.

[0057] Inflammatory conditions include acute, chronic, and relapsing inflammatory conditions. Acute inflammatory conditions are typically relatively short-lived, lasting from a few minutes to one to two days, although they can also last for weeks. Acute inflammatory conditions are characterized by increased blood flow, fluid and plasma protein exudation (edema), and migration of white blood cells (such as neutrophils). Chronic inflammatory conditions are typically longer-lasting, such as weeks to months to years or longer, and are histologically associated with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Relapsing inflammatory conditions include conditions that recur or recur periodically over a period of time. Some inflammatory conditions fall into one or more categories. Exemplary inflammatory conditions include, but are not limited to, atherosclerosis; arthritis; inflammation-promoted cancer; asthma; autoimmune uveitis; adoptive immune responses; dermatitis; multiple sclerosis; complications of diabetes; osteoporosis; Alzheimer's disease; cerebral malaria; hemorrhagic fever; autoimmune diseases; Crohn's disease; and inflammatory bowel disease. In some implementations, the inflammatory condition is an autoimmune condition, which in some respects is selected from lupus, rheumatoid arthritis, and autoimmune encephalomyelitis.

[0058] In some implementations, the inflammatory condition is a brain-related inflammatory condition. As used herein, the term "brain-related inflammatory condition" refers to a subset of inflammatory conditions that are at least partially caused by, originate from, or are exacerbated by inflammation in the subject's brain.

[0059] As used herein, a “pharmaceutical formulation” means a combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, such that the composition is suitable for diagnostic, therapeutic, or prophylactic use in vitro, in vivo, or ex vivo. As used herein, a “pharmaceutically acceptable carrier or excipient” means a carrier or excipient that can be used to prepare a pharmaceutical formulation that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers or excipients suitable for veterinary use as well as for human pharmaceutical use. As used in the specification and claims, a “pharmaceutically acceptable carrier or excipient” includes one or more such carriers or excipients. When present, the carrier may optionally be present in the pharmaceutical formulation as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutical formulation may include, for example, an active ingredient, one or more mammary vesicles, such as extracellular mammary vesicles carrying the carrier, as described in more detail elsewhere herein.

[0060] In some implementations, the carrier is present as a pharmaceutically acceptable salt of the active ingredient. As used herein, a “pharmaceutically acceptable salt” means any acid or base addition salt whose counterion is non-toxic to a subject administered the drug at the salt dose. Suitable salts include hydrobromide, iodide, nitrate, hydrogen sulfate, phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronide, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, naphthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and dihydroxynaphthyl salt.

[0061] The drug formulations described herein can be administered to subjects in need via any suitable method or route. Suitable routes of administration may include, but are not limited to, ear, cheek, conjunctiva, skin, dental, electroosmosis, intracervical, intrasinus, trachea, intestine, epidural, extraamniotic, in vitro, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarticular, intra-articular, intra-bile duct, intrabronchus, intracapsular, intracardiac, intracartilaginous, intracoccygeal, intracavitary, intracavitary, intracavitary, intracavitary, intracerebral, intracisional, intracorneal, intracoronary (dental), intracoronary artery, corpus cavernosum, intradermal, intravertebral, intraductal, intraduodenal, intradural, intraepithelial, intraepithelial, intraepithelial, intraesophageal, intragastric, intragingival, intraileum, intravenous, intraluminal, intralymphatic, intramedullary, intramedullary, intraluminal, intramedullary, intralesional, intraluminal, intramedullary, intramedullary, intramedullary, intraluminal, intramedullary, intramedullary, intramedullary, intralesional, intraluminal, intramedullary ... Intraspinal, intrasynaptic, intratendinous, intratesticular, intrasheath, intrathoracic, intracanal, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intracardiac, intravesical, intravitreal, iontophoresis, irrigation, larynx, nose, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, epidural, perineurial, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, local, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, ​​and / or vaginal administration, and / or any combination of the above routes of administration, which generally depends on the disease to be treated and / or the active ingredient and / or the delivery vehicle.

[0062] Where appropriate, one or more of the milk vesicles (such as milk vesicles carrying a delivery vehicle) described in more detail elsewhere herein may be provided to subjects in need as components (such as active ingredients or reagents) in pharmaceutical formulations. Therefore, pharmaceutical formulations containing one or more milk vesicles, such as milk vesicles carrying a delivery vehicle described in more detail elsewhere herein, comprising delivery vehicles in pharmaceutically acceptable salt forms are also described. Suitable salts include hydrobromide, iodide, nitrate, hydrogen sulfate, phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronide, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, naphthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and dihydroxynaphthylate.

[0063] As used herein, “agent” means any substance, compound, molecule, and / or analogue that may be biologically active or that, when administered to a subject in any other manner, induces a biological and / or physiological effect. As used herein, “active agent” or “active ingredient” means a substance, compound, or molecule that is biologically active or that, when administered to a subject in any other manner, induces a biological or physiological effect. In other words, “active agent” or “active ingredient” means one or more components of a composition to which all or part of the effects of the composition are attributable. An agent may be a primary active agent, or in other words, one or more components of a composition to which all or part of the effects of the composition are attributable. An agent may be a secondary agent, or in other words, one or more components of a composition to which additional part and / or other effects of the composition are attributable. In some embodiments, the active agent is a milk vesicle or a milk vesicle carrying a delivery. In some embodiments, the active agent includes a delivery vehicle of a milk vesicle carrying a delivery vehicle or a delivery vehicle of a milk vesicle carrying a delivery vehicle.

[0064] In some embodiments, the milk vesicles are prepared by any of the methods described elsewhere herein. In some embodiments, the milk vesicles carrying the delivery vehicle are prepared as described elsewhere herein and / or by the methods described elsewhere herein.

[0065] milk vesicles Mammalian milk is rich in extracellular vesicles and holds great promise as a source for the large-scale production of extracellular vesicles, which are widely used as delivery media. Exemplary species for milk production that can be used as the source of milk vesicles provided herein include cows, buffalo, goats, sheep, camels, yaks, horses, reindeer, donkeys, and humans. Cows or cow milk is produced in large quantities by the dairy industry, is widely consumed, and is generally well tolerated by the human immune system. Furthermore, milk vesicles have been reported to enter the bloodstream from the intestine and be transported to various organs, including the brain, heart, intestines, and lungs (Wolf T, BS (2015). The intestinal transport of bovine milk vesicles is mediated by endocytosis in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. JNutr, 2201-2206), which provides a fundamental property for the oral administration of vesicles carrying delivery vehicles (including, but not limited to, therapeutic agents).

[0066] The milk used as the vesicle source in the compositions and methods disclosed herein can be derived from any mammal. In some embodiments, mammalian milk is cow's milk, sheep's milk, pig's milk, camel's milk, horse's milk, goat's milk, human milk, etc. In some embodiments, the milk is not pasteurized. In some embodiments, the milk is pasteurized.

[0067] Extracellular vesicles were isolated and purified from milk.

[0068] Methods for large-scale and cost-effective separation of extracellular vesicles from body fluids are of great interest for the eventual commercial-scale use of vesicles as delivery media. Numerous methods exist for separating exosomes from biological fluids based on ultracentrifugation (UC). These methods typically involve differential centrifugation steps and / or density gradient-based UC separation. However, the ability to produce large quantities of vesicles is limited by the requirement of multiple UC steps and the fact that UC can only rotate small volumes. Furthermore, the shear forces applied during repeated UC rotations can have a detrimental effect on vesicle structural integrity (Taylor DD, SS (2015). Methods of isolating extracellular vesicles impact downstream analyses of their cargoes. Methods, 3-10). Other techniques that may impose less physical rigor during exosome separation include ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography (SEC), and methods based on polyethylene glycol precipitation.

[0069] While milk is a promising source of commercial-scale extracellular vesicles, its isolation presents unique challenges. Milk contains a diverse mixture of proteins, minerals, lipids, and other macromolecules, and this complexity of the milk composition contributes to the challenges of purifying extracellular vesicles. Casein is the major component of milk, comprising approximately 80% of all milk proteins. Casein aggregates with calcium phosphate and other milk proteins into large colloidal complexes to form so-called casein micelles. These micelles are approximately 10 nm in diameter and can further aggregate into even larger coagulated structures (Bhat, MT (2016). Casein Proteins: structural and functional aspects. Intech). Casein micelle aggregates are thought to bind to and trap vesicles through chemical interactions, hindering their separation from contaminating milk proteins; observations confirmed by transmission electron microscopy (TEM) analysis of milk-derived exosome formulations (Sedykh SE, BE (2020). Milk Exosomes: Isolation, Biochemistry, Morphology, and Perspectives of Use. In CJ De Bona AG, Extracellular Vesicles and their importance in human health. Intech Open). Therefore, existing methods for isolating high-purity extracellular vesicles from milk are limited by contaminating proteins such as casein. A close association has been noted between extracellular vesicles and casein-rich protein chains isolated from bovine milk (Figure 1). These associations are reminiscent of another observed association between extracellular vesicles and the extracellular matrix, termed extracellular matrix vesicles or matrix-bound nanovesicles (MBNs). Without being bound by theory, it is anticipated that isolating or loosening vesicles using the methods described herein (including by using free tryptophan to disrupt the chemical interactions between vesicles and their matrix, which may include extracellular, collagen, fibrin, or casein matrix) could increase the yield of purified vesicles isolated from such sources. Furthermore, in some embodiments, it is anticipated that the addition of free tryptophan to the isolated vesicles would help maintain the structure and bioactivity of the vesicles during procedures including lyophilization, sterilization, and storage as described herein.

[0070] Including the addition of Ca at a specified temperature 2+Methods involving chelation with other divalent cations, such as those described in PCT WO2022182782, result in high-yield separation of structurally and functionally intact vesicles from milk proteins. In some cases, the casein micelle solubilization and vesicle separation steps can be incorporated into methods with UC-based and / or TFF and SEC filtration steps for vesicle separation, thus providing a basis for the large-scale production of purified, high-quality vesicles from milk. Such methods involving chelating divalent cations with ethylenediaminetetraacetic acid (EDTA) and / or other chelating agents (including, but not limited to, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ethylene glycol-bis(o-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), sodium citrate, sodium tripolyphosphate, sodium hexametaphosphate, tetrasodium pyrophosphate, other phosphates and polyphosphates, and / or nitrobenzene) are effective in methods for separating extracellular vesicles, including those that do not require ultracentrifugation. Chemically immobilized EDTA on insoluble substrates can be used to chelate divalent cations from solution in the presence of a semipermeable membrane or without preventing contamination of the milk sample solution. However, in some cases, such as when vesicles are used for nutritional products, it may be desirable to avoid the use of chemicals that some people consider irritating, such as EDTA.

[0071] Therefore, one object of this disclosure is to develop a method similar to WO2022182782 that reduces or completely avoids the use of EDTA and / or similar chemicals that may be considered irritating by using alternative reagents. For example, in some embodiments of this disclosure, a method similar to PCT WO2022182782 is provided that utilizes tryptophan and / or tryptophan analogues as alternatives to EDTA. Free tryptophan is naturally present in milk and colostrum at concentrations up to 10 mg / ml and is enhanced through dairy industry practices such as fermentation. Breast milk is the sole source of tryptophan (TRP) in breastfed infants (PMC5906556). Therefore, free tryptophan is considered a fundamental component of milk, not a contaminant. Therefore, this document provides a method for separating vesicles from a biological fluid (such as milk), which may include, for example, (a) centrifuging the biological fluid under conditions suitable for separating fat from one or more other components of the biological fluid; (b) removing the separated fat from the biological fluid; (c) after step (b), centrifuging the remaining biological fluid once or multiple times, and skimming off any obviously separated fat after each centrifugation in step (c); (d) filtering the remaining biological fluid after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); and (f) contacting the resulting solution with tryptophan and / or more tryptophan analogs at about 20-60 degrees Celsius for about 15-120 minutes. After step (f), tangential flow filtration may optionally be performed to obtain a retentate (step (g)). The retentate may then optionally be ultracentrifuged via one or more ultracentrifugation steps. The retentate may then optionally be fractionated via column separation. In some embodiments, the method includes step (e) or step (g), but not both.

[0072] In some embodiments, step (f) is performed using tryptophan and / or one or more tryptophan analogs at a concentration between 10 µM and 1 mM. In some embodiments, step (f) is performed by contacting the milk vesicles with tryptophan and / or more tryptophan analogs at about 30-42 degrees Celsius (e.g., incubation) for about 15-120 minutes.

[0073] In some exemplary embodiments, step (a) includes centrifuging the biological fluid at a relative centrifugal force (RCF) of about 2,500. In some exemplary embodiments, (a) includes centrifuging the biological fluid at a relative centrifugal force (RCF) of about 2,000 to about 3,000.In some exemplary embodiments, (a) includes approximately 2000 rcf, 2010 rcf, 2020 rcf, 2030 rcf, 2040 rcf, 2050 rcf, 2060 rcf, 2070 rcf, 2080 rcf, 2090 rcf, 2100 rcf, 2110 rcf, 2120 rcf, 2130 rcf, 2140 rcf, 2150 rcf, 2160 rcf, 2170 rcf, 2180 rcf, 2190 rcf, 2200 rcf, 2210 rcf, 2220 rcf, 2230 rcf, 2240 rcf, 2250 rcf, 2260 rcf, 2270 rcf, 2280 rcf, 2290 rcf. rcf, 2300rcf, 2310 rcf, 2320 rcf, 2330 rcf, 2340 rcf, 2350 rcf, 2360 rcf, 2370 rcf, 2380 rcf, 2390 rcf, 2400 rcf, 2410 rcf, 2420 rcf, 2430 rcf, 2440 rcf, 2450 rcf, 2460 rcf, 2470 rcf, 2480 rcf, 2490 rcf, 2500 rcf, 2510 rcf, 2520 rcf, 2530 rcf, 2540 rcf, 2550 rcf, 2560 rcf, 2570 rcf, 2580 rcf, 2590 rcf, 2600 rcf, 2610 rcf, 2620 rcf, 2630 rcf, 2640 rcf, 2650 rcf, 2660 rcf, 2670 rcf, 2680 rcf, 2690 rcf, 2700 rcf, 2710 rcf, 2720 rcf, 2730 rcf, 2740 rcf, 2750 rcf, 2760 rcf, 2770 rcf, 2780 rcf, 2790 rcf, 2800 rcf, 2810rcf, 2820 rcf, 2830 rcf, 2840 rcf, 2850 rcf, 2860 rcf, 2870 rcf, 2880 rcf, 2890 rcf, 2900 rcf, 2910 Centrifuge the biological fluid at RCF, 2920 RCF, 2930 RCF, 2940 RCF, 2950 RCF, 2960 RCF, 2970 RCF, 2980 RCF, 2990 RCF to / or approximately 3000 Ref. In some embodiments, step (a) is repeated 1-3 times. In some embodiments, step (a) is repeated 1, 2, or 3 times.

