HER2 targeted polyplexes: the effect of polyplex composition and conjugation chemistry on in vitro and in vivo characteristics.

Knowledge of the influence of targeting ligands on pharmacokinetics and biodistribution of polymeric nonviral vectors is presently limited. We investigated the properties of three structurally different conjugates of polyethylenglycol-modified polyethylenimine coupled to the HER2 specific antibody Trastuzumab. Unlike polyethylenimine, conjugates formed small (100-230 nm) DNA polyplexes with zeta-potentials of +/- 2 mV at a broad range of N/P ratios. Stability as assessed by heparin displacement was slightly improved compared to unmodified copolymers. Erythrocyte aggregation and hemolysis were strongly reduced with conjugates. Conjugate polyplexes showed significant differences in specificity and transfection efficiency in vitro. These could be attributed to differences in cell binding and uptake assessed by flow cytometry. Pharmacokinetics of conjugates in mice revealed significant improvements over free plasmid DNA and polyethylenimine. Area under the plasma level-time curve of conjugates was increased up to 48% or 114% compared to that of polyethylenimine or free plasmid DNA, respectively. Deposition of conjugate polyplexes in lung and spleen was significantly reduced compared to that of polyethylenimine. Differences could be attributed to antibody conjugation since no significant differences in pharmacokinetics and biodistribution were found between conjugates. These findings demonstrate that conjugated antibodies not only confer active targeting but also significantly improve in vivo properties of polyplexes.