LPD vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA and liposomes. Here, we described a novel anionic LPD formulation containing protamine-DNA complexes and pH sensitive liposomes composed of DOPE and cholesteryl hemisuccinate (Chems). Central composite design (CCD) was employed to optimize stable LPD formulation with small particle size. A three factor, five-level CCD design was used for the optimization procedure, with the weight ratio of protamine/DNA (X1), the weight ratio of Chems/ DNA (X2) and the molar ratio of Chems/DOPE in the anionic liposomes (X3) as the independent variables. LPD size (Y1) and LPD protection efficiency against nuclease (Y2) were response variables. Zeta potential determination was utilized to define the experimental design region. Based on experimental design, responses for the 15 formulations were obtained. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The mathematical model predicted optimized X1-X3 levels that achieve the desired particle size and the protection efficiency against nuclease. According to these levels, an optimized LPD formulation was prepared, resulting in a particle size of 185.3 nm and protection efficiency of 80.22%.