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Article

Preparation of a Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex and Its Anti-Tumor Effect on Hepatocellular Carcinoma Cells

1
School of Basic Medical Science, Southeast University, Nanjing 210009, China
2
School of Clinical Medical Science, Southeast University, Nanjing 210009, China
*
Author to whom correspondence should be addressed.
Sensors 2009, 9(9), 7058-7068; https://doi.org/10.3390/s90907058
Submission received: 25 June 2009 / Revised: 17 August 2009 / Accepted: 3 September 2009 / Published: 4 September 2009

Abstract

:
Manganese-zinc-ferrite nanoparticles (Mn0.5Zn0.5Fe2O4, MZF-NPs) prepared by an improved co-precipitation method and were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectrometry (EDS). Then thermodynamic testing of various doses of MZF-NPs was performed in vitro. The cytotoxicity of the Mn0.5Zn0.5Fe2O4 nanoparticles in vitro was tested by the MTT assay. A nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex was made by an impregnation process. The complex’s shape, component, envelop rate and release rate of As2O3 were measured by SEM, EDS and atom fluorescence spectrometry, respectively. The therapeutic effect of nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex combined with magnetic fluid hyperthermia (MFH) on human hepatocelluar cells were evaluated in vitro by an MTT assay and flow cytometry. The results indicated that Mn0.5Zn0.5Fe2O4 and nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex were both prepared successfully. The Mn0.5Zn0.5Fe2O4 nanoparticles had powerful absorption capabilities in a high-frequency alternating electromagnetic field, and had strong magnetic responsiveness. Moreover, Mn0.5Zn0.5Fe2O4 didn’t show cytotoxicity in vitro. The therapeutic result reveals that the nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex can significantly inhibit the growth of hepatoma carcinoma cells.

1. Introduction

Current hyperthermia treatment strategies centre around the objective of preferential killing of malignant cells and localized heating of tissue deep inside the patient. Magnetic fluid hyperthermia (MFH) offers a means of doing this, which involves direct intratumoral injection of magnetic fluids into the target region, and then the particles are selectively heated in an externally applied alternating magnetic field (AMF). The technique uses the Curie temperature (Tc) of the magnetic material in magnetic response heating to achieve automatic temperature control and a constant temperature. Thus this technique could avoid overheating of the tissue. In vitro and in vivo experiments with magnetic fluids have documented significant antitumor effects in a murine model of liver cancer [1]. Heat also enhances the effectiveness of radiotherapy and magnifies the cytotoxicity of many anticancer drugs. For this reason, hyperthermic treatment, alone or in combination with traditional anticancer treatments, is receiving a great deal of attention.
Arsenic trioxide (As2O3), a kind of Traditional Chinese Medicine, has drawn researchers’ great interest due to its high efficacy in the treatment of acute promyelocytic leukaemia (APL). It also has been tested in some solid cancers, such as hepatocellular carcinoma [2], gastric carcinoma [3], breast cancer [4], etc. After intravenous or oral administration [5], As2O3 has been accompanied a series of side effects, such as skin reactions, gastrointestinal upset, hepatitis and even cardiotoxicity [6]. To make the best use of the drug and reduce the harmfulness to the body, it is very important to find a new form of As2O3 for clinical therapy.
In this study, As2O3 was integrated with Mn0.5Zn0.5Fe2O4 nanoparticles, which have super-paramagnetic characteristics. This feature is quite suitable for use in hyperthermia. On the one hand, a certain concentration of magnetic nanoparticles could absorb high power and transform it into heat in an alternating magnetic field, while barely damaging peripheric tissue. If used in vivo, the magnetic nanoparticles could be inducted in the target region by an external magnetic field. In this way, not only does the drug level in the tumor region rise, but also the drug dose decreases. Finally the nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex could produce chemotherapy and thermotherapy effects at the same time.

