主动脑靶向配体修饰PEG-PAMAM纳米递药系统的构建和调控及对脑缺血损伤的机制

Tanshinone ⅡA and scutellarin are widely used in treatment of cerebral ischemia, but their poor solubility, short half-life in circulation, in particular, very limited blood-brain barrier (BBB) penatration lead to lower bioavailability, which limit their curative efficacy. Pegylated polyamidoamine dendrimers (PEG-PAMAM) are biodegradable, biocompatible， low toxicity and have been considered as one of the most promising nanoscopic brain delivery system. Some active targeting peptides are exploited as the brain targeting ligands to conjugate PEG-PAMAM nanoparticles, such as Angiopep-2, which can target to the low-density lipoprotein receptor-related protein-1 (LRP1); Peptide HAIYPRH (T7 peptide) and anti-transferrin receptor antibody RI7217、OX26, which can target to transferrin receptor and cell-penetrating peptide (Tat peptide). These receptor-related peptides, are highly expressed on BBB and brain capillary endothelial cells (BCECs). With the brain targeting ligand modification, the drug delivery systems can penetrate the BBB without disrupting it through a receptor-mediated transport mechanism and further accumulate in cerebral ischemic region. As a potentil brain delivery system, active targeting ligand conjugated PEG-PAMAM nanoparticles have some advantages, eg, 1) improving BBB penatration and bioavailability of drug; 2) increasing the solubility and encapsulation efficiency of poorly water-soluble drugs; 3) reducing particle size and burst release while improving targeting; 4) avoiding recognition and uptake by the reticuloendothelial system, thereby prolonging the circulation time of drugs in the blood; and 5) good safety. In this study, we synthesize PAMAM as the principal delivery vehicle, subsequently conjugated the brain targeting ligands to PAMAM via bifunctional polyethyleneglycol (PEG), and then encapsulated drugs, designated as brain targeting ligands conjugated PEG-PAMAM nanoparticles. The pharmacokinetics and biodistribution of active targeting ligand conjugated PEG-PAMAM nanoparticles were investigated to evaluate its brain targeting property. The brain uptake efficiency of the nanopatticles loading 6-coumarin are examined using a laser scanning confocal microscope and CRi in vivo imaging system. The cerebral ischemia models are established to evaluate the treatment efficacy of brain targeting ligand conjugated PEG-PAMAM nanopatticles. The potential mechanisms of drug-loading nanoparticles on signaling pathways involved in inflammatory cascades and neuronal cell apoptosis after cerebral ischemia injury, including TLRs/MyD88/TRIF/NF-κB, 12/15-LOX/MAPK, JAK/STAT/SOCS3 and PIK3/Akt/GSK3β/Nrf2-OH-1 are evaluated by immunohistochemistry, western blotting and real-time quantitative PCR, respectively. Collectively, this study provides significant scientific basis to the preparation and regulation of the novel nanoparticles delivery system for therapy of ischemic stroke.

丹参酮ⅡA和灯盏花乙素对脑缺血疗效显著。但是，二者难溶于水，体内半衰期短，生物利用度低；注射后从血中消除迅速，对血脑屏障穿透性差，很难在脑组织中达到有效浓度。PEG-PAMAM树状大分子作为新型纳米载体，可增强难溶性药物的溶解度，提高生物利用度，促进药物透过血脑屏障。本研究合成和调控PEG-PAMAM纳米载体，并通过链接主动靶向配体，构建主动脑靶向配体修饰纳米粒，考察纳米粒的药代动力学和组织分布，以荧光探针活体成像系统和激光共聚焦显微镜评价脑靶向性，采用定量PCR、Western blotting 和免疫组化法，从脑缺血炎症反应和神经凋亡中心环节TLRs/MyD88/TRIF/NF-κB，12/15-LOX/MAPK，JAK/STAT/SOCS3和PIK3/Akt/GSK3β/Nrf2-OH-1信号通路角度，研究载药纳米粒对脑缺血损伤的机制，为新型脑靶向纳米递药系统研究提供科学依据。

丹参酮ⅡA和灯盏花乙素是治疗脑缺血中风的常用临床药物。但是，二者难溶于水，体内半衰期短，生物利用度低；注射后从血中消除迅速，对血脑屏障穿透性差，很难在脑组织中达到有效浓度。本研究合成G5.0代端羟基聚酰胺-胺树状大分子（G5.0 hydroxyl-terminated polyamidoamine dendrimers）作为新型纳米载体，采用双功能PEG (NHS-PEG3400-MAL)修饰，可增强难溶性药物的溶解度，提高生物利用度；通过马来酰亚胺基团藕连各种靶向配体多肽，如：低密度脂蛋白受体介导多肽Angiopep-2、转铁蛋白受体介导多肽HAIYPRH (T7)或脑缺血炎症介导中性粒细胞锚定肽PGP，构建主动脑靶向配体修饰纳米粒，促进药物透过血脑屏障。研究考察了纳米靶向药物载体的药代动力学和组织分布，以荧光探针活体成像系统和激光共聚焦显微镜评价脑靶向性；通过建立体外血脑屏障细胞模型，考察纳米载体的血脑屏障透过性能。药效学研究部分，主要考察纳米药物载体对实验性脑缺血动物模型TTC染色脑梗死面积、神经功能评分、HE染色病理学研究，以及脑缺血后神经细胞凋亡、细胞内Ca2+浓度、炎症细胞因子IL-12p40、IL-13、IL-17、IL-23。为了阐明主动脑靶向配体修饰PEG-PAMAM纳米递药系统对脑缺血的保护机制，本研究采用实时荧光定量PCR和Western blotting技术，考察了纳米药物载体对脑缺血炎症反应和神经凋亡中心环节HMGB1/TLRs/MyD88/TRIF/IRAK信号通路主要节点mRNA和蛋白表达的影响。研究结果表明，丹参酮ⅡA和灯盏花乙素制成纳米靶向药物载体后，可显著提高稳定性，具有明显的缓释效应。药代动力学显示，纳米药物载体可显著提高药物半衰期，延长体循环时间，提高脑组织分布率。体内靶向性研究表明，主动靶向配体修饰纳米药物载体可显著提高血脑屏障跨域效果，引导药物向脑部转运。药效学研究显示，纳米药物载体可显著降低脑梗死面积与神经功能评分，明显改善脑缺血病理特征，抑制神经细胞凋亡和细胞内Ca2+超载，显著降低炎症细胞因子IL-12p40、IL-13、IL-17、IL-23的表达，抑制炎症信号通路HMGB1/TLRs/MyD88/TRIF/IRAK主要节点mRNA和蛋白表达。研究结果为新型脑靶向纳米递药系统研究提供科学依据。