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Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles

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ABSTRACT

Purpose

This work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice.

Methods

Aerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method.

Results

Flow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group).

Conclusions

This work provides a technology platform for effective pulmonary delivery and gene silencing of RNAi therapeutics with potential use in preclinical studies of respiratory disease treatment.

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ACKNOWLEDGEMENTS

The authors thank Anne Chauchereau for providing the EGFP-expressing H1299 cell line used in this work and Bernard Arnold from DKFZ, Heidelberg, Germany for providing the B6;129P2-RAGE tm1.1 mice. We are also grateful to Helmy Rachman, Kirstin Hoffman and Susan Jackisch for excellent technical assistance with the animal experiments and Uwe Klemm for supervision of the animal work. This work was supported by the Danish Research Council and the EU-FP6 RiGHT programme.

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Authors and Affiliations

  1. Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Aarhus, Denmark

    Ebbe J. B. Nielsen, Sys Z. Glud, Flemming Besenbacher, Jørgen Kjems & Kenneth A. Howard

  2. Department of Molecular Biology, University of Aarhus, Aarhus, Denmark

    Ebbe J. B. Nielsen, Sys Z. Glud, Jørgen Kjems & Kenneth A. Howard

  3. Department of Physics and Astronomy, University of Aarhus, Aarhus, Denmark

    Jonathan Merrison & Flemming Besenbacher

  4. Department of Cardiology, Aarhus University Hospital, Skejby, Aarhus, Denmark

    Jan M. Nielsen

  5. Max-Planck-Institute for Infection Biology, Berlin, Germany

    Daniel Becker, Alexander Karlas, Hridayesh Prakash & Thomas F. Meyer

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  1. Ebbe J. B. Nielsen
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  2. Jan M. Nielsen
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  11. Kenneth A. Howard
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Correspondence to Kenneth A. Howard.

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Nielsen, E.J.B., Nielsen, J.M., Becker, D. et al. Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles. Pharm Res 27, 2520–2527 (2010). https://doi.org/10.1007/s11095-010-0255-y

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  • DOI: https://doi.org/10.1007/s11095-010-0255-y

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