Milk-derived exosomes and their use

Abstract

This specification describes milk exosomes and their use. In some embodiments, the milk exosomes can target damaged sites or cancer cells or populations thereof. The milk exosomes can contain exogenous cargo. Also described herein are formulations containing milk exosomes and, optionally, stimuli of IgG or ATP concentration, and / or targeting agents such as ADP. Further described herein is a method of delivering cargo to a target.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 389,744, entitled "MILK DERIVED EXOSMES AND USES THEREOF", filed on July 15, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant Numbers HL161237, HL141855, and HL056728 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application includes a sequence listing submitted in electronic format as an xml file named "VTIP - 0390WP_ST26.xml" with a size of 1,753 bytes, created on July 14, 2023. The content of this sequence listing is hereby incorporated by reference in its entirety.

[0004] The subject matter disclosed herein generally relates to milk - derived exosomes and their uses.

Background Art

[0005] Small extracellular vesicles (sEVs), such as exosomes, are produced from the phospholipid bilayer of almost all cells in the body and are present in large quantities in milk. Milk exosomes, such as bovine milk exosomes, can carry cargos such as therapeutic cargos. Milk exosomes provide a medium for oral drug delivery. sEVs, such as exosomes, are absorbed systemically and can pass through barriers intact. See, for example, Betker, Jamie L., et al. “The Potential of Exosomes from Cow Milk for Oral Delivery.” Journal of Pharmaceutical Sciences, vol. 108, no. 4, 2019, pp. 1496 - 1505; Manca, Sonia et al. “Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns.” Scientific reports vol. 8, 1 11321. 27 Jul 2018; and Munagala, Radha et al. “Bovine milk-derived exosomes for drug delivery.” Cancer letters vol. 371, 1 (2016): 48 - 61. doi:10.1016 / j.canlet.2015.10.020. However, it is still unclear how milk exosomes are able to do this and, furthermore, how such mechanisms can be used or modified for the development of clinically relevant delivery vehicles.

[0006] The citation or identification of any reference in this application does not constitute an admission that such reference is available as prior art to the present disclosure.

Summary of the Invention

[0007] Certain exemplary embodiments of the present specification describe milk exosomes containing exogenous cargo, which can target damaged sites or cancer cells or cancer cell populations by targeting and / or stimulating ATP secretion and / or concentration and / or the neonatal Fc receptor (FcRN).

[0008] In certain exemplary embodiments, the damage is mechanical damage or non - mechanical damage. In certain exemplary embodiments, the non - mechanical damage is chemical damage, electrical damage, radiation damage, inflammatory damage, or ischemic damage. In certain exemplary embodiments, the mechanical damage is a wound or a burn.

[0009] In certain exemplary embodiments, the damaged site has a higher concentration of ATP than the non - damaged site.

[0010] In certain exemplary embodiments, cancer cells or a cancer cell population have a microenvironment with a higher ATP concentration than the microenvironment of non - cancer cells or a non - cancer cell population.

[0011] In certain exemplary embodiments, cancer cells or a cancer cell population secrete ATP.

[0012] In certain exemplary embodiments, the milk exosomes do not contain an exogenous targeting moiety.

[0013] In certain exemplary embodiments, the milk exosomes contain connexin 43. In certain exemplary embodiments, the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43.

[0014] In certain exemplary embodiments, the exogenous cargo is a biomolecule. In certain exemplary embodiments, the biomolecule is a polypeptide, a peptide, or a nucleic acid. In certain exemplary embodiments, the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof. In certain exemplary embodiments, the milk exosome is a bovine milk exosome.

[0015] In certain exemplary embodiments, prior to containing the exogenous cargo, the milk exosome is separated by a method comprising: (a) centrifuging mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk; (b) removing the fat separated from the mammalian milk; (c) after step (b), centrifuging the remaining mammalian milk one or more times and skimming off the separated fat visible after each centrifugation in step (c); (d) filtering the biological fluid remaining after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); (f) optionally chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30 to 42 °C for about 15 to 120 minutes after (d) or optionally (e); and (g) optionally performing tangential flow filtration after (f) to obtain a retentate, wherein the retentate is optionally ultracentrifuged via one or more ultracentrifugation steps or stored at -80 °C, and after the retentate is optionally ultracentrifuged or stored at -80 °C, optionally separating the fractions of the retentate via a column, the method including step (e) or step (g), but not both.

[0016] Certain exemplary embodiments of the present specification describe a population of milk exosomes comprising one or more milk exosomes of the present disclosure described in more detail elsewhere herein. In certain exemplary embodiments, the population is enriched for connexin 43-positive milk exosomes. In certain exemplary embodiments, prior to containing exogenous cargo, the milk exosomes are (a) centrifuged mammalian milk under conditions suitable for separating fat from one or more other components of mammalian milk, (b) removing the fat separated from mammalian milk, (c) after step (b), centrifuging the remaining mammalian milk one or more times and skimming off the separated fat visible after each centrifugation in step (c), (d) filtering the biological fluid remaining after step (c), (e) optionally performing one or more ultracentrifugation steps after (d), (f) after (d) or optionally (e), and optionally chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30 to 42 °C for about 15 to 120 minutes, and (g) after (f), optionally performing tangential flow filtration to obtain a retentate, obtaining the retentate by ultracentrifuging the retentate through one or more ultracentrifugation steps or storing it at -80 °C, and after the retentate is optionally ultracentrifuged or stored at -80 °C, optionally separating fractions of the retentate through a column, separated by a method comprising, the method includes step (e) or step (g), but not both. In certain exemplary embodiments, the method further comprises enriching the milk exosomes for connexin 43-expressing milk exosomes. In certain exemplary embodiments, the milk exosomes do not contain exogenous targeting moieties. In certain exemplary embodiments, the exogenous cargo is a biomolecule. In certain exemplary embodiments, the biomolecule is a polypeptide, peptide, or nucleic acid. In certain exemplary embodiments, the exogenous cargo is an ACT1 peptide, ACT11 peptide, ACT minus peptide, selectide, or any combination thereof.

[0017] Certain exemplary embodiments of the present specification describe pharmaceutical formulations comprising the milk exosomes of the present disclosure or a population thereof, as described in more detail elsewhere in the present specification, and a pharmaceutically acceptable carrier. In certain exemplary embodiments, the pharmaceutical formulation further comprises a pharmaceutical agent capable of stimulating the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof. In certain exemplary embodiments, the pharmaceutical formulation is a dosage form capable of releasing a pharmaceutical agent capable of stimulating the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof, prior to the milk exosomes or a population thereof, thereby delivering a pharmaceutical agent capable of stimulating the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof, prior to the milk exosomes.

[0018] In certain exemplary embodiments, the pharmaceutical formulation further comprises one or more immunoglobulins. In certain exemplary embodiments, the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE. In certain exemplary embodiments, the pharmaceutical formulation is a dosage form capable of releasing immunoglobulins prior to the milk exosomes or a population thereof, thereby delivering immunoglobulins prior to the milk exosomes.

[0019] In certain exemplary embodiments of the present specification, a method for treating an injury at an injury site in a subject in need thereof or a method for treating cancer is described. The method comprises administering to a subject in need thereof a pharmaceutical formulation comprising the milk exosomes of the present disclosure or a population thereof described in more detail elsewhere in the present specification, or the milk exosomes of the present disclosure or a population thereof described in more detail elsewhere in the present specification. In certain exemplary embodiments, the milk exosomes are bovine milk exosomes. In certain exemplary embodiments, the milk exosomes do not contain an exogenous targeting moiety. In certain exemplary embodiments, the milk exosomes comprise connexin 43. In certain exemplary embodiments, the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43. In certain exemplary embodiments, the exogenous cargo is a biomolecule. In certain exemplary embodiments, the biomolecule is a polypeptide, a peptide, or a nucleic acid. In certain exemplary embodiments, the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

[0020] In certain exemplary embodiments, the method further comprises administering to a subject in need thereof an amount of one or more immunoglobulins. In certain exemplary embodiments, administering to a subject in need thereof an amount of one or more immunoglobulins is performed prior to administering an amount of a pharmaceutical formulation comprising the milk exosomes of the present disclosure or a population thereof, or the milk exosomes of the present disclosure or a population thereof, as described in more detail elsewhere herein. In certain exemplary embodiments, the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE. In certain exemplary embodiments, the one or more immunoglobulins consist of or comprise IgG. In certain exemplary embodiments, the one or more immunoglobulins increase the uptake of intracellular milk exosomes or a population thereof. In certain exemplary embodiments, the cell is a polarized cell. In certain exemplary embodiments, the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a germ cell, a cancer cell, or any combination thereof. In certain exemplary embodiments, the method further comprises administering to a subject in need thereof one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP. In certain exemplary embodiments, administering to a subject in need thereof one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP is performed prior to administering an amount of a pharmaceutical formulation comprising the milk exosomes of the present disclosure or a population thereof, or the milk exosomes of the present disclosure or a population thereof, as described in more detail elsewhere herein, or an amount of a pharmaceutical formulation comprising the milk exosomes of the present disclosure as described in more detail elsewhere herein. In certain exemplary embodiments, the one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP increase the cellular uptake of intracellular milk exosomes or a population thereof. In certain exemplary embodiments, the cell is a polarized cell.In certain exemplary embodiments, the cells are gastrointestinal cells, heart cells, liver cells, brain cells, nerve cells, germ cells, cancer cells, or any combination thereof. In certain exemplary embodiments, the injury is a mechanical injury site or a non-mechanical injury. In certain exemplary embodiments, the non-mechanical injury is a chemical injury, an electrical injury, a radiation injury, or an ischemic injury. In certain exemplary embodiments, the mechanical injury is a wound or a burn.

[0021] In certain exemplary embodiments, the milk exosomes or a population thereof and / or the pharmaceutical formulation have been subjected to radiation, sterilization, or both.

[0022] In certain exemplary embodiments of the present specification, there is described a method of treating a wound or cancer in a subject, comprising administering to the subject in need thereof a milk exosome and an agent capable of stimulating the release, secretion, and / or production of ATP. In certain exemplary embodiments, the milk exosome is the milk exosome of the present disclosure described in more detail elsewhere in the present specification.

[0023] In certain exemplary embodiments, the milk exosomes or a population thereof and / or the pharmaceutical formulation of the present disclosure have been subjected to radiation, sterilization, or both, as described in more detail elsewhere in the present specification.

[0024] These and other aspects, objects, features, and advantages of the embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the embodiments.

[0025] Understanding of the features and advantages of the present invention is obtained by reference to the following detailed description, which sets forth exemplary embodiments in which the principles of the invention may be utilized, and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

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[0027] The figures in this specification are for illustrative purposes only and are not necessarily drawn to scale.

Best Mode for Carrying Out the Invention

[0028] Before explaining the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus, it can obviously change. It should also be understood that the terms used in this specification are for the purpose of simply describing a specific embodiment and are not intended to be limiting.

[0029] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described in this specification can also be used in the practice or testing of this disclosure, the preferred methods and materials will be described hereinafter.

[0030] All publications and patents mentioned in this specification are described in order to disclose and describe the publications in the manner in which they are cited and / or the substances. All such publications and patents are incorporated herein by reference to the same extent as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is specifically limited to the methods and / or substances described in the cited publications and patents, and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definitions in the cited publications and patents that are not explicitly repeated in this application should not be so treated and should not be read as defining any term that appears in the appended claims. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that this disclosure has the right to precede such publication for purposes of prior disclosure. Further, the date of the provided publication may differ from the actual publication date and may need to be confirmed separately.

[0031] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with the features of any of several other embodiments without departing from the scope or spirit of the disclosure. Any recited method can be performed in the order of the recited events or in any other order that is logically possible.

[0032] When a range is recited, further aspects include from one particular value and / or to another particular value. When a range of values is provided, each intervening value, to the tenth of the unit of the lower limit value, between the upper and lower limit values of that range, as well as any other recited value or intervening value in the recited range, is understood to be encompassed within the disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also encompassed within the disclosure, subject to any specific exclusion limits within the recited range. When the recited range includes one or both of the limit values, ranges excluding one or both of those included limit values are also included within the disclosure. For example, if the recited range includes one or both of the limit values, ranges excluding one or both of those included limit values are also included within the disclosure; for instance, the phrase "from x to y" includes the range from "x" to "y" as well as the range greater than "x" and less than "y". Ranges may also be expressed as an upper limit, for example, "about x, y, z, or less", and should be interpreted to include the specific ranges of "about x", "about y", and "about z", as well as the ranges "less than x", "less than y", and "less than z". Similarly, the phrase "about x, y, z, or greater" should be interpreted to include the specific ranges of "about x", "about y", and "about z", as well as the ranges "greater than x", "greater than y", and "greater than z". Further, the phrase "about "x" to "y"" (where "x" and "y" are numerical values) includes "about "x" to about "y"".

[0033] Note that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is further understood that the endpoints of each range are significant both in relation to the other endpoints and independently of the other endpoints. Also, there are several values disclosed herein, and it is understood that each value is disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. In this specification, a range can be expressed as from "about" one particular value and / or to "about" another particular value. Similarly, when a value is expressed as an approximation using the preceding "about", it is understood that the particular value forms another aspect. For example, if the value "about 10" is disclosed, "10" is also disclosed.

[0034] It should be understood that such range formats are used for convenience and brevity and are thus to be interpreted flexibly to include not only the numerical values explicitly specified as the limitations of the range, but also all the individual numerical values or sub-ranges within that range as if each numerical value and sub-range were explicitly specified. By way of illustration, the numerical range of "about 0.1% to 5%" includes not only the explicitly listed values of about 0.1% to about 5%, but also the individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and sub-ranges (e.g., about 0.5% to about 1.1%, about 5% to about 2.4%, about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0035] General Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. General terms and definitions of techniques in molecular biology are found in Molecular Cloning: A Laboratory Manual, 2 nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition(2012)(Green and Sambrook);Current Protocols in Molecular Biology(1987)(F.M.Ausubel et al.eds.);the series Methods in Enzymology(Academic Press,Inc.):PCR2:A Practical Approach(1995)(MJ MacPherson,BD Hames,and GR Taylor eds.):Antibodies,A Laboratory Manual(1988)(Harlow and Lane,eds.):Antibodies A Laboratory Manual,2 ndedition 2013 (E.A. Greenfield ed.); Animal Culture Cell (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J.Wiley & Sons (New York, NY 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd edition (2011) can be found in

[0036] Definitions of common terms and techniques in chemistry and organic chemistry are from Smith. Organic Synthesis, published by Academic Press. 2016; Tinoco et al. Physical Chemistry, 5 th edition (2013) published by Pearson; Brown et al., Chemistry, The Central Science 14th ed. (2017), published by Pearson, Clayden et al., Organic Chemistry, 2 nd ed. 2012, Oxford University Press; Carey and Sunberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5 th ed. 2008, published by Springer; Carey and Sunberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, 5 th ed.2010, published by Springer, and Vollhartt and Schore, Organic Chemistry, Structure and Function; 8 th ed. (2018) published by W.H. Freeman can be found in.

[0037] Definitions, analysis, and techniques of common terms in genetics are, for example, Hartl and Clark. Principles of Population Genetics. 4 th Ed.2006, published by Oxford University Press. Published by Booker. Genetics: Analysis and Principles, 7 thEd. 2021, published by McGraw Hill; Isik et al., Genetic Data Analysis for Plant and Animal Breeding. First ed. 2017, published by Springer International Publishing AG; Green, E.L. Genetics and Probability in Animal Breeding Experiments. 2014, published by Palgrave; Bourdon, R.M. Understanding Animal Breeding. 2000 2 nd Ed. published by Prentice Hall; Pal and Chakravarty, Genetics and Breeding for Disease Resistance of Livestock. First Ed. 2019, published by Academic Press; Fasso, D. Classification of Genetic Variance in Animals. First Ed. 2015, published by Callisto Reference; Megahed, M. Handbook of Animal Breeding and Genetics, 2013, published by Omniscriptum Gmbh&Co.Kg., LAP Lambert Academic Publishing; Reece. Analysis of Genes and Genomes. 2004, published by John Wiley&Sons, Inc; Deonier et al., Computational Genome Analysis. 5 th Ed. 2005, published by Springer-Verlag, New York; Meneely, P.Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes. 3 rdIt is found in Ed.2020, published by Oxford University Press.

[0038] As used herein, unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include both singular and plural referents.

[0039] As used herein, terms such as “about,” “approximately,” “substantially,” etc., when used in connection with measurable variables such as parameters, amounts, durations of time, etc., include within the specified value those that are within experimental error, e.g., variables that are within + / −10% or less, + / −5% or less, + / −1% or less, and + / −0.1% or less of a particular numerical value or from that numerical value, as long as such variables are appropriate for practicing the disclosed invention (e.g., within a given data set, standards acceptable in the art, and / or, for example, within a given confidence interval (e.g., determinable by a 90%, 95%, or greater confidence interval from the mean value)). As used herein, the terms “about,” “approximately,” “at or about,” and “substantially” may mean that the quantity or value in question may be an exact value or a value that provides the same result or effect as that recited in the claims or described herein. That is, amounts, sizes, formulations, parameters, as well as other amounts and characteristics need not be exact and, if desired, may be approximate values and / or greater or lesser values reflecting other factors known to those of ordinary skill in the art such as tolerances, conversion factors, rounding, measurement errors, etc., such that the same result or effect is obtained. In some situations, it may not be possible to reasonably determine a value or effect that provides the same result or effect. Generally, amounts, sizes, formulations, parameters, or other amounts or characteristics are “about,” “approximately,” or “at or about,” whether or not so expressly stated. When “about,” “approximately,” or “at or about” is used before a quantitative value, it is understood that the parameter includes the particular quantitative value itself, unless otherwise specified.

[0040] The terms "optional" or "optionally" mean that the event or situation described hereinafter may or may not occur, and the description includes both the case where the event or situation occurs and the case where it does not occur.

[0041] The recitation of numerical ranges by endpoints includes all numbers and fractions within each range, as well as the recited endpoints.

[0042] As used herein, "biological sample" refers to a sample obtained from a living body, produced by a living body, secreted by a living body, excreted by a living body, or containing or derived from a part of a living body. A biological sample can include whole cells and / or living cells and / or cell debris, and / or cell products, and / or virus particles. A biological sample can include (or be derived from) "body fluid". A biological sample can be obtained from the environment (e.g., water source, soil, air, etc.). Such samples are also referred to herein as environmental samples. As used herein, "body fluid" refers to any non-solid excretion, secretion, or other fluid present in an organism, and unless otherwise specified or not apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or its components (e.g., plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (ear wax), endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, and mixtures of one or more of these. Biological samples include cell cultures, body fluids, and cell cultures from body fluids. Body fluids can be obtained from an organism, for example, by puncture or other collection or sampling procedures.

[0043] "Subject", "individual", and "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, domestic animals, sport animals, and pet animals. Also included are tissues, cells, and progeny of biological entities obtained or cultured in vitro.

[0044] As used herein, "cancer" refers to one or more types of cancer including, but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary central nervous system (CNS) lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer (including, but not limited to, Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, germ cell tumor, glioma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ of the breast, endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (including, but not limited to, intraocular melanoma and retinoblastoma), fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, central nervous system germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, pancreatic neuroendocrine tumor, kidney (renal cell) cancer, laryngeal cancer, leukemia, lip cancer, oral cancer, lung cancer (non-small cell and small cell), lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline duct carcinoma with and without NUT gene alteration, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasm, polypoid carcinoid, myelodysplastic / myeloproliferative neoplasm, chronic myelogenous leukemia, nasal cancer, paranasal sinus cancer, non-Hodgkin lymphoma, pancreatic cancer, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary cancer, peritoneal cancer, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, large intestine cancer (colon cancer), soft tissue sarcoma, T-cell lymphoma, pharyngeal cancer, oropharyngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, thymoma, thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, vaginal cancer, cervical cancer, vascular tumor and cancer, vulvar cancer, and Wilms tumor, among others.

[0045] Various embodiments are described below. Note that the specific embodiments are not intended as an exhaustive description or as a limitation to a broader aspect discussed in this specification. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be implemented in any other embodiment(s). Throughout this specification, references to "one embodiment", "an embodiment", or "exemplary embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment", "in an embodiment", or "in an exemplary embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics can be combined in any suitable manner, as will be apparent to one skilled in the art from the disclosure, in one or more embodiments. Additionally, some of the embodiments described herein include some features of other embodiments and do not include some features, but combinations of features of different embodiments are intended to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0046] As used herein, "administering" refers to any suitable administration of the agent(s) to be delivered and / or to the subject to which the agent(s) is / are to be administered, which may be oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-articular, parenteral, intra-arterial, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intra-articular, intracavitary, intrathecal, intralesional, intraviral, intracerebral, and intraventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheter, by stent, or via an implanted reservoir or other device that actively or passively (e.g., by diffusion) administers the composition to the perivascular space and adventitia. For example, a medical device such as a stent can contain a composition or formulation disposed on its surface, which can dissolve or otherwise be distributed to surrounding tissues and cells. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. The route of administration will usually depend on the disease being treated, the subject being treated, and / or the agent(s) being administered, and can be, for example, auricular (ear), buccal, conjunctival, cutaneous, dental, iontophoresis, endocervical, intranasal, intratracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-arterial, intra-articular, intra-biliary, intra-bronchial, intracapsular, intracardiac, intra-cartilaginous, intra-caudal vein, intra-cavernous body, intracavitary, intracerebral, intrasinus, intra-corneal, intracoronal (tooth), intra-coronary artery, intra-cavernous body, intradermal, intraspinal, intraductal, intra-duodenal, intradural, intra-epidermal, intra-esophageal, intra-gastric, intramuscular, intra-ocular, intra-ovarian, intra-pericardial, intraperitoneal, intra-pleural, intra-prostatic, intra-pulmonary, intranasal, intrathecal, intrasynovial, intratendinous, intra-testicular, intrathecal, intra-thoracic, intra-tubular, intra-tumoral, intratympanic, intra-uterine, intra-vascular, intravenous, intravenous bolus, intravenous infusion, intraventricular, intracapsular, intravitreal, iontophoresis, lavage, laryngeal, nasal, nasogastric, occlusive dressing, ocular, oral, oropharyngeal, other, parenteral, percutaneous, perijoint, epidural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, percutaneous, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above routes of administration.

[0047] As used herein, "agent" refers to any substance, compound, molecule, etc. that can be administered to a subject or to a subject to which it is administered. An agent can be inert. An agent can be an active agent. An agent can be a primary active agent, or in other words, a component(s) of a composition from which all or part of the effect of the composition results. An agent can be a secondary agent, or in other words, a component(s) of a composition from which additional parts of the composition and / or other effects result.

[0048] As used herein, "anti-infective agent" refers to a compound or molecule that can either kill an infectious agent and / or regulate or inhibit its activity, infectivity, replication, and / or spread, whereby its infectivity is reduced or eliminated and / or the disease associated therewith or its symptoms are lessened or disappear. Anti-infective agents include, but are not limited to, antibiotics, antibacterial agents, antifungal agents, antiviral agents, and antiprotozoal agents.

