Preparation and Characterization of Nano ...

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Journal of Nanoscience and Nanotechnology Vol. 12, 1–8, 2013

Preparation and Characterization of Nano-Hydroxyapatite Material for Liver Cancer Cell Treatment Sathiyamoorthy Ezhaveni1 , Rathinam Yuvakkumar1 , Mani Rajkumar3 , Nachiappan Meenakshi Sundaram2 , and Venkatachalam Rajendran1 ∗ 1

Center for Nanoscience and Technology, K. S. Rangasamy College of Technology, Tiruchengode 637215, Tamil Nadu, India 2 Department of Biomedical Engineering, PSG College of Technology, Coimbatore 641004, Tamil Nadu, India 3 Department of Physics, PSG College of Technology, Coimbatore 641004, Tamil Nadu, India Nano-hydroxyapatite was synthesized by means of the hydrothermal treatment. The effects of nanohydroxyapatite material on the behaviour of G2 liver cancer cells were explored. About 50% of cell viability was lost in nHAp material treated cells at 200 C@5 h, followed by ∼ 30% in nHAp treated cells at 100 C@5 h. Compared with control, nHAp material treated cells at 200 C@5 h showed 60% and nHAp material treated cells at 100 C@5 h showed 15% morphological change. Moreover, 50% of cell death was observed at 24 h incubation with nHAp material treated at 200 C@5 h cells and 56% cell death at 48 h incubation and hence alters and disturbs the growth of cancer cells. In contrast, the nHAp material treated at 100 C@5 h protects the cells and could be used for liver cancer cell treatment.

1. INTRODUCTION Tissue engineering as a multidisciplinary science has been considered a promising technology in the development of biological substitutes for failing tissues and organs. The most common approach proposed by Langer and Vacanti is based on living cells and signal molecules.1 To function efficiently, the biomaterials with adequate structure, suitable size, and interconnected pore structure for transporting cells, metabolites, nutrients, and signal molecules are highly essential. In addition, the materials should be nontoxic, nonimmunogenic, biocompatible, and biodegradable at ideal rates corresponding to the rate of new tissue formation.2–4 Therefore, the calcium phosphate based on inorganic hydroxyapatite (HAp) with a Ca/P ratio of 1.67 has been studied extensively and applied in a variety of fields because of its similarity to the mineral constituents of human bones and teeth.5 6 Synthetic HAp has excellent biocompatibility and bioactivity and is hence useful in reconstruction of damaged bone or tooth zones.7–10 HAp can be derived from either natural or synthetic sources and regarded as a bioactive substance, as it forms a strong ∗

Author to whom correspondence should be addressed.

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chemical bond with host bone tissue. In addition, HAp is recognized as a good bone graft material.11–13 HAp is not only bioactive but also osteoconductive, nontoxic, and nonimmunogenic. The structure of HAp is crystallographically similar to that of bone mineral with adequate amounts of carbonate substitution. In the past few years, it was reported that inorganic nanoparticles could kill cancer cells such as TiO2 . Moreover, calcium hydroxyapatite Ca10 (PO4 6 (OH)2 is the most widely accepted bioactive material and could inhibit the proliferation of several kinds of cancer cells, such as liver cancer cells and osteosarcoma U2-OS cells.14–17 Recently, various types of nanostructured materials have been developed18–20 and many of them have specific unique properties in physics, chemistry, and biology.21–23 Therefore, in the present study, nano-hydroxyapatite was synthesized by means of the hydrothermal method under high temperature and pressure24–26 and the biological effects of prepared nano-hydroxyapatite has been explored in liver cancer cells. The phase, composition and morphology of the nano-hydroxyapatite were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS), particle size analyser (PSA), and transmission electron

1533-4880/2013/12/001/008

doi:10.1166/jnn.2013.7135

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Keywords: Nano-Hydroxyapatite, Hydrothermal, Liver Cancer Cell.

Preparation and Characterization of Nano-Hydroxyapatite Material for Liver Cancer Cell Treatment

microscopy (TEM) studies. In addition, the effects of nano-hydroxyapatite behaviours were investigated using G2 liver cancer cells as the model cell.

