亚油酸修饰的普鲁兰自组装胶束作为药物载体的研究
发布时间：2018-10-25 12:25
【摘要】：在恶性肿瘤的化疗过程中,化疗药物缺乏对肿瘤的选择性,并且存在严重的副作用,降低了治疗效果。然而纳米药物载体能够改变药物的体内分布,对肿瘤组织具有靶向性,可延长药物的作用时间,降低毒副作用,在肿瘤的治疗领域具有良好的应用前景。 本文将亚油酸(Linoleic acid)通过酯化反应偶联到普鲁兰多糖(Pullulan)长链上,获得两亲性聚合物PULA1、PULA2和PULA3。通过傅里叶变换红外光谱(FTIR)和核磁共振氢谱(1H NMR)对聚合物的化学结构进行表征,采用透析法制备自组装纳米胶束PULA,芘荧光探针法测得纳米胶束PULA1、PULA2和PULA3的临界胶束浓度(CMC)分别为70,62和50μ.g/mL,动态光散射(DLS)及透射电镜(TEM)结果显示纳米胶束PULA呈球形且均匀分布,粒径在80.1～156.8nm之间,体外溶血实验表明该纳米胶束具有低溶血率,表现出良好的生物相容性。 以抗癌药物阿霉素(DOX)为模型药物,通过透析法制备了载药纳米胶束PULA/DOX,纳米胶束PULA3/DOX的载药量和包封率分别达8.05%和88.6%。载药纳米胶束P ULA/DOX在pH=7.4的磷酸缓冲液(PBS)中持续释放DOX达96h以上,DOX的累积释放量在43%～55%之间,并且DOX的释放速率随释放介质pH的降低逐渐加快,该纳米胶束的pH敏感性有利于提高DOX在肿瘤组织的累积释放量。 通过荧光显微镜和流式细胞仪检测MCF-7和SMMC-7721细胞对纳米胶束PULA的摄取情况,结果表明纳米胶束PULA能够携带大量的DOX进入细胞；抑制剂处理后的细胞摄取实验表明PULA纳米胶束主要通过耗能的、肌动蛋白聚合介导的内吞作用进入细胞；纳米胶束PULA2/DOX对细胞周期影响的结果显示该载药纳米胶束主要将MCF-7和SMMC-7721细胞阻滞在S期,并且致使部分细胞出现凋亡；纳米胶束PULA进入细胞后的传递途径及分布结果表明纳米胶束PULA携带DOX经过内吞作用进入细胞后,传递到溶酶体中,在溶酶体内释放出来的DOX逐步迁移到细胞核发挥抗癌效果；细胞毒性试验表明空白纳米胶束对Raw264.7和HEK293正常细胞均没有毒性,载药纳米胶束PULA/DOX对MCF-7和SMMC-7721细胞的毒性呈现时间和浓度依赖性；PULA2/DOX纳米胶束的体内药物代谢结果表明PULA纳米胶束能够延长DOX在血液中的保留时间,维持血液中DOX的含量在较高浓度。 综上所述,PULA纳米胶束是一种新型的具有良好生物相容性的载体材料,有望在癌症治疗领域作为疏水抗癌药物DOX的传递载体。
[Abstract]:In the course of chemotherapy of malignant tumor, the chemotherapeutic drugs are lack of selectivity to the tumor, and have serious side effects, which reduces the therapeutic effect. However, nanopharmaceutical carriers can change the distribution of drugs in vivo, target tumor tissues, prolong the time of action of drugs, reduce toxic side effects, and have a good application prospect in the field of tumor treatment. In this paper, linoleic acid (Linoleic acid) was coupled to the long chain of (Pullulan) by esterification, and amphiphilic polymers PULA1,PULA2 and PULA3. were obtained. The chemical structure of the polymer was characterized by Fourier transform infrared spectroscopy (FTIR) and 1H NMR. The critical micelle concentration (CMC) of PULA1,PULA2 and PULA3 were 70 ~ 62 渭 g / mL and 50 渭 路g 路mL ~ (-1), respectively. The results of dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that the PULA of nano-micelles was spherical and uniform. In vitro hemolysis test showed that the micelle had low hemolysis rate and showed good biocompatibility. Using adriamycin (DOX) as a model drug, the drug loading and encapsulation efficiency of nano-micelle PULA3/DOX were 8.05% and 88.6%, respectively. The drug loaded nano-micelle P ULA/DOX continuously released DOX in pH=7.4 phosphate buffer (PBS) for more than 96 h, and the cumulative release of DOX was between 43% and 55%, and the release rate of DOX increased with the decrease of the release medium pH. The pH sensitivity of the nano-micelle is beneficial to increase the cumulative release of DOX in tumor tissue. The uptake of nano-micelle PULA by MCF-7 and SMMC-7721 cells was detected by fluorescence microscope and flow cytometry. The results showed that nano-micelle PULA could carry a large number of DOX into the cells. The results of cell uptake test showed that PULA nanoparticles entered the cells mainly through energy-consuming, actin polymerization mediated endocytosis. The effect of PULA2/DOX on cell cycle showed that the drug loaded nano-micelles mainly blocked MCF-7 and SMMC-7721 cells in S phase and caused apoptosis in some cells. The transmission pathway and distribution of nano-micelle PULA into cells showed that nano-micelle PULA carried DOX into the cells through endocytosis, then transferred to lysosome, and gradually migrated the DOX released in lysosome to the nucleus to play an anticancer effect. The cytotoxicity test showed that the blank micelles were not toxic to Raw264.7 and HEK293 normal cells. The toxicity of drug loaded nano-micelle PULA/DOX to MCF-7 and SMMC-7721 cells was time-and concentration-dependent. The results of drug metabolism of PULA2/DOX nanoparticles in vivo showed that PULA nanoparticles could prolong the retention time of DOX in blood and maintain the content of DOX in blood at a high concentration. In conclusion, PULA nanoparticles are a new biocompatible carrier material, which is expected to be used as the delivery carrier of hydrophobic anticancer drug DOX in the field of cancer treatment.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;TQ460.1
