CpG Molecular Structure Controls Mineralization of Calcium Phosphate Nanoparticles and their Immunostimulation Efficacy as Vaccine Adjuvants

Co-precipitation of calcium phosphate nanoparticles (CaP) in the presence of nucleotide chains such as polynucleotides (i.e., plasmid DNA and siRNA) and oligonucleotides has been extensively used for pre-clinical gene or drug delivery and immunotherapy studies. However, the exact role of these molecules in mineralization and tuning the physiochemical characteristics of synthesized CaP’s are still not entirely clear. In this study, we evaluated effects of three different CpG oligodeoxynucleotides (ODN) and two representative nucleic acids (siRNA and DNA), when used as templates for formation of CaP’s. We examined the influence of CpG’s with naturally-occurring phosphodiester or modified phosphorothioate backbones on the homogenous formation of CaP’s from a modified simulated body fluid solution. Hydrodynamic size, size polydispersity, morphology and surface charge of the CaP’s were used as the most critical checkpoints to unravel the involved mechanisms. Our results show that CaP’s characteristics are highly dependent on composition, backbone, sequence and concentrations of the CpG’s. CpG type and concentration control the size distribution of the mineralized CaP’s and their immunostimulation performance as verified by activation of dendritic cells and secretion of pro-inflammatory interleukin-6 (IL-6) cytokine, type I interferon-α (IFN-α) and co-stimulatory CD80, CD86 and CD40 markers. This study paves the ways for better design of more efficient CaP’s loaded with different types of CpG’s for immunostimulation applications as vaccine adjuvants.