[0049] As used herein, the term "biocompatible" refers to a substance or object that, when introduced into a living organism, performs its desired function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity to natural cells, tissues, or organs, or to cells, tissues, or organs into which a substance or object has been introduced. For example, a biocompatible product is a product that, when introduced into a living organism, performs its desired function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity to natural cells, tissues, or organs.

[0050] As used herein, biocompatibility can be quantified using the following in vivo biocompatibility assays. A material or product is considered biocompatible if, in biocompatibility tests related to immune system reactions, it produces less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the reaction of the same biocompatibility test generated by the same material or product, other than the lack of surface modification of the test substance or product. Examples of useful biocompatibility tests include cytotoxicity in cell culture, inflammatory reactions after transplantation (such as by fluorescence detection of cathepsin activity), and measurement and evaluation of immune system cells mobilized for transplantation (e.g., macrophages and neutrophils).

[0051] As used herein, "biological agent" refers to any compound, composition, molecule, etc. made by a living organism, including but not limited to polynucleotides (e.g., DNA, RNA), peptides and polypeptides, and chemical compounds (e.g., hormones, chemokines, and cytokines).

[0052] As used herein, "chemotherapeutic agent" or "chemotherapeutic drug" refers to a therapeutic agent used for the prevention or treatment of cancer.

[0053] As used herein, "control" refers to an alternative subject or sample that is used in an experiment for comparison purposes and is included to minimize or distinguish the effects of variables other than the independent variable.

[0054] As used herein, the terms "disease" or "disorder" are used interchangeably herein and refer to any change in the state of the body or some organs that interrupts or inhibits the performance of functions and / or causes symptoms, such as discomfort, dysfunction, pain, or even death, in the person suffering therefrom or in a person in contact with that person. A disease or disorder may also be associated with distemper, being ailing, ailment, malady, disorder, sickness, illness, complaint, minor illness, or pain.

[0055] As used herein, "dosage", "unit dosage", or "dose" refers to a physically discrete unit suitable for use in a subject, and each unit contains a predetermined amount of a primary and / or secondary active agent calculated to produce a desired response(s) in relation to its administration.

[0056] As used herein, "immunomodulator" refers to an agent, such as a therapeutic agent, that can modulate or regulate one or more immune functions or responses.

[0057] As used herein, "infection" as used herein refers to the presence of an infectious agent in or on a subject, such as a microorganism, and the presence or growth of which, when inhibited, is beneficial to the subject. Thus, this term refers to the state resulting from the establishment, more specifically the invasion and growth, of an infectious agent, such as a microorganism, in or on a suitable host. An infection can cause tissue damage and progress to an overt disease through various cellular and toxic mechanisms.

[0058] As used herein, "inflammation" generally refers to the reaction of a vascularized tissue to injury of cells or tissues that is normally caused by physical, chemical, and / or biological agents, characterized by an acute form with the classical sequence of pain, heat, redness, swelling, and loss of function, and functions as a mechanism to initiate the elimination, dilution, or walling off of the noxious agent and / or the damaged tissue. Inflammation histologically involves a series of complex events including an increase in permeability and blood flow, the exudation of body fluids including plasma proteins, and the dilation of arterioles, capillaries, and venules with the migration of leukocytes to the focus of inflammation. Furthermore, the term includes inflammation caused by external physical or chemical injury or by biological agents such as viral, bacterial, fungal, protozoan, or metazoan parasite infections, as well as inflammation that does not appear to be induced, such as inflammation that occurs without demonstrable injury or infection, an inflammatory reaction to self-antigens (autoimmune inflammation), an inflammatory reaction to transplanted xenogeneic or allogeneic cells, tissues, or organs, an inflammatory reaction to allergens, etc. The term includes both acute and chronic inflammation. Also, the term includes both local or focal inflammation and systemic inflammation, i.e., one or more inflammatory processes are not limited to a particular tissue and generally occur in the vascular endothelium and / or other organ systems.

[0059] As used herein, "isolated" means separated from its components, cells, etc., where polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are normally associated with nature. Polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not occur in nature do not require "isolation" to distinguish them from their naturally occurring counterparts.

[0060] As used herein, the term "molecular weight" generally refers to the mass or average mass of a substance. In the case of a polymer or oligomer, the molecular weight may refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weights of polymers and oligomers can be estimated or characterized by various methods including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as weight average molecular weights (M n ) rather than number average molecular weights (M w ). Capillary viscometry provides an estimate of the molecular weight as the intrinsic viscosity determined from dilute polymer solutions using a specific set of concentration, temperature, and solvent conditions.

[0061] As used herein, "non-human mammal" refers to any mammal that is not a human.

[0062] As used herein, "nucleic acid," "nucleotide sequence," and "polynucleotide" may be used interchangeably herein and generally can refer to a series of at least two base-sugar-phosphate combinations, and in particular, single- and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules that can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions, including DNA and RNA. In addition, the polynucleotides used herein can refer to triple-stranded regions that contain RNA or DNA, or both RNA and DNA. The strands within such regions can be from the same molecule or from different molecules. These regions can include all of one or more of these molecules, but more typically include only some regions of some of these molecules. One of the molecules in the triple helix region is often an oligonucleotide. "Polynucleotide" and "nucleic acid" also encompass chemically, enzymatically, or metabolically modified forms of such polynucleotides, and in particular, the chemical forms characteristic of viral and cellular DNA and RNA, including simple and complex cells. For example, the term polynucleotide as used herein can include DNA or RNA described herein that contains one or more modified bases. Thus, by way of two examples, DNA or RNA containing rare bases such as inosine or modified bases such as tritiated bases is a polynucleotide as the term is used herein. "Polynucleotide," "nucleotide sequence," and "nucleic acid" also include PNA (peptide nucleic acid), phosphonothioates, and other variants of the phosphate backbone of natural nucleic acids. Natural nucleic acids have a phosphate backbone, and artificial nucleic acids can contain other types of backbones but contain the same bases. Thus, DNA or RNA whose backbone has been modified for stability or other reasons is a "nucleic acid" or "polynucleotide" as the term is intended herein. As used herein, "nucleic acid sequence" and "oligonucleotide" also encompass nucleic acids and polynucleotides as defined elsewhere herein.

[0063] As used herein, the terms "recombinant" or "engineered" may generally refer to nucleic acids, nucleic acid constructs, or polypeptides that do not occur in nature. Such non-naturally occurring nucleic acids include naturally occurring nucleic acids that have been modified, e.g., having deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origins that are joined using molecular biology techniques (e.g., nucleic acid sequences encoding fusion proteins (e.g., proteins or polypeptides formed from combinations of two different proteins or protein fragments), combinations of nucleic acids encoding polypeptides and promoter sequences, where the coding sequence and the promoter sequence are from different sources or, otherwise, do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc.). Recombinant, or engineered, may also refer to polypeptides encoded by recombinant nucleic acids. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by a human.

[0064] As used herein, the term "specific binding" refers to a non-covalent physical association between a first moiety and a second moiety, where the association between the first moiety and the second moiety is at least 5-fold stronger, at least 10-fold stronger, at least 50-fold stronger, at least 100-fold stronger, or stronger than the association of either moiety with any of the majority or all of the other moieties present in the environment where the binding occurs. The binding of two or more entities is considered specific if the equilibrium dissociation constant Kd is, for example, 10 -3 M or less, 10 -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less. In some embodiments, specific binding is due to a plurality of weaker interactions (e.g., each individual interaction being 10 -3It can be achieved by a plurality of individual interactions characterized by the Kd of M-mode ultrasound. In some embodiments, specific binding, which may be referred to as "molecular recognition", is a saturable binding interaction between two entities that depends on the complementary orientation of functional groups on each entity. Examples of specific binding interactions include primer - polynucleotide interactions, aptamer - aptamer target interactions, antibody - antigen interactions, avidin - biotin interactions, ligand - receptor interactions, metal - chelate interactions, hybridization between complementary nucleic acids, and the like.

[0065] As used herein, "tangible expression medium" refers to a physically tangible or accessible medium, not just an abstract thought or an unrecorded spoken word. Examples of "tangible expression medium" include, but are not limited to, words related to cellulose materials or plastic materials, or data stored in a suitable computer-readable memory form. The data can be stored in a unit device such as a flash memory or a CD-ROM, or on a server that can be accessed by a user, for example, via a web interface.

[0066] As used herein, "therapeutic" refers to treating, curing, and / or ameliorating a disease, disorder, condition, or side effect, or reducing the rate of progression of a disease, disorder, condition, or side effect. Thus, a "therapeutically effective amount" may refer to an amount of a compound that can bring about a therapeutic effect.

[0067] As used herein, the terms "treating" and "treatment" generally refer to obtaining a pharmacological and / or physiological effect. The effect can be prophylactic in that it prevents or partially prevents a disease, symptom or condition such as cancer, an inflammatory disease or disorder, and / or a mechanical or non-mechanical injury, but does not necessarily have to be. The effect can be therapeutic in that it partially or completely cures a disease, condition, symptom, or side effect resulting from a disease, disorder, or condition. As used herein, "treatment" encompasses any treatment of cancer, an inflammatory disease or disorder and / or a mechanical or non-mechanical injury in a subject, particularly a human or non-human animal, e.g., a non-human mammal, and can include any one or more of (a) preventing the onset of the disease in a subject who is susceptible to the disease but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., preventing its manifestation, and (c) alleviating the disease, i.e., alleviating or improving the disease and / or its symptoms or condition. As used herein, the term "treatment" can refer to treatment alone, prophylactic treatment alone, or both treatment and prophylactic treatment. Persons in need of treatment (subjects in need thereof) include those who already have a disorder and / or those in whom the disorder is to be prevented. As used herein, the term "treating" can include suppressing a disease, disorder or condition, e.g., preventing its progression, and alleviating a disease, disorder or condition, e.g., causing regression of the disease, disorder and / or condition. Treating a disease, disorder or condition can include improving at least one symptom of a particular disease, disorder or condition, such as treating a subject's pain by administering an analgesic (such agents do not treat the cause of the pain), even if the underlying pathophysiology is not affected.

[0068] As used herein, the terms “weight percent,” “wt%,” and “wt. %” are used interchangeably and, unless otherwise specified, indicate the weight percent of a given component based on the total weight of the composition of which it is a component. That is, unless otherwise specified, all wt% values are based on the total weight of the composition. It should be understood that the sum of the wt% values of all components in the disclosed compositions or formulations equals 100. Alternatively, if the wt% value is based on the total weight of a subset of components in the composition, it should be understood that the sum of the wt% values of the specific components in the disclosed composition or formulation equals 100.

[0069] All publications and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, published patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0070] Summary Oral delivery of therapeutic agents is desirable but, in many cases, cannot be achieved with conventional delivery approaches for some therapeutic agents. For example, first-pass and / or second-pass metabolism reduces the bioavailability of the therapeutic agent, such that oral delivery becomes clinically infeasible. In other cases, the therapeutic agent is susceptible or sensitive to different environments, such as an acidic pH. Additionally, labile drugs, such as insulin or heparin, are primarily administered by injection to ensure sufficient bioavailability. Thus, there is a need for improved delivery approaches, particularly oral delivery, for therapeutic agents.

[0071] However, the embodiments disclosed herein can provide milk exosomes that can target damaged sites (e.g., mechanical or non-mechanical damaged sites) and / or cancer cells, as well as their use for treating damage or cancer. In some embodiments, the milk exosomes can carry exogenous cargo. In some embodiments, the milk exosomes are enriched for connexin 43-positive exosomes. In some embodiments, the treatment includes co-administration of one or more agents that increase ATP production and / or release. In some embodiments, the treatment includes co-administration or pre-treatment with IgG. Other compositions, compounds, methods, features, and advantages of the present disclosure will be apparent to those skilled in the art upon examination of the following drawings, detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages are intended to be included herein and within the scope of the present disclosure.

[0072] Other compositions, compounds, methods, features, and advantages of the present disclosure will be apparent to those skilled in the art upon examination of the following drawings, detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages are intended to be included herein and within the scope of the present disclosure.

[0073] Targeted milk exosomes Certain exemplary embodiments describe milk exosomes that contain exogenous cargo and can target or stimulate adenosine triphosphate (ATP) secretion and / or concentration and / or the neonatal Fc receptor (FcRN) to target damaged sites or cancer cells or cell populations. Without being bound by theory, ATP can be concentrated in certain regions such as the cells (also referred to herein as "target" cells) to be targeted and / or in or near those cells. For example, mechanically or non-mechanically damaged regions or cells or regions of inflammation damaged by cancer cells and / or tumors may have high concentrations of secreted ATP. The milk exosomes of the present disclosure can home in on or target these regions (e.g., damaged regions or cellular microenvironments) and / or cells with elevated ATP concentrations. As shown in the examples herein, it has been demonstrated that milk exosomes can home in on regions and / or cells with higher ATP concentrations. Further, without being bound by theory, by stimulating cells to produce and / or release ATP, it can act to home in on or signal to the milk exosomes of the present disclosure to target those cells and / or regions (including the cells within those regions). As used in this context, "concentrated" refers to a region where the amount or number of ATP molecules per unit area or volume is greater compared to another area or volume. A concentration gradient can be formed where the region closest to the target cell(s) or region is the greatest and decreases as the distance from the target cell(s) or region increases. In some embodiments, "concentrated" in this context can refer to the amount or number of ATP per unit area or volume of a first region or volume being 0.001 to 1,000 percent or more greater compared to a second region or volume. Additionally, as shown in the examples, immunoglobulins present in the milk exosomes can interact with and / or bind to FcRN on the cells, thereby increasing the uptake of the milk exosomes in those cells.In some embodiments, the milk exosomes are bovine milk exosomes, ovine milk exosomes, equine milk exosomes, and / or human milk exosomes.

[0074] In certain exemplary embodiments, the site of injury is mechanical injury or non-mechanical injury. In certain exemplary embodiments, non-mechanical injury is chemical injury, electrical injury, radiation injury, ischemic injury (e.g., injury caused by or resulting from an ischemic event), or injury resulting from an inflammatory process or inflammation (also referred to herein as "inflammatory injury"). In some embodiments, mechanical injury is a wound (inflicted or caused by any process), a burn, etc. Other exemplary injuries are described in more detail elsewhere in this specification.

[0075] In some embodiments, the milk exosomes can target cancer cells (s), or areas or cells that are the subject of, or affected by, viral infection, bacterial infection, parasitic infection, mechanical injury (e.g., external and internal wounds and tissue damage, cuts, abrasions, burns, surgical wounds, secondary wounds resulting from an infection, disease, or condition), cancer, non-mechanical injury (e.g., chemical injury, electrical injury, radiation injury, inflammatory injury, ischemic and / or hypoxic injury (e.g., myocardial infarction, ischemic wound and / or stroke), multiple sclerosis, psoriasis, scleroderma, acne, eczema, or diseases of the skin and / or connective tissue, heart disease or disorder, neurodegenerative disease or disorder, neuropathy, atherosclerosis, conditions involving epithelial permeability, and / or angiogenesis (e.g., neovascularization or vasculogenesis), respiratory distress syndrome (RDS), reperfusion injury, injury or deformation of skin blood vessels, macular degeneration, choroidal neovascularization through Bruch's membrane, diabetic retinopathy (inflammatory and inflammation-related diseases and disorders), and radiation dermatitis).

[0076] A wound can be a chronic wound, or a wound that appears not to have healed completely. For example, a wound that has not healed within three months is said to be chronic. Chronic wounds include diabetic foot ulcers, ischemic, venous ulcers, venous lower leg ulcers, venous stasis, arterial, pressure, vasculitis, infectious, decubitus ulcers, burns, ulcers induced by trauma, gangrenous and mixed ulcers. Chronic wounds include wounds characterized by chronic inflammation, incomplete differentiation and overgrowth of granulation tissue, as well as incomplete re-epithelialization and incomplete wound closure, and longer healing times. Chronic wounds can include eye ulcers including corneal ulcers. The use of the disclosed invention in wound healing and tissue regeneration can include humans and livestock, sports animals and pets.

[0077] Tissue damage can occur due to, for example, cuts, abrasions, compression wounds, stretching injuries, laceration wounds, contusion wounds, bites, scratches, gunshot wounds, rupture wounds, body piercing, stab wounds, surgical wounds, surgical interventions, medical interventions, host rejection reactions after transplantation of cells, tissues, or organs, pharmaceutical effects, pharmaceutical side effects, bedsores, radiation injuries, radiation diseases, skin injuries caused by cosmetics, inflammatory injuries, visceral injuries, disease processes (e.g., asthma, cancer), infections, infectious agents, developmental processes, maturation processes (e.g., acne), genetic abnormalities, developmental abnormalities, environmental toxins, allergens, scalp injuries, facial injuries, jaw injuries, genital injuries, joint injuries, excretory organ injuries, foot injuries, finger injuries, toe injuries, bone injuries, eye injuries, corneal injuries, muscle injuries, adipose tissue injuries, lung injuries, airway injuries, hernias, anal injuries, hemorrhoids, ear injuries, skin injuries, abdominal injuries, retinal injuries, eye injuries, corneal injuries, wrist injuries, leg injuries, sports injuries, back injuries, birth injuries, early birth injuries, venomous bites, stings, impairment of barrier function, impairment of endothelial barrier function, impairment of epithelial barrier function, tendon injuries, ligament injuries, heart injuries, heart valve injuries, vascular system injuries, cartilage injuries, lymphatic system injuries, head and brain trauma, dislocations, esophageal perforations, fistulas, nail injuries, foreign bodies, fractures, frostbite, hand injuries, heat stress disorders, lacerations, neck injuries, self-destruction, shock, traumatic soft tissue injuries, spinal cord injuries, spinal column injuries, sprains, overexertion, tendon injuries, ligament injuries, cartilage injuries, chest injuries, dental injuries, trauma, nerve system-wide injuries, burns, burn wounds, scalds, sunburns, chemical burns, aging, aneurysms, strokes, surgical radiation injuries, gastrointestinal tract injuries, infarctions, or ischemic injuries.

[0078] Heart diseases and disorders that can result in damaged cells or that can otherwise be targeted by the disclosed milk exosomes include, but are not limited to, myocardial infarction, cardiomyopathy (e.g., hypertrophic cardiomyopathy), arrhythmia, and congestive heart failure. The regenerative effects of the provided compositions may result in beneficial changes in cardiac membrane excitability and ion transients. There are many different types of arrhythmias that can lead to abnormal cardiac function in humans. Arrhythmias include, but are not limited to, the following: bradycardia, tachycardia, alternans, automaticity disorders, reentrant arrhythmias, fibrillation, atrioventricular nodal reentrant tachycardia, atrial arrhythmias and induced flutter, QT prolongation syndrome, QT shortening syndrome, Brugada syndrome, premature atrial contractions, slow atrial pacemakers, multifocal atrial tachycardia, atrial flutter, atrial fibrillation, ventricular tachycardia, the most common cause of paroxysmal supraventricular tachycardia, atrioventricular nodal reentrant tachycardia, atrioventricular junctional rhythm, junctional tachycardia, premature junctional complexes, Wolff-Parkinson-White syndrome, Lown-Ganong-Levine syndrome, premature ventricular contractions (PVCs), also known as ventricular extra beats, alternans and discordant alternans, accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, ventricular fibrillation, first-degree atrioventricular block, manifested as PR prolongation, second-degree atrioventricular block, type 1 second-degree atrioventricular block, type 2 second-degree atrioventricular block, third-degree atrioventricular block, and some accessory pathway disorders (e.g., Wolff-Parkinson-White syndrome (WPW)).

[0079] Neurodegenerative and neurological disorders that can result in damaged cell(s), or can otherwise be targeted by the disclosed milk exosomes, include, but are not limited to, dementia, Alzheimer's disease, Parkinson's disease and related PD disorders, 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.

[0080] Inflammatory diseases and disorders that can lead to cell damage or can be targeted by the milk exosomes of the present disclosure can include asthma, eczema, rhinitis, atherosclerosis, arthritis (including but not limited to rheumatoid arthritis), inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis. As used herein, the term "inflammatory disorder" generally refers to a disease or disorder that is at least partially caused or exacerbated by inflammation characterized by increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), heat, erythema, swelling, pain, and / or loss of function in the affected tissue or organ. The cause of inflammation can be due to physical injury, chemicals, microorganisms, tissue necrosis, cancer, or other agents or conditions. Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders generally have a relatively short duration, lasting from about a few minutes to about 1 - 2 days, but can also last for several weeks. Characteristics of acute inflammatory diseases include increased blood flow, exudation of fluid and plasma proteins (edema), and migration of white blood cells such as neutrophils. Chronic inflammatory diseases generally last for a longer period, e.g., from several weeks to months to years or more, and are histologically associated with the presence of lymphocytes and macrophages, as well as proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders that recur after a certain period or disorders that have periodic attacks. Some inflammatory disorders are classified into one or more categories. Exemplary inflammatory disorders include, but are not limited to, atherosclerosis, arthritis, cancer promoted by inflammation, asthma, autoimmune uveitis, adoptive immune responses, dermatitis, multiple sclerosis, diabetic complications, osteoporosis, Alzheimer's disease, cerebral malaria, hemorrhagic fevers, autoimmune disorders, and inflammatory bowel disease. In some embodiments, the inflammatory disorder is an autoimmune disorder, and in some embodiments, it is selected from lupus, rheumatoid arthritis, and autoimmune encephalomyelitis. 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 at least partially caused by, or can occur, or worsen due to inflammation within the brain of a subject.

[0081] In certain exemplary embodiments, the milk exosomes do not contain an exogenous targeting moiety. In other words, in some embodiments, the milk exosomes do not contain a non-natural targeting moiety.

