Vectors With Zwitterionie Polvmers For The Delivery Of Small Interfering RNA

Small interfering RNA (siRNA) has promising application for the treatment of various diseases. However, the instrinsic deficiencies of siRNA remain the biggest challenges for its clinical applications. Polyethylene glycol (PEG) modified cationic liposomes have great application in the delivery of siRNA for overcoming its extracellular and intracellular obstacles. Unfortunately, the steric barrier due to PEGylation interferes with the siRNA encapsulation efficiency, cellular uptake and subsequent endosomal escape of cationic liposome/siRNA complexes (lipoplexes). Most importantly, PEGylated lipoplexes are intended to stimulate the "accelerated blood clearance (ABC)" immune response upon the first injection, which seriously decreases the target diseased site accumulation and therapeutic efficiency of siRNA. In this dissertation, novel long-circulating cationic liposomes based siRNA delivery system with the modification.of zwitterionic poly(carboxybetaine) (PCB) were developed, which could deliver the siRNA to the target site and enhance the gene silencing efficiency of siRNA in vitro and in vivo. In detail, the works mainly included the following issues:(1) Zwitterionic polymer PCB based l,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(carboxybetaine)n (DSPE-PCBn) lipid was designed and synthesized for cationic liposomes modification to construct novel long-circulating cationic liposomes. DSPE-PCB20 had the comparable ability to prevent the nonspecific protein adsorption and enhance the serum stability of cationic liposome/siRNA complexes as that of DSPE-PEG 2000. At the same time, the carboxyl acid groups of PCB could be protonation in acidic condition of endosomes, which increased the surface positive charge of lipoplexes and facilitated the destabilization of endosomal membrane by forming ion-pairs with anionic endosomal membrane lipids, and releasing siRNA into the cytoplasm. With excellent serum stability, siRNA encapsulation efficiency and endosomal escape ability, the DSPE-PCB20 lipoplexes with siRNA targeting apolipoprotein B (ApoB siRNA) could be successfully delivered to the liver and repressed the expression of ApoB, which resulted in a decrease of total cholesterol in serum, indicating that DSPE-PCB20 lipoplexes had promising application in hypercholesterolemia-related diseases.(2) DSPE-PCB20 modified novel long-circulating cationic liposomes were developed to verify whether PCBylation could avoid ABC phenomenon. Different from the DSPE-PEG 2000 lipoplexes, the blood clearance trend and hepatic uptake of DSPE-PCB20 lipoplexes were basically the same for the two injections, which indicated that DSPE-PCB20 could avoid ABC phenomenon and have no effect on the accumulation of the second injected DSPE-PCB20 cationic liposome/siRNA complexes in tumor. Additionally, the PCB modification could enhance the endosomal escape of siRNA after endocytose, which in turn lead to remarkable release of siRNA into cytoplasm. With excellent in vitro and in vivo behavior, the PCBylated cationic liposomes with siRNA targeting polo-like kinase 1 (siPlk1) significantly inhibited the expression of Plk1 protein and induced the apoptosis of tumor cells, suggesting its potential application for tumor therapy.(3) PCB based camptothecin (CPT) prodrug molecule CPT-PCBn was designed and synthesized to construct dual sensitive and temporally controlled novel long-circulating cationic liposomes for the codelivery of siRNA and CPT. The pH-sensitive zwitterionic polymer PCB was conjugated with CPT through pH and esterase-sensitive ester bond to enhance the stability and loading content of CPT. CPT-based cationic liposomes consisted of CPT-PCBn prodrug and cationic lipid DDAB were then constructed for siRNA codelivery for combination therapy. The dual sensitive CPT-PCBn cationic liposome/siRNA complexes stimultaneously delivered the two drugs to the same tumor cells and enabled a temporally controlled release of two drugs that the siRNA was quickly released after 4 h incubation due to the protonation of PCB in acidic endosomes, and CPT was released in a sustained manner in response to pH and esterase and highly accumulated in nucleus after 12 h incubation. The accelerated release of siPlkl and the inhibition of Plkl protein expression could improve the sensitivity of HeLa cancer cells to CPT and CPT could exert more cytotoxicity. Therefore, the dual sensitive and temporally controlled CPT-PCBn cationic liposome/siPlkl complexes exhibited a synergistic tumor suppression effect in vitro and in vivo, which demonstrated to be a promising codelivery system for cancer therapy.