银杏内酯K的PLGA-PEG纳米粒制备、表征和神经保护活性评价
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摘要
目的制备及表征银杏内酯K(GK)聚乙二醇-聚乳酸-羟基乙酸共聚物纳米粒(GK-m PEG-PLGA-NPs),并评价其神经保护活性。方法采用聚乙二醇-聚乳酸-羟基乙酸共聚物(PEG-PLGA-COOH)作为载体,复乳溶剂挥发法制备隐形纳米粒;HPLC法测定GK-m PEG-PLGA-NPs的包封率及载药量;动态光散射粒径仪和透射电镜测定GK-m PEG-PLGA-NPs的粒径分布、Zeta电位及表面形态;以p H 7.4磷酸盐缓冲溶液(PBS)作为释放介质,考察GK-m PEG-PLGA-NPs的体外释放行为;采用体外细胞实验,考察GK-m PEG-PLGA-NPs对H2O2诱导肾上腺嗜铬细胞瘤PC12细胞损伤的保护作用。结果 GK-m PEG-PLGA-NPs包封率和载药量分别为(83.40±2.85)%和(3.26±0.24)mg/g;GK-m PEG-PLGA-NPs平均粒径为(93.19±2.77)nm;Zeta电位为(-11.93±1.71)m V;60 h内GK-m PEG-PLGA-NPs累积释药量为(90.5±4.0)%。GK-m PEG-PLGA-NPs对H2O2诱导的PC12细胞存活率降低有明显的改善作用,对乳酸脱氢酶(LDH)释放具有抑制作用,但其保护作用明显弱于GK对PC12细胞作用。结论 GK-m PEG-PLGA-NPs体外释放具有缓释性行为,对H2O2诱导PC12细胞具有神经保护作用,表明GK-m PEG-PLGA-NPs具有应用前景,值得进一步研究。
Objective To prepare and characterize ginkgolide K-loaded m PEG-PLGA [poly(D,L-lactide-co-gly-colide)-block-poly(ethylene glycol)] polymer nanoparticles(GK-m PEG-PLGA-NPs) and to evaluate its neuroprotective effect on the H_2O_2-induced PC12 cells injury in vitro. Methods The PLGA-PEG-COOH polymer was selected as carrier and double emulsion solvent evaporation technique was employed to prepare the stealth nanoparticles. The encapsulation efficiency(EE) and drug load(DL) of GK-m PEG-PLGA-NPs were investigated by HPLC. The size distribution, zeta potential, and surface morphology of GK-m PEG-PLGA-NPs were characterized by dynamic light scattering(DLS) and transmission electron microscopy(TEM), respectively. The in vitro release of GK-m PEG-PLGA-NPs was examined using phosphate buffer solution(pH 7.4) as the releasing medium for 24 h. The H_2O_2-induced PC12 cells injury models was established for the investigation of the protective effect of GK-m PEG-PLGA-NPs on nerve cells in vitro. Results EE and DL of GK-m PEG-PLGA-NPs was(83.40 ± 2.85)% and(3.26 ± 0.24) mg/g, respectively. The average diameter of GK-m PEG-PLGA-NPs was(93.19 ± 2.77) nm and zeta potential was(-11.93 ± 1.71) m V. The cumulative rate of drug release was(90.5 ± 4.0)% after 60 h in phosphate buffer solution. GK-m PEG-PLGA-NPs significantly inhibited the apoptosis of PC12 cells and the release of lactic dehydrogenase induced by H_2O_2. However, the protective action of GK-m PEG-PLGA-NPs on the H_2O_2-iduced PC12 cells injury was significantly weaker than that of GK. Conclusion Our results proved that GK-m PEG-PLGA-NPs had a sustained release behavior in vitro and the neuroprotective effect of GK-m PEG-PLGA-NPs on H_2O_2-induced PC12 cells, which indicates that GK-m PEG-PLGA-NPs has the prospect of application and deserves further research.
