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阿尔茨海默病（Alzheimer’s disease, AD）是老年人中高发的一种神经退行性疾病，主要表现为进行性认知和记忆损伤。现有药物只能在一定程度上缓解症状，不能阻止疾病的进程。这一方面是由于药物在体内的半衰期短，而且存在血脑屏障（Blood brain barrier, BBB）的阻碍，药物很难进入脑内。更重要的是，AD发病与多种病理机制有关，单一药物无法实现有效治疗，迫切需要找到新的有效的治疗策略。本文针对AD治疗过程中“病灶部位药物富集难”和“对症治疗效果不佳”的难题，针对AD发病机制并根据药物递送过程中的问题设计合理的输递体系。在体外和体内水平对治疗效果进行考察，结果表明课题构建的药物递送体系能够将药物有效输递至脑内病灶部位，并且显著提高了AD的治疗效果。论文具体开展的研究工作如下：（1）脑内小胶质细胞功能紊乱与β-淀粉样蛋白（Amyloid-β, Aβ）之间存在的恶性循环是导致AD发生和恶化的重要致病机制。因此，我们针对该病因提出同时调节小胶质细胞的功能和减少Aβ的负荷的策略，打破恶性循环。为了达到这个目的，课题构建了基于两性离子聚合物聚羧基甜菜碱甲基丙烯酸酯（Poly(carboxybetaine), PCB）的纳米药物递送体系（MCPZFS NPs），该体系可以调节功能紊乱的小胶质细胞使其功能正常化，并且表现出募集Aβ进入小胶质细胞的特性。与基于中性材料聚乙二醇（Polyethylene glycol, PEG）的体系（MEPZFS NPs）相比，该纳米颗粒能够更有效地在脑部富集，并且显著缓解Aβ诱导的毒性、降低促炎细胞因子的水平。更重要的是，我们发现MCPZFS NPs具有募集Aβ进入小胶质细胞的特性，显著增强了Aβ的吞噬和降解。而且与中性载体介导的Aβ溶酶体/自噬体降解途径不同，MCPZFS NPs介导Aβ从溶酶体中快速逃逸出来，主要通过蛋白酶体途径降解。MCPZFS NPs治疗后的小鼠Aβ蛋白水平显著降低，其神经元损伤、记忆力减退以及神经炎症均得到明显改善，可以作为脑内小胶质细胞的调节剂和“Aβ清洁工”，为AD的治疗提供了一种新的视角和方法。（2）随着研究的深入，人们发现脑内过量的过渡金属离子可以促进Aβ的聚集增加其毒性。而且发现AD病人的神经元中出现了tau蛋白（tau）的磷酸化，可以在不依赖于Aβ的情况下发生，使神经元细胞的正常功能受到损伤，导致突触功能丧失以及氧化应激的产生。基于上述复杂病因，本章提出三重治疗机制协同治疗策略，1）首先利用组氨酸能够络合金属离子的特性络合脑内过量的金属离子，抑制细胞外Aβ的聚集；2）采用药物水杨酰水杨酸抑制神经元细胞中乙酰转移酶p300的表达，进一步降低磷酸化tau（p-tau）的水平；3）采用NF-κB小干扰RNA缓解氧化应激水平。为了实现药物在病灶部位的有效富集和可控释放，进一步构建金属离子和酶双响应型递送体系，实现逐级靶向和可控释药。实验结果表明该体系能够有效穿过BBB和靶向神经元细胞，并实现药物的可控释放。Aβ聚集诱导的神经毒性得到了明显的缓解，神经元细胞中p300、p-tau和NF-κB的表达显著下调，小鼠的记忆认知损伤得到了有效地缓解，Aβ和p-tau表达明显减少，脑内炎症水平显著降低，在AD的治疗中有非常好的应用前景。 综上所述，本文根据AD的复杂病因提出了两种治疗策略，并且将药物有效输递至了病灶部位，使AD的病理得到显著地改善，为今后AD的治疗提供了新的视野。;Alzheimer’s disease (AD) is the most common neurodegenerative disorder among the elderly, which causes the progressive cognitive and memory decline. The current drugs used in clinic can only meliorate the symptoms to some extent, which can not prevent the disease progression. This may be attributed to the short plasma half-time and the obstacle of the blood brain barrier (BBB), which result in poor drug accumulation in the brain. Furthermore, the pathogenesis of AD is very complex, and single drug may not achieve enough therapeutic result. Therefore, it is urgent to find a new and effective strategy for the AD therapy. To solve the problems of “poor enrichment of target sites” and “poor therapeutic effect of symptomatic treatment”, we aim at the pathogenesis of AD and develop rational delivery systems. The therapeutic effect was investigated in in vitro and in vivo. The results demonstrated that the drugs could be delivered to diseased sites effectively, and the therapeutic effect of AD was improved remarkably. In detail, the works mainly included the following issues:1) The vicious circle between dysfunctional microglia and amyloid-β (Aβ) is a crucial pathological event and accelerates the progression of AD. Therefore, we present the strategy of regulating the function of microglia and reducing Aβ burden simultaneously to break the negative feedback loop. To achieve this, a zwitterionic poly(carboxybetaine) (PCB) based nanoparticles (MCPZFS NPs) with normalizing the dysfunctional microglia and Aβ recruitment are established for the treatment of AD. Compared with the neural polyethylene glycol (PEG)-based nanoparticles (MEPZFS NPs), the brain enrichment of MCPZFS NPs is more effectively. And it is demonstrated that the PCB based nanoparticles significantly alleviate the priming of microglia by decreasing the Aβ induced cytotoxicity and the level of pro-inflammatory mediators. Most importantly, we discover MCPZFS NPs exhibited the behavior to recruit Aβ into microglia, which significantly enhanced the Aβ phagocytosis. Moreover, MCPZFS NPs can carry Aβ escape from lysosomes to cytoplasm rapidly and mediate Aβ degraded by proteasomes, which is quite different from the lysosomal/autophagy pathway of MEPZFS NPs. After the treatment with the nanopaticles, the Aβ burden, neuron damages, memory deficits, and neuroinflammation of AD mice are significantly attenuated in the brain. Therefore, the PCB-based nanoparticles have great potential to serve as an“Aβ cleaner” and provide a new insight into the therapeutic strategy for AD therapy. 2) As research continues, it has been found that excessive metal ions in the brain can promote the aggregation of Aβ and increase the toxicity. Moreover, tau phosphorylation in the neurons can take place independently of Aβ, which lead to the damages of neuron, loss of synaptic function and generation of oxidative stress. Based on the above complex etiology, this chapter proposes a triple strategy for the treatment. Firstly, Histidine was employed as metal ion chelating agent to inhibit the aggregation of Aβ. Secondly, Salsalate was introduced to reduce the expression of acetyltransferase p300 and phosphorylation of tau in neuron. Moreover, RNA interference was used to down-regulate the expression of NF-κB for oxidative stress modulation. In order to achieve enrichment and controlled release of the drug at the lesion site, a metal ion/enzyme dual-responsive delivery system is developed. It is demonstrated that the nanoparticles could permeate BBB and target to neuronal cells effectively. The cytotoxicity induced by Aβ aggregation, the expression of p300, phosphorylated tau and NF-κB in neuronal cells were reduced significantly. In animal experiment, the cognitive and memory damage of AD mice were significantly alleviated, the expression of Aβ and p-tau were reduced and the inflammation level decreased remarkably. Therefore, the nanoparticles with the triple-synergistic mechanism provide a new horizon to the therapeutic strategy for AD therapy.In summary, this article proposes two treatment strategies based on the complex etiology of AD, which deliver drugs to the lesion site effectively and significantly alleviate the pathology of AD. The new strategies provide new perspective for the treatment of AD in the future.
|刘瑞瑗. 靶向纳米递送体系用于阿尔茨海默病治疗的研究[D]. 中国科学院大学,2020.|
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