Hydroxyapatite Coated Iron Oxide Nanoparticles: A Promising Nanomaterial for Magnetic Hyperthermia Cancer Treatment

Targeting cancer cells without injuring normal cells is the prime objective in treatment of cancer. In this present study, solvothermal and wet chemical precipitation techniques were employed to synthesize iron oxide (IO), hydroxyapatite (HAp), and hydroxyapatite coated iron oxide (IO-HAp) nanoparticles for magnetic hyperthermia mediated cancer therapy. The synthesized well dispersed spherical IO-HAp nanoparticles, magnetite, and apatite phases were confirmed by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and Field emission transmission electron microscopy (FETEM) with Energy Dispersive X-ray spectroscopy (EDS). The non-toxic behavior of synthesized IO-HAp nanoparticles was confirmed by cytotoxicity assay (Trypan blue and MTT assay). The synthesized nanoparticles revealed a remarkable magnetic saturation of 83.2 emu/g for IO and 40.6 emu/g for IO-HAp nanoparticles in presence of 15,000 Oe (1.5 T) magnetic field at room temperature (300 K). The magnetic hyperthermia study that was performed with IO-HAp nanoparticles showed an excellent hyperthermia effect (SAR value 85 W/g) over MG-63 osteosarcoma cells. The in vitro hyperthermia temperature (~45 °C) was reached within 3 min, which shows a very high efficiency and kills nearly all of the experimental MG-63 osteosarcoma cells within 30 min exposure. These results could potentially open new perceptions for biomaterials that are aimed for anti-cancer therapies based on magnetic hyperthermia.


Introduction
Nanotechnology advanced the functional characteristics of nanoparticles for biomedical applications. The advances to treat diseases, such as cancer, enhanced bioimaging property, and controlled drug releasing ability make them very significant for biomedical research [1]. The prime difficulties are presently related to the systematic administration and biodistribution of drugs, inadequate drug concentration at the clinical site, toxicity due to nonspecific targeting, and high drug concentration related issues. Magnetic drug targeting solves many of these problems [2]. Since the

NO 3 + 20 H 2 O
The synthesized HAp nanoparticles were dried at 70 • C and calcined at 600 • C for one hour in the air atmosphere.

Synthesis of Magnetic Fe 3 O 4 Nanoparticles
To synthesize magnetic Fe 3 O 4 nanoparticles, the solvothermal method was employed [24]. A 0.6 M FeCl 3 ·6H 2 O solution was prepared by dissolving 1.621 g of ferric chloride salt in 50 mL of ethylene glycol and stirred under nitrogen flow for 30 min. Next, 1.3 M of sodium acetate was added under stirring. Finally, 0.5 g of trisodium citrate was added, followed by continuous stirring for 3 h at room temperature. The final mixture was transferred to an autoclave chamber (100-mL capacity Teflon-lined stainless steel) and heated at 190 • C for 10 h. Finally, after cooling down the autoclave to room temperature, the obtained dark black precipitate was washed several times with deionized (DI) water and ethanol and dried at 65 • C for 12 h, followed by storing under a nitrogen atmosphere. The synthesized Fe 3 O 4 nanoparticles were highly hydrophilic and could be easily dispersed in water.

Synthesis of Fe 3 O 4 -HAp (IO-HAp) Nanocomposites
The IO-HAp nanocomposites were synthesized by wet chemical precipitation technique ( Figure 1). The synthesized iron oxide (IO) nanoparticles were well dispersed in a 0.06 M calcium nitrate tetrahydrate 70 mL solution, and were stirred vigorously with a mechanical stirrer. During this the procedure, Ca 2+ ions initiate its nucleation over IO surface due to its favorable zeta potential. Next, 50 mL of a 0.07 M di-ammonium hydrogen phosphate solution was gently added dropwise to the IO-containing solution. The next step followed by nucleation of PO 4 3− ions deposition over Ca 2+ to form HAp crystals. The whole experimental study was performed at 40 • C temperature with pH 9.0. Soon after addition of the di-ammonium hydrogen phosphate, the clear solution became cloudy due to the formation of the HAp coating over the IO nanoparticles. The solution was continuously agitated with a mechanical stirrer for 5 h, and was finally washed with deionized water. The nanoparticles were dried at 80 • C for 6 h, and were finally kept in a vial for further application.