[0082] In certain exemplary embodiments, the damaged or its site has a higher concentration of ATP than the undamaged or its site. In certain exemplary embodiments, a cancer cell or a cancer cell population has a microenvironment with a higher ATP concentration than the microenvironment of a non-cancerous cell or non-cancerous cell population. In some embodiments, the concentration of ATP in the damaged or at its site or in a cancer cell or cancer cell population or its microenvironment is 0.01%, up to / or 0.02, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%、101%、102%、103%、104%、105%、106%、107%、108%、109%、110%、111%、112%、113%、114%、115%、116%、117%、118%、119%、120%、121%、122%、123%、124%、125%、126%、127%、128%、129%、130%、131%、132%、133%、134%、135%、136%、137%、138%、139%、140%、141%、142%、143%、144%、145%、146%、147%、148%、149%、150%、151%、152%、153%、154%、155%、156%、157%、158%、159%、160%、161%、162%、163%、164%、165%、166%、167%、168%、169%、170%、171%、172%、173%、174%、175%、176%、177%、178%、179%、180%、181%、182%、183%、184%、185%、186%、187%、188%、189%、190%、191%、192%、193%、194%、195%、196%、197%、198%、199%、200%、201%、202%、203%、204%、205%、206%、207%、208%、209%、210%、211%、212%、213%、214%、215%、216%、217%、218%、219%、220%、221%、222%、223%、224%、225%、226%、227%、228%、229%、230%、231%、232%、233%、234%、235%、236%、237%、238%、239%、240%、241%、242%、243%、244%、245%、246%、247%、248%、249%、250%、251%、252%、253%、254%、255%、256%、257%、258%、259%、260%、261%、262%、263%、264%、265%、266%、267%、268%、269%、270%、271%、272%、273%、274%、275%、276%、277%、278%、279%、280%、281%、282%、283%、284%、285%、286%、287%、288%、289%、290%、291%、292%、293%、294%、295%、296%、297%、298%、299%、300%、301%、302%、303%、304%、305%、306%、307%、308%、309%、310%、311%、312%、313%、314%、315%、316%、317%、318%、319%、320%、321%、322%、323%、324%、325%、326%、327%、328%、329%、330%、331%、332%、333%、334%、335%、336%、337%、338%、339%、340%、341%、342%、343%、344%、345%、346%、347%、348%、349%、350%、351%、352%、353%、354%、355%、356%、357%、358%、359%、360%、361%、362%、363%、364%、365%、366%、367%、368%、369%、370%、371%、372%、373%、374%、375%、376%、377%、378%、379%、380%、381%、382%、383%、384%、385%、386%、387%、388%、389%、390%、391%、392%、393%、394%、395%、396%、397%、398%、399%、400%、401%、402%、403%、404%、405%、406%、407%、408%、409%、410%、411%、412%、413%、414%、415%、416%、417%、418%、419%、420%、421%、422%、423%、424%、425%、426%、427%、428%、429%、430%、431%、432%、433%、434%、435%、436%、437%、438%、439%、440%、441%、442%、443%、444%、445%、446%、447%、448%、449%、450%、451%、452%、453%、454%、455%、456%、457%、458%、459%、460%、461%、462%、463%、464%、465%、466%、467%、468%、469%、470%、471%、472%、473%、474%、475%、476%、477%、478%、479%、480%、481%、482%、483%、484%、485%、486%、487%、488%、489%、490%、491%、492%、493%、494%、495%、496%、497%、498%、499%、500%、501%、502%、503%、504%、505%、506%、507%、508%、509%、510%、511%、512%、513%、514%、515%、516%、517%、518%、519%、520%、521%、522%、523%、524%、525%、526%、527%、528%、529%、530%、531%、532%、533%、534%、535%、536%、537%、538%、539%、540%、541%、542%、543%、544%、545%、546%、547%、548%、549%、550%、551%、552%、553%、554%、555%、556%、557%、558%、559%、560%、561%、562%、563%、564%、565%、566%、567%、568%、569%、570%、571%、572%、573%、574%、575%、576%、577%、578%、579%、580%、581%、582%、583%、584%、585%、586%、587%、588%、589%、590%、591%、592%、593%、594%、595%、596%、597%、598%、599%、600%、601%、602%、603%、604%、605%、606%、607%、608%、609%、610%、611%、612%、613%、614%、615%、616%、617%、618%、619%、620%、621%、622%、623%、624%、625%、626%、627%、628%、629%、630%、631%、632%、633%、634%、635%、636%、637%、638%、639%、640%、641%、642%、643%、644%、645%、646%、647%、648%、649%、650%、651%、652%、653%、654%、655%、656%、657%、658%、659%、660%、661%、662%、663%、664%、665%、666%、667%、668%、669%、670%、671%、672%、673%、674%、675%、676%、677%、678%、679%、680%、681%、682%、683%、684%、685%、686%、687%、688%、689%、690%、691%、692%、693%、694%、695%、696%、697%、698%、699%、700%、701%、702%、703%、704%、705%、706%、707%、708%、709%、710%、711%、712%、713%、714%、715%、716%、717%、718%、719%、720%、721%、722%、723%、724%、725%、726%、727%、728%、729%、730%、731%、732%、733%、734%、735%、736%、737%、738%、739%、740%、741%、742%、743%、744%、745%、746%、747%、748%、749%、750%、751%、752%、753%、754%、755%、756%、757%、758%、759%、760%、761%、762%、763%、764%、765%、766%、767%、768%、769%、770%、771%、772%、773%、774%、775%、776%、777%、778%、779%、780%、781%、782%、783%、784%、785%、786%、787%、788%、789%、790%、791%、792%、793%、794%、795%、796%、797%、798%、799%、800%、801%、802%、803%、804%、805%、806%、807%、808%、809%、810%、811%、812%、813%、814%、815%、816%、817%、818%、819%、820%、821%、822%、823%、824%、825%、826%、827%、828%、829%、830%、831%、832%、833%、834%、835%、836%、837%、838%、839%、840%、841%、842%、843%、844%、845%、846%、847%、848%、849%、850%、851%、852%、853%、854%、855%、856%、857%、858%, 859%, 860%, 861%, 862%, 863%, 864%, 865%, 866%, 867%, 868%, 869%, 870%, 871%, 872%, 873%, 874%, 875%, 876%, 877%, 878%, 879%, 880%, 881%, 88, 2%, 883%, 884%, 885%, 886%, 887%, 888%, 889%, 890%, 891%, 892%, 893%, 894%, 895%, 896%, 897%, 898%, 899%, 900%, 901%, 902%, 903%, 904%, 905%, 906%, 907%, 908%, 909%, 910%, 911%, 912%, 913%, 914%, 915%, 916%, 917%, 918%, 919%, 920%, 921%, 922%, 923%, 924%, 925%, 926%, 927%, 928%, 929%, 930%, 931%, 932%, 933%, 934%, 935%, 936%, 937%, 938%, 939%, 940%, 941%, 942%, 943%, 944%, 945%, 946%, 947%, 948%, 949%, 950%, 951%, 952%, 953%, 954%, 955%, 956%, 957%, 958%, 959%, 960%, 961%, 962%, 963%, 964%, 965%, 966%, 967%, 968%, 969%, 970%, 971%, 972%, 973%, 974%, 975%, 976%, 977%, 978%, 979%, 980%, 981%, 982%, 983%, 984%, 985%, 986%, 987%, 988%, 989%, 990%, 991%, 992%, 993%, 994%, 995%, 996%, 997%, 998%, 999%, greater than 1000%.

[0083] In certain exemplary embodiments, the cancer cell or cancer cell population secretes ATP. In certain exemplary embodiments, the damaged cell(s) secretes ATP.

[0084] In certain exemplary embodiments, the milk exosomes contain connexin 43. In certain exemplary embodiments, the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43. In some embodiments, the engineered connexin 43 is as described in WO2020 / 028439.

[0085] In certain exemplary embodiments, the exogenous cargo is a biomolecule. In certain exemplary embodiments, the biomolecule is a lipid, polypeptide, peptide, nucleic acid, or cellular metabolite. In certain exemplary embodiments, the exogenous cargo is an ACT1 peptide, ACT11 peptide, ACT minus I peptide, selectide, or any combination thereof. In certain exemplary embodiments, the exogenous cargo is any of the cargoes described in WO2020 / 028439, an ACT1 or ACT11 peptide, an ACT minus peptide (see, e.g., the ACT minus peptide described in WO2022 / 076932), Gap19, JM2 (see, e.g., U.S. Patent No. US9,345,744B2), a selectide (see, e.g., WO2022 / 076932), or any combination thereof. In certain exemplary embodiments, the exogenous cargo is a peptide, including but not limited to the ACT-11 peptide. In certain exemplary embodiments, the exogenous cargo is a peptide, including but not limited to the ACT-11-minus I peptide. Other peptide cargoes include those described in International Patent Application Publication No. WO2020 / 028439, particularly on pages 67-82 and 85 and 106-111. In some embodiments, the cargo compound is esterified as described on pages 81-86 of International Patent Application Publication No. WO2020 / 028439. In some embodiments, the cargo compound is multiply esterified as described on pages 81-86 of International Patent Application Publication No. WO2020 / 028439.

[0086] In certain exemplary embodiments, prior to containing exogenous cargo, milk exosomes are prepared by a method comprising: (a) centrifuging mammalian milk under conditions suitable to separate fat from one or more other components of the mammalian milk; (b) removing the fat separated from the mammalian milk; (c) after step (b), centrifuging the remaining mammalian milk one or more times and skimming off any separated fat visible after each centrifugation in step (c); (d) filtering the biological fluid remaining after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); (f) optionally chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30-42 °C for about 15-120 minutes after (d) or optionally after (e); and (g) optionally performing tangential flow filtration after (f) to obtain a retentate, wherein the retentate is optionally ultracentrifuged via one or more ultracentrifugation steps or stored at -80 °C, and optionally separating fractions of the retentate via a column after the retentate has been optionally ultracentrifuged or stored at -80 °C, the method including step (e) or step (g), but not both. Additional methods for isolating milk exosomes are described elsewhere herein.

[0087] Certain exemplary embodiments of the present specification describe a population of milk exosomes comprising one or more milk exosomes of the present disclosure described herein. In certain exemplary embodiments, the population is enriched for connexin 43-positive milk exosomes. In certain exemplary embodiments, prior to containing exogenous cargo, the milk exosomes are (a) centrifuged mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk, (b) removing the fat separated from the mammalian milk, (c) after step (b), centrifuging the remaining mammalian milk one or more times and skimming off the separated fat visible after each centrifugation in step (c), (d) filtering the biological fluid remaining after step (c), (e) optionally performing one or more ultracentrifugation steps after (d), (f) after (d) or optionally after (e), and optionally chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30 to 42 °C for about 15 to 120 minutes, and (g) after (f), optionally performing tangential flow filtration to obtain a retentate, obtaining a retentate, wherein the retentate is optionally ultracentrifuged via one or more ultracentrifugation steps or stored at -80 °C, and after the retentate is optionally ultracentrifuged or stored at -80 °C, optionally separating fractions of the retentate via a column, separated by a method comprising, the method includes step (e) or step (g), but not both. In certain exemplary embodiments, the method further comprises enriching the milk exosomes for connexin 43-expressing milk exosomes. In certain exemplary embodiments, the milk exosomes do not contain exogenous targeting moieties. In certain exemplary embodiments, the exogenous cargo is a biomolecule. In certain exemplary embodiments, the biomolecule is a polypeptide, a peptide, or a nucleic acid. In certain exemplary embodiments, the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

[0088] In certain exemplary embodiments, the milk exosome(s) or a solution containing the same (e.g., milk, whey, etc.) is sterilized and / or irradiated. In some embodiments, the milk exosome is sterilized. In some embodiments, the milk exosome is sterilized by irradiation. In some embodiments, the irradiation sterilizes the milk exosome(s) without substantially destroying the morphology of the milk exosome(s). In other words, the irradiation leaves the milk exosome(s) morphologically intact and thus provides an effective method for sterilizing the milk exosome(s) and / or a solution or formulation containing the milk exosome(s). In some embodiments, the milk exosome(s) or a formulation or solution containing the same is sterilized and / or irradiated prior to exosome isolation and / or preservation.

[0089] In some embodiments, milk exosomes have increased uptake in cells having concentrated ATP or in cells within a tissue or cellular microenvironment having concentrated ATP and / or secreted ATP, compared to cells that do not have concentrated levels of ATP or cells present in an environment without concentrated ATP. In some embodiments, milk exosomes have increased uptake in cells including cells having FcRN on their surface, gastrointestinal cells, heart cells, liver cells, brain cells, nerve cells, germ cells, lung cells, cancer cells, or any combination thereof. In some embodiments, the cells are polar cells. This means that the cells have different apical and basolateral membrane surfaces. In some embodiments, the cells are intestinal cells. In some embodiments, the cells are blood-brain barrier cells. In some embodiments, the uptake is 0.01%, up to / or 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1% higher in cells that are not in a region having concentrated ATP or cells that do not have FcRN.2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%、101%、102%、103%、104%、105%、106%、107%、108%、109%、110%、111%、112%、113%、114%、115%、116%、117%、118%、119%、120%、121%、122%、123%、124%、125%、126%、127%、128%、129%、130%、131%、132%、133%、134%、135%、136%、137%、138%、139%、140%、141%、142%、143%、144%、145%、146%、147%、148%、149%、150%、151%、152%、153%、154%、155%、156%、157%、158%、159%、160%、161%、162%、163%、164%、165%、166%、167%、168%、169%、170%、171%、172%、173%、174%、175%、176%、177%、178%、179%、180%、181%、182%、183%、184%、185%、186%、187%、188%、189%、190%、191%、192%、193%、194%、195%、196%、197%、198%、199%、200%、201%、202%、203%、204%、205%、206%、207%、208%、209%、210%、211%、212%、213%、214%、215%、216%、217%、218%、219%、220%、221%、222%、223%、224%、225%、226%、227%、228%、229%、230%、231%、232%、233%、234%、235%、236%、237%、238%、239%、240%、241%、242%、243%、244%、245%、246%、247%、248%、249%、250%、251%、252%、253%、254%、255%、256%、257%、258%、259%、260%、261%、262%、263%、264%、265%、266%、267%、268%、269%、270%、271%、272%、273%、274%、275%、276%、277%、278%、279%、280%、281%、282%、283%、284%、285%、286%、287%、288%、289%、290%、291%、292%、293%、294%、295%、296%、297%、298%、299%、300%、301%、302%、303%、304%、305%、306%、307%、308%、309%、310%、311%、312%、313%、314%、315%、316%、317%、318%、319%、320%、321%、322%、323%、324%、325%、326%、327%、328%、329%、330%、331%、332%、333%、334%、335%、336%、337%、338%、339%、340%、341%、342%、343%、344%、345%、346%、347%、348%、349%、350%、351%、352%、353%、354%、355%、356%、357%、358%、359%、360%、361%、362%、363%、364%、365%、366%、367%、368%、369%、370%、371%、372%、373%、374%、375%、376%、377%、378%、379%、380%、381%、382%、383%、384%、385%、386%、387%、388%、389%、390%、391%、392%、393%、394%、395%、396%、397%、398%、399%、400%、401%、402%、403%、404%、405%、406%、407%、408%、409%、410%、411%、412%、413%、414%、415%、416%、417%、418%、419%、420%、421%、422%、423%、424%、425%、426%、427%、428%、429%、430%、431%、432%、433%、434%、435%、436%、437%、438%、439%、440%、441%、442%、443%、444%、445%、446%、447%、448%、449%、450%、451%、452%、453%、454%、455%、456%、457%、458%、459%、460%、461%、462%、463%、464%、465%、466%、467%、468%、469%、470%、471%、472%、473%、474%、475%、476%、477%、478%、479%、480%、481%、482%、483%、484%、485%、486%、487%、488%、489%、490%、491%、492%、493%、494%、495%、496%、497%、498%、499%、500%、501%、502%、503%、504%、505%、506%、507%、508%、509%、510%、511%、512%、513%、514%、515%、516%、517%、518%、519%、520%、521%、522%、523%、524%、525%、526%、527%、528%、529%、530%、531%、532%、533%、534%、535%、536%、537%、538%、539%、540%、541%、542%、543%、544%、545%、546%、547%、548%、549%、550%、551%、552%、553%、554%、555%、556%、557%、558%、559%、560%、561%、562%、563%、564%、565%、566%、567%、568%、569%、570%、571%、572%、573%、574%、575%、576%、577%、578%、579%、580%、581%、582%、583%、584%、585%、586%、587%、588%、589%、590%、591%、592%、593%、594%、595%、596%、597%、598%、599%、600%、601%、602%、603%、604%、605%、606%、607%、608%、609%、610%、611%、612%、613%、614%、615%、616%、617%、618%、619%、620%、621%、622%、623%、624%、625%、626%、627%、628%、629%、630%、631%、632%、633%、634%、635%、636%、637%、638%、639%、640%、641%、642%、643%、644%、645%、646%、647%、648%、649%、650%、651%、652%、653%、654%、655%、656%、657%、658%、659%、660%、661%、662%、663%、664%、665%、666%、667%、668%、669%、670%、671%、672%、673%、674%、675%、676%、677%、678%、679%、680%、681%、682%、683%、684%、685%、686%、687%、688%、689%、690%、691%、692%、693%、694%、695%、696%、697%、698%、699%、700%、701%、702%、703%、704%、705%、706%、707%、708%、709%、710%、711%、712%、713%、714%、715%、716%、717%、718%、719%、720%、721%、722%、723%、724%、725%、726%、727%、728%、729%、730%、731%、732%、733%、734%、735%、736%、737%、738%、739%、740%、741%、742%、743%、744%、745%、746%、747%、748%、749%、750%、751%、752%、753%、754%、755%、756%、757%、758%、759%、760%、761%、762%、763%、764%、765%、766%、767%、768%、769%、770%、771%、772%、773%、774%、775%、776%、777%、778%、779%、780%、781%、782%、783%、784%、785%、786%、787%、788%、789%、790%、791%、792%、793%、794%、795%、796%、797%、798%、799%、800%、801%、802%、803%、804%、805%、806%、807%、808%、809%、810%、811%、812%、813%、814%、815%、816%、817%、818%、819%、820%、821%、822%、823%, 824%, 825%, 826%, 827%, 828%, 829%, 830%, 831%, 832%, 833%, 834%, 835%, 836%, 837%, 838%, 839%, 840%, 841%, 842%, 843%, 844%, 845%, 846%, 84, 7%, 848%, 849%, 850%, 851%, 852%, 853%, 854%, 855%, 856%, 857%, 858%, 859%, 860%, 861%, 862%, 863%, 864%, 865%, 866%, 867%, 868%, 869%, 870%, 871%, 872%, 873%, 874%, 875%, 876%, 877%, 878%, 879%, 880%, 881%, 882%, 883%, 884%, 885%, 886%, 887%, 888%, 889%, 890%, 891%, 892%, 893%, 894%, 895%, 896%, 897%, 898%, 899%, 900%, 901%, 902%, 903%, 904%, 905%, 906%, 907%, 908%, 909%, 910%, 911%, 912%, 913%, 914%, 915%, 916%, 917%, 918%, 919%, 920%, 921%, 922%, 923%, 924%, 925%, 926%, 927%, 928%, 929%, 930%, 931%, 932%, 933%, 934%, 935%, 936%, 937%, 938%, 939%, 940%, 941%, 942%, 943%, 944%, 945%, 946%, 947%, 948%, 949%, 950%, 951%, 952%, 953%, 954%, 955%, 956%, 957%, 958%, 959%, 960%, 961%, 962%, 963%, 964%, 965%, 966%, 967%, 968%, 969%, 970%, 971%, 972%, 973%, 974%, 975%, 976%, 977%, 978%, 979%, 980%, 981%, 982%, 983%, 984%, 985%, 986%, 987%, 988%, 989%, 990%, 991%, 992%, 993%, 994%, 995%, 996%, 997%, 998%, 999%, over 1000% increase.

[0090] Loaded milk exosomes In certain exemplary embodiments, the method further comprises loading one or more cargos into the exosomes of the formulation obtained from the methods described herein. The cargos can be loaded into the exosomes by any suitable method. Exemplary methods of loading into milk exosomes, such as those prepared by the methods described herein, are any of the methods described in International Patent Application Publication No. WO2020 / 028439, particularly on pages 83-87.

[0091] Exemplary exogenous cargos Any suitable or desired cargo(es) can be loaded into the milk exosomes. In some embodiments, the exogenous cargo(es) is / are a therapeutic compound or molecule. Exemplary cargos include, but are not limited to, nucleic acids (e.g., DNA, RNA), amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, anticonvulsants, anti-inflammatory agents, antihistamines, anti-infectives, radiosensitizers, chemotherapeutic agents, contrast agents, immunogens, anti-cancer drugs, nutrients, polynucleotide modification systems, or any combination thereof, and / or the like. In some embodiments, the exogenous cargo is a biomolecule. In some embodiments, the exogenous cargo contains an ester linkage group that aids in its uptake into the exosomes.

[0092] In certain exemplary embodiments, the exogenous cargo is any of the cargos described in WO2020 / 028439, the ACT1 or ACT11 peptide, the ACT minus peptide (see, e.g., the ACT minus peptides described in WO2022 / 076932, including but not limited to ACT minus I peptide), Gap19, JM2 (see, e.g., U.S. Patent No. US9,345,744B2), the selectide (see, e.g., WO2022 / 076932), or any combination thereof. In certain exemplary embodiments, the cargo is a peptide, including but not limited to the ACT-11 peptide. In certain exemplary embodiments, the cargo is a peptide, including but not limited to the ACT-11-minus I peptide. Other peptide cargos include those described in International Patent Application Publication No. WO2020 / 028439, particularly those described on pages 67-82, 85, and 106-111. In some embodiments, the cargo compound is esterified as described on pages 81-86 of International Patent Application Publication No. WO2020 / 028439. In some embodiments, the cargo compound is esterified multiple times as described on pages 81-86 of International Patent Application Publication No. WO2020 / 028439.

[0093] Exemplary nutrients include, but are not limited to, minerals (e.g., potassium, sodium, chloride, magnesium, manganese, cobalt, molybdenum, calcium, copper, zinc, iodine, iron, chromium, fluoride, selenium, etc.), vitamins (vitamins A, E, D, C, K, etc.), creatine, ATP, ADP, sugars (e.g., glucose, fructose, mannose, galactose, lactose, etc.), and fats and fatty acids.

[0094] Exemplary hormones include, but are not limited to, 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, CCK, obestatin, leptin, ghrelin, etc.), eicosanoids (e.g., arachidonic acid, lipoxin, and prostaglandin), purines (e.g., ATP), enzymes (e.g., creatine), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone, cortisol), but are not limited thereto.

[0095] Exemplary immunomodulators include, but are not limited to, 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-β, IFN-ε, IFN-K, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers).

[0096] Exemplary antipyretics include non-steroidal anti-inflammatory agents (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 kinins, but are not limited thereto.

[0097] Exemplary anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, chlorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotoninergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), temegicoluril, fabomotizole, seranac, bromantan, emoxypine, azapirones, barbituric acid, hydroxyzine, pregabalin, isovaleric acid, and beta blockers.

[0098] Exemplary antipsychotics include, but are not limited to, benperidol, bromperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, ciamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, periciazine, perphenazine, pipothiazine, prochlorperazine, promazine, promethazine, prothipendyl, thiopropazate, thioridazine, trifluoperazine, triflupromazine, clopoxene, clopentixol, flupentixol, thiothixene, zuclopentixol, 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, diprasidone, zotepine, alstonine, bifepranox, vitaperthin, brexpiprazole, cannabidiol, carbanipramine, pomaglumetadomethionyl, babiczarine, xanomeline, and diclazepine.

[0099] Exemplary analgesics include paracetamol / acetaminophen, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate, but not limited thereto).

[0100] Exemplary antispasmodics include mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, methaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene, but not limited thereto. Suitable anti-inflammatories include prednisone, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immunoselective anti-inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives), but not limited thereto.

[0101] Exemplary antihistamines include H1 receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, bucrizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimethindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromolynic acid, nedocromil, and p2-adrenergic agonists, but are not limited thereto.

