药学学报, 2019, 54(12): 2316-2325
引用本文:
郑爽, 俞建东, 陈礼迎, 范露慧, 祝露佳, 唐超园, 钱柯, 熊阳. 共载新型二甲双胍聚合物和多柔比星的核膜型脂质纳米粒制备及治疗乳腺癌的研究[J]. 药学学报, 2019, 54(12): 2316-2325.
ZHENG Shuang, YU Jian-dong, CHEN Li-ying, FAN Lu-hui, ZHU Lu-jia, TANG Chao-yuan, QIAN ke, XIONG Yang. Preparation of lipid membrane-wrapped nanoparticles loaded with metformin polymer and doxorubicin and evaluation of their therapeutic effect on breast cancer[J]. Acta Pharmaceutica Sinica, 2019, 54(12): 2316-2325.

共载新型二甲双胍聚合物和多柔比星的核膜型脂质纳米粒制备及治疗乳腺癌的研究
郑爽1, 俞建东1,2, 陈礼迎1, 范露慧1, 祝露佳1,3, 唐超园1, 钱柯1, 熊阳1
1. 浙江中医药大学药学院, 浙江 杭州 311402;
2. 浙江康佰裕生物科技有限公司, 浙江 杭州 310051;
3. 绍兴文理学院附属医院, 浙江 绍兴 312000
摘要:
本研究将二甲双胍聚合物(metformin polymer,PolyMet)与多柔比星(doxorubicin,DOX)共载制备一种静电吸附的核膜型脂质纳米粒(PolyMet-DOX-lipid-nanoparticles,PolyMet-DOX-LNPs),并对其治疗乳腺癌的效果进行评价。首先γ-聚谷氨酸(γ-polyglutamic acid,PGA)和DOX经酰胺反应合成阴离子链(PGA-DOX)并进行核磁表征;采用静电吸附法共载PolyMet与PGA-DOX,得到PolyMet-DOX-NPs;再用阳离子脂质体膜包裹纳米粒得到核膜型的PolyMet-DOX-LNPs;采用透射电子显微镜观察其结构与形态;以粒径、zeta电位、包封率、载药量和体外释放特性等对其进行表征。采用MTT法检测PolyMet联合DOX对4T-1细胞的杀伤作用。用荷4T-1Fluc乳腺癌小鼠研究PolyMet-DOX-LNPs的体内抗肿瘤疗效。所有动物实验符合伦理学标准,并获得浙江中医药大学实验动物伦理委员会批准。结果表明,PolyMet-DOX成功合成,DOX接枝率为(72.03±1.29)%。制得的PolyMet-DOX-LNPs呈良好的圆球形和分散性,粒径稳定在(159.3 ±7.4)nm,zeta电位为(+36.3 ±1.9)mV,包封率和载药量分别为(72.76 ±1.92)%和(1.16 ±0.12)%。体外释放曲线显示,PolyMet-DOX-LNPs在48 h内生理pH值下(pH 7.4)表现出缓慢且持续释放的特性。进一步研究显示,PolyMet与DOX联合能体外协同增强对4T-1细胞的杀伤作用。活体生物发光成像(bioluminescence imaging,BLI)结果显示,PolyMet-DOX-LNPs处理后4T-1Fluc细胞发光信号强度降低,抑制肿瘤体积生长。H&E染色和体重变化结果显示,PolyMet可减轻DOX的毒性。综上,PolyMet与DOX联合治疗乳腺癌具有较好的增效减毒作用,为新型二甲双胍聚合物在抗肿瘤方面的应用奠定了一定的基础。
关键词:    二甲双胍聚合物      多柔比星      纳米粒      乳腺癌      二甲双胍     
Preparation of lipid membrane-wrapped nanoparticles loaded with metformin polymer and doxorubicin and evaluation of their therapeutic effect on breast cancer
ZHENG Shuang1, YU Jian-dong1,2, CHEN Li-ying1, FAN Lu-hui1, ZHU Lu-jia1,3, TANG Chao-yuan1, QIAN ke1, XIONG Yang1
1. College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 311402, China;
2. Carbiogene Therapeutics Co., Ltd., Hangzhou 310051, China;
3. Affiliated Hospital of Shaoxing University, Shaoxing 312000, China
Abstract:
In this study, the lipid membrane-wrapped nanoparticles loaded with metformin polymer (PolyMet) and doxorubicin (DOX) was prepared and then evaluated therapeutic effect on breast cancer. An anionic chain PGA-DOX based on γ-polyglutamic acid (PGA) with DOX was synthesized via amidation reaction and characterized by 1H NMR. The PGA-DOX and PolyMet were loaded via electrostatic attraction to prepare the co-delivery nanoparticles system (PolyMet-DOX-NPs). Then, PolyMet-DOX-NPs were coated with cationic liposome membrane to form the core-membrane structural system (PolyMet-DOX-lipid-nanoparticles, PolyMet-DOX-LNPs). The structure and morphology of PolyMet-DOX-LNPs were observed by transmission electron microscope. The particle size, zeta potential, encapsulation efficiency (EE), drug loading (DL), release behavior in vitro of PolyMet-DOX-LNPs were investigated. The MTT assay was used to examine the cytotoxicity of PolyMet combined with DOX on 4T-1 cells. The 4T1Fluc tumor-bearing mice model was used to evaluate the therapeutic efficacy of PolyMet-DOX-LNPs in vivo. All animal experiments were performed in line with ethical standards and approved by the Animal Experiments Ethical Committee of Zhejiang Chinese Medical University. 