pH‐Dependent Structural Changes in Polyethyleneimine Affect its Gene Transfection Efficiency

Abstract Polyethyleneimine (PEI), a polymeric non‐viral vector for gene delivery, shows rich pH‐dependent behavior. This results in PEI exhibiting a proton‐sponge mechanism during transfection. Recent studies show PEI chains can themselves undergo a specific type of self‐assembly in low‐pH environment. Such assemblies can affect transfection specifically if PEI/PEI‐DNA polyplexes are subjected to such pH conditions. However, the understanding of the correlation between variations in solution pH in which the free PEI chains are conditioned and gene transfection remains limited. Here, it is shown that an interplay of pH‐driven protonation of amines and a pH‐specific hierarchical micro‐structuration in branched PEI alters gene transfection in a temporal fashion. While conditioning of PEI chains in an aqueous solution at pH 1.4 prior to DNA complexation at pH 7.4 renders ~ 2 times efficient transfection, PEI chains routed through pH 3.0 show spatiotemporal deterioration. The decreased transfection is attributed to the self‐assembly of PEI chains between pH 2.5–4.0 to form micron‐sized fibrils, leading to poor cellular uptake and diminished nuclear localization. This study, therefore, unfolds the pH landscape and conditioning times of free PEI chains before DNA complexation under that efficient/inefficient gene transfection can be achieved.