Characterization and Optimization of α-Polyglutamic Acid and Branched Polyethyleneimine Polyplexes for Gene Transfection Via a Combinatorial Approach

Gene transfection holds tremendous promise for treatment of a wide range of acquired and genetic diseases via human gene therapy and for the production of recombinant proteins with post-translational modifications on an industrial scale. Implementation of these techniques is hindered, however, by the lack of safe and efficient methods for delivery of genetic material. Conventional cationic polymer/DNA complexes (polyplexes) appear unlikely to perform all of the functions necessary for efficient transfection. Ternary complexes, comprising multiple polymers, provide an opportunity to incorporate additional functions. To explore this approach, 40 unique polyplexes composed of DNA, polyethyleneimine (PEI), and α-polyglutamic acid (PGA) were prepared via a combinatorial approach by simultaneously varying the PEI:DNA and PGA:DNA ratios. The particle diameter and zeta potential of each polyplex were determined. Transfection efficiency and cell viability experiments were performed in growth media with and without serum. Only a 5% increase in the average hydrodynamic radius was observed upon addition of α-PGA to the PEI/DNA complexes. In serum free media, the optimal polyplex demonstrated 2.9 times the transfection efficiency of the optimal PEI/DNA complex and 24.3 times the transfection efficiency of Lipofectamine 2000. In media with serum, the optimal polyplex exhibited a transfection efficiency 6.9 times greater than that of the optimal PEI/DNA complex and 2.6 times greater than that of Lipofectamine 2000.  Additionally, the α-PGA layer caused statistically significant increases in cell viability relative to PEI/DNA complexes and Lipofectamine 2000 in growth media with and without serum.