Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei

Atsushi Yamashita, Nobuhiko Yui, Tooru Ooya, Arihiro Kano, Atsushi Maruyama, Hidetaka Akita, Kentaro Kogure, Hideyoshi Harashima

Research output: Contribution to journalArticle

57 Citations (Scopus)

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 α-cyclodextrins (α-CDs), (iii) synthesis of polyrotaxane and (iv) modification of α-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.

Original languageEnglish
Pages (from-to)2861-2869
Number of pages9
JournalNature Protocols
Volume1
Issue number6
DOIs
Publication statusPublished - Jan 1 2007

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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