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

Preparation of lipid membrane-wrapped nanoparticles loaded with metformin polymer and doxorubicin and evaluation of their therapeutic effect on breast cancer

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。

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 onγ-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-DOXLNPs 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 antitumor application.