Chapter 8 Mechanochemical Synthesis of Magnetite / Hydroxyapatite Nanocomposites for Hyperthermia

Hydroxyapatite (Ca10(PO4)6(OH)2, HA), which is a calcium phosphate ceramic, has been widely used as a biomaterial in various applications (e.g., artificial bone and dental root, cosmetic foundation, etc.) because of its high biocompatibility and chemical stability. More‐ over, many attempts are being made to give new functions to HA by incorporating effective components into a HA matrix. In particular, magnetite (Fe3O4)-incorporated HA (Fe3O4/HA) nanocomposites have attracted much attention as a promising material for hyperthermia therapy of malignant bone tumor [1–4]. Recently, Fe3O4/HA composites have also been used as adsorbents [5–7] and catalysts [8,9].


Introduction
Hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 , HA), which is a calcium phosphate ceramic, has been widely used as a biomaterial in various applications (e.g., artificial bone and dental root, cosmetic foundation, etc.) because of its high biocompatibility and chemical stability.Moreover, many attempts are being made to give new functions to HA by incorporating effective components into a HA matrix.In particular, magnetite (Fe 3 O 4 )-incorporated HA (Fe 3 O 4 /HA) nanocomposites have attracted much attention as a promising material for hyperthermia therapy of malignant bone tumor [1][2][3][4].Recently, Fe 3 O 4 /HA composites have also been used as adsorbents [5][6][7] and catalysts [8,9].Fe 3 O 4 /HA composites can be synthesized conventionally by mixing HA powder with Fe 3 O 4 nanoparticles which are prepared individually [1][2][3][5][6][7][8][9][10].The conventional synthesis methods have disadvantages: reaction time required for completing the formation of HA and Fe 3 O 4 is relatively long, subsequent heat treatments for long periods of time are required for aging and crystallization.Thus, the synthesis of Fe 3 O 4 /HA composites generally consist of multi-step processes.Therefore, a simple method which can provide Fe 3 O 4 /HA composites rapidly is needed to be developed.
In this chapter, a mechanochemical method for the simple synthesis of Fe 3 O 4 /HA nanocomposites is presented.In this method, superparamagnetic Fe 3 O 4 nanoparticles are first prepared mechanochemically from ferric hydroxide [11], and then the mechanochemical synthesis of HA from dicalcium phosphate dihydrate (CaHPO 4 2H 2 O) and calcium carbonate (CaCO 3 , calcite) is performed [12][13][14], followed by the aging for a short period of time.These mechanochemical treatments are sequentially performed in a single horizon-tal tumbling ball mill at room temperature under wet conditions.The wet mechanochemical process can also contribute to the distribution of Fe 3 O 4 nanoparticles in the HA matrix, which can result in a good hyperthermia property.In addition, the use of horizontal tumbling ball mills is reasonable for the synthesis of Fe 3 O 4 /HA nanocomposites because the device structure is simple, the handling is easy, the energy consumption is relatively low, and the scale-up is easy [15].The influence of conditions on the formation of Fe 3 O 4 /HA nanocomposites was investigated and the hyperthermia property was examined.The details are described below.

