IL324481A - Extracellular vesicle compositions and preparation - Google Patents

Description

WO 2024/233809 PCT/US2024/028635 EXTRACELLULAR VESICLE COMPOSITIONS AND PREPARATION CROSS-REFERENCE TO RELATED APPLICATIONS [0001]The present application claims the benefit of priority to United States Provisional Application Serial No. 63/465,073, filed on May 9, 2023, and to United States Provisional Application Serial No.63/624,637, filed on January 24, 2024, and to United States Provisional Application Serial No. 63/640,825, filed on April 30, 2024, all of which are incorporated by reference in their entirety.

FIELD OF THE DISLOSURE [0002]The present disclosure relates generally to extracellular vesicles (for example milk-derived vesicles, or exosomes) as well as methods of making, modifying and using such vesicles. id="p-3"

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. HL161237, HL141855, and HL056728 awarded by the National Institutes of Health and STTR #2203330 by the NSF. The government has certain rights in the invention.

BACKGROUND id="p-4"

[0004]PCT Application Publication Number WO2022182782 discloses "methods of isolating exosomes from a biological fluid, such as those containing caseins." [0005]PCT Application Publication Number WO2020010161 discloses "milk vesicles as drug delivery vehicles, compositions comprising a therapeutic agent encapsulated within or otherwise associated with the milk vesicles, methods of producing such milk vesicles and compositions thereof, as well as methods of delivering such milk vesicles and compositions to a specific patient tissue or organ." SUMMARY id="p-6"

[0006]The instant 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.

WO 2024/233809 PCT/US2024/028635 id="p-7"

[0007]The terms "extracellular vesicle" or "EV" or "vesicle" as used herein are used interchangeably and refer to a small, lipid-bilayer membrane bound structure that exists outside of a cell. In many embodiments, extracellular vesicles are released from cells into the extracellular space. Extracellular vesicles have a lipid bilayer and are generally about to 1,000 nm in diameter. In addition to the lipid bilayer and internal aqueous lumen, extracellular vesicles have additional components such as tetraspanin proteins and IgG molecules; accordingly, detection of these components in isolated extracellular vesicles can be used to distinguish other (such as synthetic) particles having lipid bilayers. Extracellular vesicles often contain natural bioactive agents in their interior and/or in the outer bilayer membrane. Further, as discussed in more detail below, extracellular vesicles can be loaded with "cargo" such as therapeutic molecules and the cargo-loaded vesicles can enhance delivery of the cargo when administered to a subject; e.g., cargo-loaded vesicles in certain conditions can be used to effectively orally administer molecules that are otherwise not bioavailable or poorly bioavailable when administered orally. [0008]Milk has been found to be a rich source of extracellular vesicles, and recent studies have shown that isolated extracellular vesicle preparations can be made from milk can be loaded with "cargo" (such as therapeutic molecules) and can be used to successfully administer the cargo to subjects (see for example WO2022182782). [0009]Certain aspects and embodiments of the instant disclosure are based on discoveries that isolated extracellular vesicles that are modified to have ATP and/or one or more ATP analogues can increase the cellular uptake of the modified extracellular vesicles when administered to a subject, thereby increasing the effectiveness of administration of a cargo if present in the vesicles. The term ATP means adenosine triphosphate. As used herein, the term "ATP-analogue-vesicle" refers to an extracellular vesicle that has been modified to include ATP and/or one or more ATP analogues. In certain embodiments, the ATP-analogue-vesicles of the disclosure may be particularly useful for administration of a cargo via a route other than oral administration, such as parenteral administration, intranasal administration, IV administration, injection, subcutaneous injection or topical administration. [0010]Accordingly, in one aspect provided is a composition comprising isolated ATP- analogue vesicles wherein said vesicles comprise between 0.01 and 1,000 pM ATP or an ATP analogue. In a similar aspect, provided is a composition comprising isolated milk ATP-analogue vesicles wherein said vesicles comprise between 0.01 and 1,000 pM ATP WO 2024/233809 PCT/US2024/028635 or an ATP analogue. In yet another aspect a composition is provided wherein the composition includes isolated bovine milk ATP-analogue vesicles, wherein said vesicles comprise between 0.01 and 1,000 pM ATP or an ATP analogue. In some embodiments the ATP-analogue vesicles include ATP or an ATP analogue in a concentration between 0.01 and 1,000 pM; or 0.1 and 100 pM; or 0.25 and 175 pM; 0.5 and 50 pM; or 0.75 and pM; or 0.8 and 20 pM; or 0.85 and 15 pM; or 0.9 and 10 pM; or 0.1 and 5 pM; or 0.and 5 pM; or 0.1 and 2 pM; or 0.5 and 1.5 pM; or 0.75 and 1.25 pM. In some embodiments the ATP-analogue vesicles include ATP or an ATP analogue in a concentration of about 0.1 pM; 0.25 pM; 0.5 pM; 0.75 pM; 0.9 pM; 1 pM; 1.1 pM; 1.25 pM; 1.5 pM; 2 pM; pM; 10 pM; 50 pM; or 100 pM. Herein when an agent (such as ATP) in vesicles is described in terms of molarity (for example as pM), the vesicles are exposed to a solution of the agent at the indicated molarity until an equilibrium is achieved (for example using methods described herein) such that the molarity within each vesicle is congruent with the molar concentration of the solution. [0011]In another aspect provided is a composition comprising isolated ATP-analogue vesicles wherein said vesicles comprise between l ng and 1,000 pg ATP or an ATP analogue per mg of vesicle protein. In a similar aspect, provided is a composition comprising isolated milk ATP-analogue vesicles wherein said vesicles comprise between I ng and 1,000 pg ATP or an ATP analogue per mg of milk vesicle protein. In yet another aspect a composition is provided wherein the composition includes isolated bovine milk ATP-analogue vesicles, wherein said vesicles comprise between l ng and 1,000 pg ATP or an ATP analogue per mg of bovine milk ATP-analogue vesicles. In some embodiments the ATP-analogue vesicles include ATP or an ATP analogue in a concentration between ng and 1,000 pg per mg vesicle protein; or 10 ng and 100 pg per mg vesicle protein; or 1ng and 10 pg per mg vesicle protein; or 200 ng and 1 pg per mg vesicle protein; or 300 ng and 750 ng per mg vesicle protein; or 400 ng and 650 ng per mg vesicle protein; or 450 ng and 600 ng per mg vesicle protein; or 500 ng and 550 ng per mg vesicle protein. In some embodiments, the ATP-analogue vesicles include ATP or an ATP analogue in 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; 4ng 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; WO 2024/233809 PCT/US2024/028635 750 ng per mg vesicle protein; 1 pg per mg vesicle protein; 10 pg per mg vesicle protein; 100 pg per mg vesicle protein; or 1,000 pg per mg vesicle protein. [0012]In a related aspect, a method is provided that includes obtaining a composition that comprises isolated vesicles (such as, for example any isolated vesicle composition as described herein), contacting the vesicles with a solution comprising ATP and/or an ATP analogue to obtain a composition comprising ATP-analogue-vesicles. As used herein, the term "isolated" or the like refers to an agent (for example a vesicle), which has been separated from its original environment, i.e., the environment of the isolated agent (for example a vesicle) is substantially free of at least one component as found in the environment in which the "un-isolated" reference agents (for example a vesicles) exist. The term includes an agent (for example a vesicle) that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue, biopsy body fluid (such as milk) sample. The term also includes an agent (for example a vesicle) that is removed from at least one, some or all components as the agent (for example a vesicle) is found in non-naturally occurring environments, for example, isolated from a cell culture or cell suspension. Therefore, an isolated agent (for example a vesicle) is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. In some embodiments, an isolated vesicle of the disclosure is an isolated extracellular vesicle that has been isolated from milk, for example a vesicle that has 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, for example the liquid may be one or more solvents such as water, vegetable oils, terpenes, aldehydes, ketones, diacetone alcohol, ethylene glycol ethers, methanol, ethanol, isopropanol. In certain embodiments the liquid is an aqueous liquid. In certain embodiments the liquid is a physiological solution having electrolytes and or buffering agents. In certain embodiments the liquid may also include one or more solutes including sodium chloride, potassium chloride and other salts, magnesium, calcium, disaccharides, amino acids, nucleic acids, and further solute solutions known to those skilled in the art. [0014]In certain aspects and embodiments, the extracellular vesicle composition is provided in a dried, or lyophilized state. The term "dried" and "lyophilized" as used herein WO 2024/233809 PCT/US2024/028635 means a product such as a cake or powder from which water has been removed through freeze drying or sublimation, in which the final product has a lower water concentration than the original liquid starting material. In certain embodiments, other forms of drying or "lyophilizing" as stated herein may include spray drying, drug drying, crystallization, extrusion and other forms of drying known to those skilled 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 aboutl0%; or less than about 9%; or less than about 8%; or less than about 7%; or less than about 6%; or less than about 5%; or less than about 4%; or less than about 3%; or less than about 2%; or less than about 1%; or less than about 0.5%; or less than about 0.1% moisture by weight. [0016]The term "analogue" as used herein means a chemical compound that is similar to another chemical compound in structure and/or function, but differs structurally from that compound. Such difference may be by 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 function as the parental chemical. Such modifications are routine to persons skilled in the art, and include, for example, additional or substituted chemical moieties. In some embodiments, an analogue as used herein may have a chemical structure that is different, or very different, from the parental compound but have similarities in a relevant function or biological activity. In some embodiments, an analogue as used herein may have a chemical structure that is different, or very different, from the parental compound but have at least one relevant function or biological activity increased, decreased, or deleted. [0017]As used herein, the term "ATP analogue" refers to an analogue of ATP. In certain embodiments, an ATP analogue of the disclosure is a non-hydrolyzable analogue of ATP. In some embodiments an ATP analogue of the disclosure is one or more selected from the group consisting of a,P־methylene־ATP (a,PmATP); P,y-methylene-ATP (P,ymATP); 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); anN-alkyl-2 ATP; adenosine 5'-(P,y-imido)triphosphate (AMP-PNP); ATP-MgC12; 5-aminoimidazole-4- carboxamide ribonucleotide, disoproxil fumarate, ribavirin, azidothymidine, fludarabine, efavirenz and oxidized ATP (0ATP), Adenosine, ADP, AMP, and other nucleotides (e.g. GTP, CTP, UTP, TTP). [0018]As used herein, the term "Tryptophan analogue" refers to tryptophan itself or an analogue of tryptophan. In certain embodiments, a tryptophan analogue of the disclosure WO 2024/233809 PCT/US2024/028635 is Tryptophan. In some embodiments a tryptophan analogue of the disclosure is one or more selected from the group consisting of L-Tryptophan, D-Tryptophan, Tryptophan dimers, Tryptophan trimers, Tryptophan peptides, Histidine, L-Histidine, D-Histidine, Histidine dimers, Histidine trimers, Histidine peptides, Tyrosine, L-Tyrosine, D-Tyrosine, Tyrosine dimers, Tyrosine trimers, Tyrosine peptides, Arginine, L-Arginine, D-Arginine, Arginine dimers, Arginine trimers, Arginine peptides, Cysteine, L-Cysteine, D-Cysteine, Cysteine dimers, Cysteine trimers, Cysteine peptides, Lysine, L-Lysine, D-Lysine, Lysine dimers, Lysine trimers, Lysine Peptides, Phenylalanine, L- Phenylalanine, D- Phenylalanine, Phenylalanine dimers, Phenylalanine trimers, Phenylalanine peptides, D- Carnitine, L-Carnitine, Carnitine dimers, Carnitine trimers, Carnitine peptides, Acetyl L- Carnitine, L-Carnitine L-tartrate, Propionyl-L-carnitine, D-Taurine, L-Taurine, Taurine dimers, Taurine trimers, Taurine peptides. [0019]Other aspects and embodiments of the disclosure are based on discoveries that certain molecules such as tryptophan and/or tryptophan analogues can be used to disrupt associations of extracellular vesicles with casein and other milk protein molecules that can aggregate and complicate the isolation of extracellular vesicles from milk; and associated methods of isolating extracellular vesicles from milk wherein the methods involve contacting extracellular vesicle containing milk with tryptophan and/or tryptophan analogues. [0020]Accordingly, in one aspect provided is 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 separating extracellular vesicles from one or more milk components (such as for example casein-containing aggregates). In some embodiments the method includes contacting (incubating) the vesicle-containing sample with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM. In some embodiments the method includes contacting (incubating) the vesicle-containing sample with tryptophan and/or or more tryptophan analogues at a temperature between 4 and 50 degrees Celsius for at least minutes. In some embodiments the method includes contacting (incubating) the vesicle containing sample with tryptophan and/or 1 or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 50 degrees Celsius for at least 10 minutes. In some embodiments the method includes contacting (incubating) the vesicle containing sample with tryptophan and/or 1 or more tryptophan WO 2024/233809 PCT/US2024/028635 analogues at a concentration between 10 gM and 1 mM at a temperature between 10 and degrees Celsius for between 10 and 60 minutes. [0021]In various embodiments an extracellular vesicle of the compositions and methods of the disclosure has a diameter between (or in the case of a composition that includes a plurality of vesicles, the average (mean) diameter of the particles is between), 1 and 2,0nm; or 10 and 1,500 nm; or 20 and 1,000 nm; or 1 and 1,000 nm; or 1 and 500 nm; or 1 and 300 nm; or 1 and 200 nm; or 1 and 185 nm, or 1 and 175 nm; or 1 and 170 nm; or 1 and 165 nm; or 1 and 160 nm; or 1 and 155 nm; or 1 and 150 nm; or 1 and 145 nm; or 1 and 140 nm; or 1 and 135 nm; or 1 and 130 nm; or 10 and 1,000 nm; or 10 and 500 nm; or and 300 nm; or 10 and 200 nm; or 10 and 185 nm, or 10 and 175 nm; or 10 and 170 nm; or and 165 nm; or 10 and 160 nm; or 10 and 155 nm; or 10 and 150 nm; or 10 and 145 nm; or 10 and 140 nm; or 10 and 135 nm; or 10 and 130 nm; or 15 and 1,000 nm; or 15 and 5nm; or 15 and 300 nm; or 15 and 200 nm; or 15 and 185 nm, or 15 and 175 nm; or 15 and 170 nm; or 15 and 165 nm; or 15 and 160 nm; or 15 and 155 nm; or 15 and 150 nm; or and 145 nm; or 15 and 140 nm; or 15 and 135 nm; or 15 and 130 nm; or 20 and 1,000 nm; or 20 and 500 nm; or 20 and 300 nm; or 20 and 200 nm; or 20 and 185 nm, or 20 and 1nm; or 20 and 170 nm; or 20 and 165 nm; or 20 and 160 nm; or 20 and 155 nm; or 20 and 150 nm; or 20 and 145 nm; or 20 and 140 nm; or 20 and 135 nm; or 20 and 130 nm; or and 1,000 nm; or 25 and 500 nm; or 25 and 300 nm; or 25 and 200 nm; or 25 and 185 nm, or 25 and 175 nm; or 25 and 170 nm; or 25 and 165 nm; or 25 and 160 nm; or 25 and 1nm; or 25 and 150 nm; or 25 and 145 nm; or 25 and 140 nm; or 25 and 135 nm; or 25 and 130 nm; or 30 and 1,000 nm; or 30 and 500 nm; or 30 and 300 nm; or 30 and 200 nm; or and 185 nm, or 30 and 175 nm; or 30 and 170 nm; or 30 and 165 nm; or 30 and 160 nm; or 30 and 155 nm; or 30 and 150 nm; or 30 and 145 nm; or 30 and 140 nm; or 30 and 1nm; or 30 and 130 nm; or 35 and 1,000 nm; or 35 and 500 nm; or 35 and 300 nm; or 35 and 200 nm; or 35 and 185 nm, or 35 and 175 nm; or 35 and 170 nm; or 35 and 165 nm; or and 160 nm; or 35 and 155 nm; or 35 and 150 nm; or 35 and 145 nm; or 35 and 140 nm; or and 135 nm; or 35 and 130 nm; or 40 and 1,000 nm; or 40 and 500 nm; or 40 and 3nm; or 40 and 200 nm; or 40 and 185 nm, or 40 and 175 nm; or 40 and 170 nm; or 40 and165 nm; or 40 and 160 nm; or 40 and 155 nm; or 40 and 150 nm; or 40 and 145 nm; or 40and 140 nm; or 40 and 135 nm; or 40 and 130 nm; or 45 and 1,000 nm; or 45 and 500 nm; or 45 and 300 nm; or 45 and 200 nm; or 45 and 185 nm, or 45 and 175 nm; or 45 and 170nm; or 45 and 165 nm; or 45 and 160 nm; or 45 and 155 nm; or 45 and 150 nm; or 45 and145 nm; or 45 and 140 nm; or 45 and 135 nm; or 45 and 130 nm; or 50 and 1,000 nm; or WO 2024/233809 PCT/US2024/028635 50 and 500 nm; or 50 and 300 nm; or 50 and 200 nm; or 50 and 185 nm, or 50 and 175 nm; or 50 and 170 nm; or 50 and 165 nm; or 50 and 160 nm; or 50 and 155 nm; or 50 and 1nm; or 50 and 145 nm; or 50 and 140 nm; or 50 and 135 nm; or 50 and 130 nm; or 55 and 1,000 nm; or 55 and 500 nm; or 55 and 300 nm; or 55 and 200 nm; or 55 and 185 nm, or and 175 nm; or 55 and 170 nm; or 55 and 165 nm; or 55 and 160 nm; or 55 and 155 nm; or 55 and 150 nm; or 55 and 145 nm; or 55 and 140 nm; or 55 and 135 nm; or 55 and 1nm; or 60 and 1,000 nm; or 60 and 500 nm; or 60 and 300 nm; or 60 and 200 nm; or 60 and 185 nm, or 60 and 175 nm; or 60 and 170 nm; or 60 and 165 nm; or 60 and 160 nm; or and 155 nm; or 60 and 150 nm; or 60 and 145 nm; or 60 and 140 nm; or 60 and 135 nm; or and 130 nm; or 65 and 1,000 nm; or 65 and 500 nm; or 65 and 300 nm; or 65 and 2nm; or 65 and 185 nm, or 65 and 175 nm; or 65 and 170 nm; or 65 and 165 nm; or 65 and 160 nm; or 65 and 155 nm; or 65 and 150 nm; or 65 and 145 nm; or 65 and 140 nm; or and 135 nm; or 65 and 130 nm; or 70 and 1,000 nm; or 70 and 500 nm; or 70 and 300 nm; or 70 and 200 nm; or 70 and 185 nm, or 70 and 175 nm; or 70 and 170 nm; or 70 and 1nm; or 70 and 160 nm; or 70 and 155 nm; or 70 and 150 nm; or 70 and 145 nm; or 70 and 140 nm; or 70 and 135 nm; or 70 and 130 nm; or 75 and 1,000 nm; or 75 and 500 nm; or and 300 nm; or 75 and 200 nm; or 75 and 185 nm, or 75 and 175 nm; or 75 and 170 nm; or 75 and 165 nm; or 75 and 160 nm; or 75 and 155 nm; or 75 and 150 nm; or 75 and 1nm; or 75 and 140 nm; or 75 and 135 nm; or 75 and 130 nm; or 90 and 1,000 nm; or 90 and 500 nm; or 90 and 300 nm; or 90 and 200 nm; or 90 and 185 nm, or 90 and 175 nm; or and 170 nm; or 90 and 165 nm; or 90 and 160 nm; or 90 and 155 nm; or 90 and 150 nm; or and 145 nm; or 90 and 140 nm; or 90 and 135 nm; or 90 and 130 nm; or 95 and 1,0nm; or 95 and 500 nm; or 95 and 300 nm; or 95 and 200 nm; or 95 and 185 nm, or 95 and 175 nm; or 95 and 170 nm; or 95 and 165 nm; or 95 and 160 nm; or 95 and 155 nm; or and 150 nm; or 95 and 145 nm; or 95 and 140 nm; or 95 and 135 nm; or 95 and 130 nm; or 100 and 1,000 nm; or 100 and 500 nm; or 100 and 300 nm; or 100 and 200 nm; or 100 and 185 nm, or 100 and 175 nm; or 100 and 170 nm; or 100 and 165 nm; or 100 and 150 nm; or 100 and 155 nm; or 100 and 150 nm; or 100 and 145 nm; or 100 and 100 nm; or 100 and 135 nm; or 100 and 130 nm; or 105 and 1,000 nm; or 105 and 500 nm; or 105 and 300 nm; or 105 and 200 nm; or 105 and 185 nm, or 105 and 175 nm; or 105 and 170 nm; or 105 and 165 nm; or 105 and 160 nm; or 105 and 155 nm; or 105 and 150 nm; or 105 and 145 nm; or 105 and 140 nm; or 105 and 135 nm; or 105 and 130 nm; or 110 and 1,000 nm; or 1and 500 nm; or 110 and 300 nm; or 110 and 200 nm; or 110 and 185 nm, or 110 and 1nm; or 110 and 170 nm; or 110 and 165 nm; or 110 and 150 nm; or 110 and 155 nm; or WO 2024/233809 PCT/US2024/028635 110 and 150 nm; or 110 and 145 nm; or 110 and 100 nm; or 110 and 135 nm; or 110 and 130 nm; or 115 and 1,000 nm; or 115 and 500 nm; or 115 and 300 nm; or 115 and 200 nm; or 115 and 185 nm, or 115 and 175 nm; or 115 and 170 nm; or 115 and 165 nm; or 115 and 160 nm; or 115 and 155 nm; or 115 and 150 nm; or 115 and 145 nm; or 115 and 140 nm; or 115 and 135 nm; or 115 and 130 nm; or 120 and 1,000 nm; or 120 and 500 nm; or 1and 300 nm; or 120 and 200 nm; or 120 and 185 nm, or 120 and 175 nm; or 120 and 1nm; or 120 and 165 nm; or 120 and 150 nm; or 120 and 155 nm; or 120 and 150 nm; or 120 and 145 nm; or 120 and 100 nm; or 120 and 135 nm; or 120 and 130 nm. [0022]In some embodiments, it may be preferable to have all, or most of the extracellular vesicle particles in a composition to be less than about 600 nm, or in some embodiments, less than about 500 nm, in size. For example, extracellular vesicles used to deliver therapeutic cargo can, in some situations, attract an immune response in a subject when there are particle sizes over 500 or 600 nm. In certain embodiments, extracellular vesicles with particle sizes over about 600 nm, or over about 500 nm, or over about 250 nm can be removed using filtration, for example using a 0.2 pm (or 0.22 pm) filter or the like. Accordingly, in some embodiments at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the extracellular vesicles in an isolated vesicle composition of the disclosure have a particle size of less than about 600 nm; or less than about 500 nm; or less than about 2nm. [0023]In some embodiments, it may be preferable to have all, or most of the extracellular vesicle particles in a composition to be more than about 20 nm in size. For example, extracellular vesicles used to deliver therapeutic cargo can, in some situations, be less effective when there is a substantial amount of particles with a size less than about nm in size. In certain embodiments, extracellular vesicles with particle sizes below about nm can be removed using filtration, for example using a 500 kDa filter or the like. Accordingly, in some embodiments at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the vesicles in an isolated extracellular vesicle composition of the disclosure have a particle size greater than about 20 nm. [0024]In some embodiments, it may be preferable to have all, or most of the extracellular vesicle particles in a composition to be more than about 20 nm in size and also having vesicles to be less than about 600 nm (or in some embodiments, less than about 5nm) in size. Accordingly, in some embodiments at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the extracellular vesicles in an isolated vesicle composition of the disclosure have a particle size of less than about 600 nm; or less than about 500 nm; or less WO 2024/233809 PCT/US2024/028635 than about 250 nm; and at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the vesicles in an isolated vesicle composition of the disclosure have a particle size greater than about 20 nm. [0025]In some embodiments, extracellular vesicles of the disclosure (such as isolated milk extracellular vesicles; and/or ATP-analogue-vesicles) include one or more cargo molecules. As used herein the term "cargo" means a molecule (for example a biological molecule) that is added to the vesicles, for example a molecule that is intended to be administered to a subject using the vesicles as a delivery vehicle. In some embodiments a cargo molecule is a peptide, protein, nucleic acid, polysaccharide, a small molecule, or the like. In many embodiments, the cargo molecule is not bioavailable, or has poor bioavailability when administered by a particular route, but has increased or improved bioavailability when administered through a vesicle composition of the present disclosure. In some embodiments, a cargo molecule has a molecular weight that is greater than Daltons and less than 500,000 Daltons, between 2 and 1000 amino acids, or between picometer and 200 nanometers in size. In some embodiments the cargo is a molecule that is not naturally present in, or endogenous to, the vesicles in their natural source. In some embodiments, the cargo molecule may be endogenous to the milk vesicle in its natural source but is added exogenously, for example to increase the amount of the molecule in the vesicle. Non limiting exemplary molecules that may serve as cargo include an antibody, a hormone, a growth factor, an enzyme, a cytokine, a chemokine, a toxin, an antitoxin, a blood coagulation factor, an interfering RNA (iRNA), a micro RNA (miRNA), an antisense RNA, a messenger RNA (mRNA), a non-coding RNA, a single- stranded DNA (ssDNA), a double-stranded DNA (dsDNA), long non-coding RNA, and an iRNA (such as, for example, siRNA or shRNA). Any of the nucleotide molecules disclosed herein, or a fragment thereof, may comprise a naturally- occurring nucleotide sequence. Alternatively, the nucleotide molecules can be synthetic (non-naturally occurring).

