双修饰壳聚糖载Tat PTD-内皮抑素基因纳米粒靶向抑制眼底血管增生的研究

Due to the lack of efficient drugs and easy routes of administration for retinal vascular proliferation diseases, a novel fusion protein (Tat PTD-endostatin) was expressed in our previous work. It was proved to have excellent activity. Tat PTD was confirmed to carry endostatin to penetrate eye barriers and to reach the retina via eye drops and have effects in ocular fundus. However, protein therapy has some obvious unavoidable drawbacks such as high costs, poor stability, repeated administration and so on. And gene therapy might overcome these disadvantages. In our proposal, theoretical gene(Tat PTD-endostatin) will be delivered into ocular fundus via some carriers and will have persistent effects. Utilizing the ability to penetrate eye barriers of Tat PTD and the ability to target endothelial cells of targeting peptide (ATWLPPR), dual-modified chitosan nanoparticles is designed to deliver therapeutic gene. The gene will be delivered into ocular fundus and mediated by cell penetrating peptides(Tat PTD), specific receptor(targeting peptides) and nanoparticles. By this novel carrier system, Tat PTD-endostatin will prevent retinal or choroidal vascular proliferation disease via eye drops. The main contents are as follows: the preparation of dual -modified chitosan nanoparticles encapsulating gene, the evaluation of nanoparticles activity in vitro, the study on the ability of penetrating eye barriers and penetrating pathway, and the evaluation of pharmacodynamic in vivo.

针对眼底血管增生缺乏特效药物和简单方便给药途径的问题，申请者前期工作中利用穿膜肽（Tat PTD）的跨膜作用和内皮抑素（ES）抑制新生血管生成的作用，表达了Tat PTD-ES融合蛋白，其局部滴眼后能穿透眼球屏障到达眼底发挥作用。但是蛋白质治疗存在成本高、稳定性差、需反复用药等缺点，为此我们拟采用基因治疗将编码Tat PTD-ES基因的质粒导入眼底，使其在眼底持续表达Tat PTD-ES来发挥治疗作用。本课题利用Tat PTD穿透眼球屏障的能力及靶向肽ATWLPPR靶向内皮细胞的作用，设计了穿膜肽和靶向肽双修饰的载基因壳聚糖纳米粒，通过穿膜肽介导、受体介导及载体导入三重机制将Tat PTD-ES基因运送至眼底的内皮细胞表面，以期实现通过局部滴眼给药预防视网膜或脉络膜血管增生的目标。研究内容主要包括：双修饰载基因壳聚糖纳米粒的制备，纳米粒体外活性评价，穿透眼球屏障性能及通路研究，药效学评价。

针对眼底血管增生缺乏特效药物和简单方便给药途径的问题,我们设计了一种新型的纳米基因载体，旨在采用基因治疗的方法，将编码治疗基因Tat PTD-Es的质粒导入眼底，使其在眼底持续表达Tat PTD-Es来发挥治疗作用。本项目的特色之处是将Tat PTD穿透眼球屏障的能力及靶向肽ATWLPPR靶向内皮细胞的作用引入载基因壳聚糖纳米粒的表面，使其在通过穿膜肽介导、受体介导及载体导入三重机制将Tat PTD-ES基因运送至眼底的内皮细胞表面，以期实现通过局部滴眼给药预防视网膜或脉络膜血管增生的目标。.本项目取得的研究成果有以下几个方面：（1）首次成功构建了穿膜肽Tat PTD与靶向肽（ATWLPPR）双修饰载基因壳聚糖纳米粒；（2）对双修饰载基因壳聚糖纳米粒体外活性作了系统性评价，其转染效率高，能明显抑制内皮细胞的增殖；（3）证实了双修饰载基因壳聚糖纳米粒能够穿透眼球屏障到达眼底的视网膜；（4）载基因双修饰纳米粒在体内药效学评价（CNV模型）实验中表现出了优秀的抑制脉络膜血管生成的活性。. 本项目的研究结果为眼部基因药物靶向制剂的开发奠定了基础。