This pH can drop further to a lysosomal pH of 4 within an hour. Unlike most polycations with amines on the polymer side chain, PEI features amine groups within its polymer chain backbone, separated by only two methylene groups. The abundance and proximity of these amine groups contribute significantly to PEI's large buffering capacity. At physiological pH, around 20% of PEI amines are charged, increasing to about 45% under acidic conditions. Since 25-kDa bPEI consists of approximately 581 amines per polymer chain, this corresponds to ~116 amines being charged at pH 7 but ~261 amines charged at pH 5. Our studies demonstrate that the pH during condensation profoundly influences polyplex structure and functionality, likely due to the increased charge state of the PEI. Condensation at lower pH results in tighter DNA packaging across all three PEI systems. Moreover, polyplexes formed at these lower pH levels require less polymer per DNA weight to form and have enhanced stability. This finding aligns with previous research showing that acidic conditions during polyplex formation reduce transfection efficacy, while acidic growth media can enhance it. Time course studies show the PEI polyplexes become trapped in nonequilibrium states due to the strong binding of the polycation initially, but can rearrange to a more equilibrium state over time. These rearrangements, however, are pH dependent with polyplexes maintained at one pH showing significantly less rearranging than a neutral polyplex that is subsequently acidified.