靶向肝癌的多柔比星偶联嵌段共聚物纳米胶束表征及对肝癌HepG2细胞和Huh7细胞的抑制作用

Characterization of liver cancer-targeted doxorubicin coupling segmented copolymer nanomicelles and its inhibitory effect on hepatocellular carcinoma HepG2 and Huh7 cells

目的探讨制备的具有被动靶向和主动靶向肝癌双重作用的多柔比星(DOX)偶联嵌段共聚物纳米胶束的表征和体外抗肿瘤作用。方法采用化学方法共价偶联DOX和聚乳酸-羟基乙酸-聚乙二醇(PLGA-PEG)嵌段共聚物,合成DOX-PLGA-PEG,应用傅里叶变换红外光谱学和磁共振法验证酰胺键是否形成。利用透析法,在水相中自组装形成DOX-PLGA-PEG纳米胶束。通过物理吸附法,将肝癌特异性抗体HAb18 F(ab')2修饰至DOX-PLGA-PEG胶束,即靶向胶束。采用动态光散射法,使用粒度分析仪研究纳米胶束粒径大小及分散范围。扫描电镜观察胶束形态。紫外分光光度法测定DOX-PLGA-PEG胶束或靶向胶束载药率、包封率,并行模拟体外释药实验。相差显微镜下观察2 mg/ml DOX-PLGA-PEG胶束或靶向胶束作用后肝癌HepG2和Huh7细胞形态变化;应用平板克隆形成实验分析1 mg/ml DOX-PLGA-PEG胶束或靶向胶束作用后细胞存活情况。结果应用红外光谱观察到在波数1575 cm-1处出现一波峰,符合酰胺键波峰的位置,即酰胺键连接DOX的伯胺基和PLGA-PEG的末端羟基后合成DOX-PLGA-PEG。DOX-PLGA-PEG胶束和靶向胶束的粒径分别为(55.0±6.3)nm和(87.6±9.3)nm,多分散指数分别为0.098和0.142。扫描电镜观察发现纳米胶束呈圆球形,表面平滑。DOX-PLGA-PEG胶束和靶向胶束的载药率分别为(2.4±0.2)%和(2.2±1.9)%,包封率分别为(91.7±5.3)%和(87.5±4.8)%。DOX-PLGA-PEG胶束和靶向胶束的模拟体外释药曲线呈双相:在爆发释放之后为缓慢释放,5 d内DOX释药率约为30%,6 h内释药率为25%。2 mg/ml DOX-PLGA-PEG胶束或靶向胶束作用后,肝癌HepG2和Huh7细胞形态均发生损伤性改变,部分细胞死亡,克隆形成能力降低;靶向胶束较DOX-PLGA-PEG胶束作用明显。1 mg/ml DOX-PLGA-PEG胶束和靶向胶束作用后,HepG2和Huh7细胞存活率均随时间下降,且靶向胶束较DOX-PLGA-PEG胶束效果明显(均P<0.05)。DOX-PLGA-PEG胶束和靶向胶束对于Huh7细胞50%有效抑制分别出现在给药后2.4、5.5 d,DOX释放浓度分别为1.15、1.24μg/ml;DOX-PLGA-PEG胶束和靶向胶束对于HepG2细胞的50%有效抑制分别出现在给药后3.3、7.4 d,DOX释放浓度分别为1.20、1.31μg/ml。结论构建的被动靶向和主动靶向双重作用的DOX纳米胶束体外可大量释放活性药物,对肝癌HepG2和Huh7细胞增殖有明显抑制作用。

Objective To investigate the characterization of doxorubicin(DOX)coupling segmented copolymer nanomicelles with dual effects of passive and active targeting to liver cancer and its antineoplastic function in vitro.Methods DOX was covalently conjugated to a terminal hydroxyl group of poly lactic-co-glycolic acid-poly ethylene glycol(PLGA-PEG)diblock copolymer to form DOX-PLGA-PEG.The formation of amido bond was determined by using fourier transform infrared spectroscopy(FTIR)and magnetic resonance method.Amphiphatic diblock copolymer DOX-PLGA-PEG could self-assemble to form nanomicelles in an aqueous phase by dialysis method.The DOX-PLGA-PEG targeted micelles decorated with liver cancer HAb18 F(ab')2 specific antibody were prepared by using physical bonding method.The size and the scattering scope of nanomicelles was determined by using granulometer and dynamic light scattering(DLS).Micelle morphology was examined by using scanning electron microscopy(SEM).The drug loading rate and entrapment rate of DOX-PLGA-PEG micelles or targeted micelles were measured by using ultraviolet spectrophotometry method,and stimulated release in vitro experiment was done.After administration of 2 mg/ml DOX-PLGA-PEG or targeted micelles,cell morphology change of liver cancer HepG2 and Huh7 was observed by using the phase-contrast photomicrography.After administration of 1 mg/ml DOX-PLGA-PEG or targeted micelles,cell survival was analyzed by using plate clone formation assay.Results The spectrum peak was around 1575 cm-1 under the observation of FTIR,which was accord with the location of the peak of amido bond.Activating with p-nitrophenyl chloroformate,DOX was covalently conjugated to PLGA-PEG to produce DOX-PLGA-PEG via a carbamate linkage between the primary amine group in DOX and the terminal hydroxyl group in PLGA-PEG.DLS measurements showed that the diameter of DOX-PLGA-PEG micelles and targeted micelles was(55.0±6.3)nm and(87.6±9.3)nm,respectively,and polydispersity index was 0.098 and 0.142,respectively.SEM micrographs revealed that these nanomicelles had a spherical morphology and relatively smooth surface.Drug loading rate of DOX-PLGA-PEG micelles and targeted micelles was(2.4±0.2)%and(2.2±1.9)%,and the entrapment rate was(91.7±5.3)%and(87.5±4.8)%,respectively.The drug release curve in vitro of DOX-PLGA-PEG micelles and targeted micelles exhibited a biphasic pattern characterized by a fast initial release,followed by a slower and continuous release.The amount of the drug release rate was about 30%within 5 d,and 25%within 6 h.After 2 mg/ml DOX-PLGA-PEG micelles and targeted micelles,the cell morphology of liver cancer HepG2 and Huh7 had the impaired change,and the part of the cells were dead,the clonality decreased.The effect of targeted micelles was more significant compared with DOX-PLGA-PEG micelles.After DOX-PLGA-PEG micelles and targeted micelles,the survival rates of HepG2 and Huh7 cells were decreased with time,and the effect of targeted micelles was more effective compared with DOX-PLGA-PEG micelles(all P<0.05).The 50%effective inhibition of the targeted micelles and DOX-PLGA-PEG micelles was obtained in 2.4 d and 5.5 d,respectively for Huh7 cells.At these time points,DOX concentration was 1.15μg/ml and 1.24μg/ml,respectively.The 50%effective inhibition of targeted micelles and DOX-PLGA-PEG micelles was obtained in 3.3 d and 7.4 d,respectively for HepG2 cells.At these time points,DOX concentration was 1.20μg/ml and 1.31μg/ml.Conclusion DOX nanomicelles with dual effects of passive and active targeting can release a large number of active drugs in vitro,which plays an obvious inhibitory role in the cell proliferation of liver cancer HepG2 and Huh7 cells.