Next, we assessed the impact of condensation pH on the precipitation behavior and stability of the resulting polyplexes using an ethidium bromide (EtBr) displacement assay. Controls of free EtBr and uncondensed DNA/EtBr at each pH were also measured to determine pH-dependent Fo, and FMax values for the calculation of relative fluorescence. The assays (Figure ) confirmed that all polymers-bPEI, acPEI10, and zPEI10-required lower polymer/DNA weight ratios to fully condense DNA at pH 4 compared to pH 7.5, suggesting enhanced protonation of PEI at lower pH. The resulting stability of these polyplexes was assessed using a dextran sulfate displacement assay (Figure ). DNA was pretreated with ethidium bromide and polyplexes were formed with bPEI, acPEI10, and zPEI10 at a 1:1 polymer/DNA weight ratio at either pH 4 or pH 7.5. This amount of polymer is sufficient to fully condense DNA, resulting in a complete displacement of the EtBr and minimal fluorescence observed. Polyanionic dextran sulfate (DS) was introduced to the solution, where it competes with DNA for PEI binding. DS does not interact with EtBr and DS/EtBr shows no increase in fluorescence compared to free EtBr. At sufficient DS:DNA weight ratios, DNA is released from the polyplexes, and EtBr re-intercalates resulting in an increase in the relative fluorescence intensity. All three systems demonstrated that polyplexes condensed at pH 4 were more stable, requiring higher DS:DNA weight ratios to free DNA from the polyplexes.