Liposomes were prepared according to the standard thin film hydration method.
To a dry, thin membrane of EYPC and TRX (0 or 30 mol%) (total lipids; 1.25 × 10 -5 mol) and MGlu-HPG (lipids/polymer = 7/3, w/w) was added 500 µL of OVA (4 mg/mL) phosphate buffered saline (PBS) (pH 7.4) and the mixture was sonicated for 2 min using a bath-type sonicator. The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was centrifuged with the speed of 55,000 rpm for 2 h at 4 °C twice to remove free OVA and CpG-DNA. For CpG-DNA inclusion to liposomes, two complexation methods were examined. In the case of Pre-mix, mixed thin membrane was dispersed by mixture of OVA/CpG-DNA (2.5, 5, 7.5 g/mol lipid) in PBS. Lipid concentration and OVA encapsulation were determined by Test-Wako C (Wako Pure Chemical Industries, Ltd) and Coomassie (Bradford) Protein assay reagent (Thermo-Scientific). CpG-DNA amounts in liposomes were determined by Quant-iT Oligreen ssDNA assay as following procedure. Lipid dispersion was mixed with Triton-X 100 (0.2% vol) and Oligreen ssDNA assay reagent in fluorescence microtiter plate. Microplate was excited at 480 nm and fluorescence emission intensity was detected at 520 nm using Microplate Reader (SH-8000 CORONA ELECTRIC Co., Ltd.).
To a dry, thin membrane of EYPC and TRX (0 or 30 mol%) (total lipids; 1.25 × 10 -5 mol) and MGlu-HPG (lipids/polymer = 7/3, w/w) was added 500 µL of OVA (4 mg/mL) phosphate buffered saline (PBS) (pH 7.4) and the mixture was sonicated for 2 min using a bath-type sonicator. The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was centrifuged with the speed of 55,000 rpm for 2 h at 4 °C twice to remove free OVA and CpG-DNA. For CpG-DNA inclusion to liposomes, two complexation methods were examined. In the case of Pre-mix, mixed thin membrane was dispersed by mixture of OVA/CpG-DNA (2.5, 5, 7.5 g/mol lipid) in PBS. Lipid concentration and OVA encapsulation were determined by Test-Wako C (Wako Pure Chemical Industries, Ltd) and Coomassie (Bradford) Protein assay reagent (Thermo-Scientific). CpG-DNA amounts in liposomes were determined by Quant-iT Oligreen ssDNA assay as following procedure. Lipid dispersion was mixed with Triton-X 100 (0.2% vol) and Oligreen ssDNA assay reagent in fluorescence microtiter plate. Microplate was excited at 480 nm and fluorescence emission intensity was detected at 520 nm using Microplate Reader (SH-8000 CORONA ELECTRIC Co., Ltd.).