Mechanochemical synthesis of hydroxyapatite nanoparticles
First of all, the synthesis of HA nanoparticles containing no Fe 3 O 4 nanoparticles was investigated to optimize the synthesis process of Fe 3 O 4 /HA nanocomposites.In all the experiments presented in this chapter, the chemicals of analytical grade were used as received without further purification.Typically, 30 mmol of CaHPO 4 2H 2 O and 20 mmol of CaCO 3 , corresponding to the stoichiometric molar ratio in the formation reaction of HA expressed by Equation (1) [14], were added to 60 ml of deionized and deoxygenated water.
( ) ( ) The resulting suspension was subjected to a mechanochemical treatment using a horizontal tumbling ball mill, as illustrated in Figure 1.The suspension was placed in a Teflon-lined milling pot with an inner diameter of 90 mm and a capacity of 500 ml.Zirconia balls with a diameter of 3 mm were used as the milling media; the charged volume of the balls (including voids among the balls) was 40% of the pot capacity.The wet milling was performed at room temperature in air atmosphere under atmospheric pressure for a designated period of time.The rotational speed was 140 rpm, corresponding to the ideal critical rotational speed.After milling, the precipitate was isolated from the suspension by centrifugation, washed with acetone, and dried at room temperature in air.As a control experiment without milling, the starting suspension was vigorously stirred at room temperature for 24 h.
The samples obtained under various conditions were characterized according to standard methods.The powder X-ray diffraction (XRD) pattern of samples was obtained by an X-ray diffractometer (RINT-1500, Rigaku; CuKα radiation, 40 kV, 80 mA, 2θ=5°-50°, scanning rate: 1.0°/min).Figure 2 shows the XRD pattern of samples obtained in different milling times.As the milling time increased, the diffractions indicating the presence of CaHPO 4 2H 2 O and Ca-CO 3 decreased.Simultaneously, the diffractions indicating HA appeared.In particular, a drastic change was observed between 1 h and 3 h.On the contrary, when stirred for 24 h without milling, the XRD pattern (not shown) hardly changed from the beginning, which was almost the same as that before milling as shown in Figure 2a.These results indicate that the milling promoted the solid phase reaction expressed by Equation (1).However, after milling for 12 h, the XRD pattern was almost the same and the diffraction at 2θ=29.4°, indicating the presence of CaCO 3 , still remained even in 24 h.The differential scanning calorimetry (DSC) was performed using a thermal analyzer (SDT2960, TA Instrument) with an argon flow rate of 100 ml/min.The temperature was raised from ambient temperature to 900 °C at a rate of 20°C/min.Figure 3 shows the results of DSC analysis for the raw materials and the samples.In the sample obtained in 1 h (Figure 3d), the endothermic peaks were clearly observed at around 200°C and 750°C, which resulted from the elimination of water of crystallization in CaHPO 4 2H 2 O and the thermal decomposition of CaHPO 4 2H 2 O and CaCO 3 .Although the peaks relating to CaHPO 4 2H 2 O disappeared as the milling time, the peak resulted from the thermal decomposition of Ca-CO 3 remained even in 12 h.Accordingly, it was found that the milling was not sufficient to complete the formation reaction of HA.
The morphology of samples was observed by field emission scanning electron microscopy (FE-SEM; JSM-6700F, JEOL). Figure 4 shows typical SEM images of samples.In a milling time of 1 h, coarse particles coated with fine particles of about 100 nm were observed.From the particle size analysis of CaHPO 4 2H 2 O and CaCO 3 by the laser diffraction/scattering method (SALD-7100, Shimadzu), the median sizes were determined to be 16.2 µm for CaH-PO 4 2H 2 O and 2.0 µm for CaCO 3 .In general, horizontal tumbling ball mills are difficult to produce nanoparticles for short milling times.Therefore, coarse and fine particles could be the raw materials and HA, respectively.As the milling time increased, coarse particles disappeared and the number of HA nanoparticles increased.However, even after 12 h, a little number of coarse particles was found.In order to complete the formation reaction of HA, the heat treatment (aging) was performed after milling.For investigating the effect of heating on the formation of HA, the un-milled suspension of CaHPO 4 2H 2 O and CaCO 3 was heated under various conditions of temperature and time.Figures 5, 6, and 7 show the XRD patterns of samples obtained without milling after heating at 40, 60, and 80°C, respectively.When the suspension was heated at 40°C, the formation reaction of HA hardly took place.As increasing in the temperature, the reaction was promoted and could complete at 80°C in 8 h.Thus, when without milling, higher heating temperatures and longer heating times are needed for the formation of HA.Next, the effect of milling of the suspension before heating on the formation of HA was investigated.Figures 8-14 show the XRD patterns of samples obtained under various conditions of milling time, heating temperature, and heating time.It was found that longer milling times, higher heating temperatures, and longer heating times promoted the formation reaction of HA.In particular, as shown in Figure 10c, when the heating was performed at 80°C, only the milling for 1 h and the following heating for 1 h provided a single phase of HA.The SEM images of samples obtained by milling for different times under constant heating conditions of 80°C and 1 h are shown in Figure 15.When heating at 80°C for 1 h, a typical morphology of HA was observed regardless of the milling time.However, the particle size intended to decrease as the milling time increased.Consequently, the combination of milling and heating of the suspension of CaHPO 4 2H 2 O and CaCO 3 can produce efficiently HA for short periods of time.

