Marchesano, Valentina (2013) Innovative tools for RNA interference: multifunctional gold nanoparticles for silencing of myc proto-oncogene. [Tesi di dottorato]

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Abstract

Despite its simple body plan and structural anatomy, the Cnidaria Hydra vulgaris, a dipoblastic animal at the base of metazoan evolution, is an excellent model system to investigate the mechanisms controlling stem cell proliferation and differentiation and the balance between the two phenomena. In vertebrates one of many genes that participate in regulating cell homeostasis is the protooncogene c-myc. The MYC family of transcription factors controls disparate aspects of cell physiology including cell growth, cell cycle progression, biosynthetic metabolism, and apoptosis. As expected c-myc deregulated expression occurs in the majority of human cancers, making it a seemingly attractive target for therapeutic inhibition. In order to develop effective strategies of c-myc gene silencing, two methodologies based on small interfering RNAs (siRNA) were developed in this work, using Hydra as model organism and targeting the Hydra c-myc orthologue gene Hymyc1. The first methodology, based on free myc-siRNA supplied to the polyps by soaking under acidic condition was shown to induce specific RNAi and to affect proliferation of stem cells and derived cell types. The ability of Hydra cells to uptake double stranded RNA and to trigger a RNAi response laid the foundations for the second methodology, developed in the frame of a European project and based on the use of gold nanoparticles (AuNP) as innovative shuttles for safe and successful delivery of siRNA. AuNPs were conjugated to Hymyc-siRNA using different chemical strategies and tested for functionality in Hydra by molecular analysis, followed by measurement of cell cycling activity and quantitative estimation of cell ratios between stem and differentiated cells. Among eight different myc-siRNA-AuNP conjugates, a single nanodevice (presenting siRNA covalently conjugated to the gold core) was shown able to induce 80% gene silencing. The biological effect of Hymyc1 downregulation was elucidated as a strong impairment of the balance between stem cell self-renewal/differentiation, as shown by the accumulation of stem cell intermediate and terminal differentiation products in RNAi animals. The confirmation also in other model systems of the bioactivity of such nanodevice to induce efficient and specific gene silencing strongly supports its feasibility to be broadly used for therapeutic RNAi. In addition to the gene silencing performance, and thanks to the unique contrast agent behaviour of the gold nanoparticle core for Electron Microscopy, a detailed investigation of the mechanisms of internalization of AuNPs was performed by comparing different chemical functionalities (siRNA and positively charged groups) present on AuNP surface. The dynamic and kinetic of the whole journey of gold nanoparticles within a whole animal was reported, from the first interaction nanoparticle/cell membrane, to the intracellular trafficking and final extracellular clearance. Beside classical routes, new portals of entry/exit were captured by imaging Hydra treated tissue, including exosome-like structures as novel undescribed nanoparticle shuttles inward and outward the cell membrane. The conservation of the endocytic/secretory machinery through evolution extends the value of our finding to mammalian systems providing new clues to take into account when designing nanomaterials to interface with biological entities.

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