胡桃醌-PLGA纳米粒制备及对A375恶黑细胞的体外抗肿瘤作用
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摘要
目的:制备胡桃醌(juglone,Jug)聚乳酸-羟基乙酸(poly lactic-co-glycolic acid,PLGA)纳米粒(Jug-PLGA-NPs),并考察其理化性质、体外释放特征及对A375细胞的体外影响。方法:采用乳化挥发法制备Jug-PLGA-NPs,对其粒径、包封率、载药率以及体外释放特征进行考察;荧光显微镜观察PLGA-NPs在体外细胞的摄取情况,小动物活体成像仪观测PLGA-NPs在BALB/c荷瘤裸鼠尾静脉注射后体内的分布;用噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)比色法检测其对A375细胞增殖抑制作用,流式细胞仪进行细胞凋亡率及细胞周期检测;蛋白免疫印迹法检测蛋白激酶B(protein kinase B,Akt),磷酸化-Akt(p-Akt),周期蛋白D1(cyclin D1)的表达情况。结果:制备的Jug-PLGA-NPs平均粒径为(149.6±21.5) nm,包封率为(68.39±2.51)%,载药率(5.07±0.98)%,具有良好的缓释特征。PLGA-NPs在体外细胞摄取和体内活体成像中具有良好的穿透和靶向性能。不同浓度Jug-PLGA-NPs均能明显抑制A375细胞增殖、促进细胞凋亡,呈明显时间浓度依赖性(P<0.05),且48 h作用略优于等浓度Jug;其机制可能与调节Akt磷酸化水平,下调cyclin D1表达(P<0.05),阻滞细胞于G0/G1期有关(P<0.05)。结论:负载Jug的PLGA纳米微粒制备简便,具有良好的药物缓释、肿瘤靶向及抗肿瘤能力,为未来Jug的临床应用提供了一种新的药物剂型。
Objective:To prepare Juglone-loaded poly lactic-co-glycolic acid nanoparticles(Jug-PLGANPs),and investigate their physicochemical properties,release characteristics in vitro and anti-tumor activities on A375 melanoma cells in vitro.Method:Jug-PLGA-NPs were prepared by emulsification-solvent evaporation method.Then the particle size,encapsulation efficiency,drug loading rate and in vitro release characteristics were investigated.Fluorescence microscopy was used to observe the uptake of PLGA-NPs in vitro.The distribution of PLGA-NPs in BALB/c nude mice after tail vein injection was observed by the small living animal imaging system.Their inhibition effect on proliferation of A375 cells was detected by thiazolyl blue tetrazolium bromide(MTT)assay.Apoptosis rate and cell cycle detection were performed by flow cytometry.Western blot was used to determine the protein kinase B(Akt),phosphorylated Akt(p-Akt) and cyclinD1.Result:The average particle size of the prepared Jug-PLGA-NPs was(149.6 ± 21.5) nm,entrapment rate of(68.39 ± 2.51) %,and drugloading rate of(5.07 ± 0.98) %,showing good sustained-release characteristics.PLGA-NPs showed good penetration and targeting properties in cellular uptake in vitro and in vivo imaging.Different concentrations of JugPLGA-NPs could significantly inhibit the proliferation and promote apoptosis of A375 cells in a time and concentration dependent manner(P<0.05),and its 48 h effect was superior to that of the same concentration of Jug.The mechanism may be related to the regulation of Akt phosphorylation level,down-regulation of cyclinD1 expression(P<0.05) and the cell-cycle arrest in G0/G1 phase(P<0.05).Conclusion:The Jug-PLGA-NPs are easy to prepare and have good sustained-release characteristics,tumor targeting and anti-tumor ability,providing a new pharmaceutical dosage form for the future clinical application of Jug.