Synthesis of Magnetic Fe3O4 Nanoparticles
To synthesize magnetic Fe3O4 nanoparticles, the solvothermal method was employed [24]. A 0.6 M FeCl3·6H2O solution was prepared by dissolving 1.621 g of ferric chloride salt in 50 mL of ethylene glycol and stirred under nitrogen flow for 30 min. Next, 1.3 M of sodium acetate was added under stirring. Finally, 0.5 g of trisodium citrate was added, followed by continuous stirring for 3 h at room temperature. The final mixture was transferred to an autoclave chamber (100-mL capacity Teflon-lined stainless steel) and heated at 190 °C for 10 h. Finally, after cooling down the autoclave to room temperature, the obtained dark black precipitate was washed several times with deionized (DI) water and ethanol and dried at 65 °C for 12 h, followed by storing under a nitrogen atmosphere. The synthesized Fe3O4 nanoparticles were highly hydrophilic and could be easily dispersed in water.

Synthesis of Fe3O4-HAp (IO-HAp) Nanocomposites
The IO-HAp nanocomposites were synthesized by wet chemical precipitation technique ( Figure 1). The synthesized iron oxide (IO) nanoparticles were well dispersed in a 0.06 M calcium nitrate tetrahydrate 70 mL solution, and were stirred vigorously with a mechanical stirrer. During this the procedure, Ca 2+ ions initiate its nucleation over IO surface due to its favorable zeta potential. Next, 50 mL of a 0.07 M di-ammonium hydrogen phosphate solution was gently added dropwise to the IO-containing solution. The next step followed by nucleation of PO4 3− ions deposition over Ca 2+ to form HAp crystals. The whole experimental study was performed at 40 °C temperature with pH 9.0. Soon after addition of the di-ammonium hydrogen phosphate, the clear solution became cloudy due to the formation of the HAp coating over the IO nanoparticles. The solution was continuously agitated with a mechanical stirrer for 5 h, and was finally washed with deionized water. The nanoparticles were dried at 80 °C for 6 h, and were finally kept in a vial for further application.

Characterization
A powder X-ray diffraction (XRD) study was performed by a Bruker AXN XRD analyzer, (Karlsruhe, Germany). Fourier transform infrared spectra was measured by using JASCO FTIR 4100 (Tokyo, Japan), with wavenumber range of 450 to 4000 cm −1 . The Fourier-transform infrared spectroscopy (FTIR) pellets were prepared by mixing the samples with Potassium bromide (KBr). Thermogravimetric analysis was performed by Perkin-Elmer Pyris Diamond at a heating rate of 10 °C/min in a nitrogen atmosphere. Field emission transmission electron microscopy (FETEM) was

Characterization
A powder X-ray diffraction (XRD) study was performed by a Bruker AXN XRD analyzer, (Karlsruhe, Germany). Fourier transform infrared spectra was measured by using JASCO FTIR 4100 (Tokyo, Japan), with wavenumber range of 450 to 4000 cm −1 . The Fourier-transform infrared spectroscopy (FTIR) pellets were prepared by mixing the samples with Potassium bromide (KBr). Thermogravimetric analysis was performed by Perkin-Elmer Pyris Diamond at a heating rate of 10 • C/min in a nitrogen atmosphere. Field emission transmission electron microscopy (FETEM) was performed using FETEM, JEOL 2010 (Tokyo, Japan), instrument. The average hydrodynamic size of nanoparticles was determined by dynamic light scattering (DLS), using particle size analyzer (Beckman Coulter, Brea, CA, USA). The magnetic saturation study was performed by superconducting quantum interference device magnetometer (Quantum design, MPMS XL).

