Design And Development Of Lmw Pei-based Vector For Delivery Of DNA And Small Interfering Rna And Its Application In Breast Cancer Gene Therapy

Breast cancer is the most frequently diagnosed malignancy in women. Currently, the effectiveness of traditional breast cancer modalities, such as surgical resection, endocrine therapy, chemotherapy and radiation therapy, is quite limited. Up to date, cure rate of advanced or recurring breast is less than5%. Moreover, tumor heterogeneity and genetic instability make it unlikely that a single target will suffice for long-term treatment of most solid tumors. With RNA interference, we could rapidly and efficiently knock down the expression of any gene in any cell type, thus opening a door to treat cancer by targeting every cancer-causing gene. Since survivin is over-expressed in breast cancer, and the over-expression of survivin plays an active role in the initiation, progress, metastasis, and prognosis of breast cancer. Therefore, we synthesized a series of alkylated and/or disulfide cross-linked PEI derivatives based on branched PEI1800(MW:1800) and linear PEI2200(MW:2200), respectively. After systematically investigating the physicochemical properties, cytotoxicity, DNA transfection efficiency, gene silencing efficiency, we screened out the optimal DNA delivery vector (1PEI2200-SS and lPEI2200-C12-5.28-SS) and siRNA delivery vector (bPEI1800-C,2-19.72and1PEI2200-SS), respectively. Subsequently, we tested the in vitro and in vivo anti-cancer effect of survivin-targeted siRNA delivered through1PEI2200-SS for the treatment of breast cancer.In the second chapter, we synthesized-C12grafted bPEI1800with different substitution degrees, namely bPEI1800-C12. Results of MTT showed that bPEI1800exerted barely cytotoxicity on4T1and MCF-7cells. Amphiphilic bPET1800-C12could self-assembly into micelle-like particles with a core-shell structure in water solutions. bPEI1800-C12/DNA polyplexes showed high stability against anion displacement. The highly enhanced cellular uptake of bPEI1800-C12/DNA polyplexes was mainly mediated by caveolin, which could bypass the lysosomal degradation. Among the various bPEI1800-C12, bPEI1800-C12-19.72showed the highest transfection efficiency, which is comparable to that of lipofectamine2000and significantly higher than that of1PEI22000. Bioluminescence imaging showed that systematic injection of bPEI1800-C12-19.72/pGL4.50polyplexes resulted in intensive luciferase expression in vivo and luciferase was mainly expressed in organs of liver, spleen and kidney. In the third chapter, we synthesized biodegradable bPEI1800-SS and bPEI1800-C12-SS with disulfide cross-linking based on bPEI1800and bPEI1800-C12, respectively. We found that disulfide cross-linking enhanced the cytotoxicity of bPEI1800-Ci2-SS on4T1cells, and the cytotoxicity of bPEI1800-C12-SS was comparable to that of bPEI1800-C12. Among bPEI1800-SS and the various bPEI1800-C12-SS, bPEI1800-C12-25-SS showed the highest gene transfection efficiency.In the fourth chapter, we synthesized IPEI2200with PEOZs by acid hydrolysis, and fatherly grafted1PEI2200with-C12at different substitution degrees. Compared with1PEI2200,-C12grafting enhanced the cytotoxicity of1PEI2200-C12on4T1cells, and the cytotoxicity of1PEI2200-C12on MCF-7cells was comparable to that of1PEI2200. We found that the gene transfection efficiency of1PEI2200-C12was negatively related to its-C12substitution degree. Among the various1PEI2200-C12,1PEI2200-C12-5.28exhibited the highest gene transfection efficiency.In the fifth chapter, we synthesized biodegradable1PEI2200-SS and1PEI2200-C12-SS with disulfide cross-linking based on1PEI2200and1PEI2200-C12, respectively. We found that disulfide cross-linking reduced the cytotoxicity of1PEI2200-C12-SS on4T1cells, and the cytotoxicity of1PEI2200-C12-SS was comparable to that of1PEI2200-C12. Among1PEI2200-SS and various IPEI2200-C,2-SS,1PEI2200-C12-5.28-SS showed the highest gene transfection efficiency on4T1cells, and1PEI2200-SS exhibited the highest gene transfection efficiency on MCF-7cells.Based on the results of chapter II to IV, five superior PEI derivatives were screened out: bPEI1800-C12-19.72, bPEI1800-C12-25-SS,1PEI2200-C12-5.28, IPEI2200-SS and1PEI2200-C12-5.28-SS. In the sixth chapter, we tested the gene transfection efficacy of the five superior PEI derivatives on4T11. MCF-7and B16cells with parallel study, and screened out two best DNA delivery vectors:1PEI2200-SS and1PEI2200-C12-5.28-SS. Additionally, the in vivo gene transfection efficacy of1PEI2200-C12-5.28-SS was higher than that of1PEI2200-SS.In the seventh chapter, we parallel studied the gene silencing efficiency of the five superior PEI derivatives and screen out two best siRNA delivery vector:1PEI2200-SS and bPEI1800-C12-19.72,lPEI2200-SS is superior to bPEI1800-C12-19.72. Disulfide bond could be cleaved in the presence of DTT, and siRNA could be released out efficiently form lPEI2200-SS/siRNA polyplexes. lPEI2200-SS/siRNASUT exhibited great anti-proliferation effect on4T1cells, which was found to be caused by the induction of cell apoptosis. Most importantly, results of tumor volume, tumor weight and histological observation demonstrated that lPEI-SS/siRNASUT polyplexes effectively inhibited the tumor growth and metastasis of4T1murine breast cancer models.