穿膜肽修饰的PLGA载药纳米颗粒对AD转基因鼠记忆改善的研究

发布时间：2018-02-24 10:36

本文关键词： 阿尔茨海默症聚乳酸羟基乙酸共聚物 CRT β淀粉样蛋白　出处：《宁夏大学》2017年硕士论文　论文类型：学位论文

【摘要】：阿尔茨海默症(Alzheimer'sdisease,AD)作为一种目前致死率极高的疾病已经严重威胁到了人们的公共健康问题,受到越来越多的关注。其常见的病理特征包括老年斑、神经元纤维异常缠结和神经元胞外高密度β淀粉样蛋白(amyloid β,Aβ)的沉积。虽然目前已有多种治疗药物上市,但由于存在药物可溶性差,生物可溶性低,难以穿越血脑屏障(blood-brain barrier,BBB)等问题,致使AD的治疗现状仍不容乐观。纳米颗粒载体因其在体内体外实验中均易被检测到因此用于AD的诊断治疗日益受到重视。因此,我们希望设计出一种可以穿越BBB靶向脑部并积极发挥携带药物功能的新型纳米药物载体。聚乳酸轻基乙酸共聚物(Poly(lactic-co-glycolicacid),PLGA)是一种生物可降解的高分子微球,具有良好的生物相容性,应用前景广阔,因此我们选择PLGA为基础纳米颗粒药物载体,并将姜黄素与环肽S1包裹进纳米颗粒内腔,将制得的纳米颗粒命名为NP-S1+Cur。姜黄素是一种可以抑制Aβ毒性的天然化合物,而S1可以通过影响APP的剪切过程有效降低Aβ生成,二者对AD的致病机制均有一定的积极影响。随后我们选择用穿膜肽CRT修饰在PLGA纳米颗粒表面(CRT-NP-S1+Cur),因为CRT是一种与铁生物性能相似的多肽,可以靶向性结合转铁蛋白与转铁蛋白受体复合物,增强纳米颗粒穿越BBB的能力。通过透射电镜(Transmission electron microscope,TEM)观察纳米颗粒形态,动态光散射法(Dynamic light scattering,DLS)确定耦连CRT前后纳米颗粒的大小分别为128.6 nm and 139.8 nm,包封率分别为 23.2 ± 5.3%and 21.4 ± 6.9%,载药量分别为 0.54 ± 0.04%and 0.42 ±0.06%;利用MTT毒性实验分析纳米颗粒的生物毒性;同时,体外模拟血脑屏障实验和体内活体成像实验表明,CRT-NP-Sl+Cur具有更强的穿越BBB的能力。动物行为学和病理学研究表明,经过对AD转基因小鼠21天腹腔注射PLGA纳米颗粒后,能够改善AD鼠的空间记忆能力,小鼠脑内老年斑数量有所减少,小胶质细胞和星型胶质细胞活化水平、突触素水平均有改善。用试剂盒对实验小鼠脑部进行分析发现,PLGA纳米颗粒可以增强脑内超氧化物歧化酶(SOD)的活性,降低Aβ和活性氧(ROS)水平,促炎性细胞因子(TNF-α和IL-6)的表达,与NP-S1+Cur相比,CRT-NP-S1+Cur的作用效果更显著。以上结果表明,本实验设计的新型纳米药物载体具有穿越BBB并靶向脑部,从而高效发挥姜黄素抗氧化、抗炎症作用及S1抑制Aβ生成、降低Aβ毒性的作用,对AD的治疗具有潜在的应用价值。
[Abstract]:Alzheimer's disease (AD), a disease with a high mortality rate, has become a serious threat to public health and has attracted increasing attention. Its common pathological features include senile plaques. Abnormal tangles of neuron fibers and deposition of amyloid 尾 -A 尾 -amyloid 尾 -amyloid 尾 -amyloid 尾 -amyloid 尾 -amyloid 尾 -amyloid in neurons. It is difficult to cross the blood-brain barrier and BBB, so the current status of AD treatment is still not optimistic. The nanoparticles carrier is easy to be detected in vivo and in vitro, so the diagnosis and treatment of AD has been paid more and more attention. We hope to design a novel nano-drug carrier that can traverse the brain of BBB and play an active role in carrying drugs. Poly (lactic acid-co-glycolic acid) is a biodegradable polymer microsphere with good biocompatibility. Therefore, we chose PLGA as the base drug carrier and encapsulated curcumin and cyclic peptide S1 into the intracavity of nanoparticles. The prepared nanoparticles were named NP-S1 Cur.Curcumin is a natural compound that can inhibit the toxicity of A 尾, and S1 can effectively reduce A 尾 formation by affecting the shearing process of APP. Both of them have a positive effect on the pathogenesis of AD. Subsequently, we chose to modify CRT-NP-S1 Curan on the surface of PLGA nanoparticles with transmembrane peptide CRT, because CRT is a polypeptide similar to the biological properties of iron. It can target the complex of transferrin and transferrin receptor to enhance the ability of nanoparticles to cross BBB. The morphology of nanoparticles was observed by transmission electron microscopetem (TEM). The size of nanoparticles before and after coupling CRT was 128.6 nm and 139.8 nm, the entrapment efficiency was 23.2 卤5.3 and 21.4 卤6.9, and the drug loading was 0.54 卤0.04 and 0.42 卤0.06 respectively. The biotoxicity of nanoparticles was analyzed by MTT toxicity test. At the same time, in vitro and in vivo imaging experiments showed that CRT-NP-Sl Cur had a stronger ability to cross BBB. Animal behavioral and pathological studies showed that after 21 days of intraperitoneal injection of PLGA nanoparticles into AD transgenic mice, CRT-NP-Sl Cur had stronger ability to cross BBB. It could improve the spatial memory ability of AD mice. The number of senile plaques in the brain of AD mice was decreased, and the activation levels of microglia and astrocytes were decreased. The level of synaptophysin was improved. The results showed that PLGA nanoparticles could enhance the activity of superoxide dismutase (SOD), decrease the levels of A 尾 and reactive oxygen species (Ros), and promote the expression of inflammatory cytokines TNF- 伪 and IL-6. Compared with NP-S1 Cur, the effect of CRT-NP-S1 Cur was more significant. The results showed that the novel drug carrier could travel through BBB and target the brain, thus effectively exerting curcumin antioxidation, anti-inflammatory effect and S1 inhibiting A 尾 production. Reducing the toxicity of A 尾 has potential application value in the treatment of AD.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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