Skip to main content

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



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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6



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


  1. 1.

    Felgner PL, Barenholz Y, Behr JP, Cheng SH, Cullis P, Huang L, et al. Nomenclature for synthetic gene delivery systems. Hum Gene Ther. 1997;8:511–2.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2003;2:347–60.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Li SD, Huang L. Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther. 2006;13:1313–9.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Meyer M, Wagner E. Recent developments in the application of plasmid DNA-based vectors and small interfering RNA therapeutics for cancer. Hum Gene Ther. 2006;17:1062–76.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat Rev Drug Discov. 2005;4:581–93.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Schaffert D, Wagner E. Gene therapy progress and prospects: synthetic polymer-based systems. Gene Ther. 2008;15:1131–8.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Wagner E. The silent (r)evolution of polymeric nucleic acid therapeutics. Pharm Res. 2008;25:2920–3.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Love KT, Mahon KP, Levins CG, Whitehead KA, Querbes W, Dorkin JR, et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci USA. 2010;107:1864–9.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Shir A, Ogris M, Wagner E, Levitzki A. EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice. PLoS Med. 2006;3:e6.

    PubMed  Article  Google Scholar 

  10. 10.

    Blessing T, Kursa M, Holzhauser R, Kircheis R, Wagner E. Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery. Bioconjug Chem. 2001;12:529–37.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Wolschek MF, Thallinger C, Kursa M, Rossler V, Allen M, Lichtenberger C, et al. Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. Hepatology. 2002;36:1106–14.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Boeckle S, Wagner E, Ogris M. C- versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes. J Gene Med. 2005;7:1335–47.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Meyer M, Philipp A, Oskuee R, Schmidt C, Wagner E. Breathing life into polycations: functionalization with ph-responsive endosomolytic peptides and polyethylene glycol enables sirna delivery. J Am Chem Soc. 2008;130:3272–3.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Bonnet ME, Erbacher P, Bolcato-Bellemin AL. Systemic delivery of DNA or sirna mediated by linear polyethylenimine (l-PEI) does not induce an inflammatory response. Pharm Res. 2008;25:2972–82.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Ferrari S, Moro E, Pettenazzo A, Behr JP, Zacchello F, Scarpa M. Exgen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther. 1997;4:1100–6.

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Wightman L, Kircheis R, Rossler V, Carotta S, Ruzicka R, Kursa M, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med. 2001;3:362–72.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci USA. 2005;102:5679–84.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Carlsson J, Drevin H, Axen R. Protein thiolation and reversible protein-protein conjugation. N-succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent 90. Biochem J. 1978;173:723–37.

    PubMed  CAS  Google Scholar 

  19. 19.

    Brissault B, Kichler A, Leborgne C, Danos O, Cheradame H, Gau J, et al. Synthesis, characterization, and gene transfer application of poly(ethylene glycol-b-ethylenimine) with high molar mass polyamine block. Biomacromolecules. 2006;7:2863–70.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Ungaro F, De Rosa G, Miro A, Quaglia F. Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations. J Pharm Biomed Anal. 2003;31:143–9.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Snyder SL, Sobocinski PZ. An improved 2, 4, 6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem. 1975;64:284–8.

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Kloeckner J, Bruzzano S, Ogris M, Wagner E. Gene carriers based on hexanediol diacrylate linked oligoethylenimine: effect of chemical structure of polymer on biological properties. Bioconjug Chem. 2006;17:1339–45.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Hirabayashi K, Yano J, Inoue T, Yamaguchi T, Tanigawara K, Smyth GE, et al. Inhibition of cancer cell growth by polyinosinic-polycytidylic acid/cationic liposome complex: a new biological activity. Cancer Res. 1999;59:4325–33.

    PubMed  CAS  Google Scholar 

  24. 24.

    Field AK, Tytell AA, Lampson GP, Hilleman MR. Inducers of interferon and host resistance. Ii. Multistranded synthetic polynucleotide complexes. Proc Natl Acad Sci USA. 1967;58:1004–10.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Itaka K, Harada A, Yamasaki Y, Nakamura K, Kawaguchi H, Kataoka K. In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J Gene Med. 2004;6:76–84.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Erbacher P, Bettinger T, Belguise-Valladier P, Zou S, Coll JL, Behr JP, et al. Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI). J Gene Med. 1999;1:210–22.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Kircheis R, Schuller S, Brunner S, Ogris M, Heider KH, Zauner W, et al. Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J Gene Med. 1999;1:111–20.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Kursa M, Walker GF, Roessler V, Ogris M, Roedl W, Kircheis R, et al. Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer. Bioconjug Chem. 2003;14:222–31.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Merdan T, Kunath K, Petersen H, Bakowsky U, Voigt KH, Kopecek J, et al. Pegylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice. Bioconjug Chem. 2005;16:785–92.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Walker GF, Fella C, Pelisek J, Fahrmeir J, Boeckle S, Ogris M, et al. Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Mol Ther. 2005;11:418–25.

