Plasmid linearization changes shape and efficiency of transfection complexes

The ability to efficiently transfect plasmid DNA (pDNA) into eukaryotic cells has exerted major impact on scientific research in recent years, and translation to clinical application is ongoing, but challenging. In addition to the choice of the delivery vector, the topology of the DNA seems to be a key factor for efficient transfection. The nanostructured DNA/Vector complexes may differ in size, charge, and shape, for example. This study therefore investigated the transfection efficiency of circular versus linearized plasmid DNA using a GFP expressing vector with Lipofectamine2000 and linear 25 kDA polyethylenimine (PEI). Transfection efficiency and cytotoxicity were measured by flow cytometry and fluorescence microscopy. Shape was determined by transmission electron microscopy. Transfection agent concentrations were chosen below the toxicity level. We determined the optimal N/P ratio over 48 h by using two different concentrations of plasmid DNA. With the increase of DNA concentration and increasing N/P ratio, transfection efficiency also increased. Our results showed a better transfection efficiency with the circular compared to the linearized DNA, under the same experimental conditions for both Lipofectamine and PEI. In electron microscopy, there was a notable difference in the shape of the complexes: circular DNA had random coil appearance in well compacted, roughly spherical shape, while linearized DNA appeared as worm-like strands, both, when complexed with Lipofectamine or with polyethyleneimine. This generates the hypothesis that the shape of the transfection particle may be an important factor for successful gene transfer.