针对脑部Aβ42的双级靶向纳米递药系统的构建及对阿尔茨海默病的治疗研究

发布时间：2018-04-15 19:23

本文选题：双级靶向 + 阿尔茨海默病　；参考：《复旦大学》2014年硕士论文

【摘要】：阿尔茨海默病(Alzheimer's disease, AD)是一种常见的中枢神经系统退行性疾病,其发病机制复杂,治疗难度大,目前临床上缺乏有效的对因治疗药物。生物技术药物如神经生长因子、p-片层阻断肽等,能针对神经细胞的病变原因发挥作用,在治疗AD方面已显示出良好的应用前景,但要真正发挥其治疗优势还存在两大难点：首先,药物难以透过血脑屏障(blood brain barrier, BBB)入脑；其次,药物入脑后呈全脑分布,对病变部位缺乏选择性,药物在发挥疗效的同时,也给正常脑组织带来了重大安全隐患。因此,如何将治疗药物准确递送至AD的病变部位,从而增强治疗效果,降低中枢副作用,成为AD治疗关注的一大重点。为此,本课题采用“双级靶向”的策略,即在载药的递释系统表面同时修饰双重靶向功能基,其中第一级靶向功能基能够选择性亲和BBB,促进递释系统跨BBB转运；入脑后第二级靶向功能基能够亲和病变组织,赋予该系统对病灶组织继续寻靶的能力,使更多药物进一步浓集于脑病变部位,提高治疗效果,降低毒副作用。神经细胞外以p-淀粉样蛋白(β-amyloid, Ap)沉积为核心形成的老年斑是AD的主要病理特征之一,已被广泛认为是AD最恰当的分子生物标记,是AD诊断和治疗的理想靶点。老年斑的主要成分为Aβ42(约占96%),因此,本课题针对BBB和Aβ42沉积形成的老年斑,选择对其有高亲和力的两个靶向功能基：TGN肽(氨基酸序列为：TGNYKALHPHNG)和QSH肽(氨基酸序列为：QSHYRHISPAQV),将其修饰到聚乙二醇-聚乳酸(PEG-PLA)纳米粒表面,构建靶向脑部Aβ42的双级靶向纳米递释系统,并通过体内外试验对其双级靶向性进行确证。进一步包载p-片层阻断肽H102,考察双级靶向递释系统是否有较单级靶向递释系统更优的治疗AD的效果。本课题研究分为两部分。第一部分首先构建TGN、QSH修饰的PEG-PLA纳米粒(TQNP),并对该纳米粒表面TGN和QSH的修饰密度进行优化,进一步考察优化所得的TQNP的双级靶向性和细胞摄取机理,并对其细胞毒性进行评价。第一部分第一章采用乳化／溶媒蒸发法制备PEG-PLA纳米粒(NP),将具有脑靶向性的TGN肽和与Aβ42具有高亲和性的QSH肽共价连接到纳米粒表面,制得双级靶向纳米粒(TQNP)。表面元素分析结果证实,TGN和QSH均被成功修饰到纳米粒表面。利用bEnd.3细胞摄取实验和小鼠活体成像实验对纳米粒表面TGN肽的修饰密度进行优化；利用硫磺素-T实验和表面等离子共振技术(SPR)对纳米粒表面QSH肽的修饰密度进行优化。结果当纳米粒表面马来酰亚胺基与TGN或QSH的反应摩尔比均为3：1时,修饰密度最佳(T3Q3NP)。第一部分第二章首先对优化所得的T3Q3NP的双级靶向性进行确证。将纳米粒与Aβ42以及AD模型小鼠脑切片共孵育,透射电镜和荧光显微镜观察显示：T3Q3NP能显著抑制Aβ42纤维的形成,且与AD模型小鼠海马淀粉样斑块有较强的共定位,表明QSH修饰的纳米粒与Aβ42具有较高的亲和性。AD模型小鼠尾静脉给予纳米粒后,离体成像定性观察以及脑分布定量测定均显示T3Q3NP在Aβ42聚集的海马区的浓度显著高于单功能基修饰和未修饰纳米粒,其AUC分别是T3NP和NP的1.61和3.43倍,证实了T3Q3NP具有很好的靶向脑内Aβ42的能力。细胞摄取机制的初步研究表明,双级靶向纳米粒在bEnd.3细胞中的摄取是能量依赖的,且溶酶体和穴样凹陷均在细胞摄取过程中起重要作用。MTT实验结果显示,纳米粒在考察浓度范围(0.1～10 mg/mL)内无明显细胞毒性,具有较高的安全性。本课题第二部分以p片层阻断肽H102为模型药物,制备了载H102的双级靶向纳米粒(T3Q3NP/H102),并对其脑内分布、药效学以及短期毒性进行了考察。第二部分第一章采用复乳／溶媒蒸发法制备载H102的纳米粒(NP/H102),在纳米粒表面共价连接TGN、QSH肽,分别制备得到TNP/H102, QNP/H102和TQNP/H102。制得的四种纳米粒粒径约120 nm, Zeta电位约-28 mV。纳米粒的包封率约60%,载药量约0.55%。纳米粒的包载可显著提高H102在血和脑匀浆中的稳定性,载药纳米粒在血浆和PBS中24 h累积释放百分率分别为65%和39%,显示出一定的缓释作用。第二部分第二章对载H102的纳米粒的脑内递药特性进行考察。建立了H102体内分析的LC-MS方法,经方法学验证符合要求。AD模型小鼠尾静脉注射载H102的纳米粒(NP/H102、TNP/H102和TQNP/H102),结果显示,三种纳米粒在血中的药动学基本一致,TNP/H102和TQNP/H102的入脑量均显著高于NP/H102,且TQNP/H102的海马靶向指数DTI为3.67,显著高于TNP(DTI=1.94),说明TQNP能准确递送H102至Ap斑块沉积的海马区域。第二部分第三章对H102各制剂的药效学进行评价。采用小鼠双侧海马定位注射聚集态Aβ42构建AD小鼠模型,通过Morris水迷宫实验评价H102各制剂对AD模型小鼠学习能力及空间记忆障碍的改善作用。结果显示,TNP/H102和TQNP/H102的改善作用较为明显,尤其是TQNP/H102高剂量组,小鼠的学习和记忆能力得到明显提高。生化指标测定以及免疫组化观察结果可知,TGN修饰纳米粒(TNP/H102和TQNP/H102)能明显抑制Aβ斑块的沉积,对海马区神经细胞具有保护作用,且呈剂量依赖性。其中TQNP/H102中、高剂量能明显逆转Aβ42对海马神经元的损伤。并且,TQNP/H102中剂量组的治疗效果与TNP/H102高剂量组相似,表明在相同剂量下,双级靶向纳米粒较单功能基修饰纳米粒治疗AD作用更优。第二部分第四章对TQNP/H102进行了3周的短期毒性考察,结果显示小鼠的血液学指标正常,各主要脏器(心、肝、脾、肺、肾)均未发生明显的组织病理学改变,表明双级靶向纳米粒并未产生体内毒副作用,是一种安全可靠的递药系统。
[Abstract]:Alzheimer's disease (Alzheimer's disease AD) is a common neurodegenerative disease of the central nervous system, its pathogenesis is complex, difficult to cure, the lack of an effective clinical treatment for drugs. Biotech drugs such as nerve growth factor, p- layer blocking peptide, can cause the lesions of nerve cells play a role in the treatment of AD, have shown a good prospect, but to really play its advantages there are two major difficulties: first, the drug is difficult to penetrate the blood-brain barrier (blood brain, barrier, BBB) into the brain; secondly, the whole brain was behind the drug distribution, lack of selectivity to the lesion site in the therapeutic effect of drugs at the same time, but also to normal brain tissue brought great security risks. Therefore, how to treat the lesion accurately drug delivery to AD, so as to enhance the therapeutic effect, reduce the central side effects, become AD for attention A major