| New generation vaccines such as protein (subunit) vaccines and DNA vaccines are generally poorly immunogenic, although they may be potentially safer than other traditional vaccines such as live attenuated vaccines and killed vaccines. Therefore, there exists a clear need to develop alternative delivery systems and/or adjuvants to improve the resulting immune responses. We sought to address this need by developing a novel nanoparticle-based system. The nanoparticles were engineered from microemulsion precursors by simple cooling of the preformed warm microemulsions to room temperature in the same container. The microemulsions were comprised of emulsifying wax as the oil phase and different surfactants, either cationic, neutral, or anionic. Using this procedure, spherical and uniform nanoparticles (∼100 nm) with different charge on their surface were readily engineered. The nanoparticles were characterized using photon correlation spectroscopy, laser Doppler electrophoresis, differential scanning calorimetry, and transmission electron microscopy. Gel permeation chromatography was applied to purify the nanoparticles. The size of the nanoparticles increases in aqueous suspension. However, lyophilization of the nanoparticles with appropriate cryprotectant help to maintain the stability. Both Endosomolytic lipids (cholesterol and dioleoyl phosphotidylethanolemine) and potential cell targeting ligands (mannan for antigen-presenting cells and pullulan for hepatocytes) were successfully incorporated or deposited on the surface of the nanoparticles. A plasmid DNA encoding β-galactosidase gene and cationized β-galactosidase protein were then adsorbed on the surface of the cationic and anionic nanoparticles, respectively, for potential DNA- or protein-based vaccine delivery. The plasmid DNA-coated nanoparticles resulted in enhanced immune responses, both in breadth and depth, over the ‘naked’ pDNA alone when administered by subcutaneous, topical on skin, intranasal, and intradermal routes to Balb/C mice. For example, the antigen-specific IgG titer in the sera of the mice immunized with pDNA-coated nanoparticles was 16–200-fold greater than that in the mice immunized with ‘naked’ pDNA alone. In addition, it was found that co-administration of known adjuvants such as cholera toxin by non-invasive topical route and lipid A by subcutaneous route with the pDNA-coated nanoparticles can further enhance the resulting immune responses. Finally, the cationized β-galactosidase-coated nanoparticles also led to enhanced immune responses over native or cationized β-galactosidase protein alone. |