1H NMR spectrum showed that PGA-DOX was successfully synthesized with DOX grafting rate of (72.03 ±1.29)%. The EE and DL of PolyMet-DOX-LNPs was (72.76 ±1.92)% and (1.16 ±0.12)%, respectively. PolyMet-DOX-LNPs exhibited a suitable size of (159.3 ±7.4) nm and positive charge of (+36.3 ±1.9) mV with good spheroidal morphology and dispersibility. The release profiles in vitro showed that PolyMet-DOX-LNPs exhibited a slowly and maintained release behavior at physiological pH value (pH 7.4) within 48 h. Further studies showed that PolyMet combined with DOX could synergistically enhance the cytotoxicity on 4T-1 cells. Bioluminescence imaging (BLI) result showed that the luminescence signal intensity of 4T-1Fluc cells was reduced after treatment with PolyMet-DOX-LNPs and the tumor volume growth was also inhibited. Additionally, the H&E staining and changes of body weight showed that PolyMet could reduce the toxicity of DOX. To sum up, PolyMet has a good synergistic effect with DOX in the treatment of breast cancer, which provide the foundation for this novel metformin polymer on the anti-tumor application.
Key words:    metformin polymer    doxorubicin    nanoparticle    breast neoplasm    metformin   
收稿日期: 2019-08-02
DOI: 10.16438/j.0513-4870.2019-0626
基金项目: 国家自然科学基金资助项目(81774011,81473434).
通讯作者: 熊阳,Tel:86-571-61768158,E-mail:xyxnb@126.com
Email: xyxnb@126.com
相关功能
PDF(6567KB) Free
打印本文
0
作者相关文章
郑爽  在本刊中的所有文章
俞建东  在本刊中的所有文章
陈礼迎  在本刊中的所有文章
范露慧  在本刊中的所有文章
祝露佳  在本刊中的所有文章
唐超园  在本刊中的所有文章
钱柯  在本刊中的所有文章
熊阳  在本刊中的所有文章

参考文献:
[1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018[J]. CA Cancer J Clin, 2018, 68:7-30.
[2] Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016, 66:115-132.
[3] Johnson ML, Patel JD. Chemotherapy and targeted therapeutics as maintenance of response in advanced non-small cell lung cancer[J]. Semin Oncol, 2014, 41:93-100.
[4] Guillemard V, Uri Saragovi H. Prodrug chemotherapeutics bypass p-glycoprotein resistance and kill tumors in vivo with high efficacy and target-dependent selectivity[J]. Oncogene, 2004, 23:3613-3621.
[5] Cagel M, Grotz E, Bernabeu E, et al. Doxorubicin:nanotechnological overviews from bench to bedside[J]. Drug Discov Today, 2017, 22:270-281.
[6] Wang DD, Liu R, Wang Y, et al. In vitro targeting efficiency evaluation of reduction-responsive co-loaded doxorubicin/siRNA nanoparticles[J]. Acta Pharm Sin (药学学报), 2018, 53:2104-2112.
[7] Prieto-Vila M,Takahashi RU, Usuba W, et al. Drug resistance driven by cancer stem cells and their niche[J]. Int J Mol Sci, 2017, 18:2-22.
[8] Li Y, Luo J, Lin MT, et al. Co-delivery of metformin enhances the antimultidrug resistant tumor effect of doxorubicin by improving hypoxic tumor microenvironment[J]. Mol Pharm, 2019, 16:2679-2699.
[9] Sudha T, Bharali DJ, Yalcin M, et al. Targeted delivery of paclitaxel and doxorubicin to cancer xenografts via the nanoparticle of nano-diamino-tetrac[J]. Int J Nanomedicine, 2017, 12:1305-1315.
[10] Kendall DL, Amin R, Clayton PE. Metformin in the treatment of obese children and adolescents at risk of type 2 diabetes[J]. Paediatr Drugs, 2014, 16:13-20.
[11] Noto H, Goto A, Tsujimoto T, et al. Cancer risk in diabetic patients treated with metformin:a systematic review and meta-analysis[J]. PLoS One, 2012, 7:1-9.
[12] Dewaal D, Nogueira V, Terry AR, et al. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin[J]. Nat Commun, 2018, 9:446-460.
[13] Cetin M, Sahin S. Microparticulate and nanoparticulate drug delivery systems for metformin hydrochloride[J]. Drug Deliv, 2016, 23:2796-2805.
[14] Xu XN, Shi R, Ma YM. Research progress of pharmacokinetics of metformin based on transporters[J]. Acta Pharm Sin (药学学报), 2017, 52:865-870.
