Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

Abstract

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, approximately 40-45 degrees C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer "frustration" which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a "frustrated" bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex-membrane interactions and DNA release energetics.

Fig. 1

Plots of DNA/lipid stoichiometry (charge ratio) vs. cationic lipid content (arbitrary units) as reported by flow fluorometry, showing the time-course of DNA unbinding from EDOPC lipoplexes after addition of negatively charged liposomes: (A) DOPG; (B) DOPS; (C) membrane model mixture MM (MM=DOPC/DOPE/DOPS/Chol 45:20:20:15 w/w); (D) polar lipid extract from bovine liver (Avanti Polar Lipids). The stoichiometry (Y) axis represents the ratio of DNA to lipid charges in the particle. Lipoplexes contained cationic lipid labeled with 2.5% BODIPY-FL, and DNA labeled with the high affinity label, ethidium homodimer-2 (EthD-2) at 60 bp/dye. The panels show the distribution of stoichiometry vs. lipid content [15,16] after different times of incubation at room temperature, as indicated. Lipoplexes were initially prepared at near the isoelectric lipid/DNA ratio; negatively charged liposomes were added at a 1:1 weight ratio to the cationic lipid. In the usual case, particles that become more fluorescent are simply becoming larger. Since the fluorophore remains associated with the lipid, when two particles fuse or bind together, the resultant particle contains the fluorescence of both initial particles. Data in each panel were collected on 10,000 particles within less than 1 min.

Fig. 2

Plots of DNA/lipid stoichiometry (charge ratio) vs. cationic lipid content as reported by flow fluorometry, comparing the lipid/DNA stoichiometries of EDOPC lipoplexes 30 min after addition of various negatively charged liposomes, as indicated (see legend of Fig. 1 for details).

Fig. 3

Plots of DNA/lipid stoichiometry (charge ratio) vs. cationic lipid content as reported by flow fluorometry, comparing the DNA release from EDOPC lipoplexes at two different EDOPC/DNA charge ratios (upper panel: 1:0.75; lower panel: 1:1.5) initiated by addition of liposomes from liver polar lipid extract; negatively charged liposomes were added at a 1:1 weight ratio to the cationic lipid (see legend of Fig. 1 for details).

Fig. 4

Zeta potential of EDOPC lipoplexes as a function of the DNA/lipid charge ratio, as measured by a Malvern Zetasizer Nano ZS instrument.

Fig. 5

SAXD patterns of hydrated samples of polar lipid extracts from bovine brain (A), heart (B), and liver (C); brain extract : cholesterol 8:2 w/w (D), heart extract : cholesterol 8:2 w/w (E), liver extract : cholesterol 8:2 w/w (F), recorded during heating scans from 20°C to 90°C; the magenta pattern is for 37°C.

Fig. 6

SAXD patterns of hydrated samples of total lipid extracts from bovine brain (A), heart (B), and liver (C), recorded during heating and cooling scans between 20°C and 90°C; the magenta pattern was recorded at 37°C on heating.

Fig. 7

SAXD patterns of membrane mimicking (MM) lipid mixture DOPC/DOPE/DOPS/Chol 45:20:20:15 w/w, recorded upon heating from 25°C to 55°C; the bolded pattern indicates the first appearance of the inverted hexagonal H_II phase at 40°C.