focus. Therefore, this subject adopts "double targeting strategy", namely in the delivery system of drug loading and surface modification of dual targeting functional group, wherein the first level targeting functional group can promote the selective affinity of BBB delivery systems across the BBB transport into the second stage; behind targeting functional group can the affinity of lesions, enabling the system continues to focus on targeting the organization, make further more of the drug concentration in the brain lesions, improve therapeutic effect and reduce side effects. Nerve cells to p- amyloid deposition (beta -amyloid, Ap) to form the core of senile plaque is the main pathological features of AD AD, has been widely considered to be the most appropriate molecular markers, is the ideal target for the diagnosis and treatment of AD. The main component of senile plaques is A beta 42 (approximately 96%), therefore, the topic for the BBB and A beta 42 plaques, choose to have high affinity Two target and force to functional groups: TGN peptide (amino acid sequence: TGNYKALHPHNG) and QSH peptide (amino acid sequence: QSHYRHISPAQV), the modified polyethylene glycol polylactic acid (PEG-PLA) nanoparticles, which target the brain A beta 42 double targeted nano delivery system, and through the body outside test of the two-stage targeting was confirmed. Further p- loaded layer blocking peptide H102 on two level targeting delivery systems if there is a single stage targeting delivery system to better the effect of treatment of AD. This study is divided into two parts. The first part firstly constructed TGN, QSH modified PEG-PLA nanoparticles (TQNP), and on the surface of the nanoparticles modified density of TGN and QSH were optimized to further investigate the optimized TQNP double targeting and cellular uptake mechanism, and to evaluate its cytotoxicity. The first part of the first chapter by emulsification / solvent evaporation method to prepare PEG -PLA nanoparticles (NP), will have the brain targeting peptide TGN and A beta 42 has high affinity for the QSH peptide covalently attached to the surface of the nanoparticles, prepared double targeting nanoparticles (TQNP). The surface elemental analysis results confirmed that TGN and QSH were successfully modified onto the surface of nanoparticles by bEnd.3 cells. Uptake in mice and in vivo imaging experimental density on the surface of TGN nanoparticles modified peptides were optimized; using Thioflavin -T assay and surface plasmon resonance (SPR) density on the surface of QSH nanoparticles modified peptides were optimized. The reaction when the surface of the nanoparticles Moore maleimide groups with TGN or QSH ratio was 3:1, modified the optimum density (T3Q3NP). The first part of the second chapter on the T3Q3NP of the two-stage targeting was confirmed. The nanoparticles with A beta 42 and AD mouse brain slices were incubated, transmission electron microscope and fluorescence microscope observation showed that: The formation of T3Q3NP can significantly inhibit A beta 42 fiber, and AD model of mouse hippocampal amyloid plaques co localization of strong, showed that the nanoparticles modified by QSH and A beta 42 has a higher affinity for.AD mice after intravenous injection of nanoparticles in vitro imaging, qualitative observation and brain distribution determination showed that the concentration of T3Q3NP in the A beta 42 aggregation of the hippocampus was significantly higher than that of single functional groups of modified and unmodified AUC nanoparticles, which are T3NP