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Polyethylenimine is a popular DNA transfection reagent, and many approaches have been explored to further enhance its transfection efficiency. Substitution of branched polyethylenimine's primary amine groups is an attractive approach because it is amenable to a variety of chemistries and is also implicated as a primary factor in its cytotoxicity. The purpose of this work was to serially substitute saturated hydrocarbons to branched polyethylenimine and determine what structure/function relationships exist between the hydrocarbon length and its degree of substitution, relative to transfection efficiency in multiple cell lines. Specifically, acetate, butanoate and hexanoate were conjugated to branched polyethylenimine (Mw = 25,000) using an aqueous condensation protocol. Transfections were performed in culture using HeLa, NIH/3T3 and Clone 9 cell lines. Biophysical characteristics of the polyelectrolyte complexes were also measured (hydrodynamic diameter, relative binding affinity) and correlated to transfection efficiency. The results show that substitution of the primary amines generally increases transfection efficiency relative to unconjugated polyethylenimine, but increasing the degree of substitution beyond ˜ 25 mol% generally decreases transfection efficiency from the optimum. Additionally, increasing hydrocarbon length generally decreased transfection efficiency. There was little correlation between particle size and binding efficiency to transfection efficiency.