DETAILED DESCRIPTION Vesicle Sizes, Types and Determination Thereof [0026]As used herein, the terms "size" and "diameter" are used interchangeably. In embodiments that involve a composition that includes a plurality of vesicles (such as milk vesicles, exosomes, milk exosomes, milk extracellular vesicles, etc.,), the average (mean) size or diameter of the vesicles in the composition may be any of the sizes indicated herein for a single vesicle (for example, in the case a size range is indicated, the average size of WO 2024/233809 PCT/US2024/028635 the particles fall inside the specified range. Vesicles of the disclosure can be of any size specified herein. In certain embodiments, and where specifically indicated, when size (diameter) is represented as a range and in the case of a composition including a plurality of vesicles, the average size of the particles falls within the stated range and a specific percentage of the particles 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) or dynamic light scattering (DLS), or microfluidic resistive pulse sensing. [0028]As used herein, the term "exosome" refers to a subset of extracellular vesicles that are generally characterized by having a specific size but can also be characterized by additional unique features, such as relatively increased amounts of protein markers such as CD81, CD9 and syntenin. Exosomes are characterized by being of a size under 2nanometers, containing tetraspanin proteins including CD81, CD9 and CD63, luminal proteins including syntenin and TSG-101, is comprised of a lipid bilayer membrane and stably transfer cargoes between cells. In some embodiments an exosome (such as a milk exosome) of the disclosure has a diameter between (or in the case of a composition that includes a plurality of vesicles, the average (mean) diameter of the particles is between) and 200 nm. In some embodiments an exosome (such as a milk exosome) of the disclosure has a diameter between (or in the case of a composition that includes a plurality of vesicles, the average (mean) diameter of the particles is between) 10 and 200 nm; or 10 and 185 nm, or 10 and 175 nm; or 10 and 170 nm; or 10 and 165 nm; or 10 and 160 nm; or 10 and 1nm; or 10 and 150 nm; or 10 and 145 nm; or 10 and 140 nm; or 10 and 135 nm; or 10 and 130 nm; or 15 and 200 nm; or 15 and 185 nm, or 15 and 175 nm; or 15 and 170 nm; or and 165 nm; or 15 and 160 nm; or 15 and 155 nm; or 15 and 150 nm; or 15 and 145 nm; or and 140 nm; or 15 and 135 nm; or 15 and 130 nm; or 20 and 200 nm; or 20 and 185 nm, or 20 and 175 nm; or 20 and 170 nm; or 20 and 165 nm; or 20 and 160 nm; or 20 and 1nm; or 20 and 150 nm; or 20 and 145 nm; or 20 and 140 nm; or 20 and 135 nm; or 20 and 130 nm; or 25 and 200 nm; or 25 and 185 nm, or 25 and 175 nm; or 25 and 170 nm; or and 165 nm; or 25 and 160 nm; or 25 and 155 nm; or 25 and 150 nm; or 25 and 145 nm; or and 140 nm; or 25 and 135 nm; or 25 and 130 nm; or 30 and 200 nm; or 30 and 185 nm, or 30 and 175 nm; or 30 and 170 nm; or 30 and 165 nm; or 30 and 160 nm; or 30 and 1nm; or 30 and 150 nm; or 30 and 145 nm; or 30 and 140 nm; or 30 and 135 nm; or 30 and 130 nm; or 35 and 200 nm; or 35 and 185 nm, or 35 and 175 nm; or 35 and 170 nm; or WO 2024/233809 PCT/US2024/028635 and 165 nm; or 35 and 160 nm; or 35 and 155 nm; or 35 and 150 nm; or 35 and 145 nm; or and 140 nm; or 35 and 135 nm; or 35 and 130 nm; or 40 and 200 nm; or 40 and 185 nm, or 40 and 175 nm; or 40 and 170 nm; or 40 and 165 nm; or 40 and 160 nm; or 40 and 1nm; or 40 and 150 nm; or 40 and 145 nm; or 40 and 140 nm; or 40 and 135 nm; or 40 and 130 nm; or 45 and 200 nm; or 45 and 185 nm, or 45 and 175 nm; or 45 and 170 nm; or and 165 nm; or 45 and 160 nm; or 45 and 155 nm; or 45 and 150 nm; or 45 and 145 nm; or and 140 nm; or 45 and 135 nm; or 45 and 130 nm; or 50 and 200 nm; or 50 and 185 nm, or 50 and 175 nm; or 50 and 170 nm; or 50 and 165 nm; or 50 and 160 nm; or 50 and 1nm; or 50 and 150 nm; or 50 and 145 nm; or 50 and 140 nm; or 50 and 135 nm; or 50 and 130 nm; or 55 and 200 nm; or 55 and 185 nm, or 55 and 175 nm; or 55 and 170 nm; or and 165 nm; or 55 and 160 nm; or 55 and 155 nm; or 55 and 150 nm; or 55 and 145 nm; or and 140 nm; or 55 and 135 nm; or 55 and 130 nm; or 60 and 200 nm; or 60 and 185 nm, or 60 and 175 nm; or 60 and 170 nm; or 60 and 165 nm; or 60 and 160 nm; or 60 and 1nm; or 60 and 150 nm; or 60 and 145 nm; or 60 and 140 nm; or 60 and 135 nm; or 60 and 130 nm; or 65 and 200 nm; or 65 and 185 nm, or 65 and 175 nm; or 65 and 170 nm; or and 165 nm; or 65 and 160 nm; or 65 and 155 nm; or 65 and 150 nm; or 65 and 145 nm; or and 140 nm; or 65 and 135 nm; or 65 and 130 nm; or 70 and 200 nm; or 70 and 185 nm, or 70 and 175 nm; or 70 and 170 nm; or 70 and 165 nm; or 70 and 160 nm; or 70 and 1nm; or 70 and 150 nm; or 70 and 145 nm; or 70 and 140 nm; or 70 and 135 nm; or 70 and 130 nm; or 75 and 200 nm; or 75 and 185 nm, or 75 and 175 nm; or 75 and 170 nm; or and 165 nm; or 75 and 160 nm; or 75 and 155 nm; or 75 and 150 nm; or 75 and 145 nm; or and 140 nm; or 75 and 135 nm; or 75 and 130 nm; or 80 and 200 nm; or 80 and 185 nm, or 80 and 175 nm; or 80 and 170 nm; or 80 and 165 nm; or 80 and 160 nm; or 80 and 1nm; or 80 and 150 nm; or 80 and 145 nm; or 80 and 140 nm; or 80 and 135 nm; or 80 and 130 nm; or 85 and 200 nm; or 85 and 185 nm, or 85 and 175 nm; or 85 and 170 nm; or and 165 nm; or 85 and 160 nm; or 85 and 155 nm; or 85 and 150 nm; or 85 and 145 nm; or and 140 nm; or 85 and 135 nm; or 85 and 130 nm; or 90 and 200 nm; or 90 and 185 nm, or 90 and 175 nm; or 90 and 170 nm; or 90 and 165 nm; or 90 and 160 nm; or 90 and 1nm; or 90 and 150 nm; or 90 and 145 nm; or 90 and 140 nm; or 90 and 135 nm; or 90 and 130 nm; or 95 and 200 nm; or 95 and 185 nm, or 95 and 175 nm; or 95 and 170 nm; or and 165 nm; or 95 and 160 nm; or 95 and 155 nm; or 95 and 150 nm; or 95 and 145 nm; or and 140 nm; or 95 and 135 nm; or 95 and 130 nm; or 100 and 200 nm; or 100 and 1nm, or 100 and 175 nm; or 100 and 170 nm; or 100 and 165 nm; or 100 and 150 nm; or 1and 155 nm; or 100 and 150 nm; or 100 and 145 nm; or 100 and 100 nm; or 100 and 1 WO 2024/233809 PCT/US2024/028635 nm; or 100 and 130 nm; or 105 and 200 nm; or 105 and 185 nm, or 105 and 175 nm; or 1and 170 nm; or 105 and 165 nm; or 105 and 160 nm; or 105 and 155 nm; or 105 and 1nm; or 105 and 145 nm; or 105 and 140 nm; or 105 and 135 nm; or 105 and 130 nm; or 110 and 200 nm; or 110 and 185 nm, or 110 and 175 nm; or 110 and 170 nm; or 110 and 165 nm; or 110 and 150 nm; or 110 and 155 nm; or 110 and 150 nm; or 110 and 145 nm; or 110 and 100 nm; or 110 and 135 nm; or 110 and 130 nm; or 115 and 200 nm; or 115 and 185 nm, or 115 and 175 nm; or 115 and 170 nm; or 115 and 165 nm; or 115 and 160 nm; or 115 and 155 nm; or 115 and 150 nm; or 115 and 145 nm; or 115 and 140 nm; or 115 and 135 nm; or 115 and 130 nm; or 120 and 200 nm; or 120 and 185 nm, or 120 and 175 nm; or 120 and 170 nm; or 120 and 165 nm; or 120 and 150 nm; or 120 and 155 nm; or 120 and 150 nm; or 120 and 145 nm; or 120 and 140 nm; or 120 and 135 nm; or 120 and 130 nm. Vesicle Cargo Loading. [0029]In certain embodiments, the method further includes loading vesicles of compositions and methods described herein with one or more cargos. The vesicles can be loaded by any suitable method. Exemplary methods of loading the vesicles, such as those prepared by a method described herein, are any of those set forth in International Patent Application Publication W02020/028439, particularly at pages 83-87. [0030]Milk vesicles can be loaded with any suitable or desired cargo(s). In some embodiments, the cargo(s) are therapeutic compounds or molecules. Exemplary cargos include, but are not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti- infectives, radiation sensitizers, chemotherapeutics, imaging agents, immunogens, anti- cancer drugs, any combinations thereof, and/or the like. [0031]In various embodiments, the cargo may be a peptide, such as an ACT-11 peptide. In certain example embodiments, the cargo may be an ACT-11-minus I peptide. Other peptide cargos include those set forth in International Patent Application Publication W02020/028439, particularly at pages 67-82 and 85 and 106-111. In some embodiments, the cargo compound is esterified, such as described in International Patent Application Publication W02020/028439 at page 81-86. In some embodiments, the cargo compound has multiple esterifications, such as described in International Patent Application Publication W02020/028439 at page 81-86. [0001]In certain example embodiments, the exogenous cargo is a biologic molecule. In certain example embodiments, the exogenous cargo is a lipid, polypeptide, peptide, a WO 2024/233809 PCT/US2024/028635 nucleic acid or a cellular metabolite. In certain example embodiments, the exogenous cargo is a Cx43 carboxyl terminal peptide, ACT1 peptide, ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof. In certain example embodiments, the exogenous cargo is any of those cargos set forth in WO 2020/028439, an ACT1 or ACTpeptide, an ACT minus peptide (see e.g., ACT minus peptides set forth in WO 2022/076932), Gap 19, JM2 (see e.g., United States Patent No.: US 9,345,744 B2), a selectide (see e.g., WO 2022/076932), or any combination thereof. In certain example embodiments, the exogenous cargo is a peptide, including but not limited to an ACT-peptide. In certain example embodiments, the exogenous cargo is a peptide, including but not limited to an ACT-11-minus I peptide. Other peptide cargos include those set forth in International Patent Application Publication WO2020/028439, particularly at pages 67-and 85 and 106-111. In some embodiments, the cargo compound is esterified, such as described in International Patent Application Publication WO2020/028439 at page 81-86. In some embodiments, the cargo compound has multiple esterifications, such as described in International Patent Application Publication WO2020/028439 at page 81-86. Other exemplary peptidic cargos include: Corticorelin, Cosyntropin, Seractide, Sincalide, Protirelin, Sermorelin, Somatorelin, Tesamorelin, Secretin, Secretin (human), Secretin (porcine), Thymosin, Thyroid hormone, Thymalfasin, Thymopentin, Calcitonin, Salmon Calcitonin, Elcatonin, Human Calcitonin, Teriparatide, Atosiban, Carbetocin, Oxytocin, Buserelin, Ozempic, Gonadorelin, Goserelin, Histrelin, Leuprolide, Nafarelin, Triptorelin, Abarelix, Cetrorelix, Degarelix, Ganirelix, Depreotide, Edotreotide, Lanreotide, Octreotide, Pentetreotide, Somatostatin, Vapreotide, Argipressin, Desmopressin, Lypressin, Phenypressin, Terlipressin, Enfuvirtide, Ziconotide, Saralasin, Bivalirudin, Eptifibatide, Badpl, Carperitide, Nesiritide, aCTI, aCTll-I, aCTl-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, Albiglutide, Dulaglutide, Semaglutide, Pramlintide, Linasclotide, Sinaspultide, Pasireotide, Teduglutide, Peginesatide, Pentagastrin, Cecropin, moricin, Insect defensin, drosomycin Diptericins, Metchnikowin, Ponericins, Jelleines, Apisimin, Pyrrhocoricin, Persulcatusin, Melittin, Apidaecin, drosocin, lebocin, attacin, gloverin. Gap 19, Rotigaptide, Peptif, L2, JM, ACT017, CB1, AXT107 and other therapeutic or bioactive cargos in esterified and natural forms as described in WIPO Patent Application WO/2022/182782, which is incorporated herein by reference. Exemplary cargo nutrients could include, without limitation, minerals (e.g., potassium, sodium, chloride, magnesium, WO 2024/233809 PCT/US2024/028635 manganese, cobalt, molybdenum, calcium, copper, zinc, iodine, iron, chromium, fluoride, selenium, etc.), vitamins (vitamin 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. [0032]Exemplary hormones that could be used as cargo 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), eicosanoids (e.g., arachidonic acid, lipoxins, and prostaglandins), purines (e.g., ATP), pyrimidines (e.g., thymine), enzymes (e.g., creatine) and steroid hormones (e.g. estradiol, testosterone, tetrahydro testosterone, cortisol). [0033]Exemplary immunomodulators that may be used as cargo 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 phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers). [0034]Exemplary antipyretics include, but are not limited to, non-steroidal anti inflammatories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol/acetaminophen, metamizole, nabumetone, phenazone, and quinine. [0035]Exemplary anxiolytics include, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotonergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), temgicoluril, fabomotizole, selank, bromantane, emoxypine, azapirones, barbiturates, hydroxyzine, pregabalin, isovaleric acid, and beta blockers. [0036]Exemplary antipsychotics that could be used as cargo include benperidol, bromperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol.

WO 2024/233809 PCT/US2024/028635 tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, bifeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine.[0037] Exemplary analgesics that may be used as cargo include paracetamol/acetaminophen, nonsteroidal anti-inflammatories (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, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g. choline salicylate, magnesium salicylate, and sodium salicylate).[0038] Exemplary antispasmodics that may be used as cargo include mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, succinylcholine and dantrolene. Suitable anti-inflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammatories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immune selective anti- inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives).[0039] Exemplary anti-histamines that may be used as cargo include Hl -receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2- receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and p2-adrenergic agonists.[0040] Exemplary anti-infectives that could be used as cargo include amebicides (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, WO 2024/233809 PCT/US2024/028635 amphotericin b, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), antifungals (e.g., azole antifungals (e.g., itraconazole, fluconazole, posaconazole, 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 (e.g., amino salicylic acid), isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (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 alfa-2v/ribavirin, peginterferon alfa- 2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscamet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, abacavir, zidovudine, stavudine, emtricitabine, zalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir, fosamprenavir, 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, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin), glycylcy dines (e.g. tigecycline), leprostatics (e.g. clofazimine and thalidomide), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin), macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, WO 2024/233809 PCT/US2024/028635 piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procaine penicillin, oxacillin, di cl oxacillin, 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, doxy cy cline/omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti- infectives (e.g., nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue). [0041]Exemplary chemotherapeutics that may be used as cargo include paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, bebnostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, Cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprobde, epirubicin, oxabplatin, asparaginase, estramustine, cetuximab, vismodegib, asparaginase Erwinia chrysanthemin, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarebx, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylate, carmustine, eribubn, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-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, denileukin diftitox, 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 trioxide, 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.

