动态调控的DNA“纳米开关”在肿瘤诊断与治疗中的应用

This project focused on developing a multiple functional probe to meet the urgent requirement of early diagnosis and treatment of tumor. This probe was designed based on the DNA nanostructure as efficient carrier, which combined several functions through conjugating fluorophores, radioisotopes, target and drug molecules, together with a nanoswitch to control the dynamic release of drug molecules. The fluorescence and radioisotope labeling facilitated the multi-mode imaging of tumor, while the antibody mediated the tumor targeting and took the cytotoxic drug molecules close to tumor cells for effective treatment. More importantly, the fine design of DNA nanoswitch was performed to achieve the site-specific delivery and dynamic controllable release of drug molecules, which led to the high accuracy and low side effects. We will systematically investigate the key indicators including stability, affinity and kinetics of drug release etc, and the biocompatibility, targeting efficiency and efficacy on cells and animals. We will study the interaction of the probe and various kinds of cell lines, and discover the uptake of probes to understand their invasion, penetration, location and degradation in cell. The structure conversion of DNA tetrahedron will be observed by super-resolution microscopy to prove the kinetics of drug release. We will also inject probes in mice and tumor-bearing nude mice, and investigate the distribution, metabolism, and tumor targeting behavior of probes. Finally, we will establish a series of methods including design, preparation and characterization, improve the valuation system about the stability, affinity and biosafety, and construct a platform for cell imaging and tumor imaging, all of which will serve for the real clinical application.

本项目面向肿瘤的早期诊断和靶向治疗这个重大社会需求，发展集动态可控释放和诊疗一体化的纳米药物探针来满足临床需要。利用DNA纳米结构作为多功能载体，精确设计DNA纳米开关结构，结合多种标记技术和分子偶联技术，制备集治疗、靶向识别和信号报告于一体、兼具药物释放动态调控功能的智能纳米药物探针，实现多模态成像和肿瘤靶向治疗。系统研究探针的稳定性、亲和力、药物释放动力学等关键指标，评估在细胞和小动物水平的生物安全性、靶向效率、杀伤效果等。深入探讨探针与细胞的作用机制，分析摄取效率、穿膜途径、胞内定位等行为，建立胞内DNA纳米结构的构象变化与药物释放效率、动力学及细胞死亡率等的关系。研究探针在小动物肿瘤部位的靶向浓集、生物分布、血液半衰期、代谢途径等行为。最终建立一整套方法用于制备和表征多功能纳米药物探针、评估探针稳定性和生物活性，建立细胞和动物层次的活体成像和治疗平台，为临床应用提供可靠依据。

本项目的研究目标是发展集动态可控释放和诊疗一体化的纳米药物探针来满足临床需要。我们成功构建了系列基于DNA纳米结构为载体的成像探针和智能纳米药物递送体系，如DNA四面体、DNA树枝形拓扑结构、多种形状的（三角、方块、柱状）DNA折纸结构，利用AFM、电泳、电镜等分析测试手段对探针的结构、稳定性、光学性质及生物相容性进行表征和检测。并通过对纳米探针及纳米开关结构的设计和修饰，发展了一系列快速、高灵敏、高通量的检测方法，进行细胞和动物的多种分子标标检测及肿瘤特异性靶标的识别和成像，实现肿瘤标志物的早期高灵敏检测。此外构建智能纳米药物递送体系，借助这些递药体系，实现药物肿瘤靶向输送，提出肿瘤治疗的经皮、跨血脑屏障及智能肿瘤靶向递送药物的多种给药策略，为基于DNA纳米结构的肿瘤早期诊断和靶向治疗提供了可靠的实验依据。在项目资助下共发表论文60篇，其中包括Nature，Nature Materials、Nature Communications等在内的SCI论文53篇。 以通讯作者发表论文16篇, 其中Nature Communications(2篇)、Angew. Chem. Int. Edit.(1篇)、JACS(1篇)、ACS Applied Materials & Interfaces(3篇)、Nano Letters(1篇)、Nanoscale(2篇)、Small(1篇)等。期间受理发明专利9项，授权发明专利5项。培养博士后1名，博士研究生6名。