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With high potency in treatment of various diseases, gene therapy is mainly hindered by lack of safe and efficient gene delivery vectors. The current study was aimed to develop an efficient non-viral vector with adequate cytotoxicity. To this end, alkylcarboxylate chains (6C, 10C, 16C) were exploited to ameliorate the characteristics of PEI 750 kDa. Briefly, alkylcarboxylate chains with three different lengths (6C, 10C, 16C) were chemically grafted to the primary amine groups of PEI 750 kDa in three percentages (10, 50, and 100%). After evaluating the physicochemical properties of prepared vectors including surface charge, size, buffering capacity, and DNA condensing, their transfection efficiency and cytotoxicity were investigated in Neuro2A cells. The polyplexes size were 158.9–264.5 nm and their zeta potentials were 14–30 mV, while their buffering capacity and DNA condensing were not significantly …