Synthesis, Functionalisation and Characterisation of Ultrasmall Gold Nanoparticles with Targeting Properties for Applications in Nanomedicine

Gold nanoparticles (GNPs) are nanomaterials with excellent physicochemical and optical properties. Ultrasmall gold nanoparticles are usually obtained through a modified Brust-Schiffrin synthesis. Further functionalisation with moieties of interest, namely for active targeting, enables wide biotechnological and clinical applications. To obtain reliable and reproducible GNPs material, a tight control of the synthesis and abroad spectrum of analytical techniques are essential. To this end, the impact of key synthesis parameters –temperature, pH and reduction time –was thoroughly studied. Post-functionalisation with cRGD or cRAD and a fluorophore Sulfo-Cy5 was performed, optimised and thoroughly characterised. The interaction of these functionalised constructs with the integrin αVβ3 was assessed and shown to be dependent on the functionalised peptide. In vitro internalisation of cRGD GNPs was much higher than that of control GNPs, in both HUVECs and HepG2. In HUVECs, internalisation of cRGD GNPs was demonstrated to occur through both clathrin and caveolin mediated endocytosis pathways, while cRAD GNPs uptake was caveolin independent. Colocalisation with lysosomes was, in some cases, lower for cRGD than cRAD GNPs. In vivo behaviour differed based on the peptide present in the GNPs, with cRGD constructs being detected at earlier time points and deeper into the liver parenchyma. The uptake of cRGD GNPs was found to be dependent on the expression levels of integrin subunits αV and β3 in neuroblastoma cellular models SK-N-AS and Kelly. Interestingly, the higher density of peptide in the GNPs surface did not correlate with higher uptake in these cells. Overall, the developed ultrasmall GNPs provide a robust platform for further studies, namely focusing on the further functionalisation with therapeutic agents.