[0102] Exemplary anti-infective agents include amoebicides (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, 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, voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin, and amphotericin b), antimalarials (e.g., pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proguanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), anti-tuberculosis agents (e.g., aminosalicylic acid (e.g., aminosalicylic acid), isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antiviral agents (e.g., amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / ritonavir / lopinavir / tenofovir, interferon α-2v / ribavirin, peginterferon α-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, abacavir, zidovudine,Stavudine, Emtricitabine, Zalcitabine, Telbivudine, Simiprevir, Boceprevir, Telaprevir, Ritonavir / Lopinavir, fosamprenavir, darunavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, saquinavir, ribavirin, valganciclovir, acyclovir, famciclovir, ganciclovir, and brivudine), carbapenems (e.g., doripenem, meropenem, ertapenem, and cilastatin / imipenem), cephalosporins (e.g., cefadroxil, cefradine, cefazolin, cephalexin, cefepime, ceftobiprole, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, cefixime, cefdinir, cefixime, cefditoren, cefixime, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin), glycylcyclines (e.g., tigecycline), anti-leprosy drugs (e.g., clofazimine and thalidomide), lincosamine and its derivatives (e.g., clindamycin and lincomycin), macrolides and its derivatives (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, piperacillin / tazobactam, clavulanate / ticarcillin, penicillin, procaine penicillin, oxacillin, dicloxacillin, and nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin),Sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine, and sulfisoxazole), tetracyclines (e.g., doxycycline, demeclocycline, minocycline, doxycycline / salicylic acid, doxycycline / omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g., nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue) are included, but not limited to these.

[0103] Exemplary chemotherapeutic agents include paclitaxel, Herceptin, Brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, romidepsin, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, visimodegib, ErwiniaAsparaginase, amifostine, etoposide, flutamide, tamoxifen, fulvestrant, letrozole, degarelix, plerocetaxel, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylate, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon α-2a, gefitinib, romidepsin, ixabepilone, luxitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium chloride-89, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, peg-asparaginase, denileukin, diphtheria toxin, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, cetuximab, omacetaxine, thioguanine, dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, eptilom, tafurposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid, among others, are included but not limited to these.

[0104] Suitable radiosensitizers 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 blocking drugs (e.g., cetuximab, gefitinib), farnesyl transferase inhibitors (e.g., L-778-123), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), bFGF and VEGF targeting agents (e.g., bevacizumab and thalidomide), NBTXR3, Nimoral, sodium trans-croscylate, NVX-108, and combinations thereof. See, for example, Kvols, LK.., J Nucl Med 2005;46:187S-190S as well.

[0105] Other exemplary cargos include anticoagulants (e.g., heparin and heparin variants).

[0106] Exemplary immunogens carried as cargo or attached to the outer surface of isolated exosomes include keyhole limpet hemocyanin (KLH), concanavalin concanavalin hemocyanin (CCH) (also known as blue carrier immunogenic protein), bovine serum albumin (BSA), ovalbumin (OVA), and antigens used to generate an immune response against pathogens that cause diseases such as diphtheria, tetanus, pertussis, measles, mumps, rubella, hepatitis A, hepatitis B, meningococcal disease (e.g., meningitis), human papillomavirus varicella, rabies, influenza, retrovirus, HIV, malaria, and diseases caused by the coronavirus disease.

[0107] Exemplary polynucleotide modification systems include, but are not limited to, the CRISPR-Cas system, the OMEGA system, the PRIME editing system, base editors, meganucleases, zinc finger nucleases, recombinases, TALE nucleases, the CAST system, non-LTR retrotransposon systems, transposons, RNAi, antisense nucleic acids, etc. For example, Crooke ST, Liang XH, Baker BF, Crooke RM. Antisense technology: A review. J Biol Chem. 2021;296:100416. doi:10.1016 / j.jbc.2021.100416, Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, DOI:dx.doi.org / 10.1016 / j.molcel.2015.10.008; Peters et al., PNAS 114(35)(2017); DOI:10.1073 / pnas.1709035114; Makarova et al. 2018. The CRISPR Journal, v.1, n5, Figure 5; Kim, Y.G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A. 91, 883-887; 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, 1156-1160; 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 et al. 2017. Nature. 551:464 - 471; Cox et al. 2017. Science 358:1019 - 1027; Levy et al. Nature Biomedical Engineering doi.org / 10.1038 / s41441 - 019 - 0505 - 5 (2019); Gorsuch et al. (2022). Targeting the hepatitis B cccdna with a sequence - specific arcus nuclease to eliminate hepatitis B virus in vivo. Molecular Therapy, 30(9), 2909 - 2922. doi.org / 10.1016 / j.ymthe.2022.05.013; Anzalone et al. 2019. Nature. 576:149 - 157, particularly at Figures 1b, 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., FEBS Lett. 2018 Apr;592(8):1389 - 1399; Singh, et al.,Attachment Site Selection and Identity in Bxb1 Serine Integrase-Mediated Site-Specific Recombination, PLoS Genet. 2013 May; 9(5): e1003490; and Gupta, et al., Nucleic Acids Res. 2007 May; 35(10): 3407-3419; Tou et al. bioRxiv 2022.01.07.475005, doi.org / 10.1101 / 2022.01.07.475005; Klompe et al. Nature, doi:10.1038 / s41586-019-1323; Strecker et al. Science. 10 / 1126 / science.aax9181(2019); Christensen SM et al., RNA from the 5’ end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site, Proc Natl Acad Sci U S A. 2006 Nov 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 et al., The age and evolution of non-LTR retrotransposable elements, Mol Biol Evol.June 1999; 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 Nos. 2020 / 0239544, 2018 / 0346934; International Patent Application Publication Nos. WO2018 / 213708, WO2018 / 213726, WO2014 / 093622, WO2019 / 005884, WO2019 / 005886, WO2019 / 071048, WO2016 / 106236, WO2020 / 206231; WO 021 / 138469, WO2020 / 131862, WO2021 / 257997, WO2021 / 087394, WO2022 / 147321, WO2022 / 07682, WO2022 / 150651, WO2021 / 102042, WO2022 / 173830. See also.

[0108] In some embodiments, the exogenous cargo is present in milk exosomes in a non-zero amount in the range of 0~ / 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, 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, μg, mg, or g, or is any numerical value or sub-range within any of these ranges.

[0109] Exemplary methods for isolating milk exosomes Milk exosomes can be isolated from mammalian milk using any suitable technique. In some embodiments, the mammalian milk is bovine milk, ovine milk, or equine milk. In some embodiments, the milk exosomes or a population thereof are isolated by the methods described in Marsh et al., Nanotheranostics. 2021 Jul 5;5(4):488-498.doi:10.7150 / ntno.62213.eCollection 2021 and WO2022 / 182782, which are incorporated herein by reference in their entireties as if reproduced herein. In some embodiments, the milk exosome(s) is / are separated by a method comprising: (a) centrifuging mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk; (b) removing the fat separated from the mammalian milk; (c) after step (b), centrifuging the remaining mammalian milk one or more times and skimming off any separated fat visible after each centrifugation in step (c); (d) filtering the biological fluid remaining after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); (f) after (d) or optionally (e), and optionally chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30-42 °C for about 15-120 minutes; and (g) after (f), optionally performing tangential flow filtration to obtain a retention fluid that is optionally ultracentrifuged via one or more ultracentrifugation steps or stored at -80 °C, and optionally separating fractions of the retention fluid through a column after the retention fluid has been optionally ultracentrifuged or stored at -80 °C, the method including step (e) or step (g), but not both.

[0110] Milk Exosome Formulations and Kits Also described herein are pharmaceutical formulations and pharmaceutically acceptable carriers or excipients that include one or more milk exosomes in an amount, an effective amount, and / or at least an effective amount, and / or a therapeutically effective amount, such as exogenous cargo-loaded milk exosomes described in more detail elsewhere herein. Specific exemplary embodiments herein describe methods that include administering to a subject a formulation described in any one of the previous paragraph and / or elsewhere herein, such as the following examples. In some embodiments, the formulation administered to the subject includes milk exosomes, such as any of those described elsewhere herein and / or prepared by the methods described elsewhere herein. In some embodiments, the milk exosomes are exogenous cargo-loaded milk exosomes.

[0111] In some embodiments, the subject to which the milk exosomes or a formulation thereof is administered has a disease, disorder, condition, or injury (e.g., mechanical or non-mechanical injury). Exemplary diseases, disorders, conditions, and injuries include cancer, viral infection, bacterial infection, parasitic infection, mechanical injury (e.g., external and internal wounds and tissue damage, cuts, abrasions, burns, surgical wounds, secondary wounds due to infection, disease, or condition, etc.), cancer, non-mechanical injury (e.g., chemical injury, electrical injury, radiation injury, ischemic and / or hypoxic injury (e.g., myocardial infarction, ischemic wound and / or stroke), multiple sclerosis, psoriasis, scleroderma, acne, eczema, or diseases of the skin and / or connective tissue, heart disease or disorder, neurodegenerative disease or disorder, neuropathy, atherosclerosis, conditions involving epithelial permeability, and / or angiogenesis (e.g., angiogenesis or neovascularization), respiratory distress syndrome (RDS), reperfusion injury, skin blood vessel injury or deformation, macular degeneration, choroidal neovascularization through the Bruch's membrane, diabetic retinopathy (inflammatory and inflammation-related diseases and disorders), and radiation dermatitis, but are not limited thereto.

[0112] A wound can be a chronic wound or a wound that appears not to have healed completely. For example, a wound that has not healed within three months is said to be chronic. Chronic wounds include diabetic foot ulcers, ischemic, venous ulcers, venous lower limb ulcers, venous stasis, arterial, pressure, vasculitic, infectious, decubitus ulcers, burns, ulcers induced by trauma, gangrenous and mixed ulcers. Chronic wounds include wounds characterized by chronic inflammation, incomplete differentiation and overgrowth of granulation tissue, as well as incomplete re-epithelialization and incomplete wound closure, and a long repair time and / or. Chronic wounds can include eye ulcers including corneal ulcers. The use of the disclosed invention in wound healing and tissue regeneration can include humans and livestock, sports animals and pets.

[0113] Tissue damage can be caused by, for example, cuts, abrasions, compression wounds, stretching injuries, laceration wounds, crush injuries, bite wounds, grazes, bullet wounds, rupture wounds, body piercing, stab wounds, surgical wounds, surgical interventions, medical interventions, host rejection reactions after transplantation of cells, tissues, or organs, pharmaceutical effects, pharmaceutical side effects, misalignment, radiation damage, radiation diseases, inflammation, skin wounds caused by cosmetics, visceral damage, disease processes (e.g., asthma, cancer), infectious diseases, infectious agents, developmental processes, maturation processes (e.g., acne), genetic abnormalities, developmental abnormalities, environmental toxins, allergens, scalp injuries, facial injuries, jaw injuries, genital injuries, joint injuries, excretory organ injuries, foot injuries, finger injuries, toe injuries, bone injuries, eye injuries, corneal injuries, muscle injuries, adipose tissue injuries, lung injuries, airway injuries, hernias, anal injuries, hemorrhoids, ear injuries, skin injuries, abdominal injuries, retinal injuries, eye injuries, corneal injuries, wrist injuries, leg injuries, sports injuries, back injuries, birth injuries, early birth injuries, venomous bites, stings, impairment of barrier function, impairment of endothelial barrier function, impairment of epithelial barrier function, tendon injuries, ligament injuries, heart injuries, heart valve injuries, vascular system injuries, cartilage injuries, lymphatic system injuries, traumatic brain injuries, dislocations, esophageal perforations, fistulas, nail injuries, foreign bodies, fractures, frostbite, hand injuries, heat stress disorders, lacerations, neck injuries, self-destruction, shock, traumatic soft tissue injuries, spinal cord injuries, spinal column injuries, sprains, overexertion, tendon injuries, ligament injuries, cartilage injuries, chest injuries, dental injuries, traumas, nerve system-wide injuries, burns, burn wounds, scalds, sunburns, chemical burns, aging, aneurysms, strokes, surgical radiation injuries, gastrointestinal tract injuries, infarcts, or ischemic injuries.

[0114] Heart diseases and disorders can include, but are not limited to, myocardial infarction, cardiomyopathy (such as hypertrophic cardiomyopathy), arrhythmia, and congestive heart failure. The regenerative effects of the provided compositions may bring about beneficial changes in the membrane excitability and ion transients of the heart. There are many different types of arrhythmias that can lead to abnormal heart function in humans. Arrhythmias include, but are not limited to, the following: bradycardia, tachycardia, alternans, automaticity disorders, reentrant arrhythmias, fibrillation, atrioventricular nodal reentrant tachycardia, atrial arrhythmias and induced flutter, QT prolongation syndrome, QT shortening syndrome, Brugada syndrome, premature atrial contractions, slow atrial pacemakers, multifocal atrial tachycardia, atrial flutter, atrial fibrillation, ventricular tachycardia, atrioventricular nodal reentrant tachycardia which is the most common cause of paroxysmal supraventricular tachycardia, atrioventricular junctional rhythm, junctional tachycardia, early junctional complexes, Wolff-Parkinson-White syndrome, Lown-Ganong-Levine syndrome, premature ventricular contractions (PVC) also known as ventricular extra beats, alternans and discordant alternans, enhanced ventricular automaticity, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, ventricular fibrillation, first-degree atrioventricular block manifested as PR prolongation, second-degree atrioventricular block, type 1 second-degree atrioventricular block, type 2 second-degree atrioventricular block, third-degree atrioventricular block, and some accessory pathway disorders (such as Wolff-Parkinson-White syndrome (WPW)).

[0115] Neurodegenerative and neurological disorders can include, but are not limited to, dementia, Alzheimer's disease, Parkinson's disease and related PD disorders, 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.

[0116] Inflammatory diseases, inflammatory-related diseases and disorders can be asthma, eczema, rhinitis, atherosclerosis, arthritis (including but not limited to rheumatoid arthritis), inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis. As used herein, the term "inflammatory disorder" generally refers to a disease or disorder that is at least partially caused or exacerbated by inflammation characterized by an increase in blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), heat, erythema, swelling, pain, and / or loss of function in the affected tissue or organ. The cause of the inflammation can be due to physical injury, chemicals, microorganisms, tissue necrosis, cancer, or other agents or conditions.

[0117] Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders generally have a relatively short duration, lasting from about a few minutes to about 1-2 days, but can last for several weeks. Characteristics of acute inflammatory diseases include an increase in blood flow, exudation of body fluids and plasma proteins (edema), and migration of white blood cells such as neutrophils. Chronic inflammatory diseases generally last for a longer period, for example, from several weeks to several months to years or more, and are histologically associated with the presence of lymphocytes and macrophages, as well as the proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders that recur after a certain period or disorders with periodic attacks. Some inflammatory disorders are classified into one or more categories. Exemplary inflammatory disorders include atherosclerosis, arthritis, cancer promoted by inflammation, asthma, autoimmune uveitis, adoptive immune responses, dermatitis, multiple sclerosis, diabetic complications, osteoporosis, Alzheimer's disease, cerebral malaria, hemorrhagic fever, autoimmune disorders, and inflammatory bowel disease, but are not limited thereto. In some embodiments, the inflammatory disorder is an autoimmune disorder, and in some embodiments, it is selected from lupus, rheumatoid arthritis, and autoimmune encephalomyelitis.

[0118] 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 at least partially caused by, or can occur or be exacerbated by, inflammation within the brain of a subject.

[0119] As used herein, "pharmaceutical formulation" refers to a combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, rendering the composition suitable for diagnostic, therapeutic, or prophylactic use in vitro, in vivo, or ex vivo. As used herein, "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is generally safe, non-toxic, and biologically and otherwise desirable for the preparation of a pharmaceutical formulation, including carriers or excipients acceptable for veterinary use and human pharmaceutical use. "Pharmaceutically acceptable carrier or excipient," as used in this specification and the claims, encompasses one and more than one such carrier or excipient, such as such. 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 one or more milk exosomes, such as cargo-loaded milk exosomes, more particularly described elsewhere herein, as the active ingredient.

[0120] 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 in which the counterion is non-toxic to the subject to which the salt is administered at the pharmaceutical dosage of the salt. Suitable salts include hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, naphthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, pamoate.

[0121] The pharmaceutical formulations described herein can be administered to a subject in need thereof via any suitable method or route to the subject in need thereof. Suitable routes of administration typically depend on the disease being treated, the subject being treated, and / or the active agent(s) and / or cargo, and include, for example, auricular (ear), buccal, conjunctival, dermal, dental, iontophoresis, intracervical, intranasal, intratracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-arterial, intra-articular, intra-biliary, intra-bronchial, intra-capsular, intracardiac, intra-cartilaginous, intra-caudal vein, intra-cavernous penis, intracavitary, intracerebral, intra-alveolar, intra-corneal, intra-coronal (tooth), intra-coronary artery, intra-cavernous, intradermal, intraspinal, intraductal, intra-duodenal, intradural, intra-epidermal, intra-esophageal, intra-gastric, intramuscular, intra-ocular, intra-ovarian, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intranasal, intramedullary, intra-synovial, intratendinous, intra-testicular, intramedullary, intra-thoracic, intra-tubular, intra-tumoral, intra-tympanic, intra-uterine, intra-vascular, intravenous, intravenous bolus, intravenous infusion, intracerebral, intra-capsular, intravitreal, iontophoresis, lavage, laryngeal, nasal, nasogastric, occlusive dressing, ocular, oral, oro-pharyngeal, other, parenteral, percutaneous, perijoint, epidural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, sub-arachnoid, sub-conjunctival, subcutaneous, sublingual, submucosal, topical, percutaneous, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above routes of administration may be included.

[0122] When appropriate, one or more milk exosomes, such as cargo-loaded milk exosomes described in more detail elsewhere in this specification, can be provided to a subject in need thereof as an active ingredient or a component such as a drug in a pharmaceutical formulation. Accordingly, pharmaceutical formulations containing one or more milk exosomes, such as cargo-loaded milk exosomes described in more detail elsewhere in this specification, are also described and include cargo in the form of pharmaceutically acceptable salts. Suitable salts include hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbic acid, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, naphthalenesulfonate, propionate, malonate, mandelic acid, malate, phthalate, pamoate.

[0123] As used herein, "drug" refers to any substance, compound, molecule, etc., which may be biologically active or, otherwise, may induce a biological and / or physiological effect on the subject to which it is administered. As used herein, "active agent" or "active ingredient" refers to a substance, compound, or molecule that is biologically active or, otherwise, induces a biological or physiological effect on the subject to which it is administered. In other words, "active agent" or "active ingredient" refers to one or more components of a composition that are responsible, in whole or in part, for the effect of the composition. A drug can be a primary active drug, or, in other words, a component(s) of a composition responsible for the whole or part of the effect of the composition. A drug can be a secondary drug, or, in other words, a component(s) of a composition responsible for additional parts and / or other effects of the composition. In some embodiments, the active agent is a milk exosome, or a cargo-loaded milk exosome. In some embodiments, the active drug is or comprises the cargo of a cargo-loaded milk exosome.

[0124] In certain embodiments, milk exosomes are prepared by any of the methods described elsewhere in this specification. In some embodiments, cargo-loaded milk exosomes are described and / or prepared by the methods described elsewhere in this specification.

[0125] In some embodiments, milk exosomes or a formulation thereof are included on a material for administration to a subject. Non-limiting examples of materials include those used in the treatment of wounds such as bandages, sterile strips, sutures, staples, or grafts (e.g., skin grafts). Other exemplary materials include medical devices or implants (or components thereof). Non-limiting examples of medical implants include prosthetic limbs, breast implants, penile implants, testicular implants, artificial eyes, facial implants, artificial joints, heart valve prostheses, artificial blood vessels, dental prostheses, facial prostheses, tilting disc valves, caged ball valves, artificial ears, nasal prostheses, pacemakers, cochlear implants, stents, shunts, catheters, filters, meshes, fillers (e.g., fat and skin fillers), and skin substitutes (e.g., porcine xenografts / porcine skin, BIOBRANE, cultured keratinocytes), and / or the like.

[0126] Pharmaceutically acceptable carriers and secondary components and agents Pharmaceutical formulations can include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, milk and dairy products (e.g., casein, ice cream, custard, creams, and / or the like), salt solutions, alcohol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, flavoring agents, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone (which do not react detrimentally with the active composition).

[0127] Such pharmaceutical formulations can be sterilized and, if desired, mixed with substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and / or fragrances that do not react detrimentally with the active compound.

[0128] In some embodiments, the pharmaceutical formulation may also include an effective amount of a secondary active agent, including but not limited to biological agents or molecules such as polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, anticonvulsants, anti-inflammatories, antihistamines, anti-infectives, chemotherapeutic agents, and any combination thereof.

[0129] In some embodiments, the secondary active agent is one or more immunoglobulins. In some embodiments, the one or more immunoglobulins are IgG, IgM, IgA, IgD, IgE, or any combination thereof. In some embodiments, the one or more immunoglobulins are included in the pharmaceutical formulation in a dosage that releases the one or more immunoglobulins prior to the delivery or release of the milk exosomes present in the pharmaceutical formulation.

[0130] In some embodiments, the secondary active agent is one or more agents, ATP, ADP, or any combination thereof, that stimulate the release, secretion, and / or production of ATP. In some embodiments, the one or more agents, ATP, ADP, or any combination thereof, that stimulate the release, secretion, and / or production of ATP are included in the pharmaceutical formulation in a dosage form that releases the one or more agents, ATP, ADP, or any combination thereof, that stimulate the release, secretion, and / or production of ATP prior to the delivery or release of the milk exosomes present in the pharmaceutical formulation.

[0131] In some embodiments, the secondary activator comprises one or more immunoglobulins, one or more agents that stimulate the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof. In some embodiments, one or more immunoglobulins, one or more agents that stimulate the release, secretion, and / or production of ATP, the release, secretion, and / or production of ATP and / or ADP, or any combination thereof are present in a pharmaceutical formulation in a dosage form that releases one or more immunoglobulins, one or more agents that stimulate the release, secretion, and / or production of ATP, the release, secretion, and / or production of ATP and / or ADP, or any combination thereof before or upon delivery of the milk exosomes in the pharmaceutical formulation.

