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Peptide-Based Multicomponent Oligonucleotide Delivery Systems: Optimisation of Poly-l-lysine Dendrons for Plasmid DNA Delivery

  • Khairul A. Kamaruzaman
  • Peter M. Moyle
  • Istvan Toth
Article

Abstract

Gene therapy is a promising means to treat or prevent diseases either through gene silencing or expression. Some of the most effective delivery agents are polycationic dendrimers, which are highly branched constructs incorporating many positively charged groups. Two of the most effective dendrimers are polyethyleneimine (PEI) and poly(amidoamine) (PAMAM), which show high proficiency at overcoming barriers to oligonucleotide delivery. However, because of their abundance of cationic charge, they are associated with severe toxicity. We have therefore aimed to develop a low toxicity oligonucleotide delivery system, incorporating multiple components that have been selected and optimised to overcome the barriers to efficient oligonucleotide delivery. In this work we have focused on improving the toxicity, cellular uptake, and condensation of plasmid DNA (pDNA) through the fusion of synthetic poly-l-lysine (PLL) dendrons with the cell penetrating peptide TAT(48-60). A library of dendron structures, from 4+ to 16+ charge, and constructs containing six histidine residues, were synthesised. The effects of each modification on pDNA binding and condensation; cellular uptake and toxicity; and the size and zeta-potential of the complexes were assessed to identify the optimum dendron for incorporation into our systems. This work concluded that increasing the dendron charge from 4+ to 16+ significantly improved cellular uptake and pDNA condensation, with no effect on toxicity, while PLL dendrons with greater than 16+ charge could not be efficiently produced. In comparison, the incorporation of six histidines into these constructs had a variable effect on cellular uptake, and generated larger sized complexes, but did not affect toxicity.

Keywords

Branched poly-l-lysine Dendron Histidine Non-viral gene delivery Oligonucleotide delivery Plasmid DNA delivery 

Notes

Acknowledgments

This work was supported by an Australian Research Council (ARC) discovery Project Grant (DP130100952). P.M.M. was supported by an Australian National Health and Medical Research (NHMRC) postdoctoral training fellowship (569869). I.T. was supported by an ARC Professorial Research Fellowship (DP110100212). The authors acknowledge the facilities, technical and scientific assistance from Dr. Steven Mason and Mr. Michael Nefedov of the School of Chemistry and Molecular Biosciences for confocal microscopy and flow cytometry analysis.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Human and Animal Rights

No human or animal studies were performed in this paper.

Supplementary material

10989_2016_9545_MOESM1_ESM.docx (55 kb)
Supplementary material 1 (DOCX 54 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Khairul A. Kamaruzaman
    • 1
  • Peter M. Moyle
    • 2
  • Istvan Toth
    • 1
    • 2
    • 3
  1. 1.School of Chemistry and Molecular BiosciencesThe University of QueenslandSt LuciaAustralia
  2. 2.School of PharmacyThe University of QueenslandWoolloongabbaAustralia
  3. 3.Institute for Molecular Biosciencethe University of QueenslandSt LuciaAustralia

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