Relazione finale : secondo anno della Scuola di Dottorato in Scienze e Tecnologie Chimiche XXI ciclo

During my 1st year of PhD studies, my research was focused on two main arguments. The first reguarded specific design and use of 15-mer peptides as stabilizing agents in the preparation of gold nanoparticles. The research in this field was pointed at the clarification of bibding sites of the peptides on the gold surface. To such purpose, different characterization methods are applied as UV-vis, ATR FTIR and NMR spectroscopy, and TEM microscopy [1]. The second reguarded the preparation of gold nanoparticles stabilized by different organic ligands (5-aminovaleric acid, adipic acid, L-DOPA, glucose, glycolic acid, dopamine, tartronic acid) and their use as nanogold bioconiugates with different cancer cells (K562-leucemia myelogenous cronica caucasica humana, PC12-pheochromocytoma). It was found the selective entrance of these NPs into cancer cells [2]. Negative control was performed on human epithelial cells where no entrance of Au NP was found even after 8 h of incubation. The particle’s presence in the cells were verified by using Confocal microscopy. As proteins linked with gold nanoparticles have demonstrated better stability and efficiency for cellular uptake, we based our studies on peptide coated gold nanoparticles as efficient synthetic ligands for both gold particle stabilization and biological application. The previous work was extended by joining two separated projects. It was investigated the intracellular uptake of differently sized spherical water soluble peptide-coated Au NP into HeLa* cells [3]. It was made the comparison between two different preparation methods for peptide-coated Au NP; direct reduction with NaBH4 in aqueous solution and the displacement of citrate ligand on the surface of Au NP). It was found that the particles prepared by using the citrate displacement method enters HeLa cells in different fashion as compared with the particles prepared by direct NaBH4 reduction method. Intracellular uptake of Au NP into HeLa cells was investigated with TEM microscopy after incubation with cells, and we observed the accumulation of nanoparticles into membrane-bound compartment inside cells known as endosomes. We showed that smaller particles (<4nm) entered cells in agglomerated form, this phenomena was described also elsewhere [4-5]. However, all the particles were found in endosomes, whether early or late endosomes, and we have verified that the mechanism of the cellular uptake of gold particles is mediated via mechanisms of receptor-mediated endocytosys, as evidenced by TEM microfraphs of ultra thin cellular sections. Gold nanoparticles were not found in the nucleus. During the uptake NPs did not showed significant changes in size before and after the uptake. For the stabilization of Au NPs following peptides were used: GC15 [H2N-GK(GGK)4-G-COOH], GK15 [H2N-GC(GGC)4-G-COOH], GCK15 [H2N-G(CGG)2-K-(GGC)2-G-COOH]. Two peptides (GC15 and GK15) were synthetised by standard solid-phase procedure on Fmoc-L-Gly Wang resin, purified by HPLC and characterized by mass spectrometry (ESI-MS). GCK15 (purity 95%) peptide was purchased by Sigma-Genosys. Two different strategies were used for the preparation of peptide-coated gold nanoparticles. By using the NaBH4 reduction method particles of small size (