血小板膜-自组装纳米递药系统构建及其诱导星型胶质细胞靶向直接重编程研究

Nerve repair after central nervous system injury such as intracerebral hemorrhage has long been a research hot spot in the field of life science and medical research. Inspired by the inherent platelet property of binding to injured vasculature, this project focuses on establishing a brain-targeted nano delivery system for small molecular drugs after platelet membrane cloaking, in order to realize the in vivo targeting direct reprogramming of the activated astrocytes at the intracerebral hemorrhage site. The main contents include as follows: construct the nano drug delivery system cloaked by platelet membrane via optimizing the preparation techniques, and evaluate its drug release profiles in vitro; determine the drug contents at the hemorrhage site, and assess the drug targeting and release profiles of the novel nano-carrier as well as the influential factors; investigate the in vitro and in vivo direct reprogramming of astrocytes using immunofluorescence staining to observe the proliferation and transdifferentiation of astrocytes as well as migration and synapse formation of the newly formed neurons both in vitro and in vivo; explore the correlativity between the reprogramming efficiency and targeting ability, duration of drug release as well as therapeutic time window; evaluate the nerve repair effect after intracerebral hemorrhage via examining the inflammation reaction, encephaledema, and behavior assessment and investigate the possible mechanisms underlying the direct reprogramming. Altogether, this research aims at providing new strategies, methods and vehicles for central nervous system injury and in vivo targeting direct reprogramming.

脑出血等中枢神经系统损伤修复一直是生命医药研究的热点领域，本项研究借助血小板对出血部位的天然识别、募集功能，构建血小板膜介导的小分子药物脑靶向纳米传递系统，并籍此实现脑出血活化胶质细胞体内靶向直接重编程。主要内容包括：制备血小板膜包被的纳米药物传递系统，优化制备工艺，评价其体外释药动力学；测定脑出血部位药物含量，评价新纳米载体介导的药物靶向性、释药行为及影响因素；研究星型胶质细胞体内外直接重编程，免疫荧光观测体内外星型胶质细胞增殖与转分化、新生神经元细胞的迁移与突触形成，探究重编程的效率与靶向性能、持续释药时间以及治疗时间窗之间的相关关系；通过考察炎症反应、脑水肿、大鼠行为学，评价脑出血神经损伤修复效果；分析研判可能的直接重编程机制。本研究旨在为中枢神经系统损伤修复及体内靶向直接重编程研究提供新思路、新技术、新载体。

本研究旨在构建安全、高效的血小板膜-自组装纳米粒（PM-SAN）药物传递系统，进行星型胶质细胞靶向直接重编程，修复脑出血损伤。主要内容如下：（1）采用反复冻融、超声分散的方法制备血小板膜，以包膜法包裹姜黄素前药自组装纳米粒，得到球形、分散均匀、大小均一的PM-SAN，其表面阳性表达血小板膜糖蛋白包括CD9、CD41、CD61、CD62p，说明血小板膜成功包覆在姜黄素自组装纳米粒的表面；（2）考察了PM-SAN的稳定性和体外释药动力学，结果表明：PM-SAN在PBS中较稳定，适宜的离子强度、温度（37 ℃）以及GSH浓度（10 mM）有利于药物释放；（3）成功建立SD大鼠脑出血模型和体内药物HPLC分析方法，考察了不同影响因素对靶向性和滞留时间的影响，结果表明：PM-SAN在脑部具有明显的靶向和富集作用，粒径和载药量对靶向性影响不显著，但血小板膜特异蛋白表达量的升高和纳米粒表面电负性的降低利于血小板膜富集于出血部位；（4）采用EdU染色检测体内外星型胶质细胞的增殖，PCR和免疫荧光染色检测重编程后神经元标志物的表达，流式细胞术检测转分化的效率，结果表明：PM-SAN在体外和体内均能够显著抑制星型胶质细胞增殖和胶质瘢痕形成，并能成功、高效诱导星型胶质细胞直接转分化为神经元；（5）通过PCR和免疫荧光染色考察出血部位神经元再生、迁移、与突触形成，结果显示：PM-SAN能够促进神经元再生、迁移和突触形成；（6）器官成像和HE染色结果表明药物的靶向性越好、持续作用时间越长、治疗时间窗越宽，体内重编程的效果越好；（7）通过炎症水平、脑水肿体积、脑含水量检测以及mNSS评分，对脑出血损伤修复进行药效学评价，结果显示：PM-SAN在大鼠脑出血模型体内能够显著抑制炎症因子的表达，减小脑水肿体积和抑制脑含水量的增加，显著改善脑出血大鼠的行为功能；（8）采用RNA-seq进行重编程机制初步研究，结果表明：姜黄素能够激活神经元再生相关基因而抑制星型胶质相关基因的表达，通过激活PI3K-AKT pathway、Focal adhesion signaling、ECM-receptor interaction以及GABAergic synapses等神经再生相关通路实现星型胶质细胞向神经元的转分化。