Self-assembly of cationic lipoplexes from liposomes and plasmids of variable size.

摘要

Lipoplexes, often described as a condensed phase spontaneously formed between DNA and cationic lipids, have attracted much attention in recent years because of their potential use as nonviral vectors for gene therapy. Several studies have demonstrated that lipoplexes tend to exist as two-phase systems over most lipid:DNA mixing ratios. Employing sonicated liposomes and DNA of different sources, such studies show the coexistence of lipoplexes with excess liposomes (DNA) for mixing ratios above (below) charge neutrality, while lipoplexes involving all of the lipid and DNA in solution are found only in close proximity of the charge neutrality ratio. Because these studies have focused on a single liposome type, they leave open the question as to whether lipoplex self-assembly follows a similar pattern for all 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-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxyethyl)imidazolinium (DOTIM):DOPE (1:1) liposomes and plasmids of variable size. The liposomes used include small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), and sedimented multilamellar vesicles (sMLV). Their sizes vary from 25 nm up to >1 μm in diameter in the order SUV LUV sMLV. The plasmids used are 2.5, 7.2, and 20 kb in size.; Using sedimentation in sucrose density gradients, the present study shows that sMLV and LUV, 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 sMLV and LUV become involved in the assembly process up to (+/−) mixing ratios of ∼4 and 9, respectively, while only a small and constant fraction of SUV associates with the plasmid at all mixing ratios tested. Consequently, while maximum lipid:DNA ratios of ∼4.5 and 9 are found in LUV and sMLV lipoplexes, respectively, a maximum final ratio of only ∼2 is determined in the SUV lipoplexes. Isothermal titration calorimetry studies show that this ratio is the lowest possible charge ratio achieved when both SUV and sMLV are titrated with the plasmids.; Based on these observations, a simple model of lipoplex formation is proposed. Focusing primarily on the differences in size among the various types of liposomes used, the model is able to partially explain the results obtained.