Biophysical properties of CDAN/DOPE-analogue lipoplexes account for enhanced gene delivery

Typically, cationic liposomes are formulated from the combination of a synthetic cationic lipid (cytofectin) and a neutral, biologically available co-lipid. However, the use of cationic liposome formulations to mediate gene delivery to cells is hampered by a paradox. Cationic lipids, such as N(1)-cholesteryloxycarbonyl-3-7-diazanonane-1,9-diamine (CDAN), are needed to ensure the formation of cationic liposome-DNA (lipoplex, LD) particles by plasmid DNA (pDNA) condensation, as well as for efficient cell binding of LD particles and intracellular trafficking of pDNA post-intracellular delivery by endocytosis. However, the same cationic lipids can exhibit toxicity, and also promote LD particle colloidal instability, leading to aggregation. This results from electrostatic interactions with anionic agents in biological fluids, particularly in vivo. One of the most commonly used neutral, bioavailable co-lipids, dioleoyl L-alpha-phosphatidylethanolamine (DOPE), has been incorporated into many cationic liposome formulations owing to its fusogenic characteristics that are associated with a preference for the inverted hexagonal (H(II)) phase-a phase typical of membrane-membrane fusion events. However, these same fusogenic characteristics also destabilize LD particles substantially with respect to aggregation, in vitro and especially in vivo. Therefore, there is a real need to engineer more stable cationic liposome systems with lower cellular toxicity. We hypothesize that one way to achieve this goal should be to find the means to reduce the mol fraction of cationic lipid in cationic liposomes without impairing the overall transfection efficiency, by replacing DOPE with an alternative co-lipid with fusogenic properties "tuned" with a greater preference for the more stable lamellar phases than DOPE is able to achieve. Herein, we document the syntheses of triple bond variants of DOPE, and their formulation into a range of low charge, low cationic lipid containing LD systems. The first indications are that our hypothesis is correct in vitro.