Preparation of pDNA/CyD/AF-liposomes was performed according to the method reported by Hara et al. with some modifications [12] . Briefly, 2 μL of the solution containing pDNA (1 μg/μL) and CyDs (1 μM/μM lipids) dissolved in TE buffer were added to AF-or N-liposomes suspension. After mixing, the solution was freeze-dried. Then, the sample was rehydrated with THBS (10 mM, pH 7.5) for 30 min. After freezingthawing for three times, the vesicles were extruded through PVDF membranes (Nucleopore, Plesanton, CA) with pores of diameter 450 and 200 nm. The filtrates were used for further experiments as pDNA/CyDs/AF-liposomes or pDNA/CyDs/N-liposomes. The entrapment ratios of CyDs were evaluated by the anthrone-sulfuric acid method [26] . Briefly, 3 mL of anthrone reagent was added to 0.5 mL of the suspension containing CyDs/liposomes. The tube was covered with a glass ball and was heated for 10 min in boiling water. After quenching with cold water, absorbance of the suspension was measured by a U-2000A spectrophotometer (Hitachi, Tokyo, Japan) at 620 nm. The encapsulation ratios of pDNA were determined by a fluorescent spectrometer F-4500 (Hitachi, Tokyo, Japan). Briefly, 350 μL of the suspension containing pDNA/CyD/AF-liposomes in 10 mM THBS (pH 7.5) were mixed with 200 times diluted Picogreen dsDNA reagent (350 μL). After incubation for 30 min at 25 • C, fluorescent intensity (F po ) was determined. Next, after addition of 20% of Triton-X (20 μL) to the sample, fluorescent intensity (F pt ) was determined and the encapsulation ratio of pDNA was calculated as follows: encapsulation ratio (%) = [(F pt · r − F po )/F pt · r] · 100, where r is compensation coefficient (r = 1.03). Particle size and ζ-potential value of liposomes in 10 mM THBS (pH 7.5) were measured by a submicron particle analyzer N4 Plus (Beckman Coulter, Fullerton, CA) and ELS-8000 (Otsuka Electronics, Osaka, Japan), respectively.