The Studies On Nano-sized Hydroxyapatite As A Controlled Released Carrier Of Protein Drug

Hydroxyapatite, as the main mineral component of bones and teeth, is well known for its higher biocompatibility and stability than traditional polymer materials. In recent years, there has been widely interest in nano-sized hydroxyapatite (nsHAp) as a controlled carrier for bioactive drug because it has many advantages such as high solubility, big surface areas and biocompatibility compared to hydroxyapatite. Therefore, nsHAp-Protein compound have been prepared by adsorption and in-situ method respectively and the controlled release mechanism of nsHAp-Protein has been also explained. The interaction between nsHAp and protein drug has been investigated by Fourier transform infrared spectrometer (FT-IR). The associated research works are listed mainly as following:First, nano-sized hydroxyapatite has been successfully synthesized by precipitation and sol-gel method. The nsHAp is characterized by means of X-ray diffraction (XRD), FT-IR, scanning electronic microscopy (SEM), transmitting scanning microscopy (TEM) and N2 adsorption experiment. As a result, compared with the nsHAp synthesized by precipitation method (nsHApⅠ), the one synthesized by sol-gel method (nsHApⅡ) with poor crystallity has similar composition and structure to human natural bone apatite.Second, we used bovine serum albumin (BSA) and Ovalbumin (OVA) as model proteins to synthesize nsHAp-Protein compound by adsorption method. At the same time, many factors which influenced the amounts of adsorption such as adsorption time, acidity and irons were discussed in detail. Theadsorption results which were realized through the electronic interaction between adsorbent and adsorbate and"calcium bridging"showed a typical Freundlich curve. The adsorbed amounts in the presence of PO43- when maintaining the same pH show a notable decrease because Ca sites on the surface of nsHAp are occupied by PO43-. And the fact that the addition of Ca2+ dramatically increases the adsorbed amount indicates Ca2+ is bound onto P sites of nsHAp surface. The systematic research will lay a solid theoretical foundation for the preparation of nsHAp-Protein drug.In the passage, the bioactive enzyme - superoxide dismutase (SOD) was encapsulated into nano-sized hydroxyapatite by in-situ method for the first time. It is significant that SOD encapsulated into nsHAp retain its bioactivity and stability. The characteristic of nsHAp-SOD compound by means of XRD, RT-IR, SEM, TEM and N2 adsorption experiment indicates no structural change by comparing with nsHAp. In addition, the effect of initial concentration of SOD on drug-loaded amount (DLA) of the compound is examined. At the initial stage, DLA increased with increasing the initial concentration. The results show that DLA reached 61mg SOD/g nsHAp when the initial concentration of SOD is 1mg/ml.Third, the release mechanism of nsHAp-Protein compound was explained systemically through in-vitro release experiment. For nsHAp-BSA and nsHAp-OVA synthesized by adsorption method, the release rate was influenced as below: 1. pH; The protein with negative charge (pH>pI) will be desorbed from the surface of nsHAp through repulsive power; 2. Iron PO43- is bound with Ca sites of nsHAp surface in the presence of PO43-. That means iron exchange of nsHAp surface occurs to accelerate protein desorption. For nsHAp-SOD synthesized by in-situ method, the in-vitro release behavior ofthe system was determined by two factors: diffusion and ion exchange. The bursting release of SOD can be assigned to desorption of SOD molecules which are not tightly bound with nsHAp surface. The release behavior in the second stage may be attributed to the SOD molecules which have been incorporated into nsHAp. This slow release may result from the dissolution of nsHAp, which is combined with Ca2+ of nsHAp concentration.Finally, the secondary structure of protein and nsHAp-Protein compound was analyzed by FT-IR. The amideⅠband (1700～1600cm-1) was transformed to fourier self-deconvolution spectrum and second derivative spectrum. From the information obtained peak fitting procedure was applied to the original amideⅠband to analyze the secondary structure. The results suggested the transformation of secondary structure from order to disorder during the interaction between protein and nsHAp.In generally, with a view to developing a novel carrier for drug controlled release, a wide variety of research works were carried out from design, synthesis, preparation and characterization of nano-sized hydroxyapatite to in vitro release of nsHAp-Protein compound. It is expectable to provide a technique platform for a novel protein drug controlled release carrier. The most importance is that we can apply and possess the intellectual property in the fields of controlled release technique for protein drug on the base of this work.