转铁蛋白功能化的β-榄香烯-雷公藤红素共传递微乳协同靶向抗结直肠癌研究  

Transferrin-functionalized co-delivery microemulsion of β-elemene and celastrol for synergistic anti-colorectal cancer treatment

作　　者：沈展 陈文斌[1] SHEN Zhan;CHEN Wen-bin(The First Affiliated Hospital of Zhejiang University Medical College,Hangzhou 310011,China)

机构地区：[1]浙江大学医学院附属第一医院,浙江杭州310011

出　　处：《中草药》2019年第2期471-480,共10页Chinese Traditional and Herbal Drugs

摘　　要：目的验证转铁蛋白修饰的β-榄香烯-雷公藤红素共传递微乳(Tf-EC-MEs)协同靶向抗结直肠癌作用。方法采用四甲基偶氮唑盐(MTT)法检测β-榄香烯、雷公藤红素及联合给药对结直肠癌Lovo细胞和结肠癌HT-29细胞的细胞毒活性,优化最佳质量配比;采用"混匀-滴注"方法制备Tf-EC-MEs,并利用高效液相(HPLC)、激光粒度仪、透射电镜等表征粒子的制剂学及理化性质;采用MTT法、高效液相-二喹啉甲酸(HPLC-BCA)法、膜联蛋白V-PE/7-氨基放线菌素D(Annexin V-PE/7-AAD)试剂盒考察Tf-EC-MEs的体外抗肿瘤活性及对细胞摄取、细胞凋亡的影响;sc Lovo细胞制备荷瘤裸鼠模型,每隔2d分别iv给予β-榄香烯+雷公藤红素、β-榄香烯-雷公藤红素共传递微乳(EC-MEs)、Tf-EC-MEs,考察Tf-EC-MEs对小鼠肿瘤生长、体质量及生存时间的影响。结果β-榄香烯-雷公藤红素40∶1联合给药对Lovo和HT-29细胞的半数抑制浓度(IC50)分别为(17.5±2.9)、(36.4±3.6)μg/mL,联合指数(CI)分别为0.89和0.96,具有明显的协同抗结直肠癌效应;Tf-EC-MEs对Lovo和HT-29细胞的IC50分别为(11.7±0.6)和(27.4±1.2)μg/mL,CI分别为0.61和0.72。Tf-EC-MEs与Lovo细胞孵育4 h后的摄取量为7.2μg/mg,是β-榄香烯+雷公藤红素给药组的3.3倍。Tf-EC-MEs能够引发59.2%的Lovo细胞凋亡,显著高于β-榄香烯+雷公藤红素和EC-MEs组。Tf-EC-MEs对荷Lovo大肠癌裸鼠的肿瘤生长抑制率最为明显,且裸鼠60 d后生存率为37.5%。Tf-EC-MEs给药组裸鼠的肿瘤组织HE染色切片出现大量的细胞坏死,Ki-67免疫组化切片显示肿瘤细胞增殖被明显抑制。结论相较于β-榄香烯+雷公藤红素组和EC-MEs组,Tf-EC-MEs具有更优的协同靶向抗结直肠癌的潜力。Objective To verify the synergistic effect of transferrin modified β-elemene and celastrol co-loaded microemulsion(Tf-EC-MEs) on anti-colorectal cancer treatment.Methods The optimal mass ratio of β-elemene and celastrol to growth inhibition of Lovo and HT-29 colorectal cancer cells was optimized by MTT staining method in vitro.Tf-EC-MEs was prepared by "mixing-dripping" method,and the preparation and physicochemical properties of the particles were characterized by high performance liquid chromatography(HPLC),laser particle analyzer,and transmission electron microscope.The MTT staining,HPLC-BCA combined method,and Annexin V-PE/7-Aminoactinomycin D(Annexin V-PE/7-AAD) kit were used to investigate the antitumor activity of Tf-EC-MEs in vitro,and its effect on cell uptake,and apoptosis of tumor cells.The tumor-bearing nude mice model was established by subcutaneous injection of Lovo cells,and the tumor growth,weight,and survival time were observed after intravenous injection of β-elemene + celastrol,β-elemene-celastrol co-loaded microemulsion(EC-MEs),and Tf-EC-MEs.Results The combined administration of β-elemene and celastrol(40∶1) had significant synergistic effect on the anti-colorectal cancer of Lovo and HT-29 cells.IC50 of β-elemene + celastrol in Lovo and HT-29 cells were(17.5 ± 2.9) and(36.4 ± 3.6) μg/mL,with the CI as 0.89 and 0.96,respectively.IC50 of Tf-EC-MEs in Lovo and HT-29 cells were(11.7 ± 0.6) and(27.4 ± 1.2) μg/mL,with the CI as 0.61 and 0.72 respectively.The 4 h of Lovo uptake of Tf-EC-MEs was 7.2 μg/mg,which was 3.3 times higher than that of β-elemene + celastrol.Tf-EC-MEs induced apoptosis in 59.2% of Lovo cells,which was significantly higher than that in beta-elemene + celastrol and EC-MEs groups.Tf-EC-MEs showed the overwhelming inhibition of growth of Lovo tumor-bearing tumors.The survival rate of Tf-EC-MEs-treated mice was 37.5% at day 60.In Tf-EC-MEs treated group,HE staining sections of tumor tissues showed substantial cell necrosis and the Ki-67 immunohistochemical sections

关 键 词：Β-榄香烯 雷公藤红素 微乳 协同靶向治疗 结直肠癌 

分 类 号：R285.5[医药卫生—中药学]