Evidenze molecolari del ruolo antiproliferativo e pro-apoptotico della clusterina in cellule epiteliali prostatiche umane

The Clusterin (CLU) gene produces different forms of protein products which vary in their biological properties and distribution within the cell. Both the extra- and intracellular CLU forms regulate cell proliferation and apoptosis. Dis-regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over-expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC-3 prostate cancer cells. Following siRNA, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e. H3 mRNA, PCNA and cyclins A, B1 and D) as detected by RT-qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half-life is less than 2 hours. All CLU protein products were found poly-ubiquitinated by co-immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, strongly inducing the nuclear form of CLU (nCLU) and committing cells to caspase-dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumour suppressor factor.

Design and Characterization of New Luminescent Sensors and Labels

The aim of this Ph.D. project has been the design and characterization of new and more efficient luminescent tools, in particular sensors and labels, for analytical chemistry, medical diagnostics and imaging. Actually both the increasing temporal and spatial resolutions that are demanded by those branches, coupled to a sensitivity that is required to reach the single molecule resolution, can be provided by the wide range of techniques based on luminescence spectroscopy. As far as the development of new chemical sensors is concerned, as chemists we were interested in the preparation of new, efficient, sensing materials. In this context, we kept developing new molecular chemosensors, by exploiting the supramolecular approach, for different classes of analytes. In particular we studied a family of luminescent tetrapodal-hosts based on aminopyridinium units with pyrenyl groups for the detection of anions. These systems exhibited noticeable changes in the photophysical properties, depending on the nature of the anion; in particular, addition of chloride resulted in a conformational change, giving an initial increase in excimeric emission. A good selectivity for dicarboxylic acid was also found. In the search for higher sensitivities, we moved our attention also to systems able to perform amplification effects. In this context we described the metal ion binding properties of three photoactive poly-(arylene ethynylene) co-polymers with different complexing units and we highlighted, for one of them, a ten-fold amplification of the response in case of addition of Zn2+, Cu2+ and Hg2+ ions. In addition, we were able to demonstrate the formation of complexes with Yb3+ an Er3+ and an efficient sensitization of their typical metal centered NIR emission upon excitation of the polymer structure, this feature being of particular interest for their possible applications in optical imaging and in optical amplification for telecommunication purposes. An amplification effect was also observed during this research in silica nanoparticles derivatized with a suitable zinc probe. In this case we were able to prove, for the first time, that nanoparticles can work as “off-on” chemosensors with signal amplification. Fluorescent silica nanoparticles can be thus seen as innovative multicomponent systems in which the organization of photophysically active units gives rise to fruitful collective effects. These precious effects can be exploited for biological imaging, medical diagnostic and therapeutics, as evidenced also by some results reported in this thesis. In particular, the observed amplification effect has been obtained thanks to a suitable organization of molecular probe units onto the surface of the nanoparticles. In the effort of reaching a deeper inside in the mechanisms which lead to the final amplification effects, we also attempted to find a correlation between the synthetic route and the final organization of the active molecules in the silica network, and thus with those mutual interactions between one another which result in the emerging, collective behavior, responsible for the desired signal amplification. In this context, we firstly investigated the process of formation of silica nanoparticles doped with pyrene derivative and we showed that the dyes are not uniformly dispersed inside the silica matrix; thus, core-shell structures can be formed spontaneously in a one step synthesis. Moreover, as far as the design of new labels is concerned, we reported a new synthetic approach to obtain a class of robust, biocompatible silica core-shell nanoparticles able to show a long-term stability. Taking advantage of this new approach we also showed the synthesis and photophysical properties of core-shell NIR absorbing and emitting materials that proved to be very valuable for in-vivo imaging. In general, the dye doped silica nanoparticles prepared in the framework of this project can conjugate unique properties, such as a very high brightness, due to the possibility to include many fluorophores per nanoparticle, high stability, because of the shielding effect of the silica matrix, and, to date, no toxicity, with a simple and low-cost preparation. All these features make these nanostructures suitable to reach the low detection limits that are nowadays required for effective clinical and environmental applications, fulfilling in this way the initial expectations of this research project.

Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering

Tissue engineering is a discipline that aims at regenerating damaged biological tissues by using a cell-construct engineered in vitro made of cells grown into a porous 3D scaffold. The role of the scaffold is to guide cell growth and differentiation by acting as a bioresorbable temporary substrate that will be eventually replaced by new tissue produced by cells. As a matter or fact, the obtainment of a successful engineered tissue requires a multidisciplinary approach that must integrate the basic principles of biology, engineering and material science. The present Ph.D. thesis aimed at developing and characterizing innovative polymeric bioresorbable scaffolds made of hydrolysable polyesters. The potentialities of both commercial polyesters (i.e. poly-e-caprolactone, polylactide and some lactide copolymers) and of non-commercial polyesters (i.e. poly-w-pentadecalactone and some of its copolymers) were explored and discussed. Two techniques were employed to fabricate scaffolds: supercritical carbon dioxide (scCO2) foaming and electrospinning (ES). The former is a powerful technology that enables to produce 3D microporous foams by avoiding the use of solvents that can be toxic to mammalian cells. The scCO2 process, which is commonly applied to amorphous polymers, was successfully modified to foam a highly crystalline poly(w-pentadecalactone-co-e-caprolactone) copolymer and the effect of process parameters on scaffold morphology and thermo-mechanical properties was investigated. In the course of the present research activity, sub-micrometric fibrous non-woven meshes were produced using ES technology. Electrospun materials are considered highly promising scaffolds because they resemble the 3D organization of native extra cellular matrix. A careful control of process parameters allowed to fabricate defect-free fibres with diameters ranging from hundreds of nanometers to several microns, having either smooth or porous surface. Moreover, versatility of ES technology enabled to produce electrospun scaffolds from different polyesters as well as “composite” non-woven meshes by concomitantly electrospinning different fibres in terms of both fibre morphology and polymer material. The 3D-architecture of the electrospun scaffolds fabricated in this research was controlled in terms of mutual fibre orientation by properly modifying the instrumental apparatus. This aspect is particularly interesting since the micro/nano-architecture of the scaffold is known to affect cell behaviour. Since last generation scaffolds are expected to induce specific cell response, the present research activity also explored the possibility to produce electrospun scaffolds bioactive towards cells. Bio-functionalized substrates were obtained by loading polymer fibres with growth factors (i.e. biomolecules that elicit specific cell behaviour) and it was demonstrated that, despite the high voltages applied during electrospinning, the growth factor retains its biological activity once released from the fibres upon contact with cell culture medium. A second fuctionalization approach aiming, at a final stage, at controlling cell adhesion on electrospun scaffolds, consisted in covering fibre surface with highly hydrophilic polymer brushes of glycerol monomethacrylate synthesized by Atom Transfer Radical Polymerization. Future investigations are going to exploit the hydroxyl groups of the polymer brushes for functionalizing the fibre surface with desired biomolecules. Electrospun scaffolds were employed in cell culture experiments performed in collaboration with biochemical laboratories aimed at evaluating the biocompatibility of new electrospun polymers and at investigating the effect of fibre orientation on cell behaviour. Moreover, at a preliminary stage, electrospun scaffolds were also cultured with tumour mammalian cells for developing in vitro tumour models aimed at better understanding the role of natural ECM on tumour malignity in vivo.

Biomimetic Scaffolds for the Controlled Release of Bioactive Molecules for Tissue Engineering Applications

The temporospatial controlled delivery of growth factors is crucial to trigger the desired healing mechanisms in target tissues. The uncontrolled release of growth factors has been demonstrated to cause severe side effects in its surrounding tissues. Thus, the first working hypothesis was to tune and optimize a newly developed multiscale delivery platform based on a nanostructured silicon particle core (pSi) and a poly (dl-lactide-co-glycolide) acid (PLGA) outer shell. In a murine subcutaneous model, the platform was demonstrated to be fully tunable for the temporal and spatial control release of the payload. Secondly, a multiscale approach was followed in a multicompartment collagen scaffold, to selectively integrate different sets of PLGA-pSi loaded with different reporter proteins. The spatial confinement of the microspheres allowed the release of the reporter proteins in each of the layers of the scaffold. Finally, the staged and zero-order release kinetics enabled the temporal biochemical patterning of the scaffold. The last step of this PhD project was to test if by fully embedding PLGA microspheres in a highly structured and fibrous collagen-based scaffold (camouflaging), it was possible to prevent their early detection and clearance by macrophages. It was further studied whether such a camouflaging strategy was efficient in reducing the production of key inflammatory molecules, while preserving the release kinetics of the payload of the PLGA microspheres. Results demonstrated that the camouflaging allowed for a 10-fold decrease in the number of PLGA microspheres internalized by macrophages, suggesting that the 3D scaffold operated by cloaking the PLGA microspheres. When the production of key inflammatory cytokines induced by the scaffold was assessed, macrophages' response to the PLGA microspheres-integrated scaffolds resulted in a response similar to that observed in the control (not functionalized scaffold) and the release kinetic of a reporter protein was preserved.