Mechanistic studies of sequential injection of cationic liposome and plasmid DNA.

We previously reported that sequential injection of cationic liposome and plasmid DNA leads to notably reduced inflammatory toxicity and improved transfection in the lung (Y. Tan et al., 2001, Mol. Ther. 3, 673-682). The purpose of the current study was to explore the mechanism involved in sequential injection. It was observed that sequential injection resulted in dramatically lower DNA uptake by the liver and higher DNA levels in the lung than the lipoplex injection. In vitro experiments with macrophage cells further showed that sequential addition of liposomes and DNA could diminish the cellular uptake of DNA by these cells. The contributions of serum to the enhanced bioactivity and decreased toxicity were examined by injecting mice with samples of premixed liposome with serum and then DNA (LSD sample), and the resulting activities were compared to those obtained with injection of lipoplex-serum mixtures (LDS sample). LSD yielded 80% lower TNF-alpha levels and over 10-fold higher transfection than lipoplex, which is consistent with the reported findings with sequential injection. In contrast, LDS resulted in the same TNF-alpha levels and comparable transfection with lipoplex. Thus, the results suggest that the primary interaction of serum with liposome is a critical factor contributing to the superior activity and reduced toxicity of sequential injection. Studies on the interaction between mouse serum, liposomes, and DNA showed that DNA could bind negatively charged liposome-serum complex to form a ternary complex, which has a density similar to that of the ternary complex formed between lipoplex with serum. Further in vitro tests showed that LSD and LDS were similar in particle size and protein content, but different in protein composition as observed by 2-D gel electrophoresis. In addition, DNA in LSD was more readily displaced by dextran sulfate, an anionic polymer, than in LDS. The above findings suggest that the inhibition of opsonin protein binding on the particle surface with the sequential injection may contribute to the reduced macrophage uptake and cytokine induction and that the high ability of DNA release from the particles formed after sequential injection may contribute to the improved lung gene transfection.