Developing new applications for a metal chelator lipid : targeted delivery of nucleic acids and cytotoxic drugs

The ability to target the delivery of nucleic acids and cytotoxic drugs to specific cells has applications for the treatment of a number of diseases including cancer. Liposomes have previously been used to incorporate a range of different therapeutic agents, however the targeting/delivery of liposomes to specific cell types requires a means to conveniently anchor targeting molecules onto the surface of the liposomes. Recently, the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA{u2083}-DTDA) was incorporated into liposomes and membrane vesicles to enable the engraftment of histidine-tagged targeting molecules. Thus far, however, the in vivo use of targeted NTA{u2083}-DTDA-liposomes has been restricted to the delivery of antigenic proteins/peptides. Therefore, the primary focus of this thesis was to utilise liposomes containing NTA{u2083}-DTDA to target the delivery of nucleic acids and cytotoxic drugs to cells, and to investigate the potential application of these liposomes for cancer therapy. Preliminary studies during Honours indicated that liposomes containing the ionisable lipid 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) could incorporate small interfering RNA (siRNA) into lipoplexes together with NTA{u2083}-DTDA. Importantly, siRNA-lipoplexes engrafted with targeting molecules were shown to bind to cells through specific receptor-ligand interactions. Initial studies for the present work focussed on the further development and characterisation of these targeted siRNA-lipoplexes as well as the preparation of targeted lipoplexes containing plasmid DNA (pDNA). The targeted siRNA-lipoplexes and pDNA-lipoplexes developed in this work are shown to be of near-neutral surface charge, approximately 250 nm in diameter, serum-stable, non-toxic and able to efficiently transfect different cell lines when engrafted with different targeting peptides. For in vivo experiments, lipoplexes were prepared containing pDNA encoding for the immunodominant epitope (in C57BL/6 mice, H-2b) of the model antigen ovalbumin (OYA). When these lipoplexes were engrafted with the TLR5-binding peptide p9-Flg and injected into C57BL/6 mice, this treatment induced antigen-specific immunity that protected the mice from challenge with B16-0YA melanoma cells. Studies were also conducted to determine whether NTA{u2083}-DTDA could be incorporated into doxorubicin-containing liposomes. Experiments indicated that doxorubicin-containing liposomes incorporated with NTA{u2083}-DTDA and engrafted with the peptide p15-RGR could be targeted to NIH-3T3 cells in vitro with enhanced binding and cytotoxicity compared to non-targeted liposomes. The engraftment of liposomes with either of the tumour vasculature homing peptides: p46-RGD or p24-NRP-1, and injection into tumour-bearing mice resulted in a significant increase in the accumulation of the liposomes in the tumour. Importantly, treatment of mice with doxorubicin-containing liposomes engrafted with these same peptides significantly decreased the rate of tumour growth, thereby demonstrating therapeutic potential. In summary, the results presented in this thesis show that liposomes containing NTA{u2083}-DTDA can be used to deliver nucleic acids such as siRNA and pDNA as well as the cytotoxic drug doxorubicin to specific cells/tissues both in vitro and in vivo. The targeted delivery of pDNA to antigen-presenting cells and the targeting of doxorubicin to cells of the tumour vascular endothelium both resulted in significant anti-tumour effects. These findings,coupled with the convenience of the targeting method employed, suggest that the approaches developed could well find application in the quest for improving therapies against cancer