Zhang Tao, Tian Taoran, Zhou Ronghui, Li Songhang, Ma Wenjuan, Zhang Yuxin, Liu Nanxin, Shi Sirong, Li Qianshun, Xie Xueping, Ge Yichen, Liu Mengting, Zhang Qi, Lin Shiyu, Cai Xiaoxiao, Lin Yunfeng
State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, P. R. China.
Nat Protoc. 2020 Aug;15(8):2728-2757. doi: 10.1038/s41596-020-0355-z. Epub 2020 Jul 15.
Although organic nanomaterials and inorganic nanoparticles possess inherent flexibility, facilitating functional modification, increased intracellular uptake and controllable drug release, their underlying cytotoxicity and lack of specificity still cause safety concerns. Owing to their merits, which include natural biocompatibility, structural stability, unsurpassed programmability, ease of internalization and editable functionality, tetrahedral DNA nanostructures show promising potential as an alternative vehicle for drug delivery and biomedical treatment. Here, we describe the design, fabrication, purification, characterization and potential biomedical applications of a self-assembling tetrahedral DNA nanostructure (TDN)-based multifunctional delivery system. First, relying on Watson-Crick base pairing, four single DNA strands form a simple and typical pyramid structure via one hybridization step. Then, the protocol details four different modification approaches, including replacing a short sequence of a single DNA strand by an antisense peptide nucleic acid, appending an aptamer to the vertex, direct incubation with small-molecular-weight drugs such as paclitaxel and wogonin and coating with protective agents such as cationic polymers. These modified TDN-based complexes promote the intracellular uptake and biostability of the delivered molecules, and show promise in the fields of targeted therapy, antibacterial and anticancer treatment and tissue regeneration. The entire duration of assembly and characterization depends on the cargo type and modification method, which takes from 2 h to 3 d.
尽管有机纳米材料和无机纳米颗粒具有固有的灵活性，便于进行功能修饰、增加细胞内摄取和可控药物释放，但它们潜在的细胞毒性和缺乏特异性仍然引发安全担忧。由于四面体DNA纳米结构具有天然生物相容性、结构稳定性、无与伦比的可编程性、易于内化和可编辑功能等优点，其作为药物递送和生物医学治疗的替代载体显示出有前景的潜力。在此，我们描述了一种基于自组装四面体DNA纳米结构（TDN）的多功能递送系统的设计、制造、纯化、表征及潜在的生物医学应用。首先，依靠沃森-克里克碱基配对，四条单链DNA通过一步杂交形成一个简单且典型的金字塔结构。然后，该方案详细介绍了四种不同的修饰方法，包括用反义肽核酸替换单链DNA的短序列、在顶点附加适配体、与紫杉醇和汉黄芩素等小分子药物直接孵育以及用阳离子聚合物等保护剂进行包被。这些基于修饰TDN的复合物促进了所递送分子的细胞内摄取和生物稳定性，并在靶向治疗、抗菌和抗癌治疗以及组织再生领域显示出前景。组装和表征的整个持续时间取决于货物类型和修饰方法，耗时从2小时到3天不等。