2. Results and Discussion

2.1. Characteristics of Mn0.5Zn0.5Fe2O4 Nanoparticles

Figure 1a shows an image of Mn0.5Zn0.5Fe2O4 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 Mn0.5Zn0.5Fe2O4 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 Mn0.5Zn0.5Fe2O4 nanoparticles have powerful absorption capabilities in a high-frequency alternating electromagnetic field and strong magnetic responsiveness.

2.2. Cytotoxicity of Mn0.5Zn0.5Fe2O4 Nanoparticles

The morphological changes of L929 cells after treatment with different concentrations of Mn0.5Zn0.5Fe2O4 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 Mn0.5Zn0.5Fe2O4 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 Mn0.5Zn0.5Fe2O4 didn’t show cytotoxicity in vitro.

2.3. Characterization of the Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex

The self-prepared nanosized As2O3/Mn0.5Zn0.5Fe2O4 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.

2.4. Inhibition of HepG2 Proliferation after Treated with Nanosized Complex Combined with MFH

The therapeutic results of the MTT assay are shown in Table 1. The cell growth inhibitory ratio (IR) of the As2O3 alone group, the Mn0.5Zn0.5Fe2O4 magnetic nanoparticles group and the nanosized As2O3/Mn0.5Zn0.5Fe2O4 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/Mn0.5Zn0.5Fe2O4 complex in combination with MFH could more significantly inhibit the proliferation of the HepG2 cells compared to the As2O3 alone group and Mn0.5Zn0.5Fe2O4 magnetic nanoparticles in combination with MFH group (p < 0.05). So we may conclude that the therapeutic effect of nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex in combination with MFH on HepG2 cells is much better than that of As2O3 alone or Mn0.5Zn0.5Fe2O4 in combination with MFH.

2.5. Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex Combined with MFH Induces Apoptosis of HepG2 Cells

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 Mn0.5Zn0.5Fe2O4 magnetic nanoparticles group and the nanosized As2O3/Mn0.5Zn0.5Fe2O4 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/Mn0.5Zn0.5Fe2O4 in combination with MFH could induce an obvious cell cycle disturbance and apoptosis.

3. Instruments and Methods

3.1. Preparation and Characterization of Nanosized Mn0.5Zn0.5Fe2O4 Magnetic Nanoparticles

Mn-Zn ferrite of composition Mn0.5Zn0.5Fe2O4 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.

3.2. Heating Test of Nanosized Mn0.5Zn0.5Fe2O4 in vitro

Various doses of Mn0.5Zn0.5Fe2O4 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.

3.3. The cytotoxicity of Mn0.5Zn0.5Fe2O4 Nanoparticles

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.

3.4. Preparation and Characterization of Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex

The nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex was prepared using an impregnation process. As2O3 was purchased from Sigma (St Louis, MI, U.S.A.). Satis quantum Mn0.5Zn0.5Fe2O4 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/Mn0.5Zn0.5Fe2O4 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/Mn0.5Zn0.5Fe2O4 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.

3.5. The Therapeutic Effects on Cultured Hepatocellular Carcinoma Cells-HepG2

The growth of HepG2 cells treated with nanosized As2O3/Mn0.5Zn0.5Fe2O4 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 Mn0.5Zn0.5Fe2O4 magnetic nanoparticles combined with MFH group (10 mg/mL); (4) the nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex combined with MFH group (the quantity of As2O3/Mn0.5Zn0.5Fe2O4 and Mn0.5Zn0.5Fe2O4 was adjusted to 10 mg/mL of Mn0.5Zn0.5Fe2O4 and 5 μM of As2O3). Each group contained eight wells. The magnetic nanoparticles group and the nanosized As2O3/Mn0.5Zn0.5Fe2O4 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%.

3.6. Flow Cytometry Assay

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).

