Effective gene transfection using nonviral polymer carriers like polyplexes faces numerous intra-and extracellular obstacles. These carriers must efficiently navigate towards targeted cells, evade nucleases and the reticuloendothelial system, interact with cellular membranes, undergo endocytosis, escape from endosomes, traverse the cell's cytoskeletal network, and ultimately release DNA within the nucleus for transcription. Polyethylenimine (PEI) is one of the most successful polymers for gene transfection and is often considered a benchmark for polymeric gene delivery. Despite its success, PEI's high charge density contributes to cytotoxicity, which limits its clinical applicability. Modifying PEI by grafting different functional groups to the primary and secondary amines on its backbone has been a common strategy to reduce cytotoxicity and optimize gene delivery. These modifications, including acetylation and succinylation, generally weaken PEI-DNA interactions, requiring more polymer for effective DNA condensation and facilitating easier DNA release, as shown in our studies and supported by previous findings. Contrary to the assumption that primary amines are the primary sites of modification for bPEI due to less steric hindrance, our 2D-NMR analysis reveals a preference for modifying secondary amines, especially at lower modification levels, for both acPEI and zPEI, consistent with secondary amines being more nucleophilic than primary amines in water. In terms of colloidal and charge properties, acetylated PEI polyplexes maintained a size similar to bPEI/DNA, with a slight decrease in surface charge. In contrast, succinylated PEI polyplexes increased in size with higher modification levels, which might suggest increased aggregation in the highly succinylated zPEI complexes. Interestingly, the incorporation of negatively charged succinate groups into zPEI polyplexes did not significantly alter their zeta potential until a modification level of 30% was reached, which led to a charge reversal. This suggests that while acetylation systematically reduces PEI's charge density, succinylation alters the pKa of nearby amines, resulting in a polyampholytic zPEI that maintains a net positive charge similar to unmodified bPEI at low modification levels. Acetylated PEIs did not show a notable improvement in transfection efficiency in the presence of serum proteins. However, minimally succinylated zPEIs significantly outperformed unmodified bPEI, consistent with our prior studies. This charge reversal and weakened binding at higher modification levels correspond with a diminished transfection efficacy of highly succinylated zPEI across all weight ratios. Although the precise mechanism behind zPEI/DNA's enhanced performance in the presence of serum is not fully understood, a similar enhancement of transfection with serum was recently reported using a biodegradable PEI-mimetic polymer, similar to linear PEI, where the ratio of primary, secondary and tertiary amines was found to be a key factor. Polycations, including PEI, typically self-assemble with DNA into compact structures, forming hexagonal arrays with DNA helix surfaces separated by a water layer of about 5-15 Å, due to a balance of the attractive and repulsive forces within the condensed phase. Using osmotic pressure and small-angle X-ray scattering, we determined these forces for various polycation-DNA systems. For example, incorporating an uncharged alanine into a hexa-arginine chain (R3AR3) resulted in larger DNA-DNA spacings than in R6/DNA, while a negatively charged glutamic acid (R3ER3) significantly increased these spacings due to enhanced repulsions. Based on these observations, we hypothesized that acetylation or succinylation of PEI would also increase repulsions and reduce attractions, leading to less compact DNA packaging. This hypothesis was confirmed by X-ray experiments, where acPEI/DNA polyplexes demonstrated a continuous increase in DNA-DNA spacings with higher modification levels. Conversely, zPEI showed an initial increase in spacings with low levels of succinylation, which then decreased at higher levels, suggesting a complex interaction between succinylated PEI and DNA. This behavior warrants further investigation. It is notable, however, that the trends in DNA spacings do not correlate with colloidal properties nor polyplex stability for zPEI. Two possible explanations for the decreasing DNA-DNA spacings for zPEI/DNA at modifications above 5% are that DNA changed conformation upon condensation by zPEI or that zPEI is reducing the hydration layer. CD experiments confirmed that zPEI maintains B-form DNA similar to acPEI/DNA. During transfection, polyplexes experience a range of pH conditions, from near-neutral during preparation (comparable to the cytoplasm or blood, pH ~7. 35-7.5) to the acidic environment of lysosomes post-endocytosis. Early endosomes maintain a pH near 6.5, reached within minutes.