## 3 Results and Discussion
### PheoBSA Conjugate D In Vivo Biodistribution and Tumor Detection  
The toxicity of PheoBSA D was evaluated by incubating J774A.1 cells in presence of several concentrations of the melanin/BSA conjugate. As can be seen from the graph (Figure 3A), after three days, no cell toxicity was detected. The vitality of the J774A.1 cells was completely unaffected by the presence of the PheoBSA conjugate up to the concentration of 2.5 mg/mL, whereas at higher  
Figure 2: In vitro imaging characterization of different melanin-BSA conjugates. (**A**) Absorption spectra. (**B**) Photoacoustic spectra. (**C**) Photoacoustic emission registered at 730 nm at different concentrations. (**D**) Comparison of photoacoustic efficiency between PheoBSA D and MFA-PEG. concentrations, a moderate toxicity was measured, although any side effects in any of the injected animal was observed. The PheoBSA D showed also high stability in serum for several hours and titrated at different pH (Figure 3B). The distribution of PheoBSA-FITC (300 uL/mouse of a 2 mg/mL solution) after IV administration was investigated in male Athymic nude mice (\(n=6\)) which were subcutaneously inoculated with human prostate PC-3 cancer cells. Ex vivo optical imaging experiments on excised organs, i.e., tumor, liver, brain, kidneys, spleen, heart, lungs, and muscle were performed at 4 h and 24 h post-injection (Figure 3C,D). Images recorded 4 h after the injection showed an intense signal at kidneys and in the tumor regions whereas very weak signals were detected for liver, muscle, and lungs. No signal could be detected from the brain, heart, and spleen. After 24 h post-injection, the signal decreased although it remained still detectable in tumor and kidneys. All the other organs showed a signal comparable with the fluorescence observed in not treated animals (data not show). The capability of PheoBSA D for tumor imaging was further assessed by acquiring PA images in HER2 positive breast TS/A tumor models. The acquisitions were carried out either on VevoLzar 2100 (Visualsonic) and LOIS 3D (Tomowave) systems (Figures 4 and 5). A net tumor contrast enhancement during the first 30 min from the administration of the CA was observed on both scanners. The VevoLzar photoacoustic imager generates a lower tumor enhancement compared to those obtained with the LOIS 3D, as reported in Figures 4 and 5. Figure 3: **(A)** J774A.1 cell viability measured after 24 h of incubation with different concentrations of PheoBSA D. Data are shown as mean percentage \(\pm\) SD of viable cells normalized to untreated cells. **(B)** Serum stability measured at different pH. **(C)** Ex-vivo optical imaging of tumor (T), liver (L1), heart (H), brain (B), kidney (K), lung (L2), spleen (S), and muscle (M) of PC-3 tumor-bearing mice (\(n=3\) mice/group) at 4 and 24 h post-PheoBSA-FITC injection. **(D)** Ex-vivo optical signal PC-3 tumor-bearing mice after PheoBSA-FITC administration at 4 and 24 h post-injection, expressed as Average Radiant Efficiency (mean \(\pm\) SD). A possible reason for the observed findings appears related to the different instrument configuration that allows one to acquire a 3D image with the LOIS and 2D with the Vevo with different penetration depth [54,55]. Photoacoustic signal is partly limited to the epithelial tumor zone by Vevo, so the enhancement is calculated from about one-third to one-half of the entire ROI drawn on the tumor (Figure 4B). However, the VevoLazr is closer to a clinical setup with a handheld probe. Upon comparing the results obtained using the LOIS 3D system it was found that PheoBSA D photoacoustic enhancement in the prostate tumor model roughly doubled the signal enhancement  
Figure 4: Photoacoustic signal measured by Vevo Lazr before and after (arrow) IV administration of PheoBSA D at a dose of 10 mg/kg in TS/A tumors (\(n=4\)). (**A**) Photoacoustic enhancement measured at 730 nm.