The procedure was followed according to established by Rangel-Mendez and Streat in 2001 [27] . 5 g of activated carbon (AC) was placed in a three-necked flask with a solution of HNO₃ with deionized water (50:50) and this was brought to a controlled temperature of 85˚C for 3 h. After the respective time, three-necked flask was removed and placed in an ice bath to prevent oxidation further progress. The nitric acid excess was removed and the activated carbon was washed with deionized water. Finally, the activated carbon was dried in an oven at a temperature of 80˚C for 24 h.

For the synthesis of this ferrite, FeCl₂・4H₂O and FeCl₃・6H₂O (molar ratio 1:2) were mixed in 50 mL of deionized water. On the other hand, in a ball flask were placed 150 mL of deionized water and heated at 70˚C at 1000 rpm using a mechanical stirrer.

As the temperature was reached, 50 ml of concentrated ammonium hydroxide (pH 9.8) were added in order to facilitate a basic medium in the solution with low stirring and heating to a temperature of 70˚C. Subsequently, mixture of iron chloride solution was added dropwise and left under constant stirring (5000 rpm) for half an hour.

Once the time has expired, the precipitate obtained was washed with 2 L of deionized water to remove excess chloride and allowed to dry at room temperature for 3 days. Finally, the product obtained was washed with 1 L of water and 250 mL of ethanol and allowed to dry at room temperature.

Once obtained magnetite and the oxidized activated carbon, a carbon-magnetite composite (AC/Fe₃O₄) was obtained by mechanosynthesis technique. This procedure took place on the FRITSCH planetary mill brand, model Pulverisette 6. A certain amount of activated carbon and ferrite were added to the container and placed in agate mill at 400 rpm for 3 h in order to obtain a composite of ferrite and activated carbon 60%/40% respectively, due to this ratio showed the best heating properties obtained in preliminary tests, since the ferrite mediate the heating capacity. Finally, the product was washed with deionized water and allowed to dry at room temperature.

The magnetite nanoparticles and the composite AC/Fe₃O₄ were analyzed by X-ray diffraction (XRD) (Siemens Mod. D-5000). The magnetic properties of the samples were measured with a SQUID Quantum Design magnetometer (VSM) in applied fields from −12.5 to 12.5 KOe. The particle size and shape were studied by field emission scanning electron microscopy (SEM) (JOEL JSM-7401F) and energy dispersive X-ray (EDX) techniques. FT-IR spectra of the materials have been taken by a Perkin Elmer FTIR, model Spectrometer Frontier.

This technique was performed to determine whether the particles had the ability to generate heat. These tests consisted of placing certain concentration of sample, in a vial with 2 mL of water, which was stirred with a vortex. These vials were carried to an equipment of magnetic induction in solid state, which was programmed using a magnetic field (10.2 kA/m and frequency 200 kHz), during 15 minutes.

In order to determine the biocompatibility of activated carbon and AC/Fe₃O₄ composite with human erythrocytes, hemolysis tests were performed. The hemolysis test was performed using human whole blood from healthy non-smoking donors, following the proper guidelines for studies using human specimens. The procedure was conducted as reported by Muzquiz-Ramos et al. in 2014 [28] . A certain amount of activated carbon and composite (AC/Fe₃O₄) were contacted with 150 µL of human blooderythrocytes in 1850 µL of Alsever’s solution (dextrose 0.116 M, NaCl 0.071 M, sodium citrate 0.027 M and citric acid 0.002 M, pH 6.4) in order to obtain concentrations of 0.5 mg/mL, 2 mg/mL and 3 mg/mL. The hemolysis percent was obtained by the Equation (1).

where:

As: Sample absorbance;

: Negative control absorbance (erythrocytes/Alsever’s solution);

: Positive control absorbance (erythrocytes/deionized water).

XRD patterns of magnetite and the AC/Fe₃O₄ composite are shown in Figure 1. The diffraction peaks of the synthesized magnetite in Figure 1(a) can be perfectly indexed to the inverse spinel structure (JCPDS card no. 019-0629), and no characteristic peaks of impurities are detected in the XRD pattern, implying that the formation of the single phase spinel. As shown in Figure 1(b), for AC/Fe₃O₄ composite, could observe the most intense

peaks of magnetite. However, it has also been shown that activated carbon has a very broad peak between 40˚ and 60˚, peak that can be observed slightly in the composite XRD pattern. The disappearance of the other peaks may be due to overlap of signals by the amorphous structure of graphite [29] .

Furthermore, the crystallite size values were calculated by the Scherrer’s equation, taking the most intense peak and the Gaussian model. The crystallite size of the synthesized magnetite is 10.9 nm. Determining the particle size is very useful because, to use a material in magnetic hyperthermia, it must have a size between 9 and 15 nm. It has been shown that the spherical type nano-sized particles have higher diffusion speed, increasing the concentration of nanoparticles in the center of a blood vessel, thus limiting interaction of the nanoparticles with endothelial cells and prolong circulation time of the nanoparticles in the blood [30] .