2. EXPERIMENTAL PROCEDURE 2.1. Synthesis Starting materials calcium nitrate tetra hydrate (Ca(NO3 2 · 4H2 O) and diammonium hydrogen phosphate ((NH4 2 HPO4 were dissolved using 50-ml double distilled ultrapure water and were stirred for 1 h separately. Thereafter, the diammonium hydrogen phosphate solution was added dropwise to the calcium nitrate tetra hydrate solution and the pH was adjusted to 11–12 by adding ammonium hydroxide solution at room temperature, after the following reaction occurred: 10CaNO3 2 · 4H2 O + 6NH4 2 HPO4 + 8NH4 OH → Ca10 PO4 6 OH2 + 20NH4 NO3 + 46H2 O (1) The prepared mixture was hydrothermally treated and then obtained precipitates were washed with double distilled water until ammonia was removed when pH returned to 7. The product, nano-hydroxyapatite, was washed with ethanol, filtered, dried and ground into a fine powder using a mortar and pestle.

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2.2. Characterization 2.2.1. X-Ray Diffraction The phase and crystalline nature of the prepared samples were identified by X-ray powder diffraction patterns (X’Pert-PRO) using CuK as the radiation (1.54060 Å) source. The diffractometer was operated at 40 kV, and scans were performed over a 2 range from 10 to 80. The peak positions and the relative intensities of the powder pattern were identified in comparison with the reference powder diffraction data (JCPDS). The average crystallite size of all the nano-hydroxyapatite samples was calculated using Scherrer’s formula. 094 Dp = 1/2 Cos

(2)

where Dp is the crystallite size, is the wavelength of the X-ray, 1/2 is the wavelength of the full-width at halfmaximum, and is the diffraction angle. 2.2.2. Fourier Transform Infrared Spectroscopy Infrared (IR) spectra were recorded using an Fourier transform infrared spectrophotometer (Perkin Elmer, spectrum 100, Waltham, MA) in the frequency range of 4000– 400 cm−1 by means of the KBr pellet method (95 wt% KBr). The KBr discs were made by pressing the mixture, which contained 10 mg of hydroxyapatite nanoparticles with 100 mg of KBr at a pressure of 125 kg cm−2 . 2

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2.2.3. Particle Size Analyser The particle size distribution was determined using a particle size analyser (Nanophox, Sympatec) according to the dynamic light scattering technique. The particle size of all the samples was measured in the range of 1–1000 nm at the scattering angle of 90 . Three-dimensional photon cross-correlation techniques were used for the simultaneous measurement of particle size. Various dispersants have been studied for the dispersion of nano-hydroxyapatite particles. For particle size measurement, an optimized Sodium Hexa-Meta-Phosphate (SHMP) solution was prepared at a concentration of 0.1 wt%. Thereafter, 5 mg of nanohydroxyapatite was added to 10 ml of dispersant solution and kept in an ultrasonic water bath for 10 min. The prepared solutions were then measured for particle size distribution. 2.2.4. Scanning Electron Microscopy and Energy-Dispersive X-Ray Spectroscopy The surface morphology of the product was recorded using scanning electron microscopy (S-3000H; Hitachi, Japan). A high resolution of 3.5 nm was used with a secondary electron image display. An accelerating voltage of 20 kV was used with a magnification of 10,000X to scan the samples. An elemental analysis, energy-dispersive X-ray spectroscopy, was performed to evaluate the composition of the sample along with the scanning electron microscopy observation. 2.2.5. Transmission Electron Microscopy Transmission electron microscopic images were obtained using a transmission electron microscope (CM 200; Philips, USA). The images were formed using transmitted electrons, which can produce a magnification of up to 1,000,000× with a resolution better than 10 Å. The images can be resolved over a fluorescent screen or on a photographic film. 2.3. Application 2.3.1. Liver Cancer Cell Treatment The 8–10 mm in diameter of in nHAp material treated at 100 C@5 h and 200 C@5 h was incubated with HepG2 liver cancer cells. After the incubation period, the cells were harvested by trypsinization and centrifugation. The cell pellet was suspended in phosphate buffered saline (PBS) with 0.1% tryphan blue. The stained cells were observed using an inverted microscope for morphology. 2.3.2. Cell Counting Cells (2 × 103 were loaded on the nHAp material at 100 C@5 h and 200 C@5 h and then Dulbecco’s Modified Eagle Medium (DMEM) medium with 10% Fetal J. Nanosci. Nanotechnol. 12, 1–8, 2013

