Kanazawa Takanori, Takashima Yuuki, Murakoshi Motoko, Nakai Yuka, Okada Hiroaki
Laboratory of Pharmaceutics and Drug Delivery, Department of Pharmaceutical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Int J Pharm. 2009 Sep 8;379(1):187-95. doi: 10.1016/j.ijpharm.2009.06.015. Epub 2009 Jun 23.
Effective delivery of DNA encoding antigen into the dendritic cells (DCs), which are non-dividing cells, is very important for the development of DNA vaccines. In a previous study, we developed the PLGA nanospheres that contained a cationic nanomaterial and showed high transfection efficiency in COS7 cells, which divide. In the present study, to produce an effective vector for the DNA vaccines, the gene expression and intracellular trafficking of pDNA complexed with PLGA/PEI nanospheres, in combination with an NF-kappaB analog as a nuclear localization signal (NLS) and electroporation were evaluated in human monocyte-derived DCs (hMoDCs). Cellular uptake of pDNA both in COS7 cells and hMoDCs was enhanced using the PLGA/PEI nanospheres. On the other hand, the PLGA/PEI nanospheres significantly promoted the transfection in COS7 cells, but had almost no effect on transfection in hMoDCs. The intranuclear transport of pDNA by PLGA/PEI nanospheres in COS7 cells was significantly higher than that in hMoDCs. These results indicate that pDNA complexed with PLGA/PEI nanospheres cannot enter into the nuclei of non-dividing cells. However, PLGA/PEI nanospheres combinated with NLS and electroporation (experimental permeation enhancer) greatly elevated the transfection efficiency by improvement of not only intracellular uptake but also intranuclear transport of pDNA in the hMoDCs. Thus, this delivery system using nanospheres combined with synthesized NLS might be applicable to DC-based gene vaccines when much non-invasive application such as needle-free injector should be required.
将编码抗原的DNA有效递送至不分裂的树突状细胞（DC）中，对于DNA疫苗的开发非常重要。在先前的一项研究中，我们开发了含有阳离子纳米材料的聚乳酸-羟基乙酸共聚物（PLGA）纳米球，并且在分裂的COS7细胞中显示出高转染效率。在本研究中，为了制备用于DNA疫苗的有效载体，我们在人单核细胞衍生的DC（hMoDC）中评估了与PLGA/聚乙烯亚胺（PEI）纳米球复合的质粒DNA（pDNA）的基因表达和细胞内转运，同时使用一种核因子κB（NF-κB）类似物作为核定位信号（NLS）并结合电穿孔技术。使用PLGA/PEI纳米球增强了COS7细胞和hMoDC中pDNA的细胞摄取。另一方面，PLGA/PEI纳米球显著促进了COS7细胞中的转染，但对hMoDC中的转染几乎没有影响。PLGA/PEI纳米球在COS7细胞中对pDNA的核内转运显著高于hMoDC中的。这些结果表明，与PLGA/PEI纳米球复合的pDNA不能进入不分裂细胞的细胞核。然而，PLGA/PEI纳米球与NLS和电穿孔技术（实验性渗透增强剂）相结合，通过改善hMoDC中pDNA的细胞内摄取和核内转运，极大地提高了转染效率。因此，当需要诸如无针注射器等非侵入性应用时，这种使用纳米球与合成NLS相结合的递送系统可能适用于基于DC的基因疫苗。