4. Conclusions

Manganese zinc ferrites (Mn-Zn-Ferrit), a new kind of soft magnetic material, have been widely used in biomedicine applications including magnetic resonance, imaging contrast enhancement, tissue specific release of therapeutic agents, hyperthermia, and so on. Here, they are chosen for MFH according to their high sensitivity of magnetization to temperature and low Tc. From the results of characterization of TEM, XRD and EDS, it proved Mn0.5Zn0.5Fe2O4 were successfully prepared through an improved coprecipitation process. The prepared Mn0.5Zn0.5Fe2O4 magnetic nanoparticles had powerful absorption capabilities in the high frequency alternating magnetic field, rising to a steady temperature and showing strong magnetic responsiveness. Mn0.5Zn0.5Fe2O4 magnetic nanoparticles also show good biocompatibility, which proved the prepared nanopaticles could be applied in biomedicine.
As2O3 is a valuable therapeutic tool in hepatoma treatment. Its actions may include its ability to induce cellular apoptosis [1115], inhibit tumor metastasis [16], decrease the expression of vascular endothelial growth factor (VEGF) [17,18], enhance immune function [19], and so on. On the other hand it lacks targeting ability, and produces some adverse reactions. This limits its application in solid cancers. To resolve the problems, a nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex of As2O3 has also been prepared. From this study, nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex can be successfully prepared through an impregnation process and As2O3 can be released gradually from this As2O3/Mn0.5Zn0.5Fe2O4 complex.
The thermochemotherapy of nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex can significantly inhibit the proliferation of cultured hepatocellular cancer cells (HepG2) and induced apoptosis and cell cycle arrest when combined with magnetic fluid hyperthermia. This indicates that hyperthermia and chemotherapy could play a synergistic role. The mechanisms may be: 1) heat could make drug absorption increase due to the addition of blood flow in tumor region; 2) heat could decrease the tumor vessel regeneration for the reduction of synthesis and secretion of VEGF; 3) heat could enhance the cytotoxic effects of the chemotherapeutic drug. We have not confirmed these details, but this study may provide a new method for hepatocellular cancer therapy. However, a lot of work needs to be done if it is to be applied in clinical treatment.

Acknowledgments

The project was supported by following foundations: National Natural Science Foundation of China (Project code: 30770584); the Chinese National 863 Plan (Project code: 2007AA03Z356).

