2.3. Preparation of Liposomes

Liposomes were prepared by thin-film dispersion combined ammonium sulfate gradient method [30]. Conventional cholesterol liposomes (CHOL-LP) were prepared with a formulation of DSPC: cholesterol: DSPE-mPEG2000 at a molar ratio of 56:39:5, which referred to Doxil^® and Caelyx^® [31,32]. Diosgenin liposomes (Dios-LP) were prepared with DSPC: Dios: DSPE-mPEG2000 in a molar ratio of 56:39:5, which replaces the cholesterol with Dios. First, all lipid materials were dissolved in chloroform in a round-bottomed flask and dried using a rotary evaporation apparatus at 60 °C (water bath) to form a thin film. After the thin film was hydrated in ammonium sulfate solution (250 mmol/mL) at 60 °C for 1 h, the liposomal suspension was sonicated by an SCIENTZ-IID ultrasonic processor (Ningbo Scientz Biotechology Co., Ltd., Ningbo, China) in an ice bath to form the liposome. The concentration gradient of liposome was formed after dialysis. Because ammonium sulfate solution was used as the hydration medium, DOX-loaded liposomes could be prepared by adding the appropriate amount (≤2 mg/mL) of DOX into the CHOL-LP and Dios-LP separately and incubating at 60 °C (water bath) for 30 min, which used the concentration gradient of ammonium sulfate in liposomes as the driving force to drive DOX into the aqueous core of liposomes, obtaining CHOL-DOX-LP and Dios-DOX-LP. Finally, the liposome was stored at 4 °C.