MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking.
Researchers from the Madrid Institute for Advanced Studies (IMDEA) evaluated the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, the researchers show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.
Summary of the EV isolation approaches
Raw bovine milk was skimmed by differential centrifugation to remove cells, fat globules and large proteins. Ultracentrifugation of 25 mL of skimmed milk (SM) results in a solid pellet containing most of the SM casein, a supernatant of milk serum, and a viscous phospholipid rich soluble concentrate positioned adjacent to the casein pellet containing EVs (called fluffy layer). EV fluffy layers were filtered through 0.22 µm syringe filters. Afterwards, 700 µL of filtered EVs were enriched through two different approaches: (a) SEC (1U + SEC) or (b) additional ultracentrifugation followed by SEC (2U + SEC). After first ultracentrifugation, EVs loaded with exogenous miRNA by the transfection method were then isolated by (c) SEC (U + T + SEC) or (d) additional ultracentrifugation followed by SEC (U + T + U + SEC). Forty sequential fractions of 500 µL were collected and their miRNA and protein elution profiles were subsequently obtained by carrying out different methodology (BCA, Western Blot and/or RT-qPCR analysis).