Objective To prepare and characterize ginkgolide K-loaded m PEG-PLGA [poly(D,L-lactide-co-gly-colide)-block-poly(ethylene glycol)] polymer nanoparticles(GK-m PEG-PLGA-NPs) and to evaluate its neuroprotective effect on the H_2O_2-induced PC12 cells injury in vitro. Methods The PLGA-PEG-COOH polymer was selected as carrier and double emulsion solvent evaporation technique was employed to prepare the stealth nanoparticles. The encapsulation efficiency(EE) and drug load(DL) of GK-m PEG-PLGA-NPs were investigated by HPLC. The size distribution, zeta potential, and surface morphology of GK-m PEG-PLGA-NPs were characterized by dynamic light scattering(DLS) and transmission electron microscopy(TEM), respectively. The in vitro release of GK-m PEG-PLGA-NPs was examined using phosphate buffer solution(pH 7.4) as the releasing medium for 24 h. The H_2O_2-induced PC12 cells injury models was established for the investigation of the protective effect of GK-m PEG-PLGA-NPs on nerve cells in vitro. Results EE and DL of GK-m PEG-PLGA-NPs was(83.40 ± 2.85)% and(3.26 ± 0.24) mg/g, respectively. The average diameter of GK-m PEG-PLGA-NPs was(93.19 ± 2.77) nm and zeta potential was(-11.93 ± 1.71) m V. The cumulative rate of drug release was(90.5 ± 4.0)% after 60 h in phosphate buffer solution. GK-m PEG-PLGA-NPs significantly inhibited the apoptosis of PC12 cells and the release of lactic dehydrogenase induced by H_2O_2. However, the protective action of GK-m PEG-PLGA-NPs on the H_2O_2-iduced PC12 cells injury was significantly weaker than that of GK. Conclusion Our results proved that GK-m PEG-PLGA-NPs had a sustained release behavior in vitro and the neuroprotective effect of GK-m PEG-PLGA-NPs on H_2O_2-induced PC12 cells, which indicates that GK-m PEG-PLGA-NPs has the prospect of application and deserves further research.
引文
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[10]朱文静,张良珂.载和厚朴酚介孔二氧化硅包覆聚吡咯纳米粒的制备研究[J].中草药,2018,49(9):2057-2062.
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[13]徐骏军,陈丹飞,宋倩倩,等.p H值响应释药As2O3聚乙二醇-聚己内酯-聚乙烯亚胺纳米粒的制备及体外评价[J].中草药,2018,49(23):5532-5540.
[14]刘洋,谢栓栓,曾洁,等.表面修饰适配体S6的聚乳酸-羟基乙酸/聚乙二醇共聚物纳米粒制备及其运载小干扰RNA的应用研究[J].有机化学,2018,38(10):2706-2712.
[15]童晓东,范永春,严玮.姜黄素维生素E聚乙二醇琥珀酸酯-聚乙二醇硬脂酸酯15胶束对姜黄素溶解度和口服生物利用度的影响[J].中草药,2017,48(5):902-906.
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[18]刘朝勇,肖云芝,李瑞生,等.小菜蛾抗菌肽聚乳酸-羟基乙酸共聚物纳米粒的制备及体外评价[J].中草药,2015,46(3):348-352.
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[22]Kakkar V,Muppu S K,Chopra K,et al.Curcumin loaded solid lipid nanoparticles:An efficient formulation approach for cerebral ischemic reperfusion injury in rats[J].Eur J Pharm Biopharm,2013,85(3 Pt A):339-345.
[23]孙敏捷,张乐洋,平其能.长春碱PCL-PEG-PCL纳米粒的制备及质量评价[J].中国药科大学学报,2010,41(1):29-34.
[24]Essa S,Rabanel J M,Hildgen P.Effect of polyethylene glycol(PEG)chain organization on the physicochemical properties of poly(D,L-lactide)(PLA)based nanoparticles[J].Eur J Pharm Biopharm,2010,75(2):96-106.
[25]Owens III D E,Peppas N A.Opsonization,biodistribution,and pharmacokinetics of polymeric nanoparticles[J].Int J Pharm,2006,307(1):93-102.
[26]高红军.功能协同的复合胶束作为抗肿瘤纳米药物载体的研究[D].天津:南开大学,2014.
[27]王岩.不同分子量的MPEG-PLGA纳米药物载体的体外降解和药物释放行为研究[D].长春:吉林大学,2014.
[28]Thauvin C,Schwarz B,Delie F,et al.Functionalized PLA polymers to control loading and/or release properties of drug-loaded nanoparticles[J].Int J Pharm,2018,548(2):71-77.
[2]Ma S,Yin H,Chen L,et al.Neuroprotective effect of ginkgolide K against acute ischemic stroke on middle cerebral ischemia occlusion in rats[J].J Nat Med,2012,66(1):25-31.
[3]马舒伟,陈旅翼,何盛江,等.银杏内酯K对脑缺血的保护作用[J].中国现代应用药学,2011,28(10):877-880.
[4]Ma S,Liu X,Xun Q,et al.Neuroprotective effect of ginkgolide K against H2O2-induced PC12 cell cytotoxicity by ameliorating mitochondrial dysfunction and oxidative stress[J].Biol Pharm Bull,2014,37(2):217-225.
[5]Ma S,Liu H,Jiao H,et al.Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC12cells via inhibition of ROS generation and Ca(2+)influx[J].Neurotoxicology,2012,33(1):59-69.
[6]Liu Q,Li X,Li L,et al.Ginkgolide K protects SH-SY5Y cells against oxygen-glucose deprivation-induced injury by inhibiting the p38 and JNK signaling pathways[J].Mol Med Rep,2018,18(3):3185-3192.
