Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo

Abstract

Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.

MicroRNA profiling in kidney disease: Plasma versus plasma-derived exosomes

“…RT-qPCR-based measurements demonstrated that overall miRNA expression levels in plasma-derived exosomes was about 16 times lower than that in plasma samples, which is in line with recent publications showing low abundance of miRNAs in exosome preparations [19, 35]. However, we did observe that a subset of miRNAs were relatively enriched in plasma-derived exosomes, meaning the relative expression of the particular miRNA in exosomes was close to its expression in plasma.…”

Pancreatic cancer cell–derived exosomal microRNA‐27a promotes angiogenesis of human microvascular endothelial cells in pancreatic cancer via BTG2

“…Despite the progress achieved in the diagnosis and therapy of PC over the past few decades, PC remains one of the most lethal conditions, accompanied by a heavy health burden as well as a high mortality rate . More recently, cell‐secreted exosomes which are important regulators of cell‐cell communication have been identified as potentially useful tools in gene therapy and drug delivery, and miRs have been reported to be promising therapeutic targets for the treatment of various human diseases . Hence, the present study investigated the effect of PC cell–derived exosomal miR‐27a on PC through the regulation of BTG2.…”

Mesenchymal Stem Cells Reduce Corneal Fibrosis and Inflammation via Extracellular Vesicle-Mediated Delivery of miRNA

“…EVs were purified from this medium as described above. CFSE‐labeled EVs (47 μg protein in 100 μl) were loaded with nonmammalian miR159a (12 μg RNA) by incubation in PBS with 0.1 M CaCl 2 on ice for 30 minutes followed by 42°C for 5 minutes and returned to 4°C as previously described . EVs were recovered by pelleting as above.…”