2.9.2. Cytotoxicity Assay

The cytotoxicity of the prepared fibrous materials on human HeLa cancer cells and BALB/3T3 non-cancerous mouse fibroblasts was determined using the MTT proliferation assay. Briefly, cancer cells were seeded in 96-well plates in DMEM medium at a density of 1 × 10⁴ cells per well. After an incubation period of 24 h at 37 °C and 5% CO₂, the cells were incubated with fresh medium alone or in the presence of various fibrous materials (PHB, Ch/HA-coat-PHB, MO-in-PHB, (Ch,HP)/HA-coat-PHB, (Ch,HP)/HA-coat-(MO-in-PHB)); and solutions of MO, HP, and a mixture of both extracts (MO:HP extracts (1:2 w/w)) for 72 h. The effects of MO and HP on HeLa cell viability were compared with those of the standard chemotherapeutic agent doxorubicin (DOX). All MO- and HP-containing fibrous mats were investigated at an MO and HP concentration of 130 μg/mL and 260 μg/mL of culture medium, respectively. The concentrations of MO and HP in the studied solutions were 130 μg/mL and 260 μg/mL, respectively. Untreated cells cultures were used as a control. At 24 h and 72 h of incubation, the studied formulations (mats or solutions) and culture medium were removed, and MTT solution (0.5 mg mL⁻¹; 100 µL per well) and the cells were re-incubated for 3 h. At the end of the incubation, the MTT-containing medium was removed, and solubilizing solution (DMSO/EtOH, 1:1, v/v) was added to dissolve formazan crystals. Finally, the absorbance was read by an ELISA plate reader (TECAN, Sunrise^TM, Grödig/Salzburg, Austria) at a wavelength of 570 nm. The cell viability was presented as a percentage of the negative control cells.

All experiments were performed in six replicates.