For the analysis of PEI polyplex stabilities, a dextran sulfate (DS) displacement assay was performed. Samples containing 200 ng pUC19 were complexed with a fixed polymer/DNA weight ratio of 6.0 at a total volume of 5 µL in 10 mM Tris buffer (pH 7.5). This polymer/DNA weight ratio was sufficient for a complete complexation of DNA by all polymers used in this study. Polyplexes were then incubated at room temperature for 45 mins to ensure complete DNA complexation. Dextran sulfate was then added at various DS:DNA weight ratios, and the final solution volume was brought up to 12 µL by the addition of buffer. Samples were subsequently incubated for another 30 minutes to allow plasmid DNA to be released from the polyplexes. Agarose gels (0.8 w/v %) were prepared by dissolving agarose in a 0.2 µm filtered TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA). Before loading onto the agarose gel, 2 µL of 6x loading dye was added to each sample. Samples were electrophoresed for 30 min at 100 V. DNA was visualized by post-run staining with ethidium bromide (0.5 µg/mL) and imaged using the BioRad ChemiDoc MP Imaging System. For pH dependent polyplex stability experiments, DNA release by competition with DS was determined by measuring the relative fluorescent intensity resulting from ethidium bromide intercalation into released DNA under conditions identical to the EtBr displacement assay described above. Controls of DNA:EtBr without DS and various concentrations of DS and EtBr were measured at each pH to give Fo, and FMax values for the calculation of percent relative fluorescence described above.