Milk-borne small extracellular vesicles: kinetics and mechanisms of transport, distribution, and elimination

Abstract

Small extracellular vesicles (sEVs) in milk have the qualities desired for delivering therapeutics to diseased tissues. The production of bovine milk sEVs is scalable (10²¹ annually per cow), and they resist degradation in the gastrointestinal tract. Most cells studied to date internalize milk sEVs by a saturable process that follows Michaelis-Menten kinetics. The bioavailability of oral milk sEVs is approximately 50%. In addition to crossing the intestinal mucosa, milk sEVs also cross barriers such as the placenta and blood-brain barrier, thereby enabling the delivery of therapeutics to hard-to-reach tissues. In time course studies, levels of milk sEVs peaked in the intestinal mucosa, plasma, and urine approximately 6 h and returned to baseline 24 h after oral gavage in mice. In tissues, milk sEV levels peaked 12 h after gavage. Milk sEVs appear to be biologically safe. No cytokine storm was observed when milk sEVs were added to cultures of human peripheral blood mononuclear cells or administered orally to rats. Liver and kidney function and erythropoiesis were not impaired when milk sEVs were administered to rats by oral gavage for up to 15 days. Protocols for loading milk sEVs with therapeutic cargo are available. Currently, the use of milk sEVs (and other nanoparticles) in the delivery of therapeutics is limited by their rapid elimination through internalization by macrophages and lysosomal degradation in target cells. This mini review discusses the current knowledge base of sEV tissue distribution, excretion in feces and urine, internalization by macrophages, and degradation in lysosomes.

Keywords

Drug delivery / feces / lysosomes / macrophages / urine

References

[1] György B,Pásztói M.Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.Cell Mol Life Sci2011;68:2667-88. PMCID:PMC3142546

[2] Zonneveld MI,van Herwijnen MJ.Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures.J Extracell Vesicles2014;3:24215 PMCID:PMC4139932

[3] Théry C,Aikawa E.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.J Extracell Vesicles2018;7:1535750 PMCID:PMC6322352

[4] Leiferman A,Upadhyaya B,Zempleni J.Storage of extracellular vesicles in human milk, and microrna profiles in human milk exosomes and infant formulas.J Pediatr Gastroenterol Nutr2019;69:235-8 PMCID:PMC6658346

[5] National Nanotechnology Institute. Nantional nanotechnology initiative. Alexandria, VA: National Nanotechnology Coordination Office; 2022. Available from: www.nano.gov [Last accessed on 11 Jul 2023]

[6] Sukreet S,An TT.Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles.J Dairy Sci2021;104:9478-93

[7] Statista, Inc. Milk produced per cow in the United States from 1999 to 2023 (in pounds). Available from: https://www.statista.com/statistics/194935/quantity-of-milk-produced-per-cow-in-the-us-since-1999/ [Last accessed on 11 Jul 2023]

[8] Izumi H,Shimizu T,Ochiya T.Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.J Dairy Sci2012;95:4831-41

[9] Benmoussa A,Laffont B.Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions.J Nutr2016;146:2206-15

[10] Howard KM,Baier SR.Loss of miRNAs during processing and storage of cow's (Bos taurus) milk.J Agric Food Chem2015;63:588-92. PMCID:PMC4387787

[11] Khanam A,Zempleni J.Class A scavenger receptor-1/2 facilitates the uptake of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice.Am J Physiol Cell Physiol2021;321:C607-14 PMCID:PMC8461812

[12] Agrawal AK,Jeyabalan J.Milk-derived exosomes for oral delivery of paclitaxel.Nanomedicine2017;13:1627-36

[13] Manca S,Mutai E.Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns.Sci Rep2018;8:11321. PMCID:PMC6063888

[14] Aqil F,Jeyabalan J.Milk exosomes-natural nanoparticles for siRNA delivery.Cancer Lett2019;449:186-95

[15] Sterzenbach U,Low LH,Tan SS.Engineered exosomes as vehicles for biologically active proteins.Mol Ther2017;25:1269-78 PMCID:PMC5474961

[16] Ogunnaike M,Zempleni J.Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery.Int J Pharm2021;610:121263. PMCID:PMC8665143

[17] Wolf T,Zempleni J.The intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells.J Nutr2015;145:2201-6 PMCID:PMC4580964

[18] Munagala R,Jeyabalan J.Bovine milk-derived exosomes for drug delivery.Cancer Lett2016;371:48-61 PMCID:PMC4706492

[19] Mutai E,Zempleni J.MicroRNAs in bovine milk exosomes are bioavailable in humans but do not elicit a robust pro-inflammatory cytokine response.ExRNA2020;2:1-9

[20] Leiferman A,Grove R,Adamec J.A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice.J Nutr Biochem2018;59:123-8 PMCID:PMC6129415

[21] Sadri M,Kachman SD,Zempleni J.Milk exosomes and miRNA cross the placenta and promote embryo survival in mice.Reproduction2020;160:501-9.