Cell Culture
MG-63 osteosarcoma cells (Korea Cell Line Bank, Seoul, Korea) were used and cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum and 100 U mL −1 Penicillin and 100 µg mL −1 Streptomycin (Thermo Fischer Scientific, Waltham, MA, USA). The cells were incubated at 37 • C temperature in 5% CO 2 atmosphere. The growth medium was changed after each 24 h of incubation time interval.  (20,40,60, 80, 100, or 120 µg/mL) were then added to the culture medium. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and Trypan blue cytotoxicity assays were performed to examine the in vitro cytotoxicity of IO-HAp. The control was set as absence of nanoparticles. The cells were incubated at 37 • C, and the cell viability at different incubation times (12,24, and 48 h) was analyzed. 100 µL of MTT reagent was added to each well followed by 2-4 h incubation at 37 • C temperature. Finally, after incubation, the media were replaced by 100 µL of dimethyl sulfoxide and well mixed. A microplate reader measured the absorbance at the wavelength of 570 nm. The relative cell viability percentage is evaluated by the following equation: MG-63 cells were cultured (10 5 cells/cm 2 ) in the DMEM complete media at 37 • C. Synthesized IO, HAp, and IO-HAp nanoparticles were tested for in vitro cytotoxicity testing. The sample of 100 µg/mL concentration was mixed into the culture media. After 24 h, the cells were stained with trypan blue, and after washing with Phosphate buffer saline (PBS) the cells were examined under the microscope.

Nanoparticles Internalization Study by Cells
The MG-63 osteosarcoma cells were seeded at a cell density of 10 5 cells/mL in a 35-mm culture plate and incubated in DMEM medium for 24 h. Only IO-HAp nanoparticles (100 µg/mL) were incubated with the attached MG-63 cells for 12 h. Following the incubation with the nanoparticles, the cells were stained with the 0.01% (w/v) proflavine solution. After incubation for 30 min, the dye was removed and washed gently with PBS solution to remove the nanoparticles' excess and 100 µL of trypsin were added. After 2 min of incubation with trypsin, 100 µL of medium was added and finally observed under the fluorescence microscope (LEICA DMI 3000B, Wetzlar, Germany) with external magnetic force.

Magnetic Hyperthermia Experiment
The heating efficiency of the IO and IO-HAp nanoparticles was assessed in the presence of A/C magnetic hyperthermia device. The study was performed at 180 Gauss magnetic field strength with Nanomaterials 2017, 7, 426 6 of 15 409 kHz frequency. Approximately 100 µg/mL of the IO and IO-HAp nanoparticles were dispersed in water and were placed in a 6.5-cm radius copper coil.

In Vitro Magnetic Hyperthermia
To investigate the in vitro hyperthermia effects of IO and IO-HAp, nanoparticles MG-63 osteosarcoma cells were seeded at a cell density of 10 5 cells/mL in a 35-mm culture plate with 2.5 mL of medium and incubated for 24 h. The cells with no nanoparticles were considered as the control and were incubated for 12 h. For the experimental study, cells were incubated for 12 h with 100 µg/mL of IO and IO-HAp nanoparticles. The experimental cell plates were positioned inside the hyperthermia coil and a 0.6 Tesla A/C magnetic field was applied with a frequency of 307 kHz and amplitude of 628 Oe for 30 min. The temperature was recorded with an optical thermocouple (AMOTH 8000, Anritsu Meter Co. Ltd., Tokyo, Japan), and was also observed under Infrared (IR) thermal camera.

X-ray Diffraction (XRD) Analysis
The sharp peaks for the calcined HAp and IO nanoparticles reveal their high crystallinity (Figure 2a 409 kHz frequency. Approximately 100 µg/mL of the IO and IO-HAp nanoparticles were dispersed in water and were placed in a 6.5-cm radius copper coil.

In Vitro Magnetic Hyperthermia
To investigate the in vitro hyperthermia effects of IO and IO-HAp, nanoparticles MG-63 osteosarcoma cells were seeded at a cell density of 10 5 cells/mL in a 35-mm culture plate with 2.5 mL of medium and incubated for 24 h. The cells with no nanoparticles were considered as the control and were incubated for 12 h. For the experimental study, cells were incubated for 12 h with 100 µg/mL of IO and IO-HAp nanoparticles. The experimental cell plates were positioned inside the hyperthermia coil and a 0.6 Tesla A/C magnetic field was applied with a frequency of 307 kHz and amplitude of 628 Oe for 30 min. The temperature was recorded with an optical thermocouple (AMOTH 8000, Anritsu Meter Co. Ltd., Tokyo, Japan), and was also observed under Infrared (IR) thermal camera.

