Investigating antitumor therapeutic efficacy using magnetic hyperthermia of Fe3O4 nanoparticles

Four sets of Fe3O4 nanoparticles were synthesized using a one-pot hydrothermal method using ethylene glycol (EG) and water reaction mixture. The EG coated Fe3O4 nanoparticles were characterized using XRD (X-ray diffraction) and DLS (dynamic light scattering). The nanoparticles hydrodynamic sizes are in the range 60-100 nm and showed good dispersibility in water and cellular medium without any aggregation. The specific absorption rate (SAR) under an alternating magnetic field (AMF) was measured to evaluate the magneto hyperthermic effect of the nanoparticles under field amplitudes of 51.85, 43.87, 35.89, and 27.92 kA/m and field frequencies in the range 384.5-167.30 kHz. The nanoparticles synthesized using 30% EG showed the highest SAR value of 372.95 W/g, whereas the particles synthesized using 70% EG showed a SAR value of 217.12 W/g. SAR values show a linear dependency on the AC frequency whereas the field amplitude displayed a nonlinear effect on the SAR. Breast cancer cells (MDA) treated with the nanoparticles for 30 minutes under an AMF of frequency of 384.5 kHz and strength of 24.67 kA/m showed a 50% decrease in cell viability. The cellular viability further decreased to 25% after 24 h treatment under the AMF which is remarkable for the therapeutic application of the nanoparticles. The apoptotic cell death showed a dependency on the AMF frequency and strength combinations.


Introduction
The use of magnetic nanoparticles (MNPs) in an alternating magnetic field (AMF) to generate heat and selectively destroy cancer cells through hyperthermia is a potential cancer treatment method [1,2].Due to their high biocompatibility and minimal collateral damage to healthy tissue, Fe3O4 magnetic nanoparticles have drawn the most attention among different magnetic nanoparticles [3].Magnetic nanoparticles are also examined for drug delivery and targeting tumor cites using an external magnetic field, biosensors to detect specific biomolecules, contrast agents in MRI allowing for enhanced imaging of specific tissues, separation and purification of biomolecules and cells, magnetic scaffolds for tissue engineering, and magnetic cell labeling [4][5][6][7][8].Magnetic nanoparticles generate localized heating in tissues under an AMF [9].The technique has been explored as a potential treatment for cancer, as it has been shown to induce tumor cell death by hyperthermia-mediated apoptosis and necrosis [1].The saturation magnetization, shape, Neel relaxation, and Brownian relaxation of the nanoparticles are some of the factors that affect the magnetic heating capabilities of the particles [10,11].Synthetic conditions are used to optimize the properties by employing different synthesis methods like organometallic decomposition, hydrothermal, and microwave assisted synthesis [12,13].Synthesizing high efficiency nanoparticles for magnetic heating is essential to develop therapeutic agents for magnetic hyperthermia.Designing appropriate nanoparticles for the treatment of malignancies requires a greater understanding of the cell death mechanism triggered via hyperthermia.Numerous studies have shown that magnetic hyperthermia causes apoptosis in cancer cells by activating caspase, upregulating pro-apoptotic proteins, and downregulating anti-apoptotic BCL-2 proteins, as well as by heating cancer cells to high temperatures [14].Furthermore, it has been demonstrated that magnetic hyperthermia causes oxidative stress, which produces reactive oxygen species (ROS), which then causes cancer cells to undergo apoptosis [15,16].This study is intended to synthesize the nanoparticles with high heating efficiency using the hydrothermal method with EG acting as a co-solvent and surfactant.Furthermore, nanoparticles efficiency is examined for the killing of cancer cells with respect to frequency and field amplitude of AMF.The cell death mechanism is investigated using the flow cytometry and MTT method with two different AMF parameters.

Experimental 1 Materials and Methods
Fe3O4 nanoparticles were synthesized by a one-step hydrothermal method using a mixture EG and water solvent.2.8 g of anhydrous iron chloride FeCl3 (17.28 mmol) and iron chloride tetrahydrate FeCl2.4H2O (8.64 mmol) was dissolved in 100 ml of water and EG reaction mixture.Four sets of reaction mixture are used with EG concentration 10, 30, 50, and 70 %.They are labeled as S1, S2, S3, and S4, respectively.To obtain a homogenous solution, the reaction mixture of salts and solvent is stirred at room temperature for 20 minutes. 1 N NaOH solution is added dropwise under constant stirring to adjust the pH to 12-13, and the precipitate produced is stirred continuously for 20 minutes.The reaction mixture is transferred to a Teflon-lined autoclave, which is sealed and heated in an oven at 180 ºC for 6 h.After the autoclave cooled to room temperature, the produced nanoparticles were separated with an external magnet and washed using distilled water.Using an IR lamp, the nanoparticles are dried before being used for further characterization and magnetic hyperthermia investigations to kill cancer cells.

