基于线粒体靶向修饰的纳米氧化铈保护视网膜神经节细胞的作用研究

Nanoceria (cerium oxide nanoparticles, CNPs), which are rare earth oxide nanoparticles, can store and transport oxygen atoms due to cerium valence change and surface oxygen vacancies. The unique surface chemical characteristics reasonably assign CNPs remarkable antioxidative capacity to scavenge oxygen free radicals, which could be auto-catalytic and regenerative. Many studies have confirmed that CNPs could exert significant neuroprotection in several neurodegenerative diseases, also showed excellent biocompatibility. In the preliminary experiments, we completed the preparation and characterization of CNPs, and further confirmed the neuroprotective effects of CNPs against the oxidative and hypoxic injury in RGCs, which involved the scavenging of oxygen free radicals, restore of mitochondrial dysfunction and inhibition of endogenous apoptosis signaling pathway. However, the subcellular localization of CNPs showed diffuse distribution in cytoplasm, without specific localization on the certain organelles—mitochondria. Considering that mitochondria are the key organelles directly regulating oxygen free radicals, and play an important role in RGCs injury, we hypothesize that we could achieve the precision guidance and efficacy improvement of CNPs neuroprotection by the realization of the targeting transport of CNPs to the critical organelles—mitochondria. In order to improve the performance of CNPs, the mitochondria-targeting TPP-PEG-CNPs were prepared by using special surface modifications. The present study aims to elucidate the subcellular localization of mitochondria-targeting CNPs, and the mechanisms involving the morphological and functional changes of mitochondria, and also to clarify the superiority of neuroprotection exerted by TPP-PEG-CNPs in RGCs injury models in vitro and in vivo. The purpose of the present study is to further explore the function and mechanism of CNPs in RGCs neuroprotection. The present study would accurately guide and improve the neuroprotective potential of CNPs to RGCs by the construction of mitochondrial-targeting CNPs, which would also provide the methodological optimization for the translation of CNPs neuroprotection in optic neuropathies.

纳米氧化铈(CNPs)因表面氧空位衍生出可催化再生的氧自由基清除能力，能有效干预神经退行性疾病，且生物相容性良好。本项目前期已证实CNPs在视网膜神经节细胞(RGCs)氧化损伤中发挥保护作用，发现其清除氧自由基和修复线粒体功能障碍的机制。线粒体是直接调控氧自由基的关键细胞器，在RGCs损伤机制中扮演重要角色。但是，亚细胞定位显示CNPs分布于胞浆，并不聚集于线粒体，因此我们认为其抗氧化作用可以进一步提升。我们推测，通过实现线粒体的靶向运输，能达到CNPs视神经保护效能的提升。据此我们通过表面修饰构建了线粒体靶向的TPP-PEG-CNPs，拟在RGCs损伤模型中观察线粒体靶向CNPs的亚细胞定位及其调控线粒体形态功能的机制，以及视神经保护作用的优效性。本项目旨在探索线粒体靶向CNPs在视神经保护中的作用及机制，精准引导并提升CNPs对RGCs的保护潜能，为视神经保护的转化应用提供方法学优化。

视神经保护一直是眼科的临床难点和研究热点，基于此的基础研究、转化应用以及临床试验与日俱增，但至今仍然收效甚微。纳米医学的蓬勃发展影响着研究人体各个系统和器官的所有学科，而在眼科视神经保护领域的纳米医学仍然处于起步阶段。本项目致力于将具有特殊理化性质的氧化铈纳米粒子（CNPs）引入到视神经保护中来，并且根据视网膜神经节细胞（RGCs）的高代谢特性以及CNPs清除自由基的生物学功能，提出特定细胞器——线粒体靶向的CNPs能发挥更为持久、更为强大的保护作用。.基于此，我们首先设计了以线粒体靶向基团TPP装载于PEG基团修饰的CNPs，完成制备后进行了理化性质的表征工作，发现TPP-PEG-CNPs的单分散效果更佳，水化粒径在14nm左右，原子力显微镜和透射电镜均显示修饰后的CNPs形态完美，X 射线光电子能谱证实了铈元素+3/+4的混合价态，奠定了后续研究的物质基础。然后我们通过透射电镜在进一步证实TPP-PEG-CNPs在细胞和组织内的摄入情况和亚细胞器定位，以阐明TPP基团诱导的线粒体靶向输入的效果，结果较为理想。最后我们在体内体外实验中证实CNPs对RGCs氧化损伤的保护作用，并比较TPP-PEG-CNPs所带来的线粒体靶向为视神经保护作用带来的优势。结果显示在RGCs的体外氧化损伤模型中，CNPs能显著降低RGCs死亡，缓解RGCs凋亡，其中清除氧自由基、修复线粒体功能障碍、逆转内源性细胞凋亡通路均已被证实参与CNPs对RGCs的保护作用。除此之外，通过SD大鼠的视网膜缺血再灌注（RIR）模型和硅油滴前房注射慢性高眼压（SOHU）模型，发现CNPs能显著抑制RIR和SOHU诱发的RGCs丢失，其中在RIR模型中进一步证实了CNPs能显著改善RGCs的凋亡，并且p-ERK以及HIF-1a信号通路参与其中。.总而言之，本项目成功制备并表征了线粒体靶向CNPs，并证实了其对RGCs的保护作用，有望进一步临床转化，提升青光眼以及视神经疾病患者的视力预后。