[0074] In some embodiments, step (b) includes a first centrifugation followed by a second centrifugation. In some exemplary embodiments, the first centrifugation includes centrifuging the remaining biofluid at about 14,500 rcf for about 60 minutes. In some exemplary embodiments, the first centrifugation includes centrifuging the remaining biofluid at about 13,500 rcf to about 15,500 ref for about 45 to about 75 minutes. In some implementations, the first centrifugation includes centrifugation at approximately 13500 rcf, 13550 rcf, 13600 rcf, 13650 rcf, 13700 rcf, 13750 rcf, 13800 rcf, 13850 rcf, 13900 rcf, 13950 rcf, 14000 rcf, 14050 rcf, 14100 rcf, 14150 rcf, 14200 rcf, 14250 rcf, 14300 rcf, 14350 rcf, 14400 rcf, 14450 rcf, 14500 rcf, 14550 rcf, 14600 rcf, 14650 rcf, 14700 rcf, 14750 rcf, and 14800 rcf. rcf, 14850 rcf, 14900 rcf, 14950 rcf, 15000 rcf, 15050 rcf, 15100 rcf, 15150 rcf, 15200 rcf, 15250 rcf, 15300 rcf, 15350 rcf, 15400 rcf, 15450 rcf or about 15500 The remaining biofluid is centrifuged for approximately 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 minutes, or approximately 75 minutes. In some embodiments, the remaining biofluid after the first centrifugation is subjected to a second centrifugation, wherein the second centrifugation is performed at approximately 25,800 rcf for approximately 60 minutes. In some exemplary embodiments, the remaining biofluid after the first centrifugation is subjected to a second centrifugation, wherein the second centrifugation is performed at approximately 24,800 to approximately 26,800 rcf for approximately 45 to approximately 75 minutes.In some exemplary embodiments, the remaining biofluid after the first centrifugation is subjected to a second centrifugation, and the second centrifugation is performed at approximately 24800 rcf, 24850 rcf, 24900 rcf, 24950 rcf, 25000 rcf, 25050 rcf, 25100 rcf, 25150 rcf, 25200 rcf, 25250 rcf, 25300 rcf, 25350 rcf, 25400 rcf, 25450 rcf, 25500 rcf, 25550 rcf, 25600 rcf, 25650 rcf, 25700 rcf, 25750 rcf, 25800 rcf, 25850 rcf, 25900 rcf, 25950 rcf, 26000 rcf. rcf, 26050 rcf, 26100 rcf, 26150 rcf, 26200 rcf, 26250 rcf, 26300 rcf, 26350 rcf, 26400 rcf, 26450 rcf, 26500rcf, 26550 rcf, 26600 rcf, 26650 rcf, 26700 rcf, 26750 rcf, 26800 The centrifugation time is approximately 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or approximately 75 minutes. In some exemplary embodiments, the second centrifugation is repeated 1-3 times, each time performing the remaining biological fluid from the immediately preceding centrifugation. In some exemplary embodiments, the second centrifugation is repeated 1, 2, or 3 times, each time performing the remaining biological fluid from the immediately preceding centrifugation.

[0075] In some exemplary embodiments, step (d) includes filtering the remaining biofluid through one or more filters in series, ranging from about 0.45 micrometers (µm) to about 0.22 micrometers. In some embodiments, each filter in series is independently selected from 0.22 micrometers, 0.23 micrometers, 0.24 micrometers, 0.25 micrometers, 0.26 micrometers, 0.27 micrometers, 0.28 micrometers, 0.29 micrometers, 0.3 micrometers, 0.31 micrometers, 0.32 micrometers, 0.33 micrometers, 0.34 micrometers, 0.35 micrometers, 0.36 micrometers, 0.37 micrometers, 0.38 micrometers, 0.39 micrometers, 0.4 micrometers, 0.41 micrometers, 0.42 micrometers, 0.43 micrometers, 0.44 micrometers, or 0.45 micrometers. In some embodiments, the number of filters in series ranges from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 filters in series. In some embodiments, all filters in series have the same cutoff size. In some embodiments, at least two filters in series have the same cutoff size. In some embodiments, at least two filters in series have different cutoff sizes. In some embodiments, all filters in series have different cutoff sizes. In some embodiments, the size rejection decreases from large to small along a series of filters. For example, in three filters in series, the first filter may be a 0.45-micron filter, the second filter may be a 0.3-micron filter, and the last filter may be a 0.22-micron filter. Other configurations of the filters in series will be understood given the description herein. In some embodiments, all filters in series are made of the same material. In some embodiments, all filters in series are made of different materials. In some embodiments, at least two filters in series are made of the same material. In some embodiments, at least two filters are made of different materials. Exemplary filters include, but are not limited to, membrane filters (e.g., polyethersulfone membrane filters, polyvinylidene fluoride membrane filters, cellulose membrane filters, mixed cellulose ester membrane filters, cellulose acetate membrane filters, cellulose nitrate membrane filters, polyamide membrane filters, polycarbonate membrane filters, polytetrafluoroethylene membrane filters, polypropylene membrane filters, nitrocellulose membrane filters, etc.), glass fiber or bead filters, and / or the like. In some embodiments, step (d) includes filtering the remaining biofluid through a filter of about 0.45 micrometers, and then filtering the remaining biofluid through a filter of about 0.22 micrometers.

[0076] In some embodiments of the foregoing method, step (e) includes two or more consecutive ultracentrifugation steps, each step being performed on the remaining biological fluid from a previous ultracentrifugation. In some exemplary embodiments, (e) includes 2-10 consecutive ultracentrifugation steps (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), each step being performed on the remaining biological fluid from the previous ultracentrifugation. In some exemplary embodiments, (e) includes an ultracentrifugation step at about 50,000 rcf, an ultracentrifugation step at about 70,000 rcf, an ultracentrifugation step at about 100,000 rcf, or any combination thereof. In some exemplary embodiments, (e) includes an ultracentrifugation step at about 45,000 to about 55,000 rcf, an ultracentrifugation step at about 65,000 to about 75,000 rcf, an ultracentrifugation step at about 90,000 to about 110,000 rcf, or any combination thereof. In some exemplary embodiments, (e) includes approximately 45,000 to approximately 55,000 rcf (e.g., approximately 45,000 rcf, 45,100 rcf, 45,200 rcf, 45,300 rcf, 45,400 rcf, 45,500 rcf, 45,600 rcf, 45,700 rcf, 45,800 rcf, 45,900 rcf, 46,000 rcf, 46,100 rcf, 46,200 rcf, 46,300 rcf, 46,400 rcf, 46,500 rcf, 46,600 rcf, 46,700 rcf, 46,800 rcf, 46,900 rcf, 47,000 rcf, 47,100 rcf, 47,200 rcf, 47,300 rcf). rcf, 47400 rcf, 47500 rcf, 47600 rcf, 47700 rcf, 47800 rcf, 47900 rcf, 48000 rcf, 48100 rcf, 48200 rcf, 48300 rcf, 48400rcf, 48500 rcf, 48600 rcf, 48700 rcf, 48800 rcf, 48900 rcf, 49000 rcf, 49100 rcf, 49200 rcf, 49300 rcf, 49400 rcf, 49500 rcf, 49600 rcf, 49700 rcf, 49800 rcf, 49900rcf, 50000 rcf, 50100 rcf, 50200 rcf, 50300 rcf, 50400 rcf, 50500 rcf, 50600 rcf, 50700 rcf, 50800 rcf, 50900 rcf, 51000An ultracentrifugation step carried out at about 51,000 to about 55,000 rcf (e.g., at 51,100 rcf, 51,200 rcf, 51,300 rcf, 51,400 rcf, 51,500 rcf, 51,600 rcf, 51,700 rcf, 51,800 rcf, 51,900 rcf, 52,000 rcf, 52,100 rcf, 52,200 rcf, 52,300 rcf, 52,400 rcf, 52,500 rcf, 52,600 rcf, 52,700 rcf, 52,800 rcf, 52,900 rcf, 53,000 rcf, 53,100 rcf, 53,200 rcf, 53,300 rcf, 53,400 rcf, 53,500 rcf, 53,600 rcf, 53,700 rcf, 53,800 rcf, 53,900 rcf, 54,000 rcf, 54,100 rcf, 54,200 rcf, 54,300 rcf, 54,400 rcf, 54,500 rcf, 54,600 rcf, 54,700 rcf, 54,800 rcf, 54,900 rcf or about 55,000 rcf), and at about 65,000 to about 75,000 rcf (e.g., at 65,000 rcf, 65,100 rcf, 65,200 rcf, 65,300 rcf, 65,400 rcf, 65,500 rcf, 65,600 rcf, 65,700 rcf, 65,800 rcf, , 65,900 rcf, 66,000 rcf, 66,100 rcf, 66,200 rcf, 66,300 rcf, 66,400 rcf, 66,500 rcf, 66,600 rcf, 66,700 rcf, 66,800 rcf, 66,900 rcf, 67,000 rcf, 67,100 rcf, 67,200 rcf, 67,300 rcf, 67,400 rcf, 67,500 rcf, 67,600 rcf, 67,700 rcf, 67,800 rcf, 67,900 rcf, 68,000 rcf, 68,100 rcf, 68,200 rcf, 68,300 rcf, 68,400 rcf, 68,500 rcf, 68,600 rcf, 68,700 rcf, 68,800 rcf, 68,900 rcf, 69,000 rcf, 69,100 rcf, 69,200 rcf, 69,300 rcf, 69,400 rcf, 69,500 rcf, 69,600 rcf, 69,700 rcf, 69,800 rcf, 69,900 rcf, 70,000 rcf, 70,100 rcf, 70,200 rcf, 70,300 rcf, 70,400 rcf, 70,500 rcf, 70,600An ultracentrifugation step carried out at about 70,000 to about 75,000 rcf (e.g., 70700 rcf, 70800 rcf, 70900 rcf, 71000 rcf, 71100 rcf, 71200 rcf, 71300 rcf, 71400 rcf, 71500 rcf, 71600 rcf, 71700 rcf, 71800 rcf, 71900 rcf, 72000 rcf, 72100 rcf, 72200 rcf, 72300 rcf, 72400 rcf, 72500 rcf, 72600 rcf, 72700 rcf, 72800 rcf, 72900 rcf, 73000 rcf, 73100 rcf, 73200 rcf, 73300 rcf, 73400 rcf, 73500 rcf, 73600 rcf, 73700 rcf, 73800 rcf, 73900 rcf, 74000 rcf, 74100 rcf, 74200 rcf, 74300 rcf, 74400 rcf, 74500 rcf, 74600 rcf, 74700 rcf, 74800 rcf, 74900 rcf or about 75000 rcf), and at about 90,000 to about 110,000 rcf (e.g., 90000 rcf, 90100 rcf, 90200 rcf, 90300 rcf, 90400 rcf, 90500 rcf, 90600 rcf, 90700 rcf, 90800 rcf, 90900 rcf, 91000 rcf, 91100 rcf, 91200 rcf, 91300 rcf, 91400 rcf, 91500 rcf, 91600 rcf, 91700 rcf, 91800 rcf, 91900 rcf, 92000 rcf, 92100 rcf, 92200 rcf, 92300 rcf, 92400 rcf, 92500 rcf, 92600 rcf, 92700 rcf, 92800 rcf, 92900 rcf, 93000 rcf, 93100 rcf, 93200 rcf, 93300 rcf, 93400 rcf, 93500 rcf, 93600 rcf, 93700 rcf, 93800 rcf, 93900 rcf, 94000 rcf, 94100 rcf, 94200 rcf, 94300 rcf, 94400 rcf, 94500 rcf, 94600 rcf, 94700 rcf, 94800 rcf, 94900 rcf, 95000 rcf, 95100 rcf, 95200rcf、95300 rcf、95400 rcf、95500 rcf、95600 rcf、95700 rcf、95800 rcf、95900 rcf、96000 rcf、96100rcf、96200 rcf、96300 rcf、96400 rcf、96500 rcf、96600 rcf、96700 rcf、96800 rcf、96900 rcf、97000 rcf、97100 rcf、97200 rcf、97300 rcf、97400 rcf、97500 rcf、97600rcf、97700 rcf、97800 rcf、97900 rcf、98000 rcf、98100 rcf、98200 rcf、98300 rcf、98400 rcf、98500 rcf、98600 rcf、98700 rcf、98800 rcf、98900 rcf、99000 rcf、99100rcf、99200 rcf、99300 rcf、99400 rcf、99500 rcf、99600 rcf、99700 rcf、99800 rcf、99900 rcf、100000 rcf、100100 rcf、100200 rcf、100300 rcf、100400 rcf、100500 rcf、100600 rcf、100700 rcf、100800 rcf、100900 rcf、101000 rcf、101100 rcf、101200 rcf、101300 rcf、101400 rcf、101500 rcf、101600 rcf、101700 rcf、101800 rcf、101900 rcf、102000 rcf、102100 rcf、102200 rcf、102300 rcf、102400 rcf、102500 rcf、102600 rcf、102700 rcf、102800 rcf、102900 rcf、103000 rcf、103100 rcf、103200 rcf、103300 rcf、103400 rcf、103500 rcf、103600 rcf、103700 rcf、103800 rcf、103900 rcf、104000 rcf、104100 rcf、104200 rcf、104300 rcf、104400 rcf、104500 rcf、104600 rcf、104700rcf, 104800 rcf, 104900 rcf, 105000 rcf, 105100 rcf, 105200 rcf, 105300 rcf, 105400 rcf, 105500 rcf, 105600 rcf, 105700 rcf, 105800 rcf, 105900 rcf, 106000 rcf, 106100 rcf, 106200 rcf, 106300 rcf, 106400 rcf, 106500 rcf, 106600 rcf, 106700 rcf, 106800 rcf, 106900 rcf, 107000 rcf, 107100 rcf, 107200 rcf, 107300 rcf, 107400 rcf, 107500 rcf, 107600 rcf, 107700 rcf, 107800 rcf, 107900 rcf, 108000 rcf, 108100 rcf, 108200 rcf, 108300 rcf, 108400 rcf, 108500 rcf, 108600 rcf, 108700 rcf, 108800 rcf, 108900 rcf, 109000 rcf, 109100 rcf, 109200 rcf, 109300 rcf, 109400 rcf, 109500 An ultracentrifugation step or any combination thereof (109600 rcf, 109700 rcf, 109800 rcf, 109900 rcf, or about 110000 rcf). In some exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for about 60 minutes. In some exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for about 45-75 minutes. In some exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for approximately 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, or approximately 75 minutes.

