黄芩苷PEG-PE纳米胶束的制备、表征与细胞毒性研究
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摘要
目的:制备黄芩苷(BAI)聚乙二醇衍生化磷脂酰乙醇胺(BAI@PEG-PE)纳米胶束,并对其进行表征和细胞毒性研究。方法:采用薄膜水化法制备BAI@PEG-PE纳米胶束,观察其外观特征,检测其粒径、多分散系数(PDI)、Zeta电位、载药量、包封率。比较BAI原料药和BAI@PEG-PE纳米胶束在pH 7.4磷酸盐缓冲液中1~84 h内的释药情况。以香豆素6为荧光探针,观察PEG-PE纳米胶束在H9c2心肌细胞中的分布。将H9c2心肌细胞分为模型组、BAI原料药组和BAI@PEG-PE纳米胶束组,用不含药或含相应药物的无血清DMEM培养液培养0.5 h后,用异丙肾上腺素诱导心肌细胞凋亡,比较3组细胞的细胞核形态变化、细胞凋亡率和凋亡相关蛋白B淋巴细胞瘤2(Bcl-2)及其X蛋白(Bax)的蛋白表达水平。结果:所制备的BAI@PEG-PE纳米胶束呈现大小比较均一的圆球形,其粒径为(16.7±0.8)nm,PDI为0.11±0.01,Zeta电位为(-18.4±0.6)mV,载药量为(7.84±0.65)%,包封率为(85.7±4.9)%(n=3),84 h的累积释放度为76.5%,而BAI原料药在24 h内已基本释放完全。PEG-PE纳米胶束可增强H9c2心肌细胞对香豆素6的摄取,且主要聚集在线粒体周围。与模型组比较,BAI原料药组和BAI@PEG-PE纳米胶束组细胞的凋亡形态明显改善,凋亡率明显降低,Bcl-2蛋白表达明显增强,Bax蛋白表达水平明显降低,差异均有统计学意义(P<0.05或P<0.01),其中BAI@PEG-PE纳米胶束组的上述效果更明显(P<0.05或P<0.01)。结论。成功制得BAI@PEG-PE纳米胶束,其具有明显的缓释作用、心肌靶向性,可预防心肌细胞的凋亡。
OBJECTIVE:To prepare Baicalin-loaded Polyethylene glycol-derivatized phosphatidylethanolamine(BAI@PEG-PE)nanomicelles,and to characterize it and study its cytotoxicity. METHODS:BAI@PEG-PE nanomicelles were prepared by film hydration method and their appearance characteristics were observed. The particle size,polydispersity index,Zeta potential,drug-loading amount and encapsulation efficiency of the nanomicelles were detected. Drug release of BAI raw material and BAI@PEG-PE nanomicelles in pH 7.4 phosphate buffer were compared within 1-84 h. Using coumarin 6 as fluorescent probe,the distribution of PEG-PE nanomicelles in H9 c2 cardiomyocytes were observed. H9 c2 cardiomyocytes were divided into model group,BAI raw material group and BAI@PEG-PE nanomicelles group. After treated with serum-free DMEM medium containing no or corresponding drugs for 0.5 h,isoproterenol was used to induce cardiomyocyte apoptosis. Nuclear morphology,cell apoptosis rate and protein expression of Bcl-2 and Bax were compared with among 3 groups. RESULTS:Prepared BAI@PEG-PE nanomicelles were uniform globular shape. The particle size was(16.7±0.8)nm,PDI was 0.11±0.01 and Zeta-potential was(-18.4±0.6)mV;drug-loading amount was(7.84±0.65)%,encapsulation efficiency was(85.7±4.9)%(n=3). Accumulative release rate was76.5% within 84 h. BAI raw material was released completely within 24 h. PEG-PE nanomicelles could strengthen the intake of coumarin 6 in H9 c2 cardiomyocytes, mainly gathering around mitochondria. Compared with model group, the apoptosis morphology of cardiomyocytes were improved significantly in BAI raw material group and BAI@PEG-PE nanomicelles group;apoptosis rate was decreased significantly;protein expression of Bcl-2 was increased significantly;protein expression of Bax was decreased significantly with statistical significance(P<0.05 or P<0.01). Above effects of BAI@PEG-PE nanomicelles group were more significant(P<0.05 or P<0.01). CONCLUSIONS: BAI@PEG-PE nanomicelles are prepared successfully, and show significant sustained-release effect and myocardial targeting,and can prevent cardiomyocyte apoptosis.
