基于类风湿性关节炎信号网络的“功能鸡尾酒”型靶向小核酸递释系统的构建与评价

Rheumatoid arthritis is a complex chronic antoimmune disease with the main feature of synovitis inflammation, and there is still no targeted drugs toward the complex inflammatory signaling network. In the project, cocktail-type small nucleic acids with anti-inflammation and immunologic regulation were designed based on the pathophysiological features and inflammatory signaling network of rheumatoid arthritis.Then to realize the delivery of the small nucleic acids, PEI25K was modified by a hydrophobic amino acid N-acetyl-L-leucine and chemically conjugated to HAP-1 peptide via NHS-PEG5K-Mal method, which could be used as a ligand for targeting fibroblast-like synoviocytes. Through the strategy, a novel targeting gene delivery system was constructed with high transfection efficiency, low cytotoxity and long circulation time. Finally, the small nucleic acids were loaded using the gene carrier to explore its tratement efficiency of rheumatoid arthritis through regulating the signaling network at the cellular and animal level, and systematical studies on the pharmacokinetic behavior of carrier and small nucleic acids were performed. In conclusion, the project could realize the efficient, stable and targeting delivery of small nucleic acids to achieve the multi-level regulation of signaling network in rheumatoid arthritis. Thus, the project provides a new insight into the development and clinic application of targeting gene medicine for rheumatoid arthritis.

类风湿性关节炎是一种病因及发病机制复杂、以关节滑膜炎症为主要特征的慢性自身免疫性疾病，目前尚缺少针对复杂炎症信号网络的靶向型药物。本项目首先根据类风湿性关节炎的病理生理特征和炎症信号网络，设计具有抗炎、抑制免疫和破骨作用的"功能鸡尾酒"型小核酸基因药物；其次，通过疏水性氨基酸N-乙酰-L-亮氨酸对PEI25K的修饰及NHS-PEG5K-Mal为桥梁的化学键合策略，将针对纤维样滑膜细胞的靶向多肽HAP-1与之偶联，构建高基因转染效率、低毒、体内长循环的靶向型基因传输载体；最后，以该基因传输载体负载小核酸基因药物，在细胞和动物水平上探讨其调控类风湿性关节炎信号网络治疗疾病的作用机制，并系统研究载体及小核酸基因药物的药动学行为。本项目构建高效、稳定、靶向小核酸基因药物递释系统，实现对类风湿性关节炎信号网络多层面调控，为类风湿性关节炎靶向型基因治疗药物的研究开发及临床应用提供了新的思路。

本项目立足于小核酸药物所介导的类风湿性关节炎基因治疗，构建了高效安全的氟化修饰型高分子材料，实现了小核酸分子miR-23b在类风湿性关节炎动物模型中的稳定传输，从而达到治疗目的。具体研究内容包括：(1) 通过氟化试剂修饰，构建了氟化修饰型树枝状高分子PAMAM衍生物FP，该衍生物具有很高的转染效率，尤其在难转染细胞中取得了明显的转染效果，同时该载体可以实现核酸分子高效入胞及内涵体逃逸；（2）以该衍生物为载体，介导小核酸分子miR-23b的高效递送，不仅实现了其对巨噬细胞的增殖抑制，同时显著抑制了炎症病理反应，为动物实验水平系统评价奠定了坚实的基础；（3）以佐剂诱导关节炎大鼠为模型，尾静脉注射FP/miR-23b复合物后，该体系能有效改善患病关节炎大鼠病程发展，抑制炎症反应，改善大鼠运动情况。综上，该递送体系从抑制炎症反应和诱导凋亡两个层面出发，实现了类风湿性关节炎的有效治疗，对未来自身免疫类疾病研究的发展及临床应用等具有重要的意义。