Cationic lipid/pDNA complex formation as potential generic method to generate specific IRF pathway stimulators

Cationic liposome - CpG DNA complexes (lipoplexes) are known as stimulators of innate immunity via Toll-like receptor 9 (TLR9)-triggered activation of the nuclear factor kappa B (NF-κB) pathway. More recent reports suggest that DNA lipoplexes also engage DNA sensors in the cytosol leading to the stimulation of the interferon response factor (IRF) pathway. In this study a range of lipoplexes were formulated by using an invariable helper lipid, three different cationic lipids (DOTAP, DOTMA and DDA) and three different CpG-containing plasmids of different sizes. These lipoplexes exhibited similar hydrodynamic diameters, zeta-potentials and plasmid loading rates, despite the different lipid blends and CpG-containing plasmids. Binding and uptake of liposomal lipids by J774.A1 macrophages and JAWSII dendritic cells increased significantly (up to 4-fold) upon lipoplex formation. Cellular plasmid DNA uptake via lipoplexes compared to naked DNA was increased up to 18-fold. Analysis of signal transduction pathway activation in J774-DUAL™ reporter cells by liposomes or naked CpG plasmid DNA compared to their derived lipoplexes showed only minor activation of the NF-κB pathway, while the IRF pathway displayed massive activation factors of up to 46-fold. DOTAP- and DOTMA lipoplexes also led to massive interferon-alpha and -beta secretion of J774A.1 macrophages and JAWSII dendritic cells, which is a hallmark of IRF pathway activation. Cellular distribution studies on DOTAP lipoplexes suggest delivery of plasmid DNA via vesicular compartments into the cytosol. Taken together, the CpG plasmid DNA lipoplexes generated in this study appear to selectively stimulate DNA receptors activating the IRF pathway, while bypassing TLR9 and NF-κB activation.