白屈菜红碱纳米粒的制备及体外抗黑色素瘤活性评价

杨锦 韩伟 张永萍 陈晓兰 李哲 刘杰 吴静澜

中圖分类号 R283.6;R979.1 文献标志码 A 文章编号 1001-0408（2021）24-2980-07

DOI 10.6039/j.issn.1001-0408.2021.24.07

摘 要 目的：制备白屈菜红碱纳米粒（CHE-NPs），对其处方进行优化，并评价其体外释药行为及对黑色素瘤的抑制作用。方法：以甲氧基聚乙二醇-聚乳酸-羟基乙酸共聚物（mPEG-PLGA）为载体材料，采用纳米沉淀法制备CHE-NPs，采用高效液相色谱法和透析袋法测定包封率、载药量。以二者的总评归一（OD）值为因变量，以CHE投药量、mPEG-PLGA质量浓度、泊洛沙姆188（F68）浓度为自变量，采用Box-Behnken响应面设计优化CHE-NPs处方。检测最优处方所制CHE-NPs的粒径和Zeta电位，考察其体外释药特征，比较CHE原料药和CHE-NPs对小鼠B16黑色素瘤细胞存活率的影响并计算二者的半数抑制浓度。结果：最优处方为CHE投药量2 mg、mPEG-PLGA质量浓度13 mg/mL、F68浓度1.8%。以此所制CHE-NPs的平均包封率为（80.18±1.11）%，平均载药量为（11.36±0.28）%，平均OD值为0.96±0.04[与OD预测值（0.90）的相对偏差为6.67%]，粒径为（113.1±1.40）nm，Zeta电位为（-21.6±0.29）mV，多分散性指数为0.07±0.01（n=3）。CHE对照品、CHE-NPs在孵育8 h时的累积释放率分别为90.87%、68.68%，后者的体外释药行为符合Weibull动力学模型。CHE-NPs对B16黑色素瘤细胞的抑制作用显著强于CHE原料药，CHE-NPs和CHE原料药的24 h半数抑制浓度分别为69.35、107.36 μg/mL。结论：所制CHE-NPs具有缓释作用和较强的载药能力，同时增强了CHE对黑色素瘤的体外抑制作用。

关键词 白屈菜红碱;黑色素瘤;抑瘤作用;甲氧基聚乙二醇-聚乳酸-羟基乙酸共聚物;纳米粒;Box-Behnken响应面设计

Preparation of Chelerythrine Nanoparticles and Evaluation of Anti-melanoma Activity in vitro

YANG Jin1，HAN Wei1，ZHANG Yongping1，CHEN Xiaolan1，LI Zhe2，3，LIU Jie1，WU Jinglan1（1. College of Pharmacy， Guizhou University of Traditional Chinese Medicine， Guiyang 550025， China; 2. College of Basic Medicine， Guizhou University of Traditional Chinese Medicine， Guiyang 550025， China; 3. Innovation Research and Development Center of Veterinary Traditional Chinese Medicine Preparations， Guizhou University of Traditional Chinese Medicine， Guiyang 550025， China）

ABSTRACT   OBJECTIVE： To prepare chelerythrine nanoparticles （CHE-NPs）， optimize their formulation， and evaluate its drug release behavior in vitro and its inhibitory effect on melanoma. METHODS： Using methoxy polyethylene glycol-poly（lactic-co- glycolic acid） （mPEG-PLGA） as carrier， CHE-NPs were prepared by the nano-precipitation method. HPLC method and dialysis bag method were used to determine entrapment efficiency and drug loading. The formulation of CHE-NPs was optimized by Box-Behnken response surface design using overall desirability （OD） of them as dependent variables， CHE dosage， mPEG-PLGA concentration and poloxamer 188 （F68） concentration as independent variables. The particle size and Zeta potential of CHE-NPs prepared by the optimal formulation were detected; the characteristics of drug release in vitro were investigated; the effects of CHE and CHE-NPs on survival rate of mice B16 melanoma cells were compared， and median inhibition concentrations （IC50） of them were calculated. RESULTS： The optimal formulation included CHE of 2 mg， mPEG-PLGA of 13 mg/mL， F68 of 1.8%. Average entrapment efficiency rate of CHE-NPs prepared by the optimal formulation was （80.18±1.11）%， average drug loading was （11.36±0.28）%， average OD value was 0.96±0.04 [the relative deviation from predicted value （0.90） of OD was 6.67%]; particle size was （113.1±1.40） nm， and Zeta potential was （-21.6±0.29） mV; polydispersity index was 0.07±0.01 （n=3）;accumulative release rates of CHE control and CHE-NPs were 90.87% and 68.68% within 8 h， and drug release behavior in vitro of the latter was in accordance with Weibull kinetic model. Inhibitory effect of CHE-NPs on B16 melanoma cells was significantly stronger than that of CHE; the 24 h IC50 of CHE-NPs and CHE were 69.35 and 107.36 μg/mL， respectively. CONCLUSIONS： The prepared CHE-NPs show good sustained-effect and high capacity of drug loading， and strengthen the inhibitory effect of CHE on melanoma.

KEYWORDS Chelerythrine; Melanoma; Anti-tumor effect; Methoxy polyethylene glycolpolylactic acid-hydroxyacetic acid copolymer; Nanoparticles; Box-Behnken response surface design

基金项目：国家苗药工程技术研究中心组建项目（No.2014FU-125Q09）;贵州省科技厅学术新苗培养及创新探索专项项目（No.黔科合平台人才〔2018〕5766号-13）;地方病与少数民族性疾病教育部重点实验室（贵州医科大学）开放课题基金资助项目（No.黔教合KY字〔2019〕047）