WO 2024/233809 PCT/US2024/028635 id="p-105"

[0105] Suitable radiation sensitizers include, but are not limited to, 5-fluorouracil, platinum analogs (e.g., cisplatin, carboplatin, and oxaliplatin), gemcitabine, DNA topoisomerase I- targeting drugs (e.g., camptothecin derivatives (e.g., topotecan and irinotecan)), epidermal growth factor receptor blockade family agents (e.g., cetuximab, gefitinib), famesyltransferase inhibitors (e.g., L-778-123), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), bFGF and VEGF targeting agents (e.g., bevazucimab and thalidomide), NBTXR3, Nimoral, trans sodium crocetinate, NVX-108, and combinations thereof. See also e.g., Kvols, L.K. , JNucl Med 2005; 46:187S— 190S. [0042]Exemplary immunogens that may be used as carried as cargo or attached to the external surface of the isolated vesicles could include Keyhole Limpet Hemocyanin (KLH), Concholepas Concholepas Hemocyanin (CCH), (also Blue Carrier Immunogenic Protein), Bovine Serum Albumin (BSA), Ovalbumin (OVA), and antigens used to generate immune responses to pathogens causing disease including that causing diphtheria, tetanus, pertussis, measles, mumps, rubella, hepatitis A, hepatitis B, meningococcal disease (e.g., meningitis), human papillomavirus varicella, rabies, flu, rotoviral, HIV, malarial and coronaviral disease. [0043]Exemplary polynucleotide modification systems that may be used as carried as cargo or attached to the external surface of the isolated vesicles could include without limitation, CRISPR-Cas systems, OMEGA systems, PRIME editing systems, base editors, meganucleases, zinc-finger nucleases, recombinases, TALE nucleases, CAST systems, Non-LTR retrotransposon systems, transposons, RNAi, antisense nucleic acids, etc. See e.g., Crooke ST, Liang XH, Baker BF, Crooke RM. Antisense technology: A review. J Biol Chem. 2021;296:100416. doi: 10.1016/j.jbc.202L 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 1(35) (2017); DOI: 10.1073/pnas. 1709035114; Makarova et al. 2018. The CRISPR Journal, v. 1, n5, Figure 5; Kim, Y. G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A. 91, 883887־; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A. 93, 11561160־; Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and 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 etal. 2017.Nature. 551:464-471; Coxetal. 2017. Science358: 1019- 1027; Levy et al. Nature Biomedical Engineering doi.org/10.1038/s41441-019-0505- WO 2024/233809 PCT/US2024/028635 (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, particularly at Figures lb, 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, A. C. and Calos, M. P. (2004) J. Mol. Biol. 335, 667-678; Lei, et al., FEES Lett. 2018 Apr;592(8): 1389-1399; Singh, et al., Attachment Site Selection and Identity in Bxbl Serine Integrase-Mediated Site-Specific Recombination, PLoS Genet. 20May;9(5):el003490; and Gupta, et al., Nucleic Acids Res. 2007 May; 35(10): 3407-3419; ToueZa/. bioRxiv 2022.01.07.475005, doi.org/10.1101/2022.01.07.475005; Klompe etal. Nature, doi:10.1038/s41586-019-1323; Strecker et al. Science. 10/1126/science. aax91(2019); Christensen SM et al., RNA from the 5' end of the R2 retrotransposon controls Rprotein binding to and cleavage of its DNA target site, Proc Natl Acad Sci USA. 20Nov 21;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. 2010 May 12;1(1):15. doi: 10.1186/1759-8753-1-15; Malik HS etal., The age and evolution of non-LTR retrotransposable elements, Mol Biol Evol. 1999 Jun;16(6):793- 805; U.S. 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, 7,585,849, 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 No. 2020/0239544, 2018/0346934; International Patent Application Publications WO 2018/213708, WO 2018/213726, WO 2014/093622, WO 2019/005884, WO 2019/005886, WO 2019/071048, WO 2016/106236, WO 2020/206231; WO 2021/138469, WO 2020/131862, WO 2021/257997, WO 2021/087394, WO 2022/147321, WO 2022/07682, WO 2022/150651, WO 2021/102042, WO 2022/173830. [0044]In some embodiments, the exogenous cargo is present in the milk vesicles at any non-zero amount ranging from 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, WO 2024/233809 PCT/US2024/028635 490, 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, pg per milligram of vesicle protein, or be any numerical value or subrange within any of these ranges. [0045]In other embodiments therapeutic cargos, as listed in any embodiments herein, may be esterified to facilitate cargo loading, for example, by endogenous vesicle enzymes as described in WIPO Patent Application WO/2022/182782, which is incorporated herein by reference. Pharmaceutical, Therapeutic and Nutraceutical Preparations and Uses of Vesicles. [0046]Extracellular vesicles can be powerful tools for administering a therapeutic cargo to a subject due to the ability of vesicles to transport and protect biological signaling molecules to a subject together with unique ability of certain extracellular vesicle populations to cross tissue boundaries such as the cutaneous barrier, blood- brain-barrier. Extracellular vesicles also in certain situations can elude immune surveillance and are immunologically well-tolerated even when transferred autologously between individuals and species. Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display. J Nanobiotechnology) - further heightening interest in their potential for translation to the clinic as a novel means for improving the safety of drug delivery. Accordingly, also described herein include pharmaceutical formulations that can contain an amount, effective amount, and/or least effective amount, and/or therapeutically effective amount of one or vesicles, such as cargo loaded milk vesicles, as described herein and a pharmaceutically acceptable carrier or excipient. Described in certain example embodiments herein are formulations that include extracellular vesicles, where the formulation is produced at least in part by any one of the methods of any one of the preceding paragraphs and/or described elsewhere herein, such as in the Working Examples below. Described in certain example embodiments herein are methods that include administering a formulation as described in any one of the previous paragraphs and/or elsewhere herein, such as the Working Examples below, to a subject. In some embodiments, the formulation administered to the subject includes milk vesicles, such as any of those described elsewhere herein and/or prepared by a method described elsewhere herein. In some embodiments, the milk vesicles are cargo loaded milk vesicles. [0047]As used herein, the terms "treating", and "treatment" can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but WO 2024/233809 PCT/US2024/028635 does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as cancer, inflammatory disease or disorder, and/or a mechanical or non-mechanical injury. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. In some embodiments the effect can be the alleviation or intended prevention of undesirable symptoms. The term "treatment" as used herein covers any treatment of cancer, inflammatory disease or disorder, and/or a mechanical or non-mechanical injury, in a subject, particularly a human or non-human animal, such as a non-human mammal, and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. [0048]In some embodiments the subject to which the vesicles or formulation thereof is administered has a disease or disorder. Exemplary diseases or disorders include, but are not limited to, any cancer, a viral infection, a bacterial infection, a parasite infection, a external and internal wounds and tissue injuries, cancer, ischemic and/or hypoxic injuries (e.g. myocardial infarction, ischemic wounds and/or stroke), multiple sclerosis, psoriasis, scleroderma, acne, eczema, or a disease of the skin and/or connective tissues, cardiac diseases or disorders, neurodegenerative diseases or disorders, neurological disorders, atherosclerosis, pathologies involving epithelial permeablization and/or neovascularization (e.g., angiogenesis or vasculogenesis), respiratory distress syndrome (RDS), reperfusion injuries, dermal vascular blemish or malformation, macular degeneration, neovascularization of choriocapillaries through Bruch's membrane, diabetic retinopathy, WO 2024/233809 PCT/US2024/028635 (inflammatory and inflammation-related diseases and disorders), radiation injury, acute radiation syndrome, radiation therapy side-effects, radiation dermatitis, as well as diseases resulting from high dose radiation including Acute Radiation Syndrome, Chronic Radiation Syndrome, Bile duct Cancer, Bone Cancer, Brain cancer, breast cancer, colon cancer, esophageal cancer, gall bladder cancer, liver cancer, lung cancer, pancreatic cancer, pharyngeal cancer, ovarian cancer, salivary gland cancer, intestinal cancer, stomach cancer, thyroid cancer, urinary tract cancer, leukemia, lyphomas, multiple myeloma or any other cancer, non-malignant thyroid nodular disease, parathyroid adenoma or posterior sub capsular cataracts. [0049]Wounds can be chronic wounds or wounds that appear to not completely heal. Wounds that have not healed within three months, for example, are said to be chronic. Chronic wounds include, diabetic foot ulcers, ischemic, venous ulcers, venous leg ulcers, venous stasis, arterial, pressure, vasculitic, infectious, decubitis, trauma-induced, gangrenous and mixed ulcers. Chronic wounds include wounds that are characterized by and/or chronic inflammation, deficient and overprofuse granulation tissue differentiation and failure of re- epithelialization and wound closure and longer repair times. Chronic wounds can include ocular ulcers, including corneal ulcers. Use of the disclosed invention in wound healing and tissue regeneration can include in humans and agricultural, sports and pet animals. [0050]Tissue injuries can result from, for example, a cut, scrape, compression wound, stretch injury, laceration wound, crush wound, bite wound, graze, bullet wound, explosion injury, body piercing, stab wound, surgical wound, surgical intervention, medical intervention, host rejection following cell, tissue or organ grafting, pharmaceutical effect, pharmaceutical side-effect, bed sore, radiation injury, radiation illness, cosmetic skin wound, internal organ injury, disease process (e.g., asthma, cancer), infection, infectious agent, developmental process, maturational process (e.g., acne), genetic abnormality, developmental abnormality, environmental toxin, allergen, scalp injury, facial injuryjaw injury, sex organ injury, joint injury, excretory organ injury, foot injury, finger injury, toe injury, bone injury, eye injury, corneal injury, muscle injury, adipose tissue injury, lung injury, airway injury, hernia, anus injury, piles, ear injury, skin injury, abdominal injury, retinal injury, eye injury, corneal injury, arm injury, leg injury, athletic injury, back injury, birth injury, premature birth injury, toxic bite, sting, injury to barrier function, injury to endothelial barrier function, injury to epithelial barrier function, tendon injury, ligament injury, heart injury, heart valve injury, vascular system injury, cartilage injury, lymphatic WO 2024/233809 PCT/US2024/028635 system injury, craniocerebral trauma, dislocation, esophageal perforation, fistula, nail injury, foreign body, fracture, frostbite, hand injury, heat stress disorder, laceration, neck injury, self-mutilation, shock, traumatic soft tissue injury, spinal cord injury, spinal injury, sprain, strain, tendon injury, ligament injury, cartilage injury, thoracic injury, tooth injury, trauma, nervous system injury, bum, bum wound, wind bum, sun bum, chemical bum, aging, aneurism, stroke, radiation injury, surgical radiation injury, digestive tract injury, infarct, or ischemic injury. [0051]Cardiac diseases and disorders that may be treated by compositions and methods described herein can include, but are not limited to, myocardial infarction, cardio myopathies (e.g., hypertrophic cardiomyopathy), arrhythmias, congestive heart failure. The regenerative effects of the provided composition may result in beneficial changes in membrane excitability and ion transients of the heart. There are many different types of arrhythmias that can lead to abnormal function in the human heart. Arrhythmias include, but are not limited to bradycardias, tachycardias, altemans, automaticity defects, reentrant arrhythmias, fibrillation, AV nodal arrhythmias, atrial arrhythmias and triggered beats, Long QT syndrome, Short QT syndrome, Brugada syndrome, premature atrial Contractions, wandering Atrial pacemaker, Multifocal atrial tachycardia, Atrial flutter, Atrial fibrillation, Supraventricular tachycardia, AV nodal reentrant tachycardia is the most common cause of Paroxysmal Supraventricular Tachycardia, Junctional rhythm, Junctional tachycardia, Premature junctional complex, Wolff-Parkinson- White syndrome, Lown- Ganong-Levine syndrome, Premature Ventricular Contractions (PVC) sometimes called Ventricular Extra Beats, altemans and discordant altemans, Accelerated idioventricular rhythm, Monomorphic Ventricular tachycardia, Polymorphic ventricular tachycardia, Ventricular fibrillation, First degree heart block, which manifests as PR prolongation, Second degree heart block, Type 1 Second degree heart block, Type 2 Second degree heart block, Third degree heart block, and several accessory pathway disorders (e.g., Wolff- Parkinson- White syndrome (WPW)). [0052]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, prion disease, Spinal muscular atrophy (SMA), multiple sclerosis, epilepsy and other seizure disorders, and Huntington’s disease. [0053]Inflammatory diseases and inflammatory-related diseases and disorders can be asthma, eczema, sinusitis, atherosclerosis, arthritis (including but not limited to rheumatoid WO 2024/233809 PCT/US2024/028635 arthritis), inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis. As used herein, the term "inflammatory disorder" can include diseases or disorders which are caused, at least in part, or exacerbated, by inflammation, which is generally characterized by increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), heat, redness, swelling, pain and/or loss of function in the affected tissue or organ. The cause of inflammation can be due to physical damage, chemical substances, micro-organisms, tissue necrosis, cancer, or other agents or conditions. [0054]Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders are generally of relatively short duration, and last for from about a few minutes to about one to two days, although they can last several weeks. Characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema) and emigration of leukocytes, such as neutrophils. Chronic inflammatory disorders, generally, are of longer duration, e.g., weeks to months to years or longer, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders which recur after a period of time or which have periodic episodes. Some inflammatory disorders fall within one or more categories. Exemplary inflammatory disorders include but are not limited to atherosclerosis; arthritis; inflammation-promoted cancers; asthma; autoimmune uveitis; adoptive immune response; dermatitis; multiple sclerosis; diabetic complications; osteoporosis; Alzheimer's disease; cerebral malaria; hemorrhagic fever; autoimmune disorders; Crohn’s disease, and inflammatory bowel disease. In some embodiments, the inflammatory disorder is an autoimmune disorder that, in some aspects, is selected from lupus, rheumatoid arthritis, and autoimmune encephalomy eliti s. [0055]In some embodiments, the inflammatory disorder is a brain-related inflammatory disorder. The term "brain-related inflammatory" disorder is used herein to refer to a subset of inflammatory disorders that are caused, at least in part, or originate or are exacerbated, by inflammation in the brain of a subject. [0056]As used herein, "pharmaceutical formulation" refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. As used herein, "pharmaceutically acceptable carrier or excipient" WO 2024/233809 PCT/US2024/028635 refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier or excipient" as used in the specification and claims includes both one and more than one such carrier or excipient. When present, the cargo can optionally be present in the pharmaceutical formulation as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutical formulation can include, such as an active ingredient, one or more milk vesicles, such as cargo loaded milk extracellular vesicles, described in greater detail elsewhere herein. [0057]In some embodiments, the cargo is present as a pharmaceutically acceptable salt of the active ingredient. As used herein, "pharmaceutically acceptable salt" refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate. [0058]The pharmaceutical formulations described herein can be administered to a subject in need thereof via any suitable method or route to a subject in need thereof. Suitable administration routes can include, but are not limited to auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra- amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebral, intracistemal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavemosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, WO 2024/233809 PCT/US2024/028635 iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and/or vaginal administration, and/or any combination of the above administration routes, which typically depends on the disease to be treated and/or the active ingredient(s) and/or cargos. [0059]Where appropriate, one or more milk vesicles, such as cargo loaded milk vesicles, described in greater detail elsewhere herein can be provided to a subject in need thereof as an ingredient, such as an active ingredient or agent, in a pharmaceutical formulation. As such, also described are pharmaceutical formulations containing one or more milk vesicles, such as cargo loaded milk vesicles, described in greater detail elsewhere herein include a cargo that is in the form of a pharmaceutically acceptable salt. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate. [0060]As used herein, "agent" refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to. As used herein, "active agent" or "active ingredient" refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, "active agent" or "active ingredient" refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed. In some embodiments the active agent is a milk vesicle, or a cargo loaded milk vesicle. In some embodiments, the active agent includes or is the cargo of a cargo loaded milk vesicle.

WO 2024/233809 PCT/US2024/028635 id="p-61"

