Beyond stability, it is crucial for polyplexes to disassociate and release their nucleic acid payloads for transcription to occur. Hence, a delicate balance must be struck in nonviral gene delivery systems to generate particles stable enough to reach cells but not so stable as to prevent intracellular polyplex disassociation. We assessed the stability of acPEI and zPEI polyplexes using a displacement assay with negatively charged dextran sulfate (DS, 9-20 kDa). As a sulfated polysaccharide, DS mimics sulfated extracellular glycosaminoglycans (GAGs), which are thought to competitively displace DNA from polyplexes in vivo. Relative polymer-DNA interaction strengths can be quantified by determining the amount of DS required to displace polycations from DNA. Polyplexes were prepared at a polymer/DNA weight ratio of 6 to ensure complete DNA condensation by all PEIs. After equilibrating the polyplexes at room temperature for 30 minutes, each sample was then incubated with varying amounts of DS for an additional 30 minutes. DNA displacement was evaluated through agarose gel electrophoresis (Figure ). A systematic decrease in the DS concentration needed to release DNA was observed with increasing modification levels for both acPEI and zPEI polyplexes. At this PEI:DNA weight ratio, polyplexes created with unmodified bPEI require DS:DNA mass ratios of approximately 20 to initiate observable DNA displacement. Both acetylation and succinylation decrease polyplex stability, but succinylation yields polyplexes that dissociate at lower DS concentrations. For instance, significant DNA release is evident for zPEI20 at DS/DNA weight ratios of 2, while acPEI20 requires a DS/DNA weight ratio of 15. Presumably, these differences in polyplex formation and stability between zPEI and acPEI can be attributed to the nature of the modification. Acetylation reduces the net charge of bPEI by substituting some secondary or primary amine with uncharged acyl groups, while succinylation more significantly decreases the net polymer charge through conjugation of negatively charged carboxylate groups to PEI amines, resulting in a stronger reduction in polymer:DNA interactions.