A simple route to prepare stable hydroxyapatite nanoparticles suspension
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- Han, Y., Wang, X. & Li, S. J Nanopart Res (2009) 11: 1235. doi:10.1007/s11051-008-9507-8
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A simple ultrasound assisted precipitation method with addition of glycosaminoglycans (GAGs) is proposed to prepare stable hydroxyapatite (HAP) nanoparticles suspension from the mixture of Ca(H2PO4)2 solution and Ca(OH)2 solution. The product was characterized by XRD, FT-IR, TEM, HRTEM and particle size, and zeta potential analyzer. TEM observation shows that the suspension is composed of 10–20 nm × 20–50 nm short rod-like and 10–30 nm similar spherical HAP nanoparticles. The number-averaged particle size of stable suspension is about 30 nm between 11.6 and 110.5 nm and the zeta potential is −60.9 mV. The increase of stability of HAP nanoparticles suspension mainly depends on the electrostatic effect and steric effect of GAGs. The HAP nanoparticles can be easily transported into the cancer cells and exhibit good potential as gene or drug carrier system.
Hydroxyapatite (Ca10(PO4)6(OH)2, HAP), the prime constituent of tooth and bone mineral, has been used extensively as an artificial bone substitute in different medical applications (LeGeros 1991). In particular, nanoscale HAP gradually becomes the research emphasis due to the especial behaviors such as enhancing densification, strengthening fracture toughness, improving osseointegrative properties (Sung et al. 2004; Meyers et al. 2006; Ramesh et al. 2007; Li et al. 2007), and interesting potential as gene or drug carrier system (Roy et al. 2003; Zhu et al. 2004; Ferraz et al. 2007). Therefore, the preparation method of nanoscale HAP has been extensively studied, such as precipitation method (Zhang and Lu 2007), hydrothermal reaction (Zhou et al. 2007), sol–gel method (Chung et al. 2005), microemulsion synthesis (Sun et al. 2007), mechanochemical synthesis (Nakamura et al. 2001), combustion method (Han et al. 2004), microwave irradiation (Liu et al. 2004), and solvothermal method (Wang et al. 2006).
As an efficient and versatile carrier system, nanoparticles have to fulfill the following requirements: (i) particle sizes in the submicrometer range, (ii) the possibility of surface modification, (iii) high drug loading capacity, (iv) colloidal stability, and (v) the lack of toxic side effects (Fritz et al. 1997; Pang et al. 2002;). HAP has good biocompatibility and drug loading capacity (Ong et al. 2008). Therefore, for the application of HAP nanoparticles as an efficient and versatile carrier system, it is very important to obtain stable HAP nanoparticles suspension.
In this article, a simple method is utilized to prepare stable HAP nanoparticles suspension. The phase composition and morphology of product were studied by XRD, FT-IR, TEM, and HRTEM. The particle size distribution and stability of HAP nanoparticles suspension were measured by particle size and zeta potential analyzer. The potential of the HAP nanoparticles suspension as gene or drug carrier system was evaluated by cell culture.
The stable HAP nanoparticles suspension was prepared as the following. According to the Ca/P molar ratio of 1.67, the saturated Ca(OH)2 aqueous solution was rapidly poured into Ca(H2PO4)2 aqueous solution while strong stirring. Then the GAGs powders were added into the above suspension with the ultimate concentration of 0.4 g/L. Next the turbid dispersion was intensively stirred for about 5 min and irradiated by an ultrasonic cleaner (KQ-250, 40 kHz, 250 W, China). Finally the transparent and stable dispersion of nanoparticles was obtained. The stable dispersion of nanoparticles was cultured with Bel-7402 human liver cancer cells at 37 °C in humidified incubator containing 5% CO2 and 95% air.
Powder X-ray Diffraction (XRD, D/Max-IIIA, RIGAKU, Japan) was utilized to identify the crystalline phase composition. The morphology and crystal structure of product were observed by Transmission Electron Microscopy (TEM, Philips CM20, Netherlands) and High-resolution Transmission Electron Microscopy (HRTEM, JEM2100F, JEOL, Japan). The particle size distribution and the zeta potential of stable suspension were measured by particle size and zeta potential analyzer (Malvern Zetasizer 3000HS, UK). The nanoparticles cultured with Bel-7402 human liver cancer cells were observed by TEM.
Results and discussion
The aggregation and breakup processes of colloidal system are both greatly affected by interparticle forces. The colloidal interactions include extended Derjaguin-Landau-Verwey-Overbeek (DLVO) forces such as van der Walls attraction and electrostatic repulsion, and nonDLVO forces including hydrophobic attraction and steric repulsion (Ninham 1999; Adamczyk and Weroński 1999). The DLVO theory has been widely used to explain the interactions between colloidal particles. Traditionally, to quantify the interparticle forces in a system, it is necessary to determine particle properties, such as the zeta potential and Hamaker constant. Zeta potential is the important parameter to determine the stability of colloidal system. Zeta potential decides the magnitude of interparticle electrostatic force and influences the aggregation of colloidal particles and the stability of colloidal system. Generally, the increasing of absolute value of zeta potential will result in the enhancing of electrostatic repulsion, which corresponds to higher stability of particles suspension. In this article, the absolute value of zeta potential of HAP nanoparticles suspension is increased to 60.9 mV after the treatment of ultrasound with GAGs, which indicates that the stability of HAP nanoparticles suspension is improved.
The formation of stable HAP nanoparticles suspension relies on the ultrasound treatment and the addition of GAGs. The acoustic cavitation of ultrasound that is the formation, growth, and implosive collapse of bubbles in a liquid, can result in an enormous concentration of energy from the conversion of the kinetic energy of the liquid motion into heating of the contents of the bubble. This unique acoustic cavitation effect of ultrasound can disperse the aggregate HAP particles. In addition, GAGs have already been used as biomaterials due to their good biocompatibility. Moreover, GAGs are known to interact with HAP strongly by the electrostatic attraction between the negatively charged groups on GAGs such as carboxyl or sulphate and the calcium ions on the surface of HAP. Therefore, while aggregate HAP particles are irradiated by ultrasound, the dispersed HAP particles can easily adsorb the polymeric anions dissociated from GAGs in water, which results in the overall negative charge of HAP particles. The increase of charge on the surface of HAP particles can enhance the electrostatic repulsion between HAP particles. Furthermore, the linear GAGs molecules adsorbed on the surface of HAP particles can also increase the steric repulsion between HAP particles. The increases of electrostatic repulsion and steric repulsion between HAP particles yield stable HAP nanoparticles suspension.
The stable HAP nanoparticles suspension with the number-averaged particle size of about 30 nm and the zeta potential of −60.9 mV is obtained by a simple ultrasound assisted precipitation method with addition of GAGs. The suspension is composed of 10–20 nm × 20–50 nm short rod-like HAP nanoparticles and 10–30 nm similar spherical HAP nanoparticles by TEM observation. The stability of HAP nanoparticles suspension is enhanced due to the electrostatic effect and steric effect of GAGs. The HAP nanoparticles can be easily transported into the cancer cells and exhibit good potential as gene or drug carrier system.
This work was supported by the National Natural Science Foundation of P. R. China.