||A systematic approach to determine the parameters that will dictate the design of functional gene carriers has begun to be implemented. Poly(L-histidine) (PLH) was shown to induce membrane fusion at endosomal pH values, whereas poly(L-lysine) (PLL) has been shown to condense plasmid DNA into small particles. N-Ac-poly(L histidine)-graft-poly(L-lysine) (PLH-g-PLL) was synthesized by grafting poly(L-histidine) to poly(L-lysine). PLH-g PLL formed complexes with DNA, where particle sizes ranged from 117 ± 6 nm to 306 ± 77 nm. PLH-g-PLL showed higher transfection efficiency and lower toxicity in 293T cells compared to PLL at all equivalent weight ratios. For the purpose of targeting cancer cells, folate-polyethylene glycol)-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized at a range of grafting ratios of folate-polyethylene glycol)-folate (FPF) to polyethylenimine (PEI). FPF-g-PEIs have 2.3, 5.2, 9.3 and 20 FPFs grafted to PEI. Molecular weight was ?34,848, 47,266, 64,823 and 110,640 Da, respectively. Atomic force microscopy showed FPF-2.3g-PEI and PEI condensed DNA into oblique spheroid complexes with a diameter of ?150-200 nm. Transfection efficiency was different for CT-26 colon adenocarcinoma cells, KB oral epidermoid and normal smooth muscle cells (SMC). In CT-26 cells, FPF-2.3g-PEI and FPF-5.2g-PEI/pLuc complexes gave higher transfection efficiencies compared to PEI/pLuc complexes. In KB cells folate targeting was observed at low charge ratios, but nonspecific ionic association was observed at neutral charge ratio of FPF-g-PEI/pLuc. Smooth muscle cells showed no folate receptor activity, where PEI/pLuc complexes had superior transfection efficiencies. Free folic acid was shown to inhibit transfection with FPF-2.3g-PEI/pLuc in CT-26 cells. At neutral charge ratio, increase in the concentrations of complexes increased transfection. At 3/1 (+/?) ratio, increase in complex concentrations increased toxicity, and decreased transfection efficiency. Cytotoxicity was determined using an MTT assay. FPF-g-PEIs were less toxic compared to PEI. This decrease in toxicity was observed in all cell lines. Compared to PEI/pLuc complexes at neutral charge ratio, FPF-g-PEI/pLuc complexes were less toxic to the cells. When neutral complex concentrations increased, toxicity did not markedly increase. However, when charged complex concentrations increased, toxicity was increased.