2.6.4. In vitro cytotoxicity in lung cells

Cell viability was assessed using the MTT assay. H1299/GFP cells were seeded at a density of 10.000 cells/well in 100 μl medium in a 96-well-plate 24 h prior to transfection. Cells were transfected with PEI and VIPER polyplexes containing 20 pmol scrambled siRNA at different N/P ratios (6, 10 and 15 respectively) and incubated for 24 h at 37 °C and 5% CO₂. Once the incubation time was completed, medium was removed and 100 μl of a sterile 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to the cells and incubated for 3 h at 37 °C and 5% CO₂. Medium was then removed, and 200 μl of DMSO was added to each well to dissolve formazan crystals. Absorbance was then read at 570 nm using a microplate reader (Tecan, Männedorf, Switzerland). Results are given as mean of values of triplicates ± SD.

The effect of polyplexes on membrane integrity was assessed by measuring the release of lactate dehydrogenase (LDH) in the extracellular medium using the CytoTox96® Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, Wisconsin, USA) according to the manufacturer's protocol. Briefly, 5.000 H1299/cells were seeded in a 96-well-plate in 100 μl medium 24 h prior to transfection. Cells were treated with PEI and VIPER polyplexes containing 20 pmol siRNA at different N/P ratios (6, 10 and 15 respectively) and incubated for another 24 h. Afterwards, 50 μl of supernatant was transferred to a fresh 96-well-plate and 50 μl of substrate mix was added and incubated for 30 min at room temperature protected from light. Subsequently, 50 μl of stop solution was added and absorbance was measured at 490 nm using a microplate reader (Tecan, Männedorf, Switzerland). Untreated cells were used as negative control, while cells treated with lysis buffer represented 100% LDH release. Results are given as mean values of triplicates ± SD.