Relazione finale : primo anno della Scuola di Dottorato in Scienze e Tecnologie Chimiche

The research carried out in the 1st year of my PhD program was focused on two main arguments: A) Preparation and interaction studies between gold nanoparticles and peptides. Two dipeptides and two 15-merpeptides were used as capping agents in the preparation of monolayer-protected gold clusters. MPCs were characterized by TEM microscopy, UV-visible, NMR and ATR FTIR spectroscopy. B) Nanogold bioconiugates. Selective entrance into cancer cells of Au(0) nanoparticles stabilized by several organic ligands was studied. Part A The formation of Au-Peptide nanoparticles was obtained at room temperature in aqueous solution in agreement with the reaction (1) and Au to peptide weight ratios in the 1-10 range. Reddish colloid was immediately formed by reduction of the tetrachloroaurate sodium salt with NaBH4. The sol was purified by cycles of centrifugation and re-dispersion, in order to remove uncoordinated peptide. The reaction was followed by UV-vis spectroscopy observing the formation of plasmon resonance peaks indicative of Au(0) nanoparticles, and TEM microscopy for the size and morphology evaluation of the particles (table 1). The sols were lyophilised and the obtained powders were characterized by ATR FTIR spectroscopy. NMR spectra were recorded after the re-dispersion of lyophilised powder in H2O:D2O (9:1 volume ratio). Two 15-merpeptides were used as gold capping agents. One, H2N-GC(GGC)4-G-COOH (GC15), is composed of 10 glycine and 5 cysteine residues, the other H2N-GK(GGK)4-G-COOH (GK15) is composed of 10 glycine and 5 lysine residues (figure 1). Both peptides, having periodic sequences, were planned to allow a parallel binding to gold surface [1]. To achieve the target, GC15 bears several potential anchor groups like SH or NH2 that can covalently bind gold particle [2] and GK15 contains primary amines that can bind gold surface in different ways depending on pH of the sol and pI of the peptide [3]. Peptides were synthesized by a standard solid-phase procedure, purified by HPLC and characterized by mass spectrometry (ESI-MS). As model compounds two dipeptides were prepared, H2N-GC-COOH (GC) and H2N-GK-COOH (GK) (figure 2) by a coupling reaction in solution (figure 3). The optical and morphological characteristics of the obtained Au-dipeptide red sols are shown in table 1. Both Au-dipeptide samples were lyophilised and stored as dry powders. They were found re-dispersible in water. NMR spectrosopy was revealed as very useful for the characterization in aqueous solution. This is an important result since very few systems can be stored in dry state and then redispersed in water [4]. In Au-GC nanoparticle the binding site of dipeptide is the SH cysteine group, as evidenced by a strongly downfield shift of the CH2SH signal and an upfield shift of the amide NH proton. In Au-GK nanoparticle, the terminal amine group of glycine is responsible for an interaction between gold particle and dipeptide, showing a significant downfield shift and broadening of the signal due to the presence of CH2 of glycine. The reaction between Au(III) salt and BH4- in the presence of 15-merpeptides was carried out at pH 3. In acidic conditions the amine groups are protonated and the –COOH moieties undissociated. Thus the presence of eventual zwitterionic forms is limited, favouring an electrostatic binding to gold surface [5]. ATR FTIR spectra of pure GK15 peptide (Au/GK15= 1/10 w/w) show two bands in the amide A region, while in the lyophilised sol a new amide band, located at 3205 cm-1, arise. Considering also the amide I and II regions we can state the formation of GK15-capped gold nanoparticles. In particular the band close to 3200 cm-1 found in Au-GK15 (and in Au-GC15, see later) falls between amide A and B typical regions. It could be accounted for very strong H-bond interaction involving amide groups due to intra-monolayer interaction of the MPCs [6] and/or the presence of NH3+ cation(s). The 1H NMR spectra of the re-dispersed lyophilised sols did not show any peculiar differences in the chemical shifts of protons passing from of GK15 to Au-GK15. A broadening of the ε-CH2 lysine side chain signal strongly suggests the interaction of amine group of lysine(s) with gold surface. Moreover a broad signal at 7.5 ppm is indicative of NH3+ lysine groups (and glycine too). The combination of IR and NMR information strongly supports the presence of electrostatic bindings between the protonated NH2 group(s) of lysine and gold surface. A cherry red monodispersed sol (Au-GC15) was obtained by reduction of Au(III) salt in the presence of peptide (Au/GC15= 1/0.5 w/w). UV-vis spectra shows the plasmon peak at 514 nm related to the mean diameters of particles (by TEM) ranging in the 7.0-7.5 nm interval. Unlikely, the lyophilised Au-GC15 sol was not water-dispersible, thus NMR in D2O spectra are lacking. Further studies in DMSO-d6 and CD2Cl2 are in progress. For a better investigation of the interaction between gold and GC15 a different preparative procedure was applied. By adding suitable amounts of the lyophilised GC15 to a citrate-capped gold sol (Au/GC15=1:1 w/w) we obtained a bordeaux Au-citrate-GC15 colloid constituted by spherical nanoparticles (d =15-17 nm). In addition, the