微小RNA纳米胶囊的制备及其在RNA干扰治疗中的应用研究

发布时间：2018-08-23 18:50

【摘要】：RNA干扰(RNA interference, RNAi)是近年来发现的在生物体内普遍存在的一种生物学现象。RNA干扰技术作为重要的生物技术之一,被广泛地应用于重大疾病的诊断与治疗等领域。微小RNA(microRNAs, miRNAs)作为干扰RNA的一类,是在真核生物中发现的一类内源性的、进化保守的、具有调控基因表达功能的非编码RNA。它在包括发育、造血过程、器官形成、细胞增殖和凋亡、脂肪代谢等过程中参与调控并发挥重要作用。然而,由于miRNA在生理环境中极不稳定、体内半衰期短且其自身无法渗透到细胞内,亟待开发稳定、高效、无毒的投递体系以满足基于miRNA的疾病治疗。本文针对目前miRNA孰体在投递过程中面临的毒性高、投递效率低、稳定性差等困境,设计了一种基于纳米胶囊的miRNA投递平台,并研究了其在RNA干扰治疗中的应用。本文首先使用原位自由基聚合方法,设计并制备出了一种低毒、高效的细胞内miRNA投递纳米胶囊。比较了聚合单体种类和比例对纳米胶囊结构和表面性质的影响,发现高APm/AAm比例可获得粒径较小、表面电荷较高的纳米胶囊。在此基础上,我们还研究了miRNA纳米胶囊及商用的Lipofectamine转染试剂与miRNA结合的复合物(Lipo/miR)在模拟生理环境中的结构稳定性,证明较Lipo/miR,纳米胶囊可更好地保护miRNA,阻止其被肝素置换和核酸酶降解。细胞实验结果表明,纳米胶囊比Lipofectamine具有更低的细胞毒性,却可更高效地转染miRNA。利用纳米胶囊投递反义microRN A-21 (As-miR-21),可以显著地降低肿瘤细胞内miR-21表达,调控PTEN/PIK-Akt信号通路抑制相关转录因子例如β-catenin、HIF1-α和STAT3的表达以及它们的核转位,进而阻断血管内皮生长因子(VEGF)信号转导通路,从而抑制体内肿瘤血管增生和肿瘤生长。其次,本文针对目前miRNA在急性脑缺血治疗中面临的局部注射风险较大、静脉注射投递效率低等困境,设计并制备了一种可用于静脉注射并高效投递miRNA的纳米胶囊。比较了聚合单体种类和比例对纳米胶囊结构和表面性质的影响,发现高单体比例可获得粒径更小更均一、表面电荷较高的纳米胶囊；增加PEG用量可更有效地屏蔽纳米胶囊表面电荷,并降低纳米胶囊表面的非特异性蛋白吸附。利用动态光散射和凝胶电泳研究了纳米胶囊壳层的降解行为,发现miRNA纳米胶囊可在生理环境中保持结构稳定,而在酸性条件下聚合物壳层发生降解从而将miRNA释放出来。激光共聚焦和流式细胞术实验结果证明,PEG的引入显著降低了巨噬细胞对纳米胶囊的内吞,而星形来源的神经胶质细胞的内吞效率仍维持在较高水平,这显著降低了体内网状内皮系统(RES)对纳米胶囊的摄取,增强了其在脑部的富集并保证了miRNA进入细胞发挥功能。在大鼠急性脑缺血模型中,利用纳米胶囊尾静脉注射投递具有抗凋亡和促血管新生功能的miR-21模拟物,可显著改善模型大鼠的神经功能缺损。本文最后针对肿瘤治疗的多靶点联合用药要求,以纳米胶囊技术为基础,制备了同载As-miR-21和化疗药物阿霉素(DOX)的纳米胶囊和包裹肿瘤抑制因子VHL蛋白的纳米胶囊。MTT实验证明了,利用纳米胶囊联合投递DOX和AS-miR-21可协同抑制肿瘤细胞增殖。进一步的研究结果显示,联合投递As-miR-21、DOX和VHL可以实现对肿瘤细胞内信号通路的协同调控,实现对靶点因子的最大抑制。
[Abstract]:RNA interference (RNAi) is a ubiquitous biological phenomenon found in organisms in recent years. As one of the important biotechnologies, RNA interference technology is widely used in the diagnosis and treatment of major diseases and other fields. A class of endogenous, evolutionarily conserved, noncoding RNA that regulates gene expression. It is involved in regulation and plays an important role in many processes, including development, hematopoiesis, organogenesis, cell proliferation and apoptosis, and fat metabolism. However, due to the extremely unstable physiological environment, microRNAs have short half-lives and are not permeable by themselves. A stable, efficient and non-toxic delivery system is urgently needed to meet the needs of disease treatment based on microRNAs. In this paper, a microRNAs delivery platform based on nanocapsules is designed to overcome the difficulties of high toxicity, low delivery efficiency and poor stability in the delivery of microRNAs. Firstly, a low toxicity and high efficiency intracellular microRNAs delivery nanocapsules were designed and prepared by in situ free radical polymerization. The effects of the kinds and proportions of polymeric monomers on the structure and surface properties of the nanocapsules were compared. It was found that nanocapsules with small particle size and high surface charge could be obtained by high APm/AAm ratio. We also