GOLD NANOCONJUGATES: PREPARATION, CHARACTERISATION AND BIOLOGICAL APPLICATIONS

This PhD thesis is dedicated to the preparation, characterisation and biological application of gold nanoconjugates. Gold nanoparticles are prepared by various modern preparation methods, and subsequently characterised by spectroscopic (UV-vis, ATR-FTIR, NMR), microscopic (TEM) techniques. The stability of the conjugates is evaluated both by using Zeta-potential studies and UV-vis. The nanoparticles are used in cellular uptake experiments using human glioblastoma cancer cells, and are found to possess a low cytotoxicity. The nanoparticles are found taken up by the cells and distributed in different cellular compartments

Gold nanocages for imaging and therapy of prostate cancer cells

Gold nanocages (AuNCs) have been shown to be a useful tool both for imaging and hyperthermia therapy of cancer, thanks to their outstanding optical properties, low toxicity and facile functionalization with targeting molecules, including peptides and antibodies. In particular, hyperthermia is a minimally invasive therapy which takes advantage of the peculiar properties of gold nanoparticles to efficiently convert the absorbed light into heat. Here, we use AuNCs for the selective targeting and imaging of prostate cancer cells. Moreover, we report the hyperthermic effect characterization of the AuNCs both in solution and internalized in cells. Prostate cancer cells were irradiated at different exposure times, with a pulsed near infrared laser, and the cellular viability was evaluated by confocal microscopy

Theranostic nanocages for imaging and photothermal therapy of prostate cancer cells by active targeting of neuropeptide-Y receptor

Gold nanocages (AuNCs) have been shown to be a useful tool for imaging and hyperthermia therapy of cancer, thanks to their unique optical properties, low toxicity and facile surface functionalization. Herein, we use AuNCs for selective targeting of prostate cancer cells (PC3) via specific interaction between neuropeptide Y (NPY) receptor and three different NPY analogs conjugated to AuNCs (Figure 1). Localized surface plasmon band of the nanoconjugates was set around 800 nm, which is particularly promising for in vivo applications. Long-term stability of nanoconjugates in different media was confirmed by UV-vis and DLS studies. Active NPY receptor targeting was observed by confocal microscopy showing time-dependent AuNCs cellular uptake. Activation of ERK1/2 pathway was evaluated by Western blot to confirm the receptor-mediated specific interaction with PC3. Cellular uptake kinetics were compared as a function of peptide structure. Cytotoxicity of nanoconjugates was evaluated by MTS and Annexin V assays, confirming their safety within the concentration range explored. Hyperthermia studies were carried out irradiating the cells, previously incubated with AuNCs, with a pulsed laser at 808 nm wavelength, showing a heating enhancement from 6 to 35 \ub0C above the culture temperature dependent on the irradiation power (between 1.6 and 12.7 W/cm2). Only cells treated with AuNCs underwent morphological alterations in the cytoskeleton structure upon laser irradiation, leading to membrane blebbing and loss of microvilli associated to cell migration. This effect is particularly promising in view of possible inhibition of proliferation and invasion of cancer cells. In summary, our Au-peptide NCs proved to be an efficient theranostic nanosystem for targeted detection and activatable killing of prostate cancer cells

Modular assembly of proteins on nanoparticles

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold–sulfur bonds (Au–S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond

Study of chemically peculiar stars – I. High-resolution spectroscopy and K2 photometry of Am stars in the region of M44

ABSTRACT We present a study based on the high-resolution spectroscopy and K2 space photometry of five chemically peculiar stars in the region of the open cluster M44. The analysis of the high-precision photometric K2 data reveals that the light variations in HD 73045 and HD 76310 are rotational in nature and caused by spots or cloud-like co-rotating structures, which are non-stationary and short-lived. The time-resolved radial velocity measurements, in combination with the K2 photometry, confirm that HD 73045 does not show any periodic variability on time-scales shorter than 1.3 d, contrary to previous reports in the literature. In addition to these new rotational variables, we discovered a new heartbeat system, HD 73619, where no pulsational signatures are seen. The spectroscopic and spectropolarimetric analyses indicate that HD 73619 belongs to the peculiar Am class, with either a weak or no magnetic field, considering the 200-G detection limit of our study. The least-squares deconvolution profiles for HD 76310 indicate a complex structure in its spectra, suggesting that this star is either part of a binary system or surrounded by a cloud shell. When placed in the Hertzsprung–Russell diagram, all studied stars are evolved from the main sequence and situated in the δ Scuti instability strip. This work is relevant for further detailed studies of chemically peculiar stars, for example on inhomogeneities (including spots) in the absence of magnetic fields and the origin of the pulsational variability in heartbeat systems

Au-thymine, thymidine and thymidine 5\u2019-monophosphate nanoparticles: chemical characterisation and cellular uptake studies into U87 cancer cells

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)2 peptide as stabiliser. As comparison, the behaviour of the (GC)2 peptide has been studied. The (GC)2 and RGD-(GC)2 peptides were prepared ad hoc by Fmoc syntheses. The colloidal systems have been characterised by UV-visible, ATR-FTIR, mono and bidimensional NMR techniques and Confocal and TEM microscopies. The efficient cellular uptake of Au-RGD-(GC)2 and Au-(GC)2 stabilised gold particles into U87 cells (human glioblastoma cell), were investigated by confocal microscopy and compared with the behaviour of the (GC)2 capped gold nanoparticles. A quantitative determination of the uptaken nanoparticles have been carried out by measuring the pixel brightness of the images that highlighted the importance of the RGD termination of the peptide. A deep insight of the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)2 gold nanoparticles into the nucleus

Gold nanoparticles stabilised by nucleobases, nucleosides and nucleotides

Gold nanoparticles stabilized by nucleobases, nucleosides and nucleotides S. Avvakumova, Milano/I, F. Porta, Milano/I, G. Scari, Milano/I Dr. Svetlana Avvakumova, University of Milan, Dip. Chimica Inorganica Metall. Anal., L. Malatesta, Via Venezian 21, 20133 Milano, Italy Today gold nanoparticles (AuNPs) have become an important biomedical tool in cancer research. The presence of a negative charge on the surface of AuNPs helps to modify the surface of AuNPs by binding with several biomolecules. It is well established that capped AuNPs are biocompatible and non-toxic allowing their use in cancer treatment [1]. Nucleobases, nucleosides and nucleotides, used in this work, were among the first chemotherapeutic agents introduced for the medical treatment of cancer. These agents behave as antimetabolites and interact with a large number of intracellular targets. Biocompatible gold nanoparticles have been synthesized by reduction of NaAuCl4 aqueous solution by sodium borohydride as reducing agent. Adenine, adenosine, guanine, guanosine, cytosine, cytidine, thymine, guanosine 5\u2019- monophosphate and cytidine 5\u2019-monophosphate were used as stabilising agents. Au:stabiliser:NaBH4 molar ratios have been set up for obtaining red stable sols constituted by spherical gold nanoparticles with mean diameters in the 2-10 nm range. Reaction course was followed by UV-vis spectroscopy for studying the kinetic of reaction and plasmon peaks were found in the range of 513-530 nm. The colloids obtained were characterized by spectroscopic, microscopic and crystallographic methods (1H NMR, ATR-FTIR, XRD and TEM) studying the ligand interaction with the surface of gold nanoparticles. ATR-FTIR and 1H-NMR studies highlighted that the binding of ligands with the metallic surface dipends on the structure of molecules (nucleobases, nucleosides or nucleotides). Literature: [1] C. M. Galmarini, J. R. Mackey, C. Dumontet, The Lancet Oncology, 2002, Vol 3, p. 415