[22] Zhou F,Mutai E.Small extracellular vesicles in milk cross the blood-brain barrier in murine cerebral cortex endothelial cells and promote dendritic complexity in the hippocampus and brain function in C57BL/6J mice.Front Nutr2022;9:838543. PMCID:PMC9121399

[23] Thepharmaletter. PureTech out-licenses milk exosomes technology in $1 billion deal. Available from: https://www.thepharmaletter.com/article/puretech-out-licenses-milk-exosomes-technology-in-1-billion-deal. [Last accessed on 11 Jul 2023]

[24] Adriano B,Chauhan N,Chauhan SC.Milk exosomes: Nature's abundant nanoplatform for theranostic applications.Bioact Mater2021;6:2479-90 PMCID:PMC7856328

[25] Cieślik M,Bryniarski K.Extracellular vesicles-oral therapeutics of the future.Int J Mol Sci2022;23:7554 PMCID:PMC9315796

[26] Chutipongtanate S,Newburg DS.Human milk extracellular vesicles: a biological system with clinical implications.Cells2022;11:2345 PMCID:PMC9367292

[27] Ngu A,Wang H,Zempleni J.Milk exosomes in nutrition and drug delivery.Am J Physiol Cell Physiol2022;322:C865-74 PMCID:PMC9037700

[28] Tian MY,Liu Y.Milk exosomes: an oral drug delivery system with great application potential.Food Funct2023;14:1320-37

[29] Kusuma RJ,Friemel T,Nguyen C.Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.Am J Physiol Cell Physiol2016;310:C800-7 PMCID:PMC4895447

[30] Kahn S,Du X,Li J.Exosomal micrornas in milk from mothers delivering preterm infants survive in vitro digestion and are taken up by human intestinal cells.Mol Nutr Food Res2018;62:e1701050

[31] Takov K,Davidson SM.Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes.J Extracell Vesicles2017;6:1388731. PMCID:PMC5699187

[32] Roerig J,Kalwa H.A focus on critical aspects of uptake and transport of milk-derived extracellular vesicles across the Caco-2 intestinal barrier model.Eur J Pharm Biopharm2021;166:61-74

[33] Baier SR,Xie F,Zempleni J.MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers.J Nutr2014;144:1495-500 PMCID:PMC4162473

[34] Khanam A,Zempleni J.Bioavailability of orally administered small extracellular vesicles from bovine milk in C57BL/6J mice.Int J Pharm2023;639:122974 PMCID:PMC10175213

[35] van de Peppel IP,Jonker JW.Metabolic consequences of ileal interruption of the enterohepatic circulation of bile acids.Am J Physiol-Gastr L2020;319:G619-25

[36] Williams JM,Burkitt MD,Campbell BJ.Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip.Vet Pathol2015;52:445-55 PMCID:PMC4441880

[37] Rahman MM,Yamauchi M.Acidification effects on isolation of extracellular vesicles from bovine milk.PLoS One2019;14:e0222613 PMCID:PMC6746375

[38] Kleinjan M,Libregts SF,Feitsma AL.Regular industrial processing of bovine milk impacts the integrity and molecular composition of extracellular vesicles.J Nutr2021;151:1416-25

[39] Driedonks T,Carlson B.Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.J Extracell Biol2022;1:e59

[40] Wiklander OP,O'Loughlin A.Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting.J Extracell Vesicles2015;4:26316 PMCID:PMC4405624

[41] Izumi H,Sato Y.Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages.J Dairy Sci2015;98:2920-33

[42] Imai T,Nishikawa M.Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice.J Extracell Vesicles2015;4:26238 PMCID:PMC4323410

[43] He Y,Leone S.Milk exosomes transfer oligosaccharides into macrophages to modulate immunity and attenuate Adherent-Invasive E. coli (AIEC) Infection.Nutrients2021;13:3198 PMCID:PMC8472098

[44] Woodman PG.Multivesicular bodies: co-ordinated progression to maturity.Curr Opin Cell Biol2008;20:408-14 PMCID:PMC2577128

[45] Joshi BS,Giepmans BNG.Endocytosis of extracellular vesicles and release of their cargo from endosomes.ACS Nano2020;14:4444-55. PMCID:PMC7199215

[46] Perrin P,Janssen H.Retrofusion of intralumenal MVB membranes parallels viral infection and coexists with exosome release.Curr Biol2021;31:3884-3893.e4 PMCID:PMC8445322