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.
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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.
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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.
Li S, Tseng WC, Stolz DB, Wu SP, Watkins SC, Huang L (1999) Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther 6:585–594. doi:10.1038/sj.gt.3300865CrossRefPubMedGoogle Scholar
Oupicky D, Konak C, Dash PR, Seymour LW, Ulbrich K (1999) Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block. Bioconjug Chem 10:764–772. doi:10.1021/bc990007+CrossRefPubMedGoogle Scholar
Veldhoen S, Laufer SD, Trampe A, Restle T (2006) Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucleic Acids Res 34:6561–6573. doi:10.1093/nar/gkl941CrossRefPubMedPubMedCentralGoogle Scholar