A NOVEL TETRAESTER CONSTRUCT THAT REDUCES CATIONIC LIPID-ASSOCIATED CYTOTOXICITY - IMPLICATIONS FOR THE ONSET OF CYTOTOXICITY

The preparation of cationic amphiphiles that induce minor cytotoxic re sponse during polynucleotide delivery into mammalian cells has been li mited by the conventional use of ester, amide, or carbamate linkages t o tether either the polar or the hydrophobic domains. The deleterious effects of ammonium-based lipidic salts on cellular processes have bee n well-established. The present report is the first example of a linch pin tetraester construct that utilizes ester linkages to tether both t he polar and hydrophobic domains. Dimyristoyl and dioleoyl analogues w ere prepared from pentaerythritol, N,N-dimethylglycine, and their corr esponding fatty acyl groups via successive diesterifications followed by amine quaternization. The resultant cationic tetraesters were exami ned in transfection (luciferase) and cell proliferation (MTS) assays u sing NIH 3T3 and 16HBE14(0)-cells. The tetraesters exhibited transfect ion activity comparable to the well-studied lipids DOTAP and DC-choles terol (DC-chol) in both cell lines. The tetraester construct afforded no cytotoxicity in NIH3T3 cells and provided a significant lowering of cytotoxicity relative to DC-chol in the 16HBE14(0)-cells. The express ion of green fluorescent protein (GFP) in both cell lines also was exa mined using the lipid panel. Comparison of fluorescent and correspondi ng phase-contrast images confirmed the chemical cytotoxicity results a nd revealed that the cytotoxic response was not dependent on transgene expression. Phase-contrast micrographs of cells treated with the cati onic lipid panel in the absence of GFP plasmid showed identical morpho logy to the GFP-transfected cells, suggesting that the onset of a lipi d-mediated cytotoxic response might occur at a stage prior to endosoma l encapsulation.