[7]Fan Z Y,Liu X G,Guo R Z,et al.Pharmacokinetic studies of ginkgolide K in rat plasma and tissues after intravenous administration using ultra-high performance liquid chromatography-tandem mass spectrometry[J].JChromatogr B Analyt Technol Biomed Life Sci,2015,988:1-7.
[8]Yildiz T,Gu R,Zauscher S,et al.Doxorubicin-loaded protease-activated near-infrared fluorescent polymeric nanoparticles for imaging and therapy of cancer[J].Int JNanomed,2018,13:6961-6986.
[9]邱瀚弘,朱志军,张雪洁,等.药载比对和厚朴酚-分枝状聚乙二醇聚合物G2纳米粒形态及体外抗肿瘤活性的影响[J].药物评价研究,2018,41(11):1951-1957.
[10]朱文静,张良珂.载和厚朴酚介孔二氧化硅包覆聚吡咯纳米粒的制备研究[J].中草药,2018,49(9):2057-2062.
[11]Soni N,Dhiman R C.Phytochemical,anti-oxidant,larvicidal,and antimicrobial activities of castor(Ricinus communis)synthesized silver nanoparticles[J].Chin Herb Med,2017,9(3):289-294.
[12]Wang Y,Rapakousiou A,Ruiz J,et al.Metalation of polyaminedendrimers with ethynyl cobalticenium for the construction of mono-and heterobimetallic polycationicmetal lodendrimers[J].Chemistry,2014,20(35):11176-11186.
[13]徐骏军,陈丹飞,宋倩倩,等.p H值响应释药As2O3聚乙二醇-聚己内酯-聚乙烯亚胺纳米粒的制备及体外评价[J].中草药,2018,49(23):5532-5540.
[14]刘洋,谢栓栓,曾洁,等.表面修饰适配体S6的聚乳酸-羟基乙酸/聚乙二醇共聚物纳米粒制备及其运载小干扰RNA的应用研究[J].有机化学,2018,38(10):2706-2712.
[15]童晓东,范永春,严玮.姜黄素维生素E聚乙二醇琥珀酸酯-聚乙二醇硬脂酸酯15胶束对姜黄素溶解度和口服生物利用度的影响[J].中草药,2017,48(5):902-906.
[16]Veronese F M,Pasut G.PEGylation,successful approach to drug delivery[J].Drug Discov Today,2005,10(21):1451-1458.
[17]Fernandes C,Martins C,Fonseca A,et al.PEGylated PLGA nanoparticles as a smart carrier to increase the cellular uptake of a coumarin-based monoamine oxidase B inhibitor[J].ACS Appl Mater Interfaces,2018,10(46):39557-39569.
[18]刘朝勇,肖云芝,李瑞生,等.小菜蛾抗菌肽聚乳酸-羟基乙酸共聚物纳米粒的制备及体外评价[J].中草药,2015,46(3):348-352.
[19]Fahmy T M,Samstein R M,Harness C C,et al.Surface modification of biodegradable polyesters with fatty acid conjugates for improved drug targeting[J].Biomaterials,2005,26(28):5727-5736.
[20]Sonkusare S K,Kaul C L,Ramarao P.Dementia of Alzheimer’s disease and other neurodegenerative disorders-memantine,a new hope[J].Pharm Res,2005,51(1):1-17.
[21]Cano A,Ettcheto M,Espina M,et al.Epigallocatechin-3-gallate loaded PEGylated-PLGA nanoparticles:A new anti-seizure strategy for temporal lobe epilepsy[J].Nanomedicine,2018,14(4):1073-1085.
[22]Kakkar V,Muppu S K,Chopra K,et al.Curcumin loaded solid lipid nanoparticles:An efficient formulation approach for cerebral ischemic reperfusion injury in rats[J].Eur J Pharm Biopharm,2013,85(3 Pt A):339-345.
[23]孙敏捷,张乐洋,平其能.长春碱PCL-PEG-PCL纳米粒的制备及质量评价[J].中国药科大学学报,2010,41(1):29-34.
[24]Essa S,Rabanel J M,Hildgen P.Effect of polyethylene glycol(PEG)chain organization on the physicochemical properties of poly(D,L-lactide)(PLA)based nanoparticles[J].Eur J Pharm Biopharm,2010,75(2):96-106.
[25]Owens III D E,Peppas N A.Opsonization,biodistribution,and pharmacokinetics of polymeric nanoparticles[J].Int J Pharm,2006,307(1):93-102.
[26]高红军.功能协同的复合胶束作为抗肿瘤纳米药物载体的研究[D].天津:南开大学,2014.
[27]王岩.不同分子量的MPEG-PLGA纳米药物载体的体外降解和药物释放行为研究[D].长春:吉林大学,2014.
[28]Thauvin C,Schwarz B,Delie F,et al.Functionalized PLA polymers to control loading and/or release properties of drug-loaded nanoparticles[J].Int J Pharm,2018,548(2):71-77.