Luminescent and Photo-Responsive Gold Nanoparticles for Bio-Applications

In thesis work it has been given an overview of the most recent advances in the synthesis, characterization and bio-medical application of luminescent Gold nanospecies. It has been shown that the combination of photoactuators and nanostructures results in an apparent reduction of dyes PI QYs, due to the nanomaterials absorbance contribution, also in absence of actual interactions between the two components. It has been proposed a simple approach to take into consideration this contribution and to perform quantitative measurements of dyes PI efficiencies variations in presence of nanoparticles. The PI QY reduction has been found to be qualitatively proportional to the magnitude of the interaction between the dye and the particles measured by NMR. It has been shown that strong interaction between the two components result in significant decreases of the dye PI QY. The photoisomerization effectively determines a change in the repartition of the AB derivative between particle monolayer and solution. This confirms that photoisomerization reaction can probably be exploited for light triggered drug release. These results provided valuable tools and methodologies which improved the state of the art in the quantitative study of light triggered processes in nanoparticles based systems. For the first time it has been proven that GNPs luminescence can be switched on and off thanks to the photoisomerization of an azobenzene derivative covalently bound on the Gold surface. This has been proven to happen thanks to an efficient energy transfer between the trans AB derivative and the GNPs which leads to a sensitized NIR emission whose intensity can be tuned by photoisomerization. These unprecedented results are promising for the development of Gold based light responsive nanoparticles for theranostics. Finally, starting from Au25(Cys)18, it has been successfully developed a complex probe for luminescence based imaging composed by NIR emitting GNCs encapsulated inside mesoporous PEGylated silica NPs

C60bioconjugation with proteins: Towards a palette of carriers for all pH ranges

The high hydrophobicity of fullerenes and the resulting formation of aggregates in aqueous solutions hamper the possibility of their exploitation in many technological applications. Noncovalent bioconjugation of fullerenes with proteins is an emerging approach for their dispersion in aqueous media. Contrary to covalent functionalization, bioconjugation preserves the physicochemical properties of the carbon nanostructure. The unique photophysical and photochemical properties of fullerenes are then fully accessible for applications in nanomedicine, sensoristic, biocatalysis and materials science fields. However, proteins are not universal carriers. Their stability depends on the biological conditions for which they have evolved. Here we present two model systems based on pepsin and trypsin. These proteins have opposite net charge at physiological pH. They recognize and disperse C60in water. UV-Vis spectroscopy, zeta-potential and atomic force microscopy analysis demonstrates that the hybrids are well dispersed and stable in a wide range of pH's and ionic strengths. A previously validated modelling approach identifies the protein-binding pocket involved in the interaction with C60. Computational predictions, combined with experimental investigations, provide powerful tools to design tailor-made C60@proteins bioconjugates for specific applications

Comparison of Adaptive Neuroprotective Mechanisms of Sulforaphane and its Interconversion Product Erucin in in Vitro and in Vivo Models of Parkinson's Disease

Several studies suggest that an increase of glutathione (GSH) through activation of the transcriptional nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the dopaminergic neurons may be a promising neuroprotective strategy in Parkinson's disease (PD). Among Nrf2 activators, isothiocyanate sulforaphane (SFN), derived from precursor glucosinolate present in Brassica vegetables, has gained attention as a potential neuroprotective compound. Bioavailability studies also suggest the contribution of SFN metabolites, including erucin (ERN), to the neuroprotective effects of SFN. Therefore, we compared the in vitro neuroprotective effects of SFN and ERN at the same dose level (5 \u3bcM) and oxidative treatment with 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells. The pretreatment of SH-SY5Y cells with SFN recorded a higher (p < 0.05) active nuclear Nrf2 protein (12.0 \ub1 0.4 vs 8.0 \ub1 0.2 fold increase), mRNA Nrf2 (2.0 \ub1 0.3 vs 1.4 \ub1 0.1 fold increase), total GSH (384.0 \ub1 9.0 vs 256.0 \ub1 8.0 \u3bcM) levels, and resistance to neuronal apoptosis elicited by 6-OHDA compared to ERN. By contrast, the simultaneous treatment of SH-SY5Y cells with either SFN or ERN and 6-OHDA recorded similar neuroprotective effects with both the isothiocyanates (Nrf2 protein 2.2 \ub1 0.2 vs 2.1 \ub1 0.1 and mRNA Nrf2 2.1 \ub1 0.3 vs 1.9 \ub1 0.2 fold increase; total GSH 384.0 \ub1 4.8 vs 352.0 \ub1 6.4 \u3bcM). Finally, in vitro finding was confirmed in a 6-OHDA-PD mouse model. The metabolic oxidation of ERN to SFN could account for their similar neuroprotective effects in vivo, raising the possibility of using vegetables containing a precursor of ERN for systemic antioxidant benefits in a similar manner to SFN

