Impact of Acetylation, Succinylation, and pH on PEI-Based Polyplex Structure and Function

Polyethylenimine (PEI), a widely used cationic polymer for nonviral gene delivery, often undergoes modification to enhance its transfection efficiency while reducing cytotoxicity. This study systematically examines the effects of acetylation and succinylation on PEI’s physicochemical properties, transfection efficiency, and DNA packaging within polymer-DNA complexes called polyplexes. We synthesized and characterized a range of acetylated (acPEI, 10-50%) and succinylated (zPEI, 2-40%) PEIs, assessing their capacity to condense and deliver DNA effectively. Both acetylation and succinylation modify the colloidal and charge properties of PEI polyplexes, enhance DNA release, and alter transfection efficacy. In particular, low levels of succinylation substantially improve transgene expression in the presence of serum proteins. Structurally, acetylation consistently reduces DNA packaging density, while succinylation exhibits a more complex behavior where DNA packing density first decreases then increases with higher levels of modification. Furthermore, we demonstrate that the internal structure of PEI polyplexes is profoundly affected by pH during condensation and the pH environment during transfection. Lower pH conditions strengthen PEI-DNA interactions, resulting in polyplexes that are not only more stable, but also exhibit tighter DNA packaging. Critically, we show that pH changes, akin to those that occur during the endocytic pathway in mammalian cells, not only further compact DNA within PEI polyplexes, but also facilitate the release of PEI molecules from the complex, potentially aiding in endosomal escape.