穿透性载药纳米粒子MPP治疗青光眼作用机制的研究

The traditional nanoparticles have been widely used in many kinds of disease treatment. But if it could not only load the active therapic drugs, but also penetrate the vitreous humor rapidly and distributed uniformly in the whole eyeball, it will play a key role for the drugs which need most effectively to reach the retina, choroid membrane and even optic nerve. Based on mucus-penetrating nanoparticles (MPP) which investigated in the other organs in the past, we use the polyethylene glycol and polyanhydride materials to modify the physical and chemical properties and surface of the nanoparticles, make them meet the requirements of penetrating the vitreous humor quickly with high efficiency, and prepare a novel high bio-security of MPP, for the first time in the world, to treat the ocular diseases. To apply the tracer and fluorescent label technique to record the motion video of MPP in the vitreous humor and speculate its duration in the eye, in order to explore the effect of physical and chemical properties of MPP on its penetrating the vitreous humor and evenly distributed in the retina, choroid membrane and the optic nerve. Preparation of coupled two drugs Brinzolamide and Gastrodin with the MPP to build up a new drug delivery system on the mouse high intra-ocular pressure glaucoma model which mimics optic nerve damage at the same time. With immunostaining, TUNEL and real-time PCR, intra-ocular pressure and RGCs apoptosis will be tested before and after the intravitreal injection of new drug-loaded MPP to evaluate the possibility and efficiency of this new drug delivery system. Our study is to elucidate novel mechanisms of nanoparticles penetrate the vitreous and reveal the fact of distribution in the eye to treat glaucoma. It will provide a high efficient drug delivery system for ocular diseases and to explore a new path to treat glaucoma in future. It will open the door to the hope of medical therapy for ocular diseases and become the milestone for nanoparticles application in ophthalmological treatment.

传统纳米粒子材料在各种疾病治疗的应用已很广泛，但如果其不但能携载药物，还可以快速穿透玻璃体到达眼部各组织且均匀分布，将对药物有效进入视网膜、脉络膜或视神经等处发挥最优药效起到关键作用。基于以往可穿透体液纳米粒子的研究经验，通过调整其物理、化学性质来实现其快速、高效穿透玻璃体的能力，进而探索穿透性载药纳米粒子在治疗眼部疾病中的可行性。本课题拟通过运用纳米粒子示踪技术及荧光标记手段来揭示可穿透玻璃体液的纳米粒子在玻璃体内的运动规律，进而推算其在玻璃体内的滞留时间，探索其物理、化学性质是否是影响其快速穿透玻璃体液并均匀分布于眼底各组织的因素；同时制备偶联布林佐胺和天麻素的可穿透玻璃体的载药纳米粒子，在小鼠高眼压性青光眼视神经损伤模型中，探索玻璃体注射新型载药纳米粒子系统的可行性，从而为揭示纳米粒子在眼底分布规律及穿透玻璃体治疗青光眼的机制奠定实验基础及理论支持，纳米材料将成为眼科治疗的新希望。

本课题在以往研究基础上，通过小鼠玻璃体注射载有天麻素和布林佐胺的纳米粒子MPP探究其在眼内药物递送能力、保护视神经作用和降低眼压的作用，分析载药纳米粒子治疗青光眼的可行性。.下面就以上各方面开展的研究工作做详细的汇报。.1）MPP眼内穿透情况及毒性研究：随机挑选正常小鼠进行玻璃体注射荧光标记的MPP，荧光示踪显示MPP注射后在眼内快速扩散，6h可穿透视网膜神经节细胞层，24h即可穿透视网膜全层；HE染色结果显示在分别注射MPP 1天、3天后，视网膜神经节细胞层细胞密度和形态学变化在各组间没有明显差异。实验结果证实眼内注射MPP对视网膜没有明显毒性损伤。.2）MPP降眼压作用的研究：制备小鼠高眼压模型。模型制备成功后，分三组，分别进行玻璃体注射载有布林佐胺的MPP、玻璃体注射PBS和空白对照组。各组实验动物于同一时间点进行眼压测量，连续检查。结果表明MPP注射组眼压明显降低，另外两组眼压没有明显下降。.3）MPP保护视神经作用的研究：制备小鼠视神经夹伤模型，模型制备成功后，分三组，分别进行玻璃体注射载有天麻素的MPP、玻璃体注射PBS和空白对照组实验。免疫荧光结果表明，玻璃体腔注射MPP组Tuj-1阳性细胞存活率（96.09%±1.48%）高于玻璃体腔注射PBS组（84.19%±5.37%）和空白对照组（84.40%±4.93 %）；TUNEL凋亡检测结果表明，玻璃体腔注射MPP组RGCs凋亡率（18.50±6.94 %）明显低于玻璃体腔注射PBS组（43.86±5.31 %）和空白对照组（46.12±6.89 %）；荧光金逆行标记结果证实玻璃体腔注射MPP组RGCs的存活数目（3454.86±60.98 cells/mm2）明显高于玻璃体腔注射PBS组（3037.81±104.27 cells/mm2）和空白对照组（3093.34±104.80 cells/mm2）；Real-time PCR 结果表明玻璃体腔注射MPP组Nefh、thy1.1分子表达水平高于玻璃体腔注射PBS组和空白对照组。.综合以上研究结果证实，纳米粒子MPP能够快速穿透玻璃液到达视网膜，并且玻璃体注射载药MPP在控制眼压和保护视神经方面具有显著的效果，并具有较好生物安全性，有望成为治疗青光眼疾病的新手段。