Synthesis Of MicroRNA Nanocapsules For RNA Interference Therapy

RNA interference (RNAi) phenomenon is a ubiquitous biological process in organisms discovered in recent years. As one of the most important biotechnologies, RNAi technique has been widely used in the diagnosis and therapy of serious diseases. However, due to the instability in physiological environment, short in-vivo half-life and poor cellular uptake of the interfering RNAs, delivery systems with low toxicity, as well as high stability and transfection efficiency are urgently needed for RNAi-based therapy. MicroRNAs (miRNAs), a class of interfering RNAs, are endogenous and evolutionarily conserved noncoding-RNAs engaged in the regulation of gene expression. MiRNA research has revealed miRNAs are involved in the development, hematopoiesis, organogenesis, cell apoptosis and proliferation, lipid metabolism and in many other biological processes. In this dissertation, we developed a nanocapsule-based miRNA delivery platform for RNAi therapy in various diseases to address the challenges such as the high toxicity, low delivery efficiency and insufficient stability of the miRNA vectors during the delivery process.In the first part, miRNA nanocapsule with low toxicity and high delivery efficiency for intracellular delivery was developed by in situ radical polymerization. We investigated the structure stability between miRNA nanocapules and the commercial transfection reagent Lipofectamine complexed miRNAs, and the results show that nanocapules show better stability against heparin, RNase and serum. Further cell experiments results have demonstrated that nanocapules exhibit lower toxicity and higher miRNA transfection efficiency than Lipofectamine technique. Using antisense miR-21 (AS-miR-21) as a model miRNA, we have further demonstrated that delivery of AS-miR-21 nanocapsules could significantly downregulate the miR-21 level in the tumor cells to regulate the PTEN/PI3K-Akt signal pathway, thus inhibiting the expression and nuclear translocation of transcriptional factors such as β-catenin, HIF1-α and STAT3. Such results further lead to the inhibiton of the VEGF signal pathway, thus inhibiting the tumoral angiogenesis and tumor growth.In addition, we developed a nanocapsule-based technology for intravenous miRNA delivery. We have demonstrated that higher monomer ratio results in nanocapulses with more uniform and smaller size, and higher surface charge. Further results show that the polymer shell of nanocapsules stays stable under physiological pH environment, whereas degrades under acidic pH environment, leading to the release of the encapsulated miRNAs. We have further demonstrated that the incorporation of PEG to the surface of nanocapsules could significantly decrease the cellular uptake of macrophage, whereas the internalization of nanocapusles by the astrocytic tumor cells still maintain a high level, which signifanctly reduces the uptake of nanocapusles by the major organs of reticuloendothelial system (RES). Then, we have investigated the therapy efficacy of miR-21 mimics with anti-apoptosis and pro-angiogenesis functions delivered by nanocapules in the model MCAO rats, and the results show that such nanocapsules could significantly improve the neurological function defect of the model rats.In the last part, we have successfully synthesized nanocapsules co-loading AS-miR-21 and doxorubicin (DOX), and nanocapsules encapsulating a functional protein, VHL tumor suppressor. We have demonstrated that AS-miR-21 and DOX co-loaded nanocapuels show synergistic inhibition on tumor cells by MTT assay and validated the intact and efficient delivery of functional protein by nanocapsules using FACS and WB analysis. At last, we have demonstrated the combined treatment using AS-miR-21, DOX and VHL to the tumor cells facilitates the synergistic regulation of the cellular pathways, therefore accomplishing the maximum inhibitions to the therapy targets.