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

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


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.


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



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

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Supplementary material 1 (DOCX 54 kb)


  1. Biswas S, Torchilin VP (2013) Dendrimers for siRNA delivery pharmaceuticals 6:161–183. doi: 10.3390/ph6020161 PubMedGoogle Scholar
  2. Bosman AW, Janssen HM, Meijer EW (1999) About dendrimers: structure, physical properties, and applications. Chem Rev 99:1665–1688. doi: 10.1021/cr970069y CrossRefPubMedGoogle Scholar
  3. Breunig M, Lungwitz U, Liebl R, Goepferich A (2007) Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci USA 104:14454–14459. doi: 10.1073/pnas.0703882104 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Brooks H, Lebleu B, Vives E (2005) Tat peptide-mediated cellular delivery: back to basics. Adv Drug Deliv Rev 57:559–577. doi: 10.1016/j.addr.2004.12.001 CrossRefPubMedGoogle Scholar
  5. Cao S, Cripps A, Wei MQ (2010) New strategies for cancer gene therapy: prog and opportunities. Clin Exp Pharmacol Physiol 37:108–114. doi: 10.1111/j.1440-1681.2009.05268.x CrossRefPubMedGoogle Scholar
  6. Daneshvar N, Abdullah R, Shamsabadi FT, How CW, Aizat MMH, Mehrbod P (2013) PAMAM dendrimer roles in gene delivery methods and stem cell research. Cell Biol Int 37:415–419. doi: 10.1002/cbin.10051 CrossRefPubMedGoogle Scholar
  7. Dominska M, Dykxhoorn DM (2010) Breaking down the barriers: siRNA delivery and endosome escape. J Cell Sci 123:1183–1189. doi: 10.1242/jcs.066399 CrossRefPubMedGoogle Scholar
  8. Feng JWA, Kao J, Marshall GR (2009) A second look at mini-protein stability: analysis of FSD-1 using circular dichroism, differential scanning calorimetry, and simulations. Biophys J 97:2803–2810. doi: 10.1016/j.bpj.2009.08.046 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Ferreira GNM, Monteiro GA, Prazeres DMF, Cabral JMS (2000) Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. Trends Biotechnol 18:380–388. doi: 10.1016/s0167-7799(00)01475-x CrossRefPubMedGoogle Scholar
  10. Ferrer-Miralles N, Vazquez E, Villaverde A (2008) Membrane-active peptides for non-viral gene therapy: making the safest easier. Trends Biotechnol 26:267–275. doi: 10.1016/j.tibtech.2008.02.003 CrossRefPubMedGoogle Scholar
  11. Frohlich E (2012) The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomedicine 7:5577–5591. doi: 10.2147/ijn.s36111 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Jin L, Zeng X, Liu M, Deng Y, He NY (2014) Current progress in gene delivery technology based on chemical methods and nano-carriers. Theranostics 4:240–255. doi: 10.7150/thno.6914 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Klemm AR, Young D, Lloyd JB (1998) Effects of polyethyleneimine on endocytosis and lysosome stability. Biochem Pharmacol 56:41–46. doi: 10.1016/s0006-2952(98)00098-7 CrossRefPubMedGoogle Scholar
  14. Ledley FD (1996) Pharmaceutical approach to somatic gene therapy. Pharm Res 13:1595–1614. doi: 10.1023/a:1016420102549 CrossRefPubMedGoogle Scholar
  15. 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/ CrossRefPubMedGoogle Scholar
  16. Lo SL, Wang S (2008) An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials 29:2408–2414. doi: 10.1016/j.biomaterials.2008.01.031 CrossRefPubMedGoogle Scholar
  17. Lou Z et al (2015) Low molecular weight polyethylenimine as a transgenic vector for tumor gene therapy. Biotech Histochem 90:140–145. doi: 10.3109/10520295.2014.965278 CrossRefPubMedGoogle Scholar
