Synthesis of Synthetic Amorphous Magnesium-Substituted Calcium Phosphate Particles

Synthetic amorphous magnesium-substituted calcium phosphate (sAMCP) nanomineral particles were prepared as previously described (20 (link)). Briefly, for the preparation of particles incorporating PGN (Staphylococcus aureus, Fluka), PGN was added to phosphate (PO₄) solution [containing 39 mM (NH₄)HPO₄ (Sigma-Aldrich) in 0.15 M Tris buffer, adjusted to pH 8 with hydrochloric acid], prior to mixing in equal parts with calcium–magnesium–BSA solution (BSA; Sigma-Aldrich Company Ltd., Dorset, UK) dissolved in a Ca/Mg solution [containing 35 mM CaCl₂ (Sigma-Aldrich), 7.2 mM MgCl₂ (Sigma-Aldrich) in 0.15 M Tris buffer (Sigma-Aldrich)] at 1 mg/mL for a final PGN concentration of 50 μg/mL mixed solution. For particles incorporating protein purified derivative (PPD) of tuberculin (Statens Serum Institute, Denmark), PPD was added to the phosphate solution prior to mixing with the calcium–magnesium–BSA solution for a final PPD concentration of 100 μg/mL, which was then incubated with gentle rotation at room temperature for 1 h to allow particle formation. Particles were precipitated by centrifugation (1,500 rpm for 5 min), washed in pH 10 water, followed by washing in tissue culture grade water before re-suspending in tissue culture media at half of the original particle solution volume.