{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T18:12:36Z","timestamp":1792692756544,"version":"4.5.0"},"reference-count":36,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,9,27]],"date-time":"2023-09-27T00:00:00Z","timestamp":1695772800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100012818","name":"Comunidad de Madrid","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100012818","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioeng. Biotechnol."],"abstract":"\n Introduction:<\/jats:bold>\n Goat milk is notable as a cost-effective source of exosomes, also known as small extracellular vesicles (sEVs). These nanoparticle-like structures are naturally secreted by cells and have emerged as potential diagnostic agents and drug delivery systems, also supported by their proven therapeutic effects. However, the complexity of goat milk and the lack of standardized protocols make it difficult to isolate pure sEVs. This work presents an optimized approach that combines well-established physical isolation methods with the biological treatment of milk with rennet.\n <\/jats:p>\n \n Methods:<\/jats:bold>\n sEVs derived from goat milk were purified using a methodology that combines differential ultracentrifugation, rennet, and size-exclusion chromatography. This novel strategy was compared with two of the main methodologies developed for isolating extracellular vesicles from bovine and human milk by means of physico-chemical characterization of collected vesicles using Transmission Electron Microscopy, Western blot, Bradford Coomassie assay, Dynamic Light Scattering, Nanoparticle Tracking Analysis and Zeta Potential.\n <\/jats:p>\n \n Results:<\/jats:bold>\n Vesicles isolated with the optimized protocol had sEV-like characteristics and high homogeneity, while samples obtained with the previous methods were highly aggregated, with significant residual protein content.\n <\/jats:p>\n \n Discussion:<\/jats:bold>\n This work provides a novel biophysical methodology for isolating highly enriched goat milk sEVs samples with high stability and homogeneity, for their further evaluation in biomedical applications as diagnostic tools or drug delivery systems.\n <\/jats:p>","DOI":"10.3389\/fbioe.2023.1197780","type":"journal-article","created":{"date-parts":[[2023,10,2]],"date-time":"2023-10-02T05:13:01Z","timestamp":1696223581000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":14,"title":["Isolation of goat milk small extracellular vesicles by novel combined bio-physical methodology"],"prefix":"10.3389","volume":"11","author":[{"given":"Mar\u00eda Isabel","family":"Gonz\u00e1lez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bego\u00f1a","family":"Gallardo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Cer\u00f3n","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Aguilera-Jim\u00e9nez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Cortes-Canteli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H\u00e9ctor","family":"Peinado","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manuel","family":"Desco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beatriz","family":"Salinas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,9,27]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1016\/B978-0-12-813280-7.00005-0","article-title":"Application of microbial enzymes in the dairy industry","author":"Abada","year":"2019","journal-title":"Enzym. food Biotechnol. Elsevier"},{"key":"B2","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1007\/s10571-016-0366-z","article-title":"Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake","volume":"36","author":"Abels","year":"2016","journal-title":"Cell. Mol. Neurobiol."},{"key":"B3","doi-asserted-by":"publisher","first-page":"2479","DOI":"10.1016\/j.bioactmat.2021.01.009","article-title":"Milk exosomes: nature's abundant nanoplatform for theranostic applications","volume":"6","author":"Adriano","year":"2021","journal-title":"Bioact. Mat."