Lung cancer A549 cells (ATCC) were seeded in 96-well plates with 200 µL of tissue culture medium for 24 hours before transfection with the siRNA complexes. A broadly-cytotoxic siRNA from ThermoFisher (identity not revealed) was formulated with the RJH transfection reagents ALL-Fect, Prime-Fect, InVivo-Fect and an proprietary formulations of RJH-X at ratios of 1:23, 1:10, 1:23 and 1:10 (w/w), respectively. The siRNA complexes were prepared in Opti-MEMTM by incubating siRNAs with the transfection reagents for 30 min before addition to the cells.
As control treatments, (i) an equivalent concentration of the chosen transfection reagents and (ii) scrambled (non-specific) siRNA formulated with the chosen transfection reagents were used to treat the cells under similar conditions. Cells were transfected with a final concentration of 50 nM of siRNA (except the cells that received transfection reagent alone). After incubation for 96 hours in a humidified CO2/O2 (95/5%) atmosphere, the MTS viability assay (Abcom) was run to determine the total cell activity in wells. The obtained absorbance readings (490 nm) were taken as the total viable cells numbers. The cells treated with the transfection reagent alone were used as a reference (taken as 100% relative viability). The viabilities of the remaining cells were normalized with the viabilities of the cells treated with the transfection reagent alone.
The experimental results from the cell viability assay are shown below. The relative cell viabilities are summarized separately for each group of cells treated with different transfection reagents.
Cells treated with the transfection reagents alone typically yielded relative viabilities that were similar to the untreated cells in the case of transfection reagents Prime-Fect and ALL-Fect, indicating no effect of these transfection reagents alone on the cell viabilities. InVivo RNA-Fect and RJH-X was not tested in this regard. Cells treated with scrambled siRNA complexes of Prime-Fect and InVivo RNA-Fect gave relative viabilities equivalent to untreated cells, again indicating no toxicity of the non-specific complexes on the viabilities of A549 cells. Complexes from ALL-Fect and RJH-X displayed some loss of viabilities (~10%) indicating slight toxicity of the non-specific complexes compared to un-treated cells. In contrast, the cytotoxic siRNA complexes gave significant reduction of toxicity, with Prime-Fect complexes displaying ~40% relative cell viability and InVivo RNA-Fect complexes displaying ~16% relative cell viability, and ALL-Fect and RJH-X complexes displaying less than 10% relative cell viabilities. Therefore, multiple RJH reagents were found to be very effective in this particular application intended to induce death of lung cancer cells. The differential activity obtained between the scrambled siRNA and cytotoxic siRNA were very high, suggesting that effective therapeutic effects could be readily demonstrated. A relatively low concentration of siRNAs was used (50 nM) to obtain the desired therapeutic effect so that translation into animal models should be facilitated at such low doses.