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


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.

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Figure 7: Schematic diagram illustrating the approach to quantifying the subcellular distribution of a pDNA cluster.
Figure 8: Spectroscopic data on DMAE-SS-PRX and DMAE-α-CD, and the monitoring of DMAE-α-CD release from DMAE-SS-PRX.
Figure 9: Intracellular trafficking of pDNA and transfection.


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The authors acknowledge Dr. H.S. Choi, A. Yoshihiro, Y. Sugaya and R. Ito for their help in a portion of the experiments.

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N.Y., A.M. and H.H. contributed equally to this work.

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Correspondence to Nobuhiko Yui.

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The authors declare no competing financial interests.

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Yamashita, A., Yui, N., Ooya, T. et al. Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei. Nat Protoc 1, 2861–2869 (2006).

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