studied the structural stability of microRNA nanocapsules and commercial Lipofectamine-binding compounds (Lipo/microRNAs) in simulated physiological environments. The results showed that nanocapsules could protect microRNAs from heparin replacement and nuclease degradation better than Lipo/microRNAs. Nanocapsule delivery of antisense microRN A-21 (As-microRN A-21) significantly reduces the expression of microRN A-21 in tumor cells, and regulates the PTEN/PIK-Akt signaling pathway by inhibiting the expression of related transcription factors such as beta-catenin, HIF1-a and STAT3, as well as their nuclear translocation, thereby blocking blood translocation. Vascular endothelial growth factor (VEGF) signal transduction pathway inhibits tumor angiogenesis and tumor growth in vivo. Secondly, in view of the difficulties of high local injection risk and low intravenous delivery efficiency in the treatment of acute cerebral ischemia, we designed and prepared a kind of nanoparticles which can be used for intravenous injection and highly efficient delivery of microRNAs. Rice capsules. Comparing the effects of the types and proportions of polymeric monomers on the structure and surface properties of nanocapsules, it was found that nanocapsules with smaller and more uniform particle size and higher surface charge could be obtained with higher proportions of monomers. The degradation behavior of the nanocapsule shell was studied by dynamic light scattering and gel electrophoresis. It was found that the structure of the nanocapsule was stable in the physiological environment, and the degradation of the polymer shell under acidic conditions led to the release of the microRNAs. The results of laser confocal focusing and flow cytometry showed that the introduction of PEG significantly reduced macrophages. The uptake of nanocapsules by the reticuloendothelial system (RES) in vivo was significantly reduced, and the accumulation of microRNAs in the brain was enhanced and the function of microRNAs was ensured in the rat model of acute cerebral ischemia. The neurological deficits of the model rats were significantly improved by intravenous delivery of microRNAs-21 mimics with anti-apoptosis and angiogenesis functions. Finally, nanocapsules loaded with As-microRNAs-21 and doxorubicin (DOX) were prepared based on nanocapsule technology to meet the multi-target combination requirements of tumor therapy. Nanocapsules encapsulating tumor suppressor VHL proteins have been demonstrated by MTT experiments to synergistically inhibit the proliferation of tumor cells by using nanocapsules combined with DOX and AS-miR-21. Further studies have shown that as-miR-21, DOX and VHL can achieve synergistic regulation of signal pathways in tumor cells and maximize target factors. Inhibition.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R943;TB383.1
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本文编号：2199591