Single lipoplex study of cationic Lipoid-DNA, self-assembled complexes

A flow fluorometric analysis (using a commercial flow cytometer) of individual cationic lipoid-DNA complexes is presented. Such single lipoplex studies have the advantage of providing detailed characterization of heterogeneous ensembles of lipoplex preparations that cannot be obtained with methods that provide only population averages. Specifically, the composition (amount of lipoid and the lipoid-DNA ratio) was determined for statistically large ensembles (10(3)-10(4) particles) under a variety of conditions, including DNA:lipoid mixing ratio, lipoid dispersion method (extruded, vortexed), DNA morphology (linear, supercoiled), and concentration. In addition, the kinetics of formation were assessed for several conditions. Under essentially all conditions, two distinct regimes were observed, and on the basis of present and past data, these were identified as (1) coexistence of multilamellar lipoplexes and DNA-coated. vesicles and (2) highly fused multilamellar complexes. The former outcome is favored by excess of DNA, reduced vesicle size, linear DNA, high Concentration, and short incubation times. Fused multilamellar complexes represent, the structures of lipoplexes usually used for DNA transfection; these were formed by interaction and breakdown of DNA-coated vesicles. Because the composition of individual lipoplexes could be determined, it was possible to assess how much of the bulk sample heterogeneity originates within individual vesicles and how much is due to differences between lipoplexes.