[0061]In certain embodiments, the milk vesicle is prepared by any method described elsewhere herein. In some embodiments, the cargo loaded milk vesicle is as described and/or prepared by a method as described elsewhere herein. Milk Vesicles [0062]Mammalian milk is enriched in extracellular vesicles and has great promise as a source for large scale production extracellular vesicles for wide spread use as delivery vehicles, exemplary species used in milk production which can be used as a source of milk vesicles as provided herein include cows, buffalo, goats, sheep, camels, yaks, horses, reindeers, donkeys and humans. Bovine or cows milk is produced in large quantities by the dairy industry, widely consumed, and generally immunologically well-tolerated by humans. Moreover, milk vesicles have been reported to cross from the gut into the blood circulation and traffic to various organs, including brain, heart, gut and lungs (Wolf T, B. S.(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. J Nutr, 2201- 2206), which are properties that could provide a basis for oral administration of cargo (including but not limited to therapeutics) loaded vesicles. [0063]Milk used as a source for vesicles in the compositions and methods disclosed herein may be from any mammal. In some embodiments, the mammalian milk is bovine milk, ovine milk, porcine milk, camelid milk, equine milk, capra milk, human milk, and/or the like. In certain embodiments, the milk is unpasteurized. In some embodiments, the milk is pasteurized. Isolation and Purification of Extracellular Vesicles From Milk. [0064]Methods for large scale and cost-effective means for isolating extracellular vesicles from body fluids are of high interest for ultimate commercial scale use of vesicles as delivery vehicles. There are a number of methods for exosomal isolation from biological fluids, that are based on ultracentrifugation (UC). These methods typically involve differential centrifugation steps and/or density gradient UC-based separations. However, the ability to produce vesicles in large quantities is restricted by the requirement for multiple UC steps and the fact that UCs can only spin small volumes. It further remains that shearing forces imparted during repeated UC spins may have deleterious effects on vesicle structural integrity (Taylor DD, S. S. (2015). Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods, 3-10). Other techniques that may exert less physical rigor during exosomal isolation include ultrafiltration, WO 2024/233809 PCT/US2024/028635 tangential flow filtration (TFF), size exclusion chromatography (SEC), and polyethylene glycol precipitation-based methods. [0065]While milk is a promising source of commercial scale extracellular vesicles, milk has particular challenges in the isolation of the vesicles. Milk contains a diverse mixture of proteins, minerals, lipids, and other macromolecules and this complexity of the milk composition contributes to challenges to the purification of the extracellular vesicles. Casein proteins are a major constituent of milk, making up approximately 80% of all milk proteins. Caseins aggregate into large, colloidal complexes with calcium phosphate and other milk proteins to form what are referred to as casein micelles. These micelles are approximately 10 nm in diameter and can further coalesce into larger coagulated structures (Bhat, M. T. (2016). Casein Proteins: structural and functional aspects. Intech). Casein micelle aggregates are thought to bind to and ensnare vesicles via chemical interactions, impeding separation from contaminating milk proteins; observations that are confirmed by transmission electron microscopy (TEM) analysis of milk-derived exosomal preparations (Sedykh S.E., B. E. (2020). Milk Exosomes: Isolation, Biochemistry, Morphology, and Perspectives of Use. In C. J. De Bona A.G., Extracellular Vesicles and their importance in human health. Intech Open). As a consequence, present methods for isolation of high-purity extracellular vesicles from milk are limited by contaminating proteins, such as casein. A close association between extracellular vesicles and casein-rich strands of proteins isolated from milk has been noted (Figure 1). These associations recall another association between extracellular vesicles and extracellular matrix that has been observed called extracellular matrix vesicles or matrix bound nanovesicles (MBNs). Without being bound by theory, it is contemplated that detaching or loosening vesicles by methods described herein, including via use of free tryptophan to destabilize chemical interaction between vesicles and matrices that can include extracellular, collagen, fibrin or casein matrices, can increase the yield of purified vesicles isolated from such sources. Further it is contemplated that in certain embodiments the addition of free tryptophan to vesicles isolated thus will aid with maintenance of structure and bioactivity of vesicles during procedures including lyophilization, sterilization and storage as described herein. [0066]Methods, such as described in PCT WO2022182782, that include incorporating chelation of Ca2+ and other divalent cations at specified temperatures result in high-yield separation of structurally and functionally intact vesicles from milk proteins. In some situations, casein micelle solubilization and vesicle separation steps can be included in a method with UC-based and/or TFF and SEC filtration steps for vesicle isolation thereby WO 2024/233809 PCT/US2024/028635 providing a basis for large-scale production of purified high quality vesicles from milk. Such methods involving chelating divalent cations with ethylenediaminetetraacetic acid (EDTA) and/or other chelator(s), including, but not limited to, l,2-bis(0- 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 pyrophosphates, other phosphates and poly-phosphates and/or nitrophen are effective in methods for isolating extracellular vesicles, including without ultracentrifugation. EDTA chemically immobilized on insoluble substrates can be used to chelate divalent cations from solutions either in the presence of a semi-permeable membrane or not to prevent contamination of milk sample solution. However, in some situations, such as, for example uses of vesicles for nutraceutical uses, it may be desirable to avoid the use of chemicals such as EDTA that can be considered by some to be harsh. [0067]Accordingly, it is one object of the present disclosure to develop methods similar to WO2022182782 that reduce, or avoid completely, the use of EDTA and/or similar chemicals that may be considered as harsh by using alternative agents instead. For example, in some embodiments of the present disclosure methods that are similar to those of PCT WO2022182782 are provided that utilize tryptophan and/or tryptophan analogues as alternatives to EDTA. Free tryptophan is naturally present in milk and colostrum at concentrations of up to 10 mg/ml and enhanced by dairy industry practices such as fermentation. Breast milk is the only source of tryptophan (TRP) in breast-fed infants (PMC5906556). Thus, rather being a contaminant, free tryptophan is thought an essential constituent of milk. Accordingly, provided herein include methods of isolating vesicles from a biological fluid such as milk that, for example, may include (a) centrifuging a biological fluid under conditions suitable to separate fats from one or more other components of the biological fluid; (b) removing the separated fats from the biological fluid; (c) after step (b) centrifuging the remaining biological fluid one or more times and skimming any noticeable separated fats after each centrifuging in step (c); (d) filtering the remaining biological fluid after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); (f) contacting the resultant solution with tryptophan and/or or more tryptophan analogues at about 20-60 degrees Celsius for about 15-1minutes. After step (f), tangential flow filtration to obtain a retenate can optionally be performed (step (g)). The retenate can then optionally be ultracentrifuged via one or more WO 2024/233809 PCT/US2024/028635 ultracentrifugation steps. The retenate can then optionally be fractionated via column separation. In some embodiments the method includes step (e) or step (g) but not both. [0068]In some embodiments, step (f) is performed with tryptophan and/or one or more tryptophan analogues at a concentration between 10 pM and 1 mM. In some embodiments, step (f) is performed by contacting (e.g., incubating) the milk vesicles with tryptophan and/or or more tryptophan analogues at about 30-42 degrees Celsius for about 15-1minutes. [0069]In certain example embodiments, step (a) comprises centrifuging the biological fluid at about 2,500 relative centrifugal force (RCF). In certain example embodiment, (a) comprises centrifuging the biological fluid at about 2,000 ref to about 3,000 ref In certain example embodiment, (a) comprises centrifuging the biological fluid at about 2000 ref, 2010 ref, 2020 ref, 2030 ref, 2040 ref, 2050 ref, 2060 ref, 2070 ref, 2080 ref, 2090 ref, 21ref, 2110 ref, 2120 ref, 2130 ref, 2140 ref, 2150 ref, 2160 ref, 2170 ref, 2180 ref, 2190 ref, 2200 ref, 2210 ref, 2220 ref, 2230 ref, 2240 ref, 2250 ref, 2260 ref, 2270 ref, 2280 ref, 22ref, 2300 ref, 2310 ref, 2320 ref, 2330 ref, 2340 ref, 2350 ref, 2360 ref, 2370 ref, 2380 ref, 2390 ref, 2400 ref, 2410 ref, 2420 ref, 2430 ref, 2440 ref, 2450 ref, 2460 ref, 2470 ref, 24ref, 2490 ref, 2500 ref, 2510 ref, 2520 ref, 2530 ref, 2540 ref, 2550 ref, 2560 ref, 2570 ref, 2580 ref, 2590 ref, 2600 ref, 2610 ref, 2620 ref, 2630 ref, 2640 ref, 2650 ref, 2660 ref, 26ref, 2680 ref, 2690 ref, 2700 ref, 2710 ref, 2720 ref, 2730 ref, 2740 ref, 2750 ref, 2760 ref, 2770 ref, 2780 ref, 2790 ref, 2800 ref, 2810 ref, 2820 ref, 2830 ref, 2840 ref, 2850 ref, 28ref, 2870 ref, 2880 ref, 2890 ref, 2900 ref, 2910 ref, 2920 ref, 2930 ref, 2940 ref, 2950 ref, 2960 ref, 2970 ref, 2980 ref, 2990 ref, to/or about 3000 ref. In certain embodiments, step (a) is repeated 1-3 times. In some embodiments, step (a) is repeated 1, 2, or 3 times. [0070]In some embodiments, step (b) includes a first centrifugation followed by a second centrifugation. In certain example embodiments, the first centrifugation includes centrifuging the remaining biological fluid at about 14,500 ref for about 60 minutes. In certain example embodiments, the first centrifugation includes centrifuging the remaining biological fluid at about 13,500 ref to about 15,500 ref for about 45 to about 75 minutes. In some embodiments, the first centrifugation includes centrifuging the remaining biological fluid at about 13500 ref, 13550 ref, 13600 ref, 13650 ref, 13700 ref, 13750 ref, 13800 ref, 13850 ref, 13900 ref, 13950 ref, 14000 ref, 14050 ref, 14100 ref, 14150 ref, 14200 ref,14250 ref, 14300 ref, 14350 ref, 14400 ref, 14450 ref, 14500 ref, 14550 ref, 14600 ref,14650 ref, 14700 ref, 14750 ref, 14800 ref, 14850 ref, 14900 ref, 14950 ref, 15000 ref,15050 ref, 15100 ref, 15150 ref, 15200 ref, 15250 ref, 15300 ref, 15350 ref, 15400 ref, WO 2024/233809 PCT/US2024/028635 15450 ref, or at about 15500 ref for about 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, 70 min, 71 min, 72 min, min, 74 min, or about 75 min. In some embodiments, the second centrifugation is performed on the biological fluid remaining after the first centrifugation and wherein the second centrifugation is performed at about 25,800 rcf for about 60 minutes. In certain example embodiments, the second centrifugation is performed on the biological fluid remaining after the first centrifugation and the second centrifugation is performed at about 24,800 to about 26,800 rcf for about 45 to about 75 minutes. In certain example embodiments, the second centrifugation is performed on the biological fluid remaining after the first centrifugation and the second centrifugation is performed at about 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,26050 rcf, 26100 rcf, 26150 rcf, 26200 rcf, 26250 rcf, 26300 rcf, 26350 rcf, 26400 rcf,26450 rcf, 26500 rcf, 26550 rcf, 26600 rcf, 26650 rcf, 26700 rcf, 26750 rcf, 26800 ref for about 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, min, 67 min, 68 min, 69 min, 70 min, 71 min, 72 min, 73 min, 74 min, or about 75 min. In certain example embodiments, the second centrifugation is repeated 1-3 times with each repetition being performed on the remaining biological fluid from the centrifugation immediately prior. In certain example embodiments, the second centrifugation is repeated 1, 2, or 3 times with each repetition being performed on the remaining biological fluid from the centrifugation immediately prior. [0071]In certain example embodiments, step (d) includes filtering the remaining biological fluid through one or more filters in series ranging from about a 0.45 micron (pm) filter to about a 0.22 micron filter. In some embodiments, each filter in the series is independently selected from a 0.22 micron, 0.23 micron, 0.24 micron, 0.25 micron, 0.micron, 0.27 micron, 0.28 micron, 0.29 micron, 0.3 micron, 0.31 micron, 0.32 micron, 0.micron, 0.34 micron, 0.35 micron, 0.36 micron, 0.37 micron, 0.38 micron, 0.39 micron, 0.micron, 0.41 micron, 0.42 micron, 0.43 micron, 0.44 micron, or 0.45 micron filter. In some embodiments, the number of filters in series ranges 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or filters in series. In some embodiments, all filters in the series are the same size cut off. In some embodiments, at least 2 filters in the series have the same size cut off. In some WO 2024/233809 PCT/US2024/028635 embodiments, at least 2 filters in the series have different size cut offs. In some embodiments, all the filters in the series have different size cut offs. In some embodiments, the size exclusion decreases from large to small along a series of filters. For example, in a series of 3 filters, the first filter can be a 0.45 micron filter, the second filter can be a 0.micron filter and the last filter can be a 0.22 filter. Other configurations of filters in series will be appreciated in view of the description herein. In some embodiments, the filters in series are all the same material. In some embodiments, the filters in series are all different materials. In some embodiments, at least 2 filters in the series are the same material. In some embodiments, at least 2 filters are made of different materials. Exemplary filters include, but are not limited to, membrane filters (e.g., poly ethersulfone membrane filters, polyvinylidene fluoride membrane filters, cellulose membrane filters, mixed cellulose esters 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, and/or the like), glass fiber or bead filters, and/or the like. In some embodiments, step (d) includes filtering the remaining biological fluid through an about 0.45 micron filter followed by filtering the remaining biological fluid through an about 0.micron filter. [0072]In some embodiments of the aforementioned method, step (e) includes 2 or more serial ultracentrifugation steps, wherein each step is performed on the remaining biological fluid from the prior ultracentrifugation. In certain example embodiments, (e) includes 2-(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) serial ultracentrifugation steps, wherein each step is performed on the remaining biological fluid from the prior ultracentrifugation. In certain example embodiments, (e) includes an ultracentrifugation step performed at about 50,0ref, an ultracentrifugation step performed at about 70,000 rcf, an ultracentrifugation step performed at about 100,000 rcf, or any combination thereof. In certain example embodiments, (e) includes an ultracentrifugation step performed at about 45,000 to about 55,000 rcf, an ultracentrifugation step performed at about 65,000 to about 75,000 rcf, an ultracentrifugation step performed at about 90,000 to about 110,000 rcf, or any combination thereof. In certain example embodiments, (e) includes an ultracentrifugation step performed at about 45,000 to about 55,000 rcf (e.g., at about 45000 rcf, 45100 rcf, 452rcf, 45300 rcf, 45400 rcf, 45500 rcf, 45600 rcf, 45700 rcf, 45800 rcf, 45900 rcf, 46000 rcf, 46100 rcf, 46200 rcf, 46300 rcf, 46400 rcf, 46500 rcf, 46600 rcf, 46700 rcf, 46800 rcf, 46900 rcf, 47000 rcf, 47100 rcf, 47200 rcf, 47300 rcf, 47400 rcf, 47500 rcf, 47600 rcf, WO 2024/233809 PCT/US2024/028635 47700 ref, 47800 ref, 47900 ref, 48000 ref, 48100 ref, 48200 ref, 48300 ref, 48400 ref,48500 ref, 48600 ref, 48700 ref, 48800 ref, 48900 ref, 49000 ref, 49100 ref, 49200 ref,49300 ref, 49400 ref, 49500 ref, 49600 ref, 49700 ref, 49800 ref, 49900 ref, 50000 ref,50100 ref, 50200 ref, 50300 ref, 50400 ref, 50500 ref, 50600 ref, 50700 ref, 50800 ref,50900 ref, 51000 ref, 51100 ref, 51200 ref, 51300 ref, 51400 ref, 51500 ref, 51600 ref,51700 ref, 51800 ref, 51900 ref, 52000 ref, 52100 ref, 52200 ref, 52300 ref, 52400 ref,52500 ref, 52600 ref, 52700 ref, 52800 ref, 52900 ref, 53000 ref, 53100 ref, 53200 ref,53300 ref, 53400 ref, 53500 ref, 53600 ref, 53700 ref, 53800 ref, 53900 ref, 54000 ref,54100 ref, 54200 ref, 54300 ref, 54400 ref, 54500 ref, 54600 ref, 54700 ref, 54800 ref,54900 ref, or at about 55000 ref), an ultracentrifugation step performed at about 65,000 to about 75,000 ref (e.g., at about 65000 ref, 65100 ref, 65200 ref, 65300 ref, 65400 ref, 655ref, 65600 ref, 65700 ref, 65800 ref, 65900 ref, 66000 ref, 66100 ref, 66200 ref, 66300 ref, 66400 ref, 66500 ref, 66600 ref, 66700 ref, 66800 ref, 66900 ref, 67000 ref, 67100 ref,67200 ref, 67300 ref, 67400 ref, 67500 ref, 67600 ref, 67700 ref, 67800 ref, 67900 ref,68000 ref, 68100 ref, 68200 ref, 68300 ref, 68400 ref, 68500 ref, 68600 ref, 68700 ref,68800 ref, 68900 ref, 69000 ref, 69100 ref, 69200 ref, 69300 ref, 69400 ref, 69500 ref,69600 ref, 69700 ref, 69800 ref, 69900 ref, 70000 ref, 70100 ref, 70200 ref, 70300 ref,70400 ref, 70500 ref, 70600 ref, 70700 ref, 70800 ref, 70900 ref, 71000 ref, 71100 ref,71200 ref, 71300 ref, 71400 ref, 71500 ref, 71600 ref, 71700 ref, 71800 ref, 71900 ref,72000 ref, 72100 ref, 72200 ref, 72300 ref, 72400 ref, 72500 ref, 72600 ref, 72700 ref,72800 ref, 72900 ref, 73000 ref, 73100 ref, 73200 ref, 73300 ref, 73400 ref, 73500 ref,73600 ref, 73700 ref, 73800 ref, 73900 ref, 74000 ref, 74100 ref, 74200 ref, 74300 ref,74400 ref, 74500 ref, 74600 ref, 74700 ref, 74800 ref, 74900 ref, or at about 75000 rcl), an ultracentrifugation step performed at about 90,000 to about 110,000 ref (e.g., 90000 ref, 90100 ref, 90200 ref, 90300 ref, 90400 ref, 90500 ref, 90600 ref, 90700 ref, 90800 ref,90900 ref, 91000 ref, 91100 ref, 91200 ref, 91300 ref, 91400 ref, 91500 ref, 91600 ref,91700 ref, 91800 ref, 91900 ref, 92000 ref, 92100 ref, 92200 ref, 92300 ref, 92400 ref,92500 ref, 92600 ref, 92700 ref, 92800 ref, 92900 ref, 93000 ref, 93100 ref, 93200 ref,93300 ref, 93400 ref, 93500 ref, 93600 ref, 93700 ref, 93800 ref, 93900 ref, 94000 ref,94100 ref, 94200 ref, 94300 ref, 94400 ref, 94500 ref, 94600 ref, 94700 ref, 94800 ref,94900 ref, 95000 ref, 95100 ref, 95200 ref, 95300 ref, 95400 ref, 95500 ref, 95600 ref,95700 ref, 95800 ref, 95900 ref, 96000 ref, 96100 ref, 96200 ref, 96300 ref, 96400 ref,96500 ref, 96600 ref, 96700 ref, 96800 ref, 96900 ref, 97000 ref, 97100 ref, 97200 ref,97300 ref, 97400 ref, 97500 ref, 97600 ref, 97700 ref, 97800 ref, 97900 ref, 98000 ref, WO 2024/233809 PCT/US2024/028635 98100 ref, 98200 ref, 98300 ref, 98400 ref, 98500 ref, 98600 ref, 98700 ref, 98800 ref, 98900 ref, 99000 ref, 99100 ref, 99200 ref, 99300 ref, 99400 ref, 99500 ref, 99600 ref, 99700 ref, 99800 ref, 99900 ref, 100000 ref, 100100 ref, 100200 ref, 100300 ref, 1004ref, 100500 ref, 100600 ref, 100700 ref, 100800 ref, 100900 ref, 101000 ref, 101100 ref,101200 ref,101900 ref,102600 ref,103300 ref,104000 ref,104700 ref,105400 ref,106100 ref,106800 ref,107500 ref,108200 ref,108900 ref, 101300 ref,102000 ref,102700 ref,103400 ref,104100 ref,104800 ref,105500 ref,106200 ref,106900 ref,107600 ref,108300 ref,109000 ref, 101400 ref,102100 ref,102800 ref,103500 ref,104200 ref,104900 ref,105600 ref,106300 ref,107000 ref,107700 ref,108400 ref,109100 ref, 101500 ref,102200 ref,102900 ref,103600 ref,104300 ref,105000 ref,105700 ref,106400 ref,107100 ref,107800 ref,108500 ref,109200 ref, 101600 ref,102300 ref,103000 ref,103700 ref,104400 ref,105100 ref,105800 ref,106500 ref,107200 ref,107900 ref,108600 ref,109300 ref, 101700 ref,102400 ref,103100 ref,103800 ref,104500 ref,105200 ref,105900 ref,106600 ref,107300 ref,108000 ref,108700 ref,109400 ref, 101800 ref,102500 ref,103200 ref,103900 ref,104600 ref,105300 ref,106000 ref,106700 ref,107400 ref,108100 ref,108800 ref,109500 ref,109600 ref, 109700 ref, 109800 ref, 109900 ref, or at about 110000 ref), or any combination thereof. In certain example embodiments, the one or more of the one or more ultracentrifugation steps are each performed for about 60 minutes. In certain example embodiments, the one or more of the one or more ultracentrifugation steps are each performed for about 45-75 minutes. In certain example embodiments, the one or more of the one or more ultracentrifugation steps are each performed for about 45 min, 46 min, 47min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69min, 70 min, 71 min, 72 min, 73 min, 74 min, or about 75 min. [0073]In some embodiments, step (e) comprises a final ultracentrifugation step performed at about 130,000 ref for about 120 minutes, the resulting fluid is discarded, and the remaining pellet is resuspended in a suitable volume of a suitable solution prior to (1). In certain example embodiments, (e) comprises a final ultracentrifugation step performed at about 115,000 to about 145,000 ref for about 90-150 minutes, the resulting fluid is discarded, and the remaining pellet is resuspended in a suitable volume of a suitable solution prior to (1). In certain example embodiments, (e) comprises a final ultracentrifugation step performed at about 115,000 to about 145,000 ref (e.g., 115000 ref, 115100 ref, 115200 ref, 115300 ref, 115400 ref, 115500 ref, 115600 ref, 115700 ref, WO 2024/233809 PCT/US2024/028635 115800 ref,116500 ref,117200 ref,117900 ref,118600 ref,119300 ref,120000 ref,120700 ref,121400 ref,122100 ref,122800 ref,123500 ref,124200 ref,124900 ref,125600 ref,126300 ref,127000 ref,127700 ref,128400 ref,129100 ref,129800 ref,130500 ref,131200 ref,131900 ref,132600 ref,133300 ref,134000 ref,134700 ref,135400 ref,136100 ref,136800 ref,137500 ref,138200 ref,138900 ref, 115900 ref,116600 ref,117300 ref,118000 ref,118700 ref,119400 ref,120100 ref,120800 ref,121500 ref,122200 ref,122900 ref,123600 ref,124300 ref,125000 ref,125700 ref,126400 ref,127100 ref,127800 ref,128500 ref,129200 ref,129900 ref,130600 ref,131300 ref,132000 ref,132700 ref,133400 ref,134100 ref,134800 ref,135500 ref,136200 ref,136900 ref,137600 ref,138300 ref,139000 ref, 116000 ref,116700 ref,117400 ref,118100 ref,118800 ref,119500 ref,120200 ref,120900 ref,121600 ref,122300 ref,123000 ref,123700 ref,124400 ref,125100 ref,125800 ref,126500 ref,127200 ref,127900 ref,128600 ref,129300 ref,130000 ref,130700 ref,131400 ref,132100 ref,132800 ref,133500 ref,134200 ref,134900 ref,135600 ref,136300 ref,137000 ref,137700 ref,138400 ref,139100 ref, 116100 ref,116800 ref,117500 ref,118200 ref,118900 ref,119600 ref,120300 ref,121000 ref,121700 ref,122400 ref,123100 ref,123800 ref,124500 ref,125200 ref,125900 ref,126600 ref,127300 ref,128000 ref,128700 ref,129400 ref,130100 ref,130800 ref,131500 ref,132200 ref,132900 ref,133600 ref,134300 ref,135000 ref,135700 ref,136400 ref,137100 ref,137800 ref,138500 ref,139200 ref, 116200 ref,116900 ref,117600 ref,118300 ref,119000 ref,119700 ref,120400 ref,121100 ref,121800 ref,122500 ref,123200 ref,123900 ref,124600 ref,125300 ref,126000 ref,126700 ref,127400 ref,128100 ref,128800 ref,129500 ref,130200 ref,130900 ref,131600 ref,132300 ref,133000 ref,133700 ref,134400 ref,135100 ref,135800 ref,136500 ref,137200 ref,137900 ref,138600 ref,139300 ref, 116300 ref,117000 ref,117700 ref,118400 ref,119100 ref,119800 ref,120500 ref,121200 ref,121900 ref,122600 ref,123300 ref,124000 ref,124700 ref,125400 ref,126100 ref,126800 ref,127500 ref,128200 ref,128900 ref,129600 ref,130300 ref,131000 ref,131700 ref,132400 ref,133100 ref,133800 ref,134500 ref,135200 ref,135900 ref,136600 ref,137300 ref,138000 ref,138700 ref,139400 ref, 116400 ref,117100 ref,117800 ref,118500 ref,119200 ref,119900 ref,120600 ref,121300 ref,122000 ref,122700 ref,123400 ref,124100 ref,124800 ref,125500 ref,126200 ref,126900 ref,127600 ref,128300 ref,129000 ref,129700 ref,130400 ref,131100 ref,131800 ref,132500 ref,133200 ref,133900 ref,134600 ref,135300 ref,136000 ref,136700 ref,137400 ref,138100 ref,138800 ref,139500 ref, WO 2024/233809 PCT/US2024/028635 139600 ref,140300 ref,141000 ref,141700 ref,142400 ref,143100 ref,143800 ref, 139700 ref,140400 ref,141100 ref,141800 ref,142500 ref,143200 ref,143900 ref, 139800 ref,140500 ref,141200 ref,141900 ref,142600 ref,143300 ref,144000 ref, 139900 ref,140600 ref,141300 ref,142000 ref,142700 ref,143400 ref,144100 ref, 140000 ref,140700 ref,141400 ref,142100 ref,142800 ref,143500 ref,144200 ref, 140100 ref,140800 ref,141500 ref,142200 ref,142900 ref,143600 ref,144300 ref, 140200 ref,140900 ref,141600 ref,142300 ref,143000 ref,143700 ref,144400 ref,144500 ref, 144600 ref, 144700 ref, 144800 ref, 144900 ref, or at about 145000 ref) for about 90-150 minutes (e.g., 90 min, 91 min, 92 min, 93 min, 94 min, 95 min, 96 min, min, 98 min, 99 min, 100 min, 101 min, 102 min, 103 min, 104 min, 105 min, 106 min, 107 min, 108 min, 109 min, 110 min, 111 min, 112 min, 113 min, 114 min, 115 min, 1min, 117 min, 118 min, 119 min, 120 min, 121 min, 122 min, 123 min, 124 min, 125 min, 126 min, 127 min, 128 min, 129 min, 130 min, 131 min, 132 min, 133 min, 134 min, 1min, 136 min, 137 min, 138 min, 139 min, 140 min, 141 min, 142 min, 143 min, 144 min, 145 min, 146 min, 147 min, 148 min, 149 min, or about 150 min), the resulting fluid is discarded, and the remaining pellet is resuspended in a suitable volume of a suitable solution prior to (1). [0074]In certain example embodiments, the tangential flow filtration of (g) is performed using a 500 kDa ultrafiltration membrane. In certain example embodiments, the tangential flow filtration of (g) is performed using an ultrafiltration membrane with about a molecular weight cutoff ranging from about 250 kDa to about 750 kDa. In some embodiments the molecular weight cutoff of the ultrafiltration membrane with a molecular weight cutoff of about 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa,460kDa, 470kDa, 480kDa, 490kDa, 500kDa, 510kDa, 520kDa, 530kDa, 540kDa, 550kDa,560kDa, 570kDa, 580kDa, 590kDa, 600kDa, 610kDa, 620kDa, 630kDa, 640kDa, 650kDa, 660kDa, 670kDa, 680kDa, 690kDa, 700kDa, 710kDa, 720kDa, 730kDa, 740kDa, or about 750k Da. [0075]In certain example embodiments, the tangential flow filtration of step (g) is performed at a flow rate of about 10 mL per minute. In certain example embodiments, the tangential flow filtration of (g) is performed at a flow rate ranging from about 5 mL to about mL per minute. In certain example embodiments, the tangential flow filtration of (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, mL/min, 11.5 mL/min, 12 mL/min, 12.5 mL/min, 13 mL/min, 13.5 mL/min, 14 mL/min, WO 2024/233809 PCT/US2024/028635 14.5 mL/min, or about 15 mL/min. In certain example embodiments, in step (g), when the amount of remaining biological fluid reaches about ten percent of its starting volume before tangential flow filtration the retenate is diafiltered with a suitable buffer. [0076]In certain example embodiments, the method further includes ultracentrifuging the retenate when the retenate reaches about 20 percent of the starting diafiltration amount. In certain example embodiments, the ultracentrifugation of the retenate is performed at about 130,000 ref for about 120 at about 4 degrees Celsius. In certain example embodiments the ultracentrifugation of the retenate performed at about 115,000 to about 145,000 ref (e.g., 115000 ref, 115050 ref, 115100 ref, 115150 ref, 115200 ref, 115250 ref,115300 ref,115650 ref,116000 ref,116350 ref,116700 ref,117050 ref,117400 ref,117750 ref,118100 ref,118450 ref,118800 ref,119150 ref,119500 ref,119850 ref,120200 ref,120550 ref,120900 ref,121250 ref,121600 ref,121950 ref,122300 ref,122650 ref,123000 ref,123350 ref,123700 ref, 115350 ref,115700 ref,116050 ref,116400 ref,116750 ref,117100 ref,117450 ref,117800 ref,118150 ref,118500 ref,118850 ref,119200 ref,119550 ref,119900 ref,120250 ref,120600 ref,120950 ref,121300 ref,121650 ref,122000 ref,122350 ref,122700 ref,123050 ref,123400 ref,123750 ref, 115400 ref,115750 ref,116100 ref,116450 ref,116800 ref,117150 ref,117500 ref,117850 ref,118200 ref,118550 ref,118900 ref,119250 ref,119600 ref,119950 ref,120300 ref,120650 ref,121000 ref,121350 ref,121700 ref,122050 ref,122400 ref,122750 ref,123100 ref,123450 ref,123800 ref, 115450 ref,115800 ref,116150 ref,116500 ref,116850 ref,117200 ref,117550 ref,117900 ref,118250 ref,118600 ref,118950 ref,119300 ref,119650 ref,120000 ref,120350 ref,120700 ref,121050 ref,121400 ref,121750 ref,122100 ref,122450 ref,122800 ref,123150 ref,123500 ref,123850 ref, 115500 ref,115850 ref,116200 ref,116550 ref,116900 ref,117250 ref,117600 ref,117950 ref,118300 ref,118650 ref,119000 ref,119350 ref,119700 ref,120050 ref,120400 ref,120750 ref,121100 ref,121450 ref,121800 ref,122150 ref,122500 ref,122850 ref,123200 ref,123550 ref,123900 ref, 115550 ref,115900 ref,116250 ref,116600 ref,116950 ref,117300 ref,117650 ref,118000 ref,118350 ref,118700 ref,119050 ref,119400 ref,119750 ref,120100 ref,120450 ref,120800 ref,121150 ref,121500 ref,121850 ref,122200 ref,122550 ref,122900 ref,123250 ref,123600 ref,123950 ref, 115600 ref,115950 ref,116300 ref,116650 ref,117000 ref,117350 ref,117700 ref,118050 ref,118400 ref,118750 ref,119100 ref,119450 ref,119800 ref,120150 ref,120500 ref,120850 ref,121200 ref,121550 ref,121900 ref,122250 ref,122600 ref,122950 ref,123300 ref,123650 ref,124000 ref, WO 2024/233809 PCT/US2024/028635 124050 ref, 124100 ref, 124150 ref, 124200 ref, 124250 ref, 124300 ref, 124350 ref,124400 ref, 124450 ref, 124500 ref, 124550 ref, 124600 ref, 124650 ref, 124700 ref,124750 ref, 124800 ref, 124850 ref, 124900 ref, 124950 ref, 125000 ref, 125050 ref,125100 ref, 125150 ref, 125200 ref, 125250 ref, 125300 ref, 125350 ref, 125400 ref,125450 ref, 125500 ref, 125550 ref, 125600 ref, 125650 ref, 125700 ref, 125750 ref,125800 ref, 125850 ref, 125900 ref, 125950 ref, 126000 ref, 126050 ref, 126100 ref,126150 ref, 126200 ref, 126250 ref, 126300 ref, 126350 ref, 126400 ref, 126450 ref,126500 ref, 126550 ref, 126600 ref, 126650 ref, 126700 ref, 126750 ref, 126800 ref,126850 ref, 126900 ref, 126950 ref, 127000 ref, 127050 ref, 127100 ref, 127150 ref,127200 ref, 127250 ref, 127300 ref, 127350 ref, 127400 ref, 127450 ref, 127500 ref,127550 ref, 127600 ref, 127650 ref, 127700 ref, 127750 ref, 127800 ref, 127850 ref,127900 ref, 127950 ref, 128000 ref, 128050 ref, 128100 ref, 128150 ref, 128200 ref,128250 ref, 128300 ref, 128350 ref, 128400 ref, 128450 ref, 128500 ref, 128550 ref,128600 ref, 128650 ref, 128700 ref, 128750 ref, 128800 ref, 128850 ref, 128900 ref,128950 ref, 129000 ref, 129050 ref, 129100 ref, 129150 ref, 129200 ref, 129250 ref,129300 ref, 129350 ref, 129400 ref, 129450 ref, 129500 ref, 129550 ref, 129600 ref,129650 ref, 129700 ref, 129750 ref, 129800 ref, 129850 ref, 129900 ref, 129950 ref,130000 ref, 130050 ref, 130100 ref, 130150 ref, 130200 ref, 130250 ref, 130300 ref,130350 ref, 130400 ref, 130450 ref, 130500 ref, 130550 ref, 130600 ref, 130650 ref,130700 ref, 130750 ref, 130800 ref, 130850 ref, 130900 ref, 130950 ref, 131000 ref,131050 ref, 131100 ref, 131150 ref, 131200 ref, 131250 ref, 131300 ref, 131350 ref,131400 ref, 131450 ref, 131500 ref, 131550 ref, 131600 ref, 131650 ref, 131700 ref,131750 ref, 131800 ref, 131850 ref, 131900 ref, 131950 ref, 132000 ref, 132050 ref,132100 ref, 132150 ref, 132200 ref, 132250 ref, 132300 ref, 132350 ref, 132400 ref,132450 ref, 132500 ref, 132550 ref, 132600 ref, 132650 ref, 132700 ref, 132750 ref,132800 ref, 132850 ref, 132900 ref, 132950 ref, 133000 ref, 133050 ref, 133100 ref,133150 ref, 133200 ref, 133250 ref, 133300 ref, 133350 ref, 133400 ref, 133450 ref,133500 ref, 133550 ref, 133600 ref, 133650 ref, 133700 ref, 133750 ref, 133800 ref,133850 ref, 133900 ref, 133950 ref, 134000 ref, 134050 ref, 134100 ref, 134150 ref,134200 ref, 134250 ref, 134300 ref, 134350 ref, 134400 ref, 134450 ref, 134500 ref,134550 ref, 134600 ref, 134650 ref, 134700 ref, 134750 ref, 134800 ref, 134850 ref,134900 ref, 134950 ref, 135000 ref, 135050 ref, 135100 ref, 135150 ref, 135200 ref,135250 ref, 135300 ref, 135350 ref, 135400 ref, 135450 ref, 135500 ref, 135550 ref,135600 ref, 135650 ref, 135700 ref, 135750 ref, 135800 ref, 135850 ref, 135900 ref, WO 2024/233809 PCT/US2024/028635 135950 ref, 136000 ref, 136050 ref, 136100 ref, 136150 ref, 136200 ref, 136250 ref,136300 ref, 136350 ref, 136400 ref, 136450 ref, 136500 ref, 136550 ref, 136600 ref,136650 ref, 136700 ref, 136750 ref, 136800 ref, 136850 ref, 136900 ref, 136950 ref,137000 ref, 137050 ref, 137100 ref, 137150 ref, 137200 ref, 137250 ref, 137300 ref,137350 ref, 137400 ref, 137450 ref, 137500 ref, 137550 ref, 137600 ref, 137650 ref,137700 ref, 137750 ref, 137800 ref, 137850 ref, 137900 ref, 137950 ref, 138000 ref,138050 ref, 138100 ref, 138150 ref, 138200 ref, 138250 ref, 138300 ref, 138350 ref,138400 ref, 138450 ref, 138500 ref, 138550 ref, 138600 ref, 138650 ref, 138700 ref,138750 ref, 138800 ref, 138850 ref, 138900 ref, 138950 ref, 139000 ref, 139050 ref,139100 ref, 139150 ref, 139200 ref, 139250 ref, 139300 ref, 139350 ref, 139400 ref,139450 ref, 139500 ref, 139550 ref, 139600 ref, 139650 ref, 139700 ref, 139750 ref,139800 ref, 139850 ref, 139900 ref, 139950 ref, 140000 ref, 140050 ref, 140100 ref,140150 ref, 140200 ref, 140250 ref, 140300 ref, 140350 ref, 140400 ref, 140450 ref,140500 ref, 140550 ref, 140600 ref, 140650 ref, 140700 ref, 140750 ref, 140800 ref,140850 ref, 140900 ref, 140950 ref, 141000 ref, 141050 ref, 141100 ref, 141150 ref,141200 ref, 141250 ref, 141300 ref, 141350 ref, 141400 ref, 141450 ref, 141500 ref,141550 ref, 141600 ref, 141650 ref, 141700 ref, 141750 ref, 141800 ref, 141850 ref,141900 ref, 141950 ref, 142000 ref, 142050 ref, 142100 ref, 142150 ref, 142200 ref,142250 ref, 142300 ref, 142350 ref, 142400 ref, 142450 ref, 142500 ref, 142550 ref,142600 ref, 142650 ref, 142700 ref, 142750 ref, 142800 ref, 142850 ref, 142900 ref,142950 ref, 143000 ref, 143050 ref, 143100 ref, 143150 ref, 143200 ref, 143250 ref,143300 ref, 143350 ref, 143400 ref, 143450 ref, 143500 ref, 143550 ref, 143600 ref,143650 ref, 143700 ref, 143750 ref, 143800 ref, 143850 ref, 143900 ref, 143950 ref,144000 ref, 144050 ref, 144100 ref, 144150 ref, 144200 ref, 144250 ref, 144300 ref,144350 ref, 144400 ref, 144450 ref, 144500 ref, 144550 ref, 144600 ref, 144650 ref,144700 ref, 144750 ref, 144800 ref, 144850 ref, 144900 ref, 144950 ref, or aboutl450rd) for about 90 minutes to about 150 minutes e.g., (about 90 min, 91 min, 92 min, 93 min, min, 95 min, 96 min, 97 min, 98 min, 99 min, 100 min, 101 min, 102 min, 103 min, 1min, 105 min, 106 min, 107 min, 108 min, 109 min, 110 min, 111 min, 112 min, 113 min, 114 min, 115 min, 116 min, 117 min, 118 min, 119 min, 120 min, 121 min, 122 min, 1min, 124 min, 125 min, 126 min, 127 min, 128 min, 129 min, 130 min, 131 min, 132 min, 133 min, 134 min, 135 min, 136 min, 137 min, 138 min, 139 min, 140 min, 141 min, 1min, 143 min, 144 min, 145 min, 146 min, 147 min, 148 min, 149 min, or about 150 min) at about 4 degrees Celsius.