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Burke RS, Pun SH. Extracellular barriers to in vivo PEI and pegylated PEI polyplex-mediated gene delivery to the liver. Bioconjug Chem. 2008;19:693–704.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Malek A, Czubayko F, Aigner A. PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and sirna-induced gene targeting efficacy. J Drug Target. 2008;16:124–39.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995;374:546–9.

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Vollmer J, Krieg AM. Immunotherapeutic applications of CpG oligodeoxynucleotide tlr9 agonists. Adv Drug Deliv Rev. 2009;61:195–204.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Poeck H, Besch R, Maihoefer C, Renn M, Tormo D, Morskaya S, et al. 5′-triphosphate-siRNA: turning gene silencing and Rig-i activation against melanoma. Nat Med. 2008;14:1256–63.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Besch R, Poeck H, Hohenauer T, Senft D, Häcker G, Berking C, Hornung V, Endres S, Ruzicka T, Rothenfusser S, Hartmann G. Proapoptotic signaling induced by rig-i and mda-5 results in type I interferon-independent apoptosis in human melanoma cells. J Clin Invest. 2009.

  37. 37.

    Matsumoto M, Seya T. TLR3: interferon induction by double-stranded rna including poly(i:C). Adv Drug Deliv Rev. 2008;60:805–12.

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Okada H. Brain tumor immunotherapy with type-1 polarizing strategies. Ann NY Acad Sci. 2009;1174:18–23.

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Butowski N, Lamborn KR, Lee BL, Prados MD, Cloughesy T, DeAngelis LM, et al. A north american brain tumor consortium phase ii study of poly-iclc for adult patients with recurrent anaplastic gliomas. J Neurooncol. 2009;91:183–9.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Jasani B, Navabi H, Adams M. Ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer. Vaccine. 2009;27:3401–4.

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Milhaud PG, Machy P, Lebleu B, Leserman L. Antibody targeted liposomes containing poly(rI). Poly(rC) exert a specific antiviral and toxic effect on cells primed with interferons alpha/beta or gamma. Biochim Biophys Acta. 1989;987:15–20.

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Milhaud PG, Compagnon B, Bienvenue A, Philippot JR. Interferon production of l929 and hela cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes. Bioconjug Chem. 1992;3:402–7.

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    Sakurai F, Terada T, Maruyama M, Watanabe Y, Yamashita F, Takakura Y, et al. Therapeutic effect of intravenous delivery of lipoplexes containing the interferon-beta gene and poly I: Poly C in a murine lung metastasis model. Cancer Gene Ther. 2003;10:661–8.

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Kloeckner J, Boeckle S, Persson D, Roedl W, Ogris M, Berg K, et al. DNA polyplexes based on degradable oligoethylenimine-derivatives: combination with EGF receptor targeting and endosomal release functions. J Control Release. 2006;116:115–22.

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    de Bruin K, Ruthardt N, von Gersdorff K, Bausinger R, Wagner E, Ogris M, et al. Cellular dynamics of EGF receptor-targeted synthetic viruses. Mol Ther. 2007;15:1297–305.

    PubMed  Article  Google Scholar 

  46. 46.

    Kale AA, Torchilin VP. Enhanced transfection of tumor cells in vivo using “Smart” Ph-sensitive tat-modified pegylated liposomes. J Drug Target. 2007;15:538–45.

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, et al. Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Ther. 2007;14:68–77.

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    Rudolph C, Schillinger U, Plank C, Gessner A, Nicklaus P, Muller R, et al. Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine. Biochim Biophys Acta. 2002;1573:75.

    PubMed  CAS  Google Scholar 

  49. 49.

    Meyer M, Wagner E. pH-responsive shielding of non-viral gene vectors. Expert Opin Drug Deliv. 2006;3:563–71.

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    Knorr V, Allmendinger L, Walker GF, Paintner FF, Wagner E. An acetal-based pegylation reagent for pH-sensitive shielding of DNA polyplexes. Bioconjug Chem. 2007;18:1218–25.

    PubMed  Article  CAS  Google Scholar 

  51. 51.

    Wolff JA, Rozema DB. Breaking the bonds: non-viral vectors become chemically dynamic. Mol Ther. 2008;16:8–15.

    PubMed  Article  CAS  Google Scholar 

  52. 52.

    Han Y, Caday CG, Nanda A, Cavenee WK, Huang HJ. Tyrphostin AG 1478 preferentially inhibits human glioma cells expressing truncated rather than wild-type epidermal growth factor receptors. Cancer Res. 1996;56:3859–61.

    PubMed  CAS  Google Scholar 

  53. 53.

    Milas L, Fan Z, Andratschke NH, Ang KK. Epidermal growth factor receptor and tumour response to radiation: in vivo preclinical studies. Int J Radiat Oncol Biol Phys. 2004;58:966–71.

    PubMed  Article  CAS  Google Scholar 

Download references


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.

Author information



Corresponding authors

Correspondence to David Schaffert or Ernst Wagner.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

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)

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)

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)

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Schaffert, D., Kiss, M., Rödl, W. et al. Poly(I:C)-Mediated Tumor Growth Suppression in EGF-Receptor Overexpressing Tumors Using EGF-Polyethylene Glycol-Linear Polyethylenimine as Carrier. Pharm Res 28, 731–741 (2011).

Download citation


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