Objective To optimize the preparation process of nitidine chloride liposomes (NC-Lips), characterize their physicochemical properties, and evaluate their in vitro anti-cholangiocarcinoma activity. Methods NC-Lips were prepared by the thin-film dispersion method. Three key formulation variables, namely DSPE-mPEG₂₀₀₀ dosage, drug-to-lipid ratio, and phospholipid-to-cholesterol ratio, were selected. The formulation was optimized via single-factor experiments combined with Box-Behnken design-response surface methodology (BBD-RSM), with encapsulation efficiency, drug loading, PDI, and ζ potential as the evaluation indices. The drug content was determined by HPLC. The physicochemical properties of NC-Lips were characterized using a particle size analyzer and transmission electron microscopy. The in vitro drug release profile was investigated by the dialysis bag method. CCK-8 assay, flow cytometry, and cell cycle analysis were employed to evaluate the proliferation inhibition, apoptosis induction, and cell cycle arrest effects of NC-Lips on cholangiocarcinoma TFK-1 cells. Results The optimal formulation was as follows: DSPE-mPEG₂₀₀₀ dosage of 34.79 mg, drug-to-lipid ratio of 1:15.15, and phospholipid-to-cholesterol ratio of 17.88. The prepared NC-Lips exhibited typical liposomal vesicular morphology, with an average particle size of (77.67 ± 0.75) nm, PDI of 0.212 ± 0.01, ζ potential of (&8722;11.43 ± 0.12) mV, encapsulation efficiency of (85.96 ± 0.61)%, and drug loading of (4.37 ± 0.03)%. Among the in vitro drug release models, the Ritger-Peppas model showed the highest goodness of fit, indicating that NC-Lips had obvious sustained-release characteristics. The liposomes remained stable during 30 d storage at 4 ℃, with a drug leakage percentage of < 30%. In vitro cell experiments demonstrated that NC-Lips exerted significantly better effects on proliferation inhibition, apoptosis induction, and G₂/M phase arrest in TFK-1 cells than free nitidine chloride (P < 0.01). At a concentration of 12 μmol/L, the cell viability was only (8.17 ± 1.90)% after 48 h of treatment; at the same concentration, the apoptosis rate reached (28.90 ± 2.38)% at 24 h, and the proportion of cells in the G₂/M phase increased to (66.23 ± 3.31)%. Conclusion A stable and optimized NC-Lips formulation with excellent physicochemical properties was successfully prepared. NC-Lips exerted excellent in vitro anti-cholangiocarcinoma activity by inducing apoptosis and G₂/M phase arrest. These findings provide an experimental basis for formulation optimization and subsequent mechanistic and in vivo studies of nitidine chloride.

nitidine chloride  /  Box-Behnken design-response surface method  /  liposomes  /  cholangiocarcinoma  /  apoptosis