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Bovine Serum (FBS) was added; the cells were then incubated at 37 C with 5% CO2 . After the incubation period, at intervals of 12, 24, and 48 h, the cells were harvested by trypsinization and centrifugation. The obtained cell pellets were suspended in PBS with 0.1% tryphan blue. The stained cells were counted using a light microscope. 2.3.3. Cell Proliferation Cells (2 × 104 were seeded in 24-well plates and incubated with the nHAp material at 100 C@5 h and 200 C@5 h at different time intervals. Cell proliferation was assayed using the MTT assay (3-(4,5-Dimethylthiazol2-yl)-2,5-Diphenyltetrazolium Bromide) method by taking the optical density of viable cells in 570 nm in an Enzyme Linked Immuno Sorbent Assay (ELISA) reader (Bio-Rad, USA).

3. RESULTS AND DISCUSSION

Fig. 1. XRD patterns of nano hydroxyapatite hydrothermally treated at 100 C for (a) 5 h (b) 10 h and (c) 15 h.

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increased when the hydrothermal treatment was increased from 200 C to 300 C for 5 h, 10 h, and 15 h. Moreover, the average crystallite sizes of nano-hydroxyapatite for 5 h, 10 h, and 15 h treated at 200 C and 300 C were, respectively, 20, 31, and 38 nm and 32, 35, and 45 nm. The observed results reveal that as the hydrothermal treatment increases the crystallite size of nano-hydroxyapatite also increases. It is evident from the above result that hydrothermal treatment and time play important roles in reducing the crystallite size of the particle. In brief, the structural analysis of the prepared nanohydroxyapatite at different hydrothermal treatment temperatures at different time periods does not show any peaks other than crystalline nano-hydroxyapatite (JCPDS 090432). All prepared nano-hydroxyapatite samples at various temperature and time periods are highly crystalline. The crystalline size increases with an increase in temperature and time period. Hence, from the observed results,


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Figure 1 shows the XRD pattern of nano-hydroxyapatite hydrothermally treated at 100 C for 5 h, 10 h, and 15 h, which indicates the crystalline nature of nanohydroxyapatite. Figure 2 shows the XRD pattern of nanohydroxyapatite hydrothermally treated at 200 C for 5 h, 10 h, and 15 h. The observed sharp peak in Figure 2 confirms the existence of the crystalline phase. Figure 3 shows the XRD pattern of nano-hydroxyapatite hydrothermally treated at 300 C for 5 h, 10 h, and 15 h. The average crystallite size of the obtained nano-hydroxyapatite treated at 100 C was calculated from the broadening of the corresponding sharp peaks using the Scherrer formula.27–29 The average crystallite size of nano-hydroxyapatite for 5 h, 10 h, and 15 h sintered at 100 C was, respectively, 11, 22, and 24 nm. In contrast, the crystalline peaks



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Fig. 4. FTIR spectra of nano hydroxyapatite hydrothermally treated at 100 C for (a) 5 h (b) 10 h and (c) 15 h.

