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  • br The metabolic activity of cells was


    The metabolic activity of UNC1999 was assessed on days 1, 3, and 7 of culture using PrestoBlue assay (Invitrogen). As per the manufacturer recommendation, the assay reagent was diluted 1:10 in culture media and incubated with samples for 1 h. The fluorescent intensity, which is an index for metabolic activity, was measured using a BioTek Synergy 2 plate reader. In addition, the viability of the cultures was assessed employing a Live/Dead assay kit (Invitrogen, USA), where staining of live or dead cells is done by calcein-AM (ethidium homodimer-1). The live/dead solutions were diluted in phosphate buffered saline (PBS) at a ratio of 1:2000 and 1:500, respectively. The samples were incubated for 20 min at 37 °C and visualized for fluorescence signals using a fluor-escent microscope (Zeiss) where the live and dead cells would appear in green and red color, respectively.
    Each experiment was repeated three times and all data were pre-sented by means ± standard deviation (SD). Statistical analysis has been done with two-way ANOVA by Duncan. Differences were con-sidered statistically significant at p < 0.05.
    3. Results and discussion
    3.1. Characterization of powders
    than that Ca2+ (0.098 nm) which led to the lower unit cell parameters. On the other hand, the intensity of the peaks decreased in 0.2Fe-HT in comparison with the normal HT. This phenomenon can be attributed to the degree of distortion of HT structure that increased by doping Fe in HT.
    We have shown that by using the sol-gel method, Fe3+ ions can be successfully incorporated in HT structure. It is clear from XRD pattern that magnetite or maghemite do not form in the doped structure. Fig. 2(a), (b), and (c) showed TEM micrographs of obtained HT and 0.2Fe-HT, and as-received magnetite powders with the average particle size of around 40, 60, and 30 nm respectively (obtained by Image J software). Fig. 2(d), (e), and (f) presented the DLS analysis of HT, 0.2Fe-HT, and magnetite powders, respectively. Studying particle size dis-tribution in HT, 0.2Fe-HT, and magnetite powders showed that they were distributed in the size range of about 18–200 nm, 30–200 nm, and 8–80 nm, respectively with the peak maxima at 35–40 nm, 60 nm, and 25 nm, respectively. These results revealed that the TEM micrographs were consistent with the DLS analysis.
    The magnetization curves of the synthesized 0.2Fe-HT and as re-ceived magnetite powders are shown in Fig. 3(a) and (c) respectively. Magnetite and 0.2Fe-HT samples exhibited saturation magnetization of ~14.63 and ~5.15 emu/g, remnant magnetization of 0.23 and 0.11 emu/g and coercivity of 580.01 and 227.45 KA/m, respectively. Both materials showed relatively strong magnetic behavior. The sa-turation magnetization of magnetite was obviously higher than 0.2Fe-HT sample. Ferromagnetic material, as a structure with a multi-mag-netic domain, can produce heat because of hysteresis losses, which can be measured as the amount of energy dissipated during a magnetization cycle. Hysteresis losses can be measured by integrating the area of the hysteresis loop.
    As can be seen, both magnetite and 0.2Fe-HT powders show obvious hysteresis loop areas where heat generation is considerable because of the hysteresis loss.
    3.2. Hyperthermia property of magnetite and 0.2Fe-HT powders
    Fig. 3(b) and (d) show the behavior of generated heat as a function of time under AMF with a strength of 45.2 G (hyperthermia effect curve) for magnetite and 0.2Fe-HT samples. For the same exposure time of 250 s, magnetite specimens exhibited higher recorded temperature compared to 0.2Fe-HT, 53 and 40 °C for magnetite and 0.2Fe-HT, re-spectively. The conversion of magnetic energy to thermal energy for MNPs under AMF is explained by two mechanisms including Néel and Brownian relaxations. Néel relaxation is the dominant mechanism for the solid specimen without any dispersion in a liquid phase which is related to our case. For this mechanism, heat is generated by quickly changing the direction of magnetic moments in UNC1999 comparison with crystal lattice (internal dynamics) [22]. As can be seen in Fig. 3, magnetite exhibited a higher increase in temperature over a long period of time. Although the results showed a quick increase of temperature in mag-netite powder relative to the 0.2Fe-HT, the generated heat in 0.2Fe-HT powders was enough to get the desired temperature for hyperthermia application [8]. The generated heat under AMF in 0.2Fe-HT powders can be attributed to the existence of doped Fe in the structure. It is pertinent to mention here that this could not be related to the existence of magnetic side phases which has not been observed in the 0.2Fe-HT specimen.
    The heat generation in 0.2Fe-HT sample was related to its sig-nificant hysteresis loss due to exhibiting an obvious hysteresis loop (Fig. 3(c)). This figure also shows that 250 s was enough for increasing temperature to about 40 °C for 0.2Fe-HT, thereby suggesting that 0.2Fe-HT could be employed in developing materials for different hy-perthermia applications. As can be seen in Fig. 3(b) and (d), the tem-perature of the control sample (only water and without any dispersed magnetic sample) approximately remains constant during the experi-ment. It shows that magnetic field does not generate heat by itself.