载5-氟尿嘧啶壳聚糖纳米粒的制备及抗肿瘤活性研究

发布时间：2018-03-13 12:40

本文选题：壳聚糖　切入点：5-氟尿嘧啶　出处：《广东药学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的：化学合成5-氟尿嘧啶-壳聚糖-聚乙二醇单甲醚（5-FU-CS-mPEG）大分子前药；制备5-FU-CS-mPEG纳米粒；并考察其制备处方、体外释放及动物药效学行为。方法：采用两步合成法制备5-FU-CS-mPEG，首先在EDC/NHS的作用下，合成5-氟尿嘧啶-壳聚糖（5-FU-CS）前药，再引入亲水性mPEG，制备出亲水性较好的5-FU-CS-mPEG前药；采用红外、1H-NMR及差示热分析法表征合成产物。采用离子凝胶法制备5-FU-CS-mPEG纳米粒，紫外分光光度法测定其载药量，激光粒度分析仪测定粒径、粒径分布及Zeta电位，电子透射电镜观察其外观形态，动态透析法考察其体外释放情况并用各种释放模型进行拟合。动物水平药效学实验，以昆明小鼠为实验对象，建立H22肝癌移植瘤模型，考察高中低浓度5-FU-CS-mPEG纳米粒、mPEG-CS纳米粒及5-FU的抗癌活性及潜在的毒副作用，实验结束后，计算其抑瘤率、脾指数及胸腺指数，并制肿瘤HE染色切片，实验数据采用SPSS软件处理、分析。结果：经红外、1H-NMR及差示热分析法确证，已成功合成5-FU-CS-mPEG大分子前药；通过对5-FU-CS合成工艺进行优化，，得出制备5-FU-CS的最佳合成条件为EDC·HCl：5-FUA为1.5:1、EDC·HCl：NHS的为1:1、5-FUA：CS的摩尔比为1:1、反应时间为1d。紫外分光光度法测得5-FU-CS-mPEG纳米粒的载药量为4.2%，激光粒度仪测得空白纳米粒（mPEG-CS）、载药纳米粒（5-FU-CS-mPEG）的平均粒径分别为169.2nm、259.8nm，Zeta电位分别为+42.55mv、+39.27mv；透射电镜观察纳米粒形态规则、分散性较好；体外释放试验发现，载药纳米粒在pH=5.5、pH=7.2的缓冲液中均有一定的缓释作用， pH=5.5缓冲液中的释放符合零级方程，pH=7.2缓冲液中的释放符合Weibull模型。动物药效学实验显示载药纳米粒高、中、低浓度组的抑瘤率分别为62.05%、48.79%及36.14%，与阴性对照组相比具有显著性差异（P＜0.05），高剂量组抑瘤率与阳性对照组（5-FU）相当（P＞0.05）；病理组织切片显示给药组的细胞均出现不同程度地坏死、凋亡。结论：采用离子凝胶法制备的载药纳米粒粒径分布较均匀，形态规整；并且该载药纳米粒在体内外均表现较好的缓释特性，动物实验也表现出较强的抗肿瘤活性，因此该前药纳米制剂有望成为5-FU的新型载体。
[Abstract]:Aim: to synthesize 5-fluorouracil chitosan polyethylene glycol monomethyl ether 5-FU-CS-mPEG macromolecular prodrug, to prepare 5-FU-CS-mPEG nanoparticles, and to investigate the preparation of 5-FU-CS-mPEG nanoparticles. Methods: 5-FU-CS-mPEG was prepared by two-step synthesis. Firstly, 5-FU-CS-mPEG was synthesized by EDC/NHS, then hydrophilic mPEG was introduced to prepare 5-FU-CS-mPEG. 5-FU-CS-mPEG nanoparticles were prepared by ion-gel method, the drug loading was determined by ultraviolet spectrophotometry, the particle size, particle size distribution and Zeta potential were measured by laser particle size analyzer. Electron transmission electron microscopy (TEM) was used to observe its appearance, dynamic dialysis was used to investigate its release in vitro and all kinds of release models were fitted. The anticancer activity and potential side effects of 5-FU-CS-mPEG nanoparticles and 5-FU nanoparticles were investigated. After the experiment, tumor inhibition rate, spleen index and thymus index were calculated, and HE staining sections were made. The experimental data were processed by SPSS software. Analysis. Results: 5-FU-CS-mPEG prodrug was successfully synthesized by IR 1H-NMR and differential thermal analysis, and the synthesis process of 5-FU-CS was optimized. The optimum synthetic conditions for the preparation of 5-FU-CS were obtained as follows: the molar ratio of EDC 路HCl:5-FUA to 1.5: 1EDC 路HCl:NHS was 1: 1 and the reaction time was 1 day. The UV spectrophotometric method showed that the drug loading capacity of 5-FU-CS-mPEG nanoparticles was 4.2. The blank nanoparticles were obtained by laser particle size analyzer, and the drug-loaded nanoparticles 5-FU-CS-mPEG) were obtained. The average particle size is 169.2 nm ~ 259.8 nm ~ (-1) Zeta potential is 42.55 mv and 39.27 mv respectively, and the morphology of the nanoparticles is regular by transmission electron microscope (TEM). In vitro release test found that, The release of drug loaded nanoparticles in buffer solution of pH 5. 5 ~ 5 ~ (5) ~ (5) ~ (2) ~ (2) ~ (2) was consistent with the Weibull model. The results of animal pharmacodynamics experiments showed that the drug loaded nanoparticles were high and medium, and the release in pH = 5. 5 buffer solution was in accordance with the zero order equation (pH = 5. 5) and the release in pH ~ (2 +) buffer was consistent with the Weibull model. The tumor inhibition rates in the low concentration group were 48.79% and 36.14%, respectively, which were significantly different from those in the negative control group (P < 0.05). The tumor inhibition rate in the high dose group was similar to that in the positive control group (P > 0.05). Conclusion: the drug loaded nanoparticles prepared by ion-gel method have more uniform particle size distribution and regular morphology, and the drug loaded nanoparticles exhibit good sustained release characteristics in vivo and in vitro, and the animal experiments also show strong anti-tumor activity. Therefore, the prodrug nanometer preparation is expected to become a new carrier of 5-FU.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R965

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