formation of nanowire and pearl-necklace structures was observed highlighting mono and bidimensional aggregation [7]. ATR and 1H NMR spectra revealed the presence of both the citrate and the GC15 on gold surface; in particular 1H NMR confirmed the presence of these two groups suggesting that only small amounts of the citrate have been exchanged by GC15. However, NMR spectra revealed light differences between pure GC15 and Au-GC15. This confirms the feeble interaction (or at least not clear in this experimental condition) between SH group and gold surface in agreement with IR data. Further studies will be carried out in order to investigate the binding site(s) of GC15. Part B The entrance ability of differently capped gold sols into cancer cells was studied and compared with wild sane ones, in order to verify the selectivity. To achieve this it was important to choose ligand molecules, in order to preserve biocompatibility of the metallic system, by using natural and non-toxic organic molecules. Our research was stimulated by the study of Hainfield et al. [8-11] in which mice were firstly injected with cancer cells and gold nanoparticles, then treated with X-ray radiation at 250 kV. A synergistic effect was observed between gold nanoparticles and the X-ray treatment resulting in tumour reduction or eradication. Gold nanoparticles were capped by 5-aminovaleric acid [H2N(CH2)4COOH], adipic acid [HOOC(CH2)4COOH], 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA; C9H11NO4), glucose (C6H12O6),glycolic acid (HO-CH2-COOH),2-(3,4-Dihydroxyphenyl)ethylamine hydrochloride (Dopamine; C8H11NO2. HCl) and tartronic acid (HOOC-CH(OH)-COOH). The gold sols were prepared by reduction with NaBH4 of an auric precursor in the presence of capping ligand at pH7, purified and subsequently lyophilised. The characterization of gold nanoparticles was carried out by UV-vis, ATR FTIR and NMR spectroscopies, and by TEM microscopy. TEM images revealed that the red sols are constituted by monodispersed nanoparticles of 3-6 nm mean diameter. In the biological experiments the lyophilised powders were re-dispersed into aqueous D-MEM cell culture medium. The Gold-DMEM system was admixed with two different kinds of cancer cells, K562 and PC12 (Leucemia myelogenous cronica caucasica umana and pheochromocytoma). For the control, human wild type epithelial cells were exposed at the same conditions. The cultured cells were incubated for different periods of time (from 15 minutes to 8 hours) at 37°C in 5% CO2 flow in order to maintain physiological conditions. Observations were made using confocal microscopy in reflectance and transmission modes. In figure 4 the interaction between Au-5-aminovaleric acid and K562 cancer cells can be observed. In figure 4a the gold aggregates dispersed in the cell culture medium are shown highlighting the green colour of gold particles in accordance with its self-fluorescence at 488 nm. The experiments revealed that gold sols can penetrate into the cancer cell after a very short time of the incubation, from 5 to 15 minutes (figures 4b,c). The total uptake of gold into the cancer cell took place after 1 h figure 4f. The confocal microscopy cross section images are shown in figure 4e in order to reconstruct the 3D image of the Cell-gold system in which we can see that nanoparticles are located inside of the cell cytoplasm and not in the nucleus, suggesting that the cell is still able to keep its metabolism unchanged. In figure 5 can be observed the interaction between PC12 cancer cells with differently capped gold nanoparticles. Moreover, the transmission (a) and reflexion (b) visualization modes are reported as the image from the superimposed sections (c). A negative control experiments confirmed selectivity of the synthesized gold sols towards cancer and not towards wild sane human cells. The main goals of these gold nano systems are represented by: a) improvement in the therapy as gold particles are admitted into the cell by recognition instead of injection; b) selective toxicity towards the cancer cells (high) and wild sane cells (negative); c) preveservation of the metabolism in the case of wild sane cell. References 1. G. P. Drobny et al., Langmuir, 21 (2005) 3002 2. M. Brust et al., J. Am. Chem. Soc., 126 (2004) 10076 3. Z. Zhong et al., Chem. Eur. J., 11 (2005) 1473 4. Feldheim D.L., Foss, C.A., in Metal Nanoparticles: Synthesis, Characterization, and Application, Marcel Dekker, New York. (2002) 5. a) Bellino M.G., Calvo E.J., Gordillo G., Phys. Chem.Chem.Phys., 6 (2004) 424-428 ; b) Joshi H., Shirude P.S., Bansal V., Ganesh K.N., Sastry M., J.Phys.Chem.B,108 (2004) 11535-11540 6. a) Boal A.K., Rotello V.M., Langmuir, 16 (2000) 9527-9532; b) L. Fabris, S. Antonello, L. Armelao, R.L. Donkers, F. Polo, C. Toniolo, F. Maran, J. Am. Chem. Soc., 128 (2006) 326-336 7. Zhong Z, Luo J., Ang T.P., Highfield J., Lin J., Gedanken A., J. Phys. Chem. B, 108 (2004)18119-18123 8. J.F. Hainfeld, D.N. Slatkin, H.M. Smilowitz, Phys. Med. Biol., 2004,49, N309 9. D.M. Herold, I.J. Das, C.C. Strobbe, R.V. Iyer, J.D. Chapman, Int. J.Radiat. Biol., 2000, 76, 1357 10. H. Matsudaira, A.M. Ueno, I. Furuno, Radiat. Res., 1980, 84, 144 11. J.H. Rose, A. Norman, M. Ingram, C. Aoki, T. Solberg, A. Mesa, Int. J. Radiat. Oncol. Biol. Phys., 1999, 45, 1127