SYNTHESIS AND CHARACTERIZATION OF MANNOSYLATED CATIONIC POLYMERS FOR NUCLEIC ACIDS DELIVERY

Nowadays, drug delivery systems (DDS) are widely employed to achieve a higher control of the site of action and timing of drug release and to address such problems as low solubility, uncontrolled pharmacokinetics, side effects, toxicity and drug resistance. Gene therapy can be defined as the use of Nucleic Acids (RNA or DNA) for the prevention or treatment of diseases. It includes the use of DNA and RNA-based drugs featuring different molecular weights (oligo- and poly-nucleotides), structures (single or double strand) as well as mechanisms of action. Despite these strategies provide a unique therapeutic tool for the treatment of many diseases, such as cancer, the delivery of therapeutic nucleic acids (TNAs) still remains a challenge. The main issues in their delivery are that they suffer from biological instability, degradation by nucleases, low targeting efficacy and lack of capacity of endosomal escape. To address these problems, several DDS have been developed such as viral vectors, vesicles, nanoparticles and polymers. Recently, synthetic positively charged polymeric carriers have drawn increasing attention, since they provide opportunities for improved safety, greater flexibility and more facile manufacturing while preventing the enzymatic degradation of genetic material and mediating its cellular uptake. The aim of this thesis project is the development of cationic di-block copolymers as delivery systems for TNAs encoding for cancer antigens to target immune system cells, as cancer vaccines. The polymers were synthetised from two monomers derived from substituted N-Hydroxyethyl acrylamide (HEAA). The first, features an imidazole moiety which, thanks to its pKa of 6.95, displays buffering properties within the endosome, causing its destabilization and eventual breakage. Moreover, bearing a partial positive charge at neutral pH, the imidazole in this system will be crucial to complex the negatively charged TNAs. The second monomer bears a mannose unit to target immune cells, and specifically Antigen Presenting Cells, that express the mannose receptor. Both the syntheses of the monomers were successfully achieved, and the characterization was performed by 1H NMR, FT-IR and ESI-TOFF mass spectrometry. The synthesis of a library of polymers was performed by using a water-soluble RAFT agent (Reversible Addiction Fragmentation Chain Transfer). RAFT polymerization is a living free-radical polymerization technique providing polymers of controlled and predictable molecular weight with very narrow polydispersity for each block. First the synthesis of the RAFT agent was reached through a 3-step reaction and the characterization of the intermediates, and the final product was performed through NMR and ESI-TOFF mass spectrometry. The CTA agent was later used to synthetise a small library of polymers starting from 30 units block of mannose that was elongated with: 1) 30 units block of imidazole-HEAA, 2) 15 units block of unmodified HEAA and another block of 30 imidazole-HEAA, 3) 30 units block of a random mixture of 2/1 Imidazole-HEAA/unmodified HEAA. The polymers obtained were characterize by NMR and GPC showing a low PDI around 1.08 that increase to 1.88 after the addiction of the Imidazole block. However, the copolymers failed to complex either ssDNA or CpG ODN at any tested N/P ratio up to 100. 1H NMR was used to characterize the purified materials and the integrals of the imidazole protons were not the expected values, possibly indicating hydrolytic processes that could explain the lack of NA condensation. Future plans will involve the development of a new synthetic strategy for the imidazole monomer to avoid the hydrolysis during the polymerization and ensure NA condensation. Once the polyplexes will be formulated the size and morphology of the complexes will be characterized via DLS and TEM analysis as well as their stability throughout heparin displacement assays and the in vitro transfection efficacy.