WO 2024/233809 PCT/US2024/028635 id="p-77"

[0077] In certain example embodiments, the retenate is not ultracentrifuged prior to optionally fractionating the retenate. In these embodiments, the retenate is stored at about -80° C prior to fractionation that is optionally performed via column separation. Optional fractionating of the retenate can be performed via any suitable method, including but not limited to column separation (based on size, charge, affinity, avidity, or other method or separation strategy). Fractions containing the extracellular vesicles can be kept. [0078]In certain example embodiments, the method yields a vesicle concentrate that is atleast7.5, 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 percent of the starting volume of biologic fluid, such as milk. In certain example embodiments, the method yields a vesicle concentrate that is about 7 percent, 7.5 percent, 8 percent, 8.5 percent, 9 percent, 9.5 percent, 10 percent, 10.5 percent, 11 percent, 11.5 percent, 12 percent, 12.5 percent, 13 percent, 13.5 percent, percent, 14.5 percent, 15 percent, 15.5 percent, 16 percent, 16.5 percent, 17 percent, 17.5 percent, 18 percent, 18.5 percent, 19 percent, 19.5 percent, or about 20 percent, of the starting volume of biologic fluid, such as milk.

Specific Contemplated Embodiments [0079]In addition to the aspects and embodiments contemplated elsewhere herein, the following particular embodiments are specifically contemplated. [0080] A TP-Analogue-VxtxaceWuXas Vesicle Embodiments1. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.01 and 1,000 pM ATP or an ATP analogue.2. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.1 and 100 pM ATP or an ATP analogue.3. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.01 and 1,000 pM ATP or an ATP analogue.4. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.1 and 100 pM ATP or an ATP analogue.5. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.01 and 1,000 pM ATP or an ATP analogue.6. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.1 and 100 pM ATP or an ATP analogue.

WO 2024/233809 PCT/US2024/028635 7. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise between 0.25 and 75 pM; 0.5 and 50 pM; or 0.75 and 25 pM; or 0.and 20 pM; or 0.85 and 15 pM; or 0.9 and 10 pM; or 0.5 and 5 pM; or 0.1 and 2 pM; or 0.5 and 1.5 pM; or 0.75 and 1.25 pM of ATP or an ATP analogue.8. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise between 0.1 and 5 pM of ATP or an ATP analogue.9. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise between 0.1 and 2 pM of ATP or an ATP analogue.10. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise between 0.1 pM and 1 pM of ATP or an ATP analogue.11. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise about 1 pM of ATP or an ATP analogue.12. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between l ng and 1,000 pg ATP per mg extracellular vesicle protein.13. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 10 ng and 100 pg ATP per mg extracellular vesicle protein.14. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 100 ng and 10 pg ATP per mg extracellular vesicle protein.15. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 200 ng and 1 pg ATP per mg extracellular vesicle protein.16. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 300 ng and 750 ng ATP per mg extracellular vesicle protein.17. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 400 ng and 650 ng ATP per mg extracellular vesicle protein.18. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 450 ng and 600 ng ATP per mg extracellular vesicle protein.19. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 500 ng and 550 ng ATP per mg extracellular vesicle protein. [0081]A composition comprising extracellular vesicles wherein said extracellular vesicles comprise about 1 ng per mg extracellular vesicle protein; 10 ng per mgextracellular vesicle protein; 100 ng per mg extracellular vesicle protein; 200 ng per mgextracellular vesicle protein; 300 ng per mg extracellular vesicle protein; 400 ng per mgextracellular vesicle protein; 450 ng per mg extracellular vesicle protein; 500 ng per mg WO 2024/233809 PCT/US2024/028635 extracellular vesicle protein; 550 ng per mg extracellular vesicle protein; 600 ng per mg extracellular vesicle protein; 650 ng per mg extracellular vesicle protein; 750 ng per mg extracellular vesicle protein; 1 pg per mg extracellular vesicle protein; 10 pg per mg extracellular vesicle protein; 100 pg per mg extracellular vesicle protein; or 1,000 pg per mg extracellular vesicle protein.20. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between l ng and 1,000 pg ATP per mg extracellular vesicle protein.21. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 10 ng and 100 pg ATP per mg extracellular vesicle protein.22. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 100 ng and 10 pg ATP per mg extracellular vesicle protein.23. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 200 ng and 1 pg ATP per mg extracellular vesicle protein.24. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 300 ng and 750 ng ATP per mg extracellular vesicle protein. 25. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 400 ng and 650 ng ATP per mg extracellular vesicle protein.26. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 450 ng and 600 ng ATP per mg extracellular vesicle protein. 27. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 500 ng and 550 ng ATP per mg extracellular vesicle protein. 28. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise about 1 ng per mg extracellular vesicle protein; 10 ng per mg extracellular vesicle protein; 100 ng per mg extracellular vesicle protein; 200 ng per mgextracellular vesicle protein; 300 ng per mg extracellular vesicle protein; 400 ng per mgextracellular vesicle protein; 450 ng per mg extracellular vesicle protein; 500 ng per mgextracellular vesicle protein; 550 ng per mg extracellular vesicle protein; 600 ng per mgextracellular vesicle protein; 650 ng per mg extracellular vesicle protein; 750 ng per mgextracellular vesicle protein; 1 pg per mg extracellular vesicle protein; 10 pg per mg extracellular vesicle protein; 100 pg per mg extracellular vesicle protein; or 1,000 pg per mg extracellular vesicle protein.

WO 2024/233809 PCT/US2024/028635 29. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between l ng and 1,000 pg ATP per mg extracellular vesicle protein.30. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 10 ng and 100 pg ATP per mg extracellular vesicle protein.31. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 100 ng and 10 pg ATP per mg extracellular vesicle protein.32. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 200 ng and 1 pg ATP per mg extracellular vesicle protein.33. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 300 ng and 750 ng ATP per mg extracellular vesicle protein.34. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 400 ng and 650 ng ATP per mg extracellular vesicle protein.35. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 450 ng and 600 ng ATP per mg extracellular vesicle protein.36. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 500 ng and 550 ng ATP per mg extracellular vesicle protein.37. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise about 1 ng per mg extracellular vesicle protein; 10 ng per mg extracellular vesicle protein; 100 ng per mg extracellular vesicle protein; 200 ng per mg extracellular vesicle protein; 300 ng per mg extracellular vesicle protein; 400 ng per mg extracellular vesicle protein; 450 ng per mg extracellular vesicle protein; 500 ng per mg extracellular vesicle protein; 550 ng per mg extracellular vesicle protein; 600 ng per mg extracellular vesicle protein; 650 ng per mg extracellular vesicle protein; 750 ng per mg extracellular vesicle protein; 1 pg per mg extracellular vesicle protein; 10 pg per mg WO 2024/233809 PCT/US2024/028635 extracellular vesicle protein; 100 pg per mg extracellular vesicle protein; or 1,000 pg per mg extracellular vesicle protein.38. The composition of any of the preceding embodiments, wherein said ATP or an ATP analogue comprises ATP.39. The composition of any of the preceding embodiments, wherein said ATP or an ATP analogue comprises a non- hydrolyzable analogue of ATP.40. The composition of any of the preceding embodiments, wherein said ATP or an ATP analogue comprises one or more selected from the group consisting of a,P־ methylene-ATP (a,PmATP); P,y-methylene-ATP (P,ymATP); 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); an N-alkyl-2 ATP; adenosine 5'-(P,y-imido)triphosphate (AMP-PNP); ATP-MgC12; 5-aminoimidazole-4-carboxamide ribonucleotide, disoproxil fumarate, ribavirin, azidothymidine, fludarabine, efavirenz and oxidized ATP (0ATP), Adenosine, ADP, AMP, and other nucleotides (e.g. GTP, CTP, UTP, TTP).41. The composition of any of the preceding embodiments, wherein said ATP or an ATP analogue comprises one or more selected from the group consisting Adenosine, ADP, and AMP.42. The composition of any of the preceding embodiments, wherein said ATP or an ATP analogue consists essentially of one or more selected from the group consisting Adenosine, ADP, and AMP.43.44. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition are between 10 and 10nm.45. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said 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.46. The composition of any of the preceding embodiments, wherein at least 75%, or 80%; or 90% or 95% of the particles are less than 600 nm.47. The composition of any of the preceding embodiments, wherein at least 95% of the particles are less than 600 nm.

