Synthesis of gold nanoparticles (Au@ligand) containing thiolic compounds as Ligand: applications to therapy and molecular imaging

Gold nanoparticles (AuNPs) are the most stable metal nanoparticles, completely biocompatible, which have already proved their excellent properties for production of nanostructured materials, biomolecule detection and biological diagnostics1. The use of AuNPs as biological probes requires the improvement of colloidal stability. Thiolated species, e.g. dihydrolipoic acid (DHLA) (1)- a dithiol obtained by the reduction of thioctic (or lipoic) acid (LA) (2) - significantly limit the aggregation and help to overcome the lack of stability of gold nanoparticle colloids, conclusion proved by the doubtless results of the sulfur K-edge XANES study2. The molecules of thio-acid form on the surface of gold nanoparticles a self-assembled monolayer (SAM) with enhanced order and hence, stability and with two points of attachement to gold- sulfur atoms2. Lately, in the field of nucleosides, nucleotides and oligonucleotides, sulfur chemistry assisted by AuNPs appears particularly fruitful in the development of therapeutic agents and tools for biology. For example, AuNPs ensure efficient, considerably enhanced delivery and intracellular uptake of the drug by cancer cells, compared to the same drug in typically administered free form. Enhanced activity and nanoparticle carriers make possible the reduction of the overall concentration of the drug, renal clearance, and thus, side effects. The nanoparticles with drug (mercaptopurine) also showed excellent stability over 1 year without loss of inhibitory activity 3. A synergistic effect was observed between AuNPs and the X-ray treatment resulting in tumor reduction or eradication4. Aim of the project / Experimental approach The aims of our project are: - to synthesize gold nanoparticles (Au@ligand) with ligands containing thiol function by the technique of SAM-s in the frame of collaboration with Prof. Francesca Porta and Dr. Zelijca Krptevic (Dipartimento di Chimica Inorganica Metallorganica Analitica-University of Milan; - to select different strategies related to the application of the prepared NP-s; - to test the synthesized gold nanoparticles (Au@ligand) on the in vitro activity in cell cultures or in vivo molecular imaging. Pursuing the identification of compounds endowed with in vitro and in vivo anticancer activity, we have selected a modified nucleoside N6-(∆2-Isopentenyl)adenosine (IPA) as a drug to be delivered. As demonstrated by Spinola et al.5, IPA exerts a potent in vitro antitumoral activity on human epithelial cancer cell lines, but it is characterized by the lack of in vivo activity, that could be related to the short plasma half-life of IPA, as for other nucleosides. Following the observations of N.A.Kotov et al3, we suppose, that the conjugation of Au@thiolic ligand(LA or DHLA)-IPA would elevate plasma half-life of IPA. Thus, synthetic approach for obtaining of conjugates Au@thiolic ligand(LA or DHLA)-IPA has been built on the following: combination of LA with IPA, then -couple with Au-NP-s. For binding LA to IPA it was necessary to prepare an LA ester. Several trials have been done, with this target: 1. Lipoyl chloride, from LA and thionyl chloride – the polymerization of reaction product has been noticed; 2. Derivatives of LA: carbonate and mixed anhydride - reaction between LA and 1,1' –carbonyldiimidazole; LA-imidazolide has been obtained as a stable product in good yield. - reaction with isopropylideneglycerol as model reaction; slow reaction and low yields of product - reaction with ethyl chlorocarbonate or ethyl chloroformate?; Mixed anhydride has been obtained as a stable product in acceptable yield 3. Synthesis of LA-trifluoroethyl ester for lipase-catalyzed acylation of a nucleoside (IPA) - Reaction of LA mixed anhydride and trifluoroethanol (TFE); TFE-ester of LA has been prepared in good yield, it is an activated one that improves yield of a lipase-catalyzed transesterification with acceptor R-OH. Further experimental investigations will be focused on the study of acilation reaction lipase-catalized, between TFE-ester of LA and IPA. It could be selective an afford only the targeted 5'-ester to be coupled to AuNP-s. References 1. M.-C.Daniel, D.Astruc. Chem. Rev. 2004, 104, 293-346. 2. B.Garsia, M. Salome, L.Lemelle, J.-L.Bridot, Ph.Gillet, P.Perriat, S.Roux, O.Tillement. Chem. Commun., 2005, 369-371. 3. P.Podsiadlo, V.A.Sinani, J.H.Bahng, N.W.S.Kam, J.Lee, N.A.Kotov. Langmuir, 2008, 24, 568-574. 4. Z. Krpetic, F.Porta, G.Scari. Gold Bull., 2006, 39(2), 66-68. 5 Spinola M., Colombo F., Falvella S., Dragani T. A. Int. J. Cancer, 2007; 120, 2744