The development of optimized plasmid DNA delivery systems is necessary to deliver genetic material for effective gene therapy. To achieve this aim, a novel SLN: pDNA system was developed and the influence of the lyophilization procedure and the role of cryoprotectants in establishing the system's characteristics and stability were examined. SLNs were prepared by modified microemulsion dilution method. Afterward, a green fluorescent protein expression plasmid was loaded into the SLN system via electrostatic interactions. To examine the effects of cryoprotectants on the lyophilization process, sucrose, mannose, and trehalose were added at different ratios into the dilution medium; Tween 80 was also studied as an outer surfactant without cryoprotectants by incorporating it into the dilution medium. The system was characterized before and after the lyophilization procedure and tested with pDNA-loaded and unloaded SLNs. The compact form of the SLN: pDNA complexes were dissociated with cryoprotectants except %5 sucrose. Redispersing lyophilized SLNs and forming complexes with pDNA in an aseptic environment would be suitable at the application stage.