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 pH-responsive zwitterionic carboxybetaine methacrylate (CBMA) polymers. Zwitterionic materials are superlow-fouling in blood, biodegradable, 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 in an acidic environment (as in the endosome). This zwitterionic state repulses the DNA to unpackage the nanoparticle with a remaining biomimetic CBMA coproduct. Here we synthesize ester-based CBMA polymers with mixtures of differently-substituted amines and report an optimal ratio that balances the advantages of secondary, tertiary and quaternary amines.

In the first part of this work, we verified the nonfouling properties and pH sensitivity of mixed-amine copolymers. Secondary amines are potentially proton sponging, tertiary amines are proton sponging and positively charged under slightly acidic conditions for DNA condensation, and quaternary amines are positively charged under all pH conditions to ensure DNA condensation. We measured the nonspecific protein adsorption, DNA binding and release, and the pH responsiveness of CBMA-ester polymer brushes containing mixtures of secondary/tertiary and tertiary/quaternary amine groups. In the second part of this work, the biophysical properties of CBMA-ester polymers containing different ratios of secondary/tertiary amines and tertiary/quaternary amines were studied, as were the corresponding nanoparticles. Specifically, we examined how changing the amine content of the polymers affects the polymer's ability to condense DNA into a nanoparticle, the resulting nanoparticle size and surface charge, the pH responsiveness of the bulk polymer and its corresponding nanoparticle, and the buffering ability of the nanoparticles. Finally, we have successfully transfected cells with the luciferase plasmid, and here we compared the transfection efficiencies of mixed-amine CBMA-ester polymers. We have correlated the transfection efficiencies with the biophysical characteristics and chemical properties of the polymers. Here we identify tertiary/secondary and tertiary/quaternary amine ratios that most effectively balance endosomal escape and nanoparticle packaging to induce COS-7 cells to express luciferase.