In this work, the CO₂-modified polyethylenimine, as a novel delivery vector, has been validated by combining with the plasmid DNA to form plasmid DNA/CO₂-modified polyethylenimine complexes. We have modified polyethylenimine using CO₂ to partially convert amine groups to carbamic acid groups. The buffering capacity and the plasmid DNA binding ability of the CO₂-modified polyethylenimine and PEI-25 (polyethylenimine with Mw = 25 kDa) were characterized by acid–base titration and agarose gel electrophoresis, respectively. The particle size and zeta potential of the complexes were determined using a Zetasizer Nano ZS. Resistance to nuclease digestion was determined via DNase I protection assay. The cytotoxicity was measured by the MTT assay. The transfection efficiency of the complexes has been evaluated by flow cytometry. It is observed that the condensation capacity of CO₂-modified polyethylenimine is still comparable to polyethylenimine and the CO₂-modified polyethylenimine can protect plasmid DNA from degradation by DNase I. The diameter of the plasmid DNA/CO₂-modified polyethylenimine complex is around 140 nm and the zeta potential decreases. MTT assays confirm that the cytotoxicity is much lower for plasmid DNA/CO₂-modified polyethylenimine than for plasmid DNA/PEI-25. The flow cytometry found that in serum-free medium the transfection efficiency can reach a value of ∼60% for plasmid DNA/CO₂-modified polyethylenimine, and in 10% fetal bovine serum medium, the transfection efficiency is still as high as ∼40%, which is much higher than that of plasmid DNA/PEI-25. CO₂-modified polyethylenimine could be a novel and promising nonviral gene vector for gene therapy.

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Carbon dioxide-modified polyethylenimine as a novel gene delivery vector and its in vitro validation