联合转运siRNA与EPI的pH敏感长循环逆转多药耐药纳米载体的研究
发布时间：2018-03-14 15:48
本文选题：联合转运 切入点：表阿霉素 出处：《青岛大学》2017年硕士论文 论文类型：学位论文
【摘要】：在肿瘤治疗中,内源性或获得性的多药耐药是化疗过程中的主要障碍,其降低药物在细胞内的积累或增强肿瘤细胞DNA损伤修复机制,导致单种药物治疗肿瘤的效率降低。基因药物可以抑制细胞多药耐药的发生,但是递送过程面临较多困难,因此选择合适的载体,将基因与化疗药物联合共递送至肿瘤组织,以发挥药效,是治疗肿瘤的一个新策略。本课题制备了一种联合转运si RNA与表阿霉素(EPI)的p H敏感长循环逆转多药耐药多功能信封式纳米载体(Multifunction Envelop-type Nano Device,MEND)。MEND是以压缩的DNA、si RNA等为核心,用含有功能性配体的脂质体包裹而成的类似信封式外壳的纳米微球,兼具了纳米粒和脂质体的优点。选用聚乙二醇(PEG)连接至磷脂分子二硬脂酰基磷脂酰乙醇胺(DSPE)上形成的共聚物DSPE-PEG,实现长循环的目的。通过迈克尔加成反应,合成具有酸敏感性能的含缩酮键的聚β氨基酯结构(KPAE),其通过正负电结合作用,缩合si RNA;在细胞酸性环境中,KPAE的缩酮建断裂,实现si RNA的智能释放。BCL-2-si RNA通过调低P糖蛋白(P-gp)的表达和促进细胞凋亡,逆转细胞多药耐药的发生,协同表阿霉素起到治疗肿瘤的作用。各成分通过核磁共振、凝胶渗透色谱进行结构鉴定;本实验选用薄膜分散法制备复合物载体,EPI包裹在脂质体薄膜的磷脂双分子层疏水端,KPAE/si RNA缩合物溶液水化脂质体薄膜后,得到EPI/si RNA-MEND。利用透射电镜观察其形态;马尔文激光粒度仪测定其粒径及电位;琼脂糖凝胶电泳实验考察复合物对的si RNA缩合能力;分别采用滤膜法-高效液相色谱法和超滤离心法,测定EPI和si RNA的包封率;实验细胞选用人肝癌Hep G_2细胞,选用BCL-2-si RNA(si BCL-2)制备EPI/si BCL-2-MEND,采用MTT法,考察EPI/si BCL-2-MEND对肝癌Hep G_2细胞存活率的影响;采用荧光标记法,分别用FAM标记si RNA,Lyso Tracker-Blue DND-22标记溶酶体,Hoechst 33342标记细胞核,考察EPI/FAM-si RNA-MEND在肝癌Hep G_2细胞的分布以及溶酶体逃逸现象;采用流式细胞法,考察EPI/FAM-si RNA-MEND对肝癌Hep G_2细胞的转染效率;用EPI/si BCL-2-MEND,对Hep G_2细胞转染,考察P-糖蛋白(P-gp)的表达。采用小动物活体成像技术,考察EPI/Cy5-si RNA-MEND的体内分布。结果表明,成功合成了所需的各目标产物;MEND呈规则的球形且具有明显的脂质双分子层结构和指纹结构;EPI/si RNA-MEND平均粒径为121.6 nm,平均电位为+41.5 m V;载体复合物对EPI和si RNA的包封率分别为86.13%和97.07%;MEND在一定给药剂量范围内毒性较小,对细胞存活率几乎无影响;EPI/si BCL-2-MEND高于同浓度下单独药物的肿瘤细胞抑制率,并具有较高的转染效率;相比于游离的EPI组和EPI-MEND组,EPI/si BCL-2-MEND可显著下调P-gp的表达;EPI/Cy5-si RNA-MEND相比单独药物更容易富集于肝脏中,肾脏内几乎无分布,证明其具有长循环性能。所制备的载体复合物能够有效地将si RNA与EPI共递送至肿瘤细胞中,实现高效率的转染,并实现si RNA在细胞内的智能释放,具有良好的使用前景。
[Abstract]:In the treatment of cancer, or endogenous multidrug resistance is the main obstacle in the process of chemotherapy, reduce the accumulation of intracellular drug or enhance repair mechanisms of tumor cell DNA damage, which leads to the decrease of the efficiency of single drug therapy. Gene therapy can inhibit cell multidrug resistance, but the delivery process is facing more difficult, so choose the appropriate carrier gene combined with chemotherapy drugs were delivered to the tumor tissue, to take effect, is a new strategy for the treatment of cancer. The preparation of a joint Si RNA and transport of adriamycin (EPI) multidrug resistance P H sensitive long cycle reversal function envelope type nano carrier (Multifunction Envelop-type Nano Device, MEND.MEND) which is compressed with DNA, Si RNA as the core, nanoparticles similar envelope type shell with liposome containing functional ligands and the both The nanoparticles and liposomes. The advantages of polyethylene glycol (PEG) connected to the phospholipid molecules two stearoyl phosphatidyl ethanolamine (DSPE) copolymer DSPE-PEG on the formation of long circulation purposes. Through Michael addition reaction of poly beta amino ester bond structure containing ketal synthesis with acid sensitive properties (KPAE) through the combination of positive and negative electrical, Si, RNA in cell condensation; in acidic environment, KPAE ketal built fracture, realize the release of.BCL-2-si RNA by reducing the P glycoprotein (P-gp) Si intelligent RNA expression and promote apoptosis of MDR cells, play a synergistic anti-tumor effect of epirubicin. The components of the structures were identified by NMR, gel permeation chromatography; this experiment selected films by complex carrier dispersion method, EPI wrapped in liposome membrane phospholipid bilayer hydrophobic KPAE/si RNA condensate water solution Liposome film, EPI/si RNA-MEND. were observed by transmission electron microscope; Malvin laser particle size analyzer to determine the particle size and zeta potential; agarose gel electrophoresis of complexes of Si RNA condensation ability; method and centrifugal ultrafiltration HPLC respectively by membrane filtration method, determination of EPI and Si RNA encapsulation the experimental cell rate; human hepatocellular carcinoma Hep G_2 cells, using BCL-2-si RNA (Si BCL-2) EPI/si was prepared by BCL-2-MEND, using the MTT method, the effect of EPI/si BCL-2-MEND survival rate was investigated on hepatocellular carcinoma Hep G_2 cells; using fluorescence labeling, respectively labeled with FAM Si RNA, Lyso Tracker-Blue DND-22 Hoechst 33342 labeled lysosomes, marked nucleus. The effects of EPI/FAM-si RNA-MEND in the distribution of escape phenomenon hepatocellular carcinoma Hep G_2 cells and lysosomes; using flow cytometry, investigate the transfection efficiency of EPI/FAM-si RNA-MEND on hepatocellular carcinoma Hep G_2 cells; EPI/ si BCL-2-MEND,瀵笻ep G_2缁嗚優杞煋,鑰冨療P-绯栬泲鐧，
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