WO 2024/233809 PCT/US2024/028635 48. The composition of any of the preceding embodiments, wherein at least 99% of the particles are less than 600 nm.49. The composition of 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.50. The composition of any of the preceding embodiments, wherein at least 95% of the particles are less than about 500 nm.51. The composition of any of the preceding embodiments, wherein at least 99% ofthe particles are less than about 500 nm.52. The composition of 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.53. The composition of any of the preceding embodiments, wherein at least 95% of the particles are less than about 250 nm.54. The composition of any of the preceding embodiments, wherein at least 99% of the particles are less than about 250 nm.55. The composition of 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.56. The composition of any of the preceding embodiments, wherein at least 95% of the particles are greater than 20 nm.57. The composition of any of the preceding embodiments, wherein at least 99% of the particles are greater than 20 nm.58. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 10 and 500 nm; or 10 and 300 nm; or 10 and 200 nm; or 10 and 185 nm, or 10 and 175 nm; or 10 and 1nm; or 10 and 165 nm; or 10 and 160 nm; or 10 and 155 nm; or 10 and 150 nm; or 10 and 145 nm; or 10 and 140 nm; or 10 and 135 nm; or 10 and 130 nm; or 15 and 1,000 nm; or and 500 nm; or 15 and 300 nm; or 15 and 200 nm; or 15 and 185 nm, or 15 and 1nm; or 15 and 170 nm; or 15 and 165 nm; or 15 and 160 nm; or 15 and 155 nm; or 15 and150 nm; or 15 and 145 nm; or 15 and 140 nm; or 15 and 135 nm; or 15 and 130 nm; or 20and 1,000 nm; or 20 and 500 nm; or 20 and 300 nm; or 20 and 200 nm; or 20 and 185 nm, or 20 and 175 nm; or 20 and 170 nm; or 20 and 165 nm; or 20 and 160 nm; or 20 and 155nm; or 20 and 150 nm; or 20 and 145 nm; or 20 and 140 nm; or 20 and 135 nm; or 20 and130 nm; or 25 and 1,000 nm; or 25 and 500 nm; or 25 and 300 nm; or 25 and 200 nm; or and 185 nm, or 25 and 175 nm; or 25 and 170 nm; or 25 and 165 nm; or 25 and 1nm; or 25 and 155 nm; or 25 and 150 nm; or 25 and 145 nm; or 25 and 140 nm; or 25 and WO 2024/233809 PCT/US2024/028635 135 nm; or 25 and 130 nm; or 30 and 1,000 nm; or 30 and 500 nm; or 30 and 300 nm; or and 200 nm; or 30 and 185 nm, or 30 and 175 nm; or 30 and 170 nm; or 30 and 1nm; or 30 and 160 nm; or 30 and 155 nm; or 30 and 150 nm; or 30 and 145 nm; or 30 and 140 nm; or 30 and 135 nm; or 30 and 130 nm; or 35 and 1,000 nm; or 35 and 500 nm; or and 300 nm; or 35 and 200 nm; or 35 and 185 nm, or 35 and 175 nm; or 35 and 1nm; or 35 and 165 nm; or 35 and 160 nm; or 35 and 155 nm; or 35 and 150 nm; or 35 and 145 nm; or 35 and 140 nm; or 35 and 135 nm; or 35 and 130 nm; or 40 and 1,000 nm; or and 500 nm; or 40 and 300 nm; or 40 and 200 nm; or 40 and 185 nm, or 40 and 1nm; or 40 and 170 nm; or 40 and 165 nm; or 40 and 160 nm; or 40 and 155 nm; or 40 and 150 nm; or 40 and 145 nm; or 40 and 140 nm; or 40 and 135 nm; or 40 and 130 nm; or and 1,000 nm; or 45 and 500 nm; or 45 and 300 nm; or 45 and 200 nm; or 45 and 185 nm, or 45 and 175 nm; or 45 and 170 nm; or 45 and 165 nm; or 45 and 160 nm; or 45 and 1nm; or 45 and 150 nm; or 45 and 145 nm; or 45 and 140 nm; or 45 and 135 nm; or 45 and 130 nm; or 50 and 1,000 nm; or 50 and 500 nm; or 50 and 300 nm; or 50 and 200 nm; or and 185 nm, or 50 and 175 nm; or 50 and 170 nm; or 50 and 165 nm; or 50 and 1nm; or 50 and 155 nm; or 50 and 150 nm; or 50 and 145 nm; or 50 and 140 nm; or 50 and 135 nm; or 50 and 130 nm; or 55 and 1,000 nm; or 55 and 500 nm; or 55 and 300 nm; or and 200 nm; or 55 and 185 nm, or 55 and 175 nm; or 55 and 170 nm; or 55 and 1nm; or 55 and 160 nm; or 55 and 155 nm; or 55 and 150 nm; or 55 and 145 nm; or 55 and 140 nm; or 55 and 135 nm; or 55 and 130 nm; or 60 and 1,000 nm; or 60 and 500 nm; or and 300 nm; or 60 and 200 nm; or 60 and 185 nm, or 60 and 175 nm; or 60 and 1nm; or 60 and 165 nm; or 60 and 160 nm; or 60 and 155 nm; or 60 and 150 nm; or 60 and 145 nm; or 60 and 140 nm; or 60 and 135 nm; or 60 and 130 nm; or 65 and 1,000 nm; or and 500 nm; or 65 and 300 nm; or 65 and 200 nm; or 65 and 185 nm, or 65 and 1nm; or 65 and 170 nm; or 65 and 165 nm; or 65 and 160 nm; or 65 and 155 nm; or 65 and150 nm; or 65 and 145 nm; or 65 and 140 nm; or 65 and 135 nm; or 65 and 130 nm; or 70and 1,000 nm; or 70 and 500 nm; or 70 and 300 nm; or 70 and 200 nm; or 70 and 185 nm, or 70 and 175 nm; or 70 and 170 nm; or 70 and 165 nm; or 70 and 160 nm; or 70 and 155nm; or 70 and 150 nm; or 70 and 145 nm; or 70 and 140 nm; or 70 and 135 nm; or 70 and130 nm; or 75 and 1,000 nm; or 75 and 500 nm; or 75 and 300 nm; or 75 and 200 nm; or and 185 nm, or 75 and 175 nm; or 75 and 170 nm; or 75 and 165 nm; or 75 and 1nm; or 75 and 155 nm; or 75 and 150 nm; or 75 and 145 nm; or 75 and 140 nm; or 75 and 135 nm; or 75 and 130 nm; or 90 and 1,000 nm; or 90 and 500 nm; or 90 and 300 nm; or and 200 nm; or 90 and 185 nm, or 90 and 175 nm; or 90 and 170 nm; or 90 and 165 WO 2024/233809 PCT/US2024/028635 nm; or 90 and 160 nm; or 90 and 155 nm; or 90 and 150 nm; or 90 and 145 nm; or 90 and 140 nm; or 90 and 135 nm; or 90 and 130 nm; or 95 and 1,000 nm; or 95 and 500 nm; or and 300 nm; or 95 and 200 nm; or 95 and 185 nm, or 95 and 175 nm; or 95 and 1nm; or 95 and 165 nm; or 95 and 160 nm; or 95 and 155 nm; or 95 and 150 nm; or 95 and 145 nm; or 95 and 140 nm; or 95 and 135 nm; or 95 and 130 nm; or 100 and 1,000 nm; or 100 and 500 nm; or 100 and 300 nm; or 100 and 200 nm; or 100 and 185 nm, or 100 and 175 nm; or 100 and 170 nm; or 100 and 165 nm; or 100 and 150 nm; or 100 and 155 nm; or 100 and 150 nm; or 100 and 145 nm; or 100 and 100 nm; or 100 and 135 nm; or 1and 130 nm; or 105 and 1,000 nm; or 105 and 500 nm; or 105 and 300 nm; or 105 and 200 nm; or 105 and 185 nm, or 105 and 175 nm; or 105 and 170 nm; or 105 and 165 nm; or 105 and 160 nm; or 105 and 155 nm; or 105 and 150 nm; or 105 and 145 nm; or 1and 140 nm; or 105 and 135 nm; or 105 and 130 nm; or 110 and 1,000 nm; or 110 and 500 nm; or 110 and 300 nm; or 110 and 200 nm; or 110 and 185 nm, or 110 and 175 nm; or 110 and 170 nm; or 110 and 165 nm; or 110 and 150 nm; or 110 and 155 nm; or 1and 150 nm; or 110 and 145 nm; or 110 and 100 nm; or 110 and 135 nm; or 110 and 1nm; or 115 and 1,000 nm; or 115 and 500 nm; or 115 and 300 nm; or 115 and 200 nm; or 115 and 185 nm, or 115 and 175 nm; or 115 and 170 nm; or 115 and 165 nm; or 115 and 160 nm; or 115 and 155 nm; or 115 and 150 nm; or 115 and 145 nm; or 115 and 140 nm; or 115 and 135 nm; or 115 and 130 nm; or 120 and 1,000 nm; or 120 and 500 nm; or 1and 300 nm; or 120 and 200 nm; or 120 and 185 nm, or 120 and 175 nm; or 120 and 1nm; or 120 and 165 nm; or 120 and 150 nm; or 120 and 155 nm; or 120 and 150 nm; or 120 and 145 nm; or 120 and 100 nm; or 120 and 135 nm; or 120 and 130 nm.59. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 50 and 500 nm. 60. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 100 and 200 nm. 61. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 100 and 200 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 2nm.62. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is about 150 nm.

WO 2024/233809 PCT/US2024/028635 63. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is about 150 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.64. The composition of any of the preceding embodiments wherein the extracellular vesicles are milk extracellular vesicles.65. The composition of any of the preceding embodiments wherein the extracellular vesicles are bovine milk extracellular vesicles.66. The composition of any of the preceding embodiments wherein the extracellular vesicles are bovine milk extracellular vesicles; and wherein the bovine milk extracellular vesicles comprise bovine IgG.67. The composition of any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles.68. The composition of any one of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles, and wherein the extracellular vesicles are isolated from milk using a method of any of the "Isolation of Extracellular Vesicles From Milk" embodiments.69. The composition of any of the preceding embodiments wherein the extracellular vesicles comprise one or more cargo molecules.70. The composition of any of the preceding embodiments wherein the composition is a pharmaceutically acceptable composition.71. A method of preparing an ATP-analogue-extracellular vesicle composition of any of the preceding embodiments, said method comprising contacting the extracellular vesicles with a solution comprising ATP and/or an ATP analogue to obtain a composition comprising ATP-analogue-extracellular vesicles.72. A method of increasing uptake by cells, tissues and/or organs of a subject by administering to said subject extracellular vesicle composition of any of the preceding embodiments.73. A method of treating a subject, comprising administering to said subject an extracellular vesicle composition of any of the preceding embodiments.74. A method of treating a subject, comprising administering to said subject an extracellular vesicle composition of any of the preceding embodiments by oral, enteral, parenteral, intranasal, IV, topical or direct administration.

WO 2024/233809 PCT/US2024/028635 75. A method of increasing uptake of extracellular vesicles by dermal tissues of a subject, said method comprising: administering by topical application to skin an extracellular vesicle composition of any of the preceding embodiments in said subject.76. A method of providing benefit in a healthy subject by administering extracellular vesicles loaded with one or more nutraceutical or cosmetical cargos (e.g. peptides, curcumin, flavonoids, amino acids, lipids, fatty acids, calcium, vitamins, ATP, creatine and so on) of a composition of any of the preceding embodiments to said subject.77. A method of enhancing cargo loading of extracellular vesicles with an external cargo molecule including a peptide, small drug molecule, or other cargo compound. id="p-81"

[0081]Isolation of Extracellular Vesicles From Milk Embodiments1. A method of isolating extracellular vesicles from a milk sample, wherein the method involves contacting (or incubating) the milk with tryptophan and/or one or more tryptophan analogues and subsequently separating the milk extracellular vesicles from one or more milk components (such as for example casein-containing aggregates).2. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM.3. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a temperature between 10 and 50 degrees Celsius for at least 10 minutes.4. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 50 degrees Celsius for at least 10 minutes.5. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 50 degrees Celsius for between 10 and 60 minutes.

WO 2024/233809 PCT/US2024/028635 6. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM at a concentration between 10 pM and 1 mM at a temperature between 10 and 50 degrees Celsius for at least 10 minutes.7. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM at a temperature between 10 and 50 degrees Celsius for between 10 and 60 minutes.8. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with acid treatment to a pH of 4.6 at a temperature between 10 and degrees Celsius for between 10 and 60 minutes.9. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with acid treatment to a pH between 2.0 and 6.0 at a temperature between and 50 degrees Celsius for between 10 and 60 minutes.10. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM in conjunction with acid treatment to a pH of 4.6 at a temperature between 10 and degrees Celsius for between 10 and 60 minutes.11. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM in conjunction with acid treatment to a pH between 2.0 and 6.0 at a temperature between and 50 degrees Celsius for between 10 and 60 minutes.

WO 2024/233809 PCT/US2024/028635 12. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with Chymosin treatment at a temperature between 10 and 50 degrees Celsius for between 10 and 60 minutes13. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a temperature between 10 and 80 degrees Celsius for at least 10 seconds.14. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 80 degrees Celsius for at least 10 seconds.15. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 80 degrees Celsius for between 10 seconds and 60 minutes.16. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM at a temperature between 10 and 80 degrees Celsius for at least 10 seconds.17. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM at a temperature between 10 and 80 degrees Celsius for between 10 seconds and 60 minutes.18. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in WO 2024/233809 PCT/US2024/028635 conjunction with acid treatment to a pH of 4.6 at a temperature between 10 and degrees Celsius for between 10 seconds and 60 minutes.19. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with acid treatment to a pH between 2.0 and 6.0 at a temperature between and 80 degrees Celsius for between 10 seconds and 60 minutes.20. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM in conjunction with acid treatment to a pH of 4.6 at a temperature between 10 and degrees Celsius for between 10 seconds and 60 minutes.21. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM in conjunction with acid treatment to a pH between 2.0 and 6.0 at a temperature between and 80 degrees Celsius for between 10 seconds and 60 minutes.22. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with Chymosin treatment at a temperature between 10 and 80 degrees Celsius for between 10 seconds and 60 minutes.23. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues and/or Trehalose and/or Trehalose analogues at a temperature between 10 and 60 degrees Celsius for at least 10 minutes.24. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 60 degrees Celsius for at least 10 minutes.

WO 2024/233809 PCT/US2024/028635 . A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM at a temperature between 10 and 60 degrees Celsius for between 10 and 60 minutes.26. A method of isolating extracellular vesicles from a milk sample, wherein the methodcomprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM at a temperature between 10 and 60 degrees Celsius for at least 10 minutes.27. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM at a temperature between 10 and 60 degrees Celsius for between 10 and 60 minutes.28. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with acid treatment to a pH of 4.6 at a temperature between 10 and degrees Celsius for between 10 and 60 minutes.29. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with acid treatment to a pH between 2.0 and 6.0 at a temperature between and 60 degrees Celsius for between 10 and 60 minutes.30. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM in conjunction with acid treatment to a pH of 4.6 at a temperature between 10 and degrees Celsius for between 10 and 60 minutes.31. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM and/or WO 2024/233809 PCT/US2024/028635 Trehalose and/or Trehalose analogues at a concentration between 1 mM and 100 mM in conjunction with acid treatment to a pH between 2.0 and 6.0 at a temperature between and 60 degrees Celsius for between 10 and 60 minutes.32. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises contacting (incubating) the milk extracellular vesicles with tryptophan and/or more tryptophan analogues at a concentration between 10 pM and 1 mM in conjunction with Chymosin treatment at a temperature between 10 and 60 degrees Celsius for between 10 and 60 minutes.33. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition are between 10 and 1000 nm.34. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said 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.35. The method of any of the preceding embodiments, wherein at least 75%, or 80%; or 90% or 95% of the particles are less than 600 nm.36. The method of any of the preceding embodiments, wherein at least 95% of the particles are less than 600 nm.37. The method of any of the preceding embodiments, wherein at least 99% of the particles are less than 600 nm.38. The method of 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.39. The method of any of the preceding embodiments, wherein at least 95% of the particles are less than about 500 nm.40. The method of any of the preceding embodiments, wherein at least 99% of the particles are less than about 500 nm.41. The method of 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.42. The method of any of the preceding embodiments, wherein at least 95% of the particles are less than about 250 nm.43. The method of any of the preceding embodiments, wherein at least 99% of the particles are less than about 250 nm.44. The method of 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.

WO 2024/233809 PCT/US2024/028635 45. The method of any of the preceding embodiments, wherein at least 95% of theparticles are greater than 20 nm.46. The method of any of the preceding embodiments, wherein at least 99% of theparticles are greater than 20 nm.47. The method of any of the preceding embodiments, wherein the average particlesize of the extracellular vesicles in said composition is between 10 and 500 nm; or 10 and 300 nm; or 10 and 200 nm; or 10 and 185 nm, or 10 and 175 nm; or 10 and 170 nm; or and 165 nm; or 10 and 160 nm; or 10 and 155 nm; or 10 and 150 nm; or 10 and 145 nm; or 10 and 140 nm; or 10 and 135 nm; or 10 and 130 nm; or 15 and 1,000 nm; or 15 and 500 nm; or 15 and 300 nm; or 15 and 200 nm; or 15 and 185 nm, or 15 and 175 nm; or and 170 nm; or 15 and 165 nm; or 15 and 160 nm; or 15 and 155 nm; or 15 and 150 nm; or 15 and 145 nm; or 15 and 140 nm; or 15 and 135 nm; or 15 and 130 nm; or 20 and 1,000 nm; or 20 and 500 nm; or 20 and 300 nm; or 20 and 200 nm; or 20 and 185 nm, or and 175 nm; or 20 and 170 nm; or 20 and 165 nm; or 20 and 160 nm; or 20 and 1nm; or 20 and 150 nm; or 20 and 145 nm; or 20 and 140 nm; or 20 and 135 nm; or 20 and 130 nm; or 25 and 1,000 nm; or 25 and 500 nm; or 25 and 300 nm; or 25 and 200 nm; or and 185 nm, or 25 and 175 nm; or 25 and 170 nm; or 25 and 165 nm; or 25 and 1nm; or 25 and 155 nm; or 25 and 150 nm; or 25 and 145 nm; or 25 and 140 nm; or 25 and 135 nm; or 25 and 130 nm; or 30 and 1,000 nm; or 30 and 500 nm; or 30 and 300 nm; or and 200 nm; or 30 and 185 nm, or 30 and 175 nm; or 30 and 170 nm; or 30 and 1nm; or 30 and 160 nm; or 30 and 155 nm; or 30 and 150 nm; or 30 and 145 nm; or 30 and 140 nm; or 30 and 135 nm; or 30 and 130 nm; or 35 and 1,000 nm; or 35 and 500 nm; or and 300 nm; or 35 and 200 nm; or 35 and 185 nm, or 35 and 175 nm; or 35 and 1nm; or 35 and 165 nm; or 35 and 160 nm; or 35 and 155 nm; or 35 and 150 nm; or 35 and 145 nm; or 35 and 140 nm; or 35 and 135 nm; or 35 and 130 nm; or 40 and 1,000 nm; or and 500 nm; or 40 and 300 nm; or 40 and 200 nm; or 40 and 185 nm, or 40 and 1nm; or 40 and 170 nm; or 40 and 165 nm; or 40 and 160 nm; or 40 and 155 nm; or 40 and 150 nm; or 40 and 145 nm; or 40 and 140 nm; or 40 and 135 nm; or 40 and 130 nm; or and 1,000 nm; or 45 and 500 nm; or 45 and 300 nm; or 45 and 200 nm; or 45 and 185 nm, or 45 and 175 nm; or 45 and 170 nm; or 45 and 165 nm; or 45 and 160 nm; or 45 and 1nm; or 45 and 150 nm; or 45 and 145 nm; or 45 and 140 nm; or 45 and 135 nm; or 45 and 130 nm; or 50 and 1,000 nm; or 50 and 500 nm; or 50 and 300 nm; or 50 and 200 nm; or and 185 nm, or 50 and 175 nm; or 50 and 170 nm; or 50 and 165 nm; or 50 and 1nm; or 50 and 155 nm; or 50 and 150 nm; or 50 and 145 nm; or 50 and 140 nm; or 50 and WO 2024/233809 PCT/US2024/028635 135 nm; or 50 and 130 nm; or 55 and 1,000 nm; or 55 and 500 nm; or 55 and 300 nm; or and 200 nm; or 55 and 185 nm, or 55 and 175 nm; or 55 and 170 nm; or 55 and 1nm; or 55 and 160 nm; or 55 and 155 nm; or 55 and 150 nm; or 55 and 145 nm; or 55 and 140 nm; or 55 and 135 nm; or 55 and 130 nm; or 60 and 1,000 nm; or 60 and 500 nm; or and 300 nm; or 60 and 200 nm; or 60 and 185 nm, or 60 and 175 nm; or 60 and 1nm; or 60 and 165 nm; or 60 and 160 nm; or 60 and 155 nm; or 60 and 150 nm; or 60 and 145 nm; or 60 and 140 nm; or 60 and 135 nm; or 60 and 130 nm; or 65 and 1,000 nm; or and 500 nm; or 65 and 300 nm; or 65 and 200 nm; or 65 and 185 nm, or 65 and 1nm; or 65 and 170 nm; or 65 and 165 nm; or 65 and 160 nm; or 65 and 155 nm; or 65 and 150 nm; or 65 and 145 nm; or 65 and 140 nm; or 65 and 135 nm; or 65 and 130 nm; or and 1,000 nm; or 70 and 500 nm; or 70 and 300 nm; or 70 and 200 nm; or 70 and 185 nm, or 70 and 175 nm; or 70 and 170 nm; or 70 and 165 nm; or 70 and 160 nm; or 70 and 1nm; or 70 and 150 nm; or 70 and 145 nm; or 70 and 140 nm; or 70 and 135 nm; or 70 and 130 nm; or 75 and 1,000 nm; or 75 and 500 nm; or 75 and 300 nm; or 75 and 200 nm; or and 185 nm, or 75 and 175 nm; or 75 and 170 nm; or 75 and 165 nm; or 75 and 1nm; or 75 and 155 nm; or 75 and 150 nm; or 75 and 145 nm; or 75 and 140 nm; or 75 and 135 nm; or 75 and 130 nm; or 90 and 1,000 nm; or 90 and 500 nm; or 90 and 300 nm; or and 200 nm; or 90 and 185 nm, or 90 and 175 nm; or 90 and 170 nm; or 90 and 1nm; or 90 and 160 nm; or 90 and 155 nm; or 90 and 150 nm; or 90 and 145 nm; or 90 and 140 nm; or 90 and 135 nm; or 90 and 130 nm; or 95 and 1,000 nm; or 95 and 500 nm; or and 300 nm; or 95 and 200 nm; or 95 and 185 nm, or 95 and 175 nm; or 95 and 1nm; or 95 and 165 nm; or 95 and 160 nm; or 95 and 155 nm; or 95 and 150 nm; or 95 and 145 nm; or 95 and 140 nm; or 95 and 135 nm; or 95 and 130 nm; or 100 and 1,000 nm; or 100 and 500 nm; or 100 and 300 nm; or 100 and 200 nm; or 100 and 185 nm, or 100 and 175 nm; or 100 and 170 nm; or 100 and 165 nm; or 100 and 150 nm; or 100 and 155 nm; or 100 and 150 nm; or 100 and 145 nm; or 100 and 100 nm; or 100 and 135 nm; or 1and 130 nm; or 105 and 1,000 nm; or 105 and 500 nm; or 105 and 300 nm; or 105 and 200 nm; or 105 and 185 nm, or 105 and 175 nm; or 105 and 170 nm; or 105 and 165 nm; or 105 and 160 nm; or 105 and 155 nm; or 105 and 150 nm; or 105 and 145 nm; or 1and 140 nm; or 105 and 135 nm; or 105 and 130 nm; or 110 and 1,000 nm; or 110 and 500 nm; or 110 and 300 nm; or 110 and 200 nm; or 110 and 185 nm, or 110 and 175 nm; or 110 and 170 nm; or 110 and 165 nm; or 110 and 150 nm; or 110 and 155 nm; or 1and 150 nm; or 110 and 145 nm; or 110 and 100 nm; or 110 and 135 nm; or 110 and 1nm; or 115 and 1,000 nm; or 115 and 500 nm; or 115 and 300 nm; or 115 and 200 nm; or WO 2024/233809 PCT/US2024/028635 115 and 185 nm, or 115 and 175 nm; or 115 and 170 nm; or 115 and 165 nm; or 115 and 160 nm; or 115 and 155 nm; or 115 and 150 nm; or 115 and 145 nm; or 115 and 140 nm; or 115 and 135 nm; or 115 and 130 nm; or 120 and 1,000 nm; or 120 and 500 nm; or 1and 300 nm; or 120 and 200 nm; or 120 and 185 nm, or 120 and 175 nm; or 120 and 1nm; or 120 and 165 nm; or 120 and 150 nm; or 120 and 155 nm; or 120 and 150 nm; or 120 and 145 nm; or 120 and 100 nm; or 120 and 135 nm; or 120 and 130 nm.48. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 50 and 500 nm.49. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 100 and 200 nm.50. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 100 and 200 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.51. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is about 150 nm.52. The method of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is about 150 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.53. The method of any of the preceding embodiments wherein the extracellular vesicles are milk extracellular vesicles.54. The method of any of the preceding embodiments wherein the extracellular vesicles are bovine milk extracellular vesicles.55. The method of any of the preceding embodiments wherein the extracellular vesicles are bovine milk extracellular vesicles; and wherein the bovine milk extracellular vesicles comprise bovine IgG.56. The method of any of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles.57. The method of any one of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles, and wherein the extracellular vesicles are isolated from milk using a method of any of the "Isolation of Extracellular Vesicles From Milk" embodiments.58. The method of any of the preceding embodiments wherein the extracellular vesicles comprise one or more cargo molecules.

