Resumen de Dietary extracellular vesicles as delivery vehicles for exogenous RNAs with potential effects on host gene expression
The association between dietary habits and the incidence and prognosis of some highly prevalent chronic diseases is increasingly strong. In fact, recent findings provide evidence on how diet modulates host gene expression at post-transcriptional level, by influencing the consumer microRNA (miRNA) expression profile. MiRNAs are small non-coding RNA molecules involved in the regulation of essential biological processes. Since miRNAs from foodstuffs have been detected in mammalian blood and tissue samples, it has been suggested that not only food-derived compounds can produce biological effect by influencing the host miRNA profile but also, diet-derived miRNAs may regulate the host gene expression. This discovery converts them into potential powerful candidates for future pharmacological development of dietary miRNAs as therapeutic agents. In the last decade, it has been postulated that the transfer of food-derived miRNAs may be facilitated by the protection conferred by extracellular vesicles (EVs), which help miRNAs survive to gastrointestinal (GI) conditions and permits their uptake into the intestine and their subsequent transfer to the bloodstream. Indeed, in silico approaches have identified some of these miRNA targets which have been experimentally validated. However, evidence of whether food-derived miRNAs reach cells at minimal doses enough to produce a biological effect, remains scarce and controversial. Considering the above, this doctoral thesis aimed to: i) evaluate the biological effect of exogenous miRNAs carried by extracellular vesicles, particularly from bovine milk and broccoli, on host genome regulation; and ii) assess the suitability of food-derived extracellular vesicles as potential drug delivery nanocarriers for therapeutic applications, specifically, for RNA-based therapies. To address these objectives, a method for the isolation of food-derived EVs was firstly developed combining ultracentrifugation and size exclusion chromatography. Then, identification and characterization of their miRNA profile was performed. Subsequently, the stability of the most abundant miRNAs throughout the GI tract by simulating GI conditions was tested. Furthermore, the cellular uptake of EVs and the intracellular concentration reached and/or the relative expression increase of selected miRNAs after cell exposure to bovine milk or broccoli EVs were analyzed. Their possible biological effect by in silico approaches or transcriptional studies was also assessed. Finally, through an in vivo study, the stability and biodistribution of miRNAs after the oral administration of isolated bovine milk EVs to C57BL/6 mice was performed. Studies allowed to identify the most enriched miRNAs in bovine milk and broccoli EVs. Moreover, the uptake of mammalian and plant EVs by human cell lines, as well as the transfer of the miRNAs they carry were experimentally confirmed. However, the miRNA content transported by bovine milk and broccoli EVs administered at dietary doses is rather limited to provide enough amount of miRNAs to produce a measurable biological effect. Therefore, a loading strategy to enrich EVs with exogenous miRNAs was developed. The digestive stability analysis demonstrated high overall degradation of exogenous miRNAs, although EV-protected miRNAs resisted better GI digestion compared to free miRNAs. Interestingly, miRNAs transported in orally administered EVs reached mice organs, where they could exert their biological effect through regulation of their target messenger RNAs (mRNAs). To conclude, results suggested that, according to the concentrations and conditions tested in this doctoral thesis, diet consumed miRNAs do not reach a sufficient concentration in the host cells to regulate gene expression by the canonical pathway. However, despite many obstacles and challenges, extracellular vesicles can be used as potential nanocarriers of functional miRNAs. This opens new possibilities, both in pharma-nutritional (e.g., dietary supplements) and pharmacological (e.g., drug delivery) industries, although further research is needed to refine aspects of production, application and safety
