类风湿性关节炎是一种目前不能治愈的全身慢性免疫紊乱疾病，现有药物主要控制和减缓疾病的发展和恶化，临床急需高效低毒的新型疗法。类风湿关节炎部位伴有炎症信号通路和炎症细胞因子紊乱，导致关节腔炎症反应不断激化和病情的加重，所以直接靶向炎症信号通路的药物相比于传统药物有更高的靶向性和疗效。诱骗NF-κB寡聚核苷酸(NF-κB dODNs)能抑制炎症反应、降低炎症细胞因子、逆转关节损伤，但NF-κB dODNs在体内稳定性差、细胞转染效率低、靶向性低，限制了NF-κB dODNs的疗效和应用。近年来伴随DNA origami技术的革新发展起来了一系列新型DNA结构，其优势在于：结构、载体大小、功能修饰的高度精准性、可控性；良好的生物相容性；靶向性强等。本项目计划设计并折叠NF-κB dODNs形成DNA纳米结构，增强NF-κB dODNs的稳定性和抗炎效果。同时，为增强对炎症关节的靶向性，采用血管黏附分子(VCAM-1)亲和多肽(Binding Peptide，BPVCAM-1)修饰DNA纳米结构，增强其在炎症部位的渗入和炎症细胞的摄取，为类风湿性关节炎建立一种新型高效低毒的免疫疗法。

Rheumatoid arthritis (RA) is a chronic autoimmune disorder disease that is currently incurable. RA is characterized with chronic joint inflammation, cartilage destruction, and bone erosion, which leads to loss of mobility and shortened lifespan. RA pathogenesis involved activation of immune cells, disorder of complex cytokine network, and dysregulation of signaling pathways. NF-κB pathway is highly involved in the inflammation and can affect the secretion of various cytokines. Previous reports had showed that NF-κB decoy oligodeoxynucleotide (ODN) could inhibit inflammation and prevent the transactivation of pro-inflammatory genes. However, NF-κB decoy ODN is unstable in vitro and has low efficiency of cellular transfection and poor specificity. In this work, we are going to design and bend NF-κB decoy ODN into DNA nanostructures via DNA origami technology. Advantages of DNA nanostructures by DNA origami include good biocompatibility, high stability, and accurate control of structure, size, and modification of DNA nanostructures. Bended NF-κB decoy ODN nanostructures are expected to possess improved in vitro stability and higher anti-inflammatory efficacy. In addition, inflamed synovitis highly expresses specific molecules like vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells, macrophages, and synovial fibroblasts. To enhance specificity to inflamed synovitis, a binding peptide to VCAM-1 was conjugated to bended NF-κB decoy ODN nanostructures. By constructing VCAM-1 binding peptide modified NF-κB decoy ODN nanostructures, we aim to develop an efficient immunotherapy with good safety for RA.

靶向纳米生物药是高效低毒治疗类风湿性关节炎（RA）的关键，传统的生物药在治疗的同时会诱发肿瘤和严重感染等并发症，严重影响了RA的疗效。本研究用四条单链直链DNA自组装形成DNA四面体（TD），并通过碱基互补配对原则将NF-κB decoy ODNs和VCAM-1靶向多肽修饰在TD上，制备出具有靶向作用的载NF-κB decoy ODNs的TD（TD-1P-dODNs）。琼脂糖凝胶电泳显示该载药体系冻干前后能保持结构完整性，且载药体系（TD-dODNs、TD-1P-dODNs）在10%FBS存在下24h能保持一定的结构稳定性，而游离的dODNs在4h时已基本降解。CCK-8法显示载体材料无细胞毒性，ELISA法检测结果显示细胞炎症模型成功建立，载药体系（TD-dODNs、TD-1P-dODNs）相比游离dODNs，明显降低了细胞炎症因子TNF-α和IL-6的产生，乱序dODNs组（TDSODNs）和空白载体TD不能降低细胞炎症因子的产生。与游离dODNs相比，靶向载药体系明显提高了药物的摄取。4h时TD-dODNs和TD-1P-dODNs在RAW264.7的细胞摄取量分别为dODNs的2.67倍和2.81倍，在HUVEC的细胞摄取量分别为dODNs的1.91倍和2.63倍。细胞迁移结果显示，TD-dODNs和TD-1P-dODNs抑制HUVEC的迁移率分别为31.90%和42.24%，较游离dODNs有明显差异，乱序dODNs组（TDSODNs）不能抑制HUVEC细胞的迁移。活体成像结果显示，TD-dODNs和TD-1P-dODNs增加了药物在炎症部位的聚集。体内药效学结果显示，载药体系对炎症因子和小鼠关节炎指数有一定的降低作用。本实验构建的靶向载NF-κB decoy ODNs的DNA四面体纳米结构能提高游离dODNs的稳定性，发挥靶向作用，增加了药物的摄取，提高了药物的体外抗炎效果，但其体内抗炎效果需进一步优化。靶向生物治疗RA有望成为一种全新的高效低毒的治疗类风湿性关节炎的免疫疗法。