Genetic immunization using nanoparticles engineered from microemulsion precursors

Purpose. Genetic immunization using "naked" plasmid DNA (pDNA) has been shown to elicit broad humoral and cellular immune responses. However, more versatile and perhaps cell-targeted delivery systems are needed. To this end, a novel process to engineer cationic nanoparticles coated with pDNA for genetic immunization was explored. Methods. Cationic nanoparticles were engineered from warm oil-in-water microemulsion precursors composed of emulsifying wax as the oil phase and cetyltrimethylammonium bromide (CTAB) as the cationic surfactant. Plasmid DNA was coated on the surface of the cationic nanoparticles to produce pDNA-coated nanoparticles. An endosomolytic lipid and/or a dendritic cell-targeting ligand (mannan) were incorporated in or deposited on the nanoparticles to enhance the in vitro cell transfection efficiency and the in vivo immune responses after subcutaneous injection to Balb/C mice. The IgG titer to expressed beta-galactosidase and the cytokine release from isolated splenocytes after stimulation were determined on 28 days. Results. Cationic nanoparticles (around 100 nm) were engineered within minutes. The pDNA-coated nanoparticles were stable at 37degreesC over 30 min in selected biologic fluids. Transmission electron microscopy showed the nanoparticles were spherical. Plasmid DNA-coated nanoparticles, especially those with both an endosomolytic lipid and dendritic cell-targeting ligand, resulted in significant enhancement in both IgG titer (over 16-fold) and T-helper type-1 (Th1-type) cytokine release (up to 300% increase) over "naked" pDNA. Conclusion. A novel method to engineer pDNA-coated nanoparticles for enhanced in vitro cell transfection and enhanced in vivo immune responses was reported.