ULTRASTRUCTURAL CHARACTERIZATION OF CATIONIC LIPOSOME-DNA COMPLEXES SHOWING ENHANCED STABILITY IN SERUM AND HIGH TRANSFECTION ACTIVITY IN-VIVO

We have investigated the morphology and transfection activity of catio nic liposome-DNA complexes (CLDC) under conditions relevant to both in vivo and in vitro studies. Moreover we have attempted to establish st ructure-function relationships relevant for high transfection activiti es under both conditions. CLDC were composed of dimethyldioctadecylamm onium bromide with either 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DO PE) or cholesterol (Chol) interacting either with pre-condensed DNA or with uncondensed plasmid DNA. Furthermore for steric stabilization 1% poly(ethylene glycol)-phospholipid conjugate was added to CLDC contai ning Chol and plasmid DNA. The in vivo studies were carried out in mic e following i.v. injection, and the in vitro studies were performed on SK-BR-3 human breast cancer cells in the presence of media with serum . The morphology of the CLDC, monitored by freeze-fracture electron mi croscopy, was investigated after mixing with mouse serum or the medium where the cells were kept. The substitution of DOPE with Chol, and th e addition of ga-methoxypoly(oxyethylene)-alpha-oxycarbonyl-DSPE are p roducing CLDC which are stabilized with respect to time and serum, and are relatively small (100-300 nm). These stabilized complexes show hi gh expression of a marker gene in mouse lungs reaching expression valu es up to 10 ng luciferase per mg tissue protein, but relatively low ex pression in SK-BR-3 cells in vitro. Additionally, some of the complexe s containing pre-condensed DNA look like 'map-pin' structures showing heads of the size of liposomes and short, stiff and tapering tails. Th e in vivo transfection activity of these preparations is highest. Simi lar complexes containing DOPE rather than Chol as helper lipid precipi tate in the presence of serum:and especially of cell medium and conver t into hexagonal lipid (H-II) phase. Such complexes, despite their hig h transfection activity in vitro, show very little transfection activi ty in vivo. These comparisons may help us to understand the fundamenta l difference between in vitro and in vivo activity of CLDC: high in vi tro transfection activity seems to be associated with hexagonal lipid precipitates whereas high in vivo activity seems to be related with sm all, stabilized complexes, which in our case also exhibit some protrus ions (map-pin structures). (C) 1998 Elsevier Science B.V. All rights r eserved.