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Trastuzumab−Polyethylenimine−Polyethylene Glycol Conjugates for Targeting Her2-Expressing Tumors

Oliver Germershaus,^† Thomas Merdan,^† Udo Bakowsky,^† Martin Behe,^‡ and Thomas Kissel*^†

Department of Pharmaceutics and Biopharmacy, Philipps University Marburg, Ketzerbach 63, 35032 Marburg, Germany and Department of Nuclear Medicine, Philipps University Marburg, Baldingerstrasse, 35043 Marburg, Germany.

Bioconjugate Chem., 2006, 17 (5), pp 1190–1199

DOI: 10.1021/bc0601119

Publication Date (Web): August 15, 2006

†

Department of Pharmaceutics and Biopharmacy.

‡

Department of Nuclear Medicine.

Address for correspondence: Thomas Kissel, Department of Pharmaceutics and Biopharmacy, Philipps Universität Marburg, Ketzerbach 63, 35032 Marburg, Germany. Tel: +49-6421-282-5881. Fax: +49-6421-282-7016. E-mail: kissel@staff.uni-marburg.de.

Abstract

In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2000 Da)₁₀-graft-poly(ethylene imine 25 kDa) (PEG−PEI) covalently coupled to Trastuzumab (Herceptin) via N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) for specific gene delivery to Her2-expressing cell lines. The efficiency of DNA condensation was studied using an ethidium bromide exclusion assay and demonstrated negligible differences compared to PEG−PEI. Conjugate complex sizes were determined by dynamic light scattering to be in the range 130−180 nm. ζ potentials at different N/P ratios were close to neutral. Flow cytometry and confocal microscopy revealed efficient binding and uptake of Trastuzumab−PEI−PEG complexes using Her2-positive SK-BR-3 cells. In contrast, binding and uptake into Her2-negative OVCAR-3 cells was negligible. In good correlation with these findings, reporter gene expression using targeted complexes in SK-BR-3 cells was up to sevenfold higher than that of unmodified PEG−PEI complexes. With the use OVCAR-3 cells, no significant difference in expression efficiencies could be observed between conjugate and PEG−PEI complexes. Inhibition experiments with free Trastuzumab showed a significant decrease in reporter gene expression using SK-BR-3 cells but no decrease using OVCAR-3 cells, strongly supporting a specific Her2-receptor-mediated uptake mechanism. Our results suggest that Trastuzumab−PEI−PEG might be a promising new bioconjugate for targeted gene transfer to Her2-positive tumor cells in vivo.

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