AF-liposomes encapsulating calcein were prepared by the freezing and thawing method after addition of 0.1 mM calcein in 10 mM THBS (pH 7.5). The vesicles were extruded through two stacked polycarbonate membranes (Nucleopore, Plesanton, CA) with pores of diameter 1 μm. The sample was subjected to 10 passes through the filter at 40 • C. The filtrates were extruded through the polycarbonate membranes (pore size 0.2 μm) as described above. Two milliliters of CyDs solution adjusted at the appropriate concentration (5-20 mM) using 10 mM phosphate buffer were added to 20 μL of the liposomal suspension, and then the resulting suspension was incubated for 30 min at 25 • C. The fluorescence intensity of calcein (F t ) was measured with a fluorophotometer (Hitachi F-4500, Tokyo, Japan) at 25 • C; excitation and emission wavelengths were 490 and 520 nm, respectively. After addition of 20 μL of cobalt chloride solution (10 mM) to the sample to quench the fluorescence of nonencapsulated calcein, the intensity of fluorescence of encapsulated calcein (F in ) was also determined. Then, the liposomes were completely disrupted by the addition of 20 μL of Triton X-100 (20%) solution, and the intensities of fluorescence after quenching by cobalt chloride (F q ) were measured. Calcein encapsulation ratio was calculated by the equation as follows: encapsulation ratio