},{"key":"B4","doi-asserted-by":"publisher","first-page":"442","DOI":"10.3389\/fimmu.2014.00442","article-title":"Tetraspanins in extracellular vesicle formation and function","volume":"5","author":"Andreu","year":"2014","journal-title":"Front. Immunol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"2001","DOI":"10.1111\/jcmm.13479","article-title":"Physical exosome: exosome interactions","volume":"22","author":"Beit\u2010Yannai","year":"2018","journal-title":"J. Cell. Mol. Med."},{"key":"B6","doi-asserted-by":"publisher","first-page":"1039","DOI":"10.1038\/s41598-020-57497-7","article-title":"A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum","volume":"10","author":"Brennan","year":"2020","journal-title":"Sci. Rep."},{"key":"B7","doi-asserted-by":"publisher","first-page":"3061","DOI":"10.1039\/d0nr06309f","article-title":"General and mild modification of food-derived extracellular vesicles for enhanced cell targeting","volume":"13","author":"Chen","year":"2021","journal-title":"Nanoscale"},{"key":"B8","doi-asserted-by":"publisher","first-page":"92","DOI":"10.3389\/fgene.2018.00092","article-title":"\u201cExosomics\u201d\u2014a review of biophysics, biology and biochemistry of exosomes with a focus on human breast milk","volume":"9","author":"de la Torre Gomez","year":"2018","journal-title":"Front. Genet."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1363","DOI":"10.1007\/s00383-019-04562-6","article-title":"A comparison of exosomes derived from different periods breast milk on protecting against intestinal organoid injury","volume":"35","author":"Gao","year":"2019","journal-title":"Pediatr. Surg. Int."},{"key":"B10","doi-asserted-by":"publisher","first-page":"1700009","DOI":"10.1002\/mnfr.201700009","article-title":"Characterization and biological function of milk\u2010derived miRNAs","volume":"61","author":"Golan\u2010Gerstl","year":"2017","journal-title":"Mol. Nutr. Food Res."},{"key":"B11","doi-asserted-by":"publisher","first-page":"1062","DOI":"10.3390\/nano10061062","article-title":"Radioactive labeling of milk-derived exosomes with 99mTc and in vivo tracking by SPECT imaging","volume":"10","author":"Gonz\u00e1lez","year":"2020","journal-title":"Nanomaterials"},{"key":"B12","first-page":"191","article-title":"Purification of RNA from milk whey","volume-title":"Methods mol. Biol.","author":"Izumi","year":"2013"},{"key":"B13","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1186\/s12964-019-0401-z","article-title":"A novel population of extracellular vesicles smaller than exosomes promotes cell proliferation","volume":"17","author":"Lee","year":"2019","journal-title":"Cell Communv Signalv"},{"key":"B14","doi-asserted-by":"publisher","first-page":"789","DOI":"10.7150\/thno.18133","article-title":"Progress in exosome isolation techniques","volume":"7","author":"Li","year":"2017","journal-title":"Theranostics"},{"key":"B15","doi-asserted-by":"publisher","first-page":"2908","DOI":"10.3390\/nu12102908","article-title":"Anti-inflammatory potential of cow, donkey and goat milk extracellular vesicles as revealed by metabolomic profile","volume":"12","author":"Mecocci","year":"2020","journal-title":"Nutrients"},{"key":"B16","doi-asserted-by":"publisher","first-page":"16701","DOI":"10.1021\/acsomega.0c01582","article-title":"Zeta potential of extracellular vesicles: toward understanding the attributes that determine colloidal stability","volume":"5","author":"Midekessa","year":"2020","journal-title":"ACS Omega"},{"key":"B17","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1016\/j.canlet.2015.10.020","article-title":"Bovine milk-derived exosomes for drug delivery","volume":"371","author":"Munagala","year":"2016","journal-title":"Cancer Lett."},{"key":"B18","doi-asserted-by":"publisher","first-page":"883","DOI":"10.1038\/nm.2753","article-title":"Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET","volume":"18","author":"Peinado","year":"2012","journal-title":"Nat. Med."