Synthesis and hyperthermia property of magnetite/hydroxyapatite nanocomposites
In the synthesis of Fe 3 O 4 /HA nanocomposites, first a suspension of superparamagnetic Fe 3 O 4 nanoparticles was prepared according to a mechanochemical method reported in elsewhere [11].This method provides Fe 3 O 4 from ferric hydroxide (goethite) in the absence of a reducing agent; goethite is reduced to ferrous hydroxide by mechanochemical effects and the solid phase reaction between ferrous hydroxide and goethite generates Fe 3 O 4 [16].Subsequently, HA nanoparticles were synthesized in the suspension of Fe 3 O 4 nanoparticles in the same container by the mechanochemical method mentioned above.
4.5 mmol of ferric chloride hexahydrate (FeCl 3 6H 2 O) was dissolved in 60 ml of deionized and deoxygenated water.To precipitate amorphous ferric hydroxides (mostly, goethite), a proper amount of 1.0 M sodium hydroxide (NaOH) solution was dropped into the solution which was magnetically stirred under a continuous flow of argon at room temperature.The pH was adjusted to higher than 13.A brown suspension thus prepared was placed in a gas-tight milling pot (inner diameter 90 mm, capacity 500 ml) made of 18%Cr-8%Ni stainless steel.Stainless steel balls (diameter 3.2 mm) were used as the milling media.The charged volume including the voids among the balls was about 40% of the pot capacity.The pot was purged of air, filled with argon, and sealed.The milling was performed at room temperature for 11 h.The rotational speed was 140 rpm, corresponding to the ideal critical rotational speed.As shown in Figure 17, the SEM image indicated that the Fe 3 O 4 nanoparticles had a diameter of approximately 10-20 nm, which almost agreed with the average crystallite size (11.7 nm).The hydrodynamic size (number basis) was measured by dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments) for a dispersion, as shown in Figure 18.The median diameter was determined to be 16.4 nm from the size distribution, which was also near the average crystallite size.These results reveal that the Fe 3 O 4 nanoparticles have a single-crystal structure.
The magnetic property (magnetization-magnetic field hysteretic cycle) was analyzed using a superconducting quantum interference device (SQUID) magnetometer (Quantum Design model MPMS) at room temperature in the rage of magnetic field between -10 kOe and 10 kOe. Figure 19 shows the magnetization-magnetic field curve.The Fe 3 O 4 nanoparticles had a low coercivity (4 Oe), showing superparamagnetism.The saturation magnetization (78 emu/g) was a little lower than that of the corresponding bulk (=92 emu/g) because of the smaller size [17].After the suspension of Fe 3 O 4 nanoparticles was prepared, the milling pot was opened, and then predetermined amounts of CaHPO 4 2H 2 O and CaCO 3 were added to the suspension.Their amounts were adjusted so that the mass concentration of Fe 3 O 4 nanoparticles in the Fe 3 O 4 /HA nanocomposite was 10, 20, and 30 mass%.In order to prevent the oxidation of Fe 3 O 4 during milling, the pot was purged of air, filled with argon, and sealed prior to milling.The suspension was milled at a rotational speed of 140 rpm for 1 h at room temperature, followed by the heating at 80°C for 1 h.
Figure 20 shows the XRD pattern of Fe Figure 21 shows the SEM image of nanocomposite containing 30 mass% Fe 3 O 4 as an example.The Fe 3 O 4 nanoparticles with a diameter of about 20 nm were distributed homogeneously in the HA matrix without forming large aggregates.It was confirmed that nanometersized Fe 3 O 4 /HA composite particles were successfully synthesized.
The magnetic hyperthermia property was evaluated using an apparatus reported in elsewhere [18].A proper amount of Fe 3 O 4 /HA nanocomposite powder sample was placed in a polystyrene tube with a diameter of 16 mm, and packed by tapping the tube.The packing volume was constant at 0.8 cm 3 regardless of the Fe 3 O 4 concentration.The temperature increase was measured in an AC-magnetic field using an optical fiber thermometer.The frequency and amplitude of the AC-magnetic field were 600 kHz and 2.9 kA/m, respectively.
Figure 22 shows the temperature increase for the nanocomposites in the AC-magnetic field.
As the Fe 3 O 4 concentration increased, the temperature increased more rapidly.When the Fe 3 O 4 concentration was 30 mass%, the temperature increase of 40°C was achieved only after about 20 sec.This result supports that the Fe 3 O 4 /HA nanocomposites synthesized by this mechanochemical process exhibit a good hyperthermia property [1][2][3][4].