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one can obtain a reduced particle size at lower hydrothermal treatment temperature and time period for suitable biomedical applications. The major chemical groups present in the prepared samples can be identified by FTIR spectra, as shown in Figures 4–6. The FTIR spectrum of the synthesized sample shows the characteristic phosphate absorption bonds appearing at 475 ( 2), 566 and 603 ( 4), 970 ( 1), and 1032 and 1087 ( 3) cm−1 . The adsorption peak that corresponds to the carbonate absorption bonds appeared at 873 and 1415 ( 3), 1457 ( 4), and 1547 ( 4) cm−1 . This indicates that carbonate ions have substituted for certain phosphate positions in the apatite lattice. The observed absorption bonds at 3571 and 633 cm−1 reveal the stretching and liberation modes of OH−1 bonds. The water

associated with HAp is present at 3430 cm−1 . The above results confirm the formation of pure HAp structures. The particle size analyser (Nanophox, Sympatec, Germany) is used to measure the size distribution of the particles using the dynamic light scattering principle. Figure 7 shows the particle size distributions of nanohydroxyapatite dispersed in SHMP for 5 h, 10 h, and 15 h hydrothermally treated at 100 C. The maximum distribution (d50 of particles is 19 ± 3, 22 ± 3, and 38 ± 3 nm, respectively, for 5 h, 10 h, and 15 h at 100 C. Figure 8 shows the particle size distribution of nano-hydroxyapatite dispersed in SHMP for 5 h, 10 h, and 15 h at 200 C. The maximum distributions (d50 of particles are 47 ± 3 nm, 55 ± 3 nm, and 57 ± 3 nm, respectively, for 5 h, 10 h, and 15 h at 200 C. Figure 9 shows the particle size distributions of nano-hydroxyapatite dispersed in SHMP for 5 h, 10 h, and 15 h at 300 C. The maximum distributions


Fig. 7. PSA of nano hydroxyapatite hydrothermally treated at 100 C for (a) 5 h (b) 10 h and (c) 15 h.

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(d50 of particles are 60 ± 3 nm, 94 ± 3 nm, and 97 ± 3 nm, respectively, for 5 h, 10 h, and 15 h at 300 C. In the present study, the nano-hydroxyapatite samples are synthesized at three different hydrothermally treatments and time periods to investigate these effects on particle size for biomedical applications. The particle size distribution analysis reveals that the size of the particle increases because of the increase in hydrothermal treatment temperature and time period. The overall observation is that the maximum particle size distributions (d50 of the obtained hydroxyapatite samples are in the range of 19–38 nm, 47–57 nm, and 60–97 nm, respectively at 100 C, 200 C, and 300 C for 5 h, 10 h, and 15 h. Hence, it can be concluded that the lower the hydrothermal treatment temperature and lower the time period, the smaller

the particle size; in contrast, the higher the hydrothermal treatment and higher the time period, the bigger the particle size. The overall performance of the XRD, FTIR and PSA studies conclude that the nano-hydroxyapatite samples treated at low temperature and time (100 C@5 h) were consisted of smaller particles and could be used for biomedical applications. Figures 10(a)–(c) represent the SEM-EDAX image of nano-hydroxyapatite sintered at 100 C, 200 C, and 300 C for 5 h. The SEM images of the obtained nanohydroxyapatite particles reveal that the prepared powders consisted of a mixture of fine and large grains of the product particles, as shown in Figures 10(a)–(c). In addition, SEM analyses indicate that there are large differences in the size of the particles between different samples. Some samples reveal much smaller grains with irregular sizes, but others show obvious bulk particles with irregular sizes. It can be concluded from the SEM studies that higher sintering temperature results in larger particle sizes, indicating quicker grain growth. In addition, the surface morphology of the nano-hydroxyapatite particles is uniformly agglomerated among the particles, which may be because of small particle sizes, i.e., the high surface energy of nHAp particles resulting in their aggregation. Accordingly, sintering temperature is one of the important factors influencing the microstructure and grain growth of the sintered samples. Moreover, the results obtained from the SEM analysis correlated exactly with the results obtained from particle size analysis. The results of the energy-dispersive X-ray analysis (Figs. 10(a)–(c)) of the prepared powders show that the sample contained three kinds of elements: calcium, phosphate, and oxygen. In all the prepared samples, the highest ratio of Ca/P was present: 1.63. Figures 11(a)–(c) show TEM images of nanohydroxyapatite sintered at 100 C, 200 C, and 300 C for 5 h. TEM images reveal that the prepared powders consisted of relatively uniform particles with short nanorodlike morphology, as shown in Figures 11(a)–(c). It can be concluded from the TEM studies that nano-hydroxyapatite having a diameter of about 18–24 nm and length of about 33–40 nm was obtained, and the prepared particles had a rough surface area. Therefore, in the present work, the formation of HAp nanostructures was obtained using the hydrothermal technique. The prepared nHAp was interestingly crystalline in nature with a high yield. Thereafter, the nano-hydroxyapatite samples treated at low temperature and time (100 C@5 h and 200 C@5 h) were used for biomedical applications. These prepared materials along with well-defined nano-structured HAp are needed for both fundamental studies and clinical applications. Moreover, for clinical applications, the morphology of cells can be important in many contexts. In culture, the morphology indicates the status of the cells, both in terms of the health

