利用白细胞-纳米粒联合载体构建缺血性脑卒中全病程多级靶向递释系统研究

Ischemic stroke (IS) is caused by a reduction in the blood supply to a part of the brain and subsequently receives returning blood containing leukocytes that may occlude small vessels and release toxic products. This course of ischemia/reperfusion makes it difficult for drugs to be delivered to the lesions. Leukocytes provide a unique opportunity for drug delivery to lesion and target cell. N-Ac-PGPGGC shows high affinity to monocytes, neutrophiles and lymphoctyes. Hence, N-Ac-PGPGGC can be used as a ligand to be modified on the surface of PEGlyated dendrigraft poly-lysines nanoparticles which loaded with catalase (PGP-PEG-DGL-Cat-NP). To enhance the stability of Cat, it was modified with cis-Aconitic anhydride to increase the negative-charge for better adsorption with PEG-DGL. Then, PGP-PEG-DGL-Cat-NP can enhance the stability of catalase and the affinity of nanoparticles with leukocytes to obtain a nanoparticle-cell combination carrier. It can delivery neuroprotection to the targeted-cell in the ischemic area using the chemotaxis of leukocytes during all the pathogenesis process. Thus, a multi-targeting drug delivery system (DDS) of leukocyte-nanoparticles combination to brain is to be obtained for IS therapy based on inflammation response.

缺血性脑卒中治疗（IS）发病过程中脑组织“无血流”、“无复流”病理特性是IS脑内递药最大障碍。白细胞是唯一能够在IS缺血期、再灌期及恢复期全病程进入缺血脑组织的细胞，具胞吞和胞吐特性。本项目选择过氧化氢酶（Cat）为模型药，顺式乌头酸酐对其修饰增加电荷密度后将Cat载于PEG化聚左旋赖氨酸纳米粒（PEG-DGL-Cat-NP）；特异性交联剂增加PEG-DGL-Cat-NP紧密程度，提高Cat体内稳定性；然后将N-Ac-PGPGGC肽修饰于PEG-DGL-Cat-NP表面，使其通过与血循环中中性粒细胞、单核细胞及淋巴细胞发生高度结合并被内吞来构建纳米粒—白细胞联合载体，利用白细胞的趋化性携带活性药物在IS不同病程阶段进入缺血脑组织，然后通过白细胞与靶细胞之间相互作用递释药物进入靶细胞，实现IS全病程多级脑靶向递药，第一时间对抗白细胞造成的氧化损伤，创新性较强，有望真正解决IS治疗瓶颈。

本项目首先将Aco修饰于Cat表面增加其负电荷密度，通过静电吸附将Cat载于PEG-DGL纳米粒（PEG-DGL-Cat-NP）；采用特异性交联剂增加PEG-DGL-Cat-NP紧密程度，避免纳米粒在白细胞内的快速解聚及Cat的降解；然后将N-Ac-PGPGGC肽修饰于PEG-DGL-Cat-NP表面，使其通过与血循环中中性粒细胞（占白细胞比例约50%）、单核细胞（占白细胞比例约5%）及淋巴细胞（占白细胞比例约30%）发生高度结合并被内吞后构建纳米粒—白细胞联合载体，使药物在具有较强活性的前提下随白细胞在IS不同病程进入缺血脑组织，然后进一步利用白细胞与靶细胞之间的相互作用递释药物进入靶细胞，实现IS全病程多级靶向递药目的，从而第一时间对抗白细胞造成的氧化损伤。.实验结果表明cl PGP-PEG-DGL/CAT-Aco纳米粒能够有效抑制缺血脑损伤，明显提高脑缺血的治疗效果。对I/R小鼠给予cl PGP-PEG-DGL/CAT-Aco纳米粒后，尾静脉注射双氢罗丹明123（DHR）评价载药纳米粒清除细胞内ROS的能力，同时利用TUNEL染色评价载药纳米粒的神经保护作用，进而阐述纳米粒发挥药效的方式。结果表明 cl PGP-PEG-DGL/CAT-Aco纳米粒可通过抑制ROS介导的凋亡途径发挥药效。综上，本课题成功构建了cl PGP-PEG-DGL/CAT-Aco纳米粒，该纳米粒具有载药量大，包封率高的特点，可通过与细胞表面受体的特异性结合被中性粒细胞高效摄取，防止蛋白药物在细胞内的快速降解。随后中性粒细胞携带其跨越BBB浸润至病变部位，进一步通过中性粒细胞或外泌小体与神经细胞融合的方式促进纳米粒从中性粒细胞转移至神经元细胞内，实现了大分子蛋白药物脑内多级靶向递送。该纳米粒能够显著抑制缺血再灌注损伤，提高药物治疗效果。