小干扰RNA纳米递送载体及其肿瘤治疗应用研究

发布时间：2018-05-12 06:54

本文选题：小干扰RNA + 纳米递送载体　；参考：《中国科学技术大学》2017年博士论文

【摘要】：siRNA作为近年来兴起的一种核酸类药物,虽然给肿瘤治疗带来了新的机遇,但是临床siRNA药物的开发还面临着巨大的挑战。通过对肿瘤进行分类诊断,目前已经找到了大量的分子靶点,如何从众多的肿瘤相关基因中筛选出合适的靶点进行干预,以及如何设计出良好的siRNA递送载体用于克服多重给药障碍成为了目前研究的热点。本课题根据肿瘤细胞和正常细胞基因功能的差异,通过合成新型的聚合物对现有纳米载体进行优化,实现高效递送siRNA到肿瘤细胞进行肿瘤治疗,研究内容主要分为以下两个部分:1.我们根据对肿瘤低糖环境下神经胶质瘤细胞代谢功能基因的分析,找到了神经胶质瘤细胞特异性高表达的葡萄糖转运体3(GLUT3)。随后我们筛选了高效靶向GLUT3的siRNA,并用聚乙二醇-聚乳酸嵌段聚合物(PEG-b-PLA),辅以阳离子脂质(BHEM-Chol),通过双乳化法制备了包载GLUT3siRNA的纳米颗粒。在细胞实验中,我们验证了该纳米颗粒将siRNA递送到神经胶质瘤细胞和神经胶质瘤干细胞的能力,并检测了 GLUT3基因沉默对神经胶质瘤细胞葡萄糖摄取、乳酸产生等代谢指标的抑制效果,最后通过肿瘤治疗实验证明该siRNA递送载体可以通过代谢抑制下调神经胶质瘤干细胞的比例,实现对肿瘤生长的抑制。2.针对阳离子脂质辅助的PEG-b-PLA纳米颗粒在肿瘤细胞摄取和胞内siRNA释放上的不足,我们合成了肿瘤酸敏感化学键(Dlinkm)桥联的PEG-Dlinkm-PLGA,这种聚合物只在PEG和PLGA中间改变了桥联化学键,对整个聚合物的改变很小,因此同FDA批准的PEG-PLGA 一样,具有很好的生物相容性和安全性。通过PEG-Dlinkm-PLGA制备的新型纳米颗粒,可以在保留原纳米颗粒血液长循环和肿瘤富集能力的前提下,通过Dlinkm化学键实现肿瘤部位酸响应PEG脱壳,增加肿瘤细胞对纳米颗粒的摄取,并通过PLGA替代PLA,加快胞内siRNA释放,从而在整体上提高载药纳米颗粒对靶基因的沉默效果和肿瘤治疗效果。我们分别在细胞水平和动物水平验证了该纳米颗粒酸响应PEG脱壳的能力,并证明该体系在肿瘤细胞内能够更快地释放出包载的siRNA。最后在MDA-MB-231乳腺癌原位小鼠模型中,证明该体系能够以更低的siRNA给药剂量实现更好的治疗效果。
[Abstract]:As a nucleic acid drug, siRNA has brought new opportunities for tumor treatment, but the development of clinical siRNA drugs is still facing great challenges. Through the classification and diagnosis of tumors, a large number of molecular targets have been found, and how to screen suitable targets from many tumor-related genes for intervention, And how to design a good siRNA delivery vector to overcome multiple drug delivery barriers has become a hot topic. According to the difference of gene function between tumor cells and normal cells, the novel polymer was synthesized to optimize the existing nano-carriers and to deliver siRNA to tumor cells for tumor therapy. The research contents are divided into the following two parts: 1. Based on the analysis of the metabolic function genes of glioma cells in low glucose environment, we found the glucose transporter 3G UT3N specifically expressed in glioma cells. Then we screened the siRNAs targeting GLUT3 and prepared the GLUT3siRNA nanoparticles by double emulsification with PEG-b-PLAN and cationic lipids BHEM-Cholan. In cell experiments, we demonstrated the ability of the nanoparticles to deliver siRNA to glioma cells and glioma stem cells, and detected the glucose uptake of glioma cells by GLUT3 gene silencing. The inhibitory effect of lactate production and other metabolic indexes on tumor growth was demonstrated by tumor therapy. Finally, the siRNA delivery vector could down-regulate the proportion of neural glioma stem cells through metabolic inhibition, and achieve the inhibition of tumor growth. 2. In view of the deficiency of cationic lipide-assisted PEG-b-PLA nanoparticles in tumor cell uptake and intracellular siRNA release, we have synthesized PEG-Dlinkm-PLGA-bridged tumor acid-sensitive chemical bond. This polymer only changes the bridging chemical bond between PEG and PLGA. The change of the whole polymer is very small, so it has good biocompatibility and safety as PEG-PLGA approved by FDA. Under the condition of keeping the long blood circulation and tumor enrichment ability of the original nanoparticles, the novel nanoparticles prepared by PEG-Dlinkm-PLGA can realize the acid-responsive PEG demudation of tumor sites by Dlinkm chemical bond, and increase the uptake of nanoparticles by tumor cells. In order to accelerate the release of intracellular siRNA, the silencing effect of drug loaded nanoparticles to target gene and the tumor treatment effect were improved by replacing PLA with PLGA. We have verified the ability of the nanoparticles to respond to PEG demulsification at the cellular and animal levels, and demonstrated that the system can release encapsulated siRNAs more quickly in tumor cells. Finally, in MDA-MB-231 breast cancer in situ mouse model, it is proved that the system can achieve better therapeutic effect with lower dosage of siRNA.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5

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