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Fig. 10.

SEM-EDAX of nano hydroxyapatite hydrothermally treated at (a) 100 C for 5 h (b) 200 C for 5 h (c) 300 C for 5 h.

of the cells and in case of primary isolates. Therefore, the prepared nano-hydroxyapatite materials treated at low temperature and time is used in liver cancer cell treatment and the status of the cells is explored for cell morphology and cell proliferation studies. 6

3.1. Cell Counting Cell counting, cell morphology, and cell proliferation studies are carried out in nHAp material treated at 100 C@5 h and 200 C@5 h along with control, as shown in J. Nanosci. Nanotechnol. 12, 1–8, 2013

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Preparation and Characterization of Nano-Hydroxyapatite Material for Liver Cancer Cell Treatment (a)

(b)

(c)

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Fig. 11. TEM image of nano hydroxyapatite hydrothermally treated at (a) 100 C for 5 h, (b) 200 C for 5 h and (c) 300 C for 5 h.

Figures 12(a)–(c). Initially, 2 × 103 cells were loaded and the total cell count in a neubauer chamber was observed. The number of viable cell counts was reduced from their normal range (control) in nHAp material treated at 100 C@5 h and 200 C@5 h, as shown in Table I. More than 50% of cell viability was lost in the nHAp material treated at 200 C@5 h cells, followed by ∼ 30% in the nHAp material at 100 C@5 h treated cells. 3.2. Cell Morphology The nHAp material treated at 200 C@5 h cells were 60% modified from normal cells and they were square and circle shaped as shown in Figure 12(c). In contrast, the nHAp material treated at 100 C@5 h cells showed a 15% morphological change compared with normal cells (Figs. 12(a) and (b)). Therefore, in the present study, nHAp material treated at 200 C@5 h cells alters and disturbs the growth of cancer cells, whereas the nHAp material treated at 100 C@5 h cells showed compatibility with cancer cells. 3.3. Cell Proliferation Cell proliferation was observed at three different periods. The maximum inhibition of proliferation was observed at J. Nanosci. Nanotechnol. 12, 1–8, 2013

Fig. 12. Cell morphology (a) control (b) nHAp material treated cells at 100 C@5 h (c) nHAp material treated cells at 200 C@5 h.

24 and 48 h of incubation. Nearly 50% of cell death was observed at 24 h incubation with nHAp material treated at 200 C@5 h cells and 56% cell death at 48 h incubation. Moreover, nHAp material treated at 100 C@5 h cells showed cell death profiles similar to those of nHAp material treated at 200 C@5 h cells, but the cell death is not very significant compared with nHAp material treated at 200 C@5 h cells. Thus, the studies on nHAp material treated at 100 C@5 h cells and at 200 C@5 h cells suggest that the prepared nHAp material treated at 100 C@5 h cells can be used as a biomaterial in liver cancer cell treatment. 7

Preparation and Characterization of Nano-Hydroxyapatite Material for Liver Cancer Cell Treatment Table I. Cell counting comparison between control and nHAp material treated at 100 C and 200 C.

