## 4 Discussion  
The angiogenic factor VEGF is the most studied growth factor, due to its specificity and important role in the activation of all steps of angiogenesis in the endothelium vasculature [51]. The splicing variant VEGF165 is the predominant form and has been shown to be up-regulated in tumour microenvironment by hypoxia or activation of oncogenes [52]. VEGF is a glycoprotein and consists of an antiparallel homodimer structure containing inter- and intra-disulphide bonds, and it has been shown to bind to three receptor types as follows: VEGFR-1 (flt-1), VEGFR-2 (flk-1 or KDR), and neurophilin-1 (NR-1). In the endothelial cells, most of the angiogenesis signaling (proliferation, migration, and survival) proceeds via the interaction between VEGF and VEGFR-2. The latter has been identified and includes residues at a loop region formed by the anti-parallel \(\beta\)-sheets \(\beta\)5-66 in the VEGF protein [53]. Only a small number of VEGF residues are important for binding to VEGF receptors [54], and, therefore, several molecules able to modulate VEGF biological activities have been reported [55]. Some VEGF mimetic peptides with antiangiogenic activity have been described [30]. It was reported that the peptide fragment encompassing the residues 73-101 blocked at N- and C-terminus (Ac-ESNITMQIMRIKPHQQQHIGESFLQHNK-NH\({}_{2}\)), included in the loop \(\beta\)5-\(\beta\)6 region involved with VEGFR2 interaction, showed anti-proliferative activity on HUVEC cells [30]. In recent decades, nanodrug delivery system has played an important role in depressing the toxic side effects and enhancing the therapeutic efficiency [56]. Functionalization of AuNPs with biological molecules has many applications in biomedical imaging, clinical diagnosis and therapy [19]. Recently, it was reported that gold nanoparticles exhibited potentially antiangiogenic effects by interacting with the heparin-binding domain of VEGF165. Furthermore, AuNPs seem to inhibit migration, tube formation and proliferation VEGF165-induced of endothelial cells [23; 57]. It was also reported that intravenously administrated AuNPs could pass through blood-retinal barrier and induce no retinal toxicity [58]. We investigated the gold nanoparticles conjugated with VEGF fragments peptide Ac-ESNITMQIMRIKPHQGQHIGESFLQHNK-NH\({}_{2}\) and its mutated VEGF73-101-G88C 5,6-FAM labelled. The obtained results showed the enhanced intracellular uptake respect to AuNPs alone and no toxicity on HUVEC cells. Indeed, the fluorescent dye conjugated to the peptide sequence allowed for the certain simultaneous localization of both the peptide molecules and the gold NPs. To note, VEGF receptors are known to use clathrin-dependent endocytosis that ultimately leads endocytosed material to lysosomes [59]. On the other hand, different physicochemical properties of gold nanoparticles, including size, clustering and surface chemistry of nanoparticles regulate their cellular uptake and transport [60; 61]. Among the most common internalization mechanisms of gold nanoparticles, there is autophagosome accumulation through size-dependent nanoparticle uptake and lysosome impairment [62; 63]. Specifically, AuNPs are known to reduce lysosomal activity by alkalization of the lysosomal lumen [64; 65]. Further studies on the peptide-functionalized hybrid nanosystems can focus to map the trafficking process of the gold-nanoparticle bound peptides as well as to test them in cancer cells as a potentially anti-cancer drug or on retinal cells to regulate retinal neovascularization. Another potential follow-up of this study can involve cell migration and tubulogenesis studies in the HUVEC treated by VEGF peptide-modified AuNPs. Indeed, we recently demonstrated that different physi- vs. chemi-sorption processes by mutated angiogenic isoforms at the AuNP surface may offer a new strategy to modulate the angiogenesis processes in wound care [66].