X-ray Diffraction (XRD) Analysis
The sharp peaks for the calcined HAp and IO nanoparticles reveal their high crystallinity ( Figure  2a

Fourier Transform Infrared Spectroscopy
The FTIR analysis for pristine HAp, IO, and IO-HAp are shown in Figure 3a. The functional groups of the prepared samples confirm the presence of pristine IO and HAp. In IO-HAp nanoparticles, with additional peaks corresponding to HAp and IO are confirmed the close interaction between IO and HAp ( Table 1). The FTIR spectrum of HAp nanoparticles confirms the characteristic bands for PO 4 3− appear at 479, 567, 605, 1046, and 1098 cm −1 [25,26].  [29]. This peak appears possibly due to the use of ethylene glycol during the synthesis of iron oxide nanoparticles.

Thermogravimetric Analysis
The HAp, IO, and IO-HAp nanoparticles were studied for their thermal stability by thermogravimetric analysis in inert (nitrogen) atmosphere. The thermal decay curves (Figure 3b) show the differences in residual mass of the samples with controlled heating. The total weight loss from pure IO was approximately 2.64% during the experimental study due to the evaporation of adsorbed physical and chemical water. For HAp and IO-HAp nanoparticles weight losses were 6.8% and 9.07%, respectively. Moreover, for HAp and IO-HAp, the first weight loss was recorded at 80-200 • C due to entrapped physical water and at 200-500 • C temperature due to the crystallization of HAp nanoparticles, and the third weight loss that was related to the decomposition of remaining chemical complexes. At a higher temperature, no such major weight loss was detected. TG analysis of the IO-HAp nanoparticles exhibited weight loss of up to 850 • C temperature, and thereafter weight losses were negligible. The thermogravimetric study reveals the thermal stability of these nanoparticles with increased heating in a controlled atmosphere. The FETEM study revealed that the synthesized IO nanoparticles are spherical in structure and well dispersed, as presented in Figure 4a. The particle size distribution of synthesized IO nanoparticles shows spherical morphology with an average size 62.14 ± 10.8 nm. The HAp nanoparticles coating over IO nanoparticles are shown in Figure 4c,d. The EDS analysis confirmed the presence of Fe, Ca, P, and O with Ca/P ratio of 1.70 (Figure 4f). The synthesized IO and IO-HAp nanoparticles average hydrodynamic particle size diameters were studied by laser mediated dynamic light scattering method (Figure 4b,e). The average size distribution of IO nanoparticles was determined as 75.34 ± 5.56 nm, and for IO-HAp was 95.16 ± 14.92 nm, which is very close resemblance with TEM calculated data.  The FETEM study revealed that the synthesized IO nanoparticles are spherical in structure and well dispersed, as presented in Figure 4a. The particle size distribution of synthesized IO nanoparticles shows spherical morphology with an average size 62.14 ± 10.8 nm. The HAp nanoparticles coating over IO nanoparticles are shown in Figure 4c,d. The EDS analysis confirmed the presence of Fe, Ca, P, and O with Ca/P ratio of 1.70 (Figure 4f). The synthesized IO and IO-HAp nanoparticles average hydrodynamic particle size diameters were studied by laser mediated dynamic light scattering method (Figure 4b,e). The average size distribution of IO nanoparticles was determined as 75.34 ± 5.56 nm, and for IO-HAp was 95.16 ± 14.92 nm, which is very close resemblance with TEM calculated data. The FETEM study revealed that the synthesized IO nanoparticles are spherical in structure and well dispersed, as presented in Figure 4a. The particle size distribution of synthesized IO nanoparticles shows spherical morphology with an average size 62.14 ± 10.8 nm. The HAp nanoparticles coating over IO nanoparticles are shown in Figure 4c,d. The EDS analysis confirmed the presence of Fe, Ca, P, and O with Ca/P ratio of 1.70 (Figure 4f). The synthesized IO and IO-HAp nanoparticles average hydrodynamic particle size diameters were studied by laser mediated dynamic light scattering method (Figure 4b,e). The average size distribution of IO nanoparticles was determined as 75.34 ± 5.56 nm, and for IO-HAp was 95.16 ± 14.92 nm, which is very close resemblance with TEM calculated data.