Nanoparticles Characterization and Cell Culture
The diffraction pattern is obtained using an X-ray powder diffractometer (Bruker D8) with a Cu Ka radiation source (wavelength=1.5406A, generator voltage=40 kV, and generator current=40 mA).The powder sample was scanned at a rate of 0.02 o /0.1 s throughout a 2-theta range of 25-70 o .The hydrodynamic diameter and zeta potential of EG coated nanoparticles are determined using the Anton Paar Litesizer DLS 500 using forward scattering angle.Cells from the MDA-MB-231 human breast cancer cell line (ATCC® HTB-26, ATCC, Manassas, VA, USA) were grown in RPMI and Mccoy's media conditions, which were both supplemented with 10% FBS and 1% penicillin/streptomycin, respectively.For further investigation, all cell lines were cultured at 37 oC with 5% CO2 and humidified air.After being washed with PBS and transferred to a 15-ml centrifuge tube containing medium at each time point, the cells were resuspended in 500 ml of binding buffer before being mixed with 5 μl of Annexin V-FITC and 5 μl of PI.Using BD Biosciences, San Jose, California, USA, flow cytometric analysis was carried out after 15 minutes.

XRD
Figure 1 displays the XRD patterns of the nanoparticles synthesized with varied EG percentages in the reaction mixture from the hydrothermal treatment.The ferrite nanoparticles characteristic Bragg diffraction peaks had 2-theta values of 30.19° (220), 35.57° (311), 43.22° (400), 53.47° (422), 57.10° (511), and 62.70° (440).These peaks are associated with the cubic spinel structure of ferrite and the Fd3m (227) space group, and the lattice parameters a = b = c = 8.3724 Å.The observed lattice parameters are consistent with the previously reported lattice parameter of Fe3O4 nanoparticles and were determined by multiple peak fitting approach utilizing Jade XRD.[13].As indicated in Figure 1, there were no additional peaks of any other impurity phase in the XRD patterns.FWHM determined from the highest intensity peak (311) is used to determine the average crystallite sizes of the produced nanoparticles by the Debye-Scherrer formula [17].The average sizes are in the range of 15-17 nm and depend on the percentage of EG and water mixture used for the synthesis.EG acts as a solvent and surfactant for controlling particle size which is evident from the decreasing particle size with the increase in EG percentage in reaction mixture.The average size and lattice parameters of the four sets of nanoparticles are listed in Table 1.

Hydrodynamic diameter and Zeta potential using DLS.
The dispersed nanoparticle hydrodynamic diameter and zeta potential are determined using the Anton Paar Litesizer with forward scattering angle.Figure 2(a) and (b) shows the nanoparticle zeta potential and hydrodynamic size.EG coating of the nanoparticles is responsible for their extremely high hydrodynamic diameter, which is in the range of 60-120 nm compared to the crystallite sizes determined by XRD.Table 1 includes the synthetic conditions, average crystalline sizes, hydrodynamic diameter, and zeta potential of the nanoparticles.The nanoparticle in water forms a stable dispersion, as shown by a positive zeta potential value at neutral pH.

