Cytotoxicity assay

The human fibroblast cell line was purchased from the “Iranian Biological and Genetic Resource Center” and cultured in a DMEM media with the serum concentration of 10% inside the cell culture flask. Then, it was transferred to the incubator with moist environment and CO₂ concentration of 5% at 37 °C to prevent bacteria growth, penicillin and streptomycin with the ratio of 1% and FBS with the ratio of 10% were added to the culture media and maintained at 4 °C.

To investigate the toxicity of samples and their effect on the cell growth, 10 samples with drug (Table ​(Table3),3), and same samples without drug, were selected. For sterilization, all 20 samples were exposed to UV radiation for 1 h. The extraction process was done according to the ISO10993-5 (adding 1 mL of culture media per 6 cm² of each sample). Samples of 1 × 1 cm² cross-section were first prepared. Then, 160 µL of the culture medium was added to each sample. Finally, the plate containing sample and culture media were placed in the incubator at 37 °C and 50 rpm for 24 h. After this time, the culture medium was removed and added to each cell.

Components of polymer films containing metronidazole drug

The MTT assay was used to investigate the samples' toxicity. First, the cells were cultured in a 96-well plate with 10,000 cells in each well. This process was repeated three times for each sample. Then, the cells were incubated for 24 h to fully adhere to the plate. After this period and ensuring that the cells were fully attached, the culture media were removed and the extracts with different dilution rates were added to each well. After 24 h of cells exposure to the extract, MTT (5 mg/mL) was added to each well as long as it did not exceed the volume of culture media by 10%. The cell-containing plate was placed once again in the incubator for 4 h at 37 °C. Then, the culture media were removed and 100 µL of DMSO was added into each well. The plate was subjected to low shaking frequency for 15 min to dissolve the formed formazan, and the solution absorbency of each well was recorded at 570 nm (the control cell wells only contained the culture media). The optical density was higher in wells with more cells. As a result, the cell viability was obtained from the following equation: