title: Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors.
abstract: In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2,000 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. zeta 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.
authors.affiliation: Department of Pharmaceutics and Biopharmacy, Philipps University Marburg, Ketzerbach 63, 35032 Marburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Germershaus
authors.forename: Thomas
authors.initials: T
authors.lastname: Merdan
authors.forename: Udo
authors.initials: U
authors.lastname: Bakowsky
authors.forename: Martin
authors.initials: M
authors.lastname: Behe
authors.forename: Thomas
authors.initials: T
authors.lastname: Kissel
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D015394:Molecular Structure; D009369:Neoplasms / Q000378:metabolism; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab