目的 优化氯化两面针碱脂质体（nitidine chloride liposomes，NC-Lips）的制备工艺，表征其理化性质并评价体外抗胆管癌活性。方法 采用薄膜分散法制备NC-Lips，以二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000（DSPE-mPEG₂₀₀₀）用量、药脂比和膜材比为关键影响因素，通过单因素实验结合Box-Behnken设计-效应面法（Box-Behnken design-response surface methodology，BBD-RSM）优化处方，以包封率、载药量、多分散指数（polydispersity index，PDI）和ζ电位为评价指标。利用HPLC法测定药物含量，粒度分析仪和透射电子显微镜表征脂质体理化性质，透析袋法考察体外释药特性，CCK-8法、流式细胞术和细胞周期分析评价其对胆管癌TFK-1细胞的增殖抑制、凋亡诱导及周期阻滞作用。结果 最优处方为DSPE-mPEG₂₀₀₀用量34.79 mg、药脂比1∶15.15、膜材比17.88。制得的NC-Lips呈现典型囊泡状结构，平均粒径为（77.67±0.75）nm，PDI为0.212±0.01，ζ电位为（-11.43±0.12）mV，包封率为（85.96±0.61）%，载药量为（4.37±0.03）%。体外释药中Ritger-Peppas模型拟合度最高，NC-Lips具有明显缓释特性，4 ℃储存30 d稳定性良好，渗漏率＜30%。体外细胞实验表明，NC-Lips对TFK-1细胞的增殖抑制率、凋亡诱导率及G₂/M期阻滞作用均显著优于游离NC（P＜0.01），12μmol/L浓度下作用48 h细胞存活率仅为（8.17±1.90）%，相同浓度下24 h细胞凋亡率达（28.90±2.38）%，G₂/M期占比升至（66.23±3.31）%。结论 成功制备了工艺稳定、理化性质优良的NC-Lips，能够通过诱导细胞凋亡和G₂/M期阻滞来发挥优异的体外抗胆管癌活性，为NC的剂型优化与后续机制及体内研究奠定实验基础。

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.

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广西自然科学基金资助项目（2022GXNSFDA035063）；广西自然科学基金资助项目（2023GXNSFAA026366）；国家自然科学基金面上项目（82473885）；广西医科大学一流学科创新驱动人才计划