Key Molecular Parameters of PEI for Efficient DNA Delivery Into HEK293T Cells

Abstract

The development of polyplexes from natural or synthetic polycations and nucleic acids remains limited by intracellular barriers, which restrict efficient protein expression. A fundamental understanding of how polymer molecular design influences these processes is therefore essential. Here, we investigate the relationships between polymer molecular architecture, supramolecular organization and the intracellular delivery efficiency of poly(ethylenimine) (PEI)/DNA polyplexes. Four PEIs differing in molecular weight and structure were used as model polycations to correlate polyplex physicochemical properties with biological performance. We show that PEI molecular design influences polyplex stability in biological media, cellular uptake, intracellular trafficking, transfection efficiency and cytotoxicity, together with differences in the supramolecular organization of the polyplexes. A correlation between the percentage of green fluorescent protein‐positive cells and internalized nucleic acid identifies key molecular features associated with both polyplex uptake and protein expression in HEK293T cells. Notably, branched PEI of 25 kg/mol forms small, highly stable polyplexes exhibiting hierarchical inter‐complex organization, as revealed by small‐angle x‐ray scattering. This hierarchical organization is associated with enhanced cellular internalization, intracellular trafficking and transfection efficiency. Overall, this study provides new insights into structure–property–function relationships linking polymer molecular design, polyplex organization and intracellular delivery efficiency.