576. Hydrophobic Portion of Cationic Amphiphiles Are More Important Than We Think for Transfection Efficiency

Abstract

We have recently demonstrated that a combination of two cationic lipid derivatives having the same head group but tails of different chain lengths behave considerably differently than do the separate molecules. For example, the combination of the dilauroyl (12-carbon chain) and the dioleoyl (18-carbon chain) homologues of O-ethylphosphatidylcholine transfected DNA into primary human umbilical artery endothelial cells (HUAEC) more than 30-fold more efficiently than either compound separately. These findings indicate that the hydrophobic portion of cationic amphiphiles used in vitro and in vivo to delivery nucleic acids to cells, is much more important than has been appreciated heretofore and should receive attention comparable to that given to the charged portions of these kinds of molecules. Hence, we have synthesized a variety of cationic phosphatidylcholines with unusual hydrophobic moieties and have evaluated their transfection activity and that of their mixtures with the original molecule of this class, dioleoyl-O-ethylphosphatidylcholine (EDOPC). Four relations between transfection activity and composition of the mixture (plotted as percent of the new compound in EDOPC) were found. The first is a bell-shape curve, in which transfection increases up to a certain value and then decreases again. The second is saddle-shaped, in which transfection exhibits a minimum at intermediate compositions. The third one is |[ldquo]|linear|[rdquo]|, such that transfection increases monotonically with the second cationic lipid. The fourth one is |[ldquo]|horizontal|[rdquo]|, signifying the absence of a mixture effect. In addition to transfection, some relevant physical properties of the lipoplexes were examined; specifically, membrane fusion (by fluorescence resonance energy transfer) and DNA unbinding (measured as accessibility of DNA to EtBr, by electrophoresis) both following the addition of anionic liposomes. Fusion with anionic liposomes increased with increasing proportion of the addition of second cationic lipid, regardless of its effect on transfection. However, DNA unbingding assay revealed a correlation between extent of transfection by a formulations and the extent of DNA accessibility to EtBr induced by addition of anionic lipid. It suggests that, while fusion between cationic lipid membranes and anionic cellular membranes is important in gene transfer mediated by cationic lipids, the actual unbinding of DNA from cationic lipids following such fusion is likely to be critical. From the standpoint of the design of new cationic lipids for transfection, these findings also highlight the fact that there is considerable scope for fine-tuning such systems to be more effective in DNA delivery. Judicious manipulation of critical molecular design parameters such as headgroup size and lipophilic segment dimensions to generate the proper interaction between cationic lipids and DNA and between cationic lipids and cellular anionic lipids should be helpful in preparing optimal transfection recipes.