Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Abstract

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 A, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Figure 1

Structure (from top to bottom) of the lipids lipopolyamine (RPR120535), dioctadecyldiammonium bromide (DODAB), and dioleoyl trimethylammonium propane (DOTAP).

Figure 2

RPR120535 and RPR120535/DNA complexes from zones A, B, and C (see Fig. 3), visualized by light microscopy and transmission electron microscopy (TEM). (A) Cryo-TEM micrographs of an aqueous solution of RPR120535 alone. (B) Electron micrograph of uranyl acetate-stained complexes from zone A (RPR120535/DNA charge ratio: 0.3). Inset shows the same complexes at higher magnification. (C) Complexes from zone B (charge ratio: 1.65) observed by light microscopy. (D) Electron micrograph of the same complexes stained with uranyl acetate. (E) Cryo-TEM micrograph of RPR120535/DNA complexes originating from zone C (charge ratio: 6). Inset shows the visualization by cryo-TEM of the ordered domains in these complexes. (F) Electron micrograph of uranyl acetate-stained complexes from zone C. (G) Cryo-phosphotungstate-TEM micrograph of the same complexes. In this micrograph, the complexes seem to have aggregated, because the thickest part of the vitrified film allows them to be superimposed. The scale bar represents 100 nm in A, B Inset, and D–G; 500 nm in B; and 10 μm in C.

Figure 3

(A) Colloidal stability of RPR120535/DNA complexes, at 0.1 mg DNA/ml, as a function of the RPR120535/DNA charge ratio (ratios were raised from 0 to 7 by increments of 0.3), in 4, 40, 80, or 150 mM NaCl. RPR120535/DNA complexes were defined as colloidally stable if their mean diameter was below 700 nm for at least 24 hr. Complexes from zones A and C were stable, whereas those from zone B exhibited a visible precipitate. (B) In vitro luciferase activity, measured in transfected HeLa cells, of RPR120535/DNA complexes originating from zones A, B, and C. Inset shows, in transfected NIH 3T3 cells, the in vitro transfection activity of DNA complexes with RPR120535 micelles or liposomes, i.e., RPR120535/DOPE (1/1, M/M), both at a charge ratio of 6, and of DNA complexes with DMRIE/Cholesterol, at a molar ratio of 1.65.

Figure 4

X-ray scattering studies. (A) Small-angle x-ray scattering scans of RPR120535/DNA complexes at various cationic lipid-to-DNA ratios. Symbols ○, □, and • correspond to RPR120535/DNA complexes from zones A (RPR120535/DNA charge ratio: 0.33), B (charge ratio: 1.65), and C (charge ratio: 6), respectively. (B) Because complex samples were prepared at 0.5 mg DNA/ml and concentrated 10-fold by ultracentrifugation, small-angle x-ray scattering scans were performed at 5 mg/ml on both DNA alone (○) and RPR120535 aqueous solution (□).

Figure 5

Circular dichroism spectra exhibited by RPR120535/DNA complexes (ɛ, molar ellipticity). Curves 1, 2, and 3 correspond to plasmid DNA alone, and to complexes from zones A (charge ratio: 0.6) and C (charge ratio: 6), respectively. RPR120535/DNA complexes from zone B exhibited a flat CD spectrum, because of the sedimentation of the very large complexes.

Figure 6

Schematic representation of the RPR120535/DNA complexes resulting from the association of cationic micelles and supercoiled DNA, as a function of the RPR120535/DNA ratio. A, B, and C represent negatively, neutrally, and positively charged complexes, respectively. The number of cationic lipid molecules per micelle is arbitrary.