A cell impedance-based real-time in vitro assay to assess the toxicity of amphotericin B formulations
Résumé
Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergil-losis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to LAmB (50 to 400 μg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200 μg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proin-flammatory cytokines was quantified at 6 and 24 h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with LAmB or empty liposomes, even at 400 μg/ml. Only the highest concentration tested of LAmB (400 μg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50 μg/ ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6 h with 50 μg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after LAmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.