Vibrating Sample Magnetometer
The magnetic hysteresis loops for IO and IO-HAp nanoparticles are depicted in Figure 5. The IO nanoparticles show strong magnetic behavior, with the saturation magnetization of 83.2 emu/g; whereas, IO-HAp shows relatively low magnetic saturation of 40.6 emu/g, at 300 K and 1.5 T. The magnetic properties of HAp coated IO (IO-HAp) by different researchers compared with our present study are mentioned in Table 2. The saturation magnetization of pure IO is high (83.2 emu/g) whereas after coating the saturation decreases (40.6 emu/g) due to the coating of ceramic HAp. In spite of having comparatively low magnetic saturation, it is sufficient to treat for magnetic hyperthermia study, and also more than 20 emu/g magnetic saturation of IO-HAp nanoparticles are very few reported. Both IO and IO-HAp nanoparticles show the characteristic superparamagnetic behavior, with very less coercivity and remanent magnetization. The pristine HAp does not show any hysteresis loop in the magnetic field.

Vibrating Sample Magnetometer
The magnetic hysteresis loops for IO and IO-HAp nanoparticles are depicted in Figure 5. The IO nanoparticles show strong magnetic behavior, with the saturation magnetization of 83.2 emu/g; whereas, IO-HAp shows relatively low magnetic saturation of 40.6 emu/g, at 300 K and 1.5 T. The magnetic properties of HAp coated IO (IO-HAp) by different researchers compared with our present study are mentioned in Table 2. The saturation magnetization of pure IO is high (83.2 emu/g) whereas after coating the saturation decreases (40.6 emu/g) due to the coating of ceramic HAp. In spite of having comparatively low magnetic saturation, it is sufficient to treat for magnetic hyperthermia study, and also more than 20 emu/g magnetic saturation of IO-HAp nanoparticles are very few reported. Both IO and IO-HAp nanoparticles show the characteristic superparamagnetic behavior, with very less coercivity and remanent magnetization. The pristine HAp does not show any hysteresis loop in the magnetic field.     [16]. Sarath Chandra et al. reported cobalt-doped HAp nanoparticles as a promising material for drug delivery, magnetic imaging, and hyperthermia-mediated cancer treatment [17]. The cobalt co-doped HAp materials show good biocompatibility on blood cells (<5% hemolysis), with superior antimicrobial activity [18]. Hou et al. studied the in vivo hyperthermia effect of magnetic HAp nanoparticles to treat tumors. This cytotoxicity study that was conducted with blood samples from experimental mouse reveals good biocompatibility of magnetic HAp nanoparticles [22].
The MTT assay was performed for the HAp, IO, and IO-HAp nanomaterials using MG-63 (osteosarcoma) cells. Different concentrations (20,40,60,80, 100, 120 µg/mL) of the HAp, IO, and the IO-HAp nanoparticles were studied for MTT assay. The relative percentage of cell viability is shown in Figure 6. The results show that the HAp and IO-HAp nanoparticles are cytocompatiable on MG-63 cells, whereas, IO nanoparticles shows marked toxicity. The relative percentage of cell viability suggests the maximum toxicity (approx. 42% cell death) that is shown by IO nanoparticles with the concentration of 120 g/mL. The MG-63 cells well tolerate the nanoparticles loading concentration up to 100 g/mL. The study was further performed by 100 g/mL concentration nanoparticles for 48 and 72 h (Figure 6b). The time duration study for cell cytotoxicity estimation confirms the maximum toxicity effect by IO nanoparticles, whereas HAp and IO-HAp nanoparticles show nontoxic behavior. As HAp is an excellent biocompatible material for this the coating of HAp over IO nanoparticles makes the composite nontoxic. The primary biocompatibility (in vitro cytotoxicity) screening is very important for materials that are used in the biomedical field. Ajeesh et al. reported that 40 wt % of Fe3O4-HAp does not harm osteoblast cell activity [16]. Sarath Chandra et al. reported cobalt-doped HAp nanoparticles as a promising material for drug delivery, magnetic imaging, and hyperthermia-mediated cancer treatment [17]. The cobalt co-doped HAp materials show good biocompatibility on blood cells (<5% hemolysis), with superior antimicrobial activity [18]. Hou et al. studied the in vivo hyperthermia effect of magnetic HAp nanoparticles to treat tumors. This cytotoxicity study that was conducted with blood samples from experimental mouse reveals good biocompatibility of magnetic HAp nanoparticles [22].
The MTT assay was performed for the HAp, IO, and IO-HAp nanomaterials using MG-63 (osteosarcoma) cells. Different concentrations (20,40,60,80, 100, 120 µg/mL) of the HAp, IO, and the IO-HAp nanoparticles were studied for MTT assay. The relative percentage of cell viability is shown in Figure 6. The results show that the HAp and IO-HAp nanoparticles are cytocompatiable on MG-63 cells, whereas, IO nanoparticles shows marked toxicity. The relative percentage of cell viability suggests the maximum toxicity (approx. 42% cell death) that is shown by IO nanoparticles with the concentration of 120 g/mL. The MG-63 cells well tolerate the nanoparticles loading concentration up to 100 g/mL. The study was further performed by 100 g/mL concentration nanoparticles for 48 and 72 h (Figure 6b). The time duration study for cell cytotoxicity estimation confirms the maximum toxicity effect by IO nanoparticles, whereas HAp and IO-HAp nanoparticles show nontoxic behavior. As HAp is an excellent biocompatible material for this the coating of HAp over IO nanoparticles makes the composite nontoxic.

