葛根素PEG-PLGA纳米胶束的体外评价、细胞摄取及抗急性心肌缺血的研究
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摘要
PUE@PEG-PLGA纳米胶束具有粒径小,载药量高,释药缓慢等优良特点。TEM电镜结果表明PUE@PEG-PLGA纳米胶束呈核壳状圆球型结构;采用芘测定法测定PEG-PLGA纳米胶束的临界胶束浓度(CMC)大约为4.8 mg·L~(-1);激光共聚焦试验表明,PEG-PLGA纳米胶束可以增强香豆素-6细胞摄取量,且可聚集在线粒体周围,细胞外药物残留量定量结果也可间接证实,PEG-PLGA纳米胶束可以促进药物的细胞摄取。采用结扎冠状动脉制备急性心肌缺血模型大鼠,再随机分为假手术组,模型组,PUE组和PUE@PEG-PLGA高、中、低剂量组,在结扎后5 min各给药组尾iv给药。监测心电图ST段变化,测定血清肌酸激酶(CK)、乳酸脱氢酶(LDH)、天冬氨酸转氨酶(AST)和丙二醛(MDA)水平,并测量心肌梗死面积。PUE和PUE@PEG-PLGA纳米胶束均能降低升高的ST段,降低血清中CK,LDH,AST,MDA水平,减少心肌梗死面积;其中PUE@PEG-PLGA中、高剂量组的药效明显强于PUE组,低剂量组基本与PUE组等效。PUE@PEG-PLGA纳米胶束很大程度上提高PUE心肌细胞摄取量,增强药物抗急性心肌缺血作用,减少给药剂量。
PUE@PEG-PLGA micelles has excellent characteristics such as small particle size, high drug loading and slow drug release. The results of TEM electron microscopy showed that PUE@PEG-PLGA micelles had obvious core-shell structure. The critical micelle concentration(CMC) of PEG-PLGA micelles determined by pyrene assay was about 4.8 mg·L~(-1). Laser confocal experiments showed that PEG-PLGA micelles can enhance the cellular uptake of coumarin-6 and aggregate around the mitochondria; quantitative results of extracellular drug residues also indirectly confirmed that PEG-PLGA micelles can promote cellular uptake of the drug. Acute ischemic myocardial model rats were prepared by coronary artery ligation, and then the model rats were randomly divided into six groups: Sham operation group, model group, puerarin(PUE) group, as well as low-, mid-, and high-dose PUE@PEG-PLGA micelles groups. Drugs were given by iv administration 5 min after the ligation. The ST segment changes in the electrocardiogram were monitored; serum creatine kinase(CK), lactate dehydrogenase(LDH), aspartate aminotransferase(AST), and malondialdehyde(MDA) levels were detected and myocardial infarct size was also measured. Both PUE and PUE@PEG-PLGA micelles can reduce the elevated ST segment, reduce serum CK, LDH, AST and MDA levels, and reduce myocardial infarct size. The efficacy of PUE@PEG-PLGA medium and high dose groups was significantly better than that in the PUE group, and the efficacy in PUE@PEG-PLGA low dose group was basically equivalent to that in the PUE group. PUE@PEG-PLGA micelles can greatly improve the cardiomyocytes uptake of PUE, enhance the anti-acute myocardial ischemia effect of drugs, and reduce its dosage.
PUE@PEG-PLGA micelles has excellent characteristics such as small particle size, high drug loading and slow drug release. The results of TEM electron microscopy showed that PUE@PEG-PLGA micelles had obvious core-shell structure. The critical micelle concentration(CMC) of PEG-PLGA micelles determined by pyrene assay was about 4.8 mg·L~(-1). Laser confocal experiments showed that PEG-PLGA micelles can enhance the cellular uptake of coumarin-6 and aggregate around the mitochondria; quantitative results of extracellular drug residues also indirectly confirmed that PEG-PLGA micelles can promote cellular uptake of the drug. Acute ischemic myocardial model rats were prepared by coronary artery ligation, and then the model rats were randomly divided into six groups: Sham operation group, model group, puerarin(PUE) group, as well as low-, mid-, and high-dose PUE@PEG-PLGA micelles groups. Drugs were given by iv administration 5 min after the ligation. The ST segment changes in the electrocardiogram were monitored; serum creatine kinase(CK), lactate dehydrogenase(LDH), aspartate aminotransferase(AST), and malondialdehyde(MDA) levels were detected and myocardial infarct size was also measured. Both PUE and PUE@PEG-PLGA micelles can reduce the elevated ST segment, reduce serum CK, LDH, AST and MDA levels, and reduce myocardial infarct size. The efficacy of PUE@PEG-PLGA medium and high dose groups was significantly better than that in the PUE group, and the efficacy in PUE@PEG-PLGA low dose group was basically equivalent to that in the PUE group. PUE@PEG-PLGA micelles can greatly improve the cardiomyocytes uptake of PUE, enhance the anti-acute myocardial ischemia effect of drugs, and reduce its dosage.
引文
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[5] 解晓帅,董运茁,穆殿平,等.葛根素注射液临床使用安全性的评价研究[J].中国中药杂志,2018,43(19):3956.
[6] Wu J,Zhang X,Zhang B.Efficacy and safety of puerarin injection in treatment of diabetic peripheral neuropathy:a systematic review and Meta-analysis of randomized controlled trials[J].J Tradit Chin Med,2014,34:401.
[7] Zhang K,Tang X,Zhang J,et al.PEG-PLGA copolymers:their structure and structure-influenced drug delivery applications[J].J Control Release,2014,10(183):77.
[8] Lukyanov A N,Hartner W C,Torchilin V P.Increased accumulation of PEG-PLGA micelles in the area of experimental myocardial infarction in rabbits[J].J Control Release,2004,94(1):187.
[9] Xia C F,Ye Z G,Zhou X N,et al.Tissue distribution of PEGylated puerarin in acute myocardial ischemia mode rats[J].Acta Phamr Sin,2014,49(10):1413.
[10] Wu H,Zhu L,Torchilin V P.pH-sensitive poly(histidine)-PEG/DSPE-PEG co-polymer micelles for cytosolic drug delivery[J].Biomaterials,2013,34(4):1213.
[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.
[12] Hu X,Yang F F,Liu C Y,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.
[13] Li W Q,Wu J Y,Zhang J,et al.Puerarin-loaded PEG-PE micelles with enhanced anti-apoptotic effect and betterpharmacokinetic profile[J].Drug Deliv,2018,25(1):827.
[14] Qiao H Z,Li J,Wang Y,et al.Synthesis and characterization of multi-functional linear-dendritic block copolymer for intracellular delivery of antitumor drugs[J].Int J Pharm,2013,452(1/2):363.
[15] 黄安皓,韩顺平,陆燕平,等.RGDyC与PEG共修饰的PAMAM树状大分子载三氧化二砷脑胶质瘤靶向递药系统的制备及体外评价[J].中国中药杂志,2018,43(8):1618.
[16] Wang J,Wang Y,Liang W.Delivery of drugs to cell membranes by encapsulation in PEG-PE micelles[J].J Control Release,2012,160(3):637.
[17] Gao Q,Pan H Y,Qiu S,et al.Atractyloside and 5-hydroxydecanoate block the protective effect of puerarin in isolated rat heart[J].Life Sci,2006,79(3):217.
[18] 姚慧,高琴,夏强.线粒体钾通道参与葛根素抗心肌缺氧/复氧损的作用[J].中国应用生理学杂志,2010,26(4):459.