Transfer. In vitro transfection of the pDNA/CyDs/AF-liposomes was performed utilizing the luciferase expression of pDNA (pRL-CMV-Luc or pGL3control vector) in HepG2, A549, and NIH3T3 cells. The cells (2 × 10 5 cells per 24 well plate) were seeded 6 h before transfection and then washed twice with 500 μL of serum-free medium. Two hundred μL of serum-free medium containing pDNA/CyDs/AF-liposomes in the absence and presence of AF as a competitor protein or BSA as a control protein were added to each dish and then incubated at 37 • C for 3 h. After washing HepG2 cells with serum-free medium twice, 500 μL of medium containing 10% FCS were added to each dish and then incubated at 37 • C for 21 h. After transfection, the gene expression was measured as follows: Renilla and firefly luciferase contents in the cell lysate were quantified by a luminometer (Lumat LB9506, EG&G Berthold Japan, Tokyo, Japan) using the Promega Renilla and firefly luciferase assay reagent (Tokyo, Japan), respectively. It was confirmed that CyDs and AF-liposomes had no influence on the luciferase assays under the present experimental conditions. Total protein content of the supernatant was determined by Bio-Rad protein assay kit (Bio-Rad Laboratories, Tokyo, Japan). EGFP-expressing cells were determined by a confocal laser scanning microscopy (CLSM, Olympus FV300-BXCarl Zeiss LSM-410, Tokyo, Japan) with an argon laser at 488 nm after fixation. Briefly, the cells (2 × 10 5 cells per 35 mm glass bottom dish) were seeded 6 h before transfection and then washed twice with 500 μL of serum-free medium. Transfection with pEGFP N1 DNA was performed using the same protocol as described above. The EGFP expression ratio was determined by the number of EGFP-expressing cells per 100 cells. To observe the cellular uptake of Rhodamine-labeled AF-liposomes (RH-AFliposomes) and Alexa-labeled pDNA (Alexa-pDNA), HepG2 cells (2 × 10 5 cells/dish) were incubated with the Alexa-pDNA/CyDs/RH-AF-liposomes for 3 h. After incubation, the cells were rinsed with PBS (pH 7.4) twice and fixed in methanol at 4 • C for 5 min prior to observation by a CLSM. 