药学学报, 2019, 54(3): 565-573
季宇彬, 刘彪, 于润琪, 王向涛. 双硫仑纳米混悬剂的制备及体内外抗乳腺癌研究[J]. 药学学报, 2019, 54(3): 565-573.
JI Yu-bin, LIU Biao, YU Run-qi, WANG Xiang-tao. Preparation of disulfiram naonosuspensions and their anti-tumor efficacy in vitro and in vivo[J]. Acta Pharmaceutica Sinica, 2019, 54(3): 565-573.

季宇彬1,2, 刘彪1,2, 于润琪2,3, 王向涛1,2
1. 哈尔滨商业大学, 生命科学与环境科学研究中心, 黑龙江 哈尔滨 150076;
2. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193;
3. 黑龙江中医药大学药学院, 黑龙江 哈尔滨 150040
双硫仑(disulfiram,DSF)是一种传统的戒酒药物,最近研究发现其对多种癌症类型均表现出强烈的生长抑制作用,但是水溶性差、在胃肠中不稳定及血浆半衰期短的缺点限制了其药效的发挥。本研究拟将DSF制备成纳米混悬剂(DSF-NSps)来解决此问题。采用超声条件下的反溶剂沉淀法制备DSF-NSps,以纳米粒的粒径、多分散性指数(PDI)、zeta电位和放置稳定性等为指标筛选稳定剂。采用动态光散射法测定粒径、PDI和zeta电位,透射电镜观察纳米粒形态;通过DSF-NSps在不同生理介质中的粒径变化来考察DSF-NSps在介质中稳定性。HPLC测定载药量,用透析的方法测定体外药物释放,MTT比色法测定纳米粒和游离双硫仑的体外细胞毒性,进而用4T1荷瘤小鼠研究纳米粒的体内抗肿瘤药效。所有动物实验符合动物实验伦理学标准,并获得中国医学科学院药用植物研究所实验动物伦理委员会批准。结果表明,SPC(大豆磷脂)/TPGS(生育酚琥珀酸聚乙二醇酯)组合稳定剂为DSF的优良稳定剂,药载比为DSF-SPC-TPGS=24:20:4时,制得的DSF-NSps粒径较小且放置较稳定,其载药量为(45.36 ±2.09)%,平均粒径为175.0 ±0.75 nm,PDI为0.24 ±0.07,zeta电位为-14.3 mV,外观呈球形;DSF-NSps在磷酸盐缓冲液、生理盐水、5%葡萄糖溶液、人工肠液、人工胃液和血浆中均能稳定存在,满足静脉注射及灌胃的要求;体外研究显示纳米粒装载显著提高了DSF在水相体系中的稳定性,DSF-NSps可缓慢释放包封的药物,并能提高DSF对癌细胞的生长抑制作用(纳米粒和游离DSF在48 h的IC50分别为1.07和5.53 μg·mL-1P<0.01)。在荷瘤小鼠药效实验中,DSF-NSps显示出良好的量效关系,高、中、低剂量的肿瘤抑制率分别为80.22%、75.14%和66.10%,均显著高于阳性药紫杉醇注射液(55.01%,P<0.05);组织分布实验显示DSF-NSps主要分布在肝、脾和肿瘤。综上,将双硫仑制备成纳米粒后有望成为一种高效的抗肿瘤药物用于乳腺癌的治疗。
Preparation of disulfiram naonosuspensions and their anti-tumor efficacy in vitro and in vivo
JI Yu-bin1,2, LIU Biao1,2, YU Run-qi2,3, WANG Xiang-tao1,2
1. Research Center on Life Sciences and Environmental Sciences, Harbin University of Commerce China, Harbin 150076, China;
2. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China;
3. College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China
Disulfiram (DSF) is a traditional anti-alcohol drug, but it was recently found that DSF has strong inhibitory effect on the growth of a variety of cancer cells. However, its clinical application is greatly limited due to its poor solubility, instability in gastrointestinal tract and short plasma half-life. In this study, DSF is fabricated into nanosuspensions with the aim of trying to solve these problems. DSF nanosuspensions (DSF-NSps) were prepared by the anti-solvent precipitation method under ultrasonication, and the suitable stabilizer was screened according to the size, polydispersity index (PDI), and zeta potential of the resultant nanosuspensions, along with their particle size change during the storage at room temperature. The particle size, PDI, and zeta potential of DSF-NSps were determined using dynamic light scattering method, while the morphology of DSF-NSps was observed by transmission electronic microscope (TEM). The stability of DSF-NSps in media was examined according to their particle size change in different physiological media. The concentration of DSF was measured by HPLC assay. The in vitro drug release was evaluated on basis of dialysis. MTT assay was employed to evaluate the in vitro cytotoxicity of DSF-NSps against cancer cell lines. The 4T1 tumor-bearing mouse model was used to evaluate the in vivo therapeutic efficacy of DSF-NSps. All the animal experiments were acquired according to the Regulations for Animal Experiments and Guidelines for Ethical as defined by Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College. As a result, the combinational use of soyabean lecithin (SPC) and D-alpha tocopherol acid polyethyene glycol succinate (TPGS) was determined to best stabilize DSF-NSps when the ratio of DSF-SPC-TPGS was 24:20:4 (weight ratio), with small particle size and good storage stability. The resultant DSF-NSps showed a regular spherical morphology and drug loading content of (45.36 ±2.09)%, with average particle size of 175.00 ±0.75 nm, PDI of 0.24 ±0.07 and zeta potential of -14.3 mV. DSF-NSps displayed good particle size stability in a variety of biological media including phosphate buffer saline, normal saline, 5% glucose, artificial gastric fluid, artificial intestinal fluid and plasma, which would meet the demand of both intravenous and oral administration. The in vitro study demonstrated that nano-encapsulation greatly increased the stability of DSF in aqueous media, DSF-NSps exhibited sustained release of the encapsulated drug and significantly inhibited 4T1 cells compared to free DSF (IC50, 1.07 vs 5.53 μg·mL-1, P<0.01). DSF-NSps showed a good dose-response relationship on the 4T1 tumor-bearing mice with the tumor inhibition rates at the three doses being 80.22%, 75.14% and 66.10%, all higher than that of paclitaxel injections (55.01%, P<0.05). The in vivo biodistribution study displayed that DSF-NSps were mainly distributed into liver, spleen and tumor. In sum, disulfiram nanoparticles could be expected to provide an effective anti-cancer drug for the treatment of breast cancer.
Key words:   
收稿日期: 2018-09-17
DOI: 10.16438/j.0513-4870.2018-0857
基金项目: 国家自然科学基金-广东联合基金资助项目(U1401223).
通讯作者: 王向涛
Email: xtaowang@163.com
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