Drug Delivery and Translational Research

, Volume 4, Issue 1, pp 84–95 | Cite as

Gene silencing and antitumoral effects of Eg5 or Ran siRNA oligoaminoamide polyplexes

  • Daniel EdingerEmail author
  • Raphaela Kläger
  • Christina Troiber
  • Christian Dohmen
  • Ernst Wagner
Research Article


Two antitumoral siRNAs (directed against target genes Eg5 and Ran) complexed with one of three sequence-defined cationic oligomers were compared in gene silencing in vitro and antitumoral in vivo efficacy upon intratumoral injection. Two lipo-oligomers (T-shape 49, i-shape 229) and the three-arm oligomer 386 were chosen because of their high efficiency in previous marker gene silencing screens. The oligomers showed very similar target-specific gene knockdown in murine neuroblastoma cells. Silencing persisted only for a short period (maximum on day 1 at mRNA and day 2 at protein level) triggering siRNA specific in vitro tumor cell killing. The fastest onset of protein knockdown and strongest antitumoral effect was mediated by oligomer 386. Tumor growth reduction in vivo was evaluated in the subcutaneous Neuro2A mouse model. Intratumoral injections of either Eg5 or Ran siRNA/oligomer 49 polyplexes led to reduced tumor growth and prolonged survival of mice compared to control siRNA and buffer treatment. Target knockdown was evidenced in tumors by mitotic Aster formation for Eg5 knockdown and apoptotic TUNEL stain for Ran knockdown. Ran siRNA displayed better antitumoral efficacy and was chosen for in vivo comparison of the oligomers. A very clear order of antitumoral activity (oligomer 386 > 49 > 229) was observed. In summary, the similar in vitro gene silencing efficiencies on mRNA level by the tested oligomers did not correlate with the observed therapeutic effects in vivo. Oligomer 386 with the fastest onset of protein knockdown and best in vitro cell killing mediated the best in vivo antitumoral efficacy.


Bioluminescence imaging Cancer therapy Oligoamines Polyplexes siRNA 



This work was supported by the DFG Cluster “Nanosystems Intitiative Munich”, a grant from Axolabs GmbH (formerly Roche Kulmbach GmbH), and the Biotech Cluster m4 T12. We thank Naresh Badgujar and Irene Martin for help with some of the oligomers synthesis.

Conflict of interest

Daniel Edinger, Raphaela Kläger, Christina Troiber, Christian Dohmen, and Ernst Wagner declare that they have no conflict of interest.

Integrity of research and reporting

I confirm that all experiments have been performed according to the German law and the rules of the German Research Foundation (DFG). All institutional and national guidelines for the care and use of laboratory animals were followed.

Supplementary material

13346_2013_146_Fig9_ESM.jpg (24 kb)
Supporting 1

Intratumoral injection of oligomer 49 Eg5 siRNA polyplexes showed aster formation and confirmed Eg5 knockdown in Neuro2A tumor cells (JPEG 23 kb)

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High-resolution image (TIFF 1799 kb)
13346_2013_146_Fig10_ESM.jpg (50 kb)
Supporting 2

Intratumoral injection of oligomer 49 Ran siRNA polyplexes showed positive tunnel staining and confirmed that Ran knockdown led to apoptosis of Neuro2A tumor cells (JPEG 50 kb)

13346_2013_146_MOESM2_ESM.tif (6.8 mb)
High-resolution image (TIFF 6922 kb)


