The effect of liposome size on the final lipid/DNA ratio of cationic lipoplexes

Abstract

Several studies have demonstrated that lipoplexes are two-phase systems over most mixing lipid/DNA charge ratios. Because these studies have focused on small unilamellar vesicles (SUV), they leave open the question as to whether a similar pattern is followed by other liposome types. The main purpose of this work is to examine the question further by characterizing the assembly of cationic lipoplexes prepared from 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM)/dioleoylphosphatidylethanolamine (DOPE) (1:1) liposomes of various types. Sedimentation in sucrose density gradients reveals that large unilamellar vesicles (LUV) and sedimented multilamellar vesicles (sMLV), as opposed to SUV, form lipoplexes that exist as a single phase over a relatively broad range of mixing (+/-) ratios. This is indicated by observing that most of the LUV and sMLV become involved in the assembly reaction up to mixing (+/-) ratios of 4 and 9, respectively, while only a small and constant fraction of SUV associates with DNA at all mixing (+/-) ratios tested. Consequently, while maximal (+/-) ratios of approximately 4.5 and 9 are found in LUV and sMLV lipoplexes, respectively, a final (+/-) ratio of only approximately 2 is determined in SUV lipoplexes. Isothermal titration calorimetry shows that this is the lowest possible charge ratio achieved when liposomes are titrated with DNA. Based on these observations and on the size differences of the liposomes used, a model of lipoplex formation is proposed.

FIGURE 1

Particle size of liposomes. (a and b) Transmission electron micrographs of DOTIM/DOPE (1:1) SUV (a) and sMLV (b) obtained after negative staining with uranyl acetate. The sMLV samples were fixed with osmium tetroxide before staining with uranyl acetate. (c) Volume-weighted Nicomp (multimodal) particle size distributions from dynamic light scattering of DOTIM/DOPE (1:1) SUV (solid line) and sMLV (dashed line). (d) Volume-weighted Gaussian particle size distributions of LUV extruded through polycarbonate membranes with pore diameters of 50 (solid line), 100 (dashed line), or 200 nm (dotted line).

FIGURE 2

Sedimentation of DOTIM/DOPE (1:1) lipoplexes in discontinuous sucrose density gradients. Cationic lipoplexes were prepared from either SUV or sMLV at a (+/−) ratio of 3.6 and sedimented at 116,000 × g for 16 h, at 10°C, in discontinuous sucrose density gradients.

FIGURE 3

DOTIM and DNA sedimentation profiles of lipoplexes prepared from a 7.2-kb plasmid and DOTIM/DOPE (1:1) SUV or sMLV. The results shown are the amounts of DOTIM and DNA detected in each fraction collected from the gradient. The cumulative volume indicates the position of each fraction in the gradient, and the mixing (+/−) ratio used in lipoplex formation is indicated in the legends. The SUV and sMLV curves correspond to the sedimentation of DNA-free liposomes.

FIGURE 4

Final (+/−) ratios in lipoplexes prepared from a 7.2-kb plasmid and DOTIM/DOPE (1:1) SUV or sMLV. Cationic lipoplexes were prepared from each liposome type at various (+/−) ratios and sedimented in discontinuous sucrose density gradients. The final (+/−) ratios were calculated from the DNA and DOTIM content of fractions 3 and 4 for SUV lipoplexes, and fractions 2, 3, and 4 for sMLV lipoplexes. There are 4–6 replicates in each point.

FIGURE 5

DOTIM and DNA sedimentation profiles of lipoplexes prepared from a 7.2-kb plasmid and DOTIM/DOPE (1:1) LUV extruded through membranes with pore diameters of 50 (LUV 50), 100 (LUV 100), and 200 nm (LUV 200). The numbers in the legends indicate the (+/−) ratios used in lipoplex formation. The LUV curves (LUV 50, 100, and 200) correspond to the sedimentation of DNA-free LUV.

FIGURE 6

Final (+/−) ratios in lipoplexes prepared from a 7.2-kb plasmid and DOTIM/DOPE (1:1) LUV obtained by extrusion through polycarbonate membranes with pore diameters of 50 (LUV 50), 100 (LUV 100), and 200 nm (LUV 200). All fractions, except fraction one, were considered in the calculation of the final lipid/DNA ratios. There are 4–6 replicates in each point.

FIGURE 7

Mean particle size of SUV lipoplexes sedimented in fraction 4 and sMLV lipoplexes sedimented in fraction 3 as a function of the mixing lipid/DNA ratios. The values shown are the volume-weighted mean diameters of the Gaussian distributions.

FIGURE 8

(a) Isothermal titration calorimetric traces and (b) cumulative heats of reaction obtained after titration of DOTIM/DOPE (1:1) SUV or sMLV (0.4 mM in DOTIM) with 10-μl aliquots of a 7.2-kb plasmid (3 mM), at 25°C. The heat of dilution was obtained by titrating DNA into pure buffer (10 mM Hepes, 10 mM NaCl, pH 7.4). A quantity of 30 nmol DNA was added in each injection, and the calorimeter cell contained a total of 535 nmol DOTIM.

FIGURE 9

Schematic model of lipoplex formation when a 7.2-kb plasmid is mixed with (a) SUV, (b) sMLV, and (c) LUV. The top panels represent the liposome preparations (spheres) before adding the plasmid (wavy solid line), and the bottom panels represent the lipoplexes formed at an excess of cationic lipid. In each case, the mixing (+/−) ratios increase from left to right.