*3.2. PAI and CT Confirm E*ffi*cient Nanostar Uptake by Tumor Cells*

In order to test whether tumor cells efficiently take up the nanostars, SKOV3 cells were incubated with these NPs for different time periods (1, 3, 6, 12, and 24 h). Their uptake was examined by using ICP-OES, CT and PAI. ICP-OES measurements showed for the first incubation time points an almost linear increase of intracellular gold concentration while over time this seemed to change into a plateau phase as visualized by the fitting curve in Supplementary Figure S3. After 24 h, an intracellular gold amount of 11.28 ± 0.82 pg Au/cell was observed. TEM confirmed intracellular uptake of the nanostars as they were found inside vesicular structures of the cells but not at the cell membrane.

Furthermore, we evaluated whether this uptake was sufficiently high to be visualized by PAI and CT. Hereby, the tumor cells labeled with different amounts of gold nanostars were homogenously suspended in an agar phantom at 1000 cells/μL. PA images shown in Figure 3 indicated an increased PA signal (red pixels) over incubation time, where an exponential correlation was found between the PA signal and intracellular gold concentration. After 24 h, a PAI signal 1.93 ± 0.42 a.u. was measured compared to a PA signal of 0.19 ± 0.02 a.u. for the unlabeled cells. This successful nanostar-labeling resulted in an CNR of 180.73 for PAI and a limit of detection (LOD) of 3.5 pg Au/cell (17 μM).

For CT, the contrast visualized on a grey intensity scale is shown in Figure 4 where an increase of density was noticed over time. When plotting these density values over the different intracellular gold concentrations, an exponential fit was deducted. After 24 h, a CT signal of 161.33 ± 1.42 was measured for cells labeled for 24 h with nanostars compared to 130.28 ± 3.25 for unlabeled cells. The CT imaging capabilities results in a CNR of 7.36 for CT and a LOD of 5.5 pg Au/cell (28 μM).

**Figure 3. Left**: PA images of a phantom loaded with tumor cells (n = 3) incubated for different time slots with the nanostars. **Right**: PA signal plotted for the different gold masses per cell, where the dotted line suggests an exponential relation between those 2 variables.

**Figure 4. Left**: CT images of an agar phantom loaded with tumor cells (n = 3) incubated for different time points with the nanostars. **Right**: CT signal quantified as density (grey values) plotted for the different gold masses per cell, where the dotted line suggests an exponential relation between those 2 variables.
