In vitro cytotoxicity assay and photothermal efficiency

The percentage of viable MDA-MB-231 breast cancer cells and L929 human normal fibroblasts cell lines was calculated to evaluate the cytotoxicity of IR-CS–PPy NCs. The 96-well plates were used for seeding the cells at a concentration of 1 × 10⁴ cells/well. After 24 h of incubation with different concentrations of IR-CS–PPy NCs (0, 25, 50, 75, 100, and 125 µg/mL), 100 µL MTT was poured in each treated well to assess the cell viability. To evaluate the in vitro photothermal efficiency of IR-CS–PPy NCs, another group of IR-CS–PPy NCs-treated cells was irradiated with 2 W/cm² NIR laser for 5 min. Next, the cells were incubated for a further 2 h, and finally, the MTT assay was performed to quantify the cell viability post-NIR laser treatment.

For qualitative photothermal capacity evaluation, 12-well plates were used to incubate MDA-MB-231 and L929 cells (2 × 10⁵ cells per well) for 24 h at 37 °C of temperature. After removing the culture media, we separate the cells into four groups: group I (PBS treatment), group II (PBS plus NIR laser treatment), group III (125 μg/mL IR-CS–PPy NCs treatment), and group IV (125 μg/mL IR-CS–PPy NCs plus NIR laser treatment). After 5 min of laser exposure, the cells in all groups were stained with acridine orange (AO) and propidium iodide (PI). The in vitro photothermal effect was assessed based on fluorescence images of the samples obtained using a Leica DMI300B fluorescence microscope (Leica Microsystems, Wetzlar, Germany).