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靶向脑淀粉斑的载EGCG纳米递释系统的构建及其治疗阿尔茨海默病小鼠的研究

Constructing Brain Aβ-Targeting Nanoparticles Loaded with EGCG for Treating Alzheimer’s Disease in Mice

    摘要
  • 摘要:
      目的  构建一种能够进入脑组织且靶向β淀粉样蛋白（β-amyloid, Aβ）的表面修饰多肽序列PTLHTHNRRRRR（简称RD2肽）的纳米递释系统。选择荷载表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate, EGCG），以验证该靶向递释系统对阿尔茨海默病（Alzheimer’s disease, AD）治疗的可行性。
      方法  制备载EGCG的纳米粒（NP/EGCG）、表面修饰RD2肽的NP/EGCG（RD2-NP/EGCG）和表面修饰RD2肽的未载药纳米粒（RD2-NP），并对其进行表征。硫磺素T实验考察RD2-NP与Aβ的结合能力，离体成像观察RD2-NP在脑病变部位的分布。将寡聚态Aβ42注入ICR小鼠双侧海马内构建AD小鼠模型，AD小鼠分别尾静脉注射生理盐水、EGCG溶液、NP/EGCG或RD2-NP/EGCG共28 d，水迷宫实验评价各组小鼠的空间记忆能力；RT-PCR法测定小鼠海马中肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）和白介素-1β（interleukin-1β, IL-1β）的mRNA水平；尼氏染色观察海马神经元的形态变化；HE染色观察心、肝、脾、肺、肾的病理学改变。
      结果  制得的RD2-NP/EGCG粒径为（204.83±2.80） nm，Zeta电位约为−23.88 mV，包封率为94.39%，载药量为5.90%，RD2肽修饰对纳米粒的理化特性无显著影响。RD2-NP具有良好的Aβ结合能力，能浓集于海马和大脑皮质。RD2-NP/EGCG治疗4周能够显著降低AD模型小鼠脑中炎症因子TNF-α和IL-1β水平，修复神经元损伤，改善空间记忆障碍，且无器官毒性。
      结论  表面修饰RD2肽纳米递释系统可高效递送药物到AD病变部位，改善EGCG对AD的治疗效果。

     

    Abstract:
      Objective  To construct a nanodelivery system surface-modified with RD2 peptide (polypeptide sequence PTLHTHNRRRRR) for brain tissue penetration and β-amyloid (Aβ) binding. Epigallocatechin-3-gallate (EGCG) was selected for encapsulation in the targeted delivery system and its therapeutic potential for Alzheimer’s disease (AD) was investigated.
      Methods  EGCG-load nanoparticles (NP/EGCG), NP/EGCG with RD2 peptide surface modification (RD2-NP/EGCG), as well as RD2 peptide-modified blank nanoparticles (RD2-NP) were prepared and characterized. Thioflavin T assay was done to assess the ability of RD2-NP to bind with Aβ and ex vivo imaging was conducted to evaluate the distribution of RD2-NP in brain lesion sites. The AD mice model was established by injecting oligomeric Aβ42 in the bilateral hippocampi of ICR mice. Then AD mice were administered intravenously through the tail vein with normal saline, EGCG solution, NP/EGCG or RD2-NP/EGCG for 28 d, respectively, and the Morris water maze tests were performed to assess the spatial memory of mice. Subsequently, RT-PCR method was used to determine the mRNA levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the hippocampus of the mice, and the morphological changes of hippocampal neurons were observed with Nissl staining. Additionally, the pathological changes of heart, liver, spleen, lung, and kidney were characterized by hematoxylin-eosin (HE) staining.
      Results  The particle diameter of the prepared RD2-NP/EGCG was (204.83±2.80) nm and the zeta potential was −23.88 mV. The encapsulation efficiency and drug loading capacity were 94.39% and 5.90%, respectively. The RD2 peptide modification has no significant effect on the physiochemical properties of the nanoparticles. RD2-NP had good Aβ binding ability, and it could be concentrated in hippocampus and cerebral cortex, the most common Aβ deposition sites. The four-week RD2-NP/EGCG treatment significantly decreased the expression of the pro-inflammatory cytokine TNF-α and IL-1β, restored neuronal losses and hippocampal damage, and ameliorated spatial memory impairment in AD model mice. Moreover, treatment with the RD2-NP/EGCG did not present organ toxicity.
      Conclusion  Surface modified RD2 peptide nanodelivery system can efficiently deliver drugs to AD lesions and improve the therapeutic effect of EGCG on AD.

     

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