[0077] In some embodiments, step (e) includes a final ultracentrifugation at about 130,000 rcf for about 120 minutes, discarding the resulting fluid, and resuspending the remaining precipitate in a suitable volume of suitable solution prior to (1). In some exemplary embodiments, (e) includes a final ultracentrifugation at about 115,000 to about 145,000 rcf for about 90-150 minutes, discarding the resulting fluid, and resuspending the remaining precipitate in a suitable volume of suitable solution prior to (1). In some exemplary embodiments, (e) includes approximately 115,000 to approximately 145,000 refs (e.g., 115000 rcf, 115100 rcf, 115200 rcf, 115300 rcf, 115400 rcf, 115500 rcf, 115600 rcf, 115700 rcf, 115800 rcf, 115900 rcf, 116000 rcf, 116100 rcf, 116200 rcf, 116300 rcf, 116400 rcf, 116500 rcf, 116600 rcf, 116700 rcf, 116800 rcf, 116900 rcf, 117000 rcf). rcf, 117100 rcf, 117200 rcf, 117300 rcf, 117400 rcf, 117500 rcf, 117600 rcf, 117700 rcf, 117800 rcf, 117900 rcf, 118000 rcf, 118100 rcf, 118200 rcf, 118300 rcf, 118400 rcf, 118500 rcf, 118600 rcf, 118700 rcf, 118800 rcf, 118900 rcf, 119000 rcf, 119100 rcf, 119200 rcf, 119300 rcf, 119400 rcf, 119500 rcf, 119600 rcf, 119700 rcf, 119800 rcf, 119900 rcf, 120000 rcf, 120100 rcf, 120200 rcf, 120300 rcf, 120400 rcf, 120500 rcf, 120600 rcf, 120700 rcf, 120800 rcf, 120900 rcf, 121000 rcf, 121100 rcf, 121200 rcf, 121300 rcf, 121400 rcf, 121500 rcf, 121600 rcf, 121700 rcf, 121800 rcf, 121900 rcf, 122000rcf、122100 rcf、122200 rcf、122300 rcf、122400 rcf、122500 rcf、122600 rcf、122700 rcf、122800 rcf、122900 rcf、123000 rcf、123100 rcf、123200 rcf、123300 rcf、123400 rcf、123500 rcf、123600 rcf、123700 rcf、123800 rcf、123900 rcf、124000 rcf、124100 rcf、124200 rcf、124300 rcf、124400 rcf、124500 rcf、124600 rcf、124700 rcf、124800 rcf、124900 rcf、125000 rcf、125100 rcf、125200 rcf、125300 rcf、125400 rcf、125500 rcf、125600 rcf、125700 rcf、125800 rcf、125900 rcf、126000 rcf、126100 rcf、126200 rcf、126300 rcf、126400 rcf、126500 rcf、126600 rcf、126700 rcf、126800 rcf、126900 rcf、127000 rcf、127100 rcf、127200 rcf、127300 rcf、127400 rcf、127500 rcf、127600 rcf、127700 rcf、127800 rcf、127900 rcf、128000 rcf、128100 rcf、128200 rcf、128300 rcf、128400 rcf、128500 rcf、128600 rcf、128700 rcf、128800 rcf、128900 rcf、129000 rcf、129100 rcf、129200 rcf、129300 rcf、129400 rcf、129500 rcf、129600 rcf、129700 rcf、129800 rcf、129900 rcf、130000 rcf、130100 rcf、130200 rcf、130300 rcf、130400 rcf、130500 rcf、130600 rcf、130700 rcf、130800 rcf、130900 rcf、131000 rcf、131100rcf、131200 rcf、131300 rcf、131400 rcf、131500 rcf、131600 rcf、131700 rcf、131800 rcf、131900 rcf、132000 rcf、132100 rcf、132200 rcf、132300 rcf、132400 rcf、132500 rcf、132600 rcf、132700 rcf、132800 rcf、132900 rcf、133000 rcf、133100 rcf、133200 rcf、133300 rcf、133400 rcf、133500 rcf、133600 rcf、133700 rcf、133800 rcf、133900 rcf、134000 rcf、134100 rcf、134200 rcf、134300 rcf、134400 rcf、134500 rcf、134600 rcf、134700 rcf、134800 rcf、134900 rcf、135000 rcf、135100 rcf、135200 rcf、135300 rcf、135400 rcf、135500 rcf、135600 rcf、135700 rcf、135800 rcf、135900 rcf、136000 rcf、136100 rcf、136200 rcf、136300 rcf、136400 rcf、136500 rcf、136600 rcf、136700 rcf、136800 rcf、136900 rcf、137000 rcf、137100 rcf、137200 rcf、137300 rcf、137400 rcf、137500 rcf、137600 rcf、137700 rcf、137800 rcf、137900 rcf、138000 rcf、138100 rcf、138200 rcf、138300 rcf、138400 rcf、138500 rcf、138600 rcf、138700 rcf、138800 rcf、138900 rcf、139000 rcf、139100 rcf、139200 rcf、139300 rcf、139400 rcf、139500 rcf、139600 rcf、139700 rcf、139800 rcf、139900 rcf、140000 rcf、140100 rcf、140200rcf, 140300 rcf, 140400 rcf, 140500 rcf, 140600 rcf, 140700 rcf, 140800 rcf, 140900 rcf, 141000 rcf, 141100 rcf, 141200 rcf, 141300 rcf, 141400 rcf, 141500 rcf, 141600 rcf, 141700 rcf, 141800 rcf, 141900 rcf, 142000 rcf, 142100 rcf, 142200 rcf, 142300 rcf, 142400 rcf, 142500 rcf, 142600 rcf, 142700 rcf, 142800 rcf, 142900 rcf, 143000 rcf, 143100 rcf, 143200 rcf, 143300 rcf, 143400 rcf, 143500 rcf, 143600 rcf, 143700 rcf, 143800 rcf, 143900 rcf, 144000 rcf, 144100 rcf, 144200 rcf, 144300 rcf, 144400 rcf, 144500 rcf, 144600 rcf, 144700 rcf, 144800 rcf, 144900 rcf or approximately 145000 The process (rcf) will last approximately 90-150 minutes (e.g., 90 minutes, 91 minutes, 92 minutes, 93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 minutes, 99 minutes, 100 minutes, 101 minutes, 102 minutes, 103 minutes, 104 minutes, 105 minutes, 106 minutes, 107 minutes, 108 minutes, 109 minutes, 110 minutes, 111 minutes, 112 minutes, 113 minutes, 114 minutes, 115 minutes, 116 minutes, 117 minutes, 118 minutes, 119 minutes, 120 minutes, 121 minutes, 122 minutes). 123 minutes, 124 minutes, 125 minutes, 126 minutes, 127 minutes, 128 minutes, 129 minutes, 130 minutes, 131 minutes, 132 minutes, 133 minutes, 134 minutes, 135 minutes, 136 minutes, 137 minutes, 138 minutes, 139 minutes, 140 minutes, 141 minutes, 142 minutes, 143 minutes, 144 minutes, 145 minutes, 146 minutes, 147 minutes, 148 minutes, 149 minutes or about 150 minutes), discard the resulting fluid, and resuspend the remaining precipitate in a suitable volume of suitable solution before (1).

[0078] In some exemplary embodiments, tangential flow filtration (g) is performed using a 500 kDa ultrafiltration membrane. In some exemplary embodiments, tangential flow filtration (g) is performed using an ultrafiltration membrane with a molecular weight cutoff value ranging from about 250 kDa to about 750 kDa. In some embodiments, the molecular weight cutoff values ​​are about 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, 510 kDa, 520 kDa, 530 kDa, 5... Molecular weight cutoff values ​​for ultrafiltration membranes of 40 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 660 kDa, 670 kDa, 680 kDa, 690 kDa, 700 kDa, 710 kDa, 720 kDa, 730 kDa, 740 kDa, or approximately 750 kDa.

[0079] In some exemplary embodiments, the tangential flow filtration of step (g) is performed at a flow rate of about 10 mL / min. In some exemplary embodiments, the tangential flow filtration of step (g) is performed at a flow rate ranging from about 5 mL / min to about 15 mL / min. In some exemplary embodiments, the tangential flow filtration of step (g) is performed at a flow rate of about 5 mL / min, 5.5 mL / min, 6 mL / min, 6.5 mL / min, 7 mL / min, 7.5 mL / min, 8 mL / min, 8.5 mL / min, 9 mL / min, 9.5 mL / min, 10 mL / min, 10.5 mL / min, 11 mL / min, 11.5 mL / min, 12 mL / min, 12.5 mL / min, 13 mL / min, 13.5 mL / min, 14 mL / min, 14.5 mL / min, or about 15 mL / min. In some exemplary embodiments, in step (g), when the amount of remaining biofluid reaches about 10 percent of its initial volume before tangential flow filtration, the retentate is percolated with a suitable buffer solution.

[0080] In some exemplary embodiments, the method further includes ultracentrifuging the retentate when it reaches approximately 20% of the initial percolation volume. In some exemplary embodiments, the ultracentrifugation of the retentate is performed at approximately 130,000 rcf at approximately 4 degrees Celsius for approximately 120 minutes. In some exemplary embodiments, the filtrate is ultracentrifuged at approximately 115,000 to approximately 145,000 rcf (e.g., 115,000 rcf, 115,050 rcf, 115,100 rcf, 115,150 rcf, 115,200 rcf, 115,250 rcf, 115,300 rcf, 115,350 rcf, 115,400 rcf, 115,450 rcf, 115,500 rcf, 115,550 rcf, 115,600 rcf, 115,650 rcf, 115,700 rcf, 115,750 rcf, 115,800 rcf, 115,850 rcf, 115,900 rcf, 115,950 rcf, 116,000 rcf). rcf, 116050 rcf, 116100 rcf, 116150 rcf, 116200 rcf, 116250 rcf, 116300 rcf, 116350 rcf, 116400 rcf, 116450 rcf, 116500 rcf, 116550 rcf, 116600 rcf, 116650 rcf, 116700 rcf, 116750 rcf, 116800 rcf, 116850 rcf, 116900 rcf, 116950 rcf, 117000 rcf, 117050 rcf, 117100 rcf, 117150 rcf, 117200 rcf, 117250 rcf, 117300 rcf, 117350 rcf, 117400 rcf, 117450 rcf, 117500 rcf, 117550 rcf, 117600 rcf, 117650 rcf, 117700 rcf, 117750 rcf, 117800 rcf, 117850 rcf, 117900 rcf, 117950 rcf, 118000 rcf, 118050 rcf, 118100 rcf, 118150 rcf, 118200 rcf, 118250 rcf, 118300 rcf, 118350 rcf, 118400 rcf, 118450 rcf, 118500 rcf, 118550 rcf, 118600 rcf, 118650 rcf, 118700 rcf, 118750 rcf, 118800 rcf, 118850 rcf, 118900rcf、118950 rcf、119000 rcf、119050 rcf、119100 rcf、119150 rcf、119200 rcf、119250 rcf、119300 rcf、119350 rcf、119400 rcf、119450 rcf、119500 rcf、119550 rcf、119600 rcf、119650 rcf、119700 rcf、119750 rcf、119800 rcf、119850 rcf、119900 rcf、119950 rcf、120000 rcf、120050 rcf、120100 rcf、120150 rcf、120200 rcf、120250 rcf、120300 rcf、120350 rcf、120400 rcf、120450 rcf、120500 rcf、120550 rcf、120600 rcf、120650 rcf、120700 rcf、120750 rcf、120800 rcf、120850 rcf、120900 rcf、120950 rcf、121000 rcf、121050 rcf、121100 rcf、121150 rcf、121200 rcf、121250 rcf、121300 rcf、121350 rcf、121400 rcf、121450 rcf、121500 rcf、121550 rcf、121600 rcf、121650 rcf、121700 rcf、121750 rcf、121800 rcf、121850 rcf、121900 rcf、121950 rcf、122000 rcf、122050 rcf、122100 rcf、122150 rcf、122200 rcf、122250 rcf、122300 rcf、122350 rcf、122400 rcf、122450 rcf、122500 rcf、122550 rcf、122600 rcf、122650 rcf、122700 rcf、122750 rcf、122800 rcf、122850 rcf、122900 rcf、122950 rcf、123000 rcf、123050 rcf、123100 rcf、123150 rcf、123200 rcf、123250 rcf、123300 rcf、123350 rcf、123400 rcf、123450rcf、123500 rcf、123550 rcf、123600 rcf、123650 rcf、123700 rcf、123750 rcf、123800 rcf、123850 rcf、123900 rcf、123950 rcf、124000 rcf、124050 rcf、124100 rcf、124150 rcf、124200 rcf、124250 rcf、124300 rcf、124350 rcf、124400 rcf、124450 rcf、124500 rcf、124550 rcf、124600 rcf、124650 rcf、124700 rcf、124750 rcf、124800 rcf、124850 rcf、124900 rcf、124950 rcf、125000 rcf、125050 rcf、125100 rcf、125150 rcf、125200 rcf、125250 rcf、125300 rcf、125350 rcf、125400 rcf、125450 rcf、125500 rcf、125550 rcf、125600 rcf、125650 rcf、125700 rcf、125750 rcf、125800 rcf、125850 rcf、125900 rcf、125950 rcf、126000 rcf、126050 rcf、126100 rcf、126150 rcf、126200 rcf、126250 rcf、126300 rcf、126350 rcf、126400 rcf、126450 rcf、126500 rcf、126550 rcf、126600 rcf、126650 rcf、126700 rcf、126750 rcf、126800 rcf、126850 rcf、126900 rcf、126950 rcf、127000 rcf、127050 rcf、127100 rcf、127150 rcf、127200 rcf、127250 rcf、127300 rcf、127350 rcf、127400 rcf、127450 rcf、127500 rcf、127550 rcf、127600 rcf、127650 rcf、127700 rcf、127750 rcf、127800 rcf、127850 rcf、127900 rcf、127950 rcf、128000rcf、128050 rcf、128100 rcf、128150 rcf、128200 rcf、128250 rcf、128300 rcf、128350 rcf、128400 rcf、128450 rcf、128500 rcf、128550 rcf、128600 rcf、128650 rcf、128700 rcf、128750 rcf、128800 rcf、128850 rcf、128900 rcf、128950 rcf、129000 rcf、129050 rcf、129100 rcf、129150 rcf、129200 rcf、129250 rcf、129300 rcf、129350 rcf、129400 rcf、129450 rcf、129500 rcf、129550 rcf、129600 rcf、129650 rcf、129700 rcf、129750 rcf、129800 rcf、129850 rcf、129900 rcf、129950 rcf、130000 rcf、130050 rcf、130100 rcf、130150 rcf、130200 rcf、130250 rcf、130300 rcf、130350 rcf、130400 rcf、130450 rcf、130500 rcf、130550 rcf、130600 rcf、130650 rcf、130700 rcf、130750 rcf、130800 rcf、130850 rcf、130900 rcf、130950 rcf、131000 rcf、131050 rcf、131100 rcf、131150 rcf、131200 rcf、131250 rcf、131300 rcf、131350 rcf、131400 rcf、131450 rcf、131500 rcf、131550 rcf、131600 rcf、131650 rcf、131700 rcf、131750 rcf、131800 rcf、131850 rcf、131900 rcf、131950 rcf、132000 rcf、132050 rcf、132100 rcf、132150 rcf、132200 rcf、132250 rcf、132300 rcf、132350 rcf、132400 rcf、132450 rcf、132500 rcf、132550rcf、132600 rcf、132650 rcf、132700 rcf、132750 rcf、132800 rcf、132850 rcf、132900 rcf、132950 rcf、133000 rcf、133050 rcf、133100 rcf、133150 rcf、133200 rcf、133250 rcf、133300 rcf、133350 rcf、133400 rcf、133450 rcf、133500 rcf、133550 rcf、133600 rcf、133650 rcf、133700 rcf、133750 rcf、133800 rcf、133850 rcf、133900 rcf、133950 rcf、134000 rcf、134050 rcf、134100 rcf、134150 rcf、134200 rcf、134250 rcf、134300 rcf、134350 rcf、134400 rcf、134450 rcf、134500 rcf、134550 rcf、134600 rcf、134650 rcf、134700 rcf、134750 rcf、134800 rcf、134850 rcf、134900 rcf、134950 rcf、135000 rcf、135050 rcf、135100 rcf、135150 rcf、135200 rcf、135250 rcf、135300 rcf、135350 rcf、135400 rcf、135450 rcf、135500 rcf、135550 rcf、135600 rcf、135650 rcf、135700 rcf、135750 rcf、135800 rcf、135850 rcf、135900 rcf、135950 rcf、136000 rcf、136050 rcf、136100 rcf、136150 rcf、136200 rcf、136250 rcf、136300 rcf、136350 rcf、136400 rcf、136450 rcf、136500 rcf、136550 rcf、136600 rcf、136650 rcf、136700 rcf、136750 rcf、136800 rcf、136850 rcf、136900 rcf、136950 rcf、137000 rcf、137050 rcf、137100rcf、137150 rcf、137200 rcf、137250 rcf、137300 rcf、137350 rcf、137400 rcf、137450 rcf、137500 rcf、137550 rcf、137600 rcf、137650 rcf、137700 rcf、137750 rcf、137800 rcf、137850 rcf、137900 rcf、137950 rcf、138000 rcf、138050 rcf、138100 rcf、138150 rcf、138200 rcf、138250 rcf、138300 rcf、138350 rcf、138400 rcf、138450 rcf、138500 rcf、138550 rcf、138600 rcf、138650 rcf、138700 rcf、138750 rcf、138800 rcf、138850 rcf、138900 rcf、138950 rcf、139000 rcf、139050 rcf、139100 rcf、139150 rcf、139200 rcf、139250 rcf、139300 rcf、139350 rcf、139400 rcf、139450 rcf、139500 rcf、139550 rcf、139600 rcf、139650 rcf、139700 rcf、139750 rcf、139800 rcf、139850 rcf、139900 rcf、139950 rcf、140000 rcf、140050 rcf、140100 rcf、140150 rcf、140200 rcf、140250 rcf、140300 rcf、140350 rcf、140400 rcf、140450 rcf、140500 rcf、140550 rcf、140600 rcf、140650 rcf、140700 rcf、140750 rcf、140800 rcf、140850 rcf、140900 rcf、140950 rcf、141000 rcf、141050 rcf、141100 rcf、141150 rcf、141200 rcf、141250 rcf、141300 rcf、141350 rcf、141400 rcf、141450 rcf、141500 rcf、141550 rcf、141600 rcf、141650rcf、141700 rcf、141750 rcf、141800 rcf、141850 rcf、141900 rcf、141950 rcf、142000 rcf、142050 rcf、142100 rcf、142150 rcf、142200 rcf、142250 rcf、142300 rcf、142350 rcf、142400 rcf、142450 rcf、142500 rcf、142550 rcf、142600 rcf、142650 rcf、142700 rcf、142750 rcf、142800 rcf、142850 rcf、142900 rcf、142950 rcf、143000 rcf、143050 rcf、143100 rcf、143150 rcf、143200 rcf、143250 rcf、143300 rcf、143350 rcf、143400 rcf、143450 rcf、143500 rcf、143550 rcf、143600 rcf、143650 rcf、143700 rcf、143750 rcf、143800 rcf、143850 rcf、143900 rcf、143950 rcf、144000 rcf、144050 rcf、144100 rcf、144150 rcf、144200 rcf、144250 rcf、144300 rcf、144350 rcf、144400 rcf、144450 rcf、144500 rcf、144550 rcf、144600 rcf、144650 rcf、144700 rcf、144750 rcf、144800 rcf、144850 rcf、144900 rcf、144950RCF (or approximately 145,000 RCL) is applied at approximately 4 degrees Celsius for approximately 90 to approximately 150 minutes (e.g., approximately 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 minutes). 118 minutes, 119 minutes, 120 minutes, 121 minutes, 122 minutes, 123 minutes, 124 minutes, 125 minutes, 126 minutes, 127 minutes, 128 minutes, 129 minutes, 130 minutes, 131 minutes, 132 minutes, 133 minutes, 134 minutes, 135 minutes, 136 minutes, 137 minutes, 138 minutes, 139 minutes, 140 minutes, 141 minutes, 142 minutes, 143 minutes, 144 minutes, 145 minutes, 146 minutes, 147 minutes, 148 minutes, 149 minutes, or approximately 150 minutes).

