LIGUORI, FRANCESCA (2026) NANOBIOMATERIALS TARGETING THE TUMOR MICROENVIRONMENT FOR ENHANCED GENE THERAPY. [Tesi di dottorato]

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Abstract

Abstract Golgi phosphoprotein 3 (GOLPH3), also known as GPP34, GMx33, or MIDAS, is a Golgi apparatus-associated protein involved in regulating fundamental cellular processes, including tumor cell proliferation, migration, and metabolism. Numerous studies have highlighted its overexpression in various solid tumors, including lung, breast, prostate, and colon cancer, identifying GOLPH3 as a promising therapeutic target. In this context, suppression of GOLPH3 by RNA interference (RNAi) represents an effective therapeutic strategy. In this context, the development of efficient, biocompatible, and tunable delivery systems is therefore essential to fully exploit the therapeutic potential of RNAi. To this end, this thesis focused on the development and characterization of polymeric nanosystems for the targeted delivery of anti-GOLPH3 siRNA in prostate cancer. Two nanoparticle platforms were investigated. The first consists of poly(lactic-co-glycolic acid) (PLGA) nanoparticles functionalized with imidazolium-based ionic liquids (ILs), selected for their tunable amphiphilicity, cationic character, and ability to modulate nanoparticle–cell interactions. Following a comprehensive screening, formulations containing 1-hexadecyl-3-methylimidazolium chloride (C16ImCl) and 1-decylecyl-3-methylimidazolium chloride (C10ImCl) were selected, obtaining PLGA-C16 and PLGA-C10 nanoparticles. The second platform developed in this work consisted of hybrid nanoparticles obtained by conjugating PLGA with linear polyethylenimine (PEI), termed LGIP, formulated at two different polymer/siRNA ratios (LGIP-1 and LGIP-10), in order to evaluate the influence of the PEI content on the system properties and biological efficacy. All nanosystems were thoroughly characterized in terms of size, morphology, surface charge, colloidal stability, siRNA loading and release profiles. In vitro studies demonstrated that all formulations exhibited a good biocompatibility profile in both DU145 prostate cancer cells, and in healthy PNT2 prostatic epithelial cells. Cellular uptake and intracellular trafficking analyses revealed that nanoparticle surface chemistry strongly influences internalization kinetics and endocytic pathways. In particular, PLGA-C16 nanoparticles exhibited rapid and efficient uptake, predominantly via clathrin-mediated endocytosis at low concentrations, while PLGA-C10 nanoparticles showed slower internalization and a greater reliance on micropinocytosis at higher doses. LGIP nanoparticles displayed delayed but sustained cellular uptake involving multiple endocytic routes. Functional wound healing assays confirmed that effective silencing of GOLPH3, mediated primarily by PLGA-C16 nanoparticles and to a lesser extent by LGIP-1, results in significant inhibition of tumor cell migration. Finally, in three-dimensional (3D) models of tumor spheroids highlighted, the differential effect of PLGA-C16 and PLGA-C10 suggested that the alkyl chain length of ionic liquids influences spheroid morphogenesis and interaction with nanoparticles, underscoring the importance of nanosystem composition in more physiologically relevant contexts. Overall, this work demonstrates that rational modulation of nanoparticle surface chemistry through ionic liquid functionalization or polymer conjugation critically determines cellular uptake mechanisms, intracellular fate, and gene silencing efficacy. These findings support the potential of PLGA-ILs and LGIP nanoparticles as versatile and effective non-viral platforms for siRNA delivery and provide valuable design principles for the development of advanced RNAi-based therapeutic strategies targeting oncogenic pathways in cancer.

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