One-step formation of plasmid DNA-loaded lipid-inorganic salt nanoparticles optimized via two-step design of experiments.

For successful gene delivery, researchers need to consider multiple processes, such as the endosomal escape of cargos. The preparation of highly functional systems is complicated. Here, we developed a simplified preparation method for plasmid DNA-loaded galactosylated lipid-inorganic salt nanoparticles, which adopts a one-step alcohol injection method for the formation of lipid nanoparticles. The nanoparticles were designed to have a core of plasmid DNA with inorganic salts consisting of calcium carbonate and phosphate for endosomal escape and a lipid shell for the stable formation of nanoparticles. The formulation and process parameters were optimized using a two-step design of experiments, that is, a definitive screening design followed by a central composite design. We successfully established uniform nanoparticles of 120 nm in size based on micropipette mixing. Transfection efficiency of the nanoparticles prepared via micropipette mixing was equivalent to that of a commercially available transfection reagent in human hepatocellular carcinoma HepG2 cells. Using a microfluidic device, the nanoparticle size was decreased to 70 nm, while a high transfection efficiency was maintained in HepG2 cells. Moreover, the effect of inorganic salts on the nanoparticle transfection efficiency was evaluated. Notably, the formation of a calcium carbonate core was crucial for achieving an effective endosomal escape and a high transfection efficiency. Overall, this study provides valuable information for the design and simplified preparation of lipid-inorganic salt nanoparticles for efficient gene delivery.