[Abstract]:In the course of chemotherapy of malignant tumor, the chemotherapeutic drugs are lack of selectivity to the tumor, and have serious side effects, which reduces the therapeutic effect. However, nanopharmaceutical carriers can change the distribution of drugs in vivo, target tumor tissues, prolong the time of action of drugs, reduce toxic side effects, and have a good application prospect in the field of tumor treatment. In this paper, linoleic acid (Linoleic acid) was coupled to the long chain of (Pullulan) by esterification, and amphiphilic polymers PULA1,PULA2 and PULA3. were obtained. The chemical structure of the polymer was characterized by Fourier transform infrared spectroscopy (FTIR) and 1H NMR. The critical micelle concentration (CMC) of PULA1,PULA2 and PULA3 were 70 ~ 62 渭 g / mL and 50 渭 路g 路mL ~ (-1), respectively. The results of dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that the PULA of nano-micelles was spherical and uniform. In vitro hemolysis test showed that the micelle had low hemolysis rate and showed good biocompatibility. Using adriamycin (DOX) as a model drug, the drug loading and encapsulation efficiency of nano-micelle PULA3/DOX were 8.05% and 88.6%, respectively. The drug loaded nano-micelle P ULA/DOX continuously released DOX in pH=7.4 phosphate buffer (PBS) for more than 96 h, and the cumulative release of DOX was between 43% and 55%, and the release rate of DOX increased with the decrease of the release medium pH. The pH sensitivity of the nano-micelle is beneficial to increase the cumulative release of DOX in tumor tissue. The uptake of nano-micelle PULA by MCF-7 and SMMC-7721 cells was detected by fluorescence microscope and flow cytometry. The results showed that nano-micelle PULA could carry a large number of DOX into the cells. The results of cell uptake test showed that PULA nanoparticles entered the cells mainly through energy-consuming, actin polymerization mediated endocytosis. The effect of PULA2/DOX on cell cycle showed that the drug loaded nano-micelles mainly blocked MCF-7 and SMMC-7721 cells in S phase and caused apoptosis in some cells. The transmission pathway and distribution of nano-micelle PULA into cells showed that nano-micelle PULA carried DOX into the cells through endocytosis, then transferred to lysosome, and gradually migrated the DOX released in lysosome to the nucleus to play an anticancer effect. The cytotoxicity test showed that the blank micelles were not toxic to Raw264.7 and HEK293 normal cells. The toxicity of drug loaded nano-micelle PULA/DOX to MCF-7 and SMMC-7721 cells was time-and concentration-dependent. The results of drug metabolism of PULA2/DOX nanoparticles in vivo showed that PULA nanoparticles could prolong the retention time of DOX in blood and maintain the content of DOX in blood at a high concentration. In conclusion, PULA nanoparticles are a new biocompatible carrier material, which is expected to be used as the delivery carrier of hydrophobic anticancer drug DOX in the field of cancer treatment.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;TQ460.1
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