Succinic-based acyclic (non-rigid) and macrocyclic (rigid) cationic lipids were synthesized. The synthetic lipids were formulated with plasmid DNA and a neutral lipid. The in vitro transfection and cytotoxicity of formulations was evaluated using CHO-K1 cells. Introduction of the succinic unit within the hydrophobic domain improved lipid hydration. Mismatched (rigid lipid blended with a non-rigid lipid) outperformed matched formulations.