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Stabilized antisense-lipid particles with enhanced intracellular delivery properties

University of British Columbia

Effective systemic delivery systems for DNA and RNA are essential to the successful clinical application of genetic drugs. Liposomal formulations of genetic drugs, such as antisense oligonucleotides (AS-ODN) and plasmid DNA, are difficult to achieve as the large size and highly charged nature of these molecules mitigates against the formation of small, neutral, serum stable carriers, which are required to achieve the long circulation times necessary for efficient delivery to disease sites such as tumors. Stabilized antisense-lipid particles (SALPs) are liposomal carriers of antisense oligonucleotides that exhibit high antisense-to-lipid ratios, long circulation lifetimes and accumulate preferentially at disease sites. As a result of high encapsulation efficiencies, SALPs are able to deliver large quantities of AS-ODN to target tissues. However, SALPs are relatively ineffective at delivering antisense molecules inside target cells. For AS-ODN to be effective in inhibiting gene expression, they must be internalized by the target cell, effectively released from endosomal compartments and translocated to the nucleus, their primary site of action. The studies conducted in this thesis examined methods to enhance the intracellular delivery of antisense oligonucleotides contained within SALP systems. In particular, we examined the effect of a surface associated cationic PEG-lipid (CPL4) on increasing cellular uptake of SALPs. Subsequent studies investigated the ability of dioleoylphosphatidylethanolamine (DOPE) in SALP systems to enhance nuclear delivery of AS-ODN sequences by promoting endosome destabilization. It is shown that the incorporation of CPL4 into distearylphosphatidylcholine/ cholesterol/ l,2-dioleoyl-3-[N,N-dimethylamino]propane/ PEG-CeramideC₁₄ (20/45/25/10 mol%) SALP was successful in increasing the cellular internalization of these vesicles but did not result in increased nuclear delivery of AS-ODN. Nuclear accumulation of oligonucleotides was achieved through the use of a novel formulation, DODAP/DOPE/PEGCer- Ci4, in which the fusogenic lipid DOPE was substituted for DSPC. However, these liposomes are unstable. Reasons for SALP instability are discussed as are ways to improve the stability and intracellular delivery properties of SALP.

Thesis/Dissertation

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2009-07-13

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http://hdl.handle.net/2429/10688

Biochemistry and Molecular Biology

University of British Columbia

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