Magnetic hyperthermia measurements of nanoparticles dispersions
The nanoparticles are dispersed in water by probe sonication for 10 minutes to obtain the water dispersion of nanoparticles.This is important in order to determine the magneto thermic effect using nanoScale Bio magnets hyperthermia instrument.AMF with frequencies of 384.50, 330.55, 304.75, and 167.30kHz and field amplitudes of 51.85, 43.87, 35.89, and 27.92 kA/m is used to acquire the heating profiles of the nanoparticles.In order to precisely determine the heating efficiency, adiabatic conditions were maintained for the calorimetric tests.Using a thermal probe, the rise in water dispersion temperature is measured.SAR values for the dispersion of nanoparticles are obtained using the slope of the initial heating profile.The SAR values are given by equation 1 [18]: mMNP is the mass (in g) of the MNPs in the water dispersion, C (4184 Jkg -1 K -1 ) is the specific heat capacity of water, and dT/dt is the initial slope of the temperature-time curve derived from AMF treatment.Figure 3(a) displays the heating profiles of S2 (30% EG) nanoparticles produced under a field amplitude of 27.92 kA/m and different frequencies.Figure 3(b) displays the SAR values obtained for nanodispersions of 1 and 2 mg/ml as a function of EG %.S2 nanoparticles showed the highest SAR value indicating high thermal conversion ability among the four set of nanoparticles.S2 nanoparticles (30% EG) showed the highest SAR value of 372.95 W/g whereas S4 (70%) had a SAR value of 217.12 W/g under AMF.The SAR values of the nanoparticles showed a dependence on the percentage of EG used for the synthesis.The heating profiles are obtained under constant frequency and field amplitude range.The SAR values obtained from the four sets of nanoparticles at constant field amplitude and frequency are shown in Figures 3(c) and (d).SAR values increase nonlinearly with AMF strength and almost linearly with AMF frequency.Based on the linear response theory (LRT), where SAR increases linearly with AMF frequency and quadratically with AMF intensity, the results slightly deviate from those anticipated [19].The role of dipolar interactions at this particle concentration could be the cause of this deviation from the LRT.When compared to several papers that synthesis nanoparticles utilizing the coprecipitation approach, the SAR values observed in this study are significantly higher [20,21].

MTT Cytotoxicity Assay
Since S2 (30%) nanoparticles showed high thermal conversion ability, invitro studies for the killing of cancer cells are examined with two different sets of AMF parameters (AMF1: 24.67 kA/m-384.50kHz and AMF2:47.74 kA/m-167.5kHz) which generate a saturation temperature of 46 ºC for 1 mg/ml nano dispersion.The effect of magnetic field frequency and field amplitude parameters, which result in the same saturation temperature, on the mechanism of cell death is examined using these parameter combinations.Four set of cells were utilized to examine the magnetic cytotoxicity: Cells from the control group (C) were incubated at 37 °C throughout the experiment.One group of cells (F) was subjected to a magnetic field without of nanoparticles to examine the effects of the magnetic field.Another group of cells (P) were treated without the presence of a field in order to study the effects of the nanoparticles alone.For the AMF treatment cells (P+F) were treated with 1 mg/ml of S2 nanoparticles for 30 minutes exposure time and 46 ºC saturation temperature.The cytotoxicity of EG coated nanoparticles treated with two sets of AMF settings at a concentration of 1 mg/ml is determined using the MTT assay.MDA-MB-231 that was treated with AMF treatment was seeded in triplicate on a 96-well tissue culture plate (Corning, Sigma-Aldrich Co., St. Louis, MO, USA) at a density of 5000 cells per well.The absorbance of the MTT assay was calculated at 570 nm using the ELISA plate reader SynergyTM HTX microplate reader (BioTek, Winooski, VT, USA) used to evaluate cell viability.Figure 4 illustrates the cytotoxicity of 1 mg/ml nanoparticles treated with AMF (24.67 kA/m-384.50kHz) reduced the viability of MDA-MB-231 cells by about 48 and 75% after 0 and after 24 h, respectively.The results of the cell proliferation experiment for cells treated with AMF showed a significant difference for various time points for cell lines compared to the control group with a p value of 0.0001.It was shown that there was no magnetic field toxicity when the viability of cancer cells treated with the magnetic field alone was comparable to the viability of the control at various time points.

