(645f) Utilizing Hydrolysis and Mixed-Amine Ratio for Effective and Targeted Gene Delivery

A key limitation to the development of human gene therapy is the lack of safe, efficient, and controllable methods for gene delivery. Current research focuses on non-viral gene delivery agents, but these generally lack the required efficacy, cause toxicity due to both the materials used and nonspecific delivery, and are unstable in vivo when administered systemically. The engineering of specialized vehicles capable of overcoming various gene delivery barriers is critical to achieving successful gene transfection. This work describes efforts to create an integrated platform based on hydrolyzable zwitterionic carboxybetaine methacrylate (CBMA) polymers. Zwitterionic materials are superlow-fouling in blood, hydrolytically degradable, readily tunable, and biomimetic (nontoxic). The key attribute of CBMA-ester polymers that makes them ideal for gene delivery lies in the fact that they are positively charged in their native form when they condense DNA, but they become zwitterionic CBMA upon hydrolysis in an acidic environment (as in the endosome). In the zwitterionic state, the polymer repels the DNA, and unpacks the nanoparticle to leave a biomimetic pCBMA coproduct. We have previously studied the effect of differently-substituted amines and found an optimal ratio tertiary:quaternary CBMA-ethyl esters for nonspecific gene delivery to COS-7 cells. Here, we report the results of studies that introduce parameters affecting the optimal tertiary:quaternary ratio, varying the ester leaving group CBMA- esters, and target specific cells for targeted gene delivery.

In this work, we synthesized tertiary and quaternary CBMA- esters with different ester leaving groups and copolymerized them at controlled and discrete ratios. These polymers were used to condense DNA encoding for the firefly luciferase gene, and the resulting nanoparticles were tuned to create small, electrostatically neutral targeting agents. The optimal amine ratio to receptor-mediated cell uptake, endosomal escape, and gene expression was identified. The cellular uptake of our most effective amine-ratio CBMA-ester by targeted cells was compared to that by nontargeted (COS-7) cells, to test the cell-specificity of the vector. Our results suggest that the different ester leaving groups and functional groups further improves the efficacy and safety of our CBMA ester gene delivery system.