Improved application of the inverse emulsion method for generating flexible asymmetric liposomes for DNA plasmid delivery

The design of vehicles for transdermal gene delivery is at the forefront of molecular medicine, facilitating targeted therapies. Reports suggest that flexible liposomes can be a good alternative for transdermal delivery, and asymmetric liposomes may enhance gene delivery efficiency. This study aims to create flexible asymmetric-type liposomes with high encapsulation of DNA and high deformability rates. The synthesis of asymmetric liposomes was standardized using the inverse emulsion method, with lipids DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) as the inner layer, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) lipid as the outer layer, cholesterol as a stabilizing component, and Span 80 and ethanol as components that promote flexibility. The pIRES2-EGFP plasmid was used as the encapsulated genetic material. Asymmetric liposomes were characterized using transmission electron microscopy (TEM), encapsulation efficiency percentage (%EE), and the deformability index determined by the extrusion method. Results indicate that the asymmetric liposomes possess a well-defined bilayer, with bilayer deformability varying depending on the components used; for instance, liposomes containing flexible components exhibit a more deformable bilayer than those made solely of lipids. The average size of the liposomes was below 200 nm, and the %EE ranged from 75% to 90%. The liposomes containing Span 80 surfactant exhibited the highest flexibility index. This technique successfully produced asymmetric liposomes with appropriate encapsulation of the DNA plasmid without degradation during the process. Future studies are expected to evaluate the cytotoxicity, transfection, and skin permeation.