Synthesis and chemical modification of degradable polymers to enhance gene delivery

Abstract

Poly([beta]-amino ester)s are a class of cationic, degradable polymers that have shown significant promise as gene delivery agents, more effective than the state-of-the-art, commercially available non-viral systems. The main objective of this thesis is to synthesize new poly([beta]-amino ester)s and modify existing ones to further improve their gene delivery properties for clinical applications. This has been accomplished by developing both side- and end-chain chemistries for poly([beta]-amino ester)s. A series of novel poly([beta]-amino ester)s were prepared using a new amine monomer 2-(2-pyridyldithio)-ethylamine. The polymer side chains display fast and selective reactivity towards thiol ligands, as demonstrated using mercaptoethylamine (MEA) and RGDC, a ligand that binds with high affinity to certain integrin receptors on angiogenic endothelial cells. The MEA derivatives in particular, were able to self-assemble with plasmid DNA to form nano-complexes that can partially disassemble in response to intracellular glutathione concentrations. These polymers also displayed low cellular toxicity and were able to mediate transfection at high levels in human hepatocellular carcinoma cells. It is envisioned that the PDA poly([beta]-amino ester)s can serve as cationic, degradable platforms to attach targeting ligands, viral peptides and other molecules to a single chain to improve gene delivery.

(cont.) A two-step end-modification strategy is also presented to optimize the functionality at the polymer end points. Conditions were developed so that many structurally diverse end groups could be explored, without the need for polymer purification. Using a highly efficient poly(3-amino ester), C32, optimization of the terminal amine group improved in vitro gene transfection by 30% and reduced the polymer:DNA ratio 5-fold. Differences of single carbons and functional groups at the polymer ends were shown to affect many polymer-DNA properties, including the binding affinity, complex size and surface charge, levels of endocytosis, cytotoxicity and transfection. Intraperitoneal gene delivery in mice using several end-modified C32 polymers proved an order-of-magnitude more effective than unmodified C32, as measured in whole body scans and harvested organs. The end- and side-chain modification strategies presented here have led to the discovery of improved poly([beta]-amino ester)s for gene delivery and may aid in their future development into clinically useful delivery systems.