| Malignant tumors, are fatal diseases which severally threaten the health of human, According to the report of the World Health Organization, the global number of newly diagnosed and death of tumors are increasing year by year. Therefore exploring effective modalities for anti-tumor therapy have been a hot topic. siRNA, which could induce RNA interference response in vivo, provides a promising method for the treatment of tumors. However, naked siRNA is easily degraded by RNase, together with the short half-life, hydrophilic propertity, and negative charge, so naked siRNA showed poor intracellular uptake when used alone, consequently, vectors are needed to protect and deliver siRNA into cells. Up to now, the lack of appropriate vectors has become a major hurdle in the development of siRNA therapy. Therefore, we synthesized a series of non-viral vectors and screened the most efficient vector to deliver functional siRNA for anti-tumor therapy in vivo.First of all, by a chemical reaction, six kinds of cell penetrating peptides (CPPs) were covalently conjugated to the free amino groups in CS molecules through a coupling reaction. The structural composition of copolymers were confirmed by FTIR and ’HNMR analysisits. Then, MTT assays were performed to evaluate the cytotoxicity of CPP-g-CS vectors using different cell lines. The results of cytotoxicity assay showed that cell viability of all vectors at different concentrations on four cell lines all exceeded>87%. siRNA-loaded nanoparticles were prepared through the complex coacervation method, and the physicochemical properties of nanoparticles were investigated in detail. The assay results showed that the spherical nanoparticles had an average size of about200nm with a narrow polydispersity index, moreover, positive charges of18.58mV existed on the surface of nanoparticles.Luciferase reporter gene assays were performed to evaluate the silencing effects of nanoparticles prepared using different CPP-g-CS vectors. Results from the reporter gene assay showed that TAT-g-CS/siRNA and6R-g-CS/siRNA had the strongest inhibitory potential against the target gene, and the inhibitory efficiency against exogenous gene were73.7%and64.9%, respectively, and the efficiency against endogenous genes were75.3%and64.92%respectively. Then, inhibitors were used to investigate the mechanism of transfection, meanwhile, the effects of incubation time and siRNA doses on silencing efficiency of the target gene were tested. By the way, cellular uptaken of nanoparticles were observed by laser confocal. Anti-proliferation efficiency of siRNA loaded nanoparticles were measured using the Survivin and Bcl-2targeted genes by CCK-8assays. The results showed that the anti-proliferation efficiency of nanoparticles were55.6%and52.8%, respectively, and the ratios of apoptotic cells induced by the functional nanoparticles were98.77%and99.2%, respectively.Finally, nanoparticles prepared with Survivin and Bcl-2targeted siRNAs were intratumorally injected into tumor-bearing mice. Tumor volume and weight were monitored during the tumor progression to evaluate the effect of anti-tumor growth, on the other hand, the lung tissues were excised and fixed by Bouin’s solution to observe the tumor metastasis. Also, tumor specimens were fixed and subjected to the histopathological examination. It was found that the four kinds of nanoparticles can significantly inhibit the growth and metastasis of breast cancer in BLAB/c mice. At the same time, bioluminescence was also applied to observe the anti-metastasis effect of Survivin-targeted nanoparticle on tumor-bearing mice, and the survival of tumor-bearing mice were tested. The results were in accord with the former conclusions that the Survivin targeted nanoparticles could effectively inhibit tumor growth and metastasis, and moreover, the survival time of tumor-bearing mice were also prolonged.Six derivatives of chitosan were synthesized and characterized in this project, and the most efficient vector for siRNA delivery was screened out by comparing the silencing efficiency against target gene. Compared with other studies on non-viral vectors without screening process, the design of our study are more reasonable and easier to get better results. In addition, most studies on siRNA delivery are limited to reporter genes or therapeutic genes in vitro. In this study, we employed the most efficient vectors to load functional siRNAs which targeted Survivin and Bcl-2to prepare nanoparticles. More importantly, in vivo anti-tumor activities of the functional siRNA loaded nanoparticles were assessed, at the same time, the anti-metastasis effect was observed by bioluminecence. The conception and contents of our study has not been reported yet. |
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