WO 2024/233809 PCT/US2024/028635 59. The method of any of the preceding embodiments wherein the composition is a pharmaceutically acceptable composition.60. The method of any of the preceding embodiments, wherein the Tryptophan or Tryptophan analog consists of one or more from the list including L-Tryptophan, D- Tryptophan, Tryptophan, Tryptophan dimers, Tryptophan Trimers, Tryptophan peptides, Histidine, L-Histidine, D-Histidine, Histidine Dimers, Histidine Trimers, Histidine peptides, Tyrosine, L-Tyrosine, D-Tyrosine, Tyrosine Dimers, Tyrosine Trimers, Tyrosine peptides, Arginine, L-Arginine, D-Arginine, Arginine Dimers, Arginine Trimers, Arginine peptides, Cysteine, L-Cysteine, D-Cysteine, Cysteine Dimers, Cysteine Trimers, Cysteine peptides, Lysine, L-Lysine, D-Lysine, Lysine Dimers, Lysine Trimers, Lysine Peptides, Phenylalanine, L- Phenylalanine, D-Phenylalanine, Phenylalanine Dimers, Phenylalanine Trimers, Phenylalanine peptides D-Carnitine, L-Carnitine, Carnitine dimers, Carnitine trimers, Carnitine peptides, Acetyl L-Carnitine, L-Carnitine L-tartrate, Propionyl-L-carnitine, D-Taurine, L-Taurine, Taurine dimers, Taurine trimers, Taurine peptides id="p-82"

[0082]Lyophilization oJMilk^xtxaceWxxXax Vesicles Embodiments1. A composition comprising extracellular vesicles wherein said composition comprises above 0.01 pM Tryptophan or Tryptophan Analog.2. A composition comprising extracellular vesicles wherein said composition comprises above 0.001% W/W% Tryptophan or Tryptophan analog.3. A composition comprising extracellular vesicles wherein said composition comprises between 0.01 pM and 100 mM Tryptophan or Tryptophan analog.4. A composition comprising extracellular vesicles wherein said composition comprises between 0.001% W/W% and 90% W/W% Tryptophan or Tryptophan analog. 5. A composition comprising extracellular vesicles wherein said composition comprises between 0.1 pM and 10 mM Tryptophan or Tryptophan analog.6. A composition comprising extracellular vesicles wherein said composition comprises between 0.01% W/W% and 50% W/W% Tryptophan or Tryptophan analog.7. A composition comprising extracellular vesicles wherein said composition comprises between 1 pM and 1 mM Tryptophan or Tryptophan analog.8. A composition comprising extracellular vesicles wherein said composition comprises between 0.1% W/W% and 10% W/W% Tryptophan or Tryptophan analog.

WO 2024/233809 PCT/US2024/028635 9. A composition comprising extracellular vesicles wherein said composition comprises between 10 pM and 100 pM Tryptophan or Tryptophan analog.10. A composition comprising extracellular vesicles wherein said composition comprises between 0.2% W/W% and 1% W/W% Tryptophan or Tryptophan analog.11. A composition comprising milk extracellular vesicles wherein said composition comprises above 0.01 pM Tryptophan or Tryptophan Analog.12. A composition comprising milk extracellular vesicles wherein said composition comprises above 0.001% W/W% Tryptophan or Tryptophan analog.13. A composition comprising milk extracellular vesicles wherein said composition comprises between 0.01 pM and 100 mM Tryptophan or Tryptophan analog.14. A composition comprising milk extracellular vesicles wherein said composition comprises between 0.001% W/W% and 90% W/W% Tryptophan or Tryptophan analog.15. A composition comprising milk extracellular vesicles wherein said composition comprises between 0.1 pM and 10 mM Tryptophan or Tryptophan analog.16. A composition comprising milk extracellular vesicles wherein said composition comprises between 0.01% W/W% and 50% W/W% Tryptophan or Tryptophan analog.17. A composition comprising milk extracellular vesicles wherein said composition comprises between 1 pM and 1 mM Tryptophan or Tryptophan analog.18. A composition comprising milk extracellular vesicles wherein said composition comprises between 0.1% W/W% and 10% W/W% Tryptophan or Tryptophan analog.19. A composition comprising milk extracellular vesicles wherein said composition comprises between 10 pM and 100 pM Tryptophan or Tryptophan analog.20. A composition comprising milk extracellular vesicles wherein said composition comprises between 0.2% W/W% and 1% W/W% Tryptophan or Tryptophan analog.21. A composition comprising bovine milk extracellular vesicles wherein said composition comprises above 0.01 pM Tryptophan or Tryptophan Analog.22. A composition comprising bovine milk extracellular vesicles wherein said composition comprises above 0.001% W/W% Tryptophan or Tryptophan analog.23. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 0.01 pM and 100 mM Tryptophan or Tryptophan analog.24. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 0.001% W/W% and 90% W/W% Tryptophan or Tryptophan analog.

WO 2024/233809 PCT/US2024/028635 . A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 0.1 pM and 10 mM Tryptophan or Tryptophan analog. 26. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 0.01% W/W% and 50% W/W% Tryptophan or Tryptophan analog.27. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 1 pM and 1 mM Tryptophan or Tryptophan analog.28. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 0.1% W/W% and 10% W/W% Tryptophan or Tryptophan analog.29. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 10 pM and 100 pM Tryptophan or Tryptophan analog.30. A composition comprising bovine milk extracellular vesicles wherein said composition comprises between 0.2% W/W% and 1% W/W% Tryptophan or Tryptophan analog.31. The composition of any of the preceding embodiments, wherein said composition comprises between 0.1 pM and 1 mM; or 1.0 pM and 750 pM; or 10 pM and 500 pM; or pM and 300 pM; or 40 pM and 200 pM; or 50 pM and 150 pM Tryptophan or a Tryptophan analog.32. The composition of any of the preceding embodiments, wherein said composition comprises about 100 pM Tryptophan or a Tryptophan analog.33. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise above 0.01 pM Tryptophan or Tryptophan Analog.34. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise above 0.001% W/W% Tryptophan or Tryptophan analog.35. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.01 pM and 100 mM Tryptophan or Tryptophan analog.36. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.001% W/W% and 90% W/W% Tryptophan or Tryptophan analog.37. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.1 pM and 10 mM Tryptophan or Tryptophan analog.

WO 2024/233809 PCT/US2024/028635 38. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.01% W/W% and 50% W/W% Tryptophan or Tryptophan analog.39. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 1 pM and 1 mM Tryptophan or Tryptophan analog.40. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.1% W/W% and 10% W/W% Tryptophan or Tryptophan analog.41. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 10 pM and 100 pM Tryptophan or Tryptophan analog.42. A composition comprising extracellular vesicles wherein said extracellular vesicles comprise between 0.2% W/W% and 1% W/W% Tryptophan or Tryptophan analog.43. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise above 0.01 pM Tryptophan or Tryptophan Analog.44. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise above 0.001% W/W% Tryptophan or Tryptophan analog.45. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.01 pM and 100 mM Tryptophan or Tryptophan analog.46. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.001% W/W% and 90% W/W% Tryptophan or Tryptophan analog.47. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.1 pM and 10 mM Tryptophan or Tryptophan analog.48. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.01% W/W% and 50% W/W% Tryptophan or Tryptophan analog.49. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 1 pM and 1 mM Tryptophan or Tryptophan analog.50. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.1% W/W% and 10% W/W% Tryptophan or Tryptophan analog.51. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 10 pM and 100 pM Tryptophan or Tryptophan analog.

WO 2024/233809 PCT/US2024/028635 52. A composition comprising milk extracellular vesicles wherein said extracellular vesicles comprise between 0.2% W/W% and 1% W/W% Tryptophan or Tryptophan analog.53. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise above 0.01 pM Tryptophan or Tryptophan Analog.54. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise above 0.001% W/W% Tryptophan or Tryptophan analog.55. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.01 pM and 100 mM Tryptophan or Tryptophan analog.56. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.001% W/W% and 90% W/W% Tryptophan or Tryptophan analog.57. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.1 pM and 10 mM Tryptophan or Tryptophan analog.58. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.01% W/W% and 50% W/W% Tryptophan or Tryptophan analog.59. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 1 pM and 1 mM Tryptophan or Tryptophan analog.60. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.1% W/W% and 10% W/W% Tryptophan or Tryptophan analog.61. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 10 pM and 100 pM Tryptophan or Tryptophan analog.62. A composition comprising bovine milk extracellular vesicles wherein said extracellular vesicles comprise between 0.2% W/W% and 1% W/W% Tryptophan or Tryptophan analog.63. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise between 0.1 pM and 1 mM; or 1.0 pM and 750 pM; or 10 pM and 5 WO 2024/233809 PCT/US2024/028635 uM; or 20 pM and 300 uM; or 40 pM and 200 uM; or 50 pM and 150 pM Tryptophan or a Tryptophan analog.64. The composition of any of the preceding embodiments, wherein said extracellular vesicles comprise about 100 pM Tryptophan or a Tryptophan analog.65. The composition of any of the preceding embodiments, wherein said Tryptophan or Tryptophan analog comprises one or more selected from the group consisting of L- Tryptophan, D-Tryptophan, Tryptophan, Tryptophan dimers, Tryptophan Trimerstrimers, Tryptophan peptides, Histidine, L-Histidine, D-Histidine, Histidine Dimersdimers, Histidine Trimerstrimers, Histidine peptides, Tyrosine, L-Tyrosine, D-Tyrosine, Tyrosine Dimersdimers, Tyrosine Trimerstrimers, Tyrosine peptides, Arginine, L-Arginine, D- Arginine, Arginine Dimersdimers, Arginine Trimerstrimers, Arginine peptides, Cysteine, L-Cysteine, D-Cysteine, Cysteine Dimersdimers, Cysteine Trimerstrimers, Cysteine peptides, Lysine, L-Lysine, D-Lysine, Lysine Dimersdimers, Lysine Trimerstrimers, Lysine Peptides, Phenylalanine, L- Phenylalanine, D-Phenylalanine, Phenylalanine Dimersdimers, Phenylalanine Trimerstrimers, Phenylalanine peptides, D-Carnitine, L- Carnitine, Carnitine dimers, Carnitine trimers, Carnitine peptides, Acetyl L-Carnitine, L- Carnitine L-tartrate, Propionyl-L-carnitine, D-Taurine, L-Taurine, Taurine dimers, Taurine trimers, Taurine peptides66. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition are between 10 and 10nm.67. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said 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.68. The composition of any of the preceding embodiments, wherein at least 75%, or 80%; or 90% or 95% of the particles in said composition are less than 600 nm.69. The composition of any of the preceding embodiments, wherein at least 95% of the particles in said composition are less than 600 nm.70. The composition of any of the preceding embodiments, wherein at least 99% of the particles in said composition are less than 600 nm.71. The composition of any of the preceding embodiments, wherein at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the particles in said composition are less than about 500 nm.

WO 2024/233809 PCT/US2024/028635 72. The composition of any of the preceding embodiments, wherein at least 95% of the particles in said composition are less than about 500 nm.73. The composition of any of the preceding embodiments, wherein at least 99% of the particles in said composition are less than about 500 nm.74. The composition of any of the preceding embodiments, wherein at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the particles in said composition are less than about 250 nm.75. The composition of any of the preceding embodiments, wherein at least 95% of the particles in said composition are less than about 250 nm.76. The composition of any of the preceding embodiments, wherein at least 99% of the particles in said composition are less than about 250 nm.77. The composition of any of the preceding embodiments, wherein at least 75%, or 80%; or 90%, or 95%, or 98%, or 99% of the particles in said composition are greater than 20 nm.78. The composition of any of the preceding embodiments, wherein at least 95% of the particles in said composition are greater than 20 nm.79. The composition of any of the preceding embodiments, wherein at least 99% of the particles in said composition are greater than 20 nm.80. The composition of any of the preceding embodiments, wherein the average particle size of the extracellular vesicles in said composition is between 10 and 500 nm; or 10 and 300 nm; or 10 and 200 nm; or 10 and 185 nm, or 10 and 175 nm; or 10 and 1nm; or 10 and 165 nm; or 10 and 160 nm; or 10 and 155 nm; or 10 and 150 nm; or 10 and 145 nm; or 10 and 140 nm; or 10 and 135 nm; or 10 and 130 nm; or 15 and 1,000 nm; or and 500 nm; or 15 and 300 nm; or 15 and 200 nm; or 15 and 185 nm, or 15 and 1nm; or 15 and 170 nm; or 15 and 165 nm; or 15 and 160 nm; or 15 and 155 nm; or 15 and150 nm; or 15 and 145 nm; or 15 and 140 nm; or 15 and 135 nm; or 15 and 130 nm; or 20and 1,000 nm; or 20 and 500 nm; or 20 and 300 nm; or 20 and 200 nm; or 20 and 185 nm, or 20 and 175 nm; or 20 and 170 nm; or 20 and 165 nm; or 20 and 160 nm; or 20 and 155nm; or 20 and 150 nm; or 20 and 145 nm; or 20 and 140 nm; or 20 and 135 nm; or 20 and130 nm; or 25 and 1,000 nm; or 25 and 500 nm; or 25 and 300 nm; or 25 and 200 nm; or and 185 nm, or 25 and 175 nm; or 25 and 170 nm; or 25 and 165 nm; or 25 and 1nm; or 25 and 155 nm; or 25 and 150 nm; or 25 and 145 nm; or 25 and 140 nm; or 25 and 135 nm; or 25 and 130 nm; or 30 and 1,000 nm; or 30 and 500 nm; or 30 and 300 nm; or and 200 nm; or 30 and 185 nm, or 30 and 175 nm; or 30 and 170 nm; or 30 and 1 WO 2024/233809 PCT/US2024/028635 nm; or 30 and 160 nm; or 30 and 155 nm; or 30 and 150 nm; or 30 and 145 nm; or 30 and 140 nm; or 30 and 135 nm; or 30 and 130 nm; or 35 and 1,000 nm; or 35 and 500 nm; or and 300 nm; or 35 and 200 nm; or 35 and 185 nm, or 35 and 175 nm; or 35 and 1nm; or 35 and 165 nm; or 35 and 160 nm; or 35 and 155 nm; or 35 and 150 nm; or 35 and 145 nm; or 35 and 140 nm; or 35 and 135 nm; or 35 and 130 nm; or 40 and 1,000 nm; or and 500 nm; or 40 and 300 nm; or 40 and 200 nm; or 40 and 185 nm, or 40 and 1nm; or 40 and 170 nm; or 40 and 165 nm; or 40 and 160 nm; or 40 and 155 nm; or 40 and 150 nm; or 40 and 145 nm; or 40 and 140 nm; or 40 and 135 nm; or 40 and 130 nm; or and 1,000 nm; or 45 and 500 nm; or 45 and 300 nm; or 45 and 200 nm; or 45 and 185 nm, or 45 and 175 nm; or 45 and 170 nm; or 45 and 165 nm; or 45 and 160 nm; or 45 and 1nm; or 45 and 150 nm; or 45 and 145 nm; or 45 and 140 nm; or 45 and 135 nm; or 45 and 130 nm; or 50 and 1,000 nm; or 50 and 500 nm; or 50 and 300 nm; or 50 and 200 nm; or and 185 nm, or 50 and 175 nm; or 50 and 170 nm; or 50 and 165 nm; or 50 and 1nm; or 50 and 155 nm; or 50 and 150 nm; or 50 and 145 nm; or 50 and 140 nm; or 50 and 135 nm; or 50 and 130 nm; or 55 and 1,000 nm; or 55 and 500 nm; or 55 and 300 nm; or and 200 nm; or 55 and 185 nm, or 55 and 175 nm; or 55 and 170 nm; or 55 and 1nm; or 55 and 160 nm; or 55 and 155 nm; or 55 and 150 nm; or 55 and 145 nm; or 55 and 140 nm; or 55 and 135 nm; or 55 and 130 nm; or 60 and 1,000 nm; or 60 and 500 nm; or and 300 nm; or 60 and 200 nm; or 60 and 185 nm, or 60 and 175 nm; or 60 and 1nm; or 60 and 165 nm; or 60 and 160 nm; or 60 and 155 nm; or 60 and 150 nm; or 60 and 145 nm; or 60 and 140 nm; or 60 and 135 nm; or 60 and 130 nm; or 65 and 1,000 nm; or and 500 nm; or 65 and 300 nm; or 65 and 200 nm; or 65 and 185 nm, or 65 and 1nm; or 65 and 170 nm; or 65 and 165 nm; or 65 and 160 nm; or 65 and 155 nm; or 65 and150 nm; or 65 and 145 nm; or 65 and 140 nm; or 65 and 135 nm; or 65 and 130 nm; or 70and 1,000 nm; or 70 and 500 nm; or 70 and 300 nm; or 70 and 200 nm; or 70 and 185 nm, or 70 and 175 nm; or 70 and 170 nm; or 70 and 165 nm; or 70 and 160 nm; or 70 and 155nm; or 70 and 150 nm; or 70 and 145 nm; or 70 and 140 nm; or 70 and 135 nm; or 70 and130 nm; or 75 and 1,000 nm; or 75 and 500 nm; or 75 and 300 nm; or 75 and 200 nm; or and 185 nm, or 75 and 175 nm; or 75 and 170 nm; or 75 and 165 nm; or 75 and 1nm; or 75 and 155 nm; or 75 and 150 nm; or 75 and 145 nm; or 75 and 140 nm; or 75 and 135 nm; or 75 and 130 nm; or 90 and 1,000 nm; or 90 and 500 nm; or 90 and 300 nm; or and 200 nm; or 90 and 185 nm, or 90 and 175 nm; or 90 and 170 nm; or 90 and 1nm; or 90 and 160 nm; or 90 and 155 nm; or 90 and 150 nm; or 90 and 145 nm; or 90 and 140 nm; or 90 and 135 nm; or 90 and 130 nm; or 95 and 1,000 nm; or 95 and 500 nm; or WO 2024/233809 PCT/US2024/028635 95 and 300 nm; or 95 and 200 nm; or 95 and 185 nm, or 95 and 175 nm; or 95 and 1nm; or 95 and 165 nm; or 95 and 160 nm; or 95 and 155 nm; or 95 and 150 nm; or 95 and 145 nm; or 95 and 140 nm; or 95 and 135 nm; or 95 and 130 nm; or 100 and 1,000 nm; or 100 and 500 nm; or 100 and 300 nm; or 100 and 200 nm; or 100 and 185 nm, or 100 and 175 nm; or 100 and 170 nm; or 100 and 165 nm; or 100 and 150 nm; or 100 and 155 nm; or 100 and 150 nm; or 100 and 145 nm; or 100 and 100 nm; or 100 and 135 nm; or 1and 130 nm; or 105 and 1,000 nm; or 105 and 500 nm; or 105 and 300 nm; or 105 and 200 nm; or 105 and 185 nm, or 105 and 175 nm; or 105 and 170 nm; or 105 and 165 nm; or 105 and 160 nm; or 105 and 155 nm; or 105 and 150 nm; or 105 and 145 nm; or 1and 140 nm; or 105 and 135 nm; or 105 and 130 nm; or 110 and 1,000 nm; or 110 and 500 nm; or 110 and 300 nm; or 110 and 200 nm; or 110 and 185 nm, or 110 and 175 nm; or 110 and 170 nm; or 110 and 165 nm; or 110 and 150 nm; or 110 and 155 nm; or 1and 150 nm; or 110 and 145 nm; or 110 and 100 nm; or 110 and 135 nm; or 110 and 1nm; or 115 and 1,000 nm; or 115 and 500 nm; or 115 and 300 nm; or 115 and 200 nm; or 115 and 185 nm, or 115 and 175 nm; or 115 and 170 nm; or 115 and 165 nm; or 115 and 160 nm; or 115 and 155 nm; or 115 and 150 nm; or 115 and 145 nm; or 115 and 140 nm; or 115 and 135 nm; or 115 and 130 nm; or 120 and 1,000 nm; or 120 and 500 nm; or 1and 300 nm; or 120 and 200 nm; or 120 and 185 nm, or 120 and 175 nm; or 120 and 1nm; or 120 and 165 nm; or 120 and 150 nm; or 120 and 155 nm; or 120 and 150 nm; or 120 and 145 nm; or 120 and 100 nm; or 120 and 135 nm; or 120 and 130 nm.81. The composition of any of the preceding embodiments, wherein the averageparticle size of the extracellular vesicles in said composition is between 50 and 500 nm. 82. The composition of any of the preceding embodiments, wherein the averageparticle size of the extracellular vesicles in said composition is between 100 and 200 nm. 83. The composition of any of the preceding embodiments, wherein the averageparticle size of the extracellular vesicles in said composition is between 100 and 200 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 2nm.84. The composition of any of the preceding embodiments, wherein the averageparticle size of the extracellular vesicles in said composition is about 150 nm.85. The composition of any of the preceding embodiments, wherein the averageparticle size of the extracellular vesicles in said composition is about 150 nm, and wherein 95% of the extracellular vesicles have a particle size between 100 and 200 nm.

