Pharmaceutical Research

, Volume 28, Issue 4, pp 731–741 | Cite as

Poly(I:C)-Mediated Tumor Growth Suppression in EGF-Receptor Overexpressing Tumors Using EGF-Polyethylene Glycol-Linear Polyethylenimine as Carrier

  • David Schaffert
  • Melinda Kiss
  • Wolfgang Rödl
  • Alexei Shir
  • Alexander Levitzki
  • Manfred Ogris
  • Ernst Wagner
Research Paper



To develop a novel polyethylenimine (PEI)-based polymeric carrier for tumor-targeted delivery of cytotoxic double-stranded RNA polyinosinic:polycytidylic acid, poly(I:C). The novel carrier should be chemically less complex but at least as effective as a previously developed tetra-conjugate containing epidermal growth factor (EGF) as targeting ligand, polyethylene glycol (PEG) as shielding spacer, 25 kDa branched PEI as RNA binding and endosomal buffering agent, and melittin as endosomal escape agent.


Novel conjugates were designed employing a simplified synthetic strategy based on 22 kDa linear polyethylenimine (LPEI), PEG spacers, and recombinant EGF. The efficacy of various conjugates (different PEG spacers, with and without targeting EGF) in poly(I:C)-mediated cell killing was evaluated in vitro using two human U87MG glioma cell lines. The most effective polyplex was tested for in vivo activity in A431 tumor xenografts.


Targeting conjugate LPEI-PEG2 kDa-EGF was found as most effective in poly(I:C)-triggered killing of tumor cells in vitro. The efficacy correlated with glioma cell EGFR levels. Repeated intravenous administration of poly(I:C) polypexes strongly retarded growth of A431 human tumor xenograft in mice.


The optimized LPEI-PEG2 kDa-EGF conjugate displays reduced chemical complexity and efficient poly(I:C)-mediated killing of EGFR overexpressing tumors in vitro and in vivo.


epidermal growth factor polyplex receptor-mediated delivery RNA delivery tumor targeting 



Hepes buffered glucose (5% (w/v) glucose, 20 mM Hepes, pH 7.4)


N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid


ion-exchange chromatography


linear polyethylenimine with an average molecular weight of 22 kDa


orthopyridyl dithio


polyethylene glycole


poly inosinic acid


polyglutamic acid


poly inosinic-cytidylic acid


size exclusion chromatography



We thank Olga Brück for assistance in preparing the manuscript and Miriam Sindelar for skillful assistance with the syntheses. This work was supported by EC project GIANT, the DFG projects SFB 486, and SPP1230, and the excellence cluster Nanosystems Initiative Munich (NIM). AS and AL are supported by grants from the ERC : ERC/B3/JM/NL/MW/gk/D(2009) 600950 and the National Cancer Institute (USA): 1R01CA125500.

Supplementary material

11095_2010_225_MOESM1_ESM.pdf (107 kb)
Figure S1 Binding of poly(I:C) to PEI as analyzed by agarose gel shift assay. Four-hundred or eight-hundred ng poly(I:C) were complexed using either LPEI or brPEI and analyzed by gel shift assay. Both polymer backbones were able to efficiently complex poly(I:C) at a minimal N/P ratio of 6. (PDF 106 kb)
11095_2010_225_MOESM2_ESM.pdf (195 kb)
Figure S2 Binding of poly(I:C) to PEI conjugates as analyzed by heparin dissociation and agarose gel shift assay. Eight-hundred ng poly(I:C) were complexed using indicated polymers at N/P ratio of 8 and treated with indicated amounts of the polyanion heparin, resulting in partial release of poly(I:C) at higher concentrations. (PDF 194 kb)
11095_2010_225_MOESM3_ESM.pdf (106 kb)
Figure S3 Dose titration of poly(I:C) LPEI-PEG-EGF conjugates on tumor cell line U87MGwtEGFR. Poly(I) polyplexes served as negative control. (PDF 105 kb)
11095_2010_225_MOESM4_ESM.pdf (53 kb)
Figure S4 Relative EGF receptor cell surface level on tumor cell lines. U87MG (a), U87MGwtEGFR cells (b) were incubated with a mouse anti-EGFR antibody followed by treatment with an Alexa-488 conjugated secondary polyclonal goat anti-mouse antibody. Untreated cells (cells only) as well as cells, incubated only with secondary antibody (2nd AB only) served as negative control. (PDF 53 kb)


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • David Schaffert
    • 1
    • 2
  • Melinda Kiss
    • 1
    • 2
  • Wolfgang Rödl
    • 1
    • 2
  • Alexei Shir
    • 3
  • Alexander Levitzki
    • 3
  • Manfred Ogris
    • 1
    • 2
  • Ernst Wagner
    • 1
    • 2
  1. 1.Pharmaceutical Biotechnology, Department of Pharmacy Center for Drug ResearchLudwig-Maximilians-Universität MunichMunichGermany
  2. 2.Center for NanoScience (CeNS)Ludwig-Maximilians-Universität MunichMunichGermany
  3. 3.Unit of Cellular Signaling, Department of Biological ChemistryThe Hebrew University of JerusalemJerusalemIsrael

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