Before each measurement, 950 μl Hepes buffer was added to 50 μl of the lipoplexes or polyplexes. Toxicity of the lipoplexes and polyplexes was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay after transfecting the different complexes in the BGM cell line, which are kidney epithelial cells from the African Green Monkey (ATCC: CCL-26). Briefly, BGM cells were seeded in 96-well plates (100 μl/well; 3 × 105 cells/ml) and transfected 24 h later by pipetting the complexes into the culture medium (MEM supplemented

with 10% FCS, 1% vitamins, 1% l-glutamin, 1% streptomycin and 2% vancomycin, all products from Invitrogen). Cytotoxicity of all lipoplexes and polyplexes was tested in duplicate after 24 and 48 h of incubation with the complexes by adding

Entinostat order MTT (10 μl, 0.5 mg/ml) to the cells. The MTT assay was performed as described before [18] and the percentage cell survival was calculated as follows: [OD585–OD620 (transfected cells)]/[OD585–OD620 (non-transfected cells)] × 100%. Complexes inducing less than 40% cell death were selected to perform quantification of ompA expression. To determine transfection efficiencies, lipoplexes and polyplexes were transfected in duplicate in BGM cells, seeded in 24-well plates (500 μl/well; 3 × 105 cells/ml) and cultured in an atmosphere of 37 °C and 5% CO2. After 24 h, the culture medium was removed, cells were rinsed with PBS and MEM, without serum and antibiotics, was added. An appropriate amount of all different lipoplexes and polyplexes was added to the cells. After incubating 3 h at 37 °C and 5% CO2, complexes were removed, cells were rinsed again CHIR 99021 with PBS and complete culture medium was added. Naked pDNA and complexes with PolyFect® transfection

reagent (Qiagen) were used as negative and positive controls, respectively. At 24 and 48 h following transfection, cells were trypsinized and isothipendyl resuspended in 300 μl PBS. To quantify ompA expression, the percentage of transfected cells was determined by measuring EGFP fluorescence (488 nm) using a FACSCanto flow cytometer (BD Biosciences, Erembodegem, Belgium). Polyplexes and naked pDNA were aerosolised by using a Cirrus™ Nebulizer (Intersurgical Ltd., Berkshire, UK). This nebulizer, designed to provide particles up to 5 μm (mass median diameter of 3.5 μm), was connected to a pump that generated a pressure of 180 kPa and an air flow rate of 8 l/min. Aerosols were collected on a microscopic glass slide allowing the aerosol droplets to condense onto the slide. The condensation fluid was collected in a sterile tube. Afterwards, pDNA concentration, particle size and zeta potential of the nebulised polyplexes were examined. Subsequently, the transfection capability of the nebulised complexes was checked by flow cytometrical analysis of transfected BGM cells as described in Section 2.4. Plasmid DNA integrity was determined using gel electrophoresis.