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 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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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.

References

  1. 1

    Luo, D. & Saltzman, W.M. Synthetic DNA delivery systems. Nat. Biotechnol. 18, 33–37 (2000).

    CAS  Article  Google Scholar 

  2. 2

    Wolff, J.A. The 'grand' problem of synthetic delivery. Nat. Biotechnol. 20, 768–769 (2002).

    CAS  Article  Google Scholar 

  3. 3

    Godbey, W.T., Wu, K.K. & Mikos, A.G. Poly(ethylenimine) and its role in gene delivery. J. Control. Rel. 60, 149–160 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Kunath, K. et al. Low-molecular-weight polyethyleneimine as a nonviral vector for DNA delivery: comparison of the psysicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethyleneimine. J. Control. Rel. 89, 113–125 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Read, M.L. et al. Vectors based on reducible polycations facilitate intracellular release of nucleic acids. J. Gene Med. 5, 232–245 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Miyata, K. et al. Block catiomer polyplexes with regulated densities of charge and disulfide cross-linking directed to enhance gene expression. J. Am. Chem. Soc. 126, 2355–2361 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Ooya, T. et al. Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery. J. Am. Chem. Soc. 128, 3852–3853 (2006).

    CAS  Article  Google Scholar 

  8. 8

    Akita, H., Ito, R., Khalil, I.A., Futaki, S. & Harashima, H. Quantitative three-dimentional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy. Mol. Ther. 9, 443–451 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Hama, S. et al. Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Ther. 13, 786–794 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Ichi, T., Watanabe, J., Ooya, T. & Yui, N. Controllable erosion time and profile in poly(ethylene glycol) hydrogels by supramolecular structure of hydrolysable polyrotaxane. Biomacromolecules 2, 204–210 (2001).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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

Author information

Affiliations

Authors

Contributions

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

Corresponding author

Correspondence to Nobuhiko Yui.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nprot.2006.438

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing