Impact of Acetylation, Succinylation, and pH on DNA Packaging in PEI-Based Polyplexes

Polyethylenimine (PEI) is a widely used cationic polymer for nonviral gene delivery, often modified to enhance transfection efficiency and reduce cytotoxicity. This study investigates how acetylation, succinylation (acPEI and zPEI), and pH influence the internal DNA packaging of polyplexes. Both modifications alter physicochemical properties, leading to complexes that decondense more readily with increasing modification. X-ray scattering reveals that high acetylation produces loosely packed DNA, while succinylation unexpectedly tightens DNA packing at higher modification levels. Polyplexes formed at low pH (pH 4) are more stable and tightly packed than those formed at pH 7.5. Acidifying polyplexes initially formed at pH 7.5 induces structural rearrangement to tighter DNA packing accompanied by significant PEI release, providing direct evidence for models where free PEI aids endosomal escape. These findings challenge conventional assumptions about PEI behavior and offer new insights into DNA packaging, emphasizing tailored polymer modifications and pH conditions to optimize gene delivery.