The MTT (3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed using WT ADC and positively charged ADC in three cell lines with varying HER2 expression levels: N87 (high), MCF-7 (low), and MDA-MB-468 (non-expressing). Cells were seeded at a density of 10,000 cells per well in 96-well plates and incubated for 24 h before treatment. ADCs were added at concentrations ranging from 0.01 nM to 1000 nM, and cells were incubated for an additional 96 h. Following treatment, 5 mg/mL MTT solution was added to each well and incubated for 4 h, after which 10% SDS-HCl solution was added to dissolve the formazan crystals. Absorbance was measured at 570 nm the next day. Cell viability (%) was calculated as (ODtreated/ (ODcontrol) × 100%. GraphPad Prism (version 10.4.1) was used for curve fitting of the sigmoidal dose-response curves (cell viability vs. ADC concentration) under the nonlinear regression option, and the half-maximal inhibitory concentration (IC50) values were determined.

The MTT (3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed using WT ADC and positively charged ADC in three cell lines with varying HER2 expression levels: N87 (high), MCF-7 (low), and MDA-MB-468 (non-expressing). Cells were seeded at a density of 10,000 cells per well in 96-well plates and incubated for 24 h before treatment. ADCs were added at concentrations ranging from 0.01 nM to 1000 nM, and cells were incubated for an additional 96 h. Following treatment, 5 mg/mL MTT solution was added to each well and incubated for 4 h, after which 10% SDS-HCl solution was added to dissolve the formazan crystals. Absorbance was measured at 570 nm the next day. Cell viability (%) was calculated as (ODtreated/ (ODcontrol) × 100%. GraphPad Prism (version 10.4.1) was used for curve fitting of the sigmoidal dose-response curves (cell viability vs. ADC concentration) under the nonlinear regression option, and the half-maximal inhibitory concentration (IC50) values were determined.

The MTT (3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed using WT ADC and positively charged ADC in three cell lines with varying HER2 expression levels: N87 (high), MCF-7 (low), and MDA-MB-468 (non-expressing). Cells were seeded at a density of 10,000 cells per well in 96-well plates and incubated for 24 h before treatment. ADCs were added at concentrations ranging from 0.01 nM to 1000 nM, and cells were incubated for an additional 96 h. Following treatment, 5 mg/mL MTT solution was added to each well and incubated for 4 h, after which 10% SDS-HCl solution was added to dissolve the formazan crystals. Absorbance was measured at 570 nm the next day. Cell viability (%) was calculated as (ODtreated/ (ODcontrol) × 100%. GraphPad Prism (version 10.4.1) was used for curve fitting of the sigmoidal dose-response curves (cell viability vs. ADC concentration) under the nonlinear regression option, and the half-maximal inhibitory concentration (IC50) values were determined.