Trypan Blue Study
The experimental results showed that almost all of the cells are healthy and survived in the presence of synthesized HAp and IO-HAp nanoparticles. But, for IO nanoparticles, the cells died and retained the trypan blue stain due to the toxic effect of IO nanoparticles. The control well (without any nanoparticles) shows maximum cell proliferation that is almost similar with HAp nanoparticles containing culture well. The observed cells were healthy, as depicted in Figure 7. After the in vitro hyperthermia study, the cells are treated with trypan blue, but nearly all of the cells died due to hyperthermia effect and retained the trypan blue stain.

Trypan Blue Study
The experimental results showed that almost all of the cells are healthy and survived in the presence of synthesized HAp and IO-HAp nanoparticles. But, for IO nanoparticles, the cells died and retained the trypan blue stain due to the toxic effect of IO nanoparticles. The control well (without any nanoparticles) shows maximum cell proliferation that is almost similar with HAp nanoparticles containing culture well. The observed cells were healthy, as depicted in Figure 7. After the in vitro hyperthermia study, the cells are treated with trypan blue, but nearly all of the cells died due to hyperthermia effect and retained the trypan blue stain.

Nanoparticles Internalization Study by Cells
In the hyperthermia study, cells must uptake the magnetic nanoparticles inside so that the heating effect will able to more effectively to kill the target cells. In this study, only the IO-HAp nanoparticles were incubated with the MG-63 cells. Due to the HAp biomaterials coating the IO-HAp, nanoparticles do not show any toxic effect and are easily engulfed by the MG-63 cells. To confirm the IO-HAp internalization by MG-63 cells the IO-HAp treated cells are placed on a rotating magnetic field. By the effect of external magnetic force, only the IO-HAp internalized cells showed rotational movements (Video S1: Video of MG-63 IO-HAp NPs.).

