Development of Calcium Phosphate Nanoparticles and Their Use as Vaccine Adjuvants, 01-R9831
Sandra J. Drabik
Lucy M. Kimmel
Inclusive Dates: 07/01/08 04/01/11
Background — With the recent threat of bioterrorism, stabilization of antigens or enzymes used in protective vaccines or treatments for potential bioterrorist agents can be an effective method to protect both emergency response teams and the general population. Nanoparticles show several advantages over microparticles when used in immobilization/stabilization applications because of large surface area, and the ability to provide functionalized surfaces for multiple adsorption or attachment sites. The stabilization and controlled release of vaccines by nanoprecipitation could result in new vaccines or drugs. Anthrax protective antigen (rPA) was chosen as a vehicle to demonstrate the stabilization potential of the nanoprecipitation technique. Potential benefits of a successful outcome of this project would be a more stable rPA formulation for use in vaccine development. This project expanded on SwRI nanobased drug development and delivery endeavors. An additional benefit was establishing a collaborative relationship with USAMRID.
Approach — CaP (calcium phosphate) was chosen as a means of delivery for the rPA because it has the proven ability to function as an adjuvant (an adjuvant is needed for an effective immune response to subunit vaccines) and is safer and less controversial than adjuvants currently used in marketed vaccines. CaP nanoparticles were prepared by precipitation prompted by the addition of a calcium precursor solution to a phosphate precursor solution in the presence of one or more charged polymers, which provide stability to the suspension (CaP nanoparticles tend to aggregate very rapidly).
The functional activity of the CaP-rPA complex was developed with a cytotoxicity bioassay performed on a macrophage cell line. The stability of the prepared nanoparticles was determined by exposure to varying environmental conditions of elevated and reduced storage temperature. The stability of the complex was compared to the currently available vaccine formulation and to rPA alone; the complex's activity was confirmed using the cytotoxicity assay. An animal study (mice) was designed to examine the impact of adjuvanation with calcium phosphate-based nano-particles.
Accomplishments — A variety of nanoparticles has been prepared investigating the use of additives and varying formation conditions. The cell-based cytotoxicity assay capability has been added and used on generated particles. Methods for the nanoparticle isolation have been developed that allow the collection, washing and re-suspension of the material. rPA is currently in use for particle preparation; particles have been successfully prepared, washed and lyophilized. A stability study has shown that the generated particles loaded with rPA are more stable than the rPA itself at elevated temperatures. The animal study showed that the particles were non-immunogenic, although they were active in the cell based assay.