and NP 1.61 and 3.43 times, confirmed that T3Q3NP has good targeting ability in the brain of A beta 42. Preliminary study on the uptake mechanism showed that double targeting nanoparticles in bEnd.3 cell uptake is energy dependent, and lysosomes and cave in depression play an important role in the cellular uptake of.MTT nanoparticles in the experimental results show that the effects of concentration range (0.1 ~ 10 mg/mL) without obvious cytotoxicity, with high safety All of this topic. The second part of the p layer H102 blocking peptide as a model drug, preparation of H102 loaded double targeting nanoparticles (T3Q3NP/H102), and distribution on the brain, pharmacodynamics and short-term toxicity were investigated. The second part of the first chapter by double emulsion / solvent evaporation nanoparticles prepared by H102 the (NP/H102), in the surface of the nanoparticles covalently connected to TGN, QSH peptides, which were prepared by TNP/H102, QNP/H102 and TQNP/H102. four nanoparticles prepared by about 120 nm in diameter, encapsulation efficiency of the Zeta potential is about -28 mV. nanoparticles of about 60%, the drug loading of about 0.55%. loaded nanoparticles can significantly improve the stability of H102 in blood and brain homogenate, drug loaded nanoparticles in plasma and PBS in the 24 h accumulative release percentage were 65% and 39%, showing a certain sustained-release effect. The second part makes research on the second chapter of the H102 loaded in the brain delivery property. A H102 body LC-MS analysis method, the method validation conforms to the requirements of nanoparticles injected into the tail vein of.AD mice carrying H102 (NP/H102, TNP/H102 and TQNP/H102). The results show that dynamic consistent three kinds of nanoparticles in the blood of the drug, TNP/H102 and TQNP/H102 in the brain were significantly higher than that of NP/ H102, and TQNP/H102 to the hippocampus target index DTI as of 3.67, significantly higher than that of TNP (DTI=1.94), that the hippocampus TQNP can deliver H102 to Ap plaque deposition. Effects of second parts and third chapters on the preparation of H102 learning evaluation. Using the mouse hippocampus injection of aggregation of A beta 42 build AD mouse model, through the evaluation of the Morris water maze effect H102 preparation of AD model mice learning and spatial memory deficits. The results showed that the effect of TNP/H102 and TQNP/H102 is obvious, especially the TQNP/H102 high dose group, the ability of learning and memory in mice To improve observation results and determination. Immunohistochemical and biochemical indexes, TGN modified nanoparticles (TNP/H102 and TQNP/H102) can inhibit the deposition of A beta plaques, has a protective effect on neuronal cells, in a dose-dependent manner. The TQNP/H102 in high doses can significantly reverse A beta 42 on hippocampal neurons injury and the therapeutic effect of TNP/H102 dose group and TQNP/H102 high dose group is similar to that of the same dose, double targeting nanoparticles with single functional group modified nanoparticles on AD effect better. The second part of the fourth chapter investigated the short-term toxicity of 3 weeks of TQNP/H102, showed that mice blood indexes of normal. The main organs (heart, liver, spleen, lung, kidney) showed no apparent histopathologic changes, showed that double targeting nanoparticles did not produce toxic side effects in vivo, is a safe and reliable delivery system.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R96

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