(549b) Combinatorial Libraries of Poly(Acrylic Acid)-Based Vectors for Nucleic Acid Delivery

Gene therapy represents an alternative treatment for countless chronic diseases, including viral infections, genetic disorders and cancer, by the incorporation of therapeutic genetic material into cells or tissues. Depending on the therapy administered, functional genes (DNA) can be directly inserted into cells or endogenous gene expression can be regulated by means of a short interfering RNA (siRNA). Introducing nucleic acids into cells is limited by numerous challenges, predominantly the lack of effective delivery systems that can safely transport these macromolecules to their site of action while overcoming a manifold of barriers hindering the delivery pathway. For this, synthetic vectors, including polymers, have increasingly gained attention mainly due to their easily controllable molecular composition and thermodynamic stability. The goal of this research is to employ a combinatorial chemistry approach to design polymeric vectors for enhanced nucleic acid delivery. Toward this goal we synthesized poly(acrylic acid) (pAA) by reversible addition-fragmentation chain transfer (RAFT) polymerization with controlled molecular weights (5kDa, 10kDa and 25kDa) and narrow polydispersity indices (< 1.2). Based on these precursors, polymer libraries were generated by conjugating two distinct moieties, agmatine (Agm) and D-(+)-galactosamine (Gal), at various ratios to the polymer side-chains. Agm serves as a cationic source to facilitate interactions with the nucleic acid and cell membrane while Gal is used for tissue-specific targeting to hepatocytes. Biochemical characterization for the 10kDa-pAA library, including cytotoxicity affects and binding affinity of polymers to GL4 siRNA, showed that as cationic density (Agm) increases and Gal content decreases, polymers conjugates bind more strongly to GL4 siRNA but have higher cytotoxicity to HepG2/C3A-luc2 cells. Initial biophysical characterization shows that polyplexes < 180nm in diameter with positive zeta potentials are achieved which are important qualities to facilitate receptor-mediated endocytosis. Additionally, confocal fluorescence microscopy was employed to visualize internalization of fluorescently-labeled polyplexes over time. Further characterization, including transfection efficiencies and physicochemical characterization of additional pAA libraries, is essential to fully correlate the polymer structure with nucleic acid delivery.