DEVELOPMENT OF A SEMISYNTHETIC NANOSTRUCTURED SYSTEM FOR THE SELECTIVE DELIVERY OF NUCLEIC ACID THERAPEUTICS TO THE TUMOR SITE

The mechanism of RNA interference has garnered substantial attention since its discovery, owing to its capacity to achieve highly specific post-transcriptional gene silencing. This process relies on small interfering RNAs (siRNAs), short double-stranded oligonucleotides that recognize complementary mRNA sequences and promote their degradation, thereby preventing translation of the corresponding protein. Despite this therapeutic potential, the clinical translation of siRNA-based strategies remains limited by several extracellular and intracellular barriers, including nuclease-mediated degradation, potential immune stimulation and inefficient cellular uptake, all of which necessitate the development of safe, efficient, and targeted delivery systems. This PhD thesis work focuses on the design, formulation, and evaluation of a novel siRNA delivery platform based on ANANAS nanoparticles (Avidin Nucleic Acid NanoASsemblies). The physicochemical and functional properties of ANANAS have previously enabled applications in diagnostics, in the targeted delivery of chemotherapeutics, and in the hepatic delivery of steroidal anti-inflammatory molecules. In the present work, ANANAS are investigated for the first time as a modular system for the selective delivery of siRNA to tumour cells. ANANAS represent poly-avidin nanosystems derived from avidin, a tetrameric glycoprotein with dual binding capabilities: association with nucleic acids, exploited to condense avidin onto non-coding plasmid DNA into toroidal assemblies, and extremely high-affinity binding to biotin. This latter property enables precise, stoichiometrically controlled decoration of nanoparticles with biotinylated functional elements, including targeting ligands, fusogenic moieties to promote endo-lysosomal release, and biotin-modified siRNAs. In this project, siRNA sequences were designed to target mitochondrial ion channels (VDAC1 and Kv1.3), key regulators of apoptosis and metabolism in highly aggressive cancers, including pancreatic ductal adenocarcinoma (PDAC) and triple-negative breast cancer (TNBC). The initial phase of the project focused on the physicochemical and functional characterization of the formulations which confirmed efficient nanoparticle decoration with siRNA, the release of the oligonucleotide under reducing or acidic conditions, according to linker chemistry, and improved stability relative to free siRNA. Targeting performance and internalization efficiency were assessed using EGFR-overexpressing tumour cell models, and functional biological outcomes were evaluated through Western blotting, RT-qPCR, and flow cytometry analyses. These experiments underscored the pivotal role of lysosomal escape for effective gene silencing, demonstrating that intact release of siRNA into the cytoplasm is essential for the final activity. Co-treatment with the lysosome-permeabilizing agent L-leucyl-L-leucine methyl ester (LLOMe) yielded the first measurable silencing effects in GFP-expressing cells. Overall, this work, performed in collaboration with the research groups of Profs. Mattarei, Morpurgo and Szabò, demonstrates the potential of ANANAS nanoparticles as a flexible and modular platform for siRNA delivery, while also revealing critical challenges associated with lysosomal sequestration and nucleic acid instability. The results provide insights that are broadly applicable to the rational design of nucleic acid delivery systems, emphasizing the central importance of linker chemistry, oligonucleotide protection strategies, and the coordinated integration of functional components to achieve efficient gene silencing. By integrating concepts from chemistry, nanotechnology, and cell biology, the findings contribute to the development of precision nanomedicine approaches and offer a framework for designing safe and effective gene-silencing strategies against highly aggressive malignancies.