Milk Extracellular Vesicles: Isolation, Function and Future Uses

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Isolation, function, and potential future uses of extracellular vesicles derived from milk – a complex and commonly overlooked biofluid.

Despite its common position on supermarket shelves, milk is a biofluid like – and unlike - any other. Its production is under the biological control of various hormones and its composition – though standardised to a certain extent within species – is subject to the influence of disease and the environment in much the same way as blood or urine. This makes milk of interest to human health; for example, the impacts of human breastmilk on infants and animal (e.g., cow) milk on humans of all ages. Other interests include using milk biomarkers to assess animal health in agriculture, and the use of milk components as dietary drug delivery vectors. Despite its great potential, milk is a somewhat neglected biofluid, making research into milk a rich avenue for new discoveries. This research relies on understanding the components of this complex biofluid.

Components of milk  

Whilst the most famous (and perhaps infamous) components of milk are lactose and lipids, milk is far more complex than just sugar and fat. Milk contains whole cells (e.g. immune and stem1) as well as bacterial2 cells, which are theorised to work in concert to promote healthy development of the infant gut.3 Key to the composition of milk are the fat globules (nanometre to micrometre scale), which are composed of lipid cores surrounded by a specialised phospholipid trilayer membrane. Protein is also a major constituent, with free proteins (e.g., antibodies) and larger structures such as the nanosized casein complexes known as casein micelles. Micronutrients (e.g., calcium, vitamins) are also an important component of milk and are crucial to its nutritional value. Finally, there are extracellular vesicles (EVs).

EVs are heterogenous nanosized particles consisting of a phospholipid bilayer surrounding a core of proteins, RNAs and small molecules. EVs are of great interest in the study of milk as their contents are highly changeable with the cellular environment from which they originated. They also have the potential to have a large impact on the functioning of the cells with which they come into contact through the delivery of their contents into target cells.

The challenges of EV isolation from milk

The question of how to best isolate pure EVs from milk is a difficult one to answer. The removal of fat globules and whole cells is comparatively easy. Both will separate from the EV-containing portion when milk is centrifuged at around 2,000xg.4 Cells will pellet at the bottom of the tube and fat globules will settle at the top. The fat globules can then be skimmed from the top and the remaining supernatant transferred to a fresh tube. A filtration step can be added to remove remaining fat globules.4 While this part may be straightforward, it gets more complex when the remaining protein is considered.

Free protein can be efficiently separated from EVs by size using a size exclusion chromatography column such as qEV columns. However, casein micelles prove harder to remove as their size overlaps with EVs. There are multiple methodologies for removing casein micelles. The first is acidification. This precipitates casein micelles, allowing them to be removed by centrifugation; but on the downside, this method also alters the structure of EVs.5 EDTA dissociation can also be used, though this may also damage EVs.6 Though newer to the field, chymosin (otherwise known as rennin) can be used to cleave casein, causing its aggregation and enabling the removal of casein, fat globules and cells by centrifugation in a single step.7 An in-depth comparison of these methods for casein removal is required to fully determine the ideal workflow.

The role of milk EVs in health and disease

Research into the functionality of EVs from milk has focused on several areas. Here we will be focusing mainly on the impact of milk EVs on the gastrointestinal system with which they come into direct contact when consumed. The exception to that is the interesting ability of human breastmilk EVs to promote clotting through their association with tissue factor.8 As coagulation effectively seals wounds, the function of this coagulant property is likely in protecting the mother from infection during nipple trauma associated with breastfeeding.9 Interestingly, this pro-coagulation activity was found to be completely absent from cow milk.

As the survival of premature and low birthweight infants improves worldwide, there is a concurrent rise in necrotising enterocolitis (NEC) which disproportionately impacts these infants.10 NEC is an inflammatory condition of the intestines which often results in sepsis and is fatal in around 23.5% of affected neonates.11 With such a high mortality rate and the protective impact of breastmilk against developing NEC12, research into the impact of milk EVs is an important area for research. This research is especially important as there is a lower rate of this protective breastfeeding occurring for the infants most at risk of NEC, due to the impracticalities of breastfeeding in the NICU environment (e.g. parents unable to always be present when feeding is required) and problems suckling in very low birth weight and very premature babies.13 If milk EVs were protective against NEC, isolated EVs could be beneficial fortifiers to either donor or formula milk.

Indeed, this appears to be the case. In mouse models of NEC, human breastmilk EVs were protective against mortality14 and disease severity.15,16 The mechanism through which breastmilk EVs protect against NEC is likely multi-modal with increases in intestinal epithelial cell proliferation17, improved goblet cell function16 and improved barrier tightness.18 Milk EVs may also be beneficial in adult intestinal inflammatory disorders.18 Taken together, these studies highlight the importance of further research into milk EVs for the treatment and prevention of NEC and other inflammatory intestinal disorders.