[0081] In some exemplary embodiments, the retentate is not subjected to ultracentrifugation prior to optional fractionation. In these embodiments, the retentate is stored at approximately -80°C prior to optional fractionation via column separation. Optional fractionation of the retentate can be performed by any suitable method, including but not limited to column separation (based on size, charge, affinity, or other methods or separation strategies). Fractions containing extracellular vesicles may be retained.

[0082] In some exemplary embodiments, the method produces a vesicle concentrate that is at least 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or at least 20% of the initial volume of a biological fluid (such as milk). In some exemplary embodiments, the method produces a vesicle concentrate that is approximately 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or about 20% of the initial volume of a biological fluid (such as milk).

[0083] Specific expected implementation plan In addition to the aspects and implementation schemes anticipated elsewhere in this document, the following specific implementation schemes are anticipated.

[0084] ATP-analogues extracellular Vesicle embodiments 1. A composition comprising extracellular vesicles, wherein the extracellular vesicles contain 0.01 to 1,000 μM of ATP or an ATP analogue.

[0085] 2. A composition comprising extracellular vesicles, wherein the extracellular vesicles contain 0.1 to 100 μM of ATP or an ATP analogue.

[0086] 3. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles contain 0.01 to 1,000 μM of ATP or ATP analogues.

[0087] 4. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles contain 0.1 to 100 μM of ATP or an ATP analogue.

[0088] 5. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles contain 0.01 to 1,000 μM of ATP or ATP analogues.

[0089] 6. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles contain 0.1 to 100 μM of ATP or an ATP analogue.

[0090] 7. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles comprise 0.25 to 75 μM, 0.5 to 50 μM, or 0.75 to 25 μM, or 0.8 to 20 μM, or 0.85 to 15 μM, or 0.9 to 10 μM, or 0.5 to 5 μM, or 0.1 to 2 μM, or 0.5 to 1.5 μM, or 0.75 to 1.25 μM of ATP or an ATP analogue.

[0091] 8. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles contain 0.1 to 5 μM of ATP or an ATP analogue.

[0092] 9. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles comprise 0.1 to 2 μM of ATP or an ATP analogue.

[0093] 10. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles comprise 0.1 μM to 1 μM of ATP or an ATP analogue.

[0094] 11. The composition according to any one of the foregoing embodiments, wherein the extracellular vesicles contain about 1 μM of ATP or an ATP analogue.

[0095] 12. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 1 ng to 1,000 μg ATP / mg extracellular vesicle protein.

[0096] 13. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 10 ng to 100 μg ATP / mg extracellular vesicle protein.

[0097] 14. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 100 ng to 10 μg ATP / mg extracellular vesicle protein.

[0098] 15. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 200 ng to 1 μg ATP / mg extracellular vesicle protein.

[0099] 16. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 300 ng to 750 ng ATP / mg extracellular vesicle protein.

[0100] 17. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 400 ng to 650 ng ATP / mg extracellular vesicle protein.

[0101] 18. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 450 ng to 600 ng ATP / mg extracellular vesicle protein.

[0102] 19. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise 500 ng to 550 ng ATP / mg extracellular vesicle protein.

[0103] A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise about 1 ng / mg extracellular vesicle protein; 10 ng / mg extracellular vesicle protein; 100 ng / mg extracellular vesicle protein; 200 ng / mg extracellular vesicle protein; 300 ng / mg extracellular vesicle protein; 400 ng / mg extracellular vesicle protein; 450 ng / mg extracellular vesicle protein; 500 ng / mg extracellular vesicle protein; 550 ng / mg extracellular vesicle protein; 600 ng / mg extracellular vesicle protein; 650 ng / mg extracellular vesicle protein; 750 ng / mg extracellular vesicle protein; 1 μg / mg extracellular vesicle protein; 10 μg / mg extracellular vesicle protein; 100 μg / mg extracellular vesicle protein; or 1,000 μg / mg extracellular vesicle protein.

[0104] 20. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 1 ng to 1,000 μg ATP / mg extracellular vesicle protein.

[0105] 21. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 10 ng to 100 μg ATP / mg extracellular vesicle protein.

[0106] 22. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 100 ng to 10 μg ATP / mg extracellular vesicle protein.

[0107] 23. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 200 ng to 1 μg ATP / mg extracellular vesicle protein.

[0108] 24. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 300 ng to 750 ng ATP / mg extracellular vesicle protein.

[0109] 25. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 400 ng to 650 ng ATP / mg extracellular vesicle protein.

[0110] 26. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 450 ng to 600 ng ATP / mg extracellular vesicle protein.

[0111] 27. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 500 ng to 550 ng ATP / mg extracellular vesicle protein.

[0112] 28. A composition comprising extracellular vesicles of breast cells, wherein the extracellular vesicles comprise about 1 ng / mg extracellular vesicle protein; 10 ng / mg extracellular vesicle protein; 100 ng / mg extracellular vesicle protein; 200 ng / mg extracellular vesicle protein; 300 ng / mg extracellular vesicle protein; 400 ng / mg extracellular vesicle protein; 450 ng / mg extracellular vesicle protein; 500 ng / mg extracellular vesicle protein; 550 ng / mg extracellular vesicle protein; 600 ng / mg extracellular vesicle protein; 650 ng / mg extracellular vesicle protein; 750 ng / mg extracellular vesicle protein; 1 μg / mg extracellular vesicle protein; 10 μg / mg extracellular vesicle protein; 100 μg / mg extracellular vesicle protein; or 1,000 μg / mg extracellular vesicle protein.

[0113] 29. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 1 ng to 1,000 μg ATP / mg extracellular vesicle protein.

[0114] 30. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 10 ng to 100 μg ATP / mg extracellular vesicle protein.

[0115] 31. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 100 ng to 10 μg ATP / mg extracellular vesicle protein.

[0116] 32. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 200 ng to 1 μg ATP / mg extracellular vesicle protein.

[0117] 33. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 300 ng to 750 ng ATP / mg extracellular vesicle protein.

[0118] 34. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 400 ng to 650 ng ATP / mg extracellular vesicle protein.

[0119] 35. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 450 ng to 600 ng ATP / mg extracellular vesicle protein.

[0120] 36. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise 500 ng to 550 ng ATP / mg extracellular vesicle protein.

[0121] 37. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise about 1 ng / mg extracellular vesicle protein; 10 ng / mg extracellular vesicle protein; 100 ng / mg extracellular vesicle protein; 200 ng / mg extracellular vesicle protein; 300 ng / mg extracellular vesicle protein; 400 ng / mg extracellular vesicle protein; 450 ng / mg extracellular vesicle protein; 500 ng / mg extracellular vesicle protein; 550 ng / mg extracellular vesicle protein; 600 ng / mg extracellular vesicle protein; 650 ng / mg extracellular vesicle protein; 750 ng / mg extracellular vesicle protein; 1 μg / mg extracellular vesicle protein; 10 μg / mg extracellular vesicle protein; 100 μg / mg extracellular vesicle protein; or 1,000 μg / mg extracellular vesicle protein.

[0122] 38. The composition as described in any of the foregoing embodiments, wherein the ATP or ATP analogue comprises ATP.

[0123] 39. The composition of any one of the foregoing embodiments, wherein the ATP or ATP analog comprises a non-hydrolyzable analog of ATP.

[0124] 40. The composition of any one of the foregoing embodiments, wherein the ATP or ATP analog comprises one or more of the following: α,β-methylene-ATP (α,βmATP); β,γ-methylene-ATP (β,γmATP); 2-thio-ATP (2-SH-ATP); 2-methylthio-ATP (2-MeS-ATP); 2',3'-O-2,4,6'-trinitrophenyl-ATP (TNP-ATP); 2',3'-9-(4-benzoyl)-ATP (BzATP); N-alkyl-2-ATP; adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP); ATP-MgCl2; 5-aminoimidazolium-4-carboxamide ribonucleotide, dipyridoxine fumarate, ribavirin, azidothymidine, fludarabine, efavirenz, and oxidized ATP. (oATP), adenosine, ADP, AMP and other nucleotides (e.g. GTP, CTP, UTP, TTP).

[0125] 41. The composition of any one of the foregoing embodiments, wherein the ATP or ATP analog comprises one or more of the group consisting of adenosine, ADP and AMP.

[0126] 42. The composition of any one of the foregoing embodiments, wherein the ATP or ATP analogue is substantially composed of one or more of the following: adenosine, ADP and AMP. 43. 44. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 10 and 1000 nm.

[0128] 45. The composition of any one of the preceding embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 10 and 1000 nm, and wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are between 10 and 1000 nm.

[0129] 46. ​​The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95% of the particles are less than 600 nm.

[0130] 47. The composition as described in any of the preceding embodiments, wherein at least 95% of the particles are less than 600 nm.

[0131] 48. The composition as described in any of the preceding embodiments, wherein at least 99% of the particles are less than 600 nm.

[0132] 49. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are less than about 500 nm.

[0133] 50. The composition as described in any of the preceding embodiments, wherein at least 95% of the particles are smaller than about 500 nm.

[0134] 51. The composition as described in any of the preceding embodiments, wherein at least 99% of the particles are smaller than about 500 nm.

[0135] 52. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are less than about 250 nm.

[0136] 53. The composition as described in any of the preceding embodiments, wherein at least 95% of the particles are smaller than about 250 nm.

[0137] 54. The composition as described in any of the preceding embodiments, wherein at least 99% of the particles are smaller than about 250 nm.

[0138] 55. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are greater than 20 nm.

[0139] 56. The composition as described in any of the preceding embodiments, wherein at least 95% of the particles are larger than 20 nm.

[0140] 57. The composition as described in any of the preceding embodiments, wherein at least 99% of the particles are larger than 20 nm.