Apoptosis
In response to numerous physiological and pathological stimuli, apoptosis, a type of programmed cell death, occurs.It is distinguished by separate morphological and biochemical alterations, including cell shrinkage, chromatin condensation, DNA fragmentation, and caspase enzyme activation [22].As a result of cellular stress caused by magnetic hyperthermia, the MNPs' heat may stimulate a number of signalling pathways that cause apoptosis.The activation of heat shock proteins (HSPs), which are essential for controlling cellular stress responses, is one such mechanism [23].HSPs can accelerate protein breakdown, stop proteins from misfolding, and control the function of apoptotic proteins like caspases.Additionally, oxidative stress brought on by magnetic hyperthermia might result in the production of reactive oxygen species (ROS), which can harm cellular components and cause apoptosis.The mitogen-activated protein kinase (MAPK) pathway, which can cause apoptosis by activating caspases and encouraging the release of cytochrome c from the mitochondria, is one of the signalling pathways that ROS can activate [24,25].The apoptosis of cancer cells involves early apoptosis, late apoptosis, and necroptosis.Using a flow cytometer from BD Biosciences in San Jose, California, the apoptotic and necrotic cell death of MDA-MB-231 cells after treatment with two sets of AMF field and frequency parameters is determined.MDA-MB-231 cells were exposed to magnetic nanoparticles and AMF for 0 hours, and the apoptosis rate was approximately 48%.within 24 hours, it increased to 75%, as illustrated in Figure 5. Nanoparticles are biocompatible and don't have any inherent toxicity, as evidenced by the fact that cells treated with the field (F) and particle (P) alone have minimal apoptosis.The percentage of early and late apoptosis obtained from the flow cytometry are listed in Table 2. From the apoptosis data it is clear that two sets of AMF parameters used cause apoptosis in different amounts: AMF1 (24.67 kA/m-384.50kHz) shows high rate of late apoptosis compared to little early apoptosis effect, whereas AMF2 (47.74 kA/m-167.5kHz) conditions show significant apoptosis both early and late.Total apoptosis seems to be the same for both sets of parameters.The apoptotic cell death showed a dependency on the AMF frequency and field strength combinations though the cells are subjected to similar thermal treatment of 46 ºC for 30 min.This can be related to the impact of the field parameters on the uptake of nanoparticles by cells, ROS produced as a result of thermal heating, and the response of the cell thermal shock proteins, which are essential factors for cell death.Understanding the effect of field parameters on the cell death pathways will help to determine the differential effect of modulating reactive oxygen species and heat shock proteins response to the AMF parameters.These studies will help in designing the in vivo magnetic hyperthermal treatment of cancer tumors.In order to understand the impact of AMF settings on the gene and shock protein expressed by the nanoparticles, additional studies are required.Overall, these studies suggest that magnetic hyperthermia induce apoptosis in cancer cells, which may contribute to its potential as a therapeutic technique for cancer treatment.

Conclusions
Ethylene glycol and water mixtures in various percentages are used as the reaction medium in a one-step hydrothermal technique to produce Fe3O4 nanoparticles.The produced nanoparticles had average crystalline diameters between 15 and 17 nm.The nanoparticles' zeta potential shows that their dispersions are stable in aqueous medium.The proportion of EG and water employed for the synthesis determines the nanoparticles' ability for heat conversion; the 30% EG reaction mixture displayed the highest SAR value, measuring 372.95 W/g.SAR values show a linear dependency on the AC frequency whereas the field amplitude has a nonlinear effect on the SAR values.The two sets of AMF parameters used for the invitro treatment causes apoptosis in a different rate, 24.67 kA/m-384.50kHz shows high late apoptosis whereas 47.74 kA/m-167.5kHz conditions show significant early apoptosis.Though the nanoparticles showed high SAR and high magnetic heating toxicity towards cancer cells.Further studies are required to understand the field parameters dependent cellular uptake of the nanoparticles on the effect of ROS and shock protein response for thermal heating.These studies will help to improve the potential of the magnetic nanoparticles for the designing the in vivo treatment of cancer tumors using AMF.

Figure 1 .
Figure 1.XRD patterns of the nanoparticles synthesized using hydrothermal method with ethylene glycol percentage 10, 30, 50, and 70% in the reaction mixture.

Figure 2 .
Figure 2. (a) Hydrodynamic diameter of Fe3O4 nanoparticles obtained using dynamic light scattering method.(b) Zeta potential of nanoparticles at neutral pH.

Figure 3 .
Figure 3. (a) Heating profiles obtained from the S2 nano dispersion under field amplitude 27.92 kA/m and various frequencies.(b) SAR values of the S1-S4 nanoparticles obtained under AMF (384.5 kHz and 27.92 kA/m), (c) Field amplitude dependent SAR values at 167.5 kHz, (d) AC frequency dependent SAR of S1-S4 nanoparticles at 27.92 kA/m.

Figure 4 .
Figure 4. Cell viability of MDA cells treated with Fe3O4 nanoparticles under AMF with frequency of 384.5 kHz and strength of 24.67 kA/m obtained after 0 and 24 hr treatment (C-Cells, F-Cells+AMF, P-Cells+Particles, & P+F-ells+Particles+AMF).

Table 1 .
The average crystallite size, lattice constant, hydrodynamic diameter, and zeta potential of Fe3O4 nanoparticles.

Table 2 .
Apoptosis of MDA-MB-231 cells subjected to two distinct sets of frequencies and field strengths.