Functionalization of calcium phosphate nanoparticles with organig phosphates
The principal goals of this thesis were to functionalize calcium phosphate nanoparticles with organic phosphates and to stabilize the nanoparticles as stable colloid. The intention of this project was approached successfully with some organic phosphates. The results are summarized here. The calcium phosphate nanoparticles were prepared by a continuous precipitation method and immediately stabilized as colloid by rapidly adding an aqueous solution of organic phosphate. A variety of organic phosphates (porphyrin, phytic acid, phosphoamino acid and surfactants) was used to functionalize the calcium phosphate surface. The conditions were optimized to prepare stable monodisperse colloids. Organic phosphate showed a high chemical affinity to bind to the calcium phosphate surface which was confirmed by the zeta potential values (-20 mV…-35 mV). The particle size, morphology, and porosity were determined by the surface-adsorbed organic phosphates. For example, (a) p-TPPP-functionalized particles – aggregation of primary calcium phosphate particles (ca. 20-30 nm) into ca. 250 nm solid spherical particles. (b) Phytic acid-functionalized particles – solid spherical particles. (c) TyrP-functionalized particles – aggregation of primary calcium phosphate particles (ca. 20-30 nm) into ca. 250 nm hollow spherical particles (d) Alkyl phosphate-functionalized particles – needle- or flower-like microparticles. The number of phosphate groups present in organic phosphates and their concentration played a critical role in determining the nature of particle precipitation. For example, the p-TPPP molecule has four phosphate groups. It functionalized the calcium phosphate surface and stabilized the particles in solution only when the p-TPPP to calcium concentration ratio was 1:100. If an excess amount of p-TPPP was used (p-TPPP:Ca2+=1:30), only the calcium salt of p-TPPP was obtained. p-TPPP-functionalized calcium phosphate nanoparticles were prepared and used in cell culture studies to check its viability. In collaboration with the Chair of Molecular Neurochemistry at the University of Bochum, the cell culture experiments were carried out. The p-TPPP-functionalized calcium particles were taken up by the NIH 3T3 fibroblast cell and no adverse effect was observed. This showed a good prospect that the nanoparticles can be used as fluorescing agents without any adverse effects in the cells. A new and simple method to prepare calcium phytate nanoparticles was described where calcium phosphate nanoparticles were used as nuclei. This method works only in a narrow concentration range of phytic acid. The size of the calcium phytate nanoparticles can be adjusted by varying the calcium to phytate ratio. As the colloids of calcium phytate are stable and biocompatible, they may be used in biomedical studies (e.g. liver and spleen scintigraphy) without any other stabilizing agents. The one-step synthesis of hollow particles was described in our study which is easy, simple and more convenient. The hollow spherical particles are formed by the direct self-assembly of the primary calcium phosphate nanoparticles in the presence of an excess amount of O-phospho-L-tyrosine. It was found from SEM measurements that the hollow morphology was not stable under mechanical agitation and thermal conditions. The simple procedure for the preparation of hollow calcium phosphate nanoparticles makes it advantageous over template-assisted method. These hollow particles may be used as drug delivery vehicles. The alkyl phosphate-functionalized calcium phosphate colloids showed less stability. SEM measurements showed aggregated microparticles. The content of the alkyl phosphate in the precipitated particles was (about 10-30 wt%) estimated by elemental analysis. This indicated that the alkyl phosphates on the surface of hydroxyapatite underwent an ion exchange reaction with inorganic phosphate ions. As a result, the surface of the hydroxyapatite particles covered with the hydrophobic calcium salt of alkyl phosphate was found as precipitate.
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