Creation And Evaluation Of Novel Biocompatible Nanoparticles For SiRNA Delivery

RNA interference (RNAi) is a sequence specific transcription regulation that leads to silencing of gene expression. RNAi is a conserved post-transcriptional gene regulation and plays crucial role in development, cell division, differentiation, as well as various pathological processes.RNAi has great potential for clinical applications. However, creation of highly efficient novel delivery system is necessary for clinical application of RNAi. Combing our advantages on RNAi and previous experiences with preparing nanoparticles such as polysaccharides and dendrimers, we designed and synthesized novel curdlan-based, peptidomimetic-based as well as cholesterol-based nanoparticles. In this study, we have employed three distinguished nanocarriers to evaluate siRNA delivery efficiency in vitro and in vivo.We synthesized curdlan-based nanoparticles, denoted as 6AC-100PEG, which was obtained by conjugating mPEG 2000 to 6-amino-6-deoxy-curdlan. siRNA delivery efficiency of 6AC-100PEG/siRNA complex showed successfully downregulated expression of endogenous GAPDH and LXRα genes in human liver hepatocellular HepG2 cells.Systemic administration of 6AC-100PEG/siApoB complex significantly reduced the level of ApoB mRNA in mouse liver, indicating that 6AC-100PEG can efficiently deliver siRNA to mouse liver and induces RNAi. Notably, to the best of our knowledge, our study is the first report on the in vivo delivery of biotherapeutics using curdlan-based cationic polymer, revealing a potential carbohydrate based nanoparticles for systemic application of RNAi.We next developed highly efficient liposomal siRNA delivery agent, denoted as DoGo3, based on a novel peptidomimetic built from natural amino acids. The novel system has been designed to increase the stability of lipoplex/siRNA complex by tightly controlling physical chemical parameters of the resulting particle. Our preliminary study demonstrated that lipoplex with biotin labeled-siRNA can preferentially distribute to liver and efficiently decrease Apolipoprotein B (ApoB) protein level which contributes to the cholesterol level in the blood. This result leads us to evaluate potential of the lipoplex to target orthotopic lung tumor in mice. Systemic administration of DoGo3/siRNA lipoplex was able to deliver near-infrared dye labeled-siRNA to the lungs and surrounding tumors. It should be noted that DoGo3 was not modified specifically to target tumor cells.We finally examined another cholesterol-based cationic lipid, designed as Chorn-1, for siRNA delivery and in vitro Chorn-1/siRNA complex demonstrated robust downregulation of GAPDH mRNA expression in HepG2 cells. To improve in vivo biocompatibility, we formulated lipoplex containing our novel cationic lipid(Chorn-1), our previously synthesized novel nanoparticle (DoGol) and modified mPEG2000 (mPEG-OLA). The lipoplex was able to decrease the expression of ApoB in mouse liver and showed no apparent toxicity.These studies will be great interest and contribution for further applications and will be inspiration for further development of more advanced carrier systems.