Delivery Of Sirna And Chemotherapeutic Drug With Nanoparticles 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. Based on the fact that nanoparticulate drug delivery system has shown greatly enhanced chemotherapy efficiency for cancer therapy, this dissertation has focused on the development of nanoparticles as drug carriers for the combination of RNA interference therapy and chemotherapy as well as cancer stem cell therapy to improve breast cancer treatment efficacy. In the first part of the dissertation, we have synthesized a novel functionalized amphiphilic triblock copolymer consisting of monomethoxy poly(ethylene glycol), poly(ε-caprolactone) (PCL) and poly(2-aminoethyl ethylene phosphate) denoted as mPEG-b-PCL-b-PPEEA, which can self-assemble into micellar nanoparticles (MNP) in aqueous solution. The MNP are capable of simultaneously delivering small interfering RNA (siRNA) and paclitaxel into the same cancer cells after systemic administration to synergistically inhibit the growth of breast cancer xenografts. This combination therapy has provided a new strategy for more effective treatment of breast cancer. In the second part, using a gold nanoparticle grafted with doxorubicin (DOX) through pH-responsive hydrazone bond (DOX-Hyd@AuNP) as a model, we have investigated the effect of nanoparticulate drug delivery system on cancer stem cells (CSCs). Compared with free DOX, the DOX-Hyd@AuNP significantly enhances the uptake of DOX by CSCs and impairs the stemness of CSCs in both in vitro and in vivo experiments. The study in tumor-bearing mice has shown that the DOX-Hyd@AuNP can significantly inhibit tumor growth while do not enhance the proportion of CSCs. On the contrary, the free DOX treatment results in remarkable increase of CSCs in the tumor tissue. This study provides important evidence for designing novel drug delivery system.The main content and conclusions of this dissertation are summarized below:1. Downregulation of the expression of the key protein in breast cancer cells can significantly enhance sensitivity of the cells to conventional chemotherapeutics. Naoparticulate drug delivery carriers have the potential to simultaneously deliver siRNA and chemotherapeutics; however, the preparation of such carriers has been a great challenge. Moreover, whether the combination of siRNA and small molecule anticancer drugs can synergistically inhibit the tumor growth in vivo has not been effectively confirmed. To obtain the co-delivery vehicle for siRNA and chemotherapeutic, we have designed and synthesized the functionalized amphiphilic triblock copolymer mPEG-b-PCL-b-PPEEA. The polymer self-assembles into uniform core-shell-corona MNP in aqueous solution with PCL as the hydrophobic core, PPEEA as the hydrophilic shell and PEG as the hydrophilic corona. 1H NMR spectrum in D2O and transmission electron microscopy (TEM) analyses has confirmed the formation of MNP. The MNP are positively charged with the zeta potential of about 45 mV due to the presence of protonized amino groups from PPEEA block, and form stable complexes with siRNA at an appropriate N/P ratio, while the PCL core can be used to load hydrophobic anticancer drug. For in vitro study, we have found that MNP can deliver siRNA into cell cytoplasm and release it there. The targeting gene expression is significantly silenced and the silencing efficiency is closely related to the siRNA dose and N/P ratio. In addition, the MNP are highly biocompatible and have great potential as drug delivery carriers for cancer therapy.Subsequently, we have used the MNP to simultaneously load siRNA targeting Plk1 (siPlk1) and paclitaxel to form“two-in-one”delivery carrier paclitaxelmicellplexsiPlk1, and demonstrated that the ratio of the two loaded drugs can be adjusted. Several different measurements have confirmed that the paclitaxelmicellplexsiPlk1 can simultaneously deliver siPlk1 and paclitaxel into the tumor cells after in vitro co-culture with breast cancer cells. Further analyses have demonstrated that the co-delivery system is capable of inhibiting the proliferation of the cancer cells and enhancing their apoptosis, reflecting the synergies of siPlk1 and paclitaxel. Further in vivo study has demonstrated that the paclitaxelmicellplexsiPlk1 can simultaneously transport siPlk1 and paclitaxel into tumoral cells after systemic administration to synergistically prohibit tumor growth. On the contrary, separate delivery of siPlk1 and paclitaxel by the same MNP do not show significantly enhanced inhibition efficiency. This study represents the first example of systemic and simultaneous delivery of siRNA and chemotherapeutics for synergistic tumor growth inhibition, and provides novel strategy for more effective therapy against breast cancer.2. Cancer stem cells (CSCs) are a small portion of cells with features of stem cell in the tumor tissue. They have the self-renewal capability and have been considered as the root of tumor. Due to the multi-drug resistant and other properties of CSCs, common chemotherapy drugs show minimal effect on their proliferation. Instead, chemotherapy enriches CSCs in tumor tissue, making it more difficult to completely cure the tumor. Up to today, the influence of nanoparticulate drug delivery system on breast cancer stem cells is unclear. In this study, DOX-Hyd@AuNP has been used to enhance the DOX accumulation in CSCs and facilitate intercellular DOX release under the condition of low pH in lysosome to overcome the drug resistance of CSCs. We have observed that DOX can be delivered into the breast cancer stem cells by DOX-Hyd@AuNP and significantly impair the stemness of stem cells and reduce their ability in forming tumors in comparison with free DOX at the same dose. Further in vivo study has shown that the DOX-Hyd@AuNP can significantly inhibit tumor growth while do not enhance the proportion of CSCs. On the contrary, the free DOX treatment results in remarkable increase of CSCs in the tumor tissue, which may cause recurrence and metastasis of the cancer. This strategy provides evidence for designing drug delivery system for breast cancer treatment and has brought hope to completely cure breast cancer.