[0132] In some embodiments, the combination therapy results in an increase in the uptake of milk exosomes by one cell, or more than one cell. In some embodiments, the milk exosomes have no released ATP at a concentrated level, or have released and concentrated ATP, or are in an environment where the secreted ATP is concentrated, compared to cells in an environment where there is no concentrated ATP released, or no concentrated ATP secreted by stimulated cells, for example, due to the prompt by the presence of damaged or cancer cells. In some embodiments, the milk exosomes have increased uptake in cells including cells having FcRN on the surface, gastrointestinal cells, heart cells, liver cells, brain cells, nerve cells, cancer cells, or any combination thereof. In some embodiments, the cells are polar cells. This means that the cells have different apical and basolateral membrane surfaces. In some embodiments, the cells are intestinal cells. In some embodiments, the cells are blood-brain barrier cells. In some embodiments, the uptake by the cells is 0.01%, up to 0.02% or 0.02, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75% higher than that in cells that are in a region without concentrated ATP or cells without FcRN, by a secondary ATP, ADP, immunoglobulin, and / or an agent having an effect of stimulating ATP release, secretion and / or production.0.76%、0.77%、0.78%、0.79%、0.8%、0.81%、0.82%、0.83%、0.84%、0.85%、0.86%、0.87%、0.88%、0.89%、0.9%、0.91%、0.92%、0.93%、0.94%、0.95%、0.96%、0.97%、0.98%、0.99%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%、101%、102%、103%、104%、105%、106%、107%、108%、109%、110%、111%、112%、113%、114%、115%、116%、117%、118%、119%、120%、121%、122%、123%、124%、125%、126%、127%、128%、129%、130%、131%、132%、133%、134%、135%、136%、137%、138%、139%、140%、141%、142%、143%、144%、145%、146%、147%、148%、149%、150%、151%、152%、153%、154%、155%、156%、157%、158%、159%、160%、161%、162%、163%、164%、165%、166%、167%、168%、169%、170%、171%、172%、173%、174%、175%、176%、177%、178%、179%、180%、181%、182%、183%、184%、185%、186%、187%、188%、189%、190%、191%、192%、193%、194%、195%、196%、197%、198%、199%、200%、201%、202%、203%、204%、205%、206%、207%、208%、209%、210%、211%、212%、213%、214%、215%、216%、217%、218%、219%、220%、221%、222%、223%、224%、225%、226%、227%、228%、229%、230%、231%、232%、233%、234%、235%、236%、237%、238%、239%、240%、241%、242%、243%、244%、245%、246%、247%、248%、249%、250%、251%、252%、253%、254%、255%、256%、257%、258%、259%、260%、261%、262%、263%、264%、265%、266%、267%、268%、269%、270%、271%、272%、273%、274%、275%、276%、277%、278%、279%、280%、281%、282%、283%、284%、285%、286%、287%、288%、289%、290%、291%、292%、293%、294%、295%、296%、297%、298%、299%、300%、301%、302%、303%、304%、305%、306%、307%、308%、309%、310%、311%、312%、313%、314%、315%、316%、317%、318%、319%、320%、321%、322%、323%、324%、325%、326%、327%、328%、329%、330%、331%、332%、333%、334%、335%、336%、337%、338%、339%、340%、341%、342%、343%、344%、345%、346%、347%、348%、349%、350%、351%、352%、353%、354%、355%、356%、357%、358%、359%、360%、361%、362%、363%、364%、365%、366%、367%、368%、369%、370%、371%、372%、373%、374%、375%、376%、377%、378%、379%、380%、381%、382%、383%、384%、385%、386%、387%、388%、389%、390%、391%、392%、393%、394%、395%、396%、397%、398%、399%、400%、401%、402%、403%、404%、405%、406%、407%、408%、409%、410%、411%、412%、413%、414%、415%、416%、417%、418%、419%、420%、421%、422%、423%、424%、425%、426%、427%、428%、429%、430%、431%、432%、433%、434%、435%、436%、437%、438%、439%、440%、441%、442%、443%、444%、445%、446%、447%、448%、449%、450%、451%、452%、453%、454%、455%、456%、457%、458%、459%、460%、461%、462%、463%、464%、465%、466%、467%、468%、469%、470%、471%、472%、473%、474%、475%、476%、477%、478%、479%、480%、481%、482%、483%、484%、485%、486%、487%、488%、489%、490%、491%、492%、493%、494%、495%、496%、497%、498%、499%、500%、501%、502%、503%、504%、505%、506%、507%、508%、509%、510%、511%、512%、513%、514%、515%、516%、517%、518%、519%、520%、521%、522%、523%、524%、525%、526%、527%、528%、529%、530%、531%、532%、533%、534%、535%、536%、537%、538%、539%、540%、541%、542%、543%、544%、545%、546%、547%、548%、549%、550%、551%、552%、553%、554%、555%、556%、557%、558%、559%、560%、561%、562%、563%、564%、565%、566%、567%、568%、569%、570%、571%、572%、573%、574%、575%、576%、577%、578%、579%、580%、581%、582%、583%、584%、585%、586%、587%、588%、589%、590%、591%、592%、593%、594%、595%、596%、597%、598%、599%、600%、601%、602%、603%、604%、605%、606%、607%、608%、609%、610%、611%、612%、613%、614%、615%、616%、617%、618%、619%、620%、621%、622%、623%、624%、625%、626%、627%、628%、629%、630%、631%、632%、633%、634%、635%、636%、637%、638%、639%、640%、641%、642%、643%、644%、645%、646%、647%、648%、649%、650%、651%、652%、653%、654%、655%、656%、657%、658%、659%、660%、661%、662%、663%、664%、665%、666%、667%、668%、669%、670%、671%、672%、673%、674%、675%、676%、677%、678%、679%、680%、681%、682%、683%、684%、685%、686%、687%、688%、689%、690%、691%、692%、693%、694%、695%、696%、697%、698%、699%、700%、701%、702%、703%、704%、705%、706%、707%、708%、709%、710%、711%、712%、713%、714%、715%、716%、717%、718%、719%、720%、721%、722%、723%、724%、725%、726%、727%、728%、729%、730%、731%、732%、733%、734%、735%、736%、737%、738%、739%、740%、741%、742%、743%、744%、745%、746%、747%、748%、749%、750%、751%、752%、753%、754%、755%、756%、757%、758%、759%、760%、761%、762%、763%、764%、765%、766%、767%、768%、769%、770%、771%、772%、773%、774%、775%、776%、777%、778%、779%、780%、781%、782%、783%、784%、785%、786%、787%、788%、789%、790%、791%、792%、793%、794%、795%、796%、797%、798%、799%、800%、801%、802%、803%、804%、805%、806%、807%、808%、809%、810%、811%、812%、813%、814%、815%、816%、817%、81、 Increase by more than 8%, 819%, 820%, 821%, 822%, 823%, 824%, 825%, 826%, 827%, 828%, 829%, 830%, 831%, 832%, 833%, 834%, 835%, 836%, 837%, 838%, 839%, 840%, 841%, 842%, 843%, 844%, 845%, 846%, 847%, 848%, 849%, 850%, 851%, 852%, 853%, 854%, 855%, 856%, 857%, 858%, 859%, 860%, 861%, 862%, 863%, 864%, 865%, 866%, 867%, 868%, 869%, 870%, 871%, 872%, 873%, 874%, 875%, 876%, 877%, 878%, 879%, 880%, 881%, 882%, 883%, 884%, 885%, 886%, 887%, 888%, 889%, 890%, 891%, 892%, 893%, 894%, 895%, 896%, 897%, 898%, 899%, 900%, 901%, 902%, 903%, 904%, 905%, 906%, 907%, 908%, 909%, 910%, 911%, 912%, 913%, 914%, 915%, 916%, 917%, 918%, 919%, 920%, 921%, 922%, 923%, 924%, 925%, 926%, 927%, 928%, 929%, 930%, 931%, 932%, 933%, 934%, 935%, 936%, 937%, 938%, 939%, 940%, 941%, 942%, 943%, 944%, 945%, 946%, 947%, 948%, 949%, 950%, 951%, 952%, 953%, 954%, 955%, 956%, 957%, 958%, 959%, 960%, 961%, 962%, 963%, 964%, 965%, 966%, 967%, 968%, 969%, 970%, 971%, 972%, 973%, 974%, 975%, 976%, 977%, 978%, 979%, 980%, 981%, 982%, 983%, 984%, 985%, 986%, 987%, 988%, 989%, 990%, 991%, 992%, 993%, 994%, 995%, 996%, 997%, 998%, 999%, 1000%.

[0133] Effective dosage

[0134] In some embodiments, the amount of the primary active agent (e.g., milk exosomes, cargo-loaded milk exosomes, and / or cargo) and / or the optional secondary agent can be an effective amount, a minimum effective amount, and / or a therapeutically effective amount. As used herein, "effective amount" refers to the amount of the primary agent and / or the optional secondary agent included in a pharmaceutical formulation that achieves one or more therapeutic or desired effects. As used herein, "minimum effective" amount refers to the lowest amount of the primary agent and / or the optional secondary agent that achieves one or more therapeutic or other desired effects. As used herein, "therapeutically effective amount" refers to the amount of the primary agent and / or the optional secondary agent included in a pharmaceutical formulation that achieves one or more therapeutic effects.

[0135] The effective amount, minimum effective amount, and / or therapeutically effective amount of the primary and optional secondary active agents described elsewhere herein included in a pharmaceutical formulation can be any non-zero amount in the range of about 0 to 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, 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, μg, mg, or g, or can be any numerical value or sub-range within any of these ranges.

[0136] In some embodiments, the effective amount, minimum effective amount, and / or therapeutic effective amount can be an effective concentration, minimum effective concentration, and / or therapeutic effective concentration, each of which can be any non-zero amount in the range of about 0 to 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, 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 pM, nM, μM, mM, or M, or can be any numerical value or sub-range within any of these ranges.

[0137] In other embodiments, the effective amount, minimum effective amount, and / or therapeutic effective amount of the primary and optional secondary active agents is any non-zero amount in the range of about 0 to 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, 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 IU, or any numerical value or sub-range within any of these ranges.

[0138] In some embodiments, the primary active agent and / or optional secondary active agent present in the pharmaceutical formulation can be any non-zero amount in the range of about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9, ~1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v, or any numerical value or sub-range within any of these ranges.

[0139] In some embodiments where the cell or cell population is present in the pharmaceutical formulation (e.g., as a secondary active agent), the effective amount of the cell is about 1 or 2 cells to 1×10 1 / mL, 1×10 20 / mL or more, for example, about 1×10 1 / mL, 1×10 2 / mL, 1×10 3 / mL, 1×10 4 / mL, 1×10 5 / mL, 1×10 6 / mL, 1×10 7 / mL, 1×10 8 / mL, 1×10 9 / mL, 1×10 10 / mL, 1×10 11 / mL, 1×10 12 / mL, 1×10 13 / mL, 1×10 14 / mL, 1×10 15 / mL, 1×10 16 / mL, 1×10 17 / mL, 1×10 18 / mL, 1×10 19 / mL, ~ / or about 1×10 20 / mL of any amount, or any numerical or sub-range within any of these ranges.

[0140] In some embodiments, where the amount or effective amount, particularly when infectious particles are delivered (e.g., a virus or virus-like particle as a primary or secondary agent, e.g., as cargo), the effective amount of viral particles can be expressed as a titer (plaque forming units per unit volume) or as an MOI (multiplicity of infection). In some embodiments, the effective amount is about 1×10 1 particles per pL, nL, μL, mL, or L to 1×10 20 or more per pL, nL, μL, mL, or L, for example, about 1×10 1 , 1×10 2 , 1X10 3 , 1X10 4 , 1X10 5 , 1X10 6, 1X10 7 , 1X10 8 , 1X10 9 , 1X10 10 , 1X10 11 , 1X10 12 , 1X10 13 , 1X10 14 , 1X10 15 , 1X10 16 , 1X10 17 , 1X10 18 , 1X10 19 , ~ / or about 1X10 20 particles may be. In some embodiments, the effective titer is about 1×10 per pL, nL, μL, mL, or L 1 conversion unit ~ 1×10 per pL, nL, μL, mL, or L 20 conversion unit or more, for example, per pL, nL, μL, mL, or L, about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , ~ / or about 1×10 20It can be a conversion unit, or any numerical value or sub-range within these ranges. In some embodiments, the MOI of the pharmaceutical formulation is from about 0.1 to 10 or more, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 or more, or any numerical value or sub-range within these ranges.

[0141] In some embodiments, the amount or effective amount of one or more active agents(s) described herein contained in the pharmaceutical formulation can be in the range of about 1 pg / kg to about 10 mg / kg based on the body weight of the subject in need thereof or the average body weight of a particular patient population to which the pharmaceutical formulation can be administered.

[0142] In embodiments where the pharmaceutical formulation contains a secondary agent, the effective amount of the secondary active agent varies, inter alia, according to the secondary agent, the primary agent, the route of administration, the age of the subject, the disease, and the stage of the disease, which is a matter of ordinary skill.

[0143] Optionally present in the pharmaceutical formulation, the secondary active agent can be included in the pharmaceutical formulation or can be present as an independent compound or pharmaceutical formulation that can be administered simultaneously or sequentially with the compound, its derivatives, or the pharmaceutical formulation thereof.

[0144] In some embodiments, when present optionally, the effective amount of the secondary active agent is about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v of the total amount of the active agent present in the pharmaceutical formulation, or any non-zero amount within these ranges, or any numerical value or sub-range within these ranges. In additional embodiments, the effective amount of the secondary active agent is about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v of the total amount of the pharmaceutical formulation, or any non-zero amount within these ranges, or any numerical value or sub-range within these ranges.

[0145] Dosage form In some embodiments, the pharmaceutical formulations described herein can be provided in dosage forms. The dosage forms can be administered to a subject in need thereof. The dosage forms can effectively produce a specific concentration, such as an effective concentration, at a given site in a subject in need thereof. "Dosage", "unit dosage" or "dose" can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of a primary active agent calculated to provide a desired response(s) in relation to its administration and optionally present secondary active materials and / or its pharmaceutical formulation. In some embodiments, a given site is proximal to the site of administration. In some embodiments, a given site is distal to the site of administration. In some cases, the dosage form contains more of one or more active ingredients present in the pharmaceutical formulation than the final intended amount necessary to reach a specific region or location within the subject, taking into account, for example, the loss of active ingredient via first-pass and second-pass metabolism.

[0146] The dosage forms can be adapted for administration by any suitable route. Suitable routes include, but are not limited to, oral (including buccal or sublingual), rectal, intraocular, inhalation, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, intranasal, and intradermal. Other suitable routes are described elsewhere herein. Such formulations can be prepared by any method known in the art.

[0147] Dosage forms adapted for oral administration can be individual unit dosages, such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or non-aqueous liquids, edible foams or froths, or water-in-oil or oil-in-water liquid emulsions. In some embodiments, pharmaceutical formulations adapted for oral administration also include one or more agents that assist in flavoring, preserving, coloring, or dispersing the pharmaceutical formulation. Dosage forms prepared for oral administration can be in the form of solutions that can be delivered as foams, sprays, or solutions. Oral dosage forms can be administered to a subject in need thereof. Optionally, the dosage forms described herein can be microencapsulated.

[0148] The dosage form can also be prepared to extend or sustain the release of any component. In some embodiments, the milk exosomes described herein can be components with delayed release. In some embodiments, the primary active agent is a component with delayed release. In some embodiments, an optional secondary agent can be a component with delayed release. In some embodiments, an optional secondary agent is not delayed such that it is delivered before the primary active agent (e.g., the milk exosomes of the present disclosure). Suitable methods for delaying the release of a component include, but are not limited to, coating or embedding the component with a polymer, wax, gel, etc. Sustained release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules, as well as sustained release dosage forms of tablets and pellets, capsules, and granules. The release delay can be from about 1 hour to about 3 months or more.

[0149] Examples of suitable coating materials include, for example, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides, but are not limited thereto.

[0150] The coating can be formed with different ratios of water-soluble polymers, water-insoluble polymers, and / or pH-dependent polymers, with or without water-insoluble / water-soluble non-polymer excipients, to provide the desired release profile. The coating can be performed in any of (matrix or simple) "as is" materials, including but not limited to tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particulate compositions, suspension form, or sprinkle dosage form.

[0151] Where appropriate, the dosage forms described herein can be liposomes. In these embodiments, the primary active ingredient(s), and / or any secondary active ingredient(s), and / or optionally its pharmaceutically acceptable salts are incorporated into the liposomes. Thus, in embodiments where the dosage form is a liposome, the pharmaceutical formulation is a liposomal formulation. The liposomal formulation can be administered to a subject in need thereof.

[0152] Dosage forms suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for the treatment of the eye or other external tissues, such as the mouth or skin, the pharmaceutical formulation is applied as a topical ointment or cream. When formulated as an ointment, the primary active ingredient, optional secondary active ingredient, and / or, as appropriate, its pharmaceutically acceptable salt can be formulated with a paraffin-based or water-miscible ointment base. In other embodiments, the primary and / or secondary active ingredients can be formulated into a cream using a water-in-oil cream base or an oil-in-water base. Dosage forms adapted for topical administration into the mouth include lozenges, troches, and mouthwashes.

[0153] Dosage forms suitable for nasal or inhalation administration include aerosols, solutions, suspensions, gels, or dry powders. In some embodiments, the primary active ingredient, optional secondary active ingredient, and / or, as appropriate, its pharmaceutically acceptable salt that can be present in a dosage form suitable for inhalation is in a reduced particle size form obtained or obtainable by micronization. In some embodiments, the particle size of the size-reduced (e.g., micronized) compound or its salt or solvate, when measured by suitable methods known in the art, is defined by a D 50 value of from about 0.5 to about 10 microns. Dosage forms suitable for administration by inhalation also include fine particle powders or mists. Suitable dosage forms in which a carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions / suspensions of the active (primary and / or secondary) ingredient, which can be generated by various types of metered-dose pressurized aerosols, nebulizers, or insufflators. The nasal / inhalation formulation can be administered to a subject in need thereof.

[0154] In some embodiments, the dosage form is an aerosol formulation suitable for administration by inhalation. In some of these embodiments, the aerosol formulation contains, where appropriate, a solution or fine suspension of a primary active ingredient, a secondary active ingredient, and / or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable aqueous or non-aqueous solvent. The aerosol formulation can be provided in a sterile form within a sealed container, in a single-dose or multi-dose form. In some of these embodiments, the sealed container is a nasal or aerosol dispenser (e.g., a metered-dose inhaler) fitted with a metering valve for a single-dose or multi-dose, where the contents of the container are intended to be disposed of when used up.

[0155] When the aerosol dosage form is contained within an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant including but not limited to hydrofluorocarbons. The aerosol dosage form in other embodiments is contained within a pump nebulizer. The pressurized aerosol formulation can also contain a solution or suspension of a primary active ingredient, any secondary active ingredient, and / or a pharmaceutically acceptable salt thereof. In further embodiments, the aerosol formulation also contains a co-solvent and / or a modifier incorporated, for example, to improve the stability and / or taste and / or particulate mass characteristics (amount and / or profile) of the formulation. Administration of the aerosol formulation can be once or several times a day, for example, 2, 3, 4, or 8 times a day, in which case, at each occasion, 1, 2, 3, or more doses are delivered. The aerosol formulation can be administered to a subject in need thereof.

[0156] Regarding some dosage forms suitable for and / or compatible with inhalation administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the primary active agent, optional secondary active ingredients, and / or pharmaceutically acceptable salts thereof, such dosage forms can, where appropriate, contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, where appropriate, the primary active agent, secondary active material, and / or pharmaceutically acceptable salts thereof are in a reduced particle size form. In further embodiments, performance modifiers such as L-leucine or another amino acid, cellulose octaacetate, and / or a metal salt of stearic acid such as magnesium or calcium stearate are included. In some embodiments, the aerosol formulation is arranged such that each metered dose of the aerosol contains a predetermined amount of an active ingredient such as one or more of the compositions described herein, compositions, compounds, vectors, molecules, cells, and combinations thereof.

[0157] Dosage forms compatible with vaginal administration can be provided as pessaries, tampons, creams, gels, pastes, foaming agents, or spray formulations. Dosage forms compatible with rectal administration include suppositories or enemas. The vaginal formulation can be administered to a subject in need thereof.

[0158] Formulations suitable for parenteral administration and / or suitable for injection can include aqueous and non-aqueous isotonic sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the recipient subject, as well as aqueous and non-aqueous sterile suspensions that can contain suspending and thickening agents. Dosage forms compatible with parenteral administration can be provided in single-dose or multi-dose containers, including but not limited to sealed ampoules or vials. The dosage can be lyophilized and resuspended in a sterile carrier prior to administration to reconstitute the dosage. Immediate injection solutions and suspensions can, in some embodiments, be prepared from sterile powders, granules, and tablets. The parenteral formulation can be administered to a subject in need thereof.

[0159] In some embodiments, the dosage form contains, per unit dose, a predetermined amount of a primary active agent, a secondary active ingredient, and / or a pharmaceutically acceptable salt thereof, where appropriate. In one embodiment, the predetermined amount of the primary active agent, secondary active ingredient, and / or a pharmaceutically acceptable salt thereof, where appropriate, can be an effective amount, a minimum effective amount, and / or a therapeutically effective amount. In other embodiments, the predetermined amount of the primary active agent, secondary active agent, and / or a pharmaceutically acceptable salt thereof can be an appropriate portion of the effective amount of the active ingredient, where appropriate.

[0160] Combination Therapy and Combination Treatment In some embodiments, the pharmaceutical formulation(s) described herein is part of a combination treatment or combination therapy. The combination treatment can include the pharmaceutical formulation described herein and an additional treatment modality. The additional treatment modality can be chemotherapy, biological therapy, surgery, radiation, dietary modification, environmental modification, physical activity modification, and combinations thereof.

[0161] In some embodiments, the combination therapy or combination treatment includes, but is not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, anticonvulsants, anti-inflammatory agents, antihistamines, anti-infectives, chemotherapeutic agents, anti-cancer agents, immunogens, and any combination thereof.

[0162] In some embodiments, the combination therapy includes the milk exosomes of the present disclosure or a population thereof and one or more immunoglobulins. In some embodiments, the one or more immunoglobulins are IgG, IgM, IgA, IgD, IgE, or any combination thereof. In some embodiments, the one or more immunoglobulins are included in the combination therapy such that delivery of the one or more immunoglobulins is delivered to the subject or its target cells prior to delivery of the milk exosomes of the present disclosure.

[0163] In some embodiments, the combination therapy comprises the milk exosomes of the present disclosure or a population thereof and an agent, ATP, ADP, or any combination thereof that can stimulate the release, secretion, and / or production of ATP. In some embodiments, one or more agents, ATP, ADP, or any combination thereof that stimulate ATP release, secretion, and / or production are included in the combination therapy such that they are delivered to the subject and / or target cells prior to delivery of the milk exosomes of the combination therapy.

[0164] In some embodiments, the combination therapy comprises the milk exosomes of the present disclosure or a population thereof and one or more immunoglobulins, an agent, ATP, ADP, or any combination thereof that stimulates the release, secretion, and / or production of ATP. In some embodiments, one or more immunoglobulins, an agent, ATP, ADP, or any combination thereof that stimulates ATP release, secretion, and / or production are included in the combination therapy such that they are delivered to the subject or cell targeted cells in need thereof prior to delivery of the milk exosomes of the combination treatment.