Can milk EVs reach the intestine? Does this make them druggable vectors?

The positive impact of milk EVs in mouse models of NEC suggest that milk EVs likely reach the intestine intact. The infant gastrointestinal system is gentler than that of an adult, having a higher pH and a lower concentration of digestive enzymes.19,20 Artificially loaded cargo from milk EVs was found in the distal organs in mice after oral administration21,22, supporting survival of digestion in adults. Indeed, breastmilk EVs can survive in vitro digestion and are internalised into cultured intestinal epithelial cells.23 It is also worth noting here that many past uptake studies have relied upon membrane dyes to measure EV uptake, and that this labelling process may create false positives.24 Further research is needed to understand if EVs themselves cross the intestinal barrier or if sufficient levels of any specific cargo (e.g. miRNAs) could be delivered to impact upon cells.

Their ability to survive digestion could potentially make milk EVs a viable oral drug delivery vector. In vitro studies using orally-delivered drug-loaded milk EVs support this idea.25 Others have introduced drug-loaded milk EVs into mice intravenously.26 Both routes appear to improve drug effectiveness with respect to the free drug, making this a promising area for future research.

Key questions for the milk EV field

Milk EVs are of potentially immense usefulness in medicine. They have innate protective effects on the neonatal gut14-16 and the potential to act as therapeutics in their own right in neonates and, perhaps, in adults with colitis and other intestinal inflammatory disorders.18 There is also interest in their use as vectors for drug delivery both orally and intravenously. However, at the time of writing, research into milk EVs currently accounts for only 4% of biofluid EV studies on Web of Science. As such, this research area is rich in opportunities for new discoveries, yet some key questions remain:

  1. How can milk EVs be purified to a high standard without altering the EVs? Key strides have been made in this area, but standardisation and rigorous comparison and testing of workflows has yet to be conducted. The collection and initial processing of milk is also essential for this process.
  2. How can milk (and indeed any EVs) be stored to adequately retain their integrity and functionality for therapeutic purposes? The question of EV stability for storage is a rapidly evolving field. The solving of this conundrum is essential for any EVs to be a practical therapeutic.
  3. Are milk EVs safe for long-term treatment? Whilst milk EVs are non-toxic in short in vivo studies, build-up of a low-level toxin over a chronic treatment timeline cannot be ruled out. Such a toxin could originate from the donor animal’s environment (e.g., chemicals such as pesticides) or endogenous EV components which may pathologically affect cells into which they come into contact.
  4. How well will cross-species milk EVs be tolerated in neonates and adults? Studies show that milk from non-human sources is worse for neonatal health outcomes than human breastmilk.27 Many adults do not tolerate cow milk. How would cross-species milk EVs impact upon these people?
  5. Will the promising results from in vivo drug-loaded milk EV studies translate to successful clinical trials? We are still waiting for the first clinical trials relating to milk EVs.

An important step forward to answering these questions will be the implementation of guidelines for milk EV research and reporting (i.e. required details for publications) which will be set out by the upcoming publication by the Milk Task Force.  

