Lipofectin

Lipofectin Transfection Reagent (Thermofisher Scientific) with a 1:1 of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) was used to develop liposomes by the thin film hydration method. Briefly, a stock solution of Lipofectin Transfection Reagent was resuspended in 99.5% ethanol at a concentration 10 mg/ml. Ethanol was evaporated such that a lipid film developed after 1 h. The lipid film was hydrated by adding RNase-free water and gentle shakes. The final concentration of the lipid was adjusted to approximately 6 mM and incubated at 4 °C overnight. Liposomes were filtered through polycarbonate membranes with 200 nm pores. Complexes of RNA-lipoplexes with a charge ratio of 1.3:2 (lipid:RNA) were previously shown to form stable particles targeting RNA to dendritic cells13. To calculate charge ratio of the RNA-LPX complexes, each lipid head group was counted as one positive charge and each RNA phosphodiester was counted as one negative charge. RNA was resuspended in HEPES-buffered solution at the final concentration of 1 mg/ml. Lipoplexes and RNA were diluted in 1.5 M NaCl with a charge ratio of 1.3:2 and the NaCl concentration was adjusted to 150 mM by adding RNase-free water. RNA transcripts of pIVT-empty were used as RNA controls.