[0141] 58. The composition according to any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between: 10 to 500 nm; or 10 to 300 nm; or 10 to 200 nm; or 10 to 185 nm; or 10 to 175 nm; or 10 to 170 nm; or 10 to 165 nm; or 10 to 160 nm; or 10 to 155 nm; or 10 to 150 nm; or 10 to 145 nm; or 10 to 140 nm; or 10 to 135 nm; or 10 to 130 nm; or 15 to 1000 nm; or 15 to 500 nm; or 15 to 300 nm; or 15 to 200 nm; or 15 to 185 nm; or 15 to 175 nm; or 15 to 170 nm; or 15 to 165 nm; or 15 to 160 nm; or 15 to 155 nm nm; or 15 to 150 nm; or 15 to 145 nm; or 15 to 140 nm; or 15 to 135 nm; or 15 to 130 nm; or 20 to 1,000 nm; or 20 to 500 nm; or 20 to 300 nm; or 20 to 200 nm; or 20 to 185 nm; or 20 to 175 nm; or 20 to 170 nm; or 20 to 165 nm; or 20 to 160 nm; or 20 to 155 nm; or 20 to 150 nm; or 20 to 145 nm; or 20 to 140 nm; or 20 to 135 nm; or 20 to 130 nm; or 25 to 1,000 nm; or 25 to 500 nm; or 25 to 300 nm; or 25 to 200 nm; or 25 to 185 nm; or 25 to 175 nm nm; or 25 to 170 nm; or 25 to 165 nm; or 25 to 160 nm; or 25 to 155 nm; or 25 to 150 nm; or 25 to 145 nm; or 25 to 140 nm; or 25 to 135 nm; or 25 to 130 nm; or 30 to 1,000 nm; or 30 to 500 nm; or 30 to 300 nm; or 30 to 200 nm; or 30 to 185 nm; or 30 to 175 nm; or 30 to 170 nm; or 30 to 165 nm; or 30 to 160 nm; or 30 to 155 nm; or 30 to 150 nm; or 30 to 145 nm; or 30 to 140 nm; or 30 to 135 nm; or 30 to 130 nm; or 35 to 1,000 nm; or 35 to 500 nm nm; or 35 to 300 nm; or 35 to 200 nm; or 35 to 185 nm; or 35 to 175 nm; or 35 to 170 nm; or 35 to 165 nm; or 35 to 160 nm; or 35 to 155 nm; or 35 to 150 nm; or 35 to 145 nm;Or 35 to 140 nm; or 35 to 135 nm; or 35 to 130 nm; or 40 to 1,000 nm; or 40 to 500 nm; or 40 to 300 nm; or 40 to 200 nm; or 40 to 185 nm; or 40 to 175 nm; or 40 to 170 nm; or 40 to 165 nm; or 40 to 160 nm; or 40 to 155 nm; or 40 to 150 nm; or 40 to 145 nm; or 40 to 140 nm; or 40 to 135 nm; or 40 to 130 nm; or 45 to 1,000 nm; or 45 to 500 nm; or 45 to 300 nm; or 45 to 200 nm; or 45 to 185 nm; or 45 to 175 nm; or 45 to 170 nm; or 45 to 165 nm; or 45 to 160 nm nm; or 45 to 155 nm; or 45 to 150 nm; or 45 to 145 nm; or 45 to 140 nm; or 45 to 135 nm; or 45 to 130 nm; or 50 to 1,000 nm; or 50 to 500 nm; or 50 to 300 nm; or 50 to 200 nm; or 50 to 185 nm; or 50 to 175 nm; or 50 to 170 nm; or 50 to 165 nm; or 50 to 160 nm; or 50 to 155 nm; or 50 to 150 nm; or 50 to 145 nm; or 50 to 140 nm; or 50 to 135 nm; or 50 to 130 nm; or 55 to 1,000 nm; or 55 to 500 nm; or 55 to 300 nm; or 55 to 200 nm; or 55 to 185 nm nm; or 55 to 175 nm; or 55 to 170 nm; or 55 to 165 nm; or 55 to 160 nm; or 55 to 155 nm; or 55 to 150 nm; or 55 to 145 nm; or 55 to 140 nm; or 55 to 135 nm; or 55 to 130 nm; or 60 to 1000 nm; or 60 to 500 nm; or 60 to 300 nm; or 60 to 200 nm; or 60 to 185 nm; or 60 to 175 nm; or 60 to 170 nm; or 60 to 165 nm; or 60 to 160 nm; or 60 to 155 nm; or 60 to 150 nm; or 60 to 145 nm; or 60 to 140 nm; or 60 to 135 nm; or 60 to 130 nm; or 65 to 1000 nm; nm; or 65 to 500 nm; or 65 to 300 nm; or 65 to 200 nm; or 65 to 185 nm; or 65 to 175 nm; or 65 to 170 nm; or 65 to 165 nm; or 65 to 160 nm; or 65 to 155 nm; or 65 to 150 nm; or 65 to 145 nm;Or 65 to 140 nm; or 65 to 135 nm; or 65 to 130 nm; or 70 to 1,000 nm; or 70 to 500 nm; or 70 to 300 nm; or 70 to 200 nm; or 70 to 185 nm; or 70 to 175 nm; or 70 to 170 nm; or 70 to 165 nm; or 70 to 160 nm; or 70 to 155 nm; or 70 to 150 nm; or 70 to 145 nm; or 70 to 140 nm; or 70 to 135 nm; or 70 to 130 nm; or 75 to 1,000 nm; or 75 to 500 nm; or 75 to 300 nm; or 75 to 200 nm; or 75 to 185 nm; or 75 to 175 nm; or 75 to 170 nm; or 75 to 165 nm; or 75 to 160 nm nm; or 75 to 155 nm; or 75 to 150 nm; or 75 to 145 nm; or 75 to 140 nm; or 75 to 135 nm; or 75 to 130 nm; or 90 to 1,000 nm; or 90 to 500 nm; or 90 to 300 nm; or 90 to 200 nm; or 90 to 185 nm; or 90 to 175 nm; or 90 to 170 nm; or 90 to 165 nm; or 90 to 160 nm; or 90 to 155 nm; or 90 to 150 nm; or 90 to 145 nm; or 90 to 140 nm; or 90 to 135 nm; or 90 to 130 nm; or 95 to 1,000 nm; or 95 to 500 nm; or 95 to 300 nm; or 95 to 200 nm; or 95 to 185 nm nm; or 95 to 175 nm; or 95 to 170 nm; or 95 to 165 nm; or 95 to 160 nm; or 95 to 155 nm; or 95 to 150 nm; or 95 to 145 nm; or 95 to 140 nm; or 95 to 135 nm; or 95 to 130 nm; or 100 to 1000 nm; or 100 to 500 nm; or 100 to 300 nm; or 100 to 200 nm; or 100 to 185 nm; or 100 to 175 nm; or 100 to 170 nm; or 100 to 165 nm; or 100 to 150 nm; or 100 to 155 nm; or 100 to 145 nm; or 100 to 100 nm; or 100 to 135 nm; or 100 to 130 nm; nm; or 105 to 1,000 nm; or 105 to 500 nm; or 105 to 300 nm; or 105 to 200 nm; or 105 to 185 nm; or 105 to 175 nm; or 105 to 170 nm; or 105 to 165 nm; or 105 to 160 nm; or 105 to 155 nm;Or 105 to 150 nm; or 105 to 145 nm; or 105 to 140 nm; or 105 to 135 nm; or 105 to 130 nm; or 110 to 1,000 nm; or 110 to 500 nm; or 110 to 300 nm; or 110 to 200 nm; or 110 to 185 nm; or 110 to 175 nm; or 110 to 170 nm; or 110 to 165 nm; or 110 to 150 nm; or 110 to 155 nm; or 110 to 150 nm; or 110 to 145 nm; or 110 to 100 nm; or 110 to 135 nm; or 110 to 130 nm; or 115 to 1,000 nm; or 115 to 500 nm; or 115 to 300 nm; or 115 to 200 nm nm; or 115 to 185 nm; or 115 to 175 nm; or 115 to 170 nm; or 115 to 165 nm; or 115 to 160 nm; or 115 to 155 nm; or 115 to 150 nm; or 115 to 145 nm; or 115 to 140 nm; or 115 to 135 nm; or 115 to 130 nm; or 120 to 1,000 nm; or 120 to 500 nm; or 120 to 300 nm; or 120 to 200 nm; or 120 to 185 nm; or 120 to 175 nm; or 120 to 170 nm; or 120 to 165 nm; or 120 to 150 nm; or 120 to 155 nm; or 120 to 150 nm; or 120 to 145 nm; or 120 to 100 nm nm; or 120 to 135 nm; or 120 to 130 nm.

[0142] 59. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 50 and 500 nm.

[0143] 60. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 100 and 200 nm.

[0144] 61. The composition of any one of the preceding embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 100 and 200 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

[0145] 62. The composition as described in any of the preceding embodiments, wherein the extracellular vesicles in the composition have an average particle size of about 150 nm.

[0146] 63. The composition of any one of the preceding embodiments, wherein the extracellular vesicles in the composition have an average particle size of about 150 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

[0147] 64. The composition as described in any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles.

[0148] 65. The composition according to any one of the foregoing embodiments, wherein the extracellular vesicles are bovine milk extracellular vesicles.

[0149] 66. The composition of any one of the foregoing embodiments, wherein the extracellular vesicle is a bovine milk extracellular vesicle; and wherein the bovine milk extracellular vesicle comprises bovine IgG.

[0150] 67. The composition as described in any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles.

[0151] 68. The composition of any one of the foregoing embodiments, wherein the extracellular vesicle is a milk extracellular vesicle, and wherein the extracellular vesicle is isolated from milk using the method of any one of the “Isolation of extracellular vesicles from milk” embodiments.

[0152] 69. The composition of any one of the foregoing embodiments, wherein the extracellular vesicle comprises one or more carrier molecules.

[0153] 70. The composition as described in any of the preceding embodiments, wherein the composition is a pharmaceutically acceptable composition.

[0154] 71. A method for preparing an ATP-analyte-extracellular vesicle composition as described in any of the foregoing embodiments, the method comprising contacting the extracellular vesicles with a solution containing ATP and / or an ATP analog to obtain a composition containing ATP-analyte-extracellular vesicles.

[0155] 72. A method for increasing the uptake of cells, tissues and / or organs of a subject, which is carried out by administering to the subject an extracellular vesicle composition as described in any of the foregoing embodiments.

[0156] 73. A method of treating a subject, comprising administering to the subject an extracellular vesicle composition as described in any of the preceding embodiments.

[0157] 74. A method of treating a subject, comprising administering to the subject, via oral, enteral, parenteral, intranasal, IV, topical, or direct administration, an extracellular vesicle composition as described in any of the preceding embodiments.

[0158] 75. A method for increasing the uptake of extracellular vesicles by the skin tissue of a subject, the method comprising: administering an extracellular vesicle composition as described in any of the preceding embodiments by topical application to the skin of the subject.

[0159] 76. A method of providing benefit to a healthy subject by administering to the subject extracellular vesicles loaded with one or more nutritional or cosmetic carriers (e.g., peptides, curcumin, flavonoids, amino acids, lipids, fatty acids, calcium, vitamins, ATP, creatine, etc.) of a composition as described in any of the preceding embodiments.

[0160] 77. A method for enhancing the delivery loading of extracellular vesicles with external delivery molecules, said external delivery molecules comprising peptides, small drug molecules or other delivery compounds.

[0161] Embodiments for isolating extracellular vesicles from milk 1. A method for separating extracellular vesicles from a milk sample, wherein the method comprises contacting (or incubating) the milk with tryptophan and / or one or more tryptophan analogs, and subsequently separating the milk extracellular vesicles from one or more milk components, such as, for example, casein-containing aggregates.

[0162] 2. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogues at concentrations between 10 µM and 1 mM (incubation).

[0163] 3. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogues at a temperature between 10 and 50 degrees Celsius for at least 10 minutes.

[0164] 4. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogs at concentrations between 10 µM and 1 mM at a temperature between 10 and 50 degrees Celsius for at least 10 minutes.

[0165] 5. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes.

[0166] 6. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or tryptophan analogs and / or trehalose and / or trehalose analogs at a temperature between 10 and 50 degrees Celsius for at least 10 minutes.

[0167] 7. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at a concentration between 1 mM and 100 mM at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes.

[0168] 8. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises incubating the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, and acid-treated to a pH of 4.6, at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes.

[0169] 9. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises incubating the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, and acid-treated to a pH between 2.0 and 6.0, at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes.

[0170] 10. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at concentrations between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at concentrations between 1 mM and 100 mM, along with an acid-treated pH of 4.6, at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes.

[0171] 11. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at a concentration between 1 mM and 100 mM, along with acid treatment to a pH between 2.0 and 6.0, at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes.

[0172] 12. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, along with rennet treatment, at a temperature between 10 and 50 degrees Celsius for 10 to 60 minutes. 13. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogues at a temperature of 10 to 80 degrees Celsius for at least 10 seconds.

[0173] 14. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogs at a concentration between 10 µM and 1 mM at a temperature between 10 and 80 degrees Celsius for at least 10 seconds.

[0174] 15. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or more tryptophan analogs at a concentration between 10 µM and 1 mM at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0175] 16. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogs at concentrations between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at concentrations between 1 mM and 100 mM at a temperature between 10 and 80 degrees Celsius for at least 10 seconds.

[0176] 17. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or more tryptophan analogs at a concentration between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at a concentration between 1 mM and 100 mM at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0177] 18. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises incubating the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, and acid-treated to a pH of 4.6, at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0178] 19. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises incubating the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, and acid-treated to a pH between 2.0 and 6.0, at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0179] 20. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at concentrations between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at concentrations between 1 mM and 100 mM, along with an acid-treated pH of 4.6, at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0180] 21. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at a concentration between 1 mM and 100 mM, along with acid treatment to a pH between 2.0 and 6.0, at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0181] 22. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, along with rennet treatment, at a temperature between 10 and 80 degrees Celsius for 10 seconds to 60 minutes.

[0182] 23. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or more tryptophan analogs and / or trehalose and / or trehalose analogs at a temperature of 10 to 60 degrees Celsius for at least 10 minutes.

[0183] 24. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogs at concentrations between 10 µM and 1 mM at a temperature between 10 and 60 degrees Celsius for at least 10 minutes.

[0184] 25. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or more tryptophan analogs at a concentration between 10 µM and 1 mM at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0185] 26. A method for isolating extracellular vesicles from a milk sample, wherein the method... This includes contacting (incubating) extracellular vesicles of milk cells with tryptophan and / or more tryptophan analogs at concentrations between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at concentrations between 1 mM and 100 mM at a temperature between 10 and 60 degrees Celsius for at least 10 minutes.

[0186] 27. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or more tryptophan analogs at a concentration between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at a concentration between 1 mM and 100 mM at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0187] 28. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises incubating the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, and acid-treated to a pH of 4.6, at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0188] 29. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises incubating the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, and acid-treated to a pH between 2.0 and 6.0, at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0189] 30. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at concentrations between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at concentrations between 1 mM and 100 mM, along with an acid-treated pH of 4.6, at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0190] 31. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM and / or trehalose and / or trehalose analogs at a concentration between 1 mM and 100 mM, and acid-treated to a pH between 2.0 and 6.0, at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0191] 32. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and / or tryptophan analogs at a concentration between 10 µM and 1 mM, along with rennet treatment, at a temperature between 10 and 60 degrees Celsius for 10 to 60 minutes.

[0192] 33. The method of any of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 10 and 1000 nm.

[0193] 34. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 10 and 1000 nm, and wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are between 10 and 1000 nm.

[0194] 35. The method as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95% of the particles are less than 600 nm.

[0195] 36. The method as described in any of the preceding embodiments, wherein at least 95% of the particles are less than 600 nm.

[0196] 37. The method as described in any of the preceding embodiments, wherein at least 99% of the particles are less than 600 nm.

[0197] 38. The method as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are less than about 500 nm.

[0198] 39. The method as described in any of the preceding embodiments, wherein at least 95% of the particles are smaller than about 500 nm.

[0199] 40. The method as described in any of the preceding embodiments, wherein at least 99% of the particles are smaller than about 500 nm.

[0200] 41. The method as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are less than about 250 nm.

[0201] 42. The method as described in any of the preceding embodiments, wherein at least 95% of the particles are smaller than about 250 nm.

[0202] 43. The method as described in any of the preceding embodiments, wherein at least 99% of the particles are smaller than about 250 nm.

[0203] 44. The method as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are greater than 20 nm.

[0204] 45. The method as described in any of the preceding embodiments, wherein at least 95% of the particles are larger than 20 nm.

[0205] 46. ​​The method as described in any of the preceding embodiments, wherein at least 99% of the particles are larger than 20 nm.

