*3.4. In Vivo CT and PAI Confirms Nanostar Delivery into Tumors*

We evaluated if nanostars injected into tumors can be visualized by in vivo imaging. Tumor-bearing mice (n = 3) were imaged with CT and PAI before and one day after gold nanostar injection into the tumor. Before injection, only the contrast between the skeleton and the soft tissue was visible using CT (Figure 6A). After injection of the nanostars, an intense contrast of 94.63 ± 6.77 was observed at the left side of the tumor, corresponding to the place of the tumor (Figure 6A, right). No significant change in contrast was generated in the PBS-injected control tumor with a signal of 74.11 ± 1.43 (Figure 6A, left). A CNR increase of 25.31 was calculated after intratumoral injection of gold nanostars, indicating that intratumoral delivery of nanostars can be monitored with CT in vivo.

**Figure 5. Left**: Fluorescence images of the living cells after photothermal therapy (PTT) using calcein AM staining for tumor cells (n = 3) incubated for different time periods with the nanostars. **Right**: Relative green pixel numbers plotted against the gold mass per cell. Fluorescence signal intensity is expressed relative to the unlabeled control cells (0 h).

**Figure 6.** (**A**) CT images before and after injection of gold nanostars into the tumor. An increased contrast was noticed at the tumor site after gold nanostars injection as indicated by the arrow; (**B**) In vivo PA images before and 24 h after nanostar injection. The photoacoustic imaging (PAI) signal (red pixels) is overlaid over the anatomical ultrasound images (grey pixels).

Alternatively, we evaluated whether intratumoral nanostar delivery can also be followed up with PAI. Hereby, both ultrasound and photoacoustic images were overlaid as shown in Figure 6B. The ultrasound image was used for localizing and visualizing the tumor. Although some background photoacoustic contrast due to hemoglobin is present before nanostar injection, PAI could be used to visualize the nanostars as indicated by a significant increase in signal intensity compared to the background (1.20 ± 0.17 a.u. compared to the control 0.44 ± 0.05 a.u.) with a CNR of 80.31.