Objective:To prepare Juglone-loaded poly lactic-co-glycolic acid nanoparticles(Jug-PLGANPs),and investigate their physicochemical properties,release characteristics in vitro and anti-tumor activities on A375 melanoma cells in vitro.Method:Jug-PLGA-NPs were prepared by emulsification-solvent evaporation method.Then the particle size,encapsulation efficiency,drug loading rate and in vitro release characteristics were investigated.Fluorescence microscopy was used to observe the uptake of PLGA-NPs in vitro.The distribution of PLGA-NPs in BALB/c nude mice after tail vein injection was observed by the small living animal imaging system.Their inhibition effect on proliferation of A375 cells was detected by thiazolyl blue tetrazolium bromide(MTT)assay.Apoptosis rate and cell cycle detection were performed by flow cytometry.Western blot was used to determine the protein kinase B(Akt),phosphorylated Akt(p-Akt) and cyclinD1.Result:The average particle size of the prepared Jug-PLGA-NPs was(149.6 ± 21.5) nm,entrapment rate of(68.39 ± 2.51) %,and drugloading rate of(5.07 ± 0.98) %,showing good sustained-release characteristics.PLGA-NPs showed good penetration and targeting properties in cellular uptake in vitro and in vivo imaging.Different concentrations of JugPLGA-NPs could significantly inhibit the proliferation and promote apoptosis of A375 cells in a time and concentration dependent manner(P<0.05),and its 48 h effect was superior to that of the same concentration of Jug.The mechanism may be related to the regulation of Akt phosphorylation level,down-regulation of cyclinD1 expression(P<0.05) and the cell-cycle arrest in G0/G1 phase(P<0.05).Conclusion:The Jug-PLGA-NPs are easy to prepare and have good sustained-release characteristics,tumor targeting and anti-tumor ability,providing a new pharmaceutical dosage form for the future clinical application of Jug.
引文
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[12]黄可心,王晓扬,沈韵桐,等.胡桃醌叶酸修饰自微乳的体内外靶向性研究[J].中国医院药学杂志,2016,36(4):285-289.
[13]JIN J,ZHANG Y,LI Y,et al.RNA-interferencemediated downregulation of Pin1 suppresses tumorigenicity of malignant melanoma A375 cells[J].Neoplasma,2012,60(1):92-100.
[14]Arasoglu T,Derman S,Mansuroglu B,et al.Synthesis,characterization and antibacterial activity of juglone encapsulated PLGA nanoparticles[J].J Appl Microbiol,2017,123(6):1407-1419.
[15]ZHANG Z,QIAN H,YANG M,et al.Gambogic acidloaded biomimetic nanoparticles in colorectal cancer treatment[J].Int J Nanomedicine,2017,12:1593-1605.
[16]GUO J,SI L,KONG Y,et al.PhaseⅡ,open-label,single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification[J].J Clin Oncol,2011,29(21):2904-2909.
[17]Kruiswijk F,Hasenfuss S C,Sivapatham R,et al.Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling[J].Oncogene,2016,35(17):2166-2177.
[18]Kim G,Kim J Y,Lim S C,et al.SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development[J].Faseb J,2018,doi:fj201700645.
[19]XU G G,Etzkorn F A.Pin1 as an anticancer drug target[J].Drug News Perspect,2009,22(7):399-407.
[20]Hariharan N,Sussman M A.Pin1:a molecular orchestrator in the heart[J].Trends Cardiovasc Med,2014,24(6):256-262.
[21]Bartek J,Lukas J.DNA repair:Cyclin D1multitasks[J].Nature,2011,474(7350):171-172.
[22]Liou Y C,Ryo A,HUANG H K,et al.Loss of Pin1function in the mouse causes phenotypes resembling cyclin D1-null phenotypes[J].Proc Natl Acad Sci U SA,2002,99(3):1335-1340.
[23]TAO X,ZHANG H,Park S S,et al.Loss of Pin1,suppresses hedgehog-driven medulloblastoma tumorigenesis 1[J].Neoplasia,2017,19(3):216-225.
[24]陈静岚,张国屏,孙玉波.复方青龙衣配合化疗治疗急性白血病23例[J].黑龙江中医药,1996,25(4):26-27.
[25]任生,孟丽,王桂花,等.中药青龙衣注射剂复合化疗药物介入治疗肺癌13例报告[J].中国中医基础医学杂志,2000,6(7):27-29.
[26]刘薇,林文翰,季宇彬.青龙衣毒性作用及体外抗肿瘤作用的实验研究[J].中国中药杂志,2004,29(9):4-10.
[27]CHAO S H,Greenleaf A L,Price D H.Juglone,an inhibitor of the peptidyl-prolyl isomerase Pin1,also directly blocks transcription[J].Nucleic Acids Res,2001,29(3):767-773.
[2]BAO L,Kimzey A,Sauter G,et al.Prevalent overexpression of prolyl isomerase Pin1 in human cancers[J].Am J Pathol,2004,164(5):1727-1737.