OBJECTIVE:To prepare Baicalin-loaded Polyethylene glycol-derivatized phosphatidylethanolamine(BAI@PEG-PE)nanomicelles,and to characterize it and study its cytotoxicity. METHODS:BAI@PEG-PE nanomicelles were prepared by film hydration method and their appearance characteristics were observed. The particle size,polydispersity index,Zeta potential,drug-loading amount and encapsulation efficiency of the nanomicelles were detected. Drug release of BAI raw material and BAI@PEG-PE nanomicelles in pH 7.4 phosphate buffer were compared within 1-84 h. Using coumarin 6 as fluorescent probe,the distribution of PEG-PE nanomicelles in H9 c2 cardiomyocytes were observed. H9 c2 cardiomyocytes were divided into model group,BAI raw material group and BAI@PEG-PE nanomicelles group. After treated with serum-free DMEM medium containing no or corresponding drugs for 0.5 h,isoproterenol was used to induce cardiomyocyte apoptosis. Nuclear morphology,cell apoptosis rate and protein expression of Bcl-2 and Bax were compared with among 3 groups. RESULTS:Prepared BAI@PEG-PE nanomicelles were uniform globular shape. The particle size was(16.7±0.8)nm,PDI was 0.11±0.01 and Zeta-potential was(-18.4±0.6)mV;drug-loading amount was(7.84±0.65)%,encapsulation efficiency was(85.7±4.9)%(n=3). Accumulative release rate was76.5% within 84 h. BAI raw material was released completely within 24 h. PEG-PE nanomicelles could strengthen the intake of coumarin 6 in H9 c2 cardiomyocytes, mainly gathering around mitochondria. Compared with model group, the apoptosis morphology of cardiomyocytes were improved significantly in BAI raw material group and BAI@PEG-PE nanomicelles group;apoptosis rate was decreased significantly;protein expression of Bcl-2 was increased significantly;protein expression of Bax was decreased significantly with statistical significance(P<0.05 or P<0.01). Above effects of BAI@PEG-PE nanomicelles group were more significant(P<0.05 or P<0.01). CONCLUSIONS: BAI@PEG-PE nanomicelles are prepared successfully, and show significant sustained-release effect and myocardial targeting,and can prevent cardiomyocyte apoptosis.
引文
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[15]王琳,陆丹玉,方晨.去甲斑蝥素纳米胶束的制备及抑瘤作用研究[J].中国药房,2017,28(19):2680-2684.
[2] HUANG ZQ,YE BZ,DAI ZY,et al. Curcumin inhibits autophagy and apoptosis in hypoxia/reoxygenation-induced myocytes[J]. Mol Med Rep,2015,11(6):4678-4684.
[3] JIAN J,XUAN FF,QIN FZ,et al. Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardialischemia/reperfusion injury in rats[J]. Drug Des Devel Ther,2015.DOI:10.2147/DDDT.S92549.
[4]纪相福,史道华.黄芩苷的心脑血管作用研究进展[J].海峡药学,2006,18(5):10-12.
[5] WANG XB,HE F,LIAO YL,et al. Baicalin pretreatment protects against myocardial ischemia/reperfusion injury by inhibiting mitochondrial damage-mediated apoptosis[J]. Int J Cardiol,2013,168(4):4343-4345.
[6]马文转,王金铃,屠鹏飞.黄芩苷纳米胶束的制备、表征及其对MCF-7细胞抑制作用的研究[J].中草药,2015,46(4):507-512.
[7]吴玉梅,李新悦,张谦,等.单克隆抗体OX26修饰的丹酚酸B/黄芩苷纳米结构脂质载体研究[J].中草药,2018,49(12):2801-2808.
[8] LUKYANOV AN,HARTNER WC,TORCHILIN VP. Increased accumulation of PEG-PLGA micelles in the area of experimental myocardial infarction in rabbits[J]. J Control Release,2004,94(1):187-193.
[9] XIA CF,YE ZG,ZHOU XN,et al. Tissue distribution of PEGylated puerarin in acute myocardial ischemia mode rats[J]. Acta Pharmaceutica Sinica,2014,49(10):1413-1417.
[10]国家药典委员会.中华人民共和国药典:一部[S]. 2015年版.北京:中国医药科技出版社,2015:301.
[11] RIVOLTA I,PANARITI A,LETTIERO B,et al. Cellular uptake of coumarin-6 as a model drug loaded in solid lipid nanoparticles[J]. J Physiol Pharmacol,2011,62(1):45-53.
[12] HU X,YANG FF,LIU CY,et al. In vitro uptake and transport studies of PEG-PLGA polymeric micelles in respiratory epithelial cells[J]. Eur J Pharm Biopharm,2017,114:29-37.
[13]赵慧,王雄,魏晓倩.异丙肾上腺素预处理对心肌缺血再灌注损伤大鼠心肌细胞凋亡及氧化应激的影响[J].中西医结合心脑血管病杂志,2017,15(1):36-40.
[14]蔡少艾,赵甘剑,黄尹,等.血管紧张素(1-7)抑制异丙肾上腺素诱导的H9c2心肌细胞凋亡[J].中国动脉硬化杂志,2018,26(5):438-444.
[15]王琳,陆丹玉,方晨.去甲斑蝥素纳米胶束的制备及抑瘤作用研究[J].中国药房,2017,28(19):2680-2684.