References

  1. Li, S. J. et al. Characterization of Stem Cells and Immune Cells in Preterm and Term Mother's Milk. Journal of Human Lactation 35, 528-534 (2019). https://doi.org:10.1177/0890334419838986
  2. Lackey, K. A. et al. What's Normal? Microbiomes in Human Milk and Infant Feces Are Related to Each Other but Vary Geographically: The INSPIRE Study. Frontiers in Nutrition 6 (2019). https://doi.org:10.3389/fnut.2019.00045
  3. Pannaraj, P. S. et al. Association Between Breast Milk Bacterial Communities and Establishment and Development of the Infant Gut Microbiome. Jama Pediatrics 171, 647-654 (2017). https://doi.org:10.1001/jamapediatrics.2017.0378
  4. Hu, Y., Thaler, J. & Nieuwland, R. Extracellular Vesicles in Human Milk. Pharmaceuticals 14 (2021). https://doi.org:10.3390/ph14101050
  5. Rahman, M. M. et al. Acidification effects on isolation of extracellular vesicles from bovine milk. Plos One 14 (2019). https://doi.org:10.1371/journal.pone.0222613
  6. Golan-Gerst, R. & Reif, S. Extracellular vesicles in human milk. Current Opinion in Clinical Nutrition and Metabolic Care 25, 209-215 (2022). https://doi.org:10.1097/mco.0000000000000834
  7. Tong, L. J. et al. Oral Administration of Bovine Milk-Derived Extracellular Vesicles Alters the Gut Microbiota and Enhances Intestinal Immunity in Mice. Molecular Nutrition & Food Research 64 (2020). https://doi.org:10.1002/mnfr.201901251
  8. Hu, Y. et al. Human milk triggers coagulation via tissue factor-exposing extracellular vesicles. Blood Advances 4, 6274-6282 (2020). https://doi.org:10.1182/bloodadvances.2020003012
  9. Nakamura, M. & Asaka, Y. An Evaluation of the Signs of Nipple Trauma Associated With Breastfeeding: A Delphi Study. Journal of Human Lactation  https://doi.org:10.1177/08903344221076527
  10. Battersby, C., Santhalingam, T., Costeloe, K. & Modi, N. Incidence of neonatal necrotising enterocolitis in high-income countries: a systematic review. Archives of Disease in Childhood-Fetal and Neonatal Edition 103, F182-F189 (2018). https://doi.org:10.1136/archdischild-2017-313880
  11. Jones, I. H. & Hall, N. J. Contemporary Outcomes for Infants with Necrotizing Enterocolitis-A Systematic Review. Journal of Pediatrics 220, 86-+ (2020). https://doi.org:10.1016/j.jpeds.2019.11.011
  12. Ochoa, T. J. et al. Is Mother's Own Milk Lactoferrin Intake Associated with Reduced Neonatal Sepsis, Necrotizing Enterocolitis, and Death? Neonatology 117, 167-174 (2020). https://doi.org:10.1159/000505663
  13. Parker, M. G. et al. Promoting Human Milk and Breastfeeding for the Very Low Birth Weight Infant. Pediatrics 148 (2021). https://doi.org:10.1542/peds.2021-054272
  14. Pisano, C. et al. Human Breast Milk-Derived Extracellular Vesicles in the Protection Against Experimental Necrotizing Enterocolitis. Journal of Pediatric Surgery 55, 54-58 (2020). https://doi.org:10.1016/j.jpedsurg.2019.09.052
  15. Miyake, H. et al. Human breast milk exosomes attenuate intestinal damage. Pediatric Surgery International 36, 155-163 (2020). https://doi.org:10.1007/s00383-019-04599-7
  16. Li, B. et al. Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis. Plos One 14 (2019). https://doi.org:10.1371/journal.pone.0211431
  17. Hock, A. et al. Breast milk-derived exosomes promote intestinal epithelial cell growth. Journal of Pediatric Surgery 52, 755-759 (2017). https://doi.org:10.1016/j.jpedsurg.2017.01.032
  18. Stremmel, W., Weiskirchen, R. & Melnik, B. C. Milk Exosomes Prevent Intestinal Inflammation in a Genetic Mouse Model of Ulcerative Colitis: A Pilot Experiment. Inflamm Intest Dis 5, 117-123 (2020). https://doi.org:10.1159/000507626
  19. Kamstrup, D., Berthelsen, R., Sassene, P. J., Selen, A. & Mullertz, A. In Vitro Model Simulating Gastro- Intestinal Digestion in the Pediatric Population (Neonates and Young Infants). Aaps Pharmscitech 18, 317-329 (2017). https://doi.org:10.1208/s12249-016-0649-1
  20. Zoppi, G., Andreotti, G., Pajnofer.F, Njai, D. M. & Gaburro, D. Exocrine pancreas function in premature and full term neonates. Pediatric Research 6, 880-886 (1972). https://doi.org:10.1203/00006450-197212000-00005
  21. Grossen, P. et al. Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides. Eur J Pharm Biopharm 158, 198-210 (2021). https://doi.org:10.1016/j.ejpb.2020.11.012
  22. Hazas, M. C. L. et al. Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression. European Journal of Nutrition 61, 1043-1056 (2022). https://doi.org:10.1007/s00394-021-02720-y
  23. Kahn, S. et al. Exosomal MicroRNAs in Milk from Mothers Delivering Preterm Infants Survive in Vitro Digestion and Are Taken Up by Human Intestinal Cells. Molecular Nutrition & Food Research 62 (2018). https://doi.org:10.1002/mnfr.201701050
  24. Dominkus, P. P. et al. PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles. Biochimica Et Biophysica Acta-Biomembranes 1860, 1350-1361 (2018). https://doi.org:10.1016/j.bbamem.2018.03.013
  25. Agrawal, A. K. et al. Milk-derived exosomes for oral delivery of paclitaxel. Nanomedicine-Nanotechnology Biology and Medicine 13, 1627-1636 (2017). https://doi.org:10.1016/j.nano.2017.03.001
  26. Zhou, G. N. et al. Exosome Mediated Cytosolic Cisplatin Delivery Through Clathrin-Independent Endocytosis and Enhanced Anti-cancer Effect via Avoiding Endosome Trapping in Cisplatin-Resistant Ovarian Cancer. Frontiers in Medicine 9 (2022). https://doi.org:10.3389/fmed.2022.810761
  27. Shulhan, J., Dicken, B., Hartling, L. & Larsen, B. M. K. Current Knowledge of Necrotizing Enterocolitis in Preterm Infants and the Impact of Different Types of Enteral Nutrition Products. Advances in Nutrition 8, 80-91 (2017). https://doi.org:10.3945/an.116.013193

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