Effective Immobilization of Monomeric Methylene Blue on Hydroxyapatite Nanoparticles by Controlling Inorganic-Organic...

We successfully synthesized methylene blue (MB⁺)-immobilized hydroxyapatite (HM) nanoparticles by changing the initial P/Ca molar ratio. The immobilized amount of MB⁺ increased with increasing the initial P/Ca molar ratio from 0.6 to 4.0, and the HM had an elliptical shape (long length, 21-24 nm; short length, 11-13 nm) irrespective of the initial P/Ca molar ratio. Upon increasing the initial P/Ca molar ratio, the number of carbonate ions on the HM surface decreased, which would be owing to the electrostatic repulsion by the surface phosphate ions (i.e., P-O^-), the surface P-OH mainly dissociated to form P-O^-, and the electrostatic interaction of P-O^- with MB⁺ enhanced. The bonding of MB⁺ with surface P-OH and Ca²⁺ sites of hydroxyapatite would be hydrogen-bonding and Lewis acid-base interactions, respectively. The optimum synthesis condition for MB⁺ immobilization at the monomer state was found to be the initial P/Ca molar ratio of 2.0. Here, the existence percentage of the MB⁺ monomer and the molecular occupancy of the surface carbonate ion at the initial P/Ca molar ratio of 2.0 were higher than those at 4.0 with no significant difference in the immobilized amount of MB⁺, indicating that MB⁺ at the initial P/Ca molar ratio of 4.0 is more aggregated than that at 2.0. These results suggested that a part of carbonate ions has a role as a spacer to suppress MB⁺ aggregation. Accordingly, the interfacial interactions between the MB⁺ monomer and the hydroxyapatite surface were clarified, which can effectively be controlled by the initial P/Ca molar ratio. These findings will provide fundamental and useful knowledge for the design of calcium phosphate-organic nanohybrids. We believe that these particles will be the base materials to realize diagnostic and/or therapeutic functions through the molecular state control by optimizing the synthesis conditions.