2.10.2. MTT Assay

Next, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) method was used to assay the in vitro cytotoxicity of MPI nanoparticles. MTT was obtained from Merck KGaA (Darmstadt, Germany). Cells (1 × 10⁴ cells/well) were seeded in a serum-free medium in a flat-bottom 96-well microplate. They were then exposed to a 20 µL nanoprotein sample per well at different concentrations of 1.5, 3, 6.25, 12.5, 25, 50, 100, 150, 200, and 250 μg mL⁻¹ for 48 h at 37 °C in a humidified atmosphere with 5% CO₂. After incubation, the media were discarded, and 40 µL of MTT solution was added to each well, followed by a further 4 h incubation. The MTT crystals were dissolved in 180 µL of acidified isopropanol/well. The plates were shaken at room temperature, and the absorbance was measured at 570 nm using a microplate ELISA reader (FLUOstar OPTIMA, BMG LABTECH GmbH, Ortenberg, Germany). The IC₅₀ was obtained using non-linear regression analysis of the concentration-response curve and OriginPro2024 version (Learning Edition). The absorbance of wells filled with media alone was used as a blank, and untreated control wells were seeded with cells incubated without MPI nanoparticles. Cell viability percentage (Cv %) was calculated using Equation (1):

where (Ac) is the absorbance of the control (untreated cells), and (At) is the absorbance of the tested sample, as previously described [50]. The cell cytotoxicity % was calculated using Equation (2):

Each concentration was repeated three times, and the average was calculated. All phases of this experiment were carried out at the NRC and the Creative Egyptian Biotechnologists (C.E.B) Lab.