title: Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei.
abstract: 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.
authors.affiliation: 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.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamashita
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Yui
authors.forename: Tooru
authors.initials: T
authors.lastname: Ooya
authors.forename: Arihiro
authors.initials: A
authors.lastname: Kano
authors.forename: Atsushi
authors.initials: A
authors.lastname: Maruyama
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Akita
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kogure
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
mesh_terms: D002467:Cell Nucleus / Q000737:chemistry; D003505:Cyclodextrins / Q000138:chemical synthesis*; D004247:DNA / Q000138:chemical synthesis*; D010957:Plasmids / Q000737:chemistry*; D020442:Poloxamer / Q000138:chemical synthesis*; D043862:Rotaxanes / Q000138:chemical synthesis*; D014162:Transfection / Q000379:methods*
chemical_list: D003505:Cyclodextrins; D043862:Rotaxanes; C475673:polyrotaxane; D020442:Poloxamer; D004247:DNA