The morphology of the AC/Fe₃O₄ composite was investigated by SEM observations and EDX analysis. As shown in Figure 2(a), EDX analysis indicates presence of phosphorus in activated carbon surface related to phosphates due to the activation process. Furthermore, in Figure 2(b), it can be seen the composite AC/Fe₃O₄ micrograph were Fe₃O₄ particles seems to be deposited on activated carbon surface. Also the EDX analysis indicates a decrease in the amount of C atoms on the composite, in comparison with the activated carbon and moreover the presence of Fe atoms.

In addition, Figure 3 shown the nanoparticles deposited in activated carbon surface, which have a size between 8 and 22 nm. These results are comparable to those obtained by the Scherrer’s equation of crystallite size using the diffraction data.

The magnetic properties of the samples were measured in a SQUID Quantum Design magnetometer in applied fields from −12.5 to 12.5 KOe. Figure 4 shows the hysteresis loops of the synthesized magnetite (a) and AC/Fe₃O₄ composite (b). Both hysteresis loops exhibit a superparamagnetic behavior, however the results of saturation magnetization, remanent magnetization and coercivity are summarized in Table 1. Coercivity data are of great importance, as these show superparamagnetic behavior of the materials when coercivity values are near 0. In case of the synthesized magnetite coercivity value is 10.28 Oe and for AC/Fe₃O₄ composite coercivity value is 8.92 Oe. This coercivity values indicates that both samples have a superparamagnetic behavior.

Moreover, the saturation magnetization of AC/Fe₃O₄ composite decreases in comparison of the synthesized magnetite due to the presence of activated carbon, which has no magnetic properties. These results are consistent with those expected due to the presence of activated carbon, which does not show a magnetic behavior. Mendes et al. in 2014, attribute this difference in saturation magnetization due to the presence of a non-magnetic phase, for example an organic diamagnetic material as the activated carbon [31] .

In order to determine the bonds formed between AC and Fe₃O₄ in the mechanosynthesis process, infrared

spectroscopy tests were performed. FT-IR spectra of oxidized activated carbon (AC) and AC/Fe₃O₄ composite are shown in Figure 5. When activated carbon is contacted with an oxidizing agent such as nitric acid, it has been determined that the concentration of functional groups is increased [32] . Furthermore, also in Figure 4 it is possible to observe FT-IR spectra of AC/Fe₃O₄ composite in which there is clearly a decrease in the intensity of the bands between 1500 cm⁻¹ and 1700 cm⁻¹ and disappearance of OH⁻ band (between 3500 cm⁻¹ to 3000 cm⁻¹), relative to AC precursor. This may indicate that magnetite is being bound to these functional groups in mechanosynthesis process due to the high energy formed in the milling. It is also clear oxygen-metal bond, since a

band at 773 cm⁻¹ is presented.

Moreover, the main functional groups that can be observed in the FT-IR spectra are summarized in Table 2, which for activated carbon principal bands attribution are phenolics and carboxylic acid groups, formed by the oxidation process in the material surface. For AC/Fe₃O₄ composite is more clearly detailed the disappearance of phenolic groups corresponding to bands and some kind of carboxylic acid bond, due to the bond formed between this oxygenated groups and the magnetite.

Furthermore, the heating ability of the composite was also tested. Analysis for different masses of AC/Fe₃O₄ composite (16 mg, 18 mg and 20 mg) were performed. Heating curves are presented in Figure 6, where the greater the amount of mass greater heating capacity was observed. However, for the mass of 20 mg, this composite managed to generate a temperature of 48.7˚C, temperature at which cell damage occurs known as heat ablation. According to the heating curve, 18 mg of the AC/Fe₃O₄ composite is able to use in hyperthermia technique, since this mass generated a temperature of 45.6˚C in 15 minutes. The adequate temperature for hyperthermia treatment it’s above 46˚C [33] .

For determine if the materials were biocompatible to human erythrocytes, hemolysis assay were carried out. This test is important since the material can be applied directly to the bloodstream, so that in a given moment be in contact with the erythrocytes of a human blood. Figure 7 shows the hemolysis percent caused by the AC/Fe₃O₄ composite at different concentrations. Activated carbon hemolysis percent is also reported. Error bars represent the mean and standard deviation for six experiments. The results of hemolytic test (Figure 7) demonstrated that the HR of the samples were lower than 5%. According to ASTM F 756-08 (Standard Practice for Assessment of Hemolytic Properties of Materials) [34] , HR < 5% produced by any material could be considered as not hemolytic. According to these results, we can deduce that the activated carbon and the composite AC/Fe₃O₄ are not a hemolytic materials and is biocompatible with human blood erythrocytes.