To understand potential changes in PEI-based polyplex packaging as a function of pH, we first examined how the pH at condensation affects the polyplex internal structure (Figure ). Stock solutions of DNA, bPEI, acPEI10, and zPEI10 in either 10 mM sodium acetate (pH 4) or 10 mM Tris-HCl (pH 7.5) were prepared. The buffered DNA solutions were then precipitated with PEI solutions of matching pH, and the resultant condensates were labeled pH 7.5 or pH 4 to denote the pH at condensation. After centrifugation, the condensates were transferred to fresh buffer solutions and equilibrated at room temperature for 1 and 24 hours before SAXS analysis. Representative scattering intensity profiles are shown in Figure for DNA condensed by bPEI (left), acPEI10 (middle), and zPEI10 (right) at pH 7.5 (black) and pH 4 (red) after a 24-h equilibration period. For all three systems, condensing at low pH resulted in an observed Bragg peak at higher Q, indicating smaller DNA-DNA spacings, within the condensed phase relative to samples condensed in Tris-HCl. The smallest relative change (~5-6%) was exhibited by bPEI polyplexes, where Bragg spacing shifted from 24.8 Å at pH 7.5 to 23.4 Å after condensation at pH 4. Modified PEIs showed more substantial changes (~9-11%), with DBragg shifting from 26.5 to 24.0 Å for acPEI10 and from 26.3 to 23.3 Å for zPEI10 when condensed at pH 7.5 or pH 4.