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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., authors=HUANG Yanqiu1,2 , QIU Weian1,3 , CHEN Haiyan1 , PENG Shiya1 , SU Mingzhe1 , LIU Yue2 , YE Yong1,4 , authorsList=HUANG Yanqiu, QIU Weian, CHEN Haiyan, PENG Shiya, SU Mingzhe, LIU Yue, YE Yong, authorCompany=1 School of Pharmacy, Guangxi Medical University, Nanning 530021, China; 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。, authors=黄艳秋1,2 , 丘炜桉1,3 , 陈海燕1 , 彭诗雅1 , 苏明哲1 , 刘悦2 , 叶勇1,4 , authorsList=黄艳秋, 丘炜桉, 陈海燕, 彭诗雅, 苏明哲, 刘悦, 叶勇, authorCompany=1 广西医科大学药学院, 广西 南宁 530021;
中草药
|药剂与工艺
2026
, 57
(17) :
6715
-6729
Box-Behnken设计-效应面法优化氯化两面针碱脂质体处方工艺及体外抗胆管癌活性研究
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黄艳秋1,2 , 丘炜桉1,3 , 陈海燕1 , 彭诗雅1 , 苏明哲1 , 刘悦2 , 叶勇1,4
作者信息
1 广西医科大学药学院, 广西 南宁 530021;
通讯作者:
刘悦
作者简介:
黄艳秋: 黄艳秋（2000—），女，硕士研究生，研究方向为中药新剂型与新技术。E-mail:3163591319@qq.com
Study on formulation optimization of nitidine chloride liposomes by Box-Behnken design-response surface method and anti-cholangiocarcinoma activity in vitro
HUANG Yanqiu1,2 , QIU Weian1,3 , CHEN Haiyan1 , PENG Shiya1 , SU Mingzhe1 , LIU Yue2 , YE Yong1,4
Affiliations
1 School of Pharmacy, Guangxi Medical University, Nanning 530021, China;
doi: 10.7501/j.issn.0253-2670.2026.17.006
文章导航
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。
氯化两面针碱
/
Box-Behnken设计-效应面法
/
脂质体
/
胆管癌
/
细胞凋亡
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
黄艳秋, 丘炜桉, 陈海燕, 彭诗雅, 苏明哲, 刘悦, 叶勇.
Box-Behnken设计-效应面法优化氯化两面针碱脂质体处方工艺及体外抗胆管癌活性研究.
中草药,
2026
, 57
(17)
: 6715
-6729
.
DOI: 10.7501/j.issn.0253-2670.2026.17.006
HUANG Yanqiu, QIU Weian, CHEN Haiyan, PENG Shiya, SU Mingzhe, LIU Yue, YE Yong.
Study on formulation optimization of nitidine chloride liposomes by Box-Behnken design-response surface method and anti-cholangiocarcinoma activity in vitro [J].
Chinese Traditional and Herbal Drugs ,
2026
, 57
(17)
: 6715
-6729
.
DOI: 10.7501/j.issn.0253-2670.2026.17.006
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
参考文献
引证文献
Banales J M, Marin J J G, Lamarca A, et al. Cholangiocarcinoma 2020: The next horizon in mechanisms and management [J]. Nat Rev Gastroenterol Hepatol, 2020, 17(9): 557-588.
2026年第57卷第17期
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doi: 10.7501/j.issn.0253-2670.2026.17.006
接收时间：2026-03-16
首发时间：2026-10-29
https://castjournals.cast.org.cn/joweb/zcy/CN/10.7501/j.issn.0253-2670.2026.17.006
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2种不同金属材料的力学参数
科 属数 种数 占总种数比例 属 种数 占总种数比例 鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78 小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39 多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39 红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87 小菇属 Mycena 11 5.26 光柄菇属 Pluteus 5 2.39 红菇属 Russula 17 8.13 栓菌属 Trametes 5 2.39
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