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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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