Magnetic Hyperthermia on MG-63 Cells
Magnetic hyperthermia system could be used to kill cancer cells by their energy loss as heat (hysteresis loss). Only IO and IO-HAp nanoparticles are tested for the hyperthermia effect and for the specific absorption rate (SAR) value calculation. The synthesized IO and IO-HAp nanomaterials have the ability to produce heat in an alternating magnetic field. The hyperthermia experiment of IO and IO-HAp nanoparticles was initiated at 25 °C room temperature, and it reached 42 °C within 3 min after starting the experiment. The IO nanoparticles showed a very rapid temperature increase, and within 10 min it shows around 66 °C, however, IO-HAp reached a lesser temperature of about 54 °C (Figure 8a). The coating of HAp nanoparticles as an insulator over IO nanoparticles causes less of a temperature increase when compared to pristine IO nanoparticles. For efficient hyperthermia effect to kill the cancer cells, 42 °C to 47 °C temperature is ideal for a long time duration. The heating efficiency of the synthesized superparamagnetic IO-HAp nanomaterial was studied with MG-63 cells for 30 min incubation in hyperthermia field. When the temperature reaches to 47 °C, the hyperthermia instrument is controlled by power off to maintain the temperature between 42 °C and 47 °C for 30 min. All of the temperatures were measured by thermocouple and infrared thermal camera (Figure 8b).

Nanoparticles Internalization Study by Cells
In the hyperthermia study, cells must uptake the magnetic nanoparticles inside so that the heating effect will able to more effectively to kill the target cells. In this study, only the IO-HAp nanoparticles were incubated with the MG-63 cells. Due to the HAp biomaterials coating the IO-HAp, nanoparticles do not show any toxic effect and are easily engulfed by the MG-63 cells. To confirm the IO-HAp internalization by MG-63 cells the IO-HAp treated cells are placed on a rotating magnetic field. By the effect of external magnetic force, only the IO-HAp internalized cells showed rotational movements (Video S1: Video of MG-63 IO-HAp NPs.).

Magnetic Hyperthermia on MG-63 Cells
Magnetic hyperthermia system could be used to kill cancer cells by their energy loss as heat (hysteresis loss). Only IO and IO-HAp nanoparticles are tested for the hyperthermia effect and for the specific absorption rate (SAR) value calculation. The synthesized IO and IO-HAp nanomaterials have the ability to produce heat in an alternating magnetic field. The hyperthermia experiment of IO and IO-HAp nanoparticles was initiated at 25 • C room temperature, and it reached 42 • C within 3 min after starting the experiment. The IO nanoparticles showed a very rapid temperature increase, and within 10 min it shows around 66 • C, however, IO-HAp reached a lesser temperature of about 54 • C (Figure 8a). The coating of HAp nanoparticles as an insulator over IO nanoparticles causes less of a temperature increase when compared to pristine IO nanoparticles. For efficient hyperthermia effect to kill the cancer cells, 42 • C to 47 • C temperature is ideal for a long time duration. The heating efficiency of the synthesized superparamagnetic IO-HAp nanomaterial was studied with MG-63 cells for 30 min incubation in hyperthermia field. When the temperature reaches to 47 • C, the hyperthermia instrument is controlled by power off to maintain the temperature between 42 • C and 47 • C for 30 min. All of the temperatures were measured by thermocouple and infrared thermal camera (Figure 8b).
The hyperthermia mediated cancer treatment is promising due to its less susceptibility to temperatures in the range of 42-47 • C, where normal cells are unaffected. The heating by magnetic particles due to the effect of magnetic field can occur via several mechanisms. The most common heating mechanism is due to the induced Eddy currents, which occur in bulk materials. The Eddy current for nano-sized materials heating is very weak because of the poor electrical conductivity of magnetic nanoparticles. Additionally, heating due to the hysteresis loss is dominant for this type of nanomaterials. Nanoparticles especially exhibit hysteresis loss due to Néelian and Brownian relaxation. The rotation of individual magnetic moments within the nanoparticles causes heat generation, referred to as the Néelian relaxation. In contrary, the Brownian relaxation mediated heat generation is caused due to the physical rotation of particles with the alignment process of magnetic moments (Figure 8c). After the hyperthermia treatment of MG-63 cells, trypan blue and TEM analysis were performed to analyze the cellular viability. In trypan blue assay, all of the cells retained the trypan blue stain, which confirms the effectiveness of nanoparticles in hyperthermia. In TEM analysis of treated MG-63 cells, the cells were found arrested in necrosis phase. This could be attributed to the effect of heat that is generated by magnetic hyperthermia reactive oxygen species (ROS). Gu et al., 2014, reported that the generation of ROS by heat stress might be involved with transcription-independent p53-mediated mitochondrial pathways [39]. Skibba et al., 1991, reported that hyperthermia mediated oxidative stress causes irreversible hepatocellular injury [40]. Additionally, the cellular internalization of nanoparticles could have induced the ROS, which causes oxidation and denaturation in cellular system results in damage of proteins, nucleotides, and membranes and finally resulting in cell death. In this study, the TEM analysis revealed that cell necrosis results in cell degradation and formation of blebs, which causes deformities or lysis of the cells (figure in Scheme 1). The hyperthermia mediated cancer treatment is promising due to its less susceptibility to temperatures in the range of 42-47 °C, where normal cells are unaffected. The heating by magnetic particles due to the effect of magnetic field can occur via several mechanisms. The most common heating mechanism is due to the induced Eddy currents, which occur in bulk materials. The Eddy current for nano-sized materials heating is very weak because of the poor electrical conductivity of magnetic nanoparticles. Additionally, heating due to the hysteresis loss is dominant for this type of nanomaterials. Nanoparticles especially exhibit hysteresis loss due to Néelian and Brownian relaxation. The rotation of individual magnetic moments within the nanoparticles causes heat generation, referred to as the Néelian relaxation. In contrary, the Brownian relaxation mediated heat generation is caused due to the physical rotation of particles with the alignment process of magnetic moments (Figure 8c). After the hyperthermia treatment of MG-63 cells, trypan blue and TEM analysis were performed to analyze the cellular viability. In trypan blue assay, all of the cells retained the trypan blue stain, which confirms the effectiveness of nanoparticles in hyperthermia. In TEM analysis of treated MG-63 cells, the cells were found arrested in necrosis phase. This could be attributed to the effect of heat that is generated by magnetic hyperthermia reactive oxygen species (ROS). Gu et al. 2014, reported that the generation of ROS by heat stress might be involved with transcription-independent p53-mediated mitochondrial pathways [39]. Skibba et al. 1991, reported that hyperthermia mediated oxidative stress causes irreversible hepatocellular injury [40]. Additionally, the cellular internalization of nanoparticles could have induced the ROS, which causes oxidation and denaturation in cellular system results in damage of proteins, nucleotides, and membranes and finally resulting in cell death. In this study, the TEM analysis revealed that cell necrosis results in cell degradation and formation of blebs, which causes deformities or lysis of the cells (figure in Scheme 1).

