The Biological Functions Of Extracellular Vesicle And Its Utilization As Small RNA Carrier In Vivo

Extracellular vesicles (EVs) are natural transport Nano-vesicles (30-100 nm) secreted by numerous cell types. EVs are generally known in the literature as microvesicles (MVs) and exosomes. Distant cells can exchange information by sending out signals composed of a selection of proteins, lipids, and nucleic acids in EVs. Many researches indicate that EVs are promising carrier for drug delivery.In first section, to verify the possibility that use EVs as drug delivery carrier in theoretical, we analyzed the biological and physical properties of EVs derived from 293T cells. We present the first comprehensive analysis of the protein, mRNA and miRNA profiles of 293T cell-derived exosomes. Then, we characterized these data using Gene Ontology (GO) annotation and Kyoto Encyclopedia for Genes and Genomes (KEGG) pathway analysis. The result of Bioinformatics analysis shown that the protein, mRNA and miRNA profiles of 293T cell-derived exosomes did not have distinct tissue specificity and there is no evidence shown that these contents have influence on biological function of recipient cells. To evaluate whether transfection of miRNA have effect on 293T cell-derived exosomes, we compared the quantitative protein expression profiles of between exosomes secreted from 293T cells that transfected with functional small RNA and ncRNA. The results shown that transfection of miRNA into 293T did not affect the protein composition of 293T cell-derived exosomes. Our study reveals that 293T cell-derived exosomes are suitable for in vivo drug delivery.In second section, we show that miR-150 delivered by MVs to TAM and have a promotional effect on VEGF. To verify the possibility that use EVs as drug delivery carrier in vivo, we utilization MVs to transferred antisense RNA targeted to miR-150 into mice and found that the neutralization of miR-150 down-regulates VEGF levels in vivo and attenuates angiogenesis. Therefore, we found the role of miR-150 in promoting tumors growth and proposed the therapeutic potential of neutralizing miR-150 to treat cancer and demonstrated a novel, natural, MV-based method for the transfer of nucleic acids.In third section, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (OPRM) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AG02) in exosomes. These exosomes efficiently and specifically delivered OPRM siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced OPRM mRNA and protein levels and strongly inhibited morphine relapse via the down-regulation of OPRM expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system.In summary, both unmodified and engineered EVs are suitable for small RNA drug delivery in vivo. EVs have great potential as small RNA drug carriers for therapeutic use in future. Currently, the small RNA based gene therapy has become a promising treatment tool of human diseases, but deliver efficiency of small RNA in vivo is an obstacle. Our research provided a new method for siRNA drug delivery in vivo.