2.9. Evaluation of the In Vitro Anticancer Activity

HeLa and BALB/3T3 cells were grown in 25 cm² cell culture flasks in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a CO₂ incubator (Thermo Scientific, Waltham, MA, USA, HEPA Class 100) at 37 °C and humidified atmosphere with 5% CO₂. A solution containing 0.05% trypsin and 0.02% EDTA was used for cell detachment.

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.

To analyze the mechanisms underlying the cytotoxic effects of the studied formulations (fibrous materials and solutions), the cytomorphological alterations induced in the cancer and non-cancerous cells were examined by fluorescent microscopy. The HeLa and BALB/3T3 cells were plated at a density of 2 × 10⁵/mL on glass coverslips placed into 24-well plates and incubated for 24 h in an incubator at 37 °C and 5% CO₂. The cell cultures were then exposed to the tested fibrous mats (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 24 h. At the end of the treatment, the glass coverslips were rinsed with PBS, and two fluorescent staining methods were applied. Vital staining with AO/EtBr was used to discriminate between dead and viable cells in the treated cell cultures and to identify apoptotic morphology changes. The fluorescent dyes were dissolved in PBS at a concentration of 10 μg/mL and applied to the native cell preparations. For more detailed investigation of the nuclear morphology changes, staining with the DNA-binding stain DAPI was performed. The cell preparations were fixed and stained with a 10 μg/mL methanol solution of DAPI for 10 min in the dark at room temperature and then rinsed with methanol and mounted on microscope slides. The fluorescently stained cell cultures were visualized using a fluorescent microscope (Leica DM 5000B, Wetzlar, Germany).