基于改性聚乙烯亚胺的siRNA递送系统的研究

发布时间：2018-01-14 10:14

本文关键词：基于改性聚乙烯亚胺的siRNA递送系统的研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：在全世界范围内,肿瘤疾病的发病率及死亡率均逐年上升,对人类健康产生严重威胁。放化疗作为治疗癌症的主要手段,为肿瘤患者带来了不可逆转的身体损伤,而基因治疗为很多肿瘤患者带来了福音。基因治疗是应用基因工程技术将正常基因引入患者细胞内,以纠正致病基因的缺陷而根治遗传病。PEI在作为能够携带核酸类药物进入细胞的载体在肿瘤治疗领域中有着广泛的应用,是目前非常有效的阳离子载体。但是其含有大量氨基,导致其具有毒性较大、带有大量正电荷易被吞噬、转染效率低等问题。而PEG能够屏蔽电荷、延长血液循环时间,所以经PEG修饰的PEI理论上可以降低毒性、延长其血液循环时间;同时,穿膜肽TAT具有高效的穿膜效率,能够携带大分子进入细胞,转染效率极高,所以经TAT修饰后的PEI-PEG理论上能够增加转染效率。本研究在阳离子载体PEI的基础上选择PEG和TAT对其进行修饰,得到了新型的阳离子载体,并对其相关性质及递送si RNA的效果进行评价。本研究主要包括以下几个方面:1.PEI-PEG-TAT聚合物相关性质的考察在PEI基础上设计PEI-PEG-TAT聚合物,并对其缓冲容量和细胞毒性进行了考察。PEI的缓冲容量决定了其可以在进入细胞后具有质子海绵效应,所以若经修饰后的载体,仍具备质子海绵效应将有利于核酸药物在细胞内的释放。因此,通过盐酸滴定法测定聚合物的缓冲容量,结果表明,经修饰后的PEI-PEG-TAT的缓冲容量与PEI相似,所以其仍具有质子海绵效应,能够有效的释放所携带的药物进入细胞质。采用MTT法检测细胞毒性的结果表明,当浓度达到8μg/ml时,PEI-PEG-TAT细胞活力为76%,而PEI的细胞活力仅为53%,所以经修饰后的PEI-PEG-TAT聚合物的细胞毒性要显著低于PEI,低毒安全的,更有利于其作为核酸药物载体递送药物进入细胞。2.PEI-PEG-TAT/si RNA纳米复合物的制备选取靶向survivin基因的si RNA作为模型药物,再此基础上,筛选出PEI-PEG-TAT装载si RNA形成纳米复合物最佳的条件,并对筛选得到的化合物进行稳定性检测。对不同N/P和水相制备介质进行优化筛选,结合平均粒径、电位及结合度的测定结果,选择N/P为6:1,水相介质为5%葡萄糖溶液。此条件下制备的纳米复合物能在15天内保持粒度、电位及结合度的稳定。3.PEI-PEG-TAT纳米载体递送si RNA的研究PEI-PEG-TAT载药系统递送si RNA的效果进行了评价,选择了多项试验对该载药系统进行细胞水平的考察,如对肿瘤细胞的抑制效果检测、细胞摄取效果检测、抑制基因表达效果的检测等。PEI-PEG-TAT载体与si RNA按照一定比例结合后,能够形成稳定的囊状结构,并保持稳定的粒度分布和电位。细胞水平试验结果显示,PEI-PEG-TAT载体能够成功地将si RNA转染进入到肿瘤细胞内,降低survivin m RNA和蛋白的表达,进而导致肿瘤细胞的生长抑制及死亡。综上所述,经修饰的PEI-PEG-TAT聚合物是一种有潜力的核酸药物载体,与PEI相比,毒性更低,递送si RNA进入细胞发挥RNA干扰作用的效果更好。本文设计并评价的PEI-PEG-TAT聚合物,可以为今后进一步多功能药物载体系统研究等工作打下坚实地基础。
[Abstract]:Worldwide, the morbidity and mortality of tumor diseases are increasing year by year, caused a serious threat to human health. The chemotherapy is the main treatment for cancer, bring irreversible damage to the body for cancer patients, and gene therapy for many cancer patients to bring the gospel. Gene therapy is the normal gene using gene engineering technology the introduction of the patient's cells to correct the defects of pathogenic genes and genetic disease in the.PEI radical as a carrier to carry nucleic acid drugs into cells has been used widely in the field of cancer therapy, is currently very effective carrier. But it contains a lot of cationic amino, has resulted in the great toxicity, with a positive charge easily phagocytosis, problems of low transfection efficiency. PEG can shield charge, prolong blood circulation time, so the PEG modified PEI theory can decrease the toxicity of prolonged The blood circulation time; at the same time, membrane penetrating peptide TAT has efficient penetrating efficiency, capable of carrying large molecules into cells, the transfection efficiency is extremely high, so the TAT modified PEI-PEG theory can increase the transfection efficiency. The selection of PEG and TAT on the modified based on cationic vector PEI, obtained cation vector model, and evaluate its properties and the effect of delivery of Si RNA. This study mainly includes the following aspects: the properties of 1.PEI-PEG-TAT polymer polymer PEI-PEG-TAT design based on PEI, and the buffer capacity and cytotoxicity of buffer capacity of.PEI determines that it can have a proton sponge effect at after entering the cell, so the carrier if modified, still have a proton sponge effect will be conducive to the release of intracellular nucleic acid drugs. Therefore, by hydrochloric acid titration method for the determination of The buffer capacity of polymer. The results showed that the buffer capacity of the modified PEI PEI-PEG-TAT is similar, so it still has a proton sponge effect, can effectively release the carrying drugs into the cytoplasm. The cytotoxicity was detected by MTT method. The results show that when the concentration reached 8 g/ml, PEI-PEG-TAT cell activity was 76%, and the the viability of PEI is only 53%, so the cytotoxicity of PEI-PEG-TAT polymer modified significantly less than the PEI, low toxicity and safety, more conducive to the delivery of nucleic acid as drug carrier drugs into cells.2.PEI-PEG-TAT/si RNA nanocomposites were prepared from Si RNA targeting survivin gene was used as a model drug, and then on this basis, screening PEI-PEG-TAT Si RNA loaded nanocomposite formation conditions of the best, and the stability of detection of compounds screened. For different N/P and preparation of aqueous medium optimized Screening, combined with the average particle size, the determination results of N/P and combined with potential, 6:1, aqueous medium was 5% glucose solution. Under this condition, the nanocomposites can keep the size in 15 days, and the combination of potential PEI-PEG-TAT stability study of.3.PEI-PEG-TAT nanoparticles of Si RNA delivery delivery system for delivery Si RNA effect is evaluated, the investigation of a number of test cell level of the drug carrier system, detection of inhibitory effect on tumor cells such as cell uptake, detection effect, the effect of inhibition of gene expression detection.PEI-PEG-TAT vector and Si RNA according to a certain proportion of combined, can form a cystic structure stability, and maintain stable particle size distribution and zeta potential. The cellular level results show that PEI-PEG-TAT vector can successfully Si RNA transfected into tumor cells, decrease the expression of survivin m RNA and egg white, Which leads to inhibition of tumor cell growth and death. In conclusion, the PEI-PEG-TAT is a kind of polymer modified nucleic acid drugs have the potential to the carrier, compared with PEI, lower toxicity, better delivery of Si RNA into RNA cells play disruption. This paper describes the design and evaluation of PEI-PEG-TAT polymer, can be a solid foundation for further research the function of drug carrier system work.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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