The ability of small interfering RNA (siRNA) to silence specific target genes offers not only a tool to study gene function but also represents a novel approach for the treatment of various human diseases, including cancers. The clinical use of siRNA, however, has been severely hampered by the inefficient delivery of these molecules to target cell populations due to their instability, inefficient cell entry, and poor pharmacokinetic profile. Much effort has therefore been devoted to the development of efficient in vivo siRNA delivery systems, with liposomes being the most widely employed vector. The traditional methods of packaging siRNA into liposomes, however, are often quite complex and labour-intensive, with the resulting products also being unstable at room temperature which limits their wide spread application in the clinic. The main aim of this research was to develop a simple, yet efficient, formulation technique to prepare stable siRNA-loaded liposomes which could be utilized as an efficient therapy for cancer treatment. Throughout this study, cervical cancer was used as the model system to assess the efficiency of various delivery systems. It is an ideal disease for siRNA therapy due to the cancer’s reliance on the expression of a single messenger RNA sequence which encodes two essential viral oncogenes, E6 and E7. Previous research has shown that targeting E6 and E7 by siRNA in cervical cancer cells in vitro results in either cell senescence or apoptosis. This thesis investigates the feasibility of applying E6/7 siRNA both intravaginally and intravenously to model the treatment of early-stage and end-stage cervical cancer, respectively. The practicability of applying E6/7 siRNA intravaginally for the treatment of localised cervical cancer tumours was firstly evaluated by administrating liposome-complexed siRNAs directly into the vaginal cavity of transgenic E7 mice. As no knockdown of E7 in cervical epithelium was observed for mice which received repeated treatments of E6/7 siRNA, the vaginal delivery efficiency of liposomes was further examined using fluorescently-labelled oligonucleotides. Contrary to previous reports, no delivery of lipoplexes into cervicovaginal tissues was detected irrespective of the dosage, type of lipid vector used, or the mouse estrus state at the time of administration. This lack of delivery was likely due to the poor retention of lipoplexes in the vaginal cavity as well as the inefficient penetration of lipoplexes across the mucosal layer lining the cervicovaginal epithelium. Overall, these findings indicated the necessity of developing more suitable and clinically acceptable vaginal siRNA delivery systems to enable this treatment strategy to become a reality. Despite the challenges of using liposomes to deliver siRNA via vaginal administration, their successful use in delivering siRNA intravenously to tumours was demonstrated in a subcutaneous cervical cancer mouse model. These experiments were carried out using PEGylated siRNA-loaded liposomes which were formulated using a novel Hydration-of-Freeze-Dried-Matrix (HFDM) technique. Compared to the existing formulation strategies, this method of preparation is less labour-intensive and the end product is also freeze-dried, ensuring product stability. It was found that the liposomes prepared using the HFDM method were stable in the presence of serum and they also possessed high siRNA entrapment and gene-silencing efficiencies. Following intravenous administration to mice, these particles were also found to accumulate in subcutaneous tumours to a similar degree compared to formulations prepared using a previously established technique. Importantly, these HFDM-formulated preparations showed superior stability over ones prepared using the traditional formulation method, with the particles still retaining 100% of their gene-silencing ability after storage for one month at room temperature. Using HFDM-formulated liposomes loaded with siRNA against Green Fluorescence Protein (GFP), a 50% knockdown of the GFP expression was achieved in tumours following intravenous administration. Additionally, the use of E6/7-targeted siRNA also resulted in a 50% reduction in tumour size when the siRNAs were delivered using HFDM-formulated liposomes. Importantly, this level of tumour growth suppression was comparable to that achieved from cisplatin, a clinically used chemotherapeutic for cervical cancer, at the clinically used dose. Overall, this research demonstrated that while there are still some challenges to overcome for siRNA to be used vaginally for cervical cancer treatment, HFDM-formulated PEGylated liposomes showed promise in bringing E6/7 siRNA forward as a treatment option for end-stage cervical cancer. In addition, the simplicity of preparation procedure along with superior product stability obtained from the HFDM method developed in this thesis will likely facilitate the translation of siRNA technology from laboratory to clinics for a range of other medical applications.
| Identifer | oai:union.ndltd.org:ADTP/279378 |
| Creators | Sherry Wu |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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