In conclusion, PEI modifications via acetylation and succinylation profoundly impact the physicochemical properties, internal structure, and transfection efficiency of the polyplexes. These modifications influence polyplex formation, stability, and DNA packaging in distinct ways. While acetylation consistently increases DNA packing densities, succinylation leads to more complex behaviors, resulting in polyplexes that are tightly packed but less stable. Additionally, our study underscores the crucial role of pH in influencing polyplex packaging, stability, and the release of PEI chains. These insights highlight the importance of tailoring polymer-DNA interactions through strategic PEI modifications and pH adjustments to enhance the efficacy of PEI-based gene delivery systems.