磁靶向阳离子功能化碳纳米管基因载体的构建及转染研究

Low transfection efficiency and high cytotoxicity of non-viral gene vectors are among the barriers which limite their clinical application. Design and prepare biodegradable PEI functionalized carbon nanocubes with magnetic targeted function is great promising to overcome both the barriers simultaneously. A novel multifunctional carbon nanotubes with high DNA loaded ability and low cytotoxicity will be prepared in this project through grafting biodegradable PEI onto carbon nanotube. In addition, magnetic iron oxide nanoparticles will be introduced to improve the target ability and transfection efficiency. The DNA loaded ability of these multifunctional carbon nanotubes will be studied via SEM, Gel electrophoresis and size analysis. The vitro cytotoxicity will be tested by MTT assay. The plasmids with report gene and fluorescent labeling reagents will be employed to study the relationship of functionalized conditions and transfection efficiency as well as DNA complex delivery and detached mechanism. This project will explore a series novel multifunctional gene vectors with magnetic-targeted properties and low cytotoxicity which will have enormous significance in promoting non-viral vectors to clinical application.

非病毒基因载体较高的细胞毒性和较低的转染效率限制了它的临床应用。设计、制备具有磁靶向功能的可生物降解PEI功能化的碳纳米管有望同时突破这两问题。本项目拟通过可生物降解PEI接枝碳纳米管使其具有负载DNA的能力和较低的细胞毒性，通过引入磁性Fe3O4纳米粒子增强载体的靶向效果和转染效率。运用SEM、凝胶电泳实验、粒径分析等手段考察多功能碳纳米管与DNA的复合能力，用MTT法考察该碳纳米管体外细胞毒性，用含报告基因质粒考察该多功能碳纳米管的基因转染效率，探索功能化条件与转染效率之间的关系，同时用荧光标记法考察该功能碳纳米管/DNA复合物在细胞内的运转和DNA解离机制，寻找制约功能化碳纳米管转染效率的因素，探索获得高效低毒的新型非病毒基因载体，为设计制备多功能非病毒基因载体提供科学依据。对推动非病毒基因载体进入临床应用具有十分重要的意义。

非病毒基因载体是基因治疗研究的关键问题之一。但是目前非病毒基因载体还存在转染效率偏低，细胞毒性较高和缺乏靶向功能等问题，严重影响了其应用。基于此，本项目设计了一系列功能化磁性碳纳米管基因载体。一方面，通过碳纳米管负载磁性Fe3O4实现磁靶向功能，另一方面，在磁性碳纳米管表面引入可降解的PEI，降低载体的细胞毒性。本项目设计的功能化磁性碳纳米管具有超顺磁性和良好的磁响应性。通过调控功能化磁性碳纳米管中Fe3O4的含量以及可降解PEI衍生物的接枝比例等，深入地研究了该磁性载体与DNA的组装结构、细胞毒性及体外传递效率。在优化的条件下该磁性基因载体的转染效率高于25k PEI，但细胞毒性更低。我们还进一步阐明了细胞对基因载体的摄取效率和转染效率在外加磁场下的影响规律。本项目的研究成果将为今后设计高效低毒和磁靶向功能的基因载体提供理论依据。