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 (<5nm) were obtained, and these particles were purified by dialysis for 48h. By using the citrate displacement method larger particles (14-16 nm) were obtained, and these particles were purified by centrifugation. In this case it was important to establish the quantity of the peptide for each gold citrate particle, and in our case it was calculated 500 peptide molecules for the stabilization of each gold particle. The adjustment of pH value before purification to 10 was necessary to prevent the agglomeration of the particles. The particles were then incubated with HeLa cells at 37°C and 5% CO2 flow for 1h and the samples were then processed by a number of necessary steps (fixation, post fixation, staining, dexydration, embedding in epoxy resin, polymerization, ultra thin cutting and mounting on TEM grids) in order to obtain 70 nm thick sections cutted with the ultra microtrome suitable for the TEM observations. Current studies in this research area have been focused on coating biorecognition molecules on the surface of NPs to mediate cellular accumulation in different cell compartments. In bionanotechnology it is very important to have stabilizers, which could be easily functionalized with other biologically important ligands. GCK15 peptide has been designed expressely in order to obtain further bio-functionalization of the peptide chain by biotin molecule. Having a free NH2 group due to the presence of a lysine in the middle of the 15-merpeptide chains it was easy to attach the biotin molecule by using a one-step biotinylation reaction. Firstly, the particles stabilized by GCK15 molecule were prepared (by citrate displacement preparation method), and then biotin label was attached to purified particles. The reaction of biotinylation was performed by using the biotin labeling agent, N-hydroxy sulfosuccinimidobiotin (Sulfo-NHS-Biotin). The reaction was held on room temperature by mixing 300 μl of GCK15-coated gold nanoparticles in PBS and 20 μl of Sulfo-NHS-Biotin (50 mM) in DMSO. The excess of the biotin label was set up to be at least 1000 times more than lysine molecules present in the peptide. After the reaction, the excess of biotin ligand was removed by centrifugation. By reaction of Sulfo-NHS-Biotin reagent, biotin molecule was selectively attached on the amine group of lysine present in the peptide chain. It is known from literature [6] that α-amino group is less available to biotinylation, therefore we can assume that only ε-amino groups of lysine in the side peptide chain are biotinylated. The presence of biotin molecule was detected by both sptreptavidin-agarose beads assay and the dot blot biodetection reaction for biotin. Particles having biotin ligand attached on the peptide chain coloured streptavidin beads in red, while particles having a non biotinated GCK15 peptide ligand do not show any coloration of streptavidin beads, considering very specific streptavidin-biotin interaction. Particles stabilized with GC15, GK15 and GCK15 peptides prepared in two different preparation procedures were successfully uptaken into HeLa cells. Also biotinylated GCK15 Au NP were present in HeLa cells endosomes. Considering the large number of TEM micrographs obtained, it was deduced the mechanism of the uptake of peptide-coated Au NP as receptor mediated endocytosis. It is possible to observe almost all the steps of this mechanism: 1. Specific adsorption of Au NP on the cell membrane 2. Specific recognition of AuNP by receptors in the cell’s membrane 3. Invagination of cell membrane with formation of a membrane-bound compartments known as endosome 4. Formed endosomes carrying Au NPs present in cell’s cytoplasm If the uptake mechanism of Au NPs is endocytosis it is expected their exit via exocytosis. This phenomenon would restrain their leftover time in cells, and consequently the toxicity for the organism. References: 1. Porta, F., Speranza, G., Krpetic, Z., Dal Santo, V., Francescato, P., Scari, G., Materials Science and Engineering B, 140, 2007, 187 2. Krpetic, Z., Porta, F., Scarì, G., Gold Bulletin, 39, 2006, 66 3. a) E. Boatman et.al., Cell Tiss. Res., 1976,170,1, b) http://www.microbiologybytes.com, c) http://en.wikipedia.org/wiki/HeLa 4. Chitrani, B.D., Ghazani, A.A., Chan, C.W., Nano Letters, 6, 2006, 662 5. Chitrani, B.D., Chan, C.W., Nano Letters, 7, 2007, 1542 6. Smith, J.S., Miller,B.T., Kurosky, A., Analytical Biochemistry, 197, 1991, 254 ____________________________ * HeLa cells: human epithelial cells from a fatal cervical carcinoma (taken from Henrietta Lacks, who died from her cancer in 1951) transformed by human papillomavirus 18 (HPV18), classic example of an immortalized cell line used in medical research. [3]