Quantitative determination of the composition of multi-shell calcium phosphate-oligonucleotide nanoparticles and their application for the activation of dendritic cells.

Biodegradable calcium phosphate nanoparticles as carriers for the immunoactive toll-like receptor ligands CpG and polyinosinic-polycytidylic acid for the activation of dendritic cells (DC) combined with the viral antigen hemagglutinin (HA) were prepared. A purification method based on ultracentrifugation and ultrasonication was developed to separate the nanoparticles from dissolved biomolecules. The number of biomolecules, i.e., oligonucleotides and peptide, incorporated into the nanoparticles was quantitatively determined by UV-spectroscopy, using fluorescent derivatives of the biomolecules. The immunostimulatory effects of purified calcium phosphate nanoparticles on DC were studied, i.e., cytokine production and activation of the cells in terms of the upregulation of surface molecules. Purified calcium phosphate nanoparticles, i.e., without dissolved biomolecules, are capable of inducing adaptive immunity by activation of DC. Immunostimulatory effects of purified calcium phosphate nanoparticles on DC were demonstrated by increased expression of co-stimulatory molecules and MHC II and by cytokine secretion. In addition, DC treated with purified functionalized calcium phosphate nanoparticles induced an antigen-specific T-cell response in vitro.