Biochemical, Morphological, and Functional Analyses of a Cyclic Peptide, Phospholipid, and DNA Ternary Complex used for Gene Delivery

Abstract

Gene delivery and expression in mammalian cells can be facilitated by a complex composed of the cyclic amphipathic peptides, gramicidin S or tyro-cidine, dioleoylphosphatidylethanolamine (Dope) and DNA. We have characterized physical features of complexes formed from various peptide/Dope/ DNA ratios using dynamic light scattering, sedimentation, electrophoretic mobility and DNA accessibility to ethidium intercalation and correlated these with transfection. The addition of Dope dispersions to the peptide-DNA pair produces negatively-charged particles with zeta potentials between -27.5 mV and -55 mV at pH 8.5 and diameters as measured by dynamic light scattering, between 100 nm to 400 nm. The diameter of the complex depends upon the ratio of the components. The cyclic peptides, gramicidin S and tyrocidine, mediated similar transfection levels at a 1:1 peptide/ DNA charge ratio, however, tyrocidine could displace ethidium bromide from plasmid DNA at a lower peptide: DNA charge ratio than gramicidin S suggesting subtle differences in the manner these cyclic peptides interact with DNA. Fractionation of the complexes by sedimentation on sucrose gradients separated a major fraction with a density of about 1.10 g/cm3. This fraction was enriched in peptide and Dope and had a more negative zeta potential than the initial complex. It also had the highest transfection activity and the lowest toxicity. Electron microscopic examination of negatively stained peptide—DNA complexes revealed the cyclic peptides formed a compacted cylindrical tube-like structure when bound to DNA as compared to the toroidal structure obtained with polylysine. Upon addition of Dope dispersions to the peptide—DNA pair, the compacted tubes were replaced in the negative stain by spherical structures with diameters in the 200 nm to 400 nm range. The spherical structures are correlated with high transfection levels. Thus in contrast to polylysine-based systems, the cyclic peptide-Dope-DNA complex has a negative charge and the DNA is not completely compacted yet is capable of mediating high levels of transfection in cultured cells.