De Simone, Mariarosaria (2010) Modulation of integrin activity for diagnostic and therapeutic applications. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||integrins, modulation, angiogenesis, nanogolds|
|Date Deposited:||03 Dec 2010 10:33|
|Last Modified:||30 Apr 2014 19:45|
SUMMARY Over the past decade, the scientific research spent many efforts to develop therapeutic and diagnostic systems able to contact selectively the target cells and to minimize their diffusion in healthy organs. For this purpose the most diffuse approach was targeting the molecular markers overexpressed on the tissues of interest. In this contest, the integrin family of cell adhesion receptors was one of the most studied markers. (Tucker G.C., 2006). In particular, among all integrins, v3 receptor was particularly studied in the last decade. Physiologically, it mediates many different biological processes such as intracellular signalling, cell migration, proliferation, and survival through interactions with ECM proteins such as vitronectin, fibronectin and osteopontin. The receptor mediates cell adhesion to extracellular matrix by recognizing the conserved Arg-Gly-Asp sequence of several plasma and matrix proteins. It is strongly overexpressed in activated EC, melanoma, glioblastoma and prostate cancers and in granulation tissue, whereas is not detectable in quiescent blood vessels or in the dermis and epithelium of normal skin. Therefore it can be considered a tumour and activated endothelium marker. In the last decade several αvβ3 ligands able to modulate the receptor activity were developed as drugs for therapy, as tracers for diagnosis and as ligands for targeted drug delivery systems. Among these molecules, the pentapeptide c(RGDf[NMe]V), also known as Cilengitide, is the most active , αvβ3/αvβ5 antagonist reported in literature and is in phase III clinical trials as antiangiogenic drug for glioblastoma therapy (Dechantsreiter M.A., et al., 1999; Reardon D.A., 2008; Tabatabai G., et al., 2010). However, even if all v3 ligands reported in the literature have a good affinity for the receptor, they present a low selectivity and bind, even if with lower affinity, also other integrin receptors structurally homologues of v3 such as v5 integrin (Smith J.W., et al., 2003; Eskens F.A., et al., 2003). In contrast with v3 that has a relatively limited cellular distribution v5 is widely expressed by many malignant tumor cells. Therefore, to target v3-mediated processes for diagnostic or therapeutic purposes, the development of new compounds that can discriminate between v3 and v5 is required to minimize the side-effects and increase the therapeutic effectiveness. In 2006 our research group designed and synthesized a novel and selective peptide antagonist, referred to as RGDechiHCit, to visualize αvβ3 receptor on tumour cell (Del Gatto A., et al., 2006). It is a chimeric peptide containing a cyclic RGD motif and two echistatin C-terminal moieties covalently linked by spacer sequence. Cell adhesion assays showed that RGDechiHCit selectively binds, αvβ3 integrin and does not cross-react with, αvβ5 and, αIIbβ3 integrins (Del Gatto A., et al., 2006). Furthermore, PET and SPECT imaging studies confirmed that the peptide selectively localizes on αvβ3 expressing tumor cells in xenograft animal model (Zannetti A., et al., 2009). In this experimental setting, the chimeric RGDechiHCit was not able to detect a signal originating from the newly formed intratumoral blood vessels. This may be due to incomplete neovascularization and, hence, low levels of αvβ3 expression or to the murine origin of the integrin on newly formed blood vessels because the rational design of the chimeric RGD peptide was based on the crystal structure of the extracellular region of human αvβ3. Therefore the main purpose of the present PhD thesis was to evaluate in vitro and in vivo effects of RGDechiHCit on neovascularization. In particular, we first assessed the in vitro peptide properties on bovine aortic ECs, VSMC and then in vivo, in Wistar Kyoto (WKY) rats and c57BL/6 mice, the ability of this cyclic peptide to inhibit angiogenesis in comparison with Cilengitide. A major evidence that is brought up by our results is the peculiar selectivity of RGDechiHCit towards EC, as compared to c(RGDf[NMe]V). Indeed, RGDechiHCit fails to inhibit VSMC proliferation in vitro, opposite to c(RGDf[NMe]V). This feature could be due to the selectivity of such a novel compound towards , αvβ3, VSMCs indeed express αvβ3 only during embryogenesis (Eliceri B.P., et al., 1998; Illario M., et al., 2005), but express other integrins which may be blocked by c(RGDf[NMe]V). On the contrary, αvβ3 is expressed on ECs, thus conferring RGDechiHCit selectivity toward this cell type. It is only an indirect evidence, that needs further investigation in following experiments. Comparable results between the two antagonists were obtained on wound healing and Matrigel plugs invasion. Our data suggest that inhibition of the endothelial integrin system is sufficient to inhibit angiogenesis. In conclusion the potential antiangionenic activity of the peptide opens new fields of application for the treatment of pathophysiological conditions associated to angiogenesis such as cancer, proliferative retinopathy and inflammatory disease. In the nanotechnology field, gold nanoparticles (AuNPs) are playing a pivotal role in providing new types of targeted delivery systems to permit the selective entry of one or multiple drugs in the primary tumor, as well as at the site of metastasis and its microenvironment. AuNPs can be indeed used to deliver a cargo, such as an anticancer drugs, or a radionuclide to tumor sites as well they can be also employed in tumor phothermal therapy for their plasmon resonance properties (Melancon M., et al., 2009). In both cases the targeted drug approach is achieved by the exploitation of molecular markers over-expressed in cancerous tissues such as αvβ3 integrin. Unfortunately, nanogold tend to aggregate in solution and so it is difficult to preserve them for long time. To solve this issue they can be functionalized with various organic ligands to create organic-inorganic hybrids with advanced functionality. It was recently reported the use of peptide sequences based on the GC repeats as stabilizing agents for the preparation of monolayer gold nanoparticles (Krpetić Z., 2009). On the basis of this data, we designed and synthesized a new chimeric peptide (thereafter named RGD(GC)2, displaying motifs for both targeting and capping functions. A RGD-containing peptide, derived from the Cilengitide, was chosen as a targeting ligand for, αvβ3 integrin receptor and a GC including peptide was selected in order to stabilize the gold nanoparticles. AuNPs functionalised with this peptide were prepared and characterized by Uv-Vis, ATR-IR, XRD, NMR techniques and TEM microscopy. Finally we tested the ability of the obtained nanosystems, named RGD(GC)2AuNPs, to permeate the target cells membrane (U-87 MG, glioblastoma cells). In all these studies we used as negative control peptide named (GC)2 corresponding to the capping motif obtaining the gold nanoparticles named (GC)2AuNPs. Our results encourage us to retain that this system could be a good starting point to develop selective gold nanodevices useful in the field of biotechnologies for therapeutic and diagnostic applications.
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