*3.3. E*ff*ective In Vitro Photothermal Tumor Cell Ablation Using Gold Nanostars*

Since tumor cells were effectively labeled with nanostars, we investigated if the in vitro uptake was sufficient to eradicate tumor cells by PTT. First, the potential of nanostars for heat generation necessary for PTT was investigated using a glass capillary filled with either 4.6 <sup>×</sup> 1010 nanostars/mL or water as a negative control. When irradiating the sample with a 690 nm laser (7 W/cm2), a temperature increase of 25 ◦C was observed for the nanostar suspension, while no temperature increase was measured for the water filled capillary (Supplementary Figure S4).

A crucial parameter for determining the effectiveness of PTT is to calculate the cell killing capacity (IC50 value). To determine this value, the tumor cells were labeled with the nanostars using the same conditions as for the imaging experiments. After PTT, cell death was visualized by calcein AM living cells staining and quantified by calculating the total green pixels. As visualized by fluorescence microscopy a radius increase of non-viable cells at the laser spot indicates a higher PTT effectiveness with an increasing intracellular gold concentration (Figure 5). By plotting the relative green fluorescence signal compared to the fluorescence signal of the control cells, a sigmoidal curve was fitted where an IC50 of 4.8 pg Au/cell (23 μM) was calculated.
