双级靶向递释系统用于阿尔茨海黙病的治疗研究

Alzheimer's disease (AD) is the most common neurodegenerative disorder, the main cause of dementia in the elderly, and one of the main causes of death in developed countries. The few treatments used at present, such as acetylcholinesterase inhibitors and N-methyl-D-aspartate receptor antagonist, only ease the symptoms and can not cure the disease. While enormous biotech drugs have been developed regarding our understanding of the pathogenic mechanisms of AD, there are few used in clinic for treating this disease. Two obstacles exist for their use are: first is the blockage of exogenous substances entrance into the brain by the blood-brain barrier (BBB). And the second is lacking selected distribution to diseased region after drugs into brain, which may result in sever side effect on normal brain. Therefore, in this project, "dual targeting" strategy has been put forward, which is considered a very promising resolution for this dilemma. .For dual targeting, dual functional ligands are conjugated at the surface of nanosized delivery system encapsulated with biotech drugs, one ligand targeting BBB can enhance BBB penetration of delivery system by inducing and participating in vesicular transport processes in the cell, and/or modulate epithelial permeability of BBB. After in the brain, the other ligand with high affinity to the disease region of AD will mediate the delivery system to concentrate on the lesion site. High efficiency and lower side effects provided by the dual targeting system is important for AD therapy. . Here, poly[2-(dimethylamino)ethyl methacrylate (PDMAEMA) is chosen as vector to condense report gene to form polyion complex micelle, then TGN peptide (consensus peptide sequence "TGNYKALHPHNG") and QSH peptide (consensus peptide sequence "QSHYRHISPAQV") screened by phage display, specific targeting ligands of BBB and amyloid-beta (Aβ) peptide1-42 (the main component of amyloid plaque) respectively, are utilized to conjugate with micelle to establish the dual targeting delivery system (TGN/QSH-PM). After system construction, the dual targeting of TGN/QSH-PM will be investigated in in vivo and in vitro experiments. At last, nerve growth factor gene will be used as a model DNA to complex with TGN/QSH-PM, and its treatment efficiency for AD in APPswe transgenic mice will be evaluated. This project will provide experimental data and theoretical basis for the treatment of AD by employing "dual targeting" strategy.

生物技术药物在治疗阿尔茨海默病(AD)方面已显示出良好的应用前景，但要发挥其治疗优势还存在两大难点：①药物难以透过血脑屏障(BBB)入脑；②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，也给正常脑组织留下了重大安全隐患。为此，本项目拟采用"双级靶向"策略，在包载生物技术药物的纳米递释系统表面分别修饰靶向BBB和AD病变部位的双功能基，通过双功能基的介导，以期增加药物的入脑递释和AD病变部位的浓集，从而提高药物对AD的治疗效果，减少其中枢副作用。基本思路：采用聚阳离子胶束包载报告基因，在其表面同时修饰短肽TGN(靶向BBB)和QSH(靶向AD病理学特征－老年斑)，构建载基因的双级靶向递释系统，并通过体内外试验对其双级靶向性进行验证；以神经生长因子基因为模型药物考察递释系统对APPswe转基因小鼠的治疗效果。本项目旨为采用"双级靶向"策略治疗AD提供实验资料和理论依据。

阿尔茨海默病(Alzheimer’s disease, AD)是一种常见的神经系统退行性疾病，其病因复杂，迄今仍无有效药物可转归病程。一些多肽蛋白、基因药物虽能针对神经细胞的病变原因发挥作用，但要用于AD治疗还需解决两个关键问题：①血脑屏障(BBB)对药物入脑的阻碍作用；②药物入脑后呈全脑分布，难以浓集于病变部位，不仅降低了治疗效果，也给正常脑组织带来了重大安全隐患。因此，如何将治疗药物精准递送至AD病变部位，充分发挥其作用，成为AD治疗关注的一大重点。. 为此，本项目采用“双级靶向”策略，在包载生物技术药物的纳米递释系统表面分别修饰靶向BBB和AD病变部位(以淀粉斑作为标记)的双功能基，通过双功能基的介导，增加药物的入脑递释和AD病变部位的浓集。. 具体内容分为两部分：①选择对BBB和Aβ42有高亲和力的两个靶向功能基：TGN和QSH，将其修饰到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，研究发现当双功能基在纳米粒表面的修饰密度均为10%时，递释系统呈现最优的双级靶向性。将其包载β-片层阻断肽H102，药动学和药效学实验证实双靶纳米粒能准确递送H102肽至Aβ斑块沉积的海马区域(靶向指数DTI为3.67)，有效改善AD小鼠的空间记忆障碍，在相同剂量下，治疗作用较单靶纳米粒更优。②将靶向BBB和淀粉斑的双功能基CGN和QSH共价连接到阳离子聚合物PEG-PDMAEMA上制备功能化材料，并与siRNA通过静电吸附自组装形成双级靶向复合物。该复合物最优N/P为10，CGN-PEG-PDMAEMA和QSH-PEG-PDMAEMA在复合物中的最佳用量分别为50%和25%。转运机制研究证实，部分复合物能以完整形式透过BBB，并递送siRNA进入病变的神经元，入胞后复合物能从溶酶体中逃逸，在细胞浆中释放出游离siRNA，发挥基因沉默作用。以BACE1基因为靶基因，考察载BACE1-siRNA的双靶复合物对AD的治疗效果。结果显示，双靶复合物能有效沉默BACE1基因，减少BACE1相关蛋白和淀粉斑的量，显著改善AD小鼠空间记忆障碍，且具有优良的神经保护作用和较高安全性。. 本项目为采用“双级靶向”策略治疗AD提供了实验资料和理论依据，为生物技术药物在AD治疗中的应用探索了一条新途径。