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.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Hepes buffered glucose (5% (w/v) glucose, 20 mM Hepes, pH 7.4)
N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid
linear polyethylenimine with an average molecular weight of 22 kDa
poly inosinic acid
poly inosinic-cytidylic acid
size exclusion chromatography
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.
Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2003;2:347–60.
Li SD, Huang L. Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther. 2006;13:1313–9.
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.
Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat Rev Drug Discov. 2005;4:581–93.
Schaffert D, Wagner E. Gene therapy progress and prospects: synthetic polymer-based systems. Gene Ther. 2008;15:1131–8.
Wagner E. The silent (r)evolution of polymeric nucleic acid therapeutics. Pharm Res. 2008;25:2920–3.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Snyder SL, Sobocinski PZ. An improved 2, 4, 6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem. 1975;64:284–8.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Vollmer J, Krieg AM. Immunotherapeutic applications of CpG oligodeoxynucleotide tlr9 agonists. Adv Drug Deliv Rev. 2009;61:195–204.
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.
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.
Matsumoto M, Seya T. TLR3: interferon induction by double-stranded rna including poly(i:C). Adv Drug Deliv Rev. 2008;60:805–12.
Okada H. Brain tumor immunotherapy with type-1 polarizing strategies. Ann NY Acad Sci. 2009;1174:18–23.
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.
Jasani B, Navabi H, Adams M. Ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer. Vaccine. 2009;27:3401–4.
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.
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.
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.
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.
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.
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.
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.
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.
Meyer M, Wagner E. pH-responsive shielding of non-viral gene vectors. Expert Opin Drug Deliv. 2006;3:563–71.
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.
Wolff JA, Rozema DB. Breaking the bonds: non-viral vectors become chemically dynamic. Mol Ther. 2008;16:8–15.
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.
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.
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.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
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)
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)
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)
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)
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). https://doi.org/10.1007/s11095-010-0225-4
- epidermal growth factor
- receptor-mediated delivery
- RNA delivery
- tumor targeting