Nr solution

NH₄CH₃CO₂ and CH₃CN were acquired from Carlo Erba Reagents (Val de Reuil, FR). HCOOH, Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, Triton X-100, and the NR solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibiotic solution (10,000 U/mL penicillin, 10,000 μg/mL streptomycin), 0.25% trypsin/1 mM EDTA, Fetal bovine serum (FBS), and HBSS (+/+) were purchased from Gibco Laboratories (Lenexa, KS, USA).

One mL of culturing medium containing fibroblast cell lines of density equal 1 × 105 were seeded on the BC carriers fortified with the appropriate concentrations of PUB9 and PUB10 (0.25 mg/L) and cultured for 72 h at 37 °C/5% CO₂ in an incubator. Next, the procedures utilizing Neutral Red method were performed to assess the survivability of the cells. The medium for fibroblast growth was removed and 100 μL of the NR solution (40 μg/mL; Sigma-Aldrich, Dormstadt, Germany) was introduced to wells of the plate. Cells were incubated with NR for 2 h at 37 °C. After incubation, the dye was removed, wells were rinsed with phosphate buffer saline (PBS, Sigma Aldrich, Dormstadt, Germany) and left to dry at room temperature. Next, 500 μL of a de-stain solution (50% ethanol 96%, 49% deionized water, 1% glacial acetic acid; POCH, Lublin, Poland) was introduced to each well. The plate was shaken vigorously in a microtiter plate shaker (MTS4, IKA-Labortechnik, Berlin, Germany) for 30 min until NR was extracted from the cells and formed a homogenous solution. Finally, the value of NR absorbance was measured spectrometrically using a microplate reader (Multi-scan GO, Thermo Fisher Scientific, Waltham, MA, USA) at 540 nm. The absorbance value of dyed fibroblasts seeded on the BC carriers non-fortified with PUB9 and PUB10 was considered 100% of the potential cellular growth (positive control).

The cytotoxicity of the extracts was tested for the BALB/c3T3 cell line by the neutral red (NR) uptake assay. Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well. After 24 h of incubation, extracts in a concentration range of 0.078 to 10 mg/mL were added. Dilutions were made in a cell cultivation medium. Cultivation in the presence of extracts was performed for 48 h. Afterwards, the plates were washed with phosphate-buffered saline (PBS) (Sigma, D8537, Irvine, UK), and 25 µg/mL NR solution (Sigma, N2889, Irvine, UK) diluted in 5% fetal calf serum containing media was added. After 3 h incubation in a humidified 5% CO₂ atmosphere at 37 °C, the plate was washed with PBS, and the NR taken up by viable cells was extracted using desorbing fixative (50% ethanol/1% acetic acid/49% water). Absorbance at 540 nm was measured using a Tecan M200 Infinite Pro microplate reader (Tecan, Switzerland). Cytotoxicity was expressed as a concentration-dependent reduction in the uptake of NR, compared to the untreated controls, and the IC₅₀ value for each compound was calculated using GraphPad 9 software. The cell line and test method complied with OECD guidelines [57 ].