[0165] In some embodiments, milk exosomes have increased uptake in cells having concentrated ATP or in an environment having concentrated ATP, compared to cells having no concentrated ATP or cells present in an environment without concentrated ATP. In some embodiments, milk exosomes have increased uptake in cells including cells having FcRN on the surface, gastrointestinal cells, heart cells, liver cells, brain cells, lung cells, germ cells, nerve cells, cancer cells, or any combination thereof. In some embodiments, the cells are polar cells. This means that the cells have different apical and basolateral membrane surfaces. In some embodiments, the cells are intestinal cells. In some embodiments, the cells are blood-brain barrier cells. In some embodiments, uptake by combination therapy is 0.01%, up to / or 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1%, 2%, 3%, 4%, 5%,6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%、101%、102%、103%、104%、105%、106%、107%、108%、109%、110%、111%、112%、113%、114%、115%、116%、117%、118%、119%、120%、121%、122%、123%、124%、125%、126%、127%、128%、129%、130%、131%、132%、133%、134%、135%、136%、137%、138%、139%、140%、141%、142%、143%、144%、145%、146%、147%、148%、149%、150%、151%、152%、153%、154%、155%、156%、157%、158%、159%、160%、161%、162%、163%、164%、165%、166%、167%、168%、169%、170%、171%、172%、173%、174%、175%、176%、177%、178%、179%、180%、181%、182%、183%、184%、185%、186%、187%、188%、189%、190%、191%、192%、193%、194%、195%、196%、197%、198%、199%、200%、201%、202%、203%、204%、205%、206%、207%、208%、209%、210%、211%、212%、213%、214%、215%、216%、217%、218%、219%、220%、221%、222%、223%、224%、225%、226%、227%、228%、229%、230%、231%、232%、233%、234%、235%、236%、237%、238%、239%、240%、241%、242%、243%、244%、245%、246%、247%、248%、249%、250%、251%、252%、253%、254%、255%、256%、257%、258%、259%、260%、261%、262%、263%、264%、265%、266%、267%、268%、269%、270%、271%、272%、273%、274%、275%、276%、277%、278%、279%、280%、281%、282%、283%、284%、285%、286%、287%、288%、289%、290%、291%、292%、293%、294%、295%、296%、297%、298%、299%、300%、301%、302%、303%、304%、305%、306%、307%、308%、309%、310%、311%、312%、313%、314%、315%、316%、317%、318%、319%、320%、321%、322%、323%、324%、325%、326%、327%、328%、329%、330%、331%、332%、333%、334%、335%、336%、337%、338%、339%、340%、341%、342%、343%、344%、345%、346%、347%、348%、349%、350%、351%、352%、353%、354%、355%、356%、357%、358%、359%、360%、361%、362%、363%、364%、365%、366%、367%、368%、369%、370%、371%、372%、373%、374%、375%、376%、377%、378%、379%、380%、381%、382%、383%、384%、385%、386%、387%、388%、389%、390%、391%、392%、393%、394%、395%、396%、397%、398%、399%、400%、401%、402%、403%、404%、405%、406%、407%、408%、409%、410%、411%、412%、413%、414%、415%、416%、417%、418%、419%、420%、421%、422%、423%、424%、425%、426%、427%、428%、429%、430%、431%、432%、433%、434%、435%、436%、437%、438%、439%、440%、441%、442%、443%、444%、445%、446%、447%、448%、449%、450%、451%、452%、453%、454%、455%、456%、457%、458%、459%、460%、461%、462%、463%、464%、465%、466%、467%、468%、469%、470%、471%、472%、473%、474%、475%、476%、477%、478%、479%、480%、481%、482%、483%、484%、485%、486%、487%、488%、489%、490%、491%、492%、493%、494%、495%、496%、497%、498%、499%、500%、501%、502%、503%、504%、505%、506%、507%、508%、509%、510%、511%、512%、513%、514%、515%、516%、517%、518%、519%、520%、521%、522%、523%、524%、525%、526%、527%、528%、529%、530%、531%、532%、533%、534%、535%、536%、537%、538%、539%、540%、541%、542%、543%、544%、545%、546%、547%、548%、549%、550%、551%、552%、553%、554%、555%、556%、557%、558%、559%、560%、561%、562%、563%、564%、565%、566%、567%、568%、569%、570%、571%、572%、573%、574%、575%、576%、577%、578%、579%、580%、581%、582%、583%、584%、585%、586%、587%、588%、589%、590%、591%、592%、593%、594%、595%、596%、597%、598%、599%、600%、601%、602%、603%、604%、605%、606%、607%、608%、609%、610%、611%、612%、613%、614%、615%、616%、617%、618%、619%、620%、621%、622%、623%、624%、625%、626%、627%、628%、629%、630%、631%、632%、633%、634%、635%、636%、637%、638%、639%、640%、641%、642%、643%、644%、645%、646%、647%、648%、649%、650%、651%、652%、653%、654%、655%、656%、657%、658%、659%、660%、661%、662%、663%、664%、665%、666%、667%、668%、669%、670%、671%、672%、673%、674%、675%、676%、677%、678%、679%、680%、681%、682%、683%、684%、685%、686%、687%、688%、689%、690%、691%、692%、693%、694%、695%、696%、697%、698%、699%、700%、701%、702%、703%、704%、705%、706%、707%、708%、709%、710%、711%、712%、713%、714%、715%、716%、717%、718%、719%、720%、721%、722%、723%、724%、725%、726%、727%、728%、729%、730%、731%、732%、733%、734%、735%、736%、737%、738%、739%、740%、741%、742%、743%、744%、745%、746%、747%、748%、749%、750%、751%、752%、753%、754%、755%、756%、757%、758%、759%、760%、761%、762%、763%、764%、765%、766%、767%、768%、769%、770%、771%、772%、773%、774%、775%、776%、777%、778%、779%、780%、781%、782%、783%、784%、785%、786%、787%、788%、789%、790%、791%、792%、793%、794%、795%、796%、797%、798%、799%、800%、801%、802%、803%、804%、805%、806%、807%、808%、809%、810%、811%、812%、813%、814%、815%、816%、817%、818%、819%、820%、821%、822%、823%、824%、825%, 826%, 827%, 828%, 829%, 830%, 831%, 832%, 833%, 834%, 835%, 836%, 837%, 838%, 839%, 840%, 841%, 842%, 843%, 844%, 845%, 846%, 847%, 848%, 84, 9%, 850%, 851%, 852%, 853%, 854%, 855%, 856%, 857%, 858%, 859%, 860%, 861%, 862%, 863%, 864%, 865%, 866%, 867%, 868%, 869%, 870%, 871%, 872%, 873%, 874%, 875%, 876%, 877%, 878%, 879%, 880%, 881%, 882%, 883%, 884%, 885%, 886%, 887%, 888%, 889%, 890%, 891%, 892%, 893%, 894%, 895%, 896%, 897%, 898%, 899%, 900%, 901%, 902%, 903%, 904%, 905%, 906%, 907%, 908%, 909%, 910%, 911%, 912%, 913%, 914%, 915%, 916%, 917%, 918%, 919%, 920%, 921%, 922%, 923%, 924%, 925%, 926%, 927%, 928%, 929%, 930%, 931%, 932%, 933%, 934%, 935%, 936%, 937%, 938%, 939%, 940%, 941%, 942%, 943%, 944%, 945%, 946%, 947%, 948%, 949%, 950%, 951%, 952%, 953%, 954%, 955%, 956%, 957%, 958%, 959%, 960%, 961%, 962%, 963%, 964%, 965%, 966%, 967%, 968%, 969%, 970%, 971%, 972%, 973%, 974%, 975%, 976%, 977%, 978%, 979%, 980%, 981%, 982%, 983%, 984%, 985%, 986%, 987%, 988%, 989%, 990%, 991%, 992%, 993%, 994%, 995%, 996%, 997%, 998%, 999%, 1000% and above increase.

[0166] Administration of pharmaceutical preparations The pharmaceutical formulations or dosage forms described herein can be administered one or more times per hour, per day, per month, or per year (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more per hour, per day, per month, or annually). In some embodiments, the pharmaceutical formulations or dosage forms described herein can be administered continuously over a period ranging from a few minutes to a few hours to a few days. Devices and dosage forms effective to provide continuous administration of the pharmaceutical formulations described herein are known in the art and are also described herein. In some embodiments, the first one or some initial doses administered can be at a higher dose than subsequent doses. This is typically referred to in the art as a loading dose(s) and a maintenance dose, respectively. In some embodiments, the pharmaceutical formulation can be administered such that the dose over time tapers (increases or decreases) over time, such that the subject gradually moves away from or is gradually introduced to the pharmaceutical formulation.

[0167] As described above, the pharmaceutical formulation can contain, where appropriate, a predetermined amount of a primary active agent, a secondary active agent, and / or a pharmaceutically acceptable salt thereof. In some of these embodiments, the predetermined amount can be an appropriate fraction of the effective amount of the active ingredient. Thus, such unit doses may be administered one or more times per day, per month, or per year (e.g., 1, 2, 3, 4, 5, 6 or more times per day, per month, or per year). Such pharmaceutical formulations can be prepared by any of the methods well known in the art.

[0168] When combination therapies or multiple pharmaceutical formulations are delivered to a subject, different therapeutic agents or formulations can be administered sequentially or simultaneously. Sequential administration is administration where a significant length of time occurs between administrations, such as, for example, about 15, 20, 30, 45, 60 minutes or more. The time between administrations in sequential administration can be on the order of several hours, days, months, or even years, depending on the active agent included in each administration. Simultaneous administration refers to administering two or more formulations simultaneously or substantially simultaneously (e.g., within seconds or at intervals of only a few minutes), with the intent that the formulations be administered together at the same time.

[0169] Kit Any of the compounds, compositions, formulations, and / or exosomes described herein, or combinations thereof, can be presented as a combination kit. In some embodiments, the kit includes one or more filters, tubes, devices, etc. used to prepare milk exosomes according to the methods described herein. As used herein, the terms “combination kit” or “partial kit” refer to compounds, compositions, formulations, particles, cells, and any additional components used for packaging, selling, commercializing, delivering, and / or administering a combination or single element, such as the active ingredients contained therein. Such additional components include, but are not limited to, packaging, syringes, blister packages, bottles, etc. If one or more of the compounds, compositions, formulations, particles, cells, or combinations thereof (e.g., agents) described herein contained in the kit are administered simultaneously, the combination kit provides a single formulation, e.g., a pharmaceutical formulation (e.g., a tablet) or a pharmaceutical composition in a separate formulation. If the compounds, compositions, formulations, particles, and cells described herein, or combinations thereof, and / or kit components are not administered simultaneously, the combination kit can contain each agent or other component in separate pharmaceutical formulations. The separate kit components can be included in a single package or separate packages within a kit.

[0170] In some embodiments, the combination kit also includes instructions printed on or otherwise contained in a tangible expression medium. The instructions can provide information regarding the content of the compounds, compositions, formulations, particles, and / or exosomes described herein, or any combination thereof included herein, safety information regarding the content of the compounds, compositions, formulations (e.g., pharmaceutical formulations), particles, and / or exosomes described herein, and / or information regarding the dosage, use indications, and / or recommended treatment regimens (if any) of the compound(s) and / or pharmaceutical formulation contained therein. In some embodiments, the instructions can provide instructions regarding the method of preparing milk exosomes according to the methods described elsewhere herein. In some embodiments, the instructions can provide instructions for administering the compounds, compositions, formulations, particles, and cells described herein, or combinations thereof, to a subject in need thereof.

[0171] Method of using target milk exosomes In certain exemplary embodiments herein, a method of treating an injury at an injury site or a method of treating cancer in a subject in need thereof is described, the method comprising administering to a subject in need thereof the milk exosomes of the present disclosure or a population thereof described herein, or a pharmaceutical formulation comprising the milk exosomes of the present disclosure or a population thereof described herein. Without being bound by theory, in some embodiments, the bioavailability of the exogenous cargo of the milk exosomes (if any) of the present disclosure can be improved over conventional delivery approaches. In some embodiments, the mammalian milk exosomes are bovine milk exosomes, ovine milk exosomes, caprine milk exosomes, equine milk exosomes, or human milk exosomes. In certain exemplary embodiments, the milk exosomes are bovine milk exosomes. The milk exosomes can be isolated from any suitable mammalian milk source. The milk exosomes are as described elsewhere herein.

[0172] In certain exemplary embodiments, the method further comprises administering an amount of one or more immunoglobulins to a subject in need thereof. In certain exemplary embodiments, administering the amount of one or more immunoglobulins to a subject in need thereof is performed prior to administering a pharmaceutical formulation comprising an amount of the milk exosomes or a population thereof of the present disclosure, which is described in more detail elsewhere herein, or the milk exosomes or a population thereof of the present disclosure described in more detail elsewhere herein. In certain exemplary embodiments, the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE. In certain exemplary embodiments, the one or more immunoglobulins consist of or comprise IgG. In certain exemplary embodiments, the one or more immunoglobulins increase the uptake of intracellular milk exosomes or a population thereof. In certain exemplary embodiments, the cell is a polarized cell. In certain exemplary embodiments, the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a cancer cell, or any combination thereof. In some embodiments, the cell is an intestinal cell or a blood-brain barrier cell. In some embodiments, the immunoglobulin(s) is / are delivered 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, ~ / or 100) seconds, minutes, hours or days prior to the delivery of the milk exosomes. In some embodiments, the milk exosomes and the immunoglobulin(s) are delivered simultaneously.In some embodiments, milk exosomes and immunoglobulins are delivered simultaneously, but the immunoglobulins and / or milk exosomes are in a dosage form that delays the release of the milk exosomes and / or promotes the release of the immunoglobulins such that the immunoglobulins are delivered first. Delivery of the milk exosomes and / or immunoglobulins can be performed 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, ~ / or 100) times per day, per month, or per year.

[0173] In certain exemplary embodiments, the method further comprises administering to a subject in need thereof one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP. In certain exemplary embodiments, administering to a subject in need thereof one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP is performed prior to administering an amount of the milk exosomes of the present disclosure or a population thereof, or a pharmaceutical formulation comprising the milk exosomes of the present disclosure or a population thereof as described herein. In certain exemplary embodiments, one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP increase the cellular uptake of intracellular milk exosomes or a population thereof. In certain exemplary embodiments, the cell is a polarized cell. In certain exemplary embodiments, the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a cancer cell, or any combination thereof. In some embodiments, the cell is an intestinal cell or a blood-brain barrier cell. In some embodiments, one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP are delivered 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and / or 100) seconds, minutes, hours, or days prior to the delivery of the milk exosomes. In some embodiments, the milk exosomes and one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP are delivered simultaneously.In some embodiments, milk exosomes and one or more agents, ATP, ADP, or any combination thereof that stimulate ATP release, secretion, and / or production are delivered simultaneously, but immunoglobulins and / or milk exosomes delay the release of milk exosomes and / or promote the release of one or more agents, ATP, ADP, or any combination thereof that stimulate ATP release, secretion, and / or production such that immunoglobulins are delivered first. Delivery of milk exosomes and / or one or more agents, ATP, ADP, or any combination thereof that stimulate ATP release, secretion, and / or production occurs 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and / or 100) times per day, per month, or per year.

[0174] In certain exemplary embodiments, the site of injury is a mechanical injury site or a non-mechanical injury site, a radiation injury, an inflammatory injury, or an ischemic injury. In certain exemplary embodiments, non-mechanical injury is chemical injury, electrical injury, radiation injury, or ischemic injury. In some embodiments, the subject in need thereof has a disease, disorder or condition. In some embodiments, the disease, disorder, or condition results in a region of cellular injury or inflammation. Exemplary diseases or disorders include cancer, viral infection, bacterial infection, parasitic infection, mechanical injury (e.g., external and internal wounds and tissue damage, cuts, abrasions, burns, surgical wounds, secondary wounds resulting from an infection, disease, or condition, etc.), cancer, non-mechanical injury (e.g., chemical injury, electrical injury, radiation injury, ischemic and / or hypoxic injury (e.g., myocardial infarction, ischemic wound and / or stroke), multiple sclerosis, psoriasis, scleroderma, acne, eczema, or skin and / or connective tissue diseases, heart disease or disorder, neurodegenerative disease or disorder, neuropathy, atherosclerosis, conditions involving epithelial permeability, and / or angiogenesis (e.g., angiogenesis or neovascularization), respiratory distress syndrome (RDS), reperfusion injury, skin vascular injury or deformation, macular degeneration, choroidal neovascularization through the Bruch's membrane, diabetic retinopathy (inflammatory and inflammation-related diseases and disorders), and radiation dermatitis, but are not limited thereto.

[0175] The wound can be a chronic wound, or a wound that appears not to have healed completely. For example, a wound that has not healed within three months is said to be chronic. Chronic wounds include diabetic foot ulcers, ischemic, venous ulcers, venous lower extremity ulcers, venous stasis, arterial, pressure, vasculitis, infectious, decubitus ulcers, burns, ulcers induced by trauma, gangrenous and mixed ulcers. Chronic wounds include chronic inflammation, incomplete differentiation and overgrowth of granulation tissue, and incomplete re-epithelialization and incomplete wound closure, and wounds characterized by a long repair time and / or thereof. Chronic wounds can include eye ulcers including corneal ulcers. The use of the disclosed invention in wound healing and tissue regeneration can include humans and livestock, sports animals and pets.

[0176] Tissue damage can be caused by, for example, cuts, abrasions, compression wounds, stretching injuries, laceration wounds, contusion wounds, bites, scratches, bullet wounds, rupture wounds, body piercing, stab wounds, surgical wounds, surgical interventions, medical interventions, host rejection reactions after transplantation of cells, tissues, or organs, pharmaceutical effects, pharmaceutical side effects, clinical deviation, radiation injury, radiation disease, skin damage caused by cosmetics, visceral damage, disease processes (e.g., asthma, cancer), infections, infectious agents, developmental processes, maturation processes (e.g., acne), genetic abnormalities, developmental abnormalities, environmental toxins, allergens, scalp injuries, facial injuries, jaw injuries, genital injuries, joint injuries, excretory organ injuries, foot injuries, finger injuries, toe injuries, bone injuries, eye injuries, corneal injuries, muscle injuries, adipose tissue injuries, lung injuries, airway injuries, hernias, anal injuries, hemorrhoids, ear injuries, skin injuries, abdominal injuries, retinal injuries, eye injuries, corneal injuries, wrist injuries, leg injuries, sports injuries, back injuries, birth injuries, early birth injuries, toxic bites, stings, damage to the barrier function, damage to the endothelial barrier function, damage to the epithelial barrier function, tendon injuries, ligament injuries, heart injuries, heart valve injuries, vascular system injuries, cartilage injuries, lymphatic system injuries, head and brain trauma, dislocations, esophageal perforations, fistulas, nail injuries, foreign bodies, fractures, frostbite, hand injuries, heat stress disorders, lacerations, neck injuries, self-destruction, shock, traumatic soft tissue injuries, spinal cord injuries, spinal column injuries, sprains, overexertion, tendon injuries, ligament injuries, cartilage injuries, chest injuries, dental injuries, trauma, nerve system-wide injuries, burns, burn wounds, scalds, sunburns, chemical burns, aging, aneurysms, strokes, surgical radiation injuries, gastrointestinal tract injuries, infarcts, or ischemic injuries.

[0177] Cardiac diseases and disorders can include, but are not limited to, myocardial infarction, cardiomyopathy (e.g., hypertrophic cardiomyopathy), arrhythmia, and congestive heart failure. The regenerative effects of the provided compositions may result in beneficial changes in cardiac membrane excitability and ion transients. There are many different types of arrhythmias that can lead to abnormal heart function in humans. Arrhythmias include, but are not limited to, the following: bradycardia, tachycardia, alternans, automaticity disorders, reentry arrhythmias, fibrillation, atrioventricular nodal reentry tachycardia, atrial arrhythmias and induced flutter, QT prolongation syndrome, QT shortening syndrome, Brugada syndrome, premature atrial contractions, slow atrial pacemakers, multifocal atrial tachycardia, atrial flutter, atrial fibrillation, ventricular tachycardia, atrioventricular nodal reentry tachycardia (AV nodal reentrant tachycardia), the most common cause of paroxysmal supraventricular tachycardia, atrioventricular junctional rhythm, junctional tachycardia, premature junctional complexes, Wolff-Parkinson-White syndrome, Lown-Ganong-Levine syndrome, premature ventricular contractions (PVC), also known as ventricular extra beats, alternans and discordant alternans, accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, ventricular fibrillation, first-degree atrioventricular block manifested as PR prolongation, second-degree atrioventricular block, type 1 second-degree atrioventricular block, type 2 second-degree atrioventricular block, third-degree atrioventricular block, and some accessory pathway disorders (e.g., Wolff-Parkinson-White syndrome (WPW)).

[0178] Neurological and neurodegenerative disorders can include, but are not limited to, dementia, Alzheimer's disease, Parkinson's disease and related PD disorders, 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.

[0179] Inflammatory diseases, inflammatory-related diseases and disorders can be asthma, eczema, rhinitis, atherosclerosis, arthritis (including but not limited to rheumatoid arthritis), inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis. As used herein, the term "inflammatory disorder" generally includes diseases or disorders that are at least partially caused or exacerbated by inflammation characterized by increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), heat, erythema, swelling, pain and / or loss of function in the affected tissue or organ. The cause of inflammation can be due to physical injury, chemicals, microorganisms, tissue necrosis, cancer, or other agents or conditions.

[0180] Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders generally have a relatively short duration, lasting from about a few minutes to about 1 - 2 days, but can last for several weeks. Characteristics of acute inflammatory diseases include increased blood flow, exudation of body fluids and plasma proteins (edema), and migration of white blood cells such as neutrophils. Chronic inflammatory diseases generally last for a longer period, for example, from several weeks to several months to years or more, and are histologically associated with the presence of lymphocytes and macrophages, as well as proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders that recur after a certain period or disorders with periodic attacks. Some inflammatory disorders are classified into one or more categories. Exemplary inflammatory disorders include atherosclerosis, arthritis, cancer promoted by inflammation, asthma, autoimmune uveitis, adoptive immune response, dermatitis, multiple sclerosis, diabetic complications, osteoporosis, Alzheimer's disease, cerebral malaria, hemorrhagic fever, autoimmune disorders, and inflammatory bowel disease, but are not limited thereto. In some embodiments, the inflammatory disorder is an autoimmune disorder, and in some embodiments, it is selected from lupus, rheumatoid arthritis, and autoimmune encephalomyelitis.

[0181] In some embodiments, the inflammatory disorder is a brain-related inflammatory disorder. The term "brain-related inflammatory" disorder as used herein refers to a subset of inflammatory disorders that are at least partially caused by, can occur, or can be exacerbated by inflammation within the brain of a subject.

[0182] In some embodiments, the subject has cancer.

[0183] In certain exemplary embodiments, the milk exosomes or a population thereof and / or the pharmaceutical formulation has been irradiated, sterilized, or both.

[0184] In certain exemplary embodiments, the milk exosomes, milk exosome population, or pharmaceutical formulation of the present disclosure described herein is provided, and the milk exosomes or a population thereof and / or the pharmaceutical formulation has been irradiated, sterilized, or both.

[0185] Additional embodiments are described in the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Examples

[0186] Although the embodiments of the present disclosure have been described thus far, generally, in the following examples, some additional embodiments of the present disclosure will be described. The embodiments of the present disclosure are described in connection with the following examples as well as the corresponding text and figures, but it is not intended to limit the embodiments of the present disclosure to this description. On the contrary, it is intended to cover all alternatives, modifications, and equivalents within the spirit and scope of the embodiments of the present disclosure. The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to practice the methods and use the probes disclosed and claimed herein. For numbers (e.g., amounts, temperatures, etc.), efforts have been made to ensure accuracy, but some error and deviation should be considered. Unless otherwise indicated, parts are by weight, temperature is in °C, and pressure is at or near atmospheric pressure. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0187] Example 1 - Bovine milk-derived extracellular vesicles as a cargo delivery platform Bovine milk-derived extracellular vesicles (mEV) can be orally administered and can bypass important barriers in the body using the protocol described in WO2022 / 182782. When orally administered, the mEV target damaged tissues in vivo. Figures 1A - 1Q show fluorescently tagged mEV that have experienced a targeted effect on wounded skin (Figures 1A - 1D) and wounded heart tissue (Figures 1I - 1L), but do not show a tendency for accumulation in non-damaged skin (Figures 1E - 1H) or heart (Figures 1M - 1O) tissue. Particle count analysis was performed on each skin (Figure 1P) and heart tissue (Figure 1Q), showing a statistically significant increase in uptake in damaged tissues (more than 30-fold in ischemic heart tissue and approximately 10-fold in the skin injury model). To follow up the in vivo experiment, huDF was damaged with a scratch wound and then fluorescently tagged mEV were administered (Figures 2A - 2E). As shown by the fluorescent images of non-wounded cells (Figures 2A - 2B) versus wounded cells (Figures 2C - 2D), in vitro wound causes a 20-fold enhancement of mEV uptake (Figure 2E).

[0188] mEV has previously been shown to contain the gap junction protein connexin-43 (Cx43), which implies something about wound healing and cell communication. The applicant performed Western blotting on the c-tail fragment and c-terminal fragment to verify that the mEV isolated by the applicant's own process contains Cx43 (Figures 3A - 3E). Interestingly, the applicant found that while the c-tail is present, the c-terminal is clearly absent (Figure 3A). This analysis was then verified by using nanogold TEM imaging for the c-tail (Figure 3B), showing positive expression, while the c-terminal (Figure 3D) did not show positive. Negative controls for each antibody are shown in Figures 3C (c-tail) and 3E (c-terminal). This suggests that while Cx43 is present in mEV, there is significant cleavage of the protein. Without being bound by theory, it is hypothesized that this results in the formation of an endogenous c-terminal fragment retained within the mEV.

[0189] Considering that the truncated form of Cx43 is known, the applicant then modified a series of Madine-Darby canine kidney (MDCK) cells (Figures 4A - 4H) to show an increase in expression, the B5 clone strain, and a decrease in expression, the parental Lampe strain, and performed a scratch wound uptake assay. When following exactly the same method for the huDF wound experiment, the cells reacted very differently, suggesting a specific role of Cx43 in the biological activity of mEV. Specifically, when wounds were inflicted on the parental Lampe cells, as shown in Figure 4C, mEV uptake only slightly increased. Non-wounded parental Lampe cells are shown in Figure 4B. The related bar graph for particle number analysis is shown in Figure 4A above. On the other hand, when the cells of the B5 clone strain were damaged (Figure 4E), uptake increased more than 30-fold relative to the baseline. Interestingly, the B5 clone cells also showed an increase in uptake in the non-damaged model (Figure 4D). This suggests that cellular uptake of mEV is mediated by Cx43 in both the wounded and non-wounded situations, and non-wounded B5 clone cells experience uptake levels similar to those of wounded parental Lampe cells (Figure 4A). Next, the applicant tested the mechanism of uptake in these cells. B5 clone cells were scratch wounded, fluorescently tagged mEV were provided, and then, in some cases, Gap27, a known connexin channel blocker, was also provided (Figure 4F). When incubated with Gap27 (D2), mEV uptake was blocked by more than 25% compared to the untreated ones (Figure 4F). Without being bound by theory, this suggests an important role of Cx43 in cellular uptake, and by blocking Cx43, a significant portion of mEV uptake in the damaged model would be blocked. Another mechanism of mEV uptake in the wounded environment discovered by the applicant is ATP (Figures 5A - 5C). When B5 clone cells were wounded and mEV were provided (Figure 5A), normal wound uptake was seen. When the cells were incubated with apyrase, an ATP dephosphorylating agent, at 100 μM in mEV (Figure 5B), cellular uptake was completely blocked, experiencing a decrease of more than 95% in mEV uptake.This suggests a very important role of ATP in the uptake of mEV in the wounded environment.

[0190] Beyond the potential for targeting, mEVs also contain very potent wound healing properties. When mEVs are applied to huDFs in a standard scratch wound model and cell migration is measured (Figures 6A - 6G), the wound healing ability of α - CT1 (Figure 6G), a Phase III clinical trial wound healing peptide at approximately 50 μM, was observed. This suggests that mEVs are equivalent to 50% of the dose of commercially tested wound healing drugs. The scratch wound area is measured at t = 0 (Figures 6A, 6C, and 6E), and migration is measured by measuring the variation with the final scratch wound area (Figures 6B, 6D, and 6F). mEVs statistically increase the wound healing ability of huDFs in vitro. Next, to measure the effect of cell Cx43 expression on the migration ability in mEV - treated cell cultures, the applicant replicated the scratch wounds of huDFs in the MDCK model using parental Lampe and B5 clone cell lines (Figures 7A - 7L). The wound healing experiment in MDCK cells reflects the findings of the applicant's mEV targeting experiment. Parental Lampe cells showed an improved migration ability (2 - 3 times) when provided with mEVs (Figure 7D) compared to the applicant's vehicle control (Figure 7C). However, B5 clone cells treated with mEVs (Figure 7F) showed a 10 - fold enhancement of wound healing ability compared to B5 treated with vehicle (Figure 7E), again suggesting a very important role of cell Cx43 in the biological activity of mEVs in either wound targeting or wound healing ability. Next, the applicant investigated the role of Gap27 blockade of Cx43 in mEV - mediated wound healing (Figure 7G) and noted that when incubated with 100 μM Gap27, the wound healing ability of B5 clone cells treated with mEVs decreased by more than 50% (Figure 7L). Next, the applicant extended the investigation to the role of apyrase in the wound healing ability of mEVs (Figures 8A - 8G). Similar to the use of Gap27, the addition of 100 μM apyrase in parallel with mEVs resulted in a decrease in the wound healing ability of mEVs by more than 50%. The data are shown in Figure 8G. This confirms that not only do ATP and Cx43 play important roles in the wound targeting ability of mEVs, but both also play important roles in the downstream effects of mEVs on wounded tissues and cells.To extend this study to in vivo, the Applicant next orally administered mEV to mice that were given LAD-induced cardiac events and then rescued by ischemia reperfusion (Figs. 11A-11B). mEV did not statistically reduce the scar area in the heart tissue, but the scar size tended to decrease by 25%, thereby confirming the ability of mEV to target damaged tissue and provide a therapeutic effect on heart tissue. Echocardiograms of the scar size are shown below the bar graphs for each group indicated.

[0191] mEVs have shown great potential for therapeutic applications, and the Applicant wishes to promote a novel drug delivery platform by loading exogenous drugs onto the biocompatible and non-immunogenic vesicles of the Applicant. This was achieved by slightly esterifying the peptides of the Applicant, as discussed in the prior disclosure. Figures 9A - 9B show the HPLC readout when approximately 30 μM of RPRPDDLEI (SEQ ID NO: 1) was loaded onto the mEVs of the Applicant (Figure 9A). Next, this was confirmed by nanoparticle tracking analysis (NTA) for fluorescently tagged mEVs using FAM binding to RPRPDDLEI (SEQ ID NO: 1). The NTA analysis showed that for the loading of approximately 30 μM of RPRPDDLEI (SEQ ID NO: 1) (a more accurate figure is shown in Figure 9B), the efficiency of loading RPRPDDLEI (SEQ ID NO: 1) onto the mEVs of the Applicant was approximately 34%, and its use as a cargo (e.g., drug or its molecule or agent) delivery platform was confirmed. Next, these RPRPDDLEI-loaded mEVs (SEQ ID NO: 1) were tested in multiple in vitro models based on the known therapeutic effects of the Applicant's peptides through current tests on the parent peptide of RPRPDDLEI (SEQ ID NO: 1), α-CT1. α-CT1 is currently in clinical trials for the treatment of skin wound healing and skin radiation injury, and supports the use of α-CT1 for both internal wound healing applications and internal radiation-induced injury applications. Figures 10A - 10F display the results from the huDF scratch wound model performed in Figures 6A - 6G. However, this experiment included RPRPDDLEI (SEQ ID NO: 1) (Figure 10E) and RPRPDDLEI-loaded mEVs (SEQ ID NO: 1) (Figure 10F). Each component was effective on its own, resulting in a significant enhancement of wound healing ability, but when treated together as a combined therapy after loading, the effects were compounded (Figure 10F), resulting in a very significant enhancement of wound healing ability.

[0192] Due to the known therapeutic effect of α-CT1 on skin radiation damage, although not restricted thereto, the applicant also hypothesized that the combination therapy with RPRPDDLEI-loaded mEV (SEQ ID NO: 1) is very effective in reducing radiation-induced damage to internal organs. As a test of this, FIGS. 12A-12D show the results from a radiation study using a standard cell model, intestinal epithelial cells (IEC-6). The IEC-6 cells were irradiated with a 6 Gy dose of radiation from a linear accelerator and then treated with either RPRPDDLEI-loaded mEV (SEQ ID NO: 1), the known clinical therapeutic α-CT1, mEV, or a vehicle control. To test the potential radioprotection of the applicant's mEV and drug delivery system, two assays were used, namely, the mitochondrial tracking assay (MTS) and the viability assay (LD). The results of the MTS showed that the applicant's combination therapy outperformed α-CT1 and provided much higher radiation permeability to a very significant degree (p<0.1E-20), showing very significant results (FIG. 12A). The results of the LD confirmed these findings, with the applicant's treatment platform again winning by a slight margin over α-CT1 (FIG. 12B). As shown in FIGS. 12A-12B, the viability images of the applicant's cultures revealed the important fact that the vehicle-treated samples were experiencing uncontrolled cell death emanating from the central lesion (FIG. 12C). This method of cell death is known as radiation-induced bystander effect cell death, where the initial cell death triggers a cascade of death-promoting signals, which then cause additional deaths. The applicant's treatment platform completely prevents the spread of these signals, eliminates the bystander effect, and limits cell death to only those affected by the initial radiation injury. These findings confirm that the applicant's RPRPDDLEI-loaded mEV (SEQ ID NO: 1) is a clinical-grade radiation therapeutic that provides a powerful therapeutic effect when targeted to damaged tissues and taken up by resident cells and tissues.The mEV (SEQ ID NO: 1) carrying RPRPDDLEI targets damaged tissues, promotes wound healing, protects against cell death in many models (in vitro huDF, MDCK, IEC-6, and in vivo mice), and the efficacy of the applicant's treatment method is confirmed by a number of wound environments including skin injury, LAD-induced heart injury, simple scratches, or even radiation-induced injury.

[0193] Example 2 Small extracellular vesicles (EVs), such as exosomes, are produced from the phospholipid bilayer of almost all cells in the body and are present in large quantities in milk. Bovine milk exosomes can carry therapeutic agents and can serve as vehicles for oral drug delivery. Exosomes have been demonstrated to be absorbed systemically, cross barriers, and be absorbed intact. In 2018, the International Society for Extracellular Vesicles (ISEV) published guidelines for the nomenclature of EVs (Thery et al., 2018). In particular, since a consensus on specific markers for EV subtypes such as exosomes has not yet been defined, they recommend referring to EVs based on their size, density, composition, or originating cell (Thery et al., 2018). Throughout this specification, the applicant refers to EVs by their size (<200 nm [small], or >200 nm [large and / or medium]) (see also Thery et al., 2018 J Extracell Vesicles. 2018 Nov 23;7(1):1535750.doi:10.1080 / 20013078.2018.1535750). sEVs are attractive as drug delivery vehicles, especially for labile drugs such as insulin or heparin, or drugs such as chemotherapeutic agents that generally need to be administered via injection. Efforts are underway to address this need, but for some reason, these efforts have not been converted into clinically meaningful delivery vehicles or formulations. In many cases, the EVs produced are insufficient to carry out such approaches. Furthermore, many efforts are plagued by stimulating inappropriate immune responses in the subjects to whom they are administered.

[0194] The present applicant has focused on the use of bovine milk sEVs, which, as the applicant has determined, can be produced in large quantities and are generally well tolerated by human subjects. However, it is unclear whether these exosomes can be effectively taken up by cells such as intestinal cells to serve as a vehicle for effective drug delivery, particularly oral delivery. Betker et al., 2019, demonstrated an interaction between the neonatal Fc receptor (FcRN) (also referred to herein as the "Fc receptor") and IgG on exosomes. However, Betker et al. observed a decrease in uptake. FcRN is expressed throughout the intestinal tract and in many other organs. This is a misnomer as it is continuously expressed in humans throughout adulthood. The receptor binds to the Fc portion of IgG and functions as a means of transporting IgG across cells. IgG bound to the Fc receptor has been shown to avoid lysosomal degradation. The Fc receptor functions only at acidic pH. It is generally understood that most of the binding of IgG to FcRN actually occurs when IgG is already inside the cell. Here, IgG is contained within acidified endosomes where FcRN is present. The FcRN binds to its target in an acidic environment.

[0195] Fetal Fc receptors can regulate the oral bioavailability of bovine milk exosome drug delivery. Fetal Fc receptor (FcRN) on the intestinal epithelium can bind to IgG on bovine milk exosomes and mediate uptake. Without being bound by theory, when bound to the Fc receptor, IgG is transported to the basolateral side of polarized cells (such as intestinal epithelial cells or enterocytes of the blood-brain barrier). It acts bidirectionally to transport IgG from the apical to the basolateral membrane or vice versa and recycles to the membrane. In other words, it mediates transcytosis within the cell. Targeting a therapeutic agent or delivery vehicle to the Fc receptor can be a way to increase the uptake and / or bioavailability of a therapeutic agent or other agent. It can also provide protection against degradation when sEVs pass through cells. See also Pridgen et al., 2013; Hornby et al., 2013; Dickinson et al., 1999; McCarthy et al., 2000; Yoshida et al., 2004; Roopenian and Akilesh, 2007; and Agrawal et al., 2017).

[0196] This example shows targeting the Fc receptor via IgG, at least for improving exosome uptake and / or the movement of exosomes into cells and transport through cells. Without being bound by theory, this allows exosomes and / or their cargo to avoid lysosomal degradation within the cell. This can lead to improved bioavailability, such as the oral bioavailability of cargo including but not limited to therapeutic cargo.

[0197] The applicant further investigated the mechanism of uptake of milk exosomes by intestinal cells, particularly intestinal epithelial cells. Without being bound by theory, the applicant hypothesized that bovine milk exosomes are taken up by intestinal epithelial cells via the interaction between exosome-bound IgG and Fc receptors, such as fetal Fc receptors. The applicant determined that IgG is present in small EVs.

[0198] Exosomes were generated using, for example, the methods described in Pridgen, Eric M. et al. “Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery” Science translational medicine vol.5, 213(2013):213ra167 or WO2022 / 182782. These are incorporated by reference as if fully set forth herein. Briefly, raw, non-pasteurized whole bovine milk is skimmed by undergoing a series of centrifugation steps. It was then filtered using Millipore 0.45μm and 0.22μm filters. Calcium was chelated with 30 mM EDTA at approximately 37°C for 60 minutes with gentle stirring. It was then filtered into HEPES buffer using a Repligen KrosFlo TFF system. Finally, size exclusion chromatography was performed using an IZON qEV original 70nm Sepharose column and fractionated into 96-well plates. Exosome fraction 8.0 - 9.0 produced a large amount of pure exosomes as seen by negative stain electron microscopy of the final isolate, showed a high density accumulation of sEV in the peak SEC fraction, and nanoparticle tracking analysis (NTA) showed the protein concentration of the sEV-containing solution after SEC from three separate isolations. The fraction with the peak protein concentration was fraction 8.0 - 9.0. Reproducibility was observed over multiple replicate trials.

[0199] Next, the applicant evaluated bovine sEV for the presence of IgG. Bovine milk exosomes were isolated as before. Dot blots on nitrocellulose were performed using an IgG standard curve derived from bovine serum albumin. The working range of the standard curve was 5 ng IgG to 200 ng IgG. 5 μg, 10 μg, and 20 μg of isolated bovine milk exosomes were analyzed. 5 μg of F18 (where casein was seen) was also analyzed. The control was HEPES only. The blot was incubated in rabbit anti-bovine IgG HRP for approximately 1 hour. The blot was blocked with fish skin (3%) and imaged using chemiluminescent reagents via Chemidoc. Analysis of the image showed that there was approximately 1.42 μg of IgG per 1 μg of exosomes. Figures 13A - 13B show the presence of IgG in small EVs. (Figure 13A) Dynamic range of IgG tested on nitrocellulose (1.0 second). (Figure 13B) Presence of IgG on bovine milk exosomes.

[0200] The applicant also evaluated whether FcRN is present on Caco2 cells. Caco-2 cells were used because they are a standard model of in vitro cell lines for intestinal uptake, including pharmaceutical uptake. Briefly, the cells were seeded at a density of approximately 0.2×106 cells / plate and grown to confluence (N-3). The cells were fixed with paraformaldehyde, rinsed with phosphate-buffered saline containing Tris (PBST) solution, and blocked with bovine serum albumin (BSA). Next, the cells were incubated overnight in rabbit anti-FcRn antibody. They were washed again and then incubated for approximately 1 hour with the appropriate secondary antibody (e.g., donkey anti-rabbit IgG) and then washed again. The nuclei were stained with Hoechst stain. Control cells were stained with secondary antibody only. The cells were imaged by confocal microscopy and analyzed with Image J. See, for example, FIGS. 14A-14B. In preliminary tests, it was shown that small EVs were taken up by a large amount of Caco-2 cells within 30 minutes of incubation time. In preliminary tests, it was shown that pH does not affect uptake. The experimental procedure outlined in FIG. 15 provides further evaluation by blocking the interaction between FcRN and IgG present on the surface of small EVs (sEVs). Protein G was used to bind to IgG and inhibit it. Free IgG was used to bind to FcRN. Briefly, according to the manufacturer's instructions, CellTracker™ Deep Red Dye (Thermo Fischer Scientific) was loaded onto the cells. The unloaded dye was removed by centrifugation at 16,873×g for 60 minutes at 4° C. in a SpinOUT™ (G-Biosciences) 3 ml column. The sEV uptake experiment was performed in 10 treatment groups (Table 1), and each treatment group was repeated 3 times per group. In experiments requiring protein G, recombinant protein G (Pierce) was added to IgG at a 5-fold molar concentration (calculated by the dot blot in FIG. 13A). Protein G was selected because it has a higher affinity for bovine IgG than protein A. The protein G-sEV solution was incubated at room temperature for 60 minutes.Subsequently, unbound Protein G was removed using a Micron YM100 column (Sigma Aldrich) according to the manufacturer's instructions. For Treatment Groups 1 - 6, IgG derived from bovine serum (Sigma Aldrich) or AlexaFluor 488 ChromPure bovine IgG (Jackson Immuno, whole molecule) (488-IgG) was added to 0.5 mL of Hank's balanced salt solution (HBSS, pH 7.4) at 50-fold the molar concentration of sEV IgG and incubated on Caco-2 cells for the indicated times. For all groups except Group 10, after washing the cells twice with HBSS solution, the cells were incubated with 25 μg of CTDR-sEV or unlabeled EV in 0.5 mL of HBSS at 37 °C for 30 minutes. Subsequently, the cells were washed twice with DBSS, fixed with 2% paraformaldehyde for 10 minutes, and washed three times with PBS. DNA was visualized with a 1:30,000 solution of Hoechst 3342 (Thermo Fisher Scientific).

Table 1

[0201]

[0202] Figures 16A - 16F show the results of uptake in various treatment groups in Caco - 2 cells. Figure 17 shows the response of EV uptake to IgG pretreatment or direct application of Protein G. * indicates statistical significance (P < 0.05). The uptake of CTDR - sEVs into Caco - 2 cells was quantified by counting the number of particles per nucleus present in laser - scan confocal microscope images using ImageJ. The measurement criterion of "particles per cell nucleus" was selected to ensure that the average sEV uptake per cell was quantified. In this experiment, 488 - IgG was used to evaluate the functionality of FcRN. Figure 3 demonstrates that 488 - IgG was taken up by Caco - 2 cells, and thus, an FcRn - IgG interaction can occur in this assay. Figures 16A - 16F also show that there is a large amount of inter - cellular variability regarding CTDR - sEV uptake. For example, in Figure 16D, there was more sEV uptake in the cells in the center of the image compared to the cells in the lower left corner of the image. This relationship was also observed when examining the uptake of 488 - IgG. When a block was introduced, there was no significant decrease in the amount of CTDR - sEVs taken up by Caco - 2 cells (Figures 16A - 16F). No significant difference in uptake was observed between CTDR - sEVs without a block and CTDR - sEVs blocked with Protein G, or between CTDR - sEVs without a block and CTDR - sEVs co - administered with IgG.

[0203] Figure 18 shows the uptake of IgG-488 into Caco-2 cells. Figure 18 shows the particles of 488-IgG per nucleus. * indicates statistical significance (P<0.05). 488-IgG was used not only as a means to demonstrate the functionality of FcRn but also as a means to compare the uptake with that of sEV. Figure 5 compares the uptake of 488-IgG after incubating on Caco-2 cells for 30 minutes when administered alone (without CTDR-sEV), co-administered with CTDR-sEV, and administered as a pretreatment for CTDR-sEV. The uptake of 488-IgG was significantly (P<.05) less when incubated with CTDR-sEV simultaneously compared to when administered before sEV. There was no difference in CTDR-sEV uptake when incubating 488-IgG on Caco-2 cells without sEV (Figure 5). This result was unexpected because IgG was administered at a concentration 50 times that of sEV-IgG. Notably, co-administration of 488-IgG and CTDR-sEV did not affect the uptake of CTDR-sEV compared to the control (Figure 17).

[0204] Figure 19 shows the results of blocking FcRN with unlabeled IgG as compared to 488-labeled IgG. Figure 19 shows the particles of CTDR-sEV per nucleus in each group where FcRn was "blocked". The dark grey bars represent cells treated with unlabeled IgG. The light grey bars represent cells treated with 488-IgG. * indicates statistical significance (P<0.05). Interestingly, in the groups where cells were pretreated with 488-IgG for 30 minutes or 120 minutes, the amount of internalized CTDR-sEV was significantly (P<0.05) increased compared to the control (Figure 17). Notably, Figure 4 also shows a significant within-group difference when comparing cells pretreated with unlabeled IgG and cells pretreated with 488-IgG (P<0.05). The difference between these groups is not consistent in that cells pretreated with 488-IgG for 30 minutes had more uptake of CTDR-sEV than cells pretreated with unlabeled EV for 30 minutes, but cells pretreated with 488-IgG for 120 minutes had significantly lower (P<0.05) uptake of CTDR-sEV than cells pretreated with unlabeled EV for 120 minutes. The difference in molar mass between unlabeled IgG and 488-IgG was considered in the calculations.

[0205] Figure 20 shows the co-localization of IgG and EV (within the area circled in Figure 20). Figure 20 shows single Caco-2 cells with CTDR-sEV visualized with red fluorescence, 488-IgG visualized with green fluorescence (represented in greyscale), and nuclei visualized with blue fluorescence (represented in greyscale). There is a clear segregation of CTDR-sEV and 488-IgG in the upper left and lower left regions of the cell.

