Hiroko Fukano

Synthesis of uniform and dispersive calcium carbonate nanoparticles in a protein cage through control of electrostatic potential.

We have synthesized calcium carbonate nanoparticles (Ca-NPs) in the cavity of a cage-shaped protein, apoferritin, by regulating the electrostatic potential of the molecule. The electrostatic potential in the cavity was controlled by pH changes resulting from changes in the dissolved carbon dioxide (CO(2)) concentration in the reaction solution. Recombinant L-apoferritin was mixed with a suspension of calcium carbonate (CaCO(3)), and the mixture was pressurized with gaseous CO(2) at 2 MPa. The pH of the solution decreased from 9.3 to 4.4; the CaCO(3) dissolved during pressurization, and then precipitated after the pressure was reduced to ambient. After repeating the pressurization/depressurization process three times, about 70% of the apoferritin molecules were found to contain nanoparticles with an average diameter of 5.8 ± 1.2 nm in their cavity. Energy-dispersive X-ray spectroscopy and electron diffraction analysis showed that the nanoparticles were calcite, one of the most stable crystal forms of CaCO(3). Electrostatic potential calculations revealed a transition in the potential in the apoferritin cavity, from negative to positive, below pH 4.4. The electrostatic potential change because of the change in pH was crucial for ion accumulation. Since the Ca-NPs synthesized by this method were coated with a protein shell, the particles were stably dispersed in solution and did not form aggregates. These Ca-NPs may be useful for medical applications such as synthetic bone scaffolds.

In Vitro Synthesis of Calcium Nanoparticles Using the Protein Cage of Apoferritin

Recently the creation of calcium compounds with a highly controlled ultrastructure is noted as next generation materials for biomedical applications. Here we propose the novel method for synthsizing calcium nanoparticles using iron strange protein, apoferritin. Apoferritin was incubated in saturated Ca(HCO3)2 solution at 18 °C. Temperature of the reaction solution was then increased to 37 °C and left for 2 hours to make CaCO3 sedimentated. After removing the sediments in the bulk solution by centrifugation, the supernatant was concentrated. Saturated Ca(HCO3)2 was added to it and the mixed solution was incubated at 37 °C for 30 min. This process was repeated four times. With a Transmission Electron Microscope (TEM), nearly spherical particles with a diameter of about 6 nm were observed to form in the cavity of apoferritin. The nanoparticles were observed to have a lattice structure of spacing about 0.22 nm with high resolution TEM. With Energy Dispersive X-ray spectroscopy (EDS) analysis, the peak of Ca (Kα; 3.7 keV) was detected from a synthesized nanoparticle. According to the solvent condition, nanoparticles formed in the apoferritin cavity would be CaCO3.