Cell viability assay

The cytotoxicity of DOX and TEPP-DOX was assessed in MDA-MB-231 cells by determining their half-maximal inhibitory concentrations (IC₅₀s). Cells (2 × 10³) were plated in a 96-well plate and incubated for 24 h of culture. Subsequently, DOX or TEPP-DOX was added at varying concentrations (0–40 μg/mL DOX diluted in cell culture medium). After 45 h of treatment, thiazolyl blue tetrazolium bromide (MTT; M5655; Sigma) was added to the cells and incubated for 3 h. At the end of the 48-h incubation period, the culture medium was removed and replaced with 150 μL of dimethyl sulfoxide (DMSO). Absorbance (570 nm) was measured using a plate reader (PerkinElmer). The IC₅₀ was calculated by nonlinear regression analysis using GraphPad Prism 7.0.