[0206] 47. The method of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between: 10 to 500 nm; or 10 to 300 nm; or 10 to 200 nm; or 10 to 185 nm; or 10 to 175 nm; or 10 to 170 nm; or 10 to 165 nm; or 10 to 160 nm; or 10 to 155 nm; or 10 to 150 nm; or 10 to 145 nm; or 10 to 140 nm; or 10 to 135 nm; or 10 to 130 nm; or 15 to 1000 nm; or 15 to 500 nm; or 15 to 300 nm; or 15 to 200 nm; or 15 to 185 nm; or 15 to 175 nm; or 15 to 170 nm; or 15 to 165 nm; or 15 to 160 nm; or 15 to 155 nm nm; or 15 to 150 nm; or 15 to 145 nm; or 15 to 140 nm; or 15 to 135 nm; or 15 to 130 nm; or 20 to 1,000 nm; or 20 to 500 nm; or 20 to 300 nm; or 20 to 200 nm; or 20 to 185 nm; or 20 to 175 nm; or 20 to 170 nm; or 20 to 165 nm; or 20 to 160 nm; or 20 to 155 nm; or 20 to 150 nm; or 20 to 145 nm; or 20 to 140 nm; or 20 to 135 nm; or 20 to 130 nm; or 25 to 1,000 nm; or 25 to 500 nm; or 25 to 300 nm; or 25 to 200 nm; or 25 to 185 nm; or 25 to 175 nm nm; or 25 to 170 nm; or 25 to 165 nm; or 25 to 160 nm; or 25 to 155 nm; or 25 to 150 nm; or 25 to 145 nm; or 25 to 140 nm; or 25 to 135 nm; or 25 to 130 nm; or 30 to 1,000 nm; or 30 to 500 nm; or 30 to 300 nm; or 30 to 200 nm; or 30 to 185 nm; or 30 to 175 nm; or 30 to 170 nm; or 30 to 165 nm; or 30 to 160 nm; or 30 to 155 nm; or 30 to 150 nm; or 30 to 145 nm; or 30 to 140 nm; or 30 to 135 nm; or 30 to 130 nm; or 35 to 1,000 nm; or 35 to 500 nm nm; or 35 to 300 nm; or 35 to 200 nm; or 35 to 185 nm; or 35 to 175 nm; or 35 to 170 nm; or 35 to 165 nm; or 35 to 160 nm; or 35 to 155 nm; or 35 to 150 nm; or 35 to 145 nm;Or 35 to 140 nm; or 35 to 135 nm; or 35 to 130 nm; or 40 to 1,000 nm; or 40 to 500 nm; or 40 to 300 nm; or 40 to 200 nm; or 40 to 185 nm; or 40 to 175 nm; or 40 to 170 nm; or 40 to 165 nm; or 40 to 160 nm; or 40 to 155 nm; or 40 to 150 nm; or 40 to 145 nm; or 40 to 140 nm; or 40 to 135 nm; or 40 to 130 nm; or 45 to 1,000 nm; or 45 to 500 nm; or 45 to 300 nm; or 45 to 200 nm; or 45 to 185 nm; or 45 to 175 nm; or 45 to 170 nm; or 45 to 165 nm; or 45 to 160 nm nm; or 45 to 155 nm; or 45 to 150 nm; or 45 to 145 nm; or 45 to 140 nm; or 45 to 135 nm; or 45 to 130 nm; or 50 to 1,000 nm; or 50 to 500 nm; or 50 to 300 nm; or 50 to 200 nm; or 50 to 185 nm; or 50 to 175 nm; or 50 to 170 nm; or 50 to 165 nm; or 50 to 160 nm; or 50 to 155 nm; or 50 to 150 nm; or 50 to 145 nm; or 50 to 140 nm; or 50 to 135 nm; or 50 to 130 nm; or 55 to 1,000 nm; or 55 to 500 nm; or 55 to 300 nm; or 55 to 200 nm; or 55 to 185 nm nm; or 55 to 175 nm; or 55 to 170 nm; or 55 to 165 nm; or 55 to 160 nm; or 55 to 155 nm; or 55 to 150 nm; or 55 to 145 nm; or 55 to 140 nm; or 55 to 135 nm; or 55 to 130 nm; or 60 to 1000 nm; or 60 to 500 nm; or 60 to 300 nm; or 60 to 200 nm; or 60 to 185 nm; or 60 to 175 nm; or 60 to 170 nm; or 60 to 165 nm; or 60 to 160 nm; or 60 to 155 nm; or 60 to 150 nm; or 60 to 145 nm; or 60 to 140 nm; or 60 to 135 nm; or 60 to 130 nm; or 65 to 1000 nm; nm; or 65 to 500 nm; or 65 to 300 nm; or 65 to 200 nm; or 65 to 185 nm; or 65 to 175 nm; or 65 to 170 nm; or 65 to 165 nm; or 65 to 160 nm; or 65 to 155 nm; or 65 to 150 nm; or 65 to 145 nm;Or 65 to 140 nm; or 65 to 135 nm; or 65 to 130 nm; or 70 to 1,000 nm; or 70 to 500 nm; or 70 to 300 nm; or 70 to 200 nm; or 70 to 185 nm; or 70 to 175 nm; or 70 to 170 nm; or 70 to 165 nm; or 70 to 160 nm; or 70 to 155 nm; or 70 to 150 nm; or 70 to 145 nm; or 70 to 140 nm; or 70 to 135 nm; or 70 to 130 nm; or 75 to 1,000 nm; or 75 to 500 nm; or 75 to 300 nm; or 75 to 200 nm; or 75 to 185 nm; or 75 to 175 nm; or 75 to 170 nm; or 75 to 165 nm; or 75 to 160 nm nm; or 75 to 155 nm; or 75 to 150 nm; or 75 to 145 nm; or 75 to 140 nm; or 75 to 135 nm; or 75 to 130 nm; or 90 to 1,000 nm; or 90 to 500 nm; or 90 to 300 nm; or 90 to 200 nm; or 90 to 185 nm; or 90 to 175 nm; or 90 to 170 nm; or 90 to 165 nm; or 90 to 160 nm; or 90 to 155 nm; or 90 to 150 nm; or 90 to 145 nm; or 90 to 140 nm; or 90 to 135 nm; or 90 to 130 nm; or 95 to 1,000 nm; or 95 to 500 nm; or 95 to 300 nm; or 95 to 200 nm; or 95 to 185 nm nm; or 95 to 175 nm; or 95 to 170 nm; or 95 to 165 nm; or 95 to 160 nm; or 95 to 155 nm; or 95 to 150 nm; or 95 to 145 nm; or 95 to 140 nm; or 95 to 135 nm; or 95 to 130 nm; or 100 to 1000 nm; or 100 to 500 nm; or 100 to 300 nm; or 100 to 200 nm; or 100 to 185 nm; or 100 to 175 nm; or 100 to 170 nm; or 100 to 165 nm; or 100 to 150 nm; or 100 to 155 nm; or 100 to 145 nm; or 100 to 100 nm; or 100 to 135 nm; or 100 to 130 nm; nm; or 105 to 1,000 nm; or 105 to 500 nm; or 105 to 300 nm; or 105 to 200 nm; or 105 to 185 nm; or 105 to 175 nm; or 105 to 170 nm; or 105 to 165 nm; or 105 to 160 nm; or 105 to 155 nm;Or 105 to 150 nm; or 105 to 145 nm; or 105 to 140 nm; or 105 to 135 nm; or 105 to 130 nm; or 110 to 1,000 nm; or 110 to 500 nm; or 110 to 300 nm; or 110 to 200 nm; or 110 to 185 nm; or 110 to 175 nm; or 110 to 170 nm; or 110 to 165 nm; or 110 to 150 nm; or 110 to 155 nm; or 110 to 150 nm; or 110 to 145 nm; or 110 to 100 nm; or 110 to 135 nm; or 110 to 130 nm; or 115 to 1,000 nm; or 115 to 500 nm; or 115 to 300 nm; or 115 to 200 nm nm; or 115 to 185 nm; or 115 to 175 nm; or 115 to 170 nm; or 115 to 165 nm; or 115 to 160 nm; or 115 to 155 nm; or 115 to 150 nm; or 115 to 145 nm; or 115 to 140 nm; or 115 to 135 nm; or 115 to 130 nm; or 120 to 1,000 nm; or 120 to 500 nm; or 120 to 300 nm; or 120 to 200 nm; or 120 to 185 nm; or 120 to 175 nm; or 120 to 170 nm; or 120 to 165 nm; or 120 to 150 nm; or 120 to 155 nm; or 120 to 150 nm; or 120 to 145 nm; or 120 to 100 nm nm; or 120 to 135 nm; or 120 to 130 nm.

[0207] 48. The method of any of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 50 and 500 nm.

[0208] 49. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 100 and 200 nm.

[0209] 50. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 100 and 200 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

[0210] 51. The method of any one of the foregoing embodiments, wherein the extracellular vesicles in the composition have an average particle size of about 150 nm.

[0211] 52. The method of any of the preceding embodiments, wherein the extracellular vesicles in the composition have an average particle size of about 150 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

[0212] 53. The method as described in any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles.

[0213] 54. The method as described in any of the preceding embodiments, wherein the extracellular vesicles are bovine milk extracellular vesicles.

[0214] 55. The method of any of the foregoing embodiments, wherein the extracellular vesicle is a bovine milk extracellular vesicle; and wherein the bovine milk extracellular vesicle comprises bovine IgG.

[0215] 56. The method as described in any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles.

[0216] 57. The method of any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles, and wherein the extracellular vesicles are separated from milk using the method of any of the “Isolation of extracellular vesicles from milk” embodiments.

[0217] 58. The method of any of the preceding embodiments, wherein the extracellular vesicles comprise one or more carrier molecules.

[0218] 59. The method as described in any of the preceding embodiments, wherein the composition is a pharmaceutically acceptable composition.

[0219] 60. The method of any of the foregoing embodiments, wherein the tryptophan or tryptophan analogue comprises one or more selected from the list including: L-tryptophan, D-tryptophan, tryptophan, tryptophan dimer, tryptophan trimer, tryptophan peptide, histidine, L-histidine, D-histidine, histidine dimer, histidine trimer, histidine peptide, tyrosine, L-tyrosine, D-tyrosine, tyrosine dimer, tyrosine trimer, tyrosine peptide, arginine, L-arginine, D-arginine, arginine dimer, arginine trimer, arginine peptide, cysteine. Acids, L-cysteine, D-cysteine, cysteine ​​dimer, cysteine ​​trimer, cysteine ​​peptide, lysine, L-lysine, D-lysine, lysine dimer, lysine trimer, lysine peptide, phenylalanine, L-phenylalanine, D-phenylalanine, phenylalanine dimer, phenylalanine trimer, phenylalanine peptide, D-carnitine, L-carnitine, carnitine dimer, carnitine trimer, carnitine peptide, acetyl L-carnitine, L-carnitine L-tartrate, propionyl L-carnitine, D-taurine, L-taurine, taurine dimer, taurine trimer, taurine peptide Freeze-drying embodiments of milk extracellular vesicles 1. A composition comprising extracellular vesicles, wherein the composition comprises tryptophan or a tryptophan analogue at a concentration greater than 0.01 µM.

[0220] 2. A composition comprising extracellular vesicles, wherein the composition comprises more than 0.001% W / W% of tryptophan or a tryptophan analogue.

[0221] 3. A composition comprising extracellular vesicles, wherein the composition comprises 0.01 µM to 100 mM of tryptophan or a tryptophan analogue.

[0222] 4. A composition comprising extracellular vesicles, wherein the composition comprises between 0.001% W / W% and 90% W / W% of tryptophan or a tryptophan analogue.

[0223] 5. A composition comprising extracellular vesicles, wherein the composition comprises between 0.1 µM and 10 mM of tryptophan or a tryptophan analogue.

[0224] 6. A composition comprising extracellular vesicles, wherein the composition comprises between 0.01% W / W% and 50% W / W% of tryptophan or tryptophan analogues.

[0225] 7. A composition comprising extracellular vesicles, wherein the composition comprises tryptophan or a tryptophan analogue between 1 µM and 1 mM.

[0226] 8. A composition comprising extracellular vesicles, wherein the composition comprises between 0.1% W / W% and 10% W / W% of tryptophan or a tryptophan analogue.

[0227] 9. A composition comprising extracellular vesicles, wherein the composition comprises between 10 µM and 100 µM of tryptophan or a tryptophan analogue.

[0228] 10. A composition comprising extracellular vesicles, wherein the composition comprises between 0.2% W / W% and 1% W / W% of tryptophan or tryptophan analogues.

[0229] 11. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises more than 0.01 µM of tryptophan or a tryptophan analogue.

[0230] 12. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises more than 0.001% W / W% of tryptophan or a tryptophan analogue.

[0231] 13. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises 0.01 µM to 100 mM of tryptophan or a tryptophan analogue.

[0232] 14. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises between 0.001% W / W% and 90% W / W% of tryptophan or tryptophan analogues.

[0233] 15. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises between 0.1 µM and 10 mM of tryptophan or a tryptophan analogue.

[0234] 16. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises between 0.01% W / W% and 50% W / W% of tryptophan or tryptophan analogues.

[0235] 17. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises tryptophan or a tryptophan analogue between 1 µM and 1 mM.

[0236] 18. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises between 0.1% W / W% and 10% W / W% of tryptophan or tryptophan analogues.

[0237] 19. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises between 10 µM and 100 µM of tryptophan or a tryptophan analogue.

[0238] 20. A composition comprising extracellular vesicles of milk cells, wherein the composition comprises between 0.2% W / W% and 1% W / W% of tryptophan or tryptophan analogues.

[0239] 21. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises tryptophan or a tryptophan analogue at a concentration greater than 0.01 µM.

[0240] 22. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises more than 0.001% W / W% of tryptophan or a tryptophan analogue.

[0241] 23. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises 0.01 µM to 100 mM of tryptophan or a tryptophan analogue.

[0242] 24. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises between 0.001% W / W% and 90% W / W% of tryptophan or a tryptophan analogue.

[0243] 25. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises between 0.1 µM and 10 mM of tryptophan or a tryptophan analogue.

[0244] 26. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises between 0.01% W / W% and 50% W / W% of tryptophan or a tryptophan analogue.

[0245] 27. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises tryptophan or a tryptophan analogue between 1 µM and 1 mM.

[0246] 28. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises between 0.1% W / W% and 10% W / W% of tryptophan or a tryptophan analogue.

[0247] 29. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises between 10 µM and 100 µM of tryptophan or a tryptophan analogue.

[0248] 30. A composition comprising bovine milk extracellular vesicles, wherein the composition comprises between 0.2% W / W% and 1% W / W% of tryptophan or tryptophan analogues.

[0249] 31. The composition of any one of the foregoing embodiments, wherein the composition comprises tryptophan or a tryptophan analogue in the range of 0.1 µM to 1 mM, or 1.0 µM to 750 µM, or 10 µM to 500 µM, or 20 µM to 300 µM, or 40 µM to 200 µM, or 50 µM to 150 µM.

[0250] 32. The composition of any one of the foregoing embodiments, wherein the composition comprises about 100 µM of tryptophan or a tryptophan analogue.

[0251] 33. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise tryptophan or a tryptophan analogue at a concentration greater than 0.01 µM.

[0252] 34. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise more than 0.001% W / W% of tryptophan or tryptophan analogues.

[0253] 35. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise between 0.01 µM and 100 mM of tryptophan or a tryptophan analogue.

[0254] 36. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise between 0.001% W / W% and 90% W / W% of tryptophan or tryptophan analogues.

[0255] 37. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise between 0.1 µM and 10 mM of tryptophan or a tryptophan analogue.

[0256] 38. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise between 0.01% W / W% and 50% W / W% of tryptophan or tryptophan analogues.

[0257] 39. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise tryptophan or a tryptophan analogue between 1 µM and 1 mM.

[0258] 40. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise tryptophan or tryptophan analogues at a concentration between 0.1% W / W% and 10% W / W%.

[0259] 41. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise between 10 µM and 100 µM of tryptophan or a tryptophan analogue.

[0260] 42. A composition comprising extracellular vesicles, wherein the extracellular vesicles comprise between 0.2% W / W% and 1% W / W% of tryptophan or tryptophan analogues.

[0261] 43. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise more than 0.01 µM of tryptophan or a tryptophan analogue.

[0262] 44. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise more than 0.001% W / W% of tryptophan or tryptophan analogues.

[0263] 45. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise 0.01 µM to 100 mM of tryptophan or a tryptophan analogue.

[0264] 46. ​​A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 0.001% W / W% and 90% W / W% of tryptophan or tryptophan analogues.

[0265] 47. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 0.1 µM and 10 mM of tryptophan or tryptophan analogues.

[0266] 48. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 0.01% W / W% and 50% W / W% of tryptophan or tryptophan analogues.

[0267] 49. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 1 µM and 1 mM of tryptophan or a tryptophan analogue.

[0268] 50. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 0.1% W / W% and 10% W / W% of tryptophan or tryptophan analogues.

[0269] 51. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 10 µM and 100 µM of tryptophan or tryptophan analogues.

[0270] 52. A composition comprising extracellular vesicles of milk cells, wherein the extracellular vesicles comprise between 0.2% W / W% and 1% W / W% of tryptophan or tryptophan analogues.

[0271] 53. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise tryptophan or a tryptophan analogue in greater than 0.01 µM.

[0272] 54. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise more than 0.001% W / W% of tryptophan or tryptophan analogues.

[0273] 55. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 0.01 µM and 100 mM of tryptophan or a tryptophan analogue.

[0274] 56. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 0.001% W / W% and 90% W / W% of tryptophan or tryptophan analogues.

[0275] 57. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 0.1 µM and 10 mM of tryptophan or tryptophan analogues.

[0276] 58. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 0.01% W / W% and 50% W / W% of tryptophan or tryptophan analogues.

[0277] 59. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 1 µM and 1 mM of tryptophan or a tryptophan analogue.

[0278] 60. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 0.1% W / W% and 10% W / W% of tryptophan or tryptophan analogues.

[0279] 61. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 10 µM and 100 µM of tryptophan or a tryptophan analogue.

[0280] 62. A composition comprising bovine milk extracellular vesicles, wherein the extracellular vesicles comprise between 0.2% W / W% and 1% W / W% of tryptophan or tryptophan analogues.

[0281] 63. The composition of any one of the preceding embodiments, wherein the extracellular vesicles comprise 0.1 µM to 1 mM, or 1.0 µM to 750 µM; or 10 µM to 500 µM; or 20 µM to 300 µM; or 40 µM to 200 µM; or 50 µM to 150 µM of tryptophan or a tryptophan analogue.

[0282] 64. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles comprise about 100 µM of tryptophan or a tryptophan analogue.

