RNA联合化疗药物共载纳米给药系统的研究

发布时间：2018-04-30 13:22

本文选题：脂质体 + Bmil　；参考：《华中科技大学》2016年博士论文

【摘要】：由于基因的多态性,肿瘤的化学治疗并不能完全杀死所有的肿瘤细胞,因此需要联合RNA药物对化疗不敏感的细胞进行抑制,从而增加化疗药物对肿瘤的治疗效果；因而我们筛选并评估了多种RNA联合化疗药物的纳米系统抑制肿瘤的研究。在多种人类肿瘤细胞中,Bmi1基因都被发现存在过表达的现象,显示出Bmi1基因可以作为潜在的基因治疗的靶点。然而,基于靶向Bmi1基因治疗的应用受制于多种因素,其中包括小干扰RNA(siRNA)没有较好的载体、较低的生物相容性以及相对较低的疗效。为了解决上述问题,我们设计了一种叶酸靶向共递送脂质体系统用于共载阿霉素和Bmi1 siRNA (FA-DOX/siRNA-L)。FA-DOX/siRNA-L的制备是通过叶酸靶向阿霉素阳离子脂质体和Bmi1 siRNA之间的静电相互吸附而成。体外和体内研究表明,FA-DOX/siRNA-L共递送的Bmi1 siRNA和阿霉素(DOX)能够明显抑制肿瘤的生长。结果还显示,通过FA-DOX/siRNA-L共传送Bmi1 siRNA与阿霉素显示出比DOX或Bmi1 siRNA单一给药更显著的抑瘤效果。实时荧光定量PCR和Western blot分析显示,FA-DOX/siRNA-L能够有效的沉默Bmi1基因在肿瘤细胞中的表达；此外,siRNA和DOX在肿瘤细胞的高聚集也表明叶酸配体对于肿瘤细胞的靶向作用。这些结果均表明,基于siRNA通过沉默Bmi1基因抑制肿瘤细胞的生长是一种有效的治疗手段,而且,通过叶酸靶向阿霉素脂质体共递送Bmi1 siRNA能够增强其抗肿瘤的效果。进而,我们以肝癌模型为治疗对象；肝癌治疗的一个主要难题在于肝癌细胞对化疗的不敏感。在这项研究中,我们设计了二种新型的脂包被顺铂共载microRNA-375以及Bmi1 siRNA纳米系统(NPC/miR-375)和(NPC/Bmi1 siRNA).对于顺铂对肝癌化疗不敏感的治疗,这二种共载药纳米系统具有潜在的应用价值。NPC/RNA纳米载药系统通过反相微乳法制备而成；首先,分别将包含KCl溶液和高水溶性的顺铂前药悬浮于反相微乳中进行混合,通过反相微乳的微反应器离子交换得到脂包被顺铂(NPC)；然后,将RNA吸附在带正电荷脂包被顺铂纳米粒的表面即得到了NPC/miR-375以及NPC/Bmi1 siRNA。然后分别对这两种共载药纳米粒进行评估；NPC/miR-375纳米共载药系统能够抑制细胞增殖并且提高顺铂对于肝癌细胞化疗的敏感性。体外细胞实验分析显示,NPC/miR-375能够从快速从溶酶体中逃逸避免miR-375在溶酶体中降解。更重要的是,在体外NPC/miR-375能增强细胞凋亡并且阻滞肝癌细胞周期的进行。在Akt/Ras诱导的原位肝癌小鼠模型中,NPC/miR-375显著抑制肝癌肿瘤的生长,并且延迟肿瘤复发。以上的结果均表明,顺铂miR-375通过脂包被顺铂纳米粒共载药的方式联合治疗肝癌,是解决肝癌化疗不敏感的潜在治疗方法。而NPC/Bmi1 siRNA中的Bmi1 siRNA能够通过抑制肿瘤干细胞的生长从而有效的提高了顺铂的治疗效果。
[Abstract]:Because of gene polymorphism, chemotherapy can not completely kill all tumor cells, so it is necessary to combine RNA drugs to inhibit chemotherapy-insensitive cells, so as to increase the therapeutic effect of chemotherapeutic drugs on tumor. Therefore, we have screened and evaluated the study of tumor inhibition by nanosystems of various RNA chemotherapeutic drugs. Overexpression of Bmi1 gene has been found in a variety of human tumor cells, suggesting that Bmi1 gene can be used as a potential target for gene therapy. However, the application of targeted Bmi1 gene therapy is limited by many factors, including small interference RNA-siRNAs without better vectors, low biocompatibility and relatively low efficacy. To solve the above problem, we designed a folic acid targeted co-delivery liposome system for the preparation of doxorubicin and Bmi1 siRNA FA-DOX / siRNA-Ln.FA-DOX / siRNA-L by electrostatic interaction between folic acid targeting adriamycin cationic liposomes and Bmi1 siRNA. In vitro and in vivo studies showed that FA-DOX / siRNA-L co-delivered Bmi1 siRNA and doxorubicin could significantly inhibit tumor growth. The results also showed that co-delivery of Bmi1 siRNA and adriamycin via FA-DOX/siRNA-L showed more significant inhibitory effect than that of DOX or Bmi1 siRNA alone. Real-time fluorescence quantitative PCR and Western blot analysis showed that FA-DOX / siRNA-L could effectively silence the expression of Bmi1 gene in tumor cells, and the high aggregation of Bmi1 and DOX in tumor cells also indicated the targeting effect of folate ligand on tumor cells. These results suggest that the inhibition of tumor cell growth by silencing Bmi1 gene based on siRNA is an effective treatment, and the co-delivery of Bmi1 siRNA to doxorubicin liposomes by folic acid can enhance its antitumor effect. Furthermore, we take the liver cancer model as the object of treatment, and one of the main problems in the treatment of liver cancer is the insensitivity of hepatoma cells to chemotherapy. In this study, we designed two novel lipids coated with cisplatin co-loaded microRNA-375 and Bmi1 siRNA nanosystem NPC-miR-375) and NPC-bmi1 siRNAs. For cisplatin insensitive to liver cancer chemotherapy, these two co-loaded drug nanosystems have potential application value. NPC-RNA nano-carrier system is prepared by reverse microemulsion method. The KCl solution and the highly water-soluble cisplatin prodrug were mixed in the reverse microemulsion, and the lipid encapsulation was obtained by ion exchange of the reverse microemulsion. NPC/miR-375 and NPC/Bmi1 sirna were obtained by adsorbing RNA onto the surface of cisplatin nanoparticles coated with positively charged lipids. Then the two co-loaded nanoparticles were evaluated to evaluate that NPC / miR-375 nanoparticles could inhibit cell proliferation and enhance the chemosensitivity of cisplatin to hepatocellular carcinoma cells. Cell analysis in vitro showed that NPC / miR-375 could escape rapidly from lysosomes to avoid miR-375 degradation in lysosomes. More importantly, in vitro NPC/miR-375 can enhance apoptosis and block the progression of hepatoma cell cycle. NPC-miR-375 significantly inhibited tumor growth and delayed tumor recurrence in Akt/Ras induced in situ hepatoma mice. These results suggest that cisplatin miR-375 combined with cisplatin nanoparticles cocarrier in the treatment of liver cancer is a potential method to solve the insensitivity of chemotherapy for liver cancer. Bmi1 siRNA in NPC/Bmi1 siRNA can effectively improve the therapeutic effect of cisplatin by inhibiting the growth of tumor stem cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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