DIOLEOYLMELITTIN AS A NOVEL SERUM-INSENSITIVE REAGENT FOR EFFICIENT TRANSFECTION OF MAMMALIAN-CELLS

Amphipathic peptides can be useful effecters to enhance gene delivery. However, peptide/DNA complexes usually require additional effecters, such as fusogenic lipids, to mediate efficient transfection. Due to we ak and/or multiple interactions between the various components of the system, the transfecting complexes are often heterogeneous and unstabl e in biological fluids. Accordingly, a hybrid molecule resulting from the covalent coupling of an amphipathic, membrane-disturbing peptide t o a lipid moiety might create a stable and efficient peptide-based gen e transfer system. The present work describes such a novel hybrid mole cule, dioleoylmelittin, resulting from the conjugation of idylethanola mine-N-[3-(2-pyridyldithio)propionate] with [Cys(1)]melittin. Dioleoyl melittin had a lower hemolytic and membrane-disturbing activity than m elittin. Size and zeta potential measurements, DNA gel electrophoresis , and electron microscopy showed that dioleoylmelittin, unlike melitti n, was able to complex plasmid DNA to form spherical particles with a net positive charge and a diameter between 50 and 250 nm. These partic les, prepared at an optimal 10/1 dioleoylmelittin/DNA ratio (w/w), med iated efficient transient transfection of reporter genes in cultured m ammalian cells including primary cells. The luciferase activity induce d by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lip id and the cell-line. Surprisingly, the presence of 10-50% fetal calf serum during dioleoylmelittin-mediated transfection enhanced 1.5-3-fol d gene expression. Dioleoylmelittin represents a new class of efficien t peptide-based transfection reagents, especially suited for serum-sen sitive cells.