Low-magnification observation
High-magnification observation

Conclusion
A mechanochemical method for the simple synthesis of nanoparticles in the HA matrix.The Fe 3 O 4 /HA nanocomposites were confirmed to have a good hyperthermia property through the measurement of temperature increase in an ACmagnetic field.For example, the 30 mass% Fe 3 O 4 /HA nanocomposites showed the temperature increase of 40°C after about 20 sec under a frequency of 600 kHz and an amplitude of 2.9 kA/m.Consequently, the Fe 3 O 4 /HA nanocomposites thus synthesized were found to be a promising material for hyperthermia therapy.

Figure 1 .
Figure 1.Schematic illustration of horizontal tumbling ball mill used in this work.

Figure 8 .
Figure 8. XRD pattern of 1 h-milled samples (a) before heating and after heating at 40°C for (b) 1 h, and (c) 5 h.

Figure 14 .
Figure 14.XRD pattern of 12 h-milled samples (a) before heating and after heating at 80°C for (b) 30 min, and (c) 1 h.

Figure 15 .
Figure 15.SEM image of samples obtained by milling for (a) 1 h, (b) 3 h, and (c) 12 h, followed by heating at 80°C for 1 h.The XRD pattern of Fe 3 O 4 nanoparticles thus prepared is shown in Figure16.The Fe 3 O 4 nanoparticles had a high crystallinity and an average crystallite size of 11.7 nm which was calculated from the full width at half-maximum (FWHM) of the Fe 3 O 4 (311) diffraction peak at 2θ=35.5° using Scherrer's formula.The lattice constant was determined to be 8.387 Å from several diffraction angles showing high intensity peaks, which was close to the standard value of Fe 3 O 4 (8.396Å) as compared to that of maghemite (8.345 Å).Figure16also shows that no reflections indicating formation of other compounds were observed.This indicates the Fe 3 O 4 nanoparticles were high purity.

Figure 18 .
Figure 18.DLS particle size distribution of Fe 3 O 4 nanoparticles prepared by mechanochemical method.
Fe 3 O 4 /HA nanocomposites has been developed, in which superparamagnetic Fe 3 O 4 nanoparticles and HA nanoparticles are sequentially prepared in a single horizontal tumbling ball mill at room temperature under wet conditions.First, the synthesis process of HA (containing no Fe 3 O 4 ) was optimized.The obtained HA samples were characterized by XRD, DSC, and SEM.The influence of conditions on the formation of HA nanoparticles was investigated.Mechanochemical effects induced during wet milling promoted the reactions between CaHPO 4 2H 2 O and CaCO 3 forming HA even at room temperature.The combination of milling and heating (aging) of the suspension of CaHPO 4 2H 2 O and CaCO 3 can produce efficiently HA for short periods of time.The optimum operating conditions in the synthesis of HA were determined as follows: a rotational speed of 140 rpm, a milling time of 1 h, an aging temperature of 80°C, and an aging time of 1 h.Next, the synthesis of Fe 3 O 4 /HA nanocomposites was investigated.The mechanochemically synthesized Fe 3 O 4 nanoparticles, of which the median diameter was 16 nm, had a high crystallinity and a high saturation magnetization of 78 emu/g, and showed superparamagnetism.The wet mechanochemical process also contributed to the distribution of Fe 3 O 4

3 O 4 concentration Crystallite size of Fe 3 O 4 Crystallite size of HA
3 O 4 /HA nanocomposites with different Fe 3 O 4 concentrations.It was confirmed that the nanocomposites consisted of Fe 3 O 4 and HA having no byproducts regardless of the Fe 3 O 4 concentration.The average crystallite sizes of Fe 3 O 4 and HA were calculated from the FWHM of the Fe 3 O 4 (311) plane at 2θ=35.5° and the HA (002) plane at 2θ=25.9°, respectively, using Scherrer's formula, and listed in Table 1.The average crystallite sizes of Fe 3 O 4 and HA were almost constant regardless of the concentration of Fe 3 O 4 in the Fe 3 O 4 /HA nanocomposites.XRD pattern of Fe 3 O 4 /HA nanocomposites with different Fe 3 O 4 concentrations.

Table 1 .
Crystallite sizes of Fe 3 O 4 and HA in Fe 3 O 4 /HA nanocomposites.