*3.5. Gold Nanostars Mediate In Vivo Photothermal Therapy*

The effectiveness of nanostars to mediate PTT was studied in a xenograft mouse model (n = 6). The left tumor was used for injection with gold nanostars, while the right tumor acted as control after sham injection with PBS. 24 h after nanostar injection, both tumors were irradiated with a continuous laser (5 min, 690 nm and 2 W/cm2). Therapy efficacy was assessed in vivo by using MRI and BLI to evaluate tumor regression and viability. For quantification of the tumor cell viability, the BLI signal was monitored one day before and 0, 1, 5, 8 and 15 days after PTT. The day before PTT and after imaging, nanostars/PBS were injected into the tumors. A decrease in BLI signal intensity was detected for the nanostar-injected tumor at day 0, which was not observed for the PBS injected tumor (Supplementary Figure S5). This is explained by the nanostars absorbing/scattering a part of the BLI signal. This time point was used as baseline for assessment of PTT. One day after PTT, a significant decrease in relative BLI signal intensity to 10.13 ± 1.41% was observed for the nanostar-injected tumor compared to the tumor at day 0, indicating a decrease in viable tumor cells or vessel patency (Figure 7). After 5 days, an increase of the BLI signal intensity was noticed to a relative value of 45.78 ± 10.02%, which indicates partial re-growth of the PTT treated tumor as probably not all tumor cells were photothermally ablated. Although, a significant difference in signal intensity was maintained when compared to the tumor at baseline. In contrast, the right control tumor showed a constant increase in BLI signal intensity over time until a relative BLI signal of 440.24 ± 51.14% after 15 days, confirming that the viability of the control tumor was not affected by laser irradiation. When comparing the nanostar-injected with the control tumor, a significant difference in BLI signal intensity was detected at day 1, 5, and 8, indicating a significant inhibition of tumor growth by PTT.

For providing more information on the therapeutic effect, MR images were acquired on day 0, 1, 8 and 15 to monitor tumor size, anatomy, and heterogeneity. After 8 and 15 days, a relative tumor volume of 68.64 ± 18.45% and 55.25 ± 30.1% was observed for the left tumor compared to day 0, indicating effective PTT (Figure 7B). This was confirmed by the significant difference between this volume and the volume of the control tumor (PBS injection), where a relative tumor volume of 142.27 ± 28.49% at day 5 and 184.12 ± 39.13% at day 8 was measured, respectively. MRI also indicate that the nanostar-injected tumors were not affected evenly by PTT over the whole tumor volume, indicating local differences due to inhomogeneous distribution of the nanostars (Figure 7B).

For validation, mice (n = 3) were sacrificed after the first (day 1) and last (day 15) imaging time point following PTT. Tumors were resected and histologically examined. After one day, a distinct blue color was seen on these histological slices originating from the nanostars with an LSPR band of 679 nm, which were visible in the tumor. These nanostars were only noticed in the tumor region where also necrotic cells were visible (Supplementary Figure S6). As can be seen on the H&E stained images, the nuclei were either defragmented or darkened in comparison to the healthy cells (Figure 8). For the PBS-injected control tumors, the blue color due to the nanostars was absent and the majority of cells appeared healthy. At the last imaging time point, no necrotic cells were visible on the histology sections for both the PBS- and nanostar-injected tumors (Supplementary Figure S6).

Using TEM, the presence of nanostar clusters inside vesicles of the cells was confirmed (Figure 8). At the ultrastructural level, tumor cells were identified based on their pleomorphic aspects, increased nuclear/cytoplasmic ratio and distinct anaplasia. The latter was typically represented by nuclear hyperchromatism, thereby confirming that the tumor cells took up the nanostars. These nanostars were present in the cytoplasm and typically stored in membrane-bound compartments (endosomes,

arrows in Figure 8B). As the endosomes were packed with nanostars, it was difficult to identify single nanostars, demonstrating efficient intracellular uptake in vivo upon intra-tumoral injection.

**Figure 7.** (**A**) In vivo bioluminescence imaging (BLI) before (day 0) and after PTT (days 1, 5, 8) illustrated in a color-coded intensity map. A quantification of the BLI signal intensity relative to day 0 (set to 100% for each mouse) is plotted for both nanostar-injected and control (PBS-injected) tumors (RT); (**B**) MR images of the tumor-bearing hind limbs taken at corresponding time points (days) after nanostaror PBS-injection. The white arrow indicates the tumor damage (hypointense area) after PTT at the nanostar-injected tumor. The relative mass volumes were quantified relative to day 0 (set at 100% per mice). For both BLI and magnetic resonance imaging (MRI) graphs, the error bars represent SD; n = 6; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

**Figure 8.** (**A**) Bright field microscopy images of control (right) and nanostar (left) injected hematoxylin and eosin (H&E) stained tumor sections. Defragmented nuclei of the tumor cells could be visualized after therapy. (**B**) Ex vivo TEM images of tumor cells that indicate the presence of NP clusters in endosomes. The right panel shows a zoomed section of these endosomes with vesicular structures visible.
