Evaluation of Generation 2 and 3 Poly(Propylenimine) Dendrimers for the Potential Cellular Delivery of Antisense Oligonucleotides Targeting the Epidermal Growth Factor Receptor

PURPOSE: To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. METHODS: Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. RESULTS: G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. CONCLUSIONS: G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.