References and Notes

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Figure 1. Characterization of Mn0.5Zn0.5Fe2O4. (a) TEM image of the nanosized Mn0.5Zn0.5Fe2O4. (b) XRD of Mn0.5Zn0.5Fe2O4. (c) EDS of Mn0.5Zn0.5Fe2O4.
Figure 1. Characterization of Mn0.5Zn0.5Fe2O4. (a) TEM image of the nanosized Mn0.5Zn0.5Fe2O4. (b) XRD of Mn0.5Zn0.5Fe2O4. (c) EDS of Mn0.5Zn0.5Fe2O4.
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Figure 2. Heating test curve of sized Mn0.5Zn0.5Fe2O4 nanoparticle fluid.
Figure 2. Heating test curve of sized Mn0.5Zn0.5Fe2O4 nanoparticle fluid.
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Figure 3. Pictures of cytotoxicity evaluation on L929 cells, which were cultured in different concentrations of Mn0.5Zn0.5Fe2O4 leaching liquor. (a) Negative group. (b) 25% Leaching liquor. (c) 50% Leaching liquor. (d) 75% Leaching liquor. (e) 100% Leaching liquor. (f) Positive group.
Figure 3. Pictures of cytotoxicity evaluation on L929 cells, which were cultured in different concentrations of Mn0.5Zn0.5Fe2O4 leaching liquor. (a) Negative group. (b) 25% Leaching liquor. (c) 50% Leaching liquor. (d) 75% Leaching liquor. (e) 100% Leaching liquor. (f) Positive group.
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Figure 4. SEM image of the nanosizedAs2O3/Mn0.5Zn0.5Fe2O4.
Figure 4. SEM image of the nanosizedAs2O3/Mn0.5Zn0.5Fe2O4.
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Figure 5. The accumulative release rate of As2O3/Mn0.5Zn0.5Fe2O4 nanoparticles.
Figure 5. The accumulative release rate of As2O3/Mn0.5Zn0.5Fe2O4 nanoparticles.
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Figure 6. Apoptosis of HepG2 cells induced by the different methods. A) The negative control group. the cells of G2 phase account for 13.71.%, the apoptotic rate was 0.42%. B) The single As2O3 group the cells of G2 phase account for 44.75%, the apoptotic rate was 11.57%. C) The single Mn0.5Zn0.5Fe2O4magnetic nanoparticles group. the cells of G2 phase account for 36.15%, the apoptotic rate was 13.4%. D) The nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex group. The cells of G2 phase account for 15.9%, the apoptotic rate was 27.72%.
Figure 6. Apoptosis of HepG2 cells induced by the different methods. A) The negative control group. the cells of G2 phase account for 13.71.%, the apoptotic rate was 0.42%. B) The single As2O3 group the cells of G2 phase account for 44.75%, the apoptotic rate was 11.57%. C) The single Mn0.5Zn0.5Fe2O4magnetic nanoparticles group. the cells of G2 phase account for 36.15%, the apoptotic rate was 13.4%. D) The nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex group. The cells of G2 phase account for 15.9%, the apoptotic rate was 27.72%.
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Table 1. The results of cytotoxicity of Mn0.5Zn0.5Fe2O4 nanoparticles evaluated by MTT assay( ± s, n = 8).
Table 1. The results of cytotoxicity of Mn0.5Zn0.5Fe2O4 nanoparticles evaluated by MTT assay( ± s, n = 8).
GroupsOptical Density (OD)RGR/%Toxicity Grade
Negative control group1.035 ± 0.0421000
25% Mn0.5Zn0.5Fe2O4 leaching liquor1.017 ± 0.03898.31
50% Mn0.5Zn0.5Fe2O4 leaching liquor1.006 ± 0.05197.21
75% Mn0.5Zn0.5Fe2O4 leaching liquor0.973 ± 0.04294.01
100% Mn0.5Zn0.5Fe2O4 leaching liquor0.952 ± 0.03491.91
Positive group0.229 ± 0.02522.14
Table 2. Growth inhibitory rate of As2O3/Mn0.5Zn0.5Fe2O4 nanoparticles with MFH on HepG2 cells.
Table 2. Growth inhibitory rate of As2O3/Mn0.5Zn0.5Fe2O4 nanoparticles with MFH on HepG2 cells.
GroupsOptical density (OD)Inhibitory rate (%)

Negative control group1.207 ± 0.0710
single As2O3group (5μm/L)0.817 ± 0.02432.3% (1)
single Mn0.5Zn0.5Fe2O4 group (10g/L)0.836 ± 0.03430.7% (1)
As2O3/ Mn0.5Zn0.5Fe2O4 complex group0.540 ± 0.03755.2% (1) (2)
Comparison of experimental groups with negative control group;
(1)p < 0.05.
Comparison of nanosized As2O3/Mn0.5Zn0.5Fe2O4 complex group with single As2O3 group and single Mn0.5Zn0.5Fe2O4 magnetic nanoparticles combined with MFH group;
(2)p < 0.05.
Table 3. The table of RGR and toxicity grade.
Table 3. The table of RGR and toxicity grade.
Toxicity gradeRelative growth rate (RGR%)
Grade 0≥ 100
Grade 175–99
Grade 250–74
Grade 325–49
Grade 41–24
Grade 50

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Zhang, J.; Zhang, D. Preparation of a Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex and Its Anti-Tumor Effect on Hepatocellular Carcinoma Cells. Sensors 2009, 9, 7058-7068. https://doi.org/10.3390/s90907058

AMA Style

Zhang J, Zhang D. Preparation of a Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex and Its Anti-Tumor Effect on Hepatocellular Carcinoma Cells. Sensors. 2009; 9(9):7058-7068. https://doi.org/10.3390/s90907058

Chicago/Turabian Style

Zhang, Jia, and Dongsheng Zhang. 2009. "Preparation of a Nanosized As2O3/Mn0.5Zn0.5Fe2O4 Complex and Its Anti-Tumor Effect on Hepatocellular Carcinoma Cells" Sensors 9, no. 9: 7058-7068. https://doi.org/10.3390/s90907058

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