  18. Madaan K, Kumar S, Poonia N, Lather V, Pandita D (2014) Dendrimers in drug delivery and targeting: drug-dendrimer interactions and toxicity issues. J Pharm Bioallied Sci 6:139–150. doi: 10.4103/0975-7406.130965 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mintzer MA, Grinstaff MW (2011) Biomedical applications of dendrimers: a tutorial. Chem Soc Rev 40:173–190. doi: 10.1039/b901839p CrossRefPubMedGoogle Scholar
  20. Miranda LP, Alewood PF (1999) Accelerated chemical synthesis of peptides and small proteins. Proc Natl Acad Sci USA 96:1181–1186. doi: 10.1073/pnas.96.4.1181 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Morille M, Passirani C, Vonarbourg A, Clavreul A, Benoit JP (2008) Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials 29:3477–3496. doi: 10.1016/j.biomaterials.2008.04.036 CrossRefPubMedGoogle Scholar
  22. Nigg EA (1997) Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386:779–787. doi: 10.1038/386779a0 CrossRefPubMedGoogle Scholar
  23. Ohsaki M, Okuda T, Wada A, Hirayama T, Niidome T, Aoyagi H (2002) In vitro gene Transfection using dendritic poly(l-lysine). Bioconjug Chem 13:510–517. doi: 10.1021/bc015525a CrossRefPubMedGoogle Scholar
  24. 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
  25. Perry HA, Saleh AFA, Aojula H, Pluen A (2008) YOYO as a dye to track penetration of LK15 DNA complexes in spheroids: use and limits. J Fluoresc 18:155–161. doi: 10.1007/s10895-007-0254-5 CrossRefPubMedGoogle Scholar
  26. Perumal OP, Inapagolla R, Kannan S, Kannan RM (2008) The effect of surface functionality on cellular trafficking of dendrimers. Biomaterials 29:3469–3476. doi: 10.1016/j.biomaterials.2008.04.038 CrossRefPubMedGoogle Scholar
  27. Pires P, Simoes S, Nir S, Gaspar R, Duzgunes N, de Lima MCP (1999) Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells. BBA-Biomembranes 1418:71–84. doi: 10.1016/s0005-2736(99)00023-1 CrossRefPubMedGoogle Scholar
  28. Shcharbin DG, Klajnert B, Bryszewska M (2009) Dendrimers in gene transfection. Biochemistry (Moscow) 74:1070–1079. doi: 10.1134/s0006297909100022 CrossRefGoogle Scholar
  29. Shcharbin D et al (2014) How to study dendrimers and dendriplexes III. Biodistribution, pharmacokinetics and toxicity in vivo. J Control Release 181:40–52. doi: 10.1016/j.jconrel.2014.02.021 CrossRefPubMedGoogle Scholar
  30. Tam JP (1988) Synthetic peptide vaccine design—synthesis and properties of a high-density multiple antigenic peptide system. Proc Natl Acad Sci USA 85:5409–5413. doi: 10.1073/pnas.85.15.5409 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Thomas CE, Ehrhardt A, Kay MA (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4:346–358. doi: 10.1038/nrg1066 CrossRefPubMedGoogle Scholar
  32. 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/gkl941 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272:16010–16017. doi: 10.1074/jbc.272.25.16010 CrossRefPubMedGoogle Scholar
  34. Wiethoff CM, Middaugh CR (2003) Barriers to nonviral gene delivery. J Pharm Sci 92:203–217. doi: 10.1002/jps.10286 CrossRefPubMedGoogle Scholar
  35. Wu JY, Huang WZ, He ZY (2013) Dendrimers as carriers for siRNA delivery and gene silencing: a review. Sci World J 2013:630654. doi: 10.1155/2013/630654 Google Scholar
  36. Xu QX, Wang CH, Pack DW (2010) Polymeric Carriers for gene delivery: chitosan and poly(amidoamine) dendrimers. Curr Pharm Des 16:2350–2368CrossRefPubMedPubMedCentralGoogle Scholar
  37. Yang WQ, Zhang Y (2012) RNAi-mediated gene silencing in cancer therapy. Exp Opin Biol Ther 12:1495–1504. doi: 10.1517/14712598.2012.712107 CrossRefGoogle Scholar
  38. Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15:541–555. doi: 10.1038/nrg3763 CrossRefPubMedGoogle Scholar
  39. Zou SM, Erbacher P, Remy JS, Behr JP (2000) Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J Gene Med 2:128–134. doi: 10.1002/(sici)1521-2254(200003/04)2:2<128:aid-jgm95>;2-w CrossRefPubMedGoogle Scholar

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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