重组高密度脂蛋白纳米递药系统的脑内清除机制研究

The development of nanotechnology has provided powerful strategies to facilitate the brain drug delivery. However, the state of art researches majorly focus on the brain drug delivery efficiency, but pay little attention to the key scientific issue — how the nanoparticulate drug delivery systems (DDS) be cleared from the central nervous system (CNS). Based on previous works, we hypothesize that beside in-situ metabolism, the recently identified glymphatic system could be another important pathway involved in the brain clearance of nanoparticulate DDS. To test this hypothesis, in this proposal, by using reconsitituted high density lipoprotein nanoparticles (rHDL) as the model DDS, two-photon fluorescent microscopy which possesses the advantages such as deep tissue penetration, high resolution and high sensitivity will be applied as a key technique for the in-situ visualization of brain vasculature, glymphatic system and brain cells, and for the real-time imaging of the in-brain transport and clearance of nanoparticulate DDS; Radiolabeling and detection and liquid chromatography-mass spectrometry will be utilized to characterize the dynamics of the glymphatic system and in-situ metabolism-mediated brain clearance of rHDL. In addition, how the factors such as structure, particle size and zeta potential affect the pathways as well as efficiency of the brain clearance of rHDL will also be investigated. We believe the findings of this work will reveal the pathways involved in the brain clearance of nanoparticulate DDS, and provide fundamental basis for rational design and application of nanoparticulate DDS for brain-targeting drug delivery.

纳米技术的发展为跨越血脑屏障药物递送提供了重要手段，但现有研究主要关注脑内药物递送效率，而对于纳米递药系统入脑后如何清除这一关键科学问题鲜有探讨，存在较大的安全隐患。基于前期工作，我们率先提出：除脑内原位代谢外，新近发现的胶质淋巴系统可能是介导纳米递药系统脑内清除的另一重要途径。为证实该假设，本项目拟以重组高密度脂蛋白纳米递药系统（rHDL）为模型，以具有高组织穿透力、空间分辨率和信噪比的双光子显微镜为核心技术，结合脑血管、胶质淋巴系统和脑细胞原位成像，探讨纳米递药系统脑内转运和清除的动态过程；结合放射性标记和液质联用技术定量检测，表征胶质淋巴系统和脑内原位代谢介导rHDL清除的动力学特征；比较不同形状、粒径和表面电位的rHDL的脑内转运途径和清除效率，探讨制剂学因素的影响。该研究有望揭示纳米递药系统的脑内清除机制，为具有脑靶向特性的纳米递药系统的合理设计和安全应用提供新的理论和实验依据。

采用纳米技术实现跨越血脑屏障药物递送为脑部疾病诊疗提供了重要手段。然而，现有研究更多关注脑内药物递送效率，而对于纳米递药系统入脑后如何清除这一关键科学问题鲜有研究，存在较大的安全隐患。基于前期工作，我们提出：脑内原位代谢可能并非纳米递药系统脑内清除的唯一途径，而最新发现的胶质淋巴系统可能参与纳米粒的脑内清除。为证实该假设，本课题以ApoE重组高密度脂蛋白（ApoE-rHDL）为模型，以具有高组织穿透力、空间分辨率和信噪比的双光子显微镜为核心技术，结合脑血管、胶质淋巴系统和脑细胞原位成像，探讨纳米递药系统脑内转运和清除的动态过程，发现入脑后的ApoE-rHDL主要被小胶质细胞摄取，并有向血管旁的胶质淋巴系统聚集的趋势；采用放射性标记示踪法定量测定脑内125I标记的ApoE-rHDL的清除动力学，结合水通道蛋白-4（aquaporin-4, AQP4）抑制和AQP4基因敲除小鼠实验，探讨胶质淋巴系统介导的ApoE-rHDL清除动力学特征，结果表明，实验小鼠的颈淋巴结检测到较高的放射剂量，而且胶质淋巴系统被抑制后，ApoE-rHDL的脑内清除效率明显下降，提示胶质淋巴系统在ApoE-rHDL脑内清除中起重要作用；采用高灵敏度和高选择性的液相色谱质谱联用技术检测ApoE-rHDL在体外培养胶质细胞内的代谢动力学特征，发现体外培养的小胶质细胞摄取ApoE-rHDL的能力强于星形胶质细胞，而ApoE-rDHL在两种胶质细胞中的降解半衰期t1/2相似。通过比较ApoE-rHDL和单唾液酸四己糖神经节苷脂GM1修饰ApoE-rHDL的脑内清除效率和体外胶质细胞降解动力学，揭示了具有不同理化特性的纳米递药系统具有不同的清除效率。为揭示胶质淋巴系统介导纳米粒从脑内清除是否具有普适性，进一步采用双光子显微成像和液相色谱质谱联用等技术探讨胶质淋巴系统对聚乙二醇-聚乳酸（PEG-PLA）纳米粒脑内清除的影响，发现胶质淋巴系统介导清除同样是PEG-PLA纳米粒从脑内清除的重要机制。该研究在一定程度上揭示了纳米递药系统的脑内清除途径，有望为具有脑靶向特性的纳米递药系统的合理设计和应用提供新的理论和实验依据。