[3]MIN S H,ZHOU X Z,LU K P.The role of Pin1 in the development and treatment of cancer[J].Arch Pharm Res,2016,39(12):1609-1620.
[4]沈广志,邹桂华,梁婷,等.核桃楸的化学成分研究进展[J].中国实验方剂学杂志,2015,21(17):219-224.
[5]Hennig L,Christner C,Kipping M,et al.Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone[J].Biochemistry,1998,37(17):5953-5960.
[6]FANG F,QIN Y X,QI L,et al.Juglone exerts antitumor effect in ovarian cancer cells[J].Iran J Basic Med Sci,2015,18(6):544-548.
[7]JI Y B,XIN G S,QU Z Y,et al.Mechanism of juglone-induced apoptosis of MCF-7 cells by the mitochondrial pathway[J].Genet Mol Res,2016,15(3):123-131.
[8]Aithal B K,Kumar M R,RAO B N,et al.Juglone,a naphthoquinone from walnut,exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells[J].Cell Biol Int,2009,33(10):1039-1049.
[9]陈丽,张建,汪思应,等.胡桃醌对小鼠黑色素瘤细胞B16F10体内迁移的影响[J].中国临床药理学与治疗学,2012,17(9):978-982.
[10]Torchilin V.Tumor delivery of macromolecular drugs based on the EPR effect[J].Adv Drug Deliv Rev,2011,63(3):131-135.
[11]杨倩,罗国伟,彭海龙,等.胡桃醌脂质体的制备及理化性质[J].南昌大学学报:工科版,2015,37(3):234-239.
[12]黄可心,王晓扬,沈韵桐,等.胡桃醌叶酸修饰自微乳的体内外靶向性研究[J].中国医院药学杂志,2016,36(4):285-289.
[13]JIN J,ZHANG Y,LI Y,et al.RNA-interferencemediated downregulation of Pin1 suppresses tumorigenicity of malignant melanoma A375 cells[J].Neoplasma,2012,60(1):92-100.
[14]Arasoglu T,Derman S,Mansuroglu B,et al.Synthesis,characterization and antibacterial activity of juglone encapsulated PLGA nanoparticles[J].J Appl Microbiol,2017,123(6):1407-1419.
[15]ZHANG Z,QIAN H,YANG M,et al.Gambogic acidloaded biomimetic nanoparticles in colorectal cancer treatment[J].Int J Nanomedicine,2017,12:1593-1605.
[16]GUO J,SI L,KONG Y,et al.PhaseⅡ,open-label,single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification[J].J Clin Oncol,2011,29(21):2904-2909.
[17]Kruiswijk F,Hasenfuss S C,Sivapatham R,et al.Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling[J].Oncogene,2016,35(17):2166-2177.
[18]Kim G,Kim J Y,Lim S C,et al.SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development[J].Faseb J,2018,doi:fj201700645.
[19]XU G G,Etzkorn F A.Pin1 as an anticancer drug target[J].Drug News Perspect,2009,22(7):399-407.
[20]Hariharan N,Sussman M A.Pin1:a molecular orchestrator in the heart[J].Trends Cardiovasc Med,2014,24(6):256-262.
[21]Bartek J,Lukas J.DNA repair:Cyclin D1multitasks[J].Nature,2011,474(7350):171-172.
[22]Liou Y C,Ryo A,HUANG H K,et al.Loss of Pin1function in the mouse causes phenotypes resembling cyclin D1-null phenotypes[J].Proc Natl Acad Sci U SA,2002,99(3):1335-1340.
[23]TAO X,ZHANG H,Park S S,et al.Loss of Pin1,suppresses hedgehog-driven medulloblastoma tumorigenesis 1[J].Neoplasia,2017,19(3):216-225.
[24]陈静岚,张国屏,孙玉波.复方青龙衣配合化疗治疗急性白血病23例[J].黑龙江中医药,1996,25(4):26-27.
[25]任生,孟丽,王桂花,等.中药青龙衣注射剂复合化疗药物介入治疗肺癌13例报告[J].中国中医基础医学杂志,2000,6(7):27-29.
[26]刘薇,林文翰,季宇彬.青龙衣毒性作用及体外抗肿瘤作用的实验研究[J].中国中药杂志,2004,29(9):4-10.
[27]CHAO S H,Greenleaf A L,Price D H.Juglone,an inhibitor of the peptidyl-prolyl isomerase Pin1,also directly blocks transcription[J].Nucleic Acids Res,2001,29(3):767-773.