STRUCTURAL AND FUNCTIONAL-ANALYSIS OF CATIONIC TRANSFECTION LIPIDS - THE HYDROPHOBIC DOMAIN

Cationic lipids (cytofectins) have gained widespread acceptance as pha rmaceutical polynucleotide delivery agents for both cultured cell and in vivo transfection, and the cytofectins DOTAP and DC-Cholesterol are being tested in clinical human gene therapy trials. This study report s the effects of modifications in the hydrophobic domain of a prototyp ic cytofectin (DORI), including modifications in lipid side-chain leng th, saturation, and symmetry. A panel of related compounds was prepare d and analyzed using DNA transfection, electron microscopy, and differ ential scanning calorimetry (DSC). Lipid formulations were prepared wi th dioleoylphosphatidylethanolamine (DOPE) as unsonicated preparations and sonicated preparations. Transfection analyses were performed usin g cultured fibroblasts, human bronchial epithelial, and Chinese hamste r ovarian cells as well as a mouse model for pulmonary gene delivery. In general, cytofectins containing dissymmetric hydrophobic domains we re found to work as well or better than the best symmetric analogs. Op timal side-chain length and symmetry varied with cell type. Compounds with phase transitions (T-c) above and below physiological temperature (37 degrees C) were tested for DNA transfection activity. In contrast to previous reports, cytofectin T-c was not found to be predictive of transfection efficacy. Pulmonary treatment with free DNA was found to be at least as effective as treatment with commonly used cytofectin:D NA complexes. However, cytofectins that incorporate a hydroxyethylammo nium moiety in the polar domain were found to enhance in vivo gene del ivery relative to free DNA.