BIOCHEMICAL, MORPHOLOGICAL, AND FUNCTIONAL ANALYSES OF A CYCLIC PEPTIDE, PHOSPHOLIPID, AND DNA TERNARY COMPLEX USED FOR GENE DELIVERY

Gene delivery and expression in mammalian cells can be facilitated by a complex composed of the cyclic amphipathic peptides, gramicidin S or tyrocidine, dioleoylphosphatidylethanolamine (DOPE) and DNA. We have characterized physical features of complexes formed from various pepti de/DOPE/DNA ratios using dynamic light scattering, sedimentation, elec trophoretic mobility and DNA accessibility to ethidium intercalation a nd correlated these with transfection. The addition of DOPE dispersion s to the peptide-DNA pair produces negatively-charged particles with z eta 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 di ameter of the complex depends upon the ratio of the components. The cy clic peptides, gramicidin S and tyrocidine, mediated similar transfect ion levels at a 1:1 peptide/DNA charge ratio, however, tyrocidine coul d displace ethidium bromide from plasmid DNA at a lower peptide:DNA ch arge ratio than gramicidin S suggesting subtle differences in the mann er these cyclic peptides interact with DNA. Fractionation of the compl exes by sedimentation on sucrose gradients separated a major fraction with a density of about 1.10 g/cm(3). This fraction was enriched in pe ptide 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 pept ide/DNA complexes revealed the cyclic peptides formed a compacted cyli ndrical tube-like structure when bound to DNA as compared to the toroi dal structure obtained with polylysine. Upon addition of DOPE dispersi ons 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 trans fection levels. Thus in contrast to polylysine-based systems, the cycl ic peptide-DOPE-DNA complex has a negative charge and the DNA is not c ompletely compacted yet is capable of mediating high levels of transfe ction in cultured cells.