Calycosin-loaded nanoliposomes were prepared through thin film evaporation-sonication method as described previously [35, 36]: SPC, cholesterol, DSPE-mPEG2000, and calycosin were dissolved with 12 mL methanol. The molar ratio of SPC to cholesterol and the mass ratio of the calycosin to the total lipid (SPC and cholesterol) (w/w) at the condition of 5% content of DSPE-mPEG2000 were 50∶38, 1:10, respectively.

The mixture was then dried (vacuum and 55 °C) to form a thin film in the rotary evaporation apparatus. Afterward, the samples were hydrated with 20 mL double distilled water, sonicated (70 w) for 15 min in an ice bath, extruded by filtering (0.45 μm and 0.22 μm), and finally freeze-dried at − 20 °C.

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