## 3 Results
### Synthesis and Physicochemical Characterization
FT-IR spectra were utilized to study the characteristic bands of AuNPs before and after the peptide conjugations (Figure 5). Both VEGFCys FAM and VEGFGly FAM peptides exhibited very similar spectral features, with the characteristic vibrational modes of amide I and II (1666-1450 cm\({}^{-1}\)), N\(-\)H (a sharp band around 3276 cm\({}^{-1}\)), C = O (1737 cm\({}^{-1}\)), C-O (1245 cm\({}^{-1}\)), aliphatic CH\({}_{2}\) (1313 cm\({}^{-1}\)) and C-H (1278 cm\({}^{-1}\)), respectively. However, the spectrum of VEGFCys FAM did not evidence the characteristic peak of thiol group related to cysteine residue as the inset shown. FT-IR spectra were strongly affected after AuNP conjugation. Particularly, the characteristic features were still visible but less evidence; also, a shift in the position of COO\({}^{-}\) (asymmetric \(\sim\)1629 cm\({}^{-1}\) and symmetric \(\sim\)1405 cm\({}^{-1}\)) and NH\({}_{3}\)\({}^{+}\) stretching was likely due to a change in their dipole moment when peptides physisorption or chemisorption on metal surface with high electron density [39]. Figure 4: \(\zeta\)-potential values of bare AuNPs, AuNPs/VEGFCys FAM, AuNPs/VEGFGly FAM and free peptides. Figure 3: TEM images (scale bar = 50 nm) of bare AuNPs (**a**), AuNPs/VEGFCys FAM (**b**) and AuNPs/VEGFGly FAM (**c**). In order to obtain information about the functionalization and the chemistry of modified nanoparticles, XPS and ToF-SIMS measurements were performed. The atomic percentage composition of AuNPs before and after the coating with peptides showed in Table 1, demonstrate that the bare AuNPs a relatively high amount of C 1s and O 1s that hide the Au 4f signal. This finding was attributed to a thick adlayer of citrate ions coating the metal core of the nanoparticles. Accordingly, the atomic concentration for Au 4f increases due to the replacement of the citrate shell by the peptide molecules adsorbing at the interface. It must be noted that no traces of N 1s and S 2p were detected at the surface of bare AuNPs, whereas, after the coating by the peptides, comparable amounts of sulphur were found for both AuNPs/VEGFGly FAM and AuNPs/VEGFCys FAM. The S 2p high-resolution spectra of the two peptides, both free and bound to the gold nanoparticles are shown in Figure 6. The peak deconvolution has been performed according to the presence of a doublet corresponding to the S 2p\({}_{3/2}\) and S 2p\({}_{1/2}\) components, at a relative distance each from the other of 1.2 eV and with an intensity peak ratio, S 2p\({}_{3/2}\)/S 2p\({}_{1/2}\), of about 2:1. The higher binding energy (BE) component S 2p\({}_{3/2}\) has been assigned to the following chemical states of sulphur: S-Au bonds, at BE = 161.3 \(\pm\) 0.2 eV [40,41] (S0 component); S-C bonds, e.g., in cysteine and methionine residues, at BE=163.5 \(\pm\) 0.2 eV [39,42] (S1 component); S-O bonds, at increasing oxidation levels, respectively at BE = 166.3 \(\pm\) 0.2 eV [37] (S2 component); 168.4 \(\pm\) 0.2 eV (S3 component) and 170.7 \(\pm\) 0.2 eV (S4 component) [43]. \begin{table}
\begin{tabular}{c c c c c c} \hline
**Samples** & **C 1s (at.\%)** & **O 1s (at.\%)** & **S 2p (at.\%)** & **N 1s (at.\%)** & **Au 4f (at.\%)** \\ \hline \hline \multicolumn{1}{c}{Bare AuNPs} & 54.6 & 34.3 & - & - & 11.1 \\ \hline \hline \multicolumn{1}{c}{AuNPs/VEGFCy\({}_{\text{S}}\)FAM} & 8.0 & 3.6 & 1.0 & - & 87.4 \\ \hline \hline \multicolumn{1}{c}{VEGFCy\({}_{\text{S}}\)FAM} & 66.7 & 18.5 & 2.0 & 12.9 & - \\ \hline \hline \multicolumn{1}{c}{AuNPs/VEGFCy\({}_{\text{S}}\)FAM} & 9.5 & 4.5 & 0.9 & 1.5 & 83.7 \\ \hline \hline \multicolumn{1}{c}{VEGFCy\({}_{\text{S}}\)FAM} & 67.3 & 15.7 & 1.6 & 15.3 & - \\ \hline \end{tabular}
\end{table} 
Table 1: Surface atomic composition of AuNPs before and after the functionalization with peptides. Figure 5: FT-IR spectra of the bare gold nanoparticles (yellow line) and the hybrid (green line) AuNP/VEGFGly FAM (**a**) or AuNP/VEGFCys FAM (**b**). The spectra of the free, unbound peptides are included for comparison.