Cytotoxic effect of Dd-DOX conjugate determined by MTT assay. A) MES-SA and MES-SA Dx5 cells were treated with Dd (6 μg), Dd-DOX (2, 4, 6 μg delivering 50, 100, 150 nM DOX respectively) and free DOX (50, 100, 150 nM). Cell viabilities were compared to their respective non-treated controls. Data were compared by applying Student’s t-test, which indicated statistically significant differences between DOX and Dd-DOX treated cells (* P < 0.05, ** P < 0.01). Data represent mean ± s.d. (n = 3 experiments, five wells per treatment). B) Dose–response curves for Dd-DOX conjugates and free DOX for the MES-SA (left panel) and MES-SA Dx5 (right panel) cell lines. Various concentrations of Dd-DOX (20, 50, 100, 150, 260, 390 nM) and DOX (50, 100, 150, 300, 450, 600 nM) were applied, and the viability of cells was determined by the MTT assay. Non-linear regression analysis was performed using Origin software (OriginLab Corporation, USA). The averaged percentage corresponding to viability of cells was plotted against the logarithm of the concentration of Dd-DOX conjugates and free DOX. Curves were fitted to a dose–response function according to the equation y = A1 + ((A2-A1)/(1 + 10^(logx0-x)•p)). Each value is the mean ± standard error obtained for three experiments (n = 3 experiments, five wells per treatment).