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

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ABSTRACT

Purpose

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.

Methods

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.

Results

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.

Conclusions

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.

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Abbreviations

HBG:

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

HEPES:

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

IEX:

ion-exchange chromatography

LPEI:

linear polyethylenimine with an average molecular weight of 22 kDa

OPSS:

orthopyridyl dithio

PEG:

polyethylene glycole

p(I):

poly inosinic acid

poly(Glu):

polyglutamic acid

poly(I:C):

poly inosinic-cytidylic acid

SEC:

size exclusion chromatography

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ACKNOWLEDGMENTS

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.

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Authors and Affiliations

  1. Pharmaceutical Biotechnology, Department of Pharmacy Center for Drug Research, Ludwig-Maximilians-Universität Munich, Butenandtstr. 5-13, 81377, Munich, Germany

    David Schaffert, Melinda Kiss, Wolfgang Rödl, Manfred Ogris & Ernst Wagner

  2. Center for NanoScience (CeNS), Ludwig-Maximilians-Universität Munich, Munich, Germany

    David Schaffert, Melinda Kiss, Wolfgang Rödl, Manfred Ogris & Ernst Wagner

  3. Unit of Cellular Signaling, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

    Alexei Shir & Alexander Levitzki

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  1. David Schaffert
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  2. Melinda Kiss
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  3. Wolfgang Rödl
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  4. Alexei Shir
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Correspondence to David Schaffert or Ernst Wagner.

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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)

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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

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  • DOI: https://doi.org/10.1007/s11095-010-0225-4

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