[15] Yousefpour P, Ahn L, Tewksbury J, et al. Conjugate of doxorubicin to albumin-binding peptide outperforms aldoxorubicin[J]. Small, 2019, 15:1-13.
[16] Yang C, Yuan C, Liu W, et al. DPD studies on mixed micelles self-assembled from MPEG-PDEAEMA and MPEG-PCL for controlled doxorubicin release[J]. Colloids Surf B Biointerfaces, 2019, 178:56-65.
[17] Alibloandi M, Abnous K, Mohammadi M, et al. Extensive preclinical investigation of polymersomal formulation of doxorubicin versus Doxil-mimic formulation[J]. J Control Release, 2017, 264:228-236.
[18] Li J, Wang Y, Zhu Y, et al. Recent advances in delivery of drug-nucleic acid combinations for cancer treatment[J]. J Control Release, 2013, 172:589-600.
[19] Xiong Y, Zhao Y, Miao L, et al. Co-delivery of polymeric metformin and cisplatin by self-assembled core-membrane nanoparticles to treat non-small cell lung cancer[J]. J Control Release, 2016, 244:63-73.
[20] Zhao Y, Wang Y, Guo S, et al. PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery[J]. Nat Commun, 2016, 7:1-9.
[21] Liang JW, Fang ZH, Li QS. Effect of metformin combined with paclitaxel on apoptosis and AMPK signaling in human breast cancer cells[J]. Chin J Pathophys (中国病理生理杂志), 2018, 34:2186-2196.
[22] Nimesh S, Chandra R. Guanidinium-grafted polyethylenimine:an efficient transfecting agent for mammalian cells[J]. Eur J Pharm Biopharm, 2008, 68:647-655.
[23] Zhu LY, Tang CY, Yu JD, et al. Impact of Coix seed oil on fluorescence excretion pharmacokinetics and protein expression in doxorubicin-resistant cells MCF-7/DOX[J]. Acta Pharm Sin (药学学报), 2018, 53:84-89.
[24] Khan M, Ong ZY, Wiradharma N, et al. Advanced materials for co-delivery of drugs and genes in cancer therapy[J]. Adv Healthc Mater, 2012, 1:373-372.
[25] Gao H, Yang Z, Zhang S, et al. Glioma-homing peptide with a cell-penetrating effect for targeting delivery with enhanced glioma localization, penetration and suppression of glioma growth[J]. J Control Release, 2013, 172:921-928.
[26] Marinello PC, Panis C, Silva TNX, et al. Metformin prevention of doxorubicin resistance in MCF-7 and MDA-MB-231 involves oxidative stress generation and modulation of cell adaptation genes[J]. Sci Rep, 2019, 9:1-11.
[27] Daugan M, Dufay Wojcicki A, Dhayer B, et al. Metformin:an anti-diabetic drug to fight cancer[J]. Pharmacol Res, 2016, 113:675-685.
[28] Elhissi AM, Karnam KK, Danesh-Azari MR, et al. Formulations generated from ethanol-based proliposomes for delivery via medical nebulizers[J]. J Pharm Pharmacol, 2006, 58:887-894.
[29] Immordino ML, Brusa P, Rocco F, et al. Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs[J]. J Control Release, 2004, 100:331-346.
[30] Sekiguchi Y, Owada J, Oishi H, et al. Noninvasive monitoring of β-cell mass and fetal β-cell genesis in mice using bioluminescence imaging[J]. Exp Anim, 2012, 61:445-451.
[31] Li J, Chen L, Du L, et al. Cage the firefly luciferin!-a strategy for developing bioluminescent probes[J]. Chem Soc Rev, 2013, 42:662-676.
相关文献:
1.张如月, 李朵璐, 杨哲, 郭金秀, 周玉冰.外泌体介导乳腺癌MCF-7细胞对多柔比星耐药性的机制[J]. 药学学报, 2019,54(5): 861-866
2.张贝贝, 黄维兰, 梅玉影, 邵悦馨, 张璐, 李瑞芳.智能型荧光纳米递送系统用于乳腺癌细胞的示踪和增殖抑制研究[J]. 药学学报, 2019,54(6): 1123-1131
3.薛建秀, 毕洪书, 李雨爱, 陈瑶, 刘晓庆, 徐正奇, 潘虎威, 石凯.壳-核结构多柔比星脂质磷酸钙纳米粒的制备及体外性能评价[J]. 药学学报, 2018,53(8): 1364-1370
4.孟庆硕, 张鹏程, 尹琦, 张志文, 于海军, 李亚平.光敏感型盐酸多柔比星脂质体逆转乳腺癌耐药[J]. 药学学报, 2017,52(5): 809-820
5.沈爱俊, 夏登宁, 甘勇, 李娟.聚合物电解质层层组装脂质纳米粒提高多柔比星的口服吸收[J]. 药学学报, 2016,51(7): 1136-1143
6.李洁丽, 郑春丽, 刘建平, 朱家壁.多柔比星PLGA纳米粒的处方工艺优化及体外释药行为研究[J]. 药学学报, 2013,48(5): 759-766