Her2Targeted SiRNA Delivery Systems For Breast Cancer Therapy

Breast cancer is one of the most common women malignancies, and has become "the first killer" for women. The conventional chemotherapy can kill the cancer cells, but the drugs also attack normal healthy cells to generate severe side effect due to the lack of selectivity. Therefore, the development of safe and efficient treatment against breast cancer has become important and urgent. Since the discovery of RNA interference, small interfering RNA (siRNA) has attracted great interests as a revolutional therapeutic agent for breast cancer because of its ability to efficiently silence specific genes. A few of delivery systems have been developed for systemic delivery of siRNA and cancer therapy. However, targeted delivery of siRNA to specific tumor tissues and tumor cells remains as one of the key challenges in the development of RNA interference as a therapeutic application. Human epidermal growth factor receptor2(Her2/neu) occurs in25to30%of human primary breast cancers. As an important biomarker of breast cancer, Her2is also an crucial therapeutic target in Her2-overexpressed breast eancer.Here, we designed two Her2-targeted siRNA delivery systems. Our results indicated that both delivery systems can effectively deliver siRNA into Her2overexpressing tumor cells and tumor tissues. The main content and conclusions of this dissertation are summarized below:In the first part, we have successfully expressed two proteins containing anti-Her2ScFv that can target Her2. One is the fusion protein of anti-Her2ScFv with protamine, and the other one is anti-Her2ScFv with cystein residue at the C-terminal. Both proteins are expressed from E.coil expression systems.In the second part, we have developed a therapeutic delivery system using the fusion protein of anti-Her2single-chain antibody fragment with the positively charged protamine, namely F5-P, as the carrier to specifically deliver siRNA-targeting DNA methyltransferases1and/or3b genes (siDNMTs) into Her2-expressing breast tumor cells. The carrier F5-P, expressed by the E. coli system, was able to bind siRNA molecules and specifically deliver the siRNA to Her2-expressing BT474breast cancer cells but not Her2-nonexpressing MDA-MB-231breast cancer cells, while delivery of siDNMTs to BT474cells successfully silenced the expression of targeted DNA methyltransferases (DNMTs) and facilitated the de-methylation of the RASSF1A tumor suppressor gene promoter, leading to the suppression of tumor cell proliferation. Moreover, as demonstrated in the BT474xenograft murine model, F5-P successfully delivered siRNA into a Her2-expressing breast tumor, and tumor growth inhibition was mediated by an intravenous injection of F5-P/siDNMTs complex by down-regulating the expression of DNMTs and restoring tumor suppressor gene expression.In the third part, we describe anti-Her2ScFv conjugated nanoparticles (NP) with stealthy property for efficient siRNA encapsulation and delivery. The nanoparticles are fabricated with poly(ethylene glycol)-b-poly(d,l-lactide), siRNA and a cationic lipid, using a double emulsion-solvent evaporation technique, which is followed by the conjugation of anti-Her2ScFv. The siRNA retained its integrity within the resulted targeting nanoparticles (T-NP), which can be effectively internalized by cancer cells, resulting in significant gene expression downregulation. In our study, we have demonstrated that T-NP carrying siRNA can targeted deliver siRNA into Her2overexpressing breast cancer cells and induce remarkable apoptosis by silencing PLK1gene. Furthermore, systemic delivery of siPLK1by T-NP significantly suppresses tumor growth in Her2overexpressing breast cancer in the xenograft murine model.