Time in h 12 24 48

Normal (control) (103 cells/ml)

nHAp material treated at 100 C@5 h (103 cells/ml)

nHAp material treated at 200 C@5 h (103 cells/ml)

23 ± 05 35 ± 03 37 ± 04

21 ± 02 24 ± 01 29 ± 04

23 ± 01 28 ± 012 21 ± 03

4. CONCLUSIONS Nano-hydroxyapatite materials were prepared and characterized comprehensively. The effects of nanohydroxyapatite on G2 liver cancer cells were studied. Cell viability was lost by about 50% and ∼ 30%, respectively, in nHAp material treated at 200 C@5 h and at 100 C@5 h cells. The cell morphological change was 60% and 15%, respectively, in nHAp material treated at 200 C@5 h and 100 C@5 h treated cells. The cell death percentage was about 50% and 56% at 24 and 48 h incubation in in nHAp material treated at 200 C@5 h. Thus, the prepared nano-hydroxyapatite material treated at 100 C@5 h with a well-defined nanostructure can be used in liver cancer cell treatment.

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6. Y. Luo, C. Zhang, F. Xu, Y. Chen, L. Fan, and Q. Wei, J. Mater. Sci. 45, 1866 (2010). 7. M. Jarcho, Clin. Orthop. Relat. Res. 157, 259 (1981). 8. P. Ducheyne and K. De Groot, J. Biomed. Mater. Res. 15, 441 (1981). 9. R. Murugan and S. Ramakrishna, American Scientific Publishers, California (2004), p. 595. 10. R. Murugan and S. Ramakrishna, American Scientific Publishers, California (2005), p. 141. 11. J. Black and G. W. Hastings, Chapman and Hall, London (1998). 12. K. De Groot, edited by T. Yammamuro and L. L. Hench, CRC Press, Boca Raton (1990). 13. L. L. Hench, J. Am. Ceram. Soc. 81, 1705 (1990). 14. R. Z. LeGeros and J. P. LeGeros, An Introduction to Bioceramics, edited by L. L. Hench and J. Wilson, World Scientific, Singapore (1993), p. 139. 15. M. I. Kay, R. A. Young, and A. S. Posner, Nature 204, 1050 (1964). 16. M. Albarghouthi, D. Abu Fara, M. Saleem, T. El-Thaher, K. Matalka, and A. Badwan, Int. J. Pharm. 206, 23 (2000). 17. Q. Fu, N. Zhou, W. Huang, D. Wang, L. Zhang, and H. Li, J. Biomed. Mater. Res. A 74, 156 (2005). 18. K. Ariga, A. Vinu, Y. Yamauchi, Q. Ji, and J. P. Hill, Bull. Chem. Soc. Jpn. 85, 1 (2012). 19. Z. Chen, Z. Jiao, M. Wu, C. H. Shek, C. M. L. Wu, and J. K. L. Lai, J. Nanosci. Nanotechnol. 12, 26 (2012). 20. L. Dai, D. W. Chang, J. B. Baek, and W. Lu, Small 8, 1130 (2012). 21. K. Ariga, T. Mori, and J. P. Hill, Adv. Mater. 24, 158 (2012). 22. Q. He, Z. Wu, and C. Huang, J. Nanosci. Nanotechnol. 12, 2943 (2012). 23. J. Lei and H. Ju, Chem. Soc. Rev. 41, 2122 (2012). 24. X. F. Pang, H. W. Zhou, and J. L. Liu, J. Nanosci. Nanotechnol. 12, 894 (2012). 25. M. Wang, N. J. Castro, J. Li, M. Keidar, and L. G. Zhang, J. Nanosci. Nanotechnol. 12, 7692 (2012). 26. M. Bricha, Y. Belmamouni, E. M. Essassi, J. M. F. Ferreira, and K. E. Mabrouk, J. Nanosci. Nanotechnol. 12, 8042 (2012). 27. T. Takahashi, K. Takayama, Y. Machida, and T. Nagai, Int. J. Pharm. 61, 35 (1990). 28. T. Fujigaya and N. Nakashima, J. Nanosci. Nanotechnol. 12, 1717 (2012). 29. D. P. Mohapatra, F. Gassara, and S. K. Brar, J. Nanosci. Nanotechnol. 11, 899 (2011).

Received: 23 October 2012. Accepted: 11 December 2012.

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