Fig 2: Cytotoxic activity of Ganoderma aff. australe aqueous extract and Ag/Cu nanoparticles against cancer and non-cancerous cell lines evaluated by MTT assay. Cells (4,500 cells/well) were seeded in 96-well plates and allowed to adhere for 24 h before treatment. Concentrations tested were based on the amount of aqueous extract used to synthesize nanoparticles (see Table 1 ). All treatments were incubated with cells for 72 h at 37 °C with 5% CO₂. After incubation, cells were rinsed with PBS and incubated with 10 µl MTT solution (5 mg/ml) for 4 h, followed by addition of 100 µl DMSO. Absorbance was measured at 570 nm. Bar graphs show cell viability (expressed as IC₅₀ in mg/ml equivalent of extract) for five cancer cell lines and two non-cancerous control lines. Cancer cell lines: Caco-2 (colon cancer, ATCC HTB-37), HT-29 (colon cancer, ATCC HTN-38), MCF7 (breast cancer, ATCC HTB-22), A-172 (glioblastoma, ATCC CRL-1620), and U-87 MG (glioblastoma, ATCC HTB-14). Non-cancerous control lines: HDFn (human dermal fibroblasts, ATCC PCS-201-010) and Detroit 551 (normal skin fibroblasts, ATCC CCL-110). All cell lines were cultured in DMEM/F12 medium supplemented with 10% FBS, 1% antibiotic-antimycotic, 1% glutamine, 1% nonessential amino acids, and 1% sodium pyruvate. A Aqueous extract (0.5 g/50 ml) showing moderate cytotoxic activity with IC₅₀ values ranging from 1.61 ± 0.35 mg/ml (Caco-2) to 5.78 ± 1.48 mg/ml (Detroit 551), demonstrating baseline bioactivity of fungal metabolites. B M2-3-3 nanoparticles (2 ml extract + 3 ml AgNO₃ + 3 ml CuSO₄) exhibiting the highest cytotoxic efficacy across all cancer cell lines, with particularly remarkable activity against glioblastoma lines A-172 (IC₅₀: 0.26 ± 0.09 mg/ml) and U-87 MG (IC₅₀: 0.31 ± 0.12 mg/ml), and colorectal cancer lines Caco-2 (IC₅₀: 0.39 ± 0.12 mg/ml) and HT-29 (IC₅₀: 0.58 ± 0.28 mg/ml). Critically, M2-3-3 showed selective cytotoxicity with significantly higher IC₅₀ values in non-cancerous lines HDFn (2.87 ± 0.64 mg/ml) and Detroit 551 (3.45 ± 0.89 mg/ml), indicating preferential toxicity toward cancer cells. C M3-2-2 nanoparticles (3 ml extract + 2 ml AgNO₃ + 2 ml CuSO₄) demonstrating intermediate cytotoxic activity with IC₅₀ values consistently higher than M2-3-3 but lower than M5-3-3 across all cancer cell lines. D M5-3-3 nanoparticles (5 ml extract + 3 ml AgNO₃ + 3 ml CuSO₄) showing the lowest cytotoxic activity among the three nanoformulations, though still superior to the crude extract. Data represent mean ± SD of three independent experiments performed in triplicate. Statistical analysis performed using one-way ANOVA followed by Tukey’s HSD post-hoc test. Asterisks indicate significant differences: *p ≤ 0.050, **p ≤ 0.010, ***p ≤ 0.001. The hierarchical efficacy pattern (M2-3-3 > M3-2-2 > M5-3-3 > crude extract) demonstrates successful nanotechnological enhancement of anticancer properties and establishes M2-3-3 as the optimal formulation with superior therapeutic selectivity. The exceptional sensitivity of glioblastoma cell lines suggests potential application in treating brain cancers, which are notoriously difficult to treat due to blood-brain barrier penetration challenges. Complete IC₅₀ values and statistical comparisons are provided in Table 4