DNA-PEI (polyethylenimine) nanoparticles has recently been used to make non-viral vectors for gene delivery but the transfection efficiency is usually low. This is because they are conventionally produced by mixing the ingredients together in bulk hence the structure of the nanoparticles is dependent on the mixing condition. Since the structure of the nanoparticles greatly affects the cellular entry, intracellular transport, nuclear membrane penetration, and DNA release, one strategy to improve their efficiency is through controlling the structure of the nanoparticles. This can be accomplished by hydrodynamically stretching the DNA to various degrees in conjunction with PEI complexation by using a microfluidic system to produce the well-defined nanoparticles. Atomic force microscopy (AFM) study of these nanoparticles in tapping mode revealed coiled, spherical, toroidal, and other structures at different N:P ratios. In addition, we have also studied the transfection efficiency of these nanoparticles in vitro. To further improve transfection efficiency, PEI will be coupled with receptor-binding ligands, e.g. transferrin, and complexed with DNA hydrodynamically and tested.