Nanotechnology in developing multifunctional non-viral gene delivery systems

Small interfering RNA (siRNA) has emerged as a powerful technology for sequence specific suppression of genes in mammalian cells and has broad applications ranging from functional gene analysis to targeted therapy. However, its clinical success is still limited because of the inherent instability and poor intracellular internalization of siRNA. Hence, the aim of the thesis study is to develop a safe non-viral siRNA delivery vehicle for effective cancer therapy. To this end, we took advantage of various nanomaterials and nanotechnologies, combined with the traditional biological techniques to reach our goal. Chapter 1 gives a background of this research and Chapter 2 summarizes the experimental procedures and techniques. Chapter 3 evaluates different generations of PPI dendrimers as potential siRNA delivery agents for cancer therapy. We demonstrated that the higher generations of PPI dendrimers were able to effectively complex siRNA into discrete nanoparticles, which resulted in a sufficient knockdown of targeted gene expression in human cancer cells.;The mechanism of siRNA complexation by PPI dendrimers was also investigated in Chapter 4. Results suggest that PPI dendrimers first 'zip' the siRNA molecules by electrostatic interactions to form extended nanofibers, followed by secondary folding into nanoparticles. Understanding this complexation pathway is crucial in the rational design of more effective vectors for therapeutic siRNAs.;Besides siRNA complexation, attempts to develop effective non-viral siRNA vectors for in vivo delivery are hampered by difficulties that include extracellular stability, siRNA intracellular availability and target cell specificity. Chapter 5 focuses on the development of a non-viral gene delivery vector to address the above mentioned problems. We demonstrated that the human cancer cells specifically internalize the designed siRNA vector and the delivered siRNAs effectively silence the targeted mRNA.;Furthermore, new methods for timely monitoring of siRNA therapeutic responses are highly desirable for the optimization of delivery strategies. Although several approaches have been reported for siRNA delivery based on engineered inorganic nanoparticles to monitor the therapeutic effects, they have various shortcomings including not efficient siRNA complexation, endosomal escape, and dissociation from the nanoparticles. We have also proposed a new design for surface modification of nanoparticles to fit the multiple requirements of siRNA delivery. SPIO nanoparticles were used as an example to demonstrate the new modification concept, which may be extended to other engineered nanoparticles for developing multifunctional gene delivery systems. The results suggest that high effective silencing can be achieved with minimum amount of nanoparticles (Chapters 6 and 7). The outcomes of these experiments open up perspectives for the development of targeted nanomedicine platform for multifunctional siRNA delivery vector, capable of monitoring the therapeutic responses of the RNA, in situ.