[0283] 65. The composition according to any one of the foregoing embodiments, wherein the tryptophan or tryptophan analog comprises one or more of the group consisting of: L-tryptophan, D-tryptophan, tryptophan dimer, tryptophan trimer, tryptophan peptide, histidine, L-histidine, D-histidine, histidine dimer, histidine trimer, histidine peptide, tyrosine, L-tyrosine, D-tyrosine, tyrosine dimer, tyrosine trimer, tyrosine peptide, arginine, L-arginine, D-arginine, arginine dimer, arginine trimer, arginine peptide, cysteine Amino acids, L-cysteine, D-cysteine, cysteine ​​dimer, cysteine ​​trimer, cysteine ​​peptide, lysine, L-lysine, D-lysine, lysine dimer, lysine trimer, lysine peptide, phenylalanine, L-phenylalanine, D-phenylalanine, phenylalanine dimer, phenylalanine trimer, phenylalanine peptide, D-carnitine, L-carnitine, carnitine dimer, carnitine trimer, carnitine peptide, acetyl L-carnitine, L-carnitine L-tartrate, propionyl L-carnitine, D-taurine, L-taurine, taurine dimer, taurine trimer, taurine peptide.

[0284] 66. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 10 and 1000 nm.

[0285] 67. The composition of any one of the preceding embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 10 and 1000 nm, and wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles are between 10 and 1000 nm.

[0286] 68. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95% of the particles in the composition are less than 600 nm.

[0287] 69. The composition of any of the foregoing embodiments, wherein at least 95% of the particles in the composition are less than 600 nm.

[0288] 70. The composition of any of the foregoing embodiments, wherein at least 99% of the particles in the composition are less than 600 nm.

[0289] 71. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles in the composition are less than about 500 nm.

[0290] 72. The composition of any of the foregoing embodiments, wherein at least 95% of the particles in the composition are smaller than about 500 nm.

[0291] 73. The composition of any of the foregoing embodiments, wherein at least 99% of the particles in the composition are smaller than about 500 nm.

[0292] 74. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles in the composition are less than about 250 nm.

[0293] 75. The composition of any of the foregoing embodiments, wherein at least 95% of the particles in the composition are smaller than about 250 nm.

[0294] 76. The composition of any of the foregoing embodiments, wherein at least 99% of the particles in the composition are smaller than about 250 nm.

[0295] 77. The composition as described in any of the preceding embodiments, wherein at least 75%, or 80%, or 90%, or 95%, or 98%, or 99% of the particles in the composition are greater than 20 nm.

[0296] 78. The composition of any of the foregoing embodiments, wherein at least 95% of the particles in the composition are larger than 20 nm.

[0297] 79. The composition of any of the foregoing embodiments, wherein at least 99% of the particles in the composition are larger than 20 nm.

[0298] 80. The composition according to any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between: 10 to 500 nm; or 10 to 300 nm; or 10 to 200 nm; or 10 to 185 nm; or 10 to 175 nm; or 10 to 170 nm; or 10 to 165 nm; or 10 to 160 nm; or 10 to 155 nm; or 10 to 150 nm; or 10 to 145 nm; or 10 to 140 nm; or 10 to 135 nm; or 10 to 130 nm; or 15 to 1000 nm; or 15 to 500 nm; or 15 to 300 nm; or 15 to 200 nm; or 15 to 185 nm; or 15 to 175 nm; or 15 to 170 nm; or 15 to 165 nm; or 15 to 160 nm; or 15 to 155 nm nm; or 15 to 150 nm; or 15 to 145 nm; or 15 to 140 nm; or 15 to 135 nm; or 15 to 130 nm; or 20 to 1,000 nm; or 20 to 500 nm; or 20 to 300 nm; or 20 to 200 nm; or 20 to 185 nm; or 20 to 175 nm; or 20 to 170 nm; or 20 to 165 nm; or 20 to 160 nm; or 20 to 155 nm; or 20 to 150 nm; or 20 to 145 nm; or 20 to 140 nm; or 20 to 135 nm; or 20 to 130 nm; or 25 to 1,000 nm; or 25 to 500 nm; or 25 to 300 nm; or 25 to 200 nm; or 25 to 185 nm; or 25 to 175 nm nm; or 25 to 170 nm; or 25 to 165 nm; or 25 to 160 nm; or 25 to 155 nm; or 25 to 150 nm; or 25 to 145 nm; or 25 to 140 nm; or 25 to 135 nm; or 25 to 130 nm; or 30 to 1,000 nm; or 30 to 500 nm; or 30 to 300 nm; or 30 to 200 nm; or 30 to 185 nm; or 30 to 175 nm; or 30 to 170 nm; or 30 to 165 nm; or 30 to 160 nm; or 30 to 155 nm; or 30 to 150 nm; or 30 to 145 nm; or 30 to 140 nm; or 30 to 135 nm; or 30 to 130 nm; or 35 to 1,000 nm; or 35 to 500 nm nm; or 35 to 300 nm; or 35 to 200 nm; or 35 to 185 nm; or 35 to 175 nm; or 35 to 170 nm; or 35 to 165 nm; or 35 to 160 nm; or 35 to 155 nm; or 35 to 150 nm; or 35 to 145 nm;Or 35 to 140 nm; or 35 to 135 nm; or 35 to 130 nm; or 40 to 1,000 nm; or 40 to 500 nm; or 40 to 300 nm; or 40 to 200 nm; or 40 to 185 nm; or 40 to 175 nm; or 40 to 170 nm; or 40 to 165 nm; or 40 to 160 nm; or 40 to 155 nm; or 40 to 150 nm; or 40 to 145 nm; or 40 to 140 nm; or 40 to 135 nm; or 40 to 130 nm; or 45 to 1,000 nm; or 45 to 500 nm; or 45 to 300 nm; or 45 to 200 nm; or 45 to 185 nm; or 45 to 175 nm; or 45 to 170 nm; or 45 to 165 nm; or 45 to 160 nm nm; or 45 to 155 nm; or 45 to 150 nm; or 45 to 145 nm; or 45 to 140 nm; or 45 to 135 nm; or 45 to 130 nm; or 50 to 1,000 nm; or 50 to 500 nm; or 50 to 300 nm; or 50 to 200 nm; or 50 to 185 nm; or 50 to 175 nm; or 50 to 170 nm; or 50 to 165 nm; or 50 to 160 nm; or 50 to 155 nm; or 50 to 150 nm; or 50 to 145 nm; or 50 to 140 nm; or 50 to 135 nm; or 50 to 130 nm; or 55 to 1,000 nm; or 55 to 500 nm; or 55 to 300 nm; or 55 to 200 nm; or 55 to 185 nm nm; or 55 to 175 nm; or 55 to 170 nm; or 55 to 165 nm; or 55 to 160 nm; or 55 to 155 nm; or 55 to 150 nm; or 55 to 145 nm; or 55 to 140 nm; or 55 to 135 nm; or 55 to 130 nm; or 60 to 1000 nm; or 60 to 500 nm; or 60 to 300 nm; or 60 to 200 nm; or 60 to 185 nm; or 60 to 175 nm; or 60 to 170 nm; or 60 to 165 nm; or 60 to 160 nm; or 60 to 155 nm; or 60 to 150 nm; or 60 to 145 nm; or 60 to 140 nm; or 60 to 135 nm; or 60 to 130 nm; or 65 to 1000 nm; nm; or 65 to 500 nm; or 65 to 300 nm; or 65 to 200 nm; or 65 to 185 nm; or 65 to 175 nm; or 65 to 170 nm; or 65 to 165 nm; or 65 to 160 nm; or 65 to 155 nm; or 65 to 150 nm; or 65 to 145 nm;Or 65 to 140 nm; or 65 to 135 nm; or 65 to 130 nm; or 70 to 1,000 nm; or 70 to 500 nm; or 70 to 300 nm; or 70 to 200 nm; or 70 to 185 nm; or 70 to 175 nm; or 70 to 170 nm; or 70 to 165 nm; or 70 to 160 nm; or 70 to 155 nm; or 70 to 150 nm; or 70 to 145 nm; or 70 to 140 nm; or 70 to 135 nm; or 70 to 130 nm; or 75 to 1,000 nm; or 75 to 500 nm; or 75 to 300 nm; or 75 to 200 nm; or 75 to 185 nm; or 75 to 175 nm; or 75 to 170 nm; or 75 to 165 nm; or 75 to 160 nm nm; or 75 to 155 nm; or 75 to 150 nm; or 75 to 145 nm; or 75 to 140 nm; or 75 to 135 nm; or 75 to 130 nm; or 90 to 1,000 nm; or 90 to 500 nm; or 90 to 300 nm; or 90 to 200 nm; or 90 to 185 nm; or 90 to 175 nm; or 90 to 170 nm; or 90 to 165 nm; or 90 to 160 nm; or 90 to 155 nm; or 90 to 150 nm; or 90 to 145 nm; or 90 to 140 nm; or 90 to 135 nm; or 90 to 130 nm; or 95 to 1,000 nm; or 95 to 500 nm; or 95 to 300 nm; or 95 to 200 nm; or 95 to 185 nm nm; or 95 to 175 nm; or 95 to 170 nm; or 95 to 165 nm; or 95 to 160 nm; or 95 to 155 nm; or 95 to 150 nm; or 95 to 145 nm; or 95 to 140 nm; or 95 to 135 nm; or 95 to 130 nm; or 100 to 1000 nm; or 100 to 500 nm; or 100 to 300 nm; or 100 to 200 nm; or 100 to 185 nm; or 100 to 175 nm; or 100 to 170 nm; or 100 to 165 nm; or 100 to 150 nm; or 100 to 155 nm; or 100 to 145 nm; or 100 to 100 nm; or 100 to 135 nm; or 100 to 130 nm; nm; or 105 to 1,000 nm; or 105 to 500 nm; or 105 to 300 nm; or 105 to 200 nm; or 105 to 185 nm; or 105 to 175 nm; or 105 to 170 nm; or 105 to 165 nm; or 105 to 160 nm; or 105 to 155 nm;Or 105 to 150 nm; or 105 to 145 nm; or 105 to 140 nm; or 105 to 135 nm; or 105 to 130 nm; or 110 to 1,000 nm; or 110 to 500 nm; or 110 to 300 nm; or 110 to 200 nm; or 110 to 185 nm; or 110 to 175 nm; or 110 to 170 nm; or 110 to 165 nm; or 110 to 150 nm; or 110 to 155 nm; or 110 to 150 nm; or 110 to 145 nm; or 110 to 100 nm; or 110 to 135 nm; or 110 to 130 nm; or 115 to 1,000 nm; or 115 to 500 nm; or 115 to 300 nm; or 115 to 200 nm nm; or 115 to 185 nm; or 115 to 175 nm; or 115 to 170 nm; or 115 to 165 nm; or 115 to 160 nm; or 115 to 155 nm; or 115 to 150 nm; or 115 to 145 nm; or 115 to 140 nm; or 115 to 135 nm; or 115 to 130 nm; or 120 to 1,000 nm; or 120 to 500 nm; or 120 to 300 nm; or 120 to 200 nm; or 120 to 185 nm; or 120 to 175 nm; or 120 to 170 nm; or 120 to 165 nm; or 120 to 150 nm; or 120 to 155 nm; or 120 to 150 nm; or 120 to 145 nm; or 120 to 100 nm nm; or 120 to 135 nm; or 120 to 130 nm.

[0299] 81. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 50 and 500 nm.

[0300] 82. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 100 and 200 nm.

[0301] 83. The composition of any one of the foregoing embodiments, wherein the average particle size of the extracellular vesicles in the composition is between 100 and 200 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

[0302] 84. The composition as described in any of the preceding embodiments, wherein the extracellular vesicles in the composition have an average particle size of about 150 nm.

[0303] 85. The composition of any one of the preceding embodiments, wherein the extracellular vesicles in the composition have an average particle size of about 150 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

[0304] 86. The composition as described in any of the foregoing embodiments, wherein the extracellular vesicles in the composition are milk extracellular vesicles.

[0305] 87. The composition as described in any of the foregoing embodiments, wherein the vesicles in the composition are bovine milk extracellular vesicles.

[0306] 88. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles in the composition are bovine milk extracellular vesicles; and wherein the bovine milk extracellular vesicles comprise bovine IgG.

[0307] 89. The composition as described in any of the foregoing embodiments, wherein the extracellular vesicles in the composition are milk extracellular vesicles.

[0308] 90. The composition as described in any of the preceding embodiments, wherein the extracellular vesicles in the composition are milk extracellular vesicles, and wherein the extracellular vesicles are isolated.

[0309] 91. The composition as described in any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles, and wherein the extracellular vesicles are isolated from milk using the method of any of the “Isolation of extracellular vesicles from milk” embodiments.

[0310] 92. The composition of any one of the foregoing embodiments, wherein the extracellular vesicles comprise one or more carrier molecules.

[0311] 93. The composition as described in any of the foregoing embodiments, wherein the composition is a pharmaceutically acceptable composition.

[0312] 94. The composition as described in any of the preceding embodiments, wherein the composition further comprises about 50 mM of trehalose or a trehalose analogue.

[0313] 95. The composition of any one of the foregoing embodiments, wherein the trehalose or trehalose analogue comprises one or more of the following: trehalose, sucrose, lactose, glucose, disaccharide molecules.

[0314] 96. The composition as described in any of the preceding embodiments, wherein the composition is then frozen and lyophilized or dried.

[0315] 97. A method for freeze-drying bovine milk extracellular vesicles supplemented with trehalose and tryptophan.

[0316] 98. A composition of freeze-dried extracellular milk vesicles, wherein the extracellular milk vesicles are loaded with a carrier molecule and supplemented with trehalose or a trehalose analogue in addition to tryptophan or a tryptophan analogue.

[0317] 99. A method for contacting extracellular vesicles with trehalose and tryptophan, wherein the extracellular vesicles are dried and / or freeze-dried.

[0318] 100. A method for contacting extracellular vesicles with trehalose and tryptophan, wherein the extracellular vesicles are frozen and lyophilized.

[0319] 101. A composition of extracellular vesicles, said extracellular vesicles being loaded with the carrier molecules listed herein, and supplemented with trehalose or trehalose analogues in addition to tryptophan or tryptophan analogues.

[0320] Example The present disclosure will be further described in the following embodiments, which do not limit the scope of the present disclosure.

[0321] Example 1 : Isolation of extracellular vesicles from milk using tryptophan.

[0322] Raw milk was collected at 4°C and defatted under industrial standard conditions (centrifuged at 5,000 xg to remove fat) to obtain skim milk. Half of the skim milk was treated with an FDA-approved pasteurization method. Two batches of skim milk were then subjected to 2.5 µm and 0.8 µm filtration, followed by treatment of the permeate with 150 µM tryptophan on ice for 10 min, while discarding the retentate. The solution was then filtered through 0.45 µm and 0.22 µm filters, discarding the retentate after each filtration. The solution was then filtered through a 500 kDa filter until the retentate volume was 20% of the initial volume, and the solution was stored. The sample was then separated using a 70 nm agarose column. Fractions 7–9 were collected and combined, representing a purified population of extracellular vesicles from milk cells.

[0323] The solution was then used for in-depth analysis and experiments, including numerous standard and unique assays. For example, proteins were analyzed using Nanodrop for protein quantification and Western blotting for protein biomarkers. Single-molecule localization microscopy was also used to analyze proteins for protein biomarkers and subpopulation analysis, but this method was also used to gain insights into size and concentration; the SMLM method was validated using nanoparticle tracking analysis for size and concentration studies. We also utilized Zetasizer measurements of zeta potential, transmission electron microscopy for visualization, confocal microscopy for studying vesicle patency, and in vitro bioactivity measurements of vesicles using an automated electrical damage protocol (ECIS).

