Novel ethanol-in-fluorocarbon microemulsions for topical genetic immunization

Purpose. Traditionally, vaccines have been administered by needle injection. Topical immunization through the intact skin with either protein- or DNA-based vaccines has attracted much attention recently. We sought to enhance the immune responses induced by DNA-based vaccines after topical application by developing novel ethanol-in-fluorocarbon (E/F) microemulsion systems to aid in the delivery of plasmid DNA (pDNA). Methods. Ten different fluorosurfactants were selected or synthesized and screened by pseudo-phase-diagram construction for their ability to form E/F microemulsions. Plasmid DNA was successfully incorporated into E/F microemulsions using several different fluorosurfactants and perfluorooctyl bromide as the continuous fluorocarbon phase. For several reasons, Zonyl(R) FSN-100 (an ethoxylated nonionic fluorosurfactant) was selected for further studies. In vivo studies were performed in mice to assess pDNA expression in skin and immunologic responses after topical application of this system using a luciferase-encoding plasmid (CMV-luciferase) and a CMV-beta-galactosidase-encoding plasmid, respectively. Results. Plasmid DNA incorporated into E/F microemulsion using FSN-100 as the surfactant was found to be stable. After topical application of this E/F microemulsion system, significant enhancements in luciferase expression and antibody and T-helper type-1 biased immune responses were observed relative to those of "naked" pDNA in saline or ethanol. For example, with the E/F microemulsion system, the specific serum IgG and IgA titers were increased by 45-fold and over 1000-fold, respectively. Conclusion. A novel fluorocarbon-based microemulsion system for potential DNA vaccine delivery was developed.