Preparation Of Novel Curdlan Nanoparticles For Intracellular Sirna Delivery

RNA interference (RNAi) is a kind of gene regulatory mechanism on post-transcription level.The gene silencing induced by RNAi is able to be applied on clinical treatment of diseases such as cancer. Concerning clinical application of RNAi, one of the most important issues is to construct gene carrier with high safety and efficiency.In this thesis, taking natural curdlan as a starting reactant, the 6-amino-6-deoxy-curdlan (6AC) with different amination degree (30%,50%,70% and 100%) has been prepared by azidation and reduction of C6 in constitutional units of curdlan.The structures of obtained compounds were confirmed by IR and 3CNMR. and the morphology of nanoparticles made of 6AC were examined with DLS(size distribution), Zeta potential and SEM. The buffering capacity of 6AC on H+ was also determined by the acid-base titration method. Then, cytotoxicity and siRNA delivery efficiency of 6AC nanoparticles have been systematically studied in vitro. The results indicates that the novel aminated curdlan can form nanoparticles with approximate diameter of 120nm, and the size is about 130nm after combination with siRNA. Their aqueous solution shows high capacity of H+ buffering, and zeta potential value is positive.6AC show low cytotoxicity in HepG2 and Hela cell lines, and is able to efficiently deliver siRNA to A549, H727, HCT116, THP-1 and MEF cells, down-regulating the targeting mRNA level by 70-90%. In addition, GFP protein level significantly decreased in mES cell lines due to delivered siRNA targeting eGFP. These finding demonstrate that this novel amine functionalized curdlan nanoparticle has the potential to further research and development value as a siRNA carrier.In conclusion, we synthesized a novel water soluble curdlan and used them as nano-carriers of siRNA successfully, and were applied to siRNA delivery as a gene vector, their safety and deliverying efficiency have been studied on cytotoxicity, gene level and protein level. The results provide us with a novel cationic polysaccharide siRNA carrier different from chitosan in gene therapy field, and layed the foundation for further study about exploring relationship between structure and activity of siRNA carrier to a certain degree.