一种可生物裂解的聚轮烷-质粒DNA（pDNA）复合纳米粒的合成及其用于将pDNA快速非病毒递送至细胞核的应用。
Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei.
作者信息
Yamashita Atsushi, Yui Nobuhiko, Ooya Tooru, Kano Arihiro, Maruyama Atsushi, Akita Hidetaka, Kogure Kentaro, Harashima Hideyoshi
机构信息
School of Materials Science and the 21st Century COE Program, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
出版信息
Nat Protoc. 2006;1(6):2861-9. doi: 10.1038/nprot.2006.438.
This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.
本方案提供了一种合成可生物裂解的聚轮烷/质粒DNA（pDNA）多聚体并将其用于将pDNA递送至细胞核的方法。可生物裂解的聚轮烷通过四个步骤合成：（i）在聚乙二醇（PEG）的两端引入二硫键；（ii）制备含二硫键的PEG与α-环糊精（α-CD）之间的包合物；（iii）合成聚轮烷；（iv）用二甲基乙二胺对聚轮烷中的α-CD进行修饰。当这两种化合物一起溶解在磷酸盐缓冲液中时，会形成pDNA与聚轮烷的多聚体。通过共聚焦激光扫描显微镜捕获的Z系列共聚焦图像，对罗丹明标记的pDNA在荧光标记细胞器中的亚细胞定位进行定量分析。预计可将大量pDNA递送至细胞核，且该多聚体具有高转染活性。本方案可在23至32天内完成。
Zotero 插件