Figure 1a shows an image of Mn_0.5Zn_0.5Fe₂O₄ nanoparticles acquired by TEM. It shows that they were nearly spherical, with high electron-density and uniform in size. The X-ray diffraction pattern of the ferrite sample is shown in Figure 1b. The observed diffraction lines were found to correspond to those of a standard manganese ferrite pattern, thereby indicating that the samples have spinel structure. The particle size of the samples had been estimated from the broadening of the X-ray diffraction peaks, using the Scherrer equation for Lorentzian peak: d = 0.9 λ/(w−w₁)/cosθ. The average particle size was found to be between 6 and 22 nm. EDS reports showed At% of Mn, Zn, Fe were 17.58%, 16.44% and 65.98%, respectively, which is consistent with a molar ratio of 0.5:0.5:1. Figure 2 shows the results of thermodynamic test of various doses of Mn_0.5Zn_0.5Fe₂O₄ nanoparticles fluid. The nanoparticles were dispersed in 0.9% NaCl and exposed to a high-frequency alternating electromagnetic field (output current equal to 30 A) for 60 min. As the concentrations of magnetic fluid (MF) increase, its temperature rose from 39.5 to 50 °C, and the temperature was stable after exposure to the magnetic field for 40 minutes. Our study thus shows that Mn_0.5Zn_0.5Fe₂O₄ nanoparticles have powerful absorption capabilities in a high-frequency alternating electromagnetic field and strong magnetic responsiveness.

The morphological changes of L929 cells after treatment with different concentrations of Mn_0.5Zn_0.5Fe₂O₄ leaching liquor were observed by inverted microscopy. As shown in Figure 3, the shapes and growth of the treated cells were similar to that of cells in the negative group. They exhibited normal features, such as clear edges, homogeneous staining and no cell fragments, while the cells of the positive group became small and globular, and even parts of cells were suspended 48 hours later. Only small amounts of cells survived. The results of the MTT assay are shown in Table 1. According to RGR and toxicity grade conversion table (see Section 3.3), the toxicity of Mn_0.5Zn_0.5Fe₂O₄ leaching liquor was classified as grade 1, which was safe to the cells. This is in agreement with the findings from inverted microscopy and demonstrated that Mn_0.5Zn_0.5Fe₂O₄ didn’t show cytotoxicity in vitro.

The self-prepared nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex particles are approximately spherical and uniform in size (Figure 4). The EDS result confirmed that the prepared complex only contained As, Mn, Zn, Fe and O. The envelop rate of As₂O₃ was 0.260%. The release rate of As₂O₃ is shown in Figure 5. As time elapsed, the release of as increased gradually.

The therapeutic results of the MTT assay are shown in Table 1. The cell growth inhibitory ratio (IR) of the As₂O₃ alone group, the Mn_0.5Zn_0.5Fe₂O₄ magnetic nanoparticles group and the nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex group were 32.3%, 30.7% and 55.2%, respectively. The negative control group and the experimental groups had a great difference (p < 0.05). Due to the combined advantages of the thermotherapy and the chemotherapy, the nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex in combination with MFH could more significantly inhibit the proliferation of the HepG2 cells compared to the As₂O₃ alone group and Mn_0.5Zn_0.5Fe₂O₄ magnetic nanoparticles in combination with MFH group (p < 0.05). So we may conclude that the therapeutic effect of nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex in combination with MFH on HepG2 cells is much better than that of As₂O₃ alone or Mn_0.5Zn_0.5Fe₂O₄ in combination with MFH.

The present study disclosed that the combination of thermotherapy and chemotherapy resulted in synergistic inhibition of hepatoma [7]. Both As₂O₃ [8] and ferromagnetic fluid thermotherapy [9] could induce apoptosis of HepG2 cells.

In our study, the flow cytometry assay showed that the apoptotic indexes of HepG2 cells of the single As₂O₃ group, the single Mn_0.5Zn_0.5Fe₂O₄ magnetic nanoparticles group and the nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex group after treatment were 11.57%, 13.48% and 27.72%, respectively. However, the apoptotic index of the control group was 0.42%. In the experimental groups, we found a significant hypodiploid peak before the G1 phase, which was the apoptotic peak (Figure 6). At the same time, there was a prominent cell cycle blockage in the G2/M phase in these groups. Other studies also found As₂O₃ and hyperthermia could arrest the cell cycle at S or G2/M phase in tumor cells respectively [9,10], so As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ in combination with MFH could induce an obvious cell cycle disturbance and apoptosis.

