Unmodified bPEI/DNA (w/w 3) yielded nanoparticles averaging 83.5 (± 5) nm in size, with a zeta potential of +12.3 (± 1.8) mV, suggesting a compact polyplex formation with a positive surface charge. The size of acetylated PEI polyplexes did not significantly vary with increased levels of modification, resulting in particle sizes between 87.4 and 97.1 nm for 10 to 50% acetylation. A modest decrease in zeta potential was observed, from approximately +18.9 mV for acPEI10 to +13.2 mV for acPEI50. Succinylation levels in zPEI, however, more strongly influenced polyplex size. The diameter of the polyplexes for zPEI/DNA (w/w 3) increased proportionally with the percentage of modification, ranging from 98 nm for zPEI2/DNA to 181 nm for zPEI20/DNA. Fully condensed zPEI30/DNA (w/w 6) also formed polyplex nanoparticles averaging 200 nm in diameter. Despite succinylation levels varying between 2% and 20%, the polyplex charge was not significantly altered, with zPEI20/DNA maintaining a positive zeta potential of +14.5 mV. A notable shift in particle charge was evident at 30% succinylation (w/w 6), as particles displayed a net negative surface charge of -7.8 mV. These unexpected results, however, are in line with our prior zPEI study. Lastly, we assessed the transfection efficacy of these newly synthesized PEIs in the presence of serum proteins. HeLa cells were transfected in the presence of 10% FBS with polyplexes of unmodified bPEI, zPEI, or acPEI to determine luciferase expression at varying PEI:DNA weight ratios (Figure ). Gene expression from unmodified bPEI typically decreases by one to two orders of magnitude in the presence of serum proteins compared to serum-free transfections. Here, bPEI/DNA demonstrated gene expression levels in HeLa cells with serum proteins present, ranging between 10 5 -10 6 RLU/mg protein across polymer/DNA weight ratios from 1 to 6. As expected from earlier results, zPEI/DNA with low levels of succinylation (< 10%) exhibited the highest transgene expressions, especially at polymer:DNA weight ratios of 3 or higher. For instance, zPEI5 (w/w 3) demonstrated a 73-fold higher transgene expression than bPEI (w/w 3), comparable to the transgene expression observed in bPEI transfected under serum-free conditions.