SISTEMI INNOVATIVI PER LA SOMMINISTRAZIONE DI FARMACI A BASE DI ACIDI NUCLEICI

This thesis investigates the design, synthesis, and evaluation of polymer-based systems for the delivery of therapeutic nucleic acids, with emphasis on how polymer architecture and terminal-group organisation govern interactions with nucleic acids and behaviour in biological environments. While viral vectors and lipid nanoparticles have reached clinical application, both platforms retain limitations related to safety, formulation sensitivity, and restricted design flexibility, motivating the development of non-viral polymeric vectors whose molecular structure can be deliberately engineered to modulate biological performance. A critical analysis of polymeric gene delivery systems highlights how charge density, topology, hydrophobicity, and end-group chemistry influence nucleic acid condensation, complex stability, intracellular processing, and barrier interactions. Particular attention is given to terminal cationic groups as primary interaction sites, where subtle chemical modifications can significantly alter binding thermodynamics and functional activity. Two gaps are identified: limited understanding of how clustering of cationic groups at polymer termini affects delivery efficiency, and the need for polymeric systems capable of penetrating mucus barriers relevant to pulmonary delivery. To address these gaps, a series of oligocationic polymers (G4-OCP, G8-OCP, D4-OCP, D8-OCP) were synthesised with precise control over terminal architecture, confining all cationic groups to one polymer end. Guanidyl-terminated and dimethylethanamino-terminated variants were prepared with either four or eight terminal charges to decouple total charge from charge accessibility. Polymer structure and polyplex formation with siRNA were characterised using spectroscopic, electrophoretic, scattering, and microscopic techniques. The results demonstrate that the spatial organisation and accessibility of terminal cationic groups strongly influence nucleic acid binding and functional performance. Guanidyl-terminated polymers formed compact, enthalpically driven complexes, while dimethylethanamino variants relied on multivalent interactions. Notably, polymers bearing four terminal charges often outperformed their eight-charge counterparts, forming more compact polyplexes and achieving stronger functional outcomes due to reduced steric congestion and improved charge accessibility. Polyplexes were stable under physiological conditions, exhibited pH-responsive dissociation, and efficiently released siRNA in the presence of competing anions. All formulations showed high cytocompatibility, efficient cellular uptake, and preliminary gene silencing. A second system comprising near-neutral polymer–mRNA nanoparticles was developed to address mucus penetration. These nanoparticles remained below 100 nm with low polydispersity and minimal surface charge, enabling diffusion through an artificial mucus model and delivery of mRNA to airway epithelial cells under submerged and air–liquid interface conditions, while retaining structural integrity following nebulisation and inducing minimal inflammatory responses. Overall, this thesis demonstrates that polymer architecture, particularly terminal-group organisation, is a decisive determinant of nucleic acid delivery performance. The findings challenge the assumption that increased charge density necessarily improves efficacy and highlight the importance of molecular geometry and functional group accessibility in the design of polymeric gene delivery systems.