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.
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 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.
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.
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.
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:
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.