## 3 Results and Discussion  
### Synthesis and Characterization of Melanin-BSA Conjugates  
The spontaneous oxidation of DA yields an untreatable, insoluble polymeric material (synthetic melanin or eumelanin). When DA oxidation is carried out in the presence of enough BSA equivalents, water-soluble melanin-BSA conjugates could be obtained. Melanin-BSA was prepared according to a modified version of a reported procedure [53], through the autoxidation of dopamine (Figure 1). The quinone compounds formed by the oxidation of DA react with the nucleophilic groups present in the protein (cysteine, histidine, lysine) and the growing melanic oligomers remain covalently linked to the protein, generating eumelanin-type conjugates (EuBSA). PheoBSA-type conjugates were prepared under the same conditions from dopamine and L-cysteine (DA:Cys molar ratio of 5:1). Different melaninBSA conjugates were prepared by varying the ratio DA/BSA _w/w_ (2:1; 1:1; 1:2) and the time of reaction as reported in Table S1 (Supplementary Material). After dialysis and lyophilization, the solubility of the conjugates was tested by adding 10 mg of solid powder to 1 mL of water. The solubility of the conjugates depends on the ratio between the melanic and protein contents, which reflect the used ratio of DA/BSA _w/w_ reagents and the applied reaction time. Conjugates A, B, C, D, E resulted to be well soluble at the tested concentration of 10 mg/mL. Most of them were prepared in the presence of cysteine, and hence are classified as Pheo-BSA conjugates, with smaller DA oligomers bound to the protein.  
Each melanin-BSA conjugates displayed the typical absorption profile of melanin extending from the UV to the visible range (Figure 2A). Their sizes (diameter) were as follows: 18.8 \(\pm\) 0.5 nm (conjugate A); 2.3 \(\pm\) 0.5 nm (conjugate B); 5.3 \(\pm\) 0.5 nm (conjugate C); 5.2 \(\pm\) 0.5 nm (conjugate D);\(4.6\pm 0.5\) nm (conjugate E) (Figure S1). The smallest sizes were observed for the Pheo-BSA conjugates, as expected based on a lower DA polymerization in the presence of cysteine. The quantification of BSA in the PheoBSA D was estimated after complete hydrolysis of the protein by NMR, according to a reported procedure [53]. The method is based on the quantification of the aromatic protons of tyrosine (20 residues in BSA) and the methyl groups of threonine (33 residues in BSA) that give easily recognizable \({}^{1}\)H NMR signals, using sucrose as standard. The result suggested an averaged ratio of 20% of the protein (_w/w_).  
FITC was conjugated with PheoBSA D for in vivo biodistribution studies. A FITC calibration curve was used for the quantification of FITC conjugated to PheoBSA D using PheoBSA D as a blank at the same concentration in mg/mL. We calculated an amount of 2 umol of FITC per g of PheoBSA D.  
Photoacoustic measurements of the conjugates showed broad absorption spectra, typical of natural melanin (Figure 2B). PheoBSA D showed the highest PA contrast properties at a concentration of 2.5 mg/mL. Despite PheoBSA C displayed comparable absorbance properties to PheoBSA D, a lower PA emission efficiency was observed, likely reflecting different PA efficiency due to a different polymerization degree when compared to PheoBSA D. Photoacoustic emission showed a linear dependence with the concentration for all the investigated systems (Figure 2C). When compared with a previously reported system, melanin free acid (MFA-PEG) [47], it was found that PheoBSA D displayed almost a two-fold increase in PA contrast capability (Figure 2D).