Preparation of Calcium Hydroxyapatite Nanoparticles Using Microreactor and Their Characteristics of Protein Adsorption

Abstract

The calcium hydroxyapatite Ca(10)(PO(4))(6)(OH)(2) (Hap) nanoparticles were prepared by using microreactor and employed these Hap nanoparticles to clarify the adsorption behavior of proteins. The size of Hap particles produced by the microreactor reduced in the order of a hardness of the reaction conditions for mixing Ca(OH)(2) and H(3)PO(4) aqueous solutions, such as flow rates of both solutions and temperature. Finally, the size of the smallest Hap nanoparticle became 2 × 15 nm(2), similar to that of BSA molecule (4 × 14 nm(2)). It is noteworthy that the smallest Hap nanoparticles still possesses rodlike shape, suggesting that particles are grown along c-axis even though the reactants mixed very rapidly in narrow channels of the microreactors. The X-ray diffraction patterns of the Hap nanoparticles revealed that the crystallinity of the materials produced by the microreactor is low. The FTIR measurement indicated that the Hap nanoparticles produced by microreactor were carbonate-substituted type B Hap, where the carbonate ions replace the phosphate ions in the crystal lattice. All the adsorption isotherms of acidic bovine serum albumin (BSA), neutral myoglobin (MGB), and basic lysozyme (LSZ) onto Hap nanoparticles from 1 × 10(-4) mol/dm(3) KCl solution were the Langmuirian type. The saturated amounts of adsorbed BSA (n(S)(BSA)) for the Hap nanoparticles produced by microreactor were decreased with decrease in the mean particle length, and finally it reduced to zero for the smallest Hap nanoparticles. Similar results were observed for the adsorption of LSZ; the saturated amounts of adsorbed LSZ (n(S)(LSZ)) also reduced to zero for the smallest Hap nanoparticles. However, in the case of MGB, the saturated mounts of adsorbed MGB (n(S)(MGB)) are also depressed with decreased in their particle size, but about half of MGB molecules still adsorbed onto the smallest Hap nanoparticles. This difference in the protein adsorption behavior was explained by the difference in the size and flexibility of three kinds of proteins. The reduction of n(S)(BSA) is due to the decrease in the fraction of C sites on the side face of each Hap nanoparticle; i.e., there is not enough area left on the nanoparticle surface to adsorb large BSA molecules even though the BSA molecules are soft and their conformations are alterable. The reduction of n(S)(LSZ) was explained by the reduction of P sites. Further, rigidity of the LSZ molecules was given another possibility of the depression of n(S)(LSZ) for the Hap nanoparticles. However, MGB molecules with small and soft structure were adsorbed on the Hap nanoparticle surface by changing their conformation. We could control the amounts of adsorbed proteins by changing the particle size of Hap in the nanometer range and kinds of proteins. These obtained results may be useful for developing biomimetic materials for bone grafts and successful surgical devices in the biochemical field.

Hydroxylapatite nanoparticles: fabrication methods and medical applications

“…They reported that the particle size decreased sharply with increasing the continuous-phase flow rate, and a narrow size distribution was achieved at a high flow rate. Kandori et al [103] recently prepared smaller HAp nanoparticles (2 nm in width, 15 nm in length) by using a multichannel microreactor.…”

Biomimetic mineralization of nano-sized, needle-like hydroxyapatite with ultrahigh capacity for lysozyme adsorption

“…Accordingly, the maximum adsorption capacity of HA/CMC towards lysozyme was determined to be around 930-940 mg/g. Note that such lysozyme adsorption capacity is higher than that reported in literature [14,22]. This thus signifies the dual role of CMC, that is, as the template to regulate the biomimetic mineralization of HA with high affinity to proteins and itself as an effective adsorbent to immobilize the lysozyme.…”

“…The solubility of HA increases with the decrease in the pH value of solution, that is, HA can be dissolved into nontoxic Ca 2+ and PO 4 3− ions in aqueous solution with a low pH value, e.g., in lysosome after cellular uptake or in acidic environment of solid tumors. The adsorption and release of proteins are strongly affected by the chemical and structural characteristics of HA [6,14], For instance, it has been shown that the needle-like HA exhibits a higher protein adsorption capacity than the spherical counterpart due to an increased adsorption sites on the surface [15]. Therefore, development of new HA with a high protein adsorption capacity remains attractive for protein drug delivery.…”

Fabrication of calcium phosphate nanoparticles in a continuous flow tube reactor

“…3) Tricalcium phosphate, Ca 3 (PO 4 ) 2 (TCP; Ca/P = 1.5) and Hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 (HAp; Ca/P = 1.67) are stable in the basic region. 4) HAp has been studied frequently among those phases, because it is excellently biocompatible and it is similar in structure and bio-chemical properties to inorganic materials involved in human bone and tooth as the primary inorganic component.…”

“…Some researchers have reported that HAp was prepared with using a micro-reactor system. 10)12) The micro-reactor system may suppress such homogeneity or heterogeneity within a controllable limit of primary factors such as diffusion of solution, pH, reaction temperature and aging time after the reaction. The primary factors directly affect both the phase and the morphology of the yielded CPs crystallites.…”