传统pDNA/载体复合物由胞吞-内涵体逃逸与细胞核的分裂作用使pDNA进入细胞核，该递送方式效率低且受细胞周期的限制。核孔复合体(NPC)是细胞质与细胞核之间进行物质交换的有效通道，其构象的改变能使核孔暂时扩大至300nm，使其成为实现pDNA核定位的潜在靶点。本课题拟构建非内吞依赖-NPC靶向-核定位球蛋白-阳离子聚合物/pDNA复合物纳米系统。有望实现：(1) 载体以非内吞机制直接进入细胞质，避免溶酶体对pDNA的降解；(2) 利用球蛋白的NPC靶向性增强核定位能力，提高转染效率。采用现代制剂学手段对其进行优化和表征；运用活细胞与活体成像术等手段评价其靶向性与转染效率，探讨入胞、核转运与定位机理；采用蛋白组学等技术研究其安全性。该系统将为pDNA的有效递送提供一种全新的策略，具有重大科学意义。本思路国内外未见报道，新颖性好，研究力量与科研条件具备，工作基础扎实且已有结果表明可行性良好。

Traditional gen carriers deliver pDNA into the nuclear by a mechanism of endocytic dependence, endosome-lysosome system escape and nuclear division, however, the transfection for the pathway is very low and limited by the cell cycle. Nuclear pore complexes (NPC) are embedded into the nuclear envelope and allow passive as well as active exchange between the nuclear and the cytoplasmic compartments. The conformational changes of NPC protein induced by the interaction between FG-domains of nucleoporins and amphiphatic or hydrophobic molecules temporarily enlarges the size of NPC channels up to 300nm, thereby presenting a potential target for delivering the pDNA into the nucleus. Herein, a nanosystem of globular protein-cationic polymer/pDNA complex with a machenism of nonendocytic dependence-NPC mediation-nuclear targeting will be developed. Its potential advantages may be included as follows: (1) directly delivering the system into cytosol by nonendocytic mechanism and thus avoiding the degradation of pDNA by endosome-lysosome system and (2) enhancing the nuclear uptake by specifically binding the globular protein to FG-domains of nucleoporins of NPC and thereby improving the transfection of pDNA. The system will be optimized and characterized by current pharmaceutical technology; moreover, the targeting and gene delivery, as well as the mechanism of cellular and nuclear uptake, will be evaluated by live-cell CLSM imaging, in vivo imaging, etc; additionally, the compatibility will be studied in the global mapping of protein expression by proteomics technology. The work will provide a new strategy for the pDNA delivery, which is of great scientific significance for the gene delivery. The design idea of the system is original since it has not been reported at home and abroad. We not only have strong research-force, but also possess perfect research-conditions for the project. Moreover, the base of relevant work for the project is solid and the primary results indicate its feasibility.

基因治疗是将外源性基因导入靶细胞中，在分子水平利用表达的产物实现对疾病治疗的方法，是治疗临床各种疾病的有效方法之一。传统的基因载体必须经过溶酶体内化-逃逸才能进入细胞，实现转染，但效率非常低，只有 < 2% 的基因能实现溶酶体逃逸。（1）以阳离子化球蛋白为压缩材料，构建了脂质纳米粒与活性药物为载体的基因、大分子药物递送体系，完全区别于目前已报道的载体。载体生物相容性非常理想，而传统的基因载体毒性非常大。成果主要发表于：Sci Rep. 2016 Jun 9;6:27559. doi: 10.1038/srep27559; International Journal of Pharmaceutics 2015, 478, 762–772; Molecular Pharmaceutics. 2015, 12.1485-1500; Part. Part. Syst. Charact. 2017, 34, 6, 201600371n/a等。（2）让我们非常惊喜的是构建的载体在4T1和Caco-2细胞的摄取与小窝蛋白介导的内吞途径以及胆固醇依赖的内吞途径密切相关；载体与小窝蛋白介导的内吞途径标记物CTB以及CAV-1的高度共定位，证明了CNCs依赖小窝蛋白介导的内吞途径被细胞摄取，避免了溶酶体对药物和miRNA的降解，极大地提高了基因药物的胞内递送效率。我们认为该项成果的发现将会给基因、多肽的胞内递送带来极大的促进作用，是一项开创新性的研究。成果主要发表于：Scientific Reports 2017, 7, 46186，Advanced Science 2017, DOI: 10.1002/advs.201700324（IF9.034）。本项目为基因、大分子多肽药物的高效递送提供了一种全新的思路，打开了新的研究方向，对于疾病特别是重大慢性疾病的治疗带来了新的希望。