Although numerous studies have investigated transfection efficiency and the colloidal properties of the resulting polyplexes, the exploration of how PEI modifications influence internal DNA packaging and their responses to external stimuli, such as pH, has been comparatively limited. In this study, we examined the physiochemical properties and internal structure of polyplexes formed through the condensation of DNA with both unmodified and modified branched PEIs (acPEI and zPEI). Two series of modified PEIs were synthesized by modification of 25-kDa bPEI through acetylation (10-50%) or succinylation (2-40%) and characterized using FT-IR and 1D-and 2D-NMR. We show that both primary and secondary amines undergo modification. DNA condensation and stability, polyplex size and charge, and gene expression efficacy in the presence of serum proteins were measured for these systems. Small-angle X-ray scattering (SAXS) experiments were conducted to ascertain the internal packaging of the resulting polyplexes as a function of the degree of modification. We found that acetylation consistently weakens polycation-DNA interactions and results in looser DNA packaging. In contrast, succinylation shows a crossover behavior with increasing modification levels: initially, lowering DNA packaging density, but then increasing DNA packing at higher modification levels. Time-course SAXS studies reveal that structural rearrangements in the DNA packaging of PEI and modified PEI polyplexes occur within an hour. Furthermore, we found that pH significantly impacts the resulting DNA packaging in all PEI polyplexes. Specifically, condensation at low pH (pH 4) leads to tighter DNA packaging within both unmodified and modified PEI polyplexes, compared to polyplexes condensed at near-neutral pH. Moreover, polyplexes initially formed at neutral pH, when acidified, exhibit substantial structural rearrangements within one hour, resulting in a more densely packaged complex. In addition, we demonstrate that PEI is expelled from these systems upon acidification, facilitating this tighter DNA packaging.