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Poly(amidoamine)s with pendant primary amine (polymer 1a-1c) were evaluated as in vitro non-viral gene delivery vectors for bone marrow stromal cells (BMSCs). The cytotoxicity of these poly(amidoamine)s, measured by MTT assay, increased with increasing length of side chain, however, they were less toxic than branched polyethylenimine (PEI) 25 kDa. Using pGL-3 and pEGFP-C1 as luciferase gene and green fluorescent protein (GFP) gene, among all polycations including polymer 1a-1c and PEI, polymer 1b at optimal N/P ratio showed highest luciferase expression (1.92 x 10(8) RLU/mg protein) as well as percentage of cells expressing GFP (29.01 +/- 2.33%). For all polycations, intracellular trafficking of Cy3-labelled plasmid DNA (pDNA) was similar. Fluorescent particles attached to cell membrane at 0.5 h after adding the polycation/DNA complexes, aggregated in cytoplasm after 2 h. and then stayed around the perinuclear region after 4 h. pDNA nuclear localization appeared at 4 h post-transfection, but much more pDNA entered into nucleus at 24 h. At high N/P ratio, polymer 1a-1c could deliver pDNA into 70-80% of BMSCs after 24 h transfection, however, labelled pDNA was observed in only 4-25% of cells at the same time. Compared to PEI, polymer 1b showed comparable or even higher percentage of pDNA uptake and nuclear localization. We concluded that poly(amidoamine)s with pendant primary amine, especially polymer 1b, are new kind of promising candidates of less toxic and highly efficient non-viral gene delivery vectors for BMSCs.