Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk

Mukhopadhya, Anindya; Santoro, Jessie; Moran, Barry; Useckaite, Zivile; O’Driscoll, Lorraine

doi:10.1016/j.foodchem.2021.129309

Cited by 18 publications

(17 citation statements)

References 52 publications

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“…Processes such as fermentation may induce exosome lysis and increase RNase levels due to microbial production [ 107 ]. In the same line, infant formulas are also subjected to harsh conditions during their production, and thus, the addition of exosomes as an ingredient to resemble the characteristics of human milk must overcome challenges related to industrial processing and storage [ 111 ]. These aspects are even more relevant in hydrolyzed formulas, which are subjected to more severe processing conditions [ 112 ].…”

Section: Milk-derived Exosomesmentioning

confidence: 99%

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

et al. 2022

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Extracellular vesicles are membrane-enclosed secreted vesicles involved in cell-to-cell communication processes, identified in virtually all body fluids. Among extracellular vesicles, exosomes have gained increasing attention in recent years as they have unique biological origins and deliver different cargos, such as nucleic acids, proteins, and lipids, which might mediate various health processes. In particular, milk-derived exosomes are proposed as bioactive compounds of breast milk, which have been reported to resist gastric digestion and reach systemic circulation, thus being bioavailable after oral intake. In the present manuscript, we critically discuss the available evidence on the health benefits attributed to milk exosomes, and we provide an outlook for the potential future uses of these compounds. The use of milk exosomes as bioactive ingredients represents a novel avenue to explore in the context of human nutrition, and they might exert important beneficial effects at multiple levels, including but not limited to intestinal health, bone and muscle metabolism, immunity, modulation of the microbiota, growth, and development.

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Section: Milk-derived Exosomesmentioning

confidence: 99%

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

et al. 2022

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“…Samples were prepared from transmission electron microscopy (TEM) analysis following our published protocol [20,21] that was adapted from a previous publication [22]. Briefly, 10 µL of the sample was placed onto carbon-coated grids (Ted-Pella B 300 M, Mason Technology Ltd., Dublin, Ireland; Cat.…”

Section: Transmission Electron Microscopymentioning

confidence: 99%

Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods

Martinez-Pacheco

O’Driscoll

2021

Cancers

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To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes.

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“…In relation to MSC EVs, a protocol was deemed suitable for separating EVs from the conditioned medium (CM) of MSC for subsequent successful administration, as therapy in the clinic in their natural (non-loaded) form has been reported [ 20 ]. For milk EVs, we recently developed an effective protocol to separate relatively pure EVs from milk, which involved the removal of caseins, which are highly abundant in milk and overlap in size with EVs [ 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

Doxorubicin Loading into Milk and Mesenchymal Stem Cells’ Extracellular Vesicles as Drug Delivery Vehicles

et al. 2023

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Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs’ CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.

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Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk

Cited by 18 publications

References 52 publications

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods

Doxorubicin Loading into Milk and Mesenchymal Stem Cells’ Extracellular Vesicles as Drug Delivery Vehicles

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