2.4. Cell Viability Assay

Breast cancer (MCF-7, MDA-MB-468), colon cancer (HCT-116), Melanoma (A-375) and macrophage cell lines come from the Biobank (Sistema Sanitario Publico Andaluz, Granada, Spain). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Sigma, St. Louis, MO, USA). The cells (5 × 10³ cells) were incubated in 96-well plates at 37 °C in a 5% CO₂ atmosphere and were treated with different concentrations of biosynthesized nanoparticles AgNPs-PE and AgNPs-LE (100, 50, 25, 12.5, 6.25, 3125, 1556 µg/mL) and different concentrations of extracts PE and LE (2000, 1750, 1500, 1250, 1000, 750, 500, 250 µg for 3 days. Then, the medium was removed and 100 μL of 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-tetrazoyl bromide (MTT) (at a concentration of 0.2 mg/mL) was added to each well and incubated for three hours. Subsequently, the MTT reagent was removed, and the formazan crystals formed were dissolved in 100 μL of dimethyl sulfoxide (DMSO) and analyzed at 570 nm in a multi-well ELISA plate reader. The inhibitory concentration 50 (IC₅₀) was calculated with the GraphPad Prism program (GraphPad 6 Software San Diego, CA, USA). All of the experiments were plated in triplicate and were carried out at least twice. In addition, non-treated cells were used as controls.

Morphological findings were assessed using SEM. The cells were grown on sterile coverslips, induced for 6 days with the IC₅₀ concentration of the extracts (PE and LE) and nanoparticles (AgNPs-PE and AgNPs-LE), and treated as described by Cáceres et al., 2019 [26]. A Hitachi S-800 scanning electron microscope (Hitachi, Tokyo, Japan) was used for observations. Counting the number of pores per cell was performed using ImageJ with quantification for each cell type from 8 random photos from three independent sample preparations.

The collection of the data from the different biological studies represents the mean ± standard deviation. Two-tailed Student t-test was used to compare differences between two groups. A two-tailed p-values < 0.05 was considered statistically significant.