Journal of Nanoparticle Research

, Volume 11, Issue 5, pp 1235–1240

A simple route to prepare stable hydroxyapatite nanoparticles suspension

Authors

    • Biomedical Materials and Engineering CenterWuhan University of Technology
  • Xinyu Wang
    • Biomedical Materials and Engineering CenterWuhan University of Technology
  • Shipu Li
    • Biomedical Materials and Engineering CenterWuhan University of Technology
Brief Communication

DOI: 10.1007/s11051-008-9507-8

Cite this article as:
Han, Y., Wang, X. & Li, S. J Nanopart Res (2009) 11: 1235. doi:10.1007/s11051-008-9507-8

Abstract

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.

Keywords

HydroxyapatiteNanoparticleStable suspensionUltrasoundSynthesisColloids

Introduction

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.

Experimental procedure

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

Figure 1 shows the XRD pattern of the nanoparticles dried by freeze-drying. As shown in Fig. 1, some major diffraction peaks emerge, which indicates that the crystals form. It is verified that the major polycrystalline structure of the nanoparticles is characteristics of HAP. The diffraction peaks broaden and three nearby major peaks merge into one broad peak due to their nanocrystalline nature. The lattice parameters of HAP nanoparticles were obtained from the Rietveld refinement analysis of XRD data. The a and c axes of HAP nanoparticles are 9.405 and 6.892 Å, respectively, which are close to the lattice parameters of stoichiometric HAP (Powder Diffraction File ICDD 09-0432, a = 9.418 Å and c = 6.884 Å). The crystalline size of HAP nanoparticles can be calculated by Scherrer’s formula as follows:
$$ X_{\text{hkl}} = k\lambda /\beta_{1/2} \cos \theta , $$
where Xhkl is the crystallite size (nm), λ is the wavelength of monochromatic X-ray beam (nm) (λ = 0.15418 nm for CuKα radiation), β1/2 is the full width at half maximum for the diffraction peak under consideration (rad), θ is the diffraction angle (°), and k is a constant varying with crystal habit and chosen to be 0.9. The (002) peak was the most distinct reflection in the XRD pattern. Therefore, the line broadening of the (002) reflection was used to calculate the mean crystalline size by the Scherrer’s formula. The crystalline size along the c-axis (long dimension) of the HAP crystals is about 14.4 nm.
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Fig. 1

XRD pattern of the nanoparticles dried by freeze-drying

Figure 2 is the FT-IR spectrum of the nanoparticles. As shown in Fig. 2, the intensive bands at 1,092 and 1,041 cm−1 are due to ν3 vibrational mode of phosphate group. Further, the band at 962 cm−1 is corresponding to ν1 vibrational mode of phosphate group, which can be observed in all spectra of HAP and carbonated apatites, and the weak band at 473 cm−1 is attributed to ν2 vibrational mode of phosphate group. Especially, phosphate ν4 bands will appear in different sites for carbonated apatite and HAP. Two sites appear in the case of carbonated apatite, centered at 603 and 567 cm−1, and three sites are observed in HAP at 633, 603 and 565 cm−1 (Rehman and Bonfield 1997; Habelitz et al. 1999). Therefore, the bands at 632, 602 and 566 cm−1 show that the nanoparticles should be HAP. Furthermore, the small sharp peak at 3,569 cm−1 assigned to the OH stretching vibration of HAP is also observed, which is partly overlapped by adsorbed water bands. The broad envelope between 3,700 and 2,700 cm−1 centered at 3,448 cm−1 is due to the O–H stretching vibration of adsorbed water. The band at 1,629 cm−1 is attributed to the O–H bending vibration of adsorbed water although the band at 1,629 cm−1 is also a strong C=O stretching band from carboxylate anions of GAGs. Moreover, the vibration bands of other groups of GAGs are also detected, which are the C–H variable-angle vibration of –CH2– group at 1,456 cm−1, the C–H stretching vibration at 2,927 cm−1, the C–O stretching vibration of –COOH group at 1,421 cm−1, the S=O and C–O–S stretching vibration of –O–SO3– group at 1,233 cm−1 and 900–796 cm−1.
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Fig. 2

FT-IR spectrum of the nanoparticles dried by freeze-drying

Figure 3 shows the TEM and HRTEM images of HAP nanoparticles. As shown in Fig. 3a, the nanoparticles are short rod-like particles with 10–20 nm in width and 20–50 nm in length, and similar spherical particles with 10–30 nm in diameter. The HRTEM image in Fig. 3b provides further insight into the structure of the products. The observed interplanar spacing is about 0.348 nm, which corresponds to the 0.344 nm separation between (002) lattice planes of hexagonal HAP.
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Fig. 3

TEM image (a) and HRTEM image (b) of the nanoparticles suspension

The particle size distribution and zeta potential of HAP nanoparticles suspension measured by the particle size and zeta potential analyzer are shown in Fig. 4. As shown in Fig. 4a, the number-averaged particle size is about 30 nm and the size distribution is from 11.6 to 110.5 nm. This result is in accordance with the TEM, which indicates that the HAP nanoparticles are well dispersed and there is no serious agglomeration. As shown in Fig. 4b, the mean value of zeta potential is negative 60.9 mV. In order to further check the stability of HAP nanoparticles suspension, the particle size distribution of HAP nanoparticles suspension after 15 days was measured. As shown in Fig. 5, after 15 days the number-averaged particle size is about 39.3 nm and the size distribution is from 20.5 to 163.1 nm. The particle size of HAP nanoparticles increases a little due to agglomeration, but obvious precipitation does not appear in the HAP nanoparticles suspension. That is to say, the HAP nanoparticles suspension is still stable after 15 days.
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Fig. 4

Size distribution curve (a) and zeta potential curve (b) of the original nanoparticles suspension

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Fig. 5

Size distribution of the nanoparticles suspension after 15 days

In order to evaluate the potential of HAP nanoparticles suspension as gene or drug carrier system, the HAP nanoparticles suspension was cultured with Bel-7402 human liver cancer cells. The distribution of HAP nanoparticles in Bel-7402 human liver cancer cells was observed by TEM as shown in Fig. 6. As shown in Fig. 6a, a large number of nanoparticles are englobed by cancer cells. The villiform protuberances on the surface of cancer cells catch the HAP nanoparticles and form closed structure. By this procedure, the HAP nanoparticles are transported into the cancer cells and distributed in vacuoles. Moreover, the size and morphology of HAP nanoparticles in the cells (Fig. 6b) are same as the original HAP nanoparticles. This demonstrates that the HAP nanoparticles suspension exhibits good potential as gene or drug carrier system.
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Fig. 6

TEM image of the nanoparticles suspension cultured with Bel-7402 human liver cancer cells. NP, nanoparticles; N, nucleus; CM, cell membrane; MV, microvilli; Cy, cytoplasm

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.

Conclusions

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.

Acknowledgment

This work was supported by the National Natural Science Foundation of P. R. China.

Copyright information

© Springer Science+Business Media B.V. 2008