  1. 1.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411(6836):494–8.CrossRefPubMedGoogle Scholar
  2. 2.
    de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov. 2007;6(6):443–53. doi: 10.1038/nrd2310.CrossRefPubMedGoogle Scholar
  3. 3.
    Schroeder A, Heller DA, Winslow MM, Dahlman JE, Pratt GW, Langer R, et al. Treating metastatic cancer with nanotechnology. Nat Rev Cancer. 2012;12(1):39–50. doi: 10.1038/nrc3180.CrossRefGoogle Scholar
  4. 4.
    Bumcrot D, Manoharan M, Koteliansky V, Sah DW. RNAi therapeutics: a potential new class of pharmaceutical drugs. NatChem Biol. 2006;2(12):711–9.Google Scholar
  5. 5.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806–11.CrossRefPubMedGoogle Scholar
  6. 6.
    Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 2005;12(5):461–6.CrossRefPubMedGoogle Scholar
  7. 7.
    Davis ME. The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic. Mol Pharm. 2009;6(3):659–68. doi: 10.1021/mp900015y.CrossRefPubMedGoogle Scholar
  8. 8.
    Schaffert D, Troiber C, Salcher EE, Frohlich T, Martin I, Badgujar N, et al. Solid-phase synthesis of sequence-defined T-, i-, and U-shape polymers for pDNA and siRNA delivery. Angew Chem Int Ed Engl. 2011;50(38):8986–9. doi: 10.1002/anie.201102165.CrossRefPubMedGoogle Scholar
  9. 9.
    Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N, et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol. 2008;26(5):561–9.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, et al. RNAi-mediated gene silencing in non-human primates. Nature. 2006;441(7089):111–4.CrossRefPubMedGoogle Scholar
  11. 11.
    Whitehead KA, Matthews J, Chang PH, Niroui F, Dorkin JR, Severgnini M, et al. In vitro–in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery. ACS Nano. 2012;6(8):6922–9. doi: 10.1021/nn301922x.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Morgan-Lappe SE, Tucker LA, Huang X, Zhang Q, Sarthy AV, Zakula D, et al. Identification of Ras-related nuclear protein, targeting protein for xenopus kinesin-like protein 2, and stearoyl-CoA desaturase 1 as promising cancer targets from an RNAi-based screen. Cancer Res. 2007;67(9):4390–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Pai SI, Lin YY, Macaes B, Meneshian A, Hung CF, Wu TC. Prospects of RNA interference therapy for cancer. Gene Ther. 2006;13(6):464–77.CrossRefPubMedGoogle Scholar
  14. 14.
    Pecot CV, Calin GA, Coleman RL, Lopez-Berestein G, Sood AK. RNA interference in the clinic: challenges and future directions. Nat Rev Cancer. 2011;11(1):59–67. doi: 10.1038/nrc2966.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Sarli V, Giannis A. Targeting the kinesin spindle protein: basic principles and clinical implications. Clin Cancer Res. 2008;14(23):7583–7. doi: 10.1158/1078-0432.CCR-08-0120.CrossRefPubMedGoogle Scholar
  16. 16.
    Judge AD, Robbins M, Tavakoli I, Levi J, Hu L, Fronda A, et al. Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice. J Clin Invest. 2009;119(3):661–73. doi: 10.1172/JCI37515.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Dasso M. Running on Ran: nuclear transport and the mitotic spindle. Cell. 2001;104(3):321–4.CrossRefPubMedGoogle Scholar
  18. 18.
    Tietze N, Pelisek J, Philipp A, Roedl W, Merdan T, Tarcha P, et al. Induction of apoptosis in murine neuroblastoma by systemic delivery of transferrin-shielded siRNA polyplexes for downregulation of Ran. Oligonucleotides. 2008;18(2):161–74.CrossRefPubMedGoogle Scholar
  19. 19.
    Schaffert D, Badgujar N, Wagner E. Novel Fmoc-polyamino acids for solid-phase synthesis of defined polyamidoamines. Org Lett. 2011;13(7):1586–9. doi: 10.1021/ol200381z.CrossRefPubMedGoogle Scholar
  20. 20.
    Fröhlich T, Edinger D, Kläger R, Troiber C, Salcher E, Badgujar N, et al. Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides. J Control Release. 2012. doi: 10.1016/j.jconrel.2012.03.018.Google Scholar
  21. 21.
    Philipp A, Zhao X, Tarcha P, Wagner E, Zintchenko A. Hydrophobically modified oligoethylenimines as highly efficient transfection agents for siRNA delivery. Bioconjug Chem. 2009;20(11):2055–61. doi: 10.1021/bc9001536.CrossRefPubMedGoogle Scholar
  22. 22.
    Zintchenko A, Philipp A, Dehshahri A, Wagner E. Simple modifications of branched PEI lead to highly efficient siRNA carriers with Low toxicity. Bioconjug Chem. 2008;19(7):1448–55.CrossRefPubMedGoogle Scholar
  23. 23.
    Dohmen C, Edinger D, Frohlich T, Schreiner L, Lachelt U, Troiber C, et al. Nanosized multifunctional polyplexes for receptor-mediated siRNA delivery. ACS Nano. 2012;6(6):5198–208. doi: 10.1021/nn300960m.CrossRefPubMedGoogle Scholar
  24. 24.
    Troiber C, Edinger D, Kos P, Schreiner L, Klager R, Herrmann A, et al. Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes. Biomaterials. 2013;34(5):1624–33. doi: 10.1016/j.biomaterials.2012.11.021.CrossRefPubMedGoogle Scholar
  25. 25.
    Bartlett DW, Davis ME. Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res. 2006;34(1):322–33.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Dohmen C, Frohlich T, Lachelt U, Rohl I, Vornlocher H-P, Hadwiger P, et al. Defined folate-PEG-siRNA conjugates for receptor-specific gene silencing. Mol Ther Nucleic Acids. 2012;1:e7. doi: 10.1038/mtna.2011.10.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    White PJ. Barriers to successful delivery of short interfering RNA after systemic administration. Clin Exp Pharmacol Physiol. 2008;35(11):1371–6. doi: 10.1111/j.1440-1681.2008.04992.x.CrossRefPubMedGoogle Scholar
  28. 28.
    Martin I, Dohmen C, Mas-Moruno C, Troiber C, Kos P, Schaffert D, et al. Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery. Org Biomol Chem. 2012;10(16):3258–68. doi: 10.1039/c2ob06907e.CrossRefPubMedGoogle Scholar

Copyright information

© Controlled Release Society 2013

Authors and Affiliations

  • Daniel Edinger
    • 1
    Email author
  • Raphaela Kläger
    • 1
  • Christina Troiber
    • 1
  • Christian Dohmen
    • 1
  • Ernst Wagner
    • 1
  1. 1.Department of Pharmacy, Pharmaceutical Biotechnology, Center for System-based Drug Research and Center for NanoScience (CeNS)Ludwig-Maximilians-UniversityMunichGermany

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