},{"key":"B19","doi-asserted-by":"publisher","first-page":"e0222613","DOI":"10.1371\/journal.pone.0222613","article-title":"Acidification effects on isolation of extracellular vesicles from bovine milk","volume":"14","author":"Rahman","year":"2019","journal-title":"PLoS One"},{"key":"B20","doi-asserted-by":"publisher","first-page":"2105421","DOI":"10.1002\/smll.202105421","article-title":"Goat milk exosomes as natural nanoparticles for detecting inflammatory processes by optical imaging","volume":"18","author":"Santos\u2010Coquillat","year":"2022","journal-title":"Small"},{"key":"B21","doi-asserted-by":"publisher","first-page":"11","DOI":"10.3390\/proteomes8020011","article-title":"Milk-derived extracellular vesicles in inter-organism, cross-species communication and drug delivery","volume":"8","author":"Sanwlani","year":"2020","journal-title":"Proteomes"},{"key":"B22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.5772\/intechopen.85416","article-title":"Milk exosomes: isolation, biochemistry, morphology, and perspectives of use","volume-title":"Extracellular vesicles and their importance in human health","author":"Sedykh","year":"2020"},{"key":"B23","doi-asserted-by":"publisher","first-page":"6466","DOI":"10.3390\/ijms21186466","article-title":"A review of exosomal isolation methods: is size exclusion chromatography the best option?","volume":"21","author":"Sidhom","year":"2020","journal-title":"Int. J. Mol. Sci."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1440132","DOI":"10.1080\/20013078.2018.1440132","article-title":"Biocompatibility of highly purified bovine milk-derived extracellular vesicles","volume":"7","author":"Somiya","year":"2018","journal-title":"J. Extracell. Vesicles"},{"key":"B25","doi-asserted-by":"publisher","first-page":"Unit 3.22","DOI":"10.1002\/0471143030.cb0322s30","article-title":"Isolation and characterization of exosomes from cell culture supernatants and biological fluids","volume":"30","author":"Th\u00e9ry","year":"2006","journal-title":"Curr. Protoc. Cell Biol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1038\/nri2567","article-title":"Membrane vesicles as conveyors of immune responses","volume":"9","author":"Th\u00e9ry","year":"2009","journal-title":"Nat. Rev. Immunol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"1535750","DOI":"10.1080\/20013078.2018.1535750","article-title":"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","volume":"7","author":"Th\u00e9ry","year":"2018","journal-title":"J. Extracell. Vesicles"},{"key":"B28","doi-asserted-by":"publisher","first-page":"436","DOI":"10.3390\/jcm9020436","article-title":"Inclusion biogenesis, methods of isolation and clinical application of human cellular exosomes","volume":"9","author":"Tschuschke","year":"2020","journal-title":"J. Clin. Med."},{"key":"B29","unstructured":"University of Guelph enzymic coagulation of milk2021"},{"key":"B30","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/j.jconrel.2014.07.049","article-title":"Extracellular vesicles as drug delivery systems: lessons from the liposome field","volume":"195","author":"van der Meel","year":"2014","journal-title":"J. Control. Release"},{"key":"B31","doi-asserted-by":"publisher","first-page":"341","DOI":"10.1016\/j.repbio.2017.09.007","article-title":"A method for the isolation and enrichment of purified bovine milk exosomes","volume":"17","author":"Vaswani","year":"2017","journal-title":"Reprod. Biol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"12","DOI":"10.1186\/s12575-020-00125-5","article-title":"Combination of size-exclusion chromatography and ultracentrifugation improves the proteomic profiling of plasma-derived small extracellular vesicles","volume":"22","author":"Wei","year":"2020","journal-title":"Biol. Proced. Online"},{"key":"B33","doi-asserted-by":"publisher","first-page":"359","DOI":"10.1080\/03639045.2018.1539743","article-title":"Efficient method for isolation of exosomes from raw bovine milk","volume":"45","author":"Yamauchi","year":"2019","journal-title":"Drug Dev. Ind. Pharm."},{"key":"B34","doi-asserted-by":"publisher","first-page":"3684","DOI":"10.7150\/thno.41580","article-title":"Progress, opportunity, and perspective on exosome isolation-efforts for efficient exosome-based theranostics","volume":"10","author":"Yang","year":"2020","journal-title":"Theranostics"},{"key":"B35","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1155\/2018\/3634563","article-title":"A comparison of traditional and novel methods for the separation of exosomes from human samples","volume":"2018","author":"Yu","year":"2018","journal-title":"Biomed. Res. Int."},{"key":"B36","doi-asserted-by":"publisher","first-page":"6917","DOI":"10.2147\/IJN.S264498","article-title":"