Enhanced Uptake and Phototoxicity of C60@albumin Hybrids by Folate Bioconjugation

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C-60@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C-60 molecules in a physiological environment, (ii) HSA, upon C-60 binding, maintains its biological identity and biocompatibility, (iii) the C-60@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C-60@HSA complex in HeLa cells

Self-Assembled Biocompatible Fluorescent Nanoparticles for Bioimaging

Fluorescence is a powerful tool for mapping biological events in real-time with high spatial resolution. Ultra-bright probes are needed in order to achieve high sensitivity: these probes are typically obtained by gathering a huge number of fluorophores in a single nanoparticle (NP). Unfortunately this assembly produces quenching of the fluorescence because of short-range intermolecular interactions. Here we demonstrate that rational structural modification of a well-known molecular fluorophore N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD) produces fluorophores that self-assemble in nanoparticles in the biocompatible environment without any dramatic decrease of the fluorescence quantum yield. Most importantly, the resulting NP show, in an aqueous environment, a brightness which is more than six orders of magnitude higher than the molecular component in the organic solvent. Moreover, the NP are prepared by nanoprecipitation and they are stabilized only via non-covalent interaction, they are surprisingly stable and can be observed as individual bright spots freely diffusing in solution at a concentration as low as 1 nM. The suitability of the NP as biocompatible fluorescent probes was demonstrated in the case of HeLa cells by fluorescence confocal microscopy and MTS assays

A modular phage vector platform for targeted photodynamic therapy of Gram-negative bacterial pathogens

: Growing antibiotic resistance has encouraged the revival of phage-inspired antimicrobial approaches. On the other hand, photodynamic therapy (PDT) is considered a very promising research domain for the protection against infectious diseases. Yet, very few efforts have been made to combine the advantages of both approaches in a modular, retargetable platform. Here, we foster the M13 bacteriophage as a multifunctional scaffold, enabling the selective photodynamic killing of bacteria. We took advantage of the well-defined molecular biology of M13 to functionalize its capsid with hundreds of photo-activable Rose Bengal sensitizers and contemporarily target this light-triggerable nanobot to specific bacterial species by phage display of peptide targeting moieties fused to the minor coat protein pIII of the phage. Upon light irradiation of the specimen, the targeted killing of diverse Gram(-) pathogens occurred at subnanomolar concentrations of the phage vector. Our findings contribute to the development of antimicrobials based on targeted and triggerable phage-based nanobiotherapeutics

Evidence of iguanodontian dinosaurs from the Lower Cretaceous of Tunisia

The fossil record of ornithischian dinosaurs from Africa is particularly scarce and limited to a few historic localities. In this study we describe new ornithischian remains from the Albian deposits of southern Tunisia (Tataouine Governorate), represented by isolated teeth of large-bodied iguanodontians. Teeth display a wide, diamond-shaped crown with a primary ridge dividing the occlusal surface in two unequal parts and two or more secondary ridges. Hook-like denticles are present on both mesial and distal crown margins and do not display mammillae. In overall morphology, specimens are comparable to those of many Early Cretaceous basal hadrosauriforms, including isolated ornithopodan teeth from comparably-aged levels of Niger. Transversal sections of the crowns permitted identification of dental tissues, which include a thick enamel, and well developed longitudinal and transverse giant tubules. Their relative extents appear to be related to the size, thus developmental age, of the tooth. Teeth are representative of the Oum ed Diab Member, a unit characterized by coastal deposits accumulated under arid to xeric climatic conditions and dominated by fish, crocodilians, and hydraulically transported rebbachisaurid and spinosaurid remains. Sedimentological data and preservation bias strongly support selective taphonomic causes for the fossil distribution of ornithischians in southern Tunisia questioning the purported geographic and paleoecologic distribution of isolated Saharan dinosaurs

Photo-tunable multicolour fluorescence imaging based on self-assembled fluorogenic nanoparticles

Non-fluorescent nanoparticles resulting from the self-assembly of a new perylene diimide behave as fluorogenic probes for biological cells under physiological conditions giving a dosage-dependent green or red fluorescence and showing very low cytotoxicity. The emission colour can be tuned by photo-irradiation to achieve multicolour labelling

Associazione tra un isotiocianato e levodopa per il trattamento della malattia di Parkinson

Si descrive una combinazione comprendente levodopa e un estratto vegetale ottenuto da una pianta della famiglia delle Cruciferae o del genere Brassica. Tale combinazione è utile come medicamento, in particolare per il trattamento del morbo di Parkinson e per il trattamento del fenomeno detto "on-off" e/o del fenomeno detto "wearing-off"