Among the numerous challenges in gene delivery with polyplexes, efficient endosomal escape is thought pivotal for successful transfection. Although debated, one of the leading hypotheses of the effectiveness of PEI for gene delivery is based on the idea of the "proton sponge" effect, which relies on PEI's capacity to buffer pH. This effect posits that the H + -buffering amines of PEI enhances the accumulation of Cl-and other counterions in endosomes during their acidification, potentially causing osmotic swelling or lysis that facilitates the escape of PEI polyplexes from endosomes. While not required for the 'proton sponge' hypothesis, it is also commonly proposed that acidic conditions cause PEI within polyplexes to overcharge, enhancing repulsive forces and altering the structure of the polyplexes within the endosome, either by causing them to swell or loosen. This hypothesis is contentious, with experimental and theoretical evidence providing mixed support. Due to this, more recently others have argued that PEI, whether free or associated within polyplexes, might interact with the endosomal membrane in a way that leads to enhanced endosomal rupture. We find that both unmodified and modified PEI polyplexes, once acidified, undergo structural reorganization leading to tighter, rather than looser, DNA packaging. We find that the resulting DNA packaging density of polyplexes formed at neutral pH and then acidified (pH 7.5-4) lies intermediate between DNA packing when condensed at pH 7.5 or pH 4 presumably due to bound PEIs having different pKa's than unbound PEI chains. Molecular dynamics simulations support this, proposing that tighter spacing at low pH might lead PEI polyplexes to release some PEI chains. Our experiments confirm a significant release of PEI from the complexes post-acidification, possibly aiding endosomal escape through interactions of the free polymer with the endo-lysosomal membrane, as has been proposed by recent free polymer-mediated escape theories.