细胞核靶向的荧光氧化石墨烯用于基因传递与同步示踪

The low transfection efficiency and lacking of spontaneous fluorescence for real-time tracking are the main problems of nonviral vector. The time-consuming and complicated processes of the additional labeling both of targeting and fluorescence will further affect the nature of vector. Based on our previous works of photoluminescence graphene oxide and the analysis of metabolism of DNA antagonist drugs using cell imaging, in this project we will develop a new nonviral vector with nucleus-targeting ability and spontaneous fluorescence based on the huge conjugate surface of graphene oxide to achieve the high transfection efficience by delivering the gene into nucleus effectively and real-time tracking of the gene delivery synchronously. (1) synthesizing the fluorescent graphene oxide with high quantum yields and strong nucleus-targeting ability by exploring the chemical grafting method on the surface of graphene oxide. (2) developing the method of grafting superbranched cationic polymer with small molecular weight onto the surface of graphene oxide to enhance its capacity of double stranded plasmid DNA by improving the charge density of the surface of graphene oxide. And then, analysis the affection of quantum yields of graphene oxide after the loading of plasmid DNA. (3) Profiling the intracellular transfer path of the nonviral vector by real-time fluorescence imaging and establishing a method to evaluate the transfection efficiency of nonviral gene vector. This project will provide a new means for improving the transfection efficiency efficiently and simplifying the targeting and tracing labeling processes of nonviral gene vector.

基因转染效率低和缺乏自发荧光示踪是非病毒载体面临的主要问题，而额外的靶向修饰及荧光标记带来的耗时、复杂操作易对载体自身性质造成影响。本项目拟在我们前期发光氧化石墨烯和DNA拮抗药物细胞代谢成像研究基础上，基于氧化石墨烯大的π共轭表面，开发具有细胞核靶向性和自发荧光示踪功能的新型基因载体，实现基因的高效细胞核靶向传递与同步示踪，提高基因转染效率。1）探索氧化石墨烯的发光及细胞核靶向双功能化修饰方法，制备具有高荧光量子产率和强细胞核靶向能力的氧化石墨烯。2）在此基础上，发展小分子量超支化阳离子聚合物接枝方法，提高氧化石墨烯基因载体电荷密度，增强其对双链质粒DNA的负载能力，并研究DNA负载对氧化石墨烯发光效率的影响。3）通过荧光实时成像，描绘该基因载体的细胞转运路径，并建立该体系基因转染效率评价方法。本项目的研究将为有效提高非病毒基因载体的转染效率和简化其靶向及示踪修饰过程提供新的手段。

针对目前的非病毒基因载体缺乏细胞核靶向性造成基因转染效率低的问题，本项目通过尺寸调控和表面修饰等手段，成功合成了具有细胞核靶向功能的阳离子碳量子点（cQD）荧光探针，并研究了其与DNA和RNA的相互作用原理，实现了活细胞及生物体内核酸结构的同步荧光成像，为基因和药物的细胞核靶向传递提供了新的载体。主要创新点和成果如下：（1）针对细胞核孔直径小于9nm和核酸带负电荷的特点，首先合成了对苯二胺修饰的尺寸2-3nm的碳量子点，进一步偶联带正电荷的4-羧丁基三苯基溴化膦来增加碳量子点的正电荷，合成了细胞核靶向的阳离子cQD探针。（2）研究了cQD探针表面电荷密度与细胞核膜穿透性的相关性，发现随着表面正电荷密度的增加，其进入细胞核的能力增强。进一步阐明了cQD探针与细胞核内DNA和RNA的不同作用方式，实现了活细胞内DNA和RNA的同步光谱区分成像。（3）基于cQD探针强的光稳定性和低的细胞毒性，实现了细胞有丝分裂过程中染色体和核仁的动态成像以及STED超高分辨成像。进一步探索了该探针在活的线虫体内的传输途径，发现了该探针具有跨越多重组织的高效生物膜穿透能力，证实了cQD探针作为细胞核靶向的新型非病毒载体的可行性。（4）同时，探索了端粒酶活性相关DNA核酸探针的设计，开发了一种端粒酶活性特异性的球形核酸探针基因运载体系，实现了肿瘤的早期诊断。本项目研究成果在Angew. Chem. Int. Ed.、ACS Nano、Nanoscale等高影响力国际期刊上发表SCI论文6篇，申请国家发明专利4项。