Polyethyleneimine (PEI) is considered a promising cationic polymer in non-viral gene delivery. DNA binding properties and other biochemical characteristics of PEI such as the proton sponge phenomenon, offered the branched 25 kDa PEI to be widely used for therapeutic DNA delivery, although the possible cytotoxic effects and the best conditions of PEI preparation are not still well recognized. While higher PEI/Plasmid ratios have increased transfection efficiencies, it induces more cell stress and toxicity. Considering that the PEI particle size and resulting cytotoxicity are affected by media ions, we used Neuro2A cells to assess the cell stress properties of PEI/Plasmid complexes prepared in a HEPES-buffered saline medium. Delivery of a plasmid containing EGFP happened in all increasing ratios of PEI/plasmid from 0.5, 2, 4, and 6, while higher ratios induced less unfolded protein response as evidenced by lower transcription of ER stress markers Grp78, Atf4, Chop, Xbp1, and induced Xbp1 splicing. These data were also supported by MTT cytotoxicity assay results. These findings indicate that preparing higher PEI/plasmid ratio complexes (using the equivalent of 200 ng DNA) in the HBS medium leads to less cytotoxicity.

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