[0206] In contrast to Betker et al., 2019 in vivo, the applicant determined that the uptake of sEVs in Caco-2 cells did not decrease even when the interaction between sEV-IgG and FcRN was blocked. Without being bound by theory, there may be multiple mechanisms that cause the internal translocation of sEVs by cells. These results also suggest an interaction between sEVs and FcRN in the intestinal model. Furthermore, these results suggest that pretreatment of intestinal cells with IgG can increase the uptake of sEVs. Without being bound by theory, these results suggest that pretreatment, co-administration, or incorporation of sEVs into IgG may be a feasible approach to increase the uptake of sEVs, such as bovine EVs loaded with cargo in the intestine. This may lead to an improvement in the oral bioavailability of the cargo.

[0207] References for Example 2 Betker, Jamie L., et al. “The Potential of Exosomes From Cow Milk for Oral Delivery.” Journal of Pharmaceutical Sciences, vol. 108, no. 4, 2019, pp. 1496 - 1505.

[0208] Manca, Sonia et al. “Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns.” Scientific reports vol. 8, 1 11321. 27 Jul. 2018

[0209] Munagala, Radha et al. “Bovine milk-derived exosomes for drug delivery.” Cancer letters vol. 371, 1(2016): 48 - 61. doi:10.1016 / j.canlet.2015.10.020

[0210] Pridgen, Eric M et al. “Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery.” Science translational medicine vol.5,213(2013):213ra167.

[0211] Sockolosky, Jonathan T, and Francis C Szoka. “The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy.” Advanced drug delivery reviews vol.91(2015):109 - 24.

[0212] Dickinson, B L et al. “Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line.” The Journal of clinical investigation vol.104,7(1999):903 - 11.

[0213] Pyzik, Michal et al. “The Neonatal Fc Receptor (FcRn): A Misnomer?.” Frontiers in immunology vol.10 1540.10 Jul.2019

[0214] Pridgen, Eric M et al. “Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery.” Science translational medicine vol.5,213(2013):213ra167.

[0215] Agrawal et al., Nanomedicine. 2017 Jul;13(5):1627 - 1636. doi:10.1016 / j.nano.2017.03.001. Epub 2017 Mar 11.

[0216] Adriano et al., Bioactive Materials. 6(8):2479 - 2490(2021)

[0217] McCarthy et al., 2000. J. Cell Sci., 113(2000), pp.1277 - 1285.

[0218] Yoshida et al., 2004. Immunity. 20(6):769 - 783

[0219] Hornby et al., Pharm Res. 2013 doi:10.1007 / s11095 - 013 - 1212 - 3

[0220] Roopenian DC, Akilesh S. FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol. 2007;7(9):715 - 725

[0221] Example 3 - Expression and Function of Connexin 43 in Exosomes Derived from Bovine Milk Small extracellular vesicles known as exosomes (nanovesicles with a diameter of 150 nm or less) are secreted by almost all cell types and are thought to play an important role in intercellular signaling. Reports that exosomes tend to avoid immune detection, cross tissue boundaries (e.g., the blood-brain barrier), and can be administered orally have also increased interest in therapeutic applications, including drug delivery devices. The present applicant has recently developed a method for large-scale purification of bovine milk-derived extracellular vesicles (Marsh et al., Nanotheranostics. 2021 Jul 5;5(4):488-498. doi:10.7150 / ntno.62213.eCollection 2021). From 1000 mL of milk, this method yields ultra-high density concentrations (>1×10 12resulting in more than 150 mL of purified microvesicles (mEV) at 100 mEV / mL (Figure 21A). Girao et al. confirmed that connexin 43 (Cx43) is present in exosomes derived from cultured cells expressing Cx43 and that this gap junction protein has an assignment in the cellular uptake of exosome cargo (Soares et al., Sci Rep. 2015 Aug 19;5:13243. doi:10.1038 / srep13243). The present applicant used Western blotting with multiple antibodies against different Cx43 epitopes and determined that a carboxyl-terminal truncated Cx43 isoform is likely to be present in bovine mEV. Immunoelectron microscopy also suggested that a significant proportion of mEV showed signals consistent with Cx43. To explore the role of Cx43 in milk-derived exosomes, the inventors conducted tests on the response of mEV in scratch-wounded monolayers (Figure 21B) of human skin fibroblasts and Madine-Darby canine kidney (MDCK) cell lines with high expression (MDCK Cx43-high) and low levels of Cx43 (MDCK Cx43-low). These results show that scratch injury (Figure 21C) in all three cell lines resulted in a significant (p<0.05) increase in mEV uptake compared to uninjured cells (Figure 21D). Interestingly, the baseline level of mEV uptake in uninjured MDCK Cx43-high cells was an order of magnitude higher than that of MDCK Cx43-low cells, and this difference was further increased by scratch wounding. In ongoing studies, the present applicant attempts to further explore the function of mEV Cx43 using an in vitro model of cell injury and to determine whether mEV uptake is also upregulated in vivo after injury. ***

[0222] Various modifications and variations of the methods, pharmaceutical compositions, and kits of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it is to be understood that additional modifications are possible and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the invention. This application has been described in connection with its specific embodiments, but further modifications are possible and this application is generally directed to any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and as may be applied to the essential features set forth above.

[0223] Further attributes, features, and embodiments of the present invention can be understood by reference to the following numbered aspects of the disclosed invention. References to the disclosure in any of the preceding aspects are applicable to any preceding numbered aspect, and any combination of any number of preceding aspects, as recognized by appropriate prior disclosure in any combination of preceding aspects that can be made. The following numbered aspects are provided.

[0224] 1. A milk exosome comprising exogenous cargo, wherein the milk exosome can target a damaged site or its location, or a cancer cell or cancer cell population by targeting or stimulating ATP secretion and / or concentration and / or the neonatal Fc receptor (FcRN).

[0225] 2. The milk exosome according to aspect 1, wherein the damage is mechanical damage or non-mechanical damage.

[0226] 3. The milk exosome according to aspect 2, wherein the non-mechanical damage is chemical damage, electrical damage, radiation damage, inflammatory damage, or ischemic damage.

[0227] 4. The milk exosome according to embodiment 2, wherein the mechanical damage is a wound or a burn.

[0228] 5. The milk exosome according to any one of embodiments 1 to 4, wherein the damaged area or site has a higher concentration of ATP than the non-damaged area or site.

[0229] 6. The milk exosome according to any one of claims 1 to 5, wherein the cancer cell or cancer cell population has a microenvironment with a higher ATP concentration than the microenvironment of non-cancer cells or non-cancer cell populations.

[0230] 7. The milk exosome according to any one of embodiments 1 to 6, wherein the cancer cell or the cancer cell population secretes ATP.

[0231] 8. The milk exosome according to any one of embodiments 1 to 7, wherein the milk exosome does not contain an exogenous targeting moiety.

[0232] 9. The milk exosome according to any one of embodiments 1 to 8, wherein the milk exosome contains connexin 43.

[0233] 10. The milk exosome according to embodiment 9, wherein the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43.

[0234] 11. The milk exosome according to any one of embodiments 1 to 10, wherein the exogenous cargo is a biomolecule.

[0235] 12. The milk exosome according to embodiment 11, wherein the biomolecule is a polypeptide, a peptide, or a nucleic acid.

[0236] 13. The milk exosome according to any one of embodiments 1 to 12, wherein the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

[0237] 14. The milk exosome according to any one of aspects 1 to 13, wherein the milk exosome is a bovine milk exosome.

[0238] 15. Before containing the exogenous cargo, the milk exosome a. centrifuging mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk; b. removing the separated fat from the mammalian milk; c. after step (b), centrifuging the remaining mammalian milk one or more times and skimming off the separated fat visible after each centrifugation in step (c); d. filtering the biological fluid remaining after step (c); e. optionally performing one or more ultracentrifugation steps after (d); f. after (d) or optionally (e), and optionally chelating divalent cations with about 10 mM to about 100 mM of EDTA at about 30 to 42 °C for about 15 to 120 minutes, and g. after (f), optionally performing tangential flow filtration to obtain a retentate, obtaining the retentate by ultracentrifuging the retentate through one or more ultracentrifugation steps or storing it at -80 °C, and after ultracentrifuging or storing the retentate at -80 °C, separating the fraction of the retentate by column separation optionally, separating by a method comprising: The method according to any one of aspects 1 to 15, which includes step (e) or step (g), but not both.

[0239] 16. A milk exosome population comprising one or more milk exosomes according to any one of aspects 1 to 15.

[0240] 17. The milk exosome population according to aspect 16, wherein the population is enriched for connexin 43-positive milk exosomes.

[0241] 18. Before including the exogenous cargo, the milk exosomes are a. centrifuging mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk; b. removing the separated fat from the mammalian milk; c. after step (b), centrifuging the remaining mammalian milk one or more times and skimming off any separated fat visible after each centrifugation in step (c); d. filtering the biological fluid remaining after step (c); e. optionally performing one or more ultracentrifugation steps after (d); f. after (d) or optionally (e), and optionally chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30 - 42°C for about 15 - 120 minutes, and g. after (f), optionally performing tangential flow filtration to obtain a retentate, obtaining the retentate by ultracentrifuging the retentate, optionally via one or more ultracentrifugation steps, or storing it at -80°C, and after ultracentrifuging or storing the retentate at -80°C, separating the fractions of the retentate, optionally by column separation, by a method comprising The method includes step (e) or step (g), but not both. Furthermore, a population of milk exosomes according to aspect 16, comprising concentrating the milk exosomes for connexin 43-expressing milk exosomes.

[0242] 19. Furthermore, a population of milk exosomes according to aspect 18, comprising concentrating the milk exosomes for connexin 43-expressing milk exosomes.

[0243] 20. A population of milk exosomes according to any one of aspects 16 - 19, wherein the milk exosomes do not contain an exogenous targeting moiety.

[0244] 21. The milk exosome according to any one of aspects 16 to 20, wherein the exogenous cargo is a biomolecule.

[0245] 22. The milk exosome population according to aspect 21, wherein the biomolecule is a polypeptide, a peptide, or a nucleic acid.

[0246] 23. The milk exosome according to any one of aspects 16 to 22, wherein the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

[0247] 24. A pharmaceutical preparation comprising the milk exosome or a population thereof according to any one of aspects 1 to 15, and a pharmaceutically acceptable carrier.

[0248] 25. The pharmaceutical preparation according to aspect 24, further comprising an agent, ATP, ADP, or any combination thereof that can stimulate the release, secretion, and / or production of ATP.

[0249] 26. The pharmaceutical preparation according to aspect 25, wherein the pharmaceutical preparation is in a dosage form that can release the agent, ATP, ADP, or any combination thereof that can stimulate the release, secretion, and / or production of ATP before the milk exosome or a population thereof, thereby delivering the agent, ATP, ADP, or any combination thereof that can stimulate the release, secretion, and / or production of ATP before the milk exosome.

[0250] 27. The pharmaceutical preparation according to any one of aspects 24 to 26, further comprising one or more immunoglobulins.

[0251] 28. The pharmaceutical preparation according to aspect 25, wherein the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE.

[0252] 29. The pharmaceutical preparation according to any one of aspects 27 to 28, which is a dosage form for delivering the immunoglobulin before the milk exosomes or a population thereof by releasing the immunoglobulin before the milk exosomes or a population thereof.

[0253] 30. A method for treating an injury at an injury site or treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof the milk exosomes or a population thereof according to claim 1, or a pharmaceutical preparation comprising the milk exosomes or a population thereof according to claim 1.

[0254] 31. The method according to aspect 30, wherein the milk exosomes are bovine milk exosomes.

[0255] 32. The milk exosomes according to any one of aspects 30 to 31, wherein the milk exosomes do not contain an exogenous targeting moiety.

[0256] 33. The method according to any one of aspects 30 to 32, wherein the milk exosomes contain connexin 43.

[0257] 34. The method according to aspect 33, wherein the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43.

[0258] 35. The method according to any one of aspects 30 to 34, wherein the exogenous cargo is a biomolecule.

[0259] 36. The method according to aspect 35, wherein the biomolecule is a polypeptide, a peptide, or a nucleic acid.

[0260] 37. The milk exosomes according to any one of aspects 30 to 36, wherein the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

[0261] 38. The method according to any one of aspects 30 to 38, further comprising administering to the subject in need thereof an amount of one or more immunoglobulins.

[0262] 39. The method according to aspect 38, wherein administering the amount of one or more immunoglobulins to the subject in need thereof is performed before administering an amount of the milk exosomes or a population thereof according to claim 1 or a pharmaceutical formulation comprising the milk exosomes or a population thereof according to claim 1.

[0263] 40. The method according to any one of aspects 38 to 39, wherein the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE.

[0264] 41. The method according to any one of aspects 38 to 40, wherein the one or more immunoglobulins consist of or comprise IgG.

[0265] 42. The method according to any one of aspects 38 to 41, wherein the one or more immunoglobulins increase the uptake of the intracellular milk exosomes or a population thereof.

[0266] 43. The method according to aspect 42, wherein the cell is a polarized cell.

[0267] 44. The method according to any one of aspects 42 to 43, wherein the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a germ cell, a cancer cell, or any combination thereof.

[0268] 45. The method according to any one of aspects 30 to 44, further comprising administering to the subject in need thereof one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP.

[0269] 46. Administering to a subject in need thereof the one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP, prior to administering an amount of the milk exosomes or a population thereof according to claim 1, or a pharmaceutical formulation comprising the milk exosomes or a population thereof according to claim 1, or a pharmaceutical formulation comprising the milk exosomes according to claim 1, is the method according to embodiment 45.

[0270] 47. The one or more agents, ATP, ADP, or any combination thereof that stimulate the release, secretion, and / or production of ATP increase the cellular uptake of the milk exosomes or a population thereof within the cell, according to any one of embodiments 45 to 46.

[0271] 48. The method according to embodiment 47, wherein the cell is a polarized cell.

[0272] 49. The method according to any one of embodiments 47 to 48, wherein the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a germ cell, a cancer cell, or any combination thereof.

[0273] 50. The method according to any one of embodiments 30 to 49, wherein the injury is a mechanical injury site or a non-mechanical injury.

[0274] 51. The method according to embodiment 50, wherein the non-mechanical injury is a chemical injury, an electrical injury, a radiation injury, or an ischemic injury.

[0275] 52. The method according to embodiment 50, wherein the mechanical injury is a wound or a burn.

[0276] 53. The method according to any one of embodiments 30 to 52, wherein the milk exosomes or a population thereof and / or the pharmaceutical formulation have been subjected to radiation, sterilization, or both.

[0277] 54. A method for treating a wound or cancer in a subject, comprising: The method comprising administering to the subject in need thereof milk exosomes and an agent capable of stimulating ATP.

[0278] 55. The method according to claim 54, wherein the milk exosomes are those according to any one of claims 1 to 15.

[0279] 56. The milk exosomes, milk exosome population, or pharmaceutical preparation according to any one of claims 1 to 29, wherein the milk exosomes or a population thereof and / or the pharmaceutical preparation thereof has been subjected to radiation, sterilization, or both.

Claims

1. A milk exosome, comprising an exogenous cargo, wherein the milk exosome can target damaged or its site, or cancer cells or cancer cell populations by targeting ATP secretion and / or concentration and / or neonatal Fc receptor (FcRn).

2. The milk exosome according to claim 1, wherein the damage is mechanical damage or non-mechanical damage.

3. The milk exosome according to claim 2, wherein the non-mechanical damage is chemical damage, electrical damage, radiation damage, inflammatory damage, or ischemic damage.

4. The milk exosome according to claim 2, wherein the mechanical damage is a wound or a burn.

5. The milk exosome according to claim 1, wherein the damaged or its site has a higher concentration of ATP than non-damaged or its site.

6. The milk exosome according to claim 1, wherein the cancer cells or cancer cell populations have a microenvironment with a higher ATP concentration than the microenvironment of non-cancer cells or non-cancer cell populations.

7. The milk exosome according to claim 1, wherein the cancer cells or cancer cell populations secrete ATP.

8. The milk exosome according to claim 1, wherein the milk exosome does not contain an exogenous target moiety.

9. The milk exosome according to claim 1, wherein the milk exosome contains connexin 43.

10. The milk exosome according to claim 9, wherein the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43.

11. The milk exosome according to claim 1, wherein the exogenous cargo is a biomolecule.

12. The milk exosome according to claim 11, wherein the biomolecule is a polypeptide, a peptide, or a nucleic acid.

13. The milk exosome according to claim 1, wherein the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

14. The milk exosome according to claim 1, wherein the milk exosome is a bovine milk exosome.

15. Before containing the exogenous cargo, the milk exosome is a. centrifuged mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk, b. removing the separated fat from the mammalian milk, c. After step (b), centrifuging the remaining mammalian milk one or more times and skimming off the separated fat visible after each centrifugation in step (c); d. Filtering the biological fluid remaining after step (c); e. Optionally performing one or more ultracentrifugation steps after (d); f. Optionally, after (d) or optionally after (e), and optionally for about 15 to 120 minutes, chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30 to 42 °C, and g. After (f), optionally performing tangential flow filtration to obtain a retentate, obtaining the retentate by ultracentrifuging the retentate optionally through one or more ultracentrifugation steps or storing it at -80 °C, and after ultracentrifuging or storing the retentate at -80 °C optionally separating the fractions of the retentate, optionally by column separation, separating by a method comprising; The method includes step (e) or step (g), but not both, the milk exosomes according to claim 1.

16. A milk exosome population comprising one or more of the milk exosomes according to any one of claims 1 to 15.

17. The milk exosome population according to claim 16, wherein the population is enriched for connexin 43-positive milk exosomes.

18. Before containing the exogenous cargo, the milk exosomes are a. Centrifuging mammalian milk under conditions suitable for separating fat from one or more other components of the mammalian milk; b. Removing the separated fat from the mammalian milk; c. After step (b), centrifuging the remaining mammalian milk one or more times and skimming off the separated fat visible after each centrifugation in step (c); d. Filtering the biological fluid remaining after step (c); e. Optionally performing one or more ultracentrifugation steps after (d); f. Optionally, after (d) or optionally after (e), and optionally for about 15 to 120 minutes, chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30 to 42 °C, and g. After (f), optionally performing tangential flow filtration to obtain a retention liquid, centrifuging the retention liquid optionally via one or more ultracentrifugation steps or storing it at -80 °C, obtaining the retention liquid, and after optionally ultracentrifuging or storing the retention liquid at -80 °C, separating the fraction of the retention liquid, optionally by column separation, by a method comprising: The milk exosome population according to claim 16, wherein the method comprises step (e) or step (g), but not both.

19. The milk exosome population according to claim 18, further comprising concentrating the milk exosomes for connexin 43 expressing the milk exosomes.

20. The milk exosome population according to claim 16, wherein the milk exosomes do not contain exogenous target moieties.

21. The milk exosome population according to claim 16, wherein the exogenous cargo is a biomolecule.

22. The milk exosome population according to claim 21, wherein the biomolecule is a polypeptide, a peptide, or a nucleic acid.

23. The milk exosome population according to claim 16, wherein the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

24. A pharmaceutical preparation comprising: The milk exosomes or a population thereof according to claim 1, and A pharmaceutically acceptable carrier.

25. The pharmaceutical preparation according to claim 24, further comprising an agent capable of stimulating the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof.

26. The pharmaceutical preparation according to claim 25, wherein the pharmaceutical preparation is in a dosage form that releases an agent capable of stimulating the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof prior to the milk exosomes or a population thereof, thereby delivering an agent capable of stimulating the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof prior to the milk exosomes.

27. The pharmaceutical preparation according to claim 24, further comprising one or more immunoglobulins.

28. The pharmaceutical preparation according to claim 25, wherein the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE.

29. The pharmaceutical dosage form according to claim 27, wherein the pharmaceutical preparation is a dosage form that releases the immunoglobulin prior to the milk exosome or a population thereof, thereby delivering the immunoglobulin prior to the milk exosome.

30. A method of treating an injury at an injury site or treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof the milk exosome or a population thereof according to claim 1, or a pharmaceutical preparation comprising the milk exosome or a population thereof according to claim 1.

31. The method according to claim 30, wherein the milk exosome is a bovine milk exosome.

32. The method according to claim 30, wherein the milk exosome does not contain an exogenous target moiety.

33. The method according to claim 30, wherein the milk exosome contains connexin 43.

34. The method according to claim 33, wherein the connexin 43 is native connexin 43 or the connexin 43 is engineered connexin 43.

35. The method according to claim 30, wherein the exogenous cargo is a biomolecule.

36. The method according to claim 35, wherein the biomolecule is a polypeptide, a peptide, or a nucleic acid.

37. The method according to claim 30, wherein the exogenous cargo is an ACT1 peptide, an ACT11 peptide, an ACT minus peptide, a selectide, or any combination thereof.

38. The method according to claim 30, further comprising administering to the subject in need thereof an amount of one or more immunoglobulins.

39. The method according to claim 38, wherein administering to the subject in need thereof the amount of one or more immunoglobulins is performed prior to administering an amount of the milk exosome or a population thereof according to claim 1, or a pharmaceutical preparation comprising the milk exosome or a population thereof according to claim 1.

40. The method according to claim 38, wherein the one or more immunoglobulins are selected from IgG, IgM, IgA, IgD, and / or IgE.

41. The method according to claim 38, wherein the one or more immunoglobulins consist of or comprise IgG.

42. The method of claim 38, wherein the one or more immunoglobulins increase the uptake of the milk exosomes or a population thereof within the cell.

43. The method of claim 42, wherein the cell is a polarized cell.

44. The method of claim 42, wherein the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a germ cell, a cancer cell, or any combination thereof.

45. The method of claim 30, further comprising administering to the subject in need thereof one or more agents that stimulate the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof.

46. The administration of the one or more agents that stimulate the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof to the subject in need thereof is performed prior to administering an amount of the milk exosomes or a population thereof of claim 1, or a pharmaceutical formulation comprising the milk exosomes or a population thereof of claim 1, or a pharmaceutical formulation comprising the milk exosomes of claim 1, the method of claim 45.

47. The method of claim 45, wherein the one or more agents that stimulate the release, secretion, and / or production of ATP, ATP, ADP, or any combination thereof increase the cellular uptake of the milk exosomes or a population thereof within the cell.

48. The method of claim 47, wherein the cell is a polarized cell.

49. The method of claim 47, wherein the cell is a gastrointestinal cell, a heart cell, a liver cell, a brain cell, a nerve cell, a germ cell, a cancer cell, or any combination thereof.

50. The method of claim 30, wherein the injury is a mechanical injury site or a non-mechanical injury.

51. The method of claim 50, wherein the non-mechanical injury is a chemical injury, an electrical injury, a radiation injury, or an ischemic injury.

52. The method of claim 50, wherein the mechanical injury is a wound or a burn.

53. The method of claim 30, wherein the milk exosomes or a population thereof and / or the pharmaceutical formulation have been subjected to radiation, sterilization, or both.

54. A method for treating a wound or cancer in a subject, comprising administering to the subject in need thereof a milk exosome and an agent capable of stimulating ATP.

55. The method according to claim 54, wherein the milk exosome is as defined in any one of claims 1 to 15.

56. The milk exosome, milk exosome population, or pharmaceutical preparation according to any one of claims 1 to 29, wherein the milk exosome or population thereof and / or the pharmaceutical preparation thereof has been subjected to radiation, sterilization, or both.

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