Mn-Zn ferrite of composition Mn_0.5Zn_0.5Fe₂O₄ was prepared by the precipitation method (for details see [6]). Its shape was observed by H-600 transmission electron microscope (TEM). X-ray diffraction (XRD) was used to analyse its crystal structure and diameter. An energy dispersive spectrometer (EDS) was employed to assay its composition.

Various doses of Mn_0.5Zn_0.5Fe₂O₄ nanoparticles were dispersed in 5 mL 0.9% NaCl, to concentrations of 8, 10, 12.5 and 15 g/L, respectively. Then the nanoparticles fluids were placed in a flat-bottomed cuvette. This in turn was placed 5 mm from the bottom of the cuvette to the center of hyper-thermia-coil of high frequency electromagnetic field (SP-04C, Shenzhen, China). The output frequency was 230 KHz and the output current was 30 ampere, heating 1 h and the temperature was measured at 5 min intervals.

The sterile nanoparticles were diffused in PRPMI 1640 medium (containing 10% fetal calf serum) for 72 h at 37 °C. After centrifugalization, the supernatant was filtered to get leaching liquor of 100% concentration. L929 cells in a 96-well plate, 6,000 cells per well, were treated after 24 hours incubation with various concentrations of leaching liquor (100%, 75%, 50% and 25% leaching liquor), and incubation was continued for 48 hours. At the same time, cells cultured with RPMI1640 medium containing 10% fetal calf serum as the negative control group, and cells with 0.7% polyacrylamide as positive group were also prepared. Inverted microscopy was used to observe general morphological changes of the L929 cells, then the MTT assay was performed and the OD value was measured at 492 nm. The cell relative growth rate was calculated as follows: RGR% = OD of experimental group/OD of control group × 100%. According to the biological evaluation of medical devices (test for in vitro cytotixicity (ISO10993-5:1999, IDT)), the toxicity grade is precise when the experimental results show grade 0 and grade 1. The results show grade 2, which needs evaluate by RGR% and the morphological changes of cultured cells. The toxicity grade is uncertian when it belongs to other grades.

The nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex was prepared using an impregnation process. As₂O₃ was purchased from Sigma (St Louis, MI, U.S.A.). Satis quantum Mn_0.5Zn_0.5Fe₂O₄ nanoparticles were added into the solution of As₂O₃ (0.01 mg/mL, pH = 5, adjusted by acetic acid) under ultrasonic sound dispersion conditions. After 30 min standing at 80 °C, the production (nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex) was centrifuged at 2,000 g/min for 10 min and rinsed twice with absolute alcohol, then dried. Their shape, component and the envelop ratio were measured with SEM, EDS and atom spectrophotometer, respectively. Subsequently the release rate of As₂O₃ was measured. One hundred mg of As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex were put in a visking bag filter and 50 mL 0.9% NaCl was placed outside as dialysis mediator. The system was agitated at the speed of 50 rpm in 37 °C. In first 5 hours, we sampled every 0.5 h intervals and supplemented the same volume of 0.9% NaCl. Then we sampled at the following 24, 36 and 48 h, respectively. The amount of As₂O₃ of the sample was measured by atom fluorescence spectrometry.

The growth of HepG2 cells treated with nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex combined with MFH was examined by an MTT assay. HepG2 cells were seeded in a 96-well plate with 6,000 cells per well, and after 24 hours incubation they were divided into 4 groups: (1) the negative control group (RPMI1640 medium containing 10% fetal calf serum); (2) the As₂O₃ alone group (5 μM of As₂O₃); (3) the Mn_0.5Zn_0.5Fe₂O₄ magnetic nanoparticles combined with MFH group (10 mg/mL); (4) the nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex combined with MFH group (the quantity of As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ and Mn_0.5Zn_0.5Fe₂O₄ was adjusted to 10 mg/mL of Mn_0.5Zn_0.5Fe₂O₄ and 5 μM of As₂O₃). Each group contained eight wells. The magnetic nanoparticles group and the nanosized As₂O₃/Mn_0.5Zn_0.5Fe₂O₄ complex group were treated with MFH for 60 minutes under a high frequency alternating electro-magnetic field (f = 230 KHz, I = 30 A). Then incubation of all groups was continued for 48 hours, the MTT assay was performed and OD value was measured at 492 nm. The cell growth inhibition rate was calculated as follows: (1-OD of experimental group / OD of control group)×100%.

The cells of control group and experimental groups were collected and rinsed in 0.1 PBS (pH 7.2–7.4) three times, resuspended and fixed in 70% ethanol at 4 °C overnight. Cells were centrifuged and resuspended in 0.1 g/L RNase A at 37 °C for 30 min and in 0.05 g/L propidium iodide at 4 °C for 30 min. The cell cycle was analyzed by flow cytometer (FACS Vantage SE, Becton-Dickson Co).