Measurement of SAR Effect
The heating efficiency of the nanoparticles in presence of A/C magnetic field was calculated as the specific absorption rate (SAR), and defined as the amount of energy transformed to heat per time and mass [41,42].
SAR is calculated by the initial linear temperature increase (∆T) per time interval (∆t).
where, c (JL/K) denotes the water, specific heat capacity (4185 J L/K), and m (g/L) is the concentration of magnetic materials in water or liquid media. The slope ∆T/∆t curve was calculated up to first 60 s only (Figure 8c). The estimated SAR value for the IO and IO-HAp nanoparticles were calculated as 103 W/g and 85 W/g, respectively. The hyperthermia mediated heating effectiveness recommends the potential application of synthesized IO-HAp nanoparticles as nano heater for magnetic hyperthermia-mediated cancer therapy.

Conclusions
The synthesis and fabrication of magnetic nanomaterials with high biocompatibility are important for application in the biomedicine. HAp coated IO nanoparticles (IO-HAp) were fabricated as an effective nano heater for hyperthermia cancer therapy. Due to the unique surface activity of HAp, the composite nanoparticles did not require any further chemical modification. The synthesized composite IO-HAp nanoparticles were hydrophilic and superparamagnetic with good magnetic saturation of 40.6 emu/g. Without the magnetic field, the synthesized nanoparticles show minimal or no cytotoxic effect on cell lines. The cytotoxicity assay study revealed that the hyperthermia (~45 • C) mediated cell death to the cancer cells. The synthesized nanoparticles show an enhanced heating efficiency as nano heater when compared to conventional HAp coated iron oxide nanoparticles with a SAR value of 85 W/g when placed to A/C magnetic field. IO-HAp combined with magnetic hyperthermia can be a safe and effective therapeutic tool for different types of cancer treatment.