WO 2024/233809 PCT/US2024/028635 86. The composition of any of the preceding embodiments wherein the extracellular vesicles in said composition are milk extracellular vesicles.87. The composition of any of the preceding embodiments wherein the vesicles in said composition are bovine milk extracellular vesicles.88. The composition of any of the preceding embodiments wherein the extracellular vesicles in said composition are bovine milk extracellular vesicles; and wherein the bovine milk extracellular vesicles comprise bovine IgG.89. The composition of any of the preceding embodiments, wherein the extracellular vesicles in said composition are milk extracellular vesicles.90. The composition of any one of the preceding embodiments, wherein the extracellular vesicles in said composition are milk extracellular vesicles, wherein the extracellular vesicles are isolated.91. The composition of any one of the preceding embodiments, wherein the extracellular vesicles are milk extracellular vesicles, and wherein the extracellular vesicles are isolated from milk using a method of any of the "Isolation of Extracellular Vesicles From Milk" embodiments.92. The composition of any of the preceding embodiments wherein the extracellular vesicles comprise one or more cargo molecules.93. The composition of any of the preceding embodiments wherein the composition is a pharmaceutically acceptable composition.94. The composition of any of the preceding embodiments wherein the composition also comprises Trehalose or a Trehalose analog at about 50 mM concentration.95. The composition of any of the preceding embodiments wherein the Trehalose or Trehalose analog consists of one or more from the list including Trehalose, Sucrose, Lactose, Glucose, Disaccharide molecules.96. The composition of any of the preceding embodiments wherein the composition is then frozen and lyophilized, or dried.97. A method of lyophilizing drying bovine milk extracellular vesicles supplemented with Trehalose and Tryptophan.98. A composition of lyophilized dried milk extracellular vesicles loaded with cargo molecules and supplemented with Trehalose or a Trehalose analog in addition to Tryptophan or a Tryptophan analog.99. A method of contacting extracellular vesicles with Trehalose and Tryptophan wherein said extracellular vesicles are dried and/or frozen and lyophilized.

WO 2024/233809 PCT/US2024/028635 100. A method of contacting extracellular vesicles with Trehalose and Tryptophan wherein said extracellular vesicles are frozen and lyophilized.101. A composition of extracellular vesicles loaded with cargo molecules listed herein and supplemented with Trehalose or a Trehalose analog in addition to Tryptophan or a Tryptophan analog.

EXAMPLES id="p-83"

[0083]The present disclosure will be further described in the following examples, which do not limit the scope of the present disclosure. id="p-84"

[0084] Example 1: Isolationof Extracellular Vesicles From Milk Using Tryptophan. [0085]Raw bovine milk was collected at 4C and defatted under industry standard conditions (centrifuged at 5,000xg to remove fat), resulting in skim milk. Half of the skim milk was treated with an FDA approved method of pasteurization. The two batches of skim milk were then subjected to 2.5 pm filtration and 0.8 pm filtration, then the permeate was treated with 150 pM tryptophan for 10 minutes on ice, while the retenate was discarded. The solution was then filtered through 0.45 pm and 0.22 pm filters, with the retenate being discarded following each filtration. The solution was then filtered on a 500 kDa filter until the retenate volume was 20% of the starting volume, and this solution was stored. This sample was then separated using a 70 nm sepharose column. Fractions 7-9 were collected and pooled, representing a purified milk extracellular vesicle population. [0086]This solution was then used for in depth analysis and experimentation including a number of standard and unique assays. For example, the protein was analyzed using Nanodrop for protein quantification, and Western blotting for protein markers. Protein is also analyzed using single molecule localization microscopy for protein markers and subpopulation analysis, but also gain insights into size and concentration using this approach; and SMLM approaches were validated using Nanoparticle Tracking Analysis for size and concentration investigation. We also utilize Zetasizer measurement for zeta potential, Transmission Electron Microscopy for visualization, confocal microscopy for investigation of vesicle patency, and in vitro bioactivity measurements of vesicles using an automated electrical wounding protocol (ECIS).

WO 2024/233809 PCT/US2024/028635 id="p-87"

[0087]ECIS is "Electric Cell-Substrate Impedance Sensing"- protocol used provided: The cellular monolayer’s confluence was measured in real time with an electric cell- substrate impedance sensing (ECIS) instrument (Applied Biophysics, NY, USA) to monitor their migratory response. Briefly, 8W1E or 96W1E+ electrode ECIS arrays were coated with lOmM L-cysteine, followed by 1% gelatin according to manufacturer guidelines. Electrodes were then stabilized in HuDF complete media, and subsequently seeded with HuDF cells at a density of IxlO5 cells/well. Cells were incubated for 8-hours to achieve at least 80% confluency, which was confirmed by previously performed growth-phase profiles. 8 hours before wound induction, media was changed to complete media supplemented with 10 ug/mL mitomycin C to limit cell proliferation. A 25 kHz, 2600 pA, 20 second electric wound was then applied for cells and impedance was monitored until cell migration was complete. Cell migration rate was determined by a custom Python script that tracked changes in impedance over time. The impedance of cell-covered electrodes was measured with the multiple frequencies over time (MFT) mode at eleven frequencies between 62.5 to 64000 Hz (62.5, 125, 250, 500, 1000, 2000, 4000, 8000,16000, 32000, 64000). Cell monolayers were treated with mEVs at optimized concentrations determined via UV-Vis intensity at 260/280nm. Each result was normalized to a control well present in each electrode array. [0088]Nanodrop analysis was performed using the Nanodrop 2000 as per the manufacturer’s instructions under 260/280 wavelength stimulation to quantify protein expression in the sample. Basic western blotting protocols are followed using antibodies including CD81, CD9, Calnexin, Syntenin-1, TSG-101, Pannexin-1, Connexin-43, Lactadherin and Casein. Single Molecule Localization Microscopy was performed using the EV profiler kit using procedures from the kits instructions, with antibodies including CD81, CD9, CD63, Pannexin-1, P2X7 and Connexin-43. Nanoparticle Tracking analysis was performed on a Nanosight NS300 using a dilution of 1:10,000 in a standard buffer solution and analyzed using a 405 nm laser. Zetasizer measurements were performed on a Horiba Scientific SZ-100V2 nanoparticle analyzer using the manufacturer’s instructions. Transmission electron microscopy was performed using formvar coated copper grids coated with 0.1% lysine. Samples were adhered to grids for 10 minutes then counterstained with uranlyess solution for 1 minute, then left overnight prior to imaging. Confocal microscopy was performed on milk vesicles loaded with Calcein AM dye on a Leica SP8. Vesicles were administered to standard microscope slides prior to coverslipping and imaging at 63x under 488 nm laser stimulation. In vitro bioactivity WO 2024/233809 PCT/US2024/028635 measurements were performed using an electric cell-substrate impedance sensing (ECIS) instrument from Applied Biophysics. [0089] Example 2: Isolation of Extracellular Vesicles From Milk Using Tryptophan and EDTA. [0090]Raw bovine milk was collected at 4C and defatted under industry standard conditions (centrifuged at 5,000xg to remove fat), resulting in skim milk. Skim milk was then subjected to 1.2 pm filtration, then the permeate was treated with 30 mM EDTA and 150 pM tryptophan for 30 minutes at room (ambient, 20C) temperature, while the retenate was discarded. The solution was then filtered through 0.45 pm and 0.22 pm filters, with the retenate being discarded following each filtration. The solution was then filtered on a 500 kDa filter until the retenate volume was 20% of the starting volume, and this solution was stored. This sample was then separated using a 70 nm sepharose column. Fractions 7- were collected and pooled, representing a purified milk extracellular vesicle population. Optionally, this sample was then buffered with 50 mM trehalose and lyophilized as described in example 7. This solution was then used for in depth analysis and experimentation, as described in Example 1. [0091] Example 3: Isolation of Extracellular Vesicles From Milk Using Tryptophan and acid pH [0092]Raw bovine milk was collected at 4C and defatted under industry standard conditions resulting in skim milk as in example 1. Skim milk was then subjected to 1.pm filtration, then the permeate was treated with 150 pM tryptophan for 60 minutes at room (ambient, 20°C) temperature, while the retenate was discarded. The permeate was then acidified to pH 4.6 by addition of HCL or citric acid, which was then removed as a pellet by centrifugation at 5,000xg, or alternatively may be removed by raising temperature to 45-50 degrees Celsius for 5-15 minutes to form a hard curd precipitate, which may then be strained from the solution. The supernatant solution following pelleting at 5,000 xg was then filtered through 0.45 and 0.22 filters, with the retenate being discarded following each filtration. The solution was then filtered on a 500 kDa filter until the retenate volume was 20% of the starting volume, and this solution was stored. This sample was then separated using a 70 nm sepharose column. Fractions 7-were collected and pooled, representing a purified milk extracellular vesicle population. Optionally, this sample was then buffered with 50 mM Trehalose and lyophilized as described in example 7. This solution was then used for in depth analysis and experimentation, as described in Example 1.

WO 2024/233809 PCT/US2024/028635 id="p-93"

[0093] Example 4: Isolation of Extracellular Vesicles from Milk Using Tryptophan, Acid pH, and Temperature [0094]Raw bovine milk was collected at 4C and defatted under industry standard conditions resulting in skim milk as in examples 1 and 3. Skim milk was then treated with 150 pM Tryptophan and was acidified by addition of citric acid, sulfuric acid, or hydrochloric acid and treated at 5 5 °C for 30 minutes to form a hard curd precipitate, which was then strained from the solution. The supernatant solution following curd formation was then filtered through 0.45 and 0.22 micron filters, with the retenate being discarded following each filtration, and the permeate being collected. The permeate solution was then filtered on a 500 kDa filter until the retenate volume was -20% of the starting volume, and this solution was stored. This sample was then separated on a 70 nm sepharose column. Fractions 7-9 were collected and pooled, representing a purified milk extracellular vesicle population. Optionally, this sample was then buffered with 50 mM Trehalose and 100 pM Tryptophan and lyophilized as described in example 7. This solution was then used for in depth analysis and experimentation, as described in Example 1. [0095] Example 5: ATP-Milk Extracellular Vesicles. [0096]Milk vesicles, or extracellular vesicles, or exosomes as collected by examples described herein or as described previously (US patent application for isolation?) were collected and incubated with adenosine triphosphate (ATP) at concentrations including 1nm, 500 nm, 1.0 pM, 2.0 pM, 5.0 pM, 10 pM, and 50 pM for 30 minutes at 37°C. Optimal concentrations of ATP for enhanced bioactivity were found at 2.0 pM and 1.0 pM, with final ATP concentrations in the milk vesicle composition ranging from 100 nM to 10 pM.. [0097] Example 6: Assessment of ATP-Extracellular Vesicles. [0098]ATP-Extracellular Vesicle combinations were then analyzed using a number of methods. Bioactivity was first measured using an in vitro analysis procedure. Cells used were human dermal fibroblasts (HuDF; ATCC, PCS-201-012) as well as two lineages of Madin Darby Canine Kidney (MDCK) Cells- one being Cx43 enhanced (Cx43+), and the other being Cx43 reduced (Cx43-). Medium used for HuDF was DMEM HG (4.5 g/L Glucose) with 2% Normal Calf Serum (NCS; Thermo Fisher/Gibco, 16010-159) and 4% Fetal Bovine Serum (FBS; Thermo Fisher/Gibco, 26140-079). Medium used for the Cx43+ MDCK cells was M199 (Millipore Sigma M4530) supplemented with 10% FBS and 1% Hygromycin B (Sigma H0654), while Cx43- MDCKs used Ml99 with 10% FBS and 1:1Sodium Pyruvate (Invitrogen 11360-070). Cells were expanded to confluency, then WO 2024/233809 PCT/US2024/028635 passaged onto sterile coverslips in a 12-well plate, then given 2 days to adhere and grow prior to assay. Assay was performed by using a sterile pipette tip to scratch the surface of the cells, then cells were rinsed lx in dPBS (Invitrogen) and provided fresh culture medium supplemented with CTDR-tagged milk EVs. Cells were given 15 minutes to take up ATP- Vesicle combinations, then were rinsed in lx dPBS and fixed in 2% Paraformaldehyde (Fisher 04042-500). Cells were rinsed 4x in PBS, then stained in 1:20,000 Hoechst (Life Technologies, H3569) and rinsed one additional time in PBS. Coverslips were then removed and adhered to microscope slide, and imaged on a Leica SP8. Images were analyzed in Image! by saving the individual red channel and converting to 8-bit, thresholding, and counting particle numbers. [0099]ATP-Extracellular Vesicles may also be analyzed by Mass-Spectrometry for ATP concentrations to validate the presence of ATP within the EVs. Samples can be prepared by using trichloroacetic acid/acetone steps from a 2-D clean up kit from Bio-Rad. Following protein precipitation, samples are resolubilized in 10 uL 8 M urea, 50 mM NH4HCO3, reduced and alkylated using dithiothritol and iodoacetamide, then digested for three hours at room temperature using endo-Lys-C. Samples are then diluted to 2M urea using 50 mM NH4HCO3, and digestion is continued overnight by the addition of sequencing-grade modified trypsin from Promega. Samples are then acidified by 2% formic acid, desalted on pre-equilibrated columns, and washed using an EASY nLC-1200 system. Column is maintained at constant temperature and interfaced online. Peptides may be eluted from the column using a binary gradient that steps from 5% solvent to 95% solvent over mins. MS data is acquired by recording full scan spectra. [0100]Extracellular Vesicle concentrations of ATP may be readily determined in a number of manners- there are a wide range of ATP concentration determination assays, with the following example provided as the basic mechanism of ATP detection in a solution. The first step is to administer firefly luciferase enzyme and substrate to activate luciferin with present ATP, which then gives luciferyl-adenylate and pyrophosphate. The following chemical reaction results in a photoelectric stimulation of oxyluciferin, which emits a green/yellow luminescent signal (550-570 nm). The solution luminescence is then read using a luminometer, and is sensitive to concentrations of ATP <50 pg/mL. Other methods utilize fluorescent capture, which enables longer term signal capture periods, and are additionally viable methods to detect and measure ATP concentration.

WO 2024/233809 PCT/US2024/028635 id="p-101"

[0101] Example 7: Lyophilization of Milk Extracellular Vesicles. Milk extracellular vesicles, or extracellular vesicles, or exosomes as collected by examples described herein or as described previously (for example as in WO2022182782) were collected and incubated in a number of chemical excipients. These include concentrations of 10 mM, 50 mM and 200 mM of Sucrose, lactose, Trehalose, TMAO and tryptophan, as well as mixtures thereof, prior to freezing, in order to result in a mixture with a glass transition temperature between 40 and 80°C. Samples were frozen at -80°C and subjected to shelf lyophilization at a condenser temperature of -100C under a vacuum pressure of 10 mTorr. Additionally, methods used for lyophilizing a composition of vesicles include A) freezing the composition by either cooling at a controlled rate of 1° C per minute to a frozen temperature, or; flash freezing the composition using liquid nitrogen, dry ice, or other flash freezing method. B) Primary Drying the composition by adjusting temperature and pressure according to the specific container and constitution of the composition, such adjustments being within the realm of common knowledge for those skilled in the art. C) Secondary Drying the composition by further reducing the moisture content of the composition by modulating temperature and vacuum conditions post-sublimation, and; Tailoring the secondary drying phase parameters, including temperature and pressure ramping, based on the specific properties of the container and the composition, such tailoring being obvious to those skilled in the art. D) Vacuum capping samples post-lyophilization. Dried samples were stored at room temperature or at 4°C under vacuum or protected from moisture by desiccant. This solution was then used for in depth analysis and experimentation including a number of standard and unique assays. We first analyze protein using Nanodrop for protein quantification, then use Nanoparticle Tracking Analysis for size and concentration investigation. We also utilize Zetasizer measurement for zeta potential, Transmission Electron Microscopy for visualization, confocal microscopy for investigation of vesicle patency, and in vitro bioactivity measurements using an automated electrical wounding protocol. Optimized formulation for lyophilization protection of milk extracellular vesicles was found to be mM Trehalose buffered with 100 pM Tryptophan, enabling 6+ month shelf stability of our formulations. LC-MS analysis of samples will elucidate the presence of Tryptophan, a novel excipient in the lyophilization of milk extracellular vesicles.

WO 2024/233809 PCT/US2024/028635 id="p-102"

[0102] Example 8: Storage Materials & Methods for storage stability testing of Lyophilized Milk Extracellular Vesicles [0103]Isolated milk extracellular vesicles, or extracellular vesicles, or exosomes loaded with actll peptide using the protocols previously described were lyophilized in the presence of 50mM Trehalose or Trehalose analog and 100 pM Tryptophan or Tryptophan analog and stored in a closed container with desiccant at room temperature. At given time points, samples were reconstituted using an equal volume of deionized H20 to the original amount of mEV volume within the composition. Equivalent doses of each sample were added to a single ECIS cell with a final volume of 400 uL post-wound as previously described in section 83. At 0 months and 6 months, samples were analyzed via NT A and particles/mL were compared. [0104]While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein. [0105]The term "about" or "approximately" as used herein means within an acceptable error range for the 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, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to ±%, or up to ± 10 %, or up to ± 5 %, or up to ± 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 2-fold, of a value. For the avoidance of doubt, a value described herein with an "about" modifier specifically includes the stated value itself, for example if a value is expressed as "about 10" - the value of exactly 10 is specifically contemplated. As used herein, ranges can be expressed as from "about" one particular value, or "about" one value to "about" another particular value. It is also understood that each unit between two particular units are also disclosed. For example, if the range of "10- 15" is disclosed, then 11, 12, 13, and 14 are also disclosed. [0106]All references referred to in the present disclosure are hereby incorporated by reference in their entirety. Various embodiments of the present disclosure may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims WO 2024/233809 PCT/US2024/028635 form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public. [0107]The embodiments of the disclosure described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present disclosure as defined in any appended claims.

Claims (19)

WO 2024/233809 PCT/US2024/028635 CLAIMS What is claimed is:

1. A composition comprising extracellular vesicles, wherein said extracellular vesicles comprise between 0.1 and 2 pM ATP or an ATP analogue.

2. The composition of claim 1, wherein said extracellular vesicles comprise between 0.5 and 1.5 pM ATP or an ATP analogue.

3. The composition of claim 2, wherein said extracellular vesicles comprise between 0.5 and 1.5 pM ATP.

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

5. The composition of claim 4, wherein said 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 that is greater than 20 nm and less than 600 nm.

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

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

10. The composition of claim 9, wherein cargo molecules comprise one or more molecule types selected from the group consisting of a nucleic acid, a polypeptide, a carbohydrate and a steroid.

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

12. The method of claim 11, wherein said composition is administered to said subject orally, intranasally, parenterally, or intravenously. WO 2024/233809 PCT/US2024/028635

13. A method of isolating extracellular vesicles from a milk sample, wherein the method comprises (i) contacting the milk with tryptophan or a tryptophan analog at a concentration between 50 pM and 300 pM for between 10 and minutes at a pH between 2.0 and 8.0 and a temperature between 10 degrees Celsius and 80 degrees Celsius, and (ii) separating said extracellular vesicles from other components of 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 either raw orpasteurized.

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

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

18. The composition of Claim 17 wherein the composition is lyophilized by either flash freezing or controlled freeze, followed by primary drying, secondary drying, and vacuum capping of samples.

19. A composition comprising Lyophilized milk extracellular vesicles obtained from the composition of Claim 18, supplemented with 100 pM Tryptophan and 50 mM Trehalose