Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression

This study was conducted in order to develop amphiphilic, low molecular weight polymeric carriers for nonviral gene delivery. Caprylic, myristic, palmitic, stearic, oleic and linoleic acids were grafted onto the 2 kDa polyethylenimine (PEI) and properties critical for gene delivery were investigated using 293T and bone marrow stromal cells. The extent of lipid substitution on the polymers was controlled by the lipid:PEI feed ratio during the synthesis. The toxicity of the native and lipid-substituted 2 kDa PEI was relatively lower than the 25 kDa PEI, although lipid substitution generally increased the toxicity of the polymers in vitro. Lipid substitution reduced the ability of the polymers to complex DNA, as well as the stability of final complexes, as measured by heparin-induced dissociation. Once fully complexed to a plasmid DNA, however, the lipid-substituted polymers increased the plasmid DNA delivery to the cells. In 293T cells, the lipid-substituted polymers displayed a transfection ability that was equivalent to highly effective 25 kDa PEI, but without the toxic effect associated with the latter polymer. Among the lipids explored, no particular lipid emerged as the ideal substituent for transgene expression, although linoleic acid appeared to be superior to other lipid substituents. No correlation was evident between the level of substitution and DNA delivery efficiency of the polymers, and as little as 1 lipid substitution per PEI was effective in transforming the ineffective 2 kDa PEI into an effective carrier. The current structure-function studies are providing important clues about the properties critical for gene delivery and providing carriers effective for nonviral plasmid delivery.