[0324] ECIS stands for "Cell-Substrate Impedance Sensing"—the protocol provided is as follows: Real-time measurement of cell monolayer confluence to monitor their migration responses using a Cell-Substrate Impedance Sensing (ECIS) instrument (Applied Biophysics, NY, USA). In short, an 8W1E or 96W1E+ electrode ECIS array is coated with 10 mM L-cysteine ​​and then coated with 1% gelatin according to the manufacturer's guidelines. The electrodes are then stabilized in HuDF complete medium, followed by 1×10⁻⁶... 5 HuDF cells were seeded at a density of 10 cells / well. Cells were incubated for 8–10 hours to achieve at least 80% confluence, as confirmed by previous growth phase profiles. Eight hours prior to wound induction, the medium was replaced with complete medium supplemented with 10 µg / mL mitomycin C to limit cell proliferation. Cells were then subjected to electrical trauma at 25 kHz, 2600 µA, for 20 seconds, and impedance was monitored until cell migration was complete. Cell migration rate was determined using a custom Python script that tracked impedance changes over time. Impedance of the cell-covered electrode was measured in time-varying multi-frequency (MFT) mode at eleven frequencies between 62.5 and 64000 Hz (62.5, 125, 250, 500, 1000, 2000, 4000, 8000, 16000, 32000, 64000). Cell monolayers were treated with optimized concentrations of mEV determined via UV-Vis intensity at 260 / 280 nm. Each result was normalized to the control wells present in each electrode array.

[0325] Nanodrop analysis was performed using a Nanodrop 2000 at 260 / 280 nm wavelength stimulation, following the manufacturer's instructions, to quantify protein expression in the samples. A basic Western blot protocol was followed using antibodies including CD81, CD9, cadherin, intercalin-1, TSG-101, panconnexin-1, connexin-43, lactobacin, and casein. Single-molecule localization microscopy was performed using the EVprofiler kit, following the procedures outlined in the kit's instructions, with antibodies including CD81, CD9, CD63, panconnexin-1, P2X7, and connexin-43. Nanoparticle tracking analysis was performed on a Nanosight NS300 using a 405 nm laser at a 1:10,000 dilution in standard buffer. Zetasizer measurements were performed on a Horiba Scientific SZ-100V2 nanoparticle analyzer, following the manufacturer's instructions. Transmission electron microscopy was performed using a copper grid coated with 0.1% lysine-coated polymethyl methacrylate (formvar). Samples were adhered to the grid for 10 minutes, counterstained with uranlyess solution for 1 minute, and then left overnight before imaging. Milk vesicles loaded with calcein AM dye were examined by confocal microscopy on a Leica SP8. Vesicles were applied to a standard microscope slide before coverslipping and imaged at 63x under 488 nm laser stimulation. In vitro bioactivity measurements were performed using a cell-substrate impedance sensing (ECIS) instrument from Applied Biophysics.

[0326] Example 2: Isolation of extracellular vesicles from milk using tryptophan and EDTA.

[0327] Raw milk was collected at 4°C and defatted under industrial standard conditions (centrifuged at 5,000 xg to remove fat) to obtain skim milk. The skim milk was then subjected to a 1.2 µm filtration, followed by treatment of the permeate with 30 mM EDTA and 150 µM tryptophan at room temperature (ambient temperature, 20°C) for 30 min, discarding the residue. The solution was then filtered through 0.45 µm and 0.22 µm filters, discarding the residue after each filtration. The solution was then filtered through a 500 kDa filter until the residue volume was 20% of the initial volume, and the solution was stored. The sample was then separated using a 70 nm agarose column. Fractions 7-9 were collected and combined, representing a purified population of extracellular vesicles from milk cells. Optionally, the sample was then buffered with 50 mM trehalose and lyophilized as described in Example 7. The solution was then used for depth analysis and experiments as described in Example 1.

[0328] Example 3: Isolation of extracellular vesicles from milk using tryptophan and acidic pH Raw milk was collected at 4°C and defatted under industrial standard conditions to obtain skim milk as described in Example 1. The skim milk was then subjected to a 1.2 µm filtration, followed by treatment of the permeate with 150 µM tryptophan at room temperature (ambient temperature, 20°C) for 60 minutes, while discarding the residue. The permeate was then acidified to pH 4.6 by adding HCl or citric acid, and removed as a precipitate by centrifugation at 5,000 x g, or alternatively, by raising the temperature to 45–50°C for 5–15 minutes to form a hard curd precipitate, which could then be filtered from the solution. The supernatant solution after precipitation was then filtered through 0.45 and 0.22 filters at 5,000 x g, discarding the residue after each filtration. The solution was then filtered through a 500 kDa filter until the residue volume was 20% of the initial volume, and the solution was stored. The sample was then separated using a 70 nm agarose column. Fractions 7–9 were collected and combined, representing the purified extracellular vesicle population of milk cells. Optionally, the sample was then buffered with 50 mM trehalose and lyophilized as described in Example 7. The solution was then used for further analysis and experiments as described in Example 1.

[0329] Example 4: Isolation of extracellular vesicles from milk using tryptophan, acidic pH, and temperature Raw milk was collected at 4°C and defatted under industrial standard conditions to obtain skim milk as described in Examples 1 and 3. The skim milk was then treated with 150 µM tryptophan and acidified by the addition of citric acid, sulfuric acid, or hydrochloric acid, and treated at 55°C for 30 minutes to form a hard curd precipitate, which was then filtered from the solution. The supernatant solution after curd formation was then filtered through 0.45 and 0.22 μm filters, discarding the residue after each filtration and collecting the permeate. The permeate solution was then filtered through a 500 kDa filter until the residue volume was approximately 20% of the initial volume, and the solution was stored. The sample was then separated on a 70 nm agarose column. Fractions 7-9 were collected and combined, representing a purified population of extracellular vesicles from milk cells. Optionally, the sample was then buffered with 50 mM trehalose and 100 µM tryptophan and lyophilized as described in Example 7. The solution was then used for further analysis and experiments as described in Example 1.

[0330] Example 5: ATP-milk extracellular vesicles.

[0331] Milk vesicles, extracellular vesicles, or exogenous bodies (isolated by U.S. patent application?) collected through the embodiments described herein or as previously described were incubated together with adenosine triphosphate (ATP) at 37°C for 30 minutes at concentrations including 100 nm, 500 nm, 1.0 µM, 2.0 µM, 5.0 µM, 10 µM, and 50 µM. The optimal ATP concentrations for enhancing bioactivity were found to be 2.0 µM and 1.0 µM, with the final ATP concentration range in the milk vesicle composition from 100 nM to 10 µM.

[0332] Example 6: Evaluation of ATP-extracellular vesicles.

[0333] The ATP-extracellular vesicle assemblage was then analyzed using multiple methods. Bioactivity was first measured using an in vitro analytical procedure. The cells used were two lineages of human dermal fibroblasts (HuDF; ATCC, PCS-201-012) and Madin Darby canine kidney (MDCK) cells, one Cx43-enhanced (Cx43+) and the other Cx43-decreased (Cx43-). The medium used for HuDF was DMEM HG (4.5 g / L glucose) containing 2% normal fetal bovine serum (NCS; Thermo Fisher / Gibco, 16010-159) and 4% fetal bovine serum (FBS; Thermo Fisher / Gibco, 26140-079). The culture medium used for Cx43+MDCK cells was M199 (MilliporeSigma M4530) supplemented with 10% FBS and 1% hygromycin B (Sigma H0654), while Cx43-MDCK used M199 with 10% FBS and 1:125 sodium pyruvate (Invitrogen 11360-070). Cells were expanded to confluence and then passaged onto sterile coverslips in 12-well plates, and given 2 days for adhesion and growth before assay. Assay was performed by scraping the cell surface using a sterile pipette tip, followed by a single wash in dPBS (Invitrogen) and fresh medium supplemented with CTDR-labeled milk EVs. Cells were given 15 minutes to absorb the ATP-vesicle combination, then washed in 1x dPBS and fixed in 2% paraformaldehyde (Fisher O4042-500). Cells were washed four times in PBS, stained with 1:20,000 Hoechst (Life Technologies, H3569), and washed once more in PBS. The coverslips were then removed, and the cells were adhered to a microscope slide and imaged on a Leica SP8. Images were analyzed in ImageJ by saving a single red channel, converting to 8-bit, thresholding, and counting the number of particles.

[0334] The presence of ATP within EVs can be verified by mass spectrometry analysis of ATP concentration in extracellular vesicles. Samples can be prepared using the trichloroacetic acid / acetone step of the Bio-Rad 2-D Purification Kit. After protein precipitation, the sample is redissolved in 10 μL 8 M urea and 50 mM NH4HCO3, reduced and alkylated with dithiothreitol and iodoacetamide, and then digested with endo-Lys-C at room temperature for three hours. The sample is then diluted to 2 M urea with 50 mM NH4HCO3 and digested overnight with the addition of sequencing-grade modified trypsin from Promega. The sample is then acidified with 2% formic acid, desalted on a pre-equilibrated column, and washed using the EASY nLC-1200 system. The column is kept at a constant temperature and connected online. Peptides can be eluted from the column using a binary gradient from 5% to 95% solvent over 60 minutes. MS data are obtained by recording full-scan spectra.

[0335] The extracellular vesicle concentration of ATP can be readily determined in a variety of ways, and a wide range of ATP concentration assays exist. The following example provides a basic mechanism for the detection of ATP in solution. The first step involves administering firefly luciferase and a substrate to activate luciferin with the present ATP, resulting in luciferinyl adenosine and pyrophosphate. The following chemical reaction leads to photoelectrostimulation of oxidized luciferin, which emits a green / yellow light signal (550-570 nm). The luminescence in the solution is then read using a photometer, and the method is sensitive to ATP concentrations <50 pg / mL. Other methods utilize fluorescence capture, which allows for longer signal capture periods and is another feasible method for detecting and measuring ATP concentration.

[0336] Example 7: Freeze-drying of milk extracellular vesicles.

[0337] Extracellular vesicles or exogenous bodies of milk cells are collected as described herein or as previously described (e.g., in WO2022182782) and incubated in a variety of chemical excipients. These include sucrose, lactose, trehalose, TMAO, and tryptophan, and mixtures thereof, at concentrations of 10 mM, 50 mM, and 200 mM prior to freezing to produce mixtures with glass transition temperatures between 40 and 80 °C. The samples are frozen at -80 °C and subjected to shelf-drying at a condenser temperature of -100 °C and a vacuum pressure of 10 mTorr. Alternatively, methods for lyophilizing the vesicle compositions include a) freezing the composition by cooling it to the freezing temperature at a controlled rate of 1 °C / min, or; or rapidly freezing the composition using liquid nitrogen, dry ice, or other rapid freezing methods. b) initially drying the composition by adjusting the temperature and pressure according to the specific container and composition of the composition, such adjustment being within the scope of knowledge of those skilled in the art. C) The composition was further dried by adjusting the temperature and vacuum conditions after sublimation to reduce the moisture content of the composition, and the parameters of the secondary drying stage, including temperature and pressure ramps, were adjusted based on the specific properties of the container and the composition, such adjustments being readily apparent to those skilled in the art. D) The sample was vacuum-capped after lyophilization. The dried sample was stored at room temperature or 4°C under vacuum, or protected from moisture by a desiccant. The solution was then used for in-depth analysis and experiments, including a number of standard and unique assays. We first used Nanodrop analysis for protein quantification, followed by nanoparticle tracking analysis for size and concentration studies. We also utilized Zetasizer measurements of zeta potential, transmission electron microscopy for visualization, confocal microscopy for studying vesicle patency, and in vitro bioactivity measurements using an automated electrical damage protocol. An optimized formulation for lyophilizing extracellular vesicles was found to be 50 mM trehalose buffered with 100 µM tryptophan, enabling our formulation to maintain shelf stability for more than 6 months. LC-MS analysis of the sample will elucidate the presence of tryptophan, a novel excipient in the lyophilization of extracellular vesicles.

[0338] Example 8: Storage materials and methods for testing the storage stability of lyophilized extracellular vesicles of milk. Isolated extracellular vesicles or exogenous bodies loaded with act11 peptide were lyophilized using the previously described protocol in the presence of 50 mM trehalose or a trehalose analogue and 100 µM tryptophan or a tryptophan analogue and stored at room temperature in a sealed container with a desiccant. At a given time point, the sample was reconstituted to the original amount of mEV volume within the composition using an equal volume of deionized H2O. As previously described in Section 83, an equivalent dose of each sample was added to a single ECIS cell with a final volume of 400 µL after trauma. The samples were analyzed via NTA and compared at 0 and 6 months.

[0339] Although this disclosure has been specifically shown and described with reference to particular embodiments, some of which are preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of this disclosure.

[0340] As used herein, the terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may mean within an acceptable standard deviation. Alternatively, “about” may mean a range of at most ±20%, or at most ±10%, or at most ±5%, or at most ±1% for a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, such as within 2 times. To avoid ambiguity, values ​​described herein with the modifier “about” specifically include the stated value itself; for example, if a value is expressed as “about 10,” a value exactly 10 is specifically expected. As used herein, a range may be expressed as from “about” one particular value, or from “about” one value to “about” another particular value. It should also be understood that each unit between two particular units is also disclosed. For example, if a range of “10–15” is disclosed, 11, 12, 13, and 14 are also disclosed.

[0341] All references cited in this disclosure are incorporated herein by reference in their entirety. Various embodiments of this disclosure may be characterized by potential claims listed in paragraphs following this one (and before the actual claims provided at the end of this application). These potential claims form part of the written description of this application. Therefore, the subject matter of the following potential claims may be presented as actual claims in subsequent proceedings relating to this application or any application claiming priority based on this application. The inclusion of such potential claims should not be construed as meaning that the subject matter of the actual claims is not covered. Therefore, a decision not to present these potential claims in subsequent proceedings should not be construed as a donation of the subject matter to the public.

[0342] The embodiments disclosed above are intended to be exemplary only; many variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to fall within the scope of this disclosure as defined by any of the appended claims.

Claims

1. A composition comprising extracellular vesicles, wherein the extracellular vesicles contain 0.1 to 2 µM of ATP or an ATP analogue.

2. The composition of claim 1, wherein the extracellular vesicles contain 0.5 to 1.5 µM of ATP or an ATP analogue.

3. The composition of claim 2, wherein the extracellular vesicles contain between 0.5 and 1.5 µM of ATP.

4. The composition of claim 3, wherein the extracellular vesicles are milk extracellular vesicles.

5. The composition of claim 4, wherein the extracellular vesicles are bovine milk extracellular vesicles.

6. The composition of claim 5, wherein the average particle size of the extracellular vesicles is between 100 and 200 nm.

7. The composition of claim 6, wherein 99% of the extracellular vesicles have a particle size greater than 20 nm and less than 600 nm.

8. A method for preparing the composition of claim 7, wherein the method comprises contacting bovine milk extracellular vesicles with ATP.

9. The composition of claim 8, wherein the extracellular vesicles are loaded with one or more carrier molecules.

10. The composition of claim 9, wherein the carrier molecule comprises one or more molecular types selected from the group consisting of nucleic acids, polypeptides, carbohydrates and steroids.

11. A method comprising administering the composition of claim 10 to a subject.

12. The method of claim 11, wherein the composition is administered orally, intranasally, parenterally, or intravenously to the subject.

13. A method for isolating extracellular vesicles from a milk sample, wherein the method comprises (i) contacting the milk with tryptophan or a tryptophan analogue at a concentration between 50 µM and 300 µM at a pH between 2.0 and 8.0 and a temperature between 10°C and 80°C for 10 to 60 minutes, and (ii) separating the extracellular vesicles from other components of the milk by filtration and / or centrifugation.

14. The method of claim 13, wherein the milk used in step (i) is defatted.

15. The method of claim 14, wherein the milk used in step (i) is raw or pasteurized.

16. The method of claim 15, wherein the method further comprises subjecting the extracellular vesicles obtained from step (ii) to filtration to remove extracellular vesicles with a particle size less than 20 nm and separate filtration to remove vesicles with a particle size greater than 600 nm.

17. A composition comprising extracellular vesicles of breast cells obtained from the method of claim 16.

18. The composition of claim 17, wherein the composition is subjected to rapid freezing or controlled freeze-drying, followed by primary drying, secondary drying and vacuum sealing of the sample.

19. A composition comprising lyophilized extracellular vesicles of milk cells obtained from the composition of claim 18, supplemented with 100 µM tryptophan and 50 mM trehalose.