超分子纳米体系联合递送药物和基因靶向肝癌的研究

发布时间：2018-06-20 23:55

本文选题：肝癌靶向 + 纳米粒子　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的肝细胞肝癌(hepatocellular carcinoma,HCC,简称肝癌)是占据全球癌症死因第三位的常见恶性肿瘤。大多数病人确诊时已处晚期,失去手术机会。传统的化疗方法效果差且毒副作用较大,因此,提高药物的治疗作用同时减少其副反应成为研究的重点。肝癌的发生、发展和转移是一个多基因共同参与的过程,将一些特定的基因如p DNA、si RNA或micro RNA导入肝癌细胞继而在基因层面干扰肿瘤的发生发展已成为肝癌治疗的新思路。将化疗药物与基因疗法相结合,利用两者的不同机制可以发挥协同作用。然而,这种联合疗法最大的挑战在于如何实现药物和基因的共载并将二者靶向运送至肝癌组织细胞。多功能纳米载体可以作为药物/基因的共载介质。本研究以阿霉素(doxorubicin,DOX)和寡聚核苷酸(oligo RNA)分别作为药物和基因模型,构建了一种同时携载DOX和oligo RNA的超分子纳米体系,并探讨了其在体内外实验中的肝癌靶向性,为肝癌的临床治疗提供新的方向和方法。方法通过希夫碱反应制备出β-环糊精(β-cyclodextrin,β-CD)和多聚-L-赖氨酸(poly-L-lysine,PLL)结合的天然生物材料β-环糊精接枝聚赖氨酸(命名为PLCD),采用傅立叶红外分析及核磁共振氢谱表征了PLCD的化学结构;应用芘的荧光实验及琼脂糖凝胶电泳阻滞实验分别考察了不同β-CD取代度的PLCD携载疏水性分子及复合oligo RNA的能力;采用透析法制备PLCD/DOX复合物,将其与oligo RNA混合,形成PLCD/DOX/oligo RNA复合物(简称PDR);采用共孵育的方法将透明质酸(hyaluronic acid,HA)引入到PDR纳米粒子表面,形成具有肝癌靶向性的超分子纳米粒子(简称为HPDR)。应用动态透析法测定PDR和HPDR两种纳米体系中的DOX在不同p H值环境下的体外释放;应用流式细胞术(flow cytometry)和激光共聚焦扫描显微镜(confocal laser scanning microscope,CLSM)检测CD44分子在肝癌细胞系MHCC-97H和Hep G2表面的表达情况及HPDR纳米粒子靶向递送DOX和荧光标记的oligo RNA(FAM-RNA)至肝癌细胞的能力;应用CCK-8(Cell Counting Kit-8)试剂检测PLCD和HA/PLCD的生物安全性,并比较PDR和HPDR纳米粒子的细胞毒性作用。构建MHCC-97H皮下荷瘤裸鼠模型,经尾静脉注射给药,采用小动物活体成像系统检测HPDR在小鼠体内的分布。结果1.成功制备了三种不同β-CD取代度的PLCD,取代度分别为7.3%、12.9%和25.1%。2.PLCD可以有效包载DOX,其载药量为11.0%,包封率为41.2%。3.当N/P为30/1时,PLCD/DOX与oligo RNA复合形成稳定的纳米粒子,其粒径为168.9 nm,表面电荷为+38.7 m V。4.当HA/PDR的质量比为3/6时,HA可成功包裹于PDR纳米粒子表面形成负电性的壳层,形成的HPDR纳米粒子成规则球形,具有典型的核-壳结构,粒径为195.8 nm,表面电荷为-22.7 m V。5.肝癌细胞系MHCC-97H和Hep G2表面均高表达CD44分子。6.HPDR纳米粒子可通过HA-CD44介导的内吞作用将DOX和RNA靶向递送至肝癌细胞,并表现出较强的细胞毒性。7.体内实验表明,与PDR纳米粒子相比,HPDR在肿瘤部位蓄积更多而在肝脏组织中蓄积较少,说明HPDR纳米粒子在体内具有良好的肝癌靶向性。结论本研究中制备的HPDR超分子纳米粒子结构稳定,在体内外均表现出良好的肝癌靶向性,有望在药物与基因联合治疗肝癌方面发挥巨大的潜力。
[Abstract]:Objective: hepatocellular carcinoma (hepatocellular, carcinoma, HCC, HCC) is a common malignant tumor in the world occupy the third cause of cancer death. Most patients are diagnosed at advanced stage and lost the chance of operation. The effect of the traditional chemotherapy and toxic side effects, therefore, provided high therapeutic effects of drugs and reduce the side-effects become. The key. The occurrence of liver cancer, development and transfer process is a multi gene involved, some specific genes such as P DNA, Si RNA or micro RNA into liver cancer cell and gene level interference on the occurrence and development of tumor has become a new way of thinking in the treatment of liver cancer. The chemotherapy drug and gene therapy combination the use of different machine. We can play a synergistic role. However, the biggest challenge is how to realize the combination therapy of drugs and genes were loaded and the two targeted delivery to liver cancer cells. Multifunctional nanocarriers can be used as drug / gene carrying medium. In this study, adriamycin (doxorubicin, DOX and oligonucleotide (oligo RNA) as) respectively. Drug and gene model, constructed a simultaneously carrying DOX and oligo RNA nano supramolecular system, and discusses the in vivo and in the liver targeting, provide new directions and methods for clinical treatment of hepatocellular carcinoma. Methods by Schiff base reaction for the preparation of beta cyclodextrin (3 -cyclodextrin. Beta -CD) and poly -L- lysine (poly-L-lys Ine, PLL) with the natural biological materials of beta cyclodextrin grafted poly lysine (named PLCD), were used to characterize the chemical structure of PLCD by Fu Liye infrared analysis and NMR; fluorescence experiments and agarose gel electrophoresis retardation test application of pyrene were investigated with different degree of substitution of beta -CD PLCD carrying hydrophobic molecular and composite oligo RNA The preparation of PLCD/DOX composite material; by the dialysis method, the oligo and RNA mixed formation of PLCD/DOX/oligo RNA complex (PDR); using the method of CO incubation of hyaluronic acid (hyaluronic acid HA) is introduced to the surface of PDR nanoparticles, forming a supramolecular of liver targeted nanoparticles (HPDR) the application of dynamic dialysis method. Determination of PDR and HPDR in the system of two kinds of nano DOX in different P H release environment in vitro; application of flow cytometry (flow cytometry) and laser scanning confocal microscope (confocal laser scanning microscope, CLSM) detection of CD44 molecules to deliver DO expression in target MHCC-97H and Hep and HPDR nanoparticles on the surface of G2 hepatocellular carcinoma cell lines Oligo RNA X (FAM-RNA) and fluorescence labeling ability to hepatocellular carcinoma cells; application of CCK-8 (Cell Counting Kit-8) biological safety detection reagent PLCD and HA/PLCD, cytotoxicity and comparison of PDR and HPDR nanoparticles. Construct MHCC-97H subcutaneous xenograft models in nude mice by tail vein injection, administered by small animal in vivo the imaging detection system HPDR Tissue distribution in mice. Results 1. were successfully prepared three different beta -CD substituted PLCD, the degree of substitution were 7.3%, 12.9% and 25.1%.2.PLCD can be effectively encapsulated DOX, the drug loading was 11%, the encapsulation rate of 41.2%.3. when N/P is 30/1, PLCD/DOX and oligo RNA to form stable composite nanoparticles. The diameter is 168.9 NM, the surface charge is +38. 7 m V.4. when the mass ratio of HA/PDR to 3/6, HA can be successfully encapsulated in PDR nanoparticles formed on the surface of the negatively charged shell, the formation of HPDR nanoparticles into regular spherical core-shell structure typical of the particle size of 195.8 nm -22.7 m V.5., the surface charge of hepatocellular carcinoma cell line MHCC-97H and Hep G2 were high surface the expression of CD44.6.HPDR nanoparticles Endocytosis can be mediated by HA-CD44 DOX and RNA targeted delivery to liver cancer cells, and showed that the cytotoxicity of.7. in vivo is stronger, compared with PDR nanoparticles, HPDR at the tumor site and accumulated more in liver tissue accumulation less, indicating that HPDR nanoparticles in vivo hepatocellular carcinoma has good target. The conclusions of this study HPDR nanoparticles were prepared by supramolecular structure stability, in vitro and in vivo showed good liver targeting, is expected to play a huge potential in drug and gene therapy of liver cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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