Synthesis and characterization of glycyrrhizic acid ...

Materials Science and Engineering C 77 (2017) 1060–1067

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Synthesis and characterization of glycyrrhizic acid coated iron oxide nanoparticles for hyperthermia applications Mostafa Saeedi a, Omid Vahidi a,⁎, Shahin Bonakdar b a b

School of Chemical, Petroleum and Gas Engineering, Iran University of Science and Technology, Narmak, Tehran 16846, Iran National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 4 February 2017 Received in revised form 31 March 2017 Accepted 2 April 2017 Available online 04 April 2017 Keywords: Iron oxide magnetic nanoparticles Oxidative precipitation Cytotoxicity Glycyrrhizic acid Magnetic hyperthermia

a b s t r a c t In the present study, iron oxide magnetic nanoparticles (IONPs) were synthesized using the oxidative precipitation method for biomedical applications. Glycyrrhizic acid (GA) extracted from the roots of licorice plant was used as the coating agent for the synthesized nanoparticles (GAIONPs). The crystal phase, morphology and size were investigated by XRD, FE-SEM and TEM. The saturation magnetization (ms) value of the nanoparticles was measured by VSM indicating lowered ms of the GAIONPs with respect to that of the IONPs due to the presence of GA. In addition, the specific loss power of nanoparticles in a solution and in a tissue mimicking phantom was measured using an alternating magnetic field generator. The presence of the GA on the crystal surface was further confirmed using FT-IR and TG/DTA measurements. The specific surface area of the nanoparticles was measured by BET indicating that GA coating agent increases the available active surface area of the nanoparticles for about 25% making it more appropriate for drug loading purposes. The cytotoxicity of the nanoparticles was investigated using MTT assay on L929 fibroblast cell line and the results demonstrated that the coating agent enhances the biocompatibility of the IONPs. The effectiveness of the nanoparticles in inducing cell death was also assessed in an in vitro hyperthermia process and the results showed that the nanoparticles are appropriate to be used for cancer treatment based on hyperthermia. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Magnetic nanoparticles (MNPs) are progressively and extensively used in numerous biomedical applications such as magnetic resonance imaging (MRI) [1–3], bioimaging [4], hyperthermia for cancer therapy [5,6], medical drug targeting [7,8], and tissue recovery [9,10]. Magnetic nanoparticle hyperthermia is an emerging thermotherapy for cancer treatment with least invasive procedure and is usually used in combination with other cancer treatment options, mostly chemotherapy and irradiation [11]. In magnetic nanoparticle hyperthermia treatment, a nano-fluid, a fluid containing dispersed MNPs, is injected into the cancer tissue and exposed to an alternating magnetic field leading to the heat generation due to the magnetic hysteresis loss mechanism and Brownian and Néel relaxation phenomena [12,13]. Cancer tissues are more vulnerable to the elevated temperature compared to the healthy tissues. Healthy tissues are resilient against temperature rise up to 5–6 °C whereas it leads to the cancer cells necrosis and apoptosis [14,15]. Simultaneously, the MNPs are able to be functionalized and deliver drugs to the targeted tissue and generate a more destructive effect on the cancer cells by releasing the drugs in the biophase [16]. ⁎ Corresponding author. E-mail address: [email protected] (O. Vahidi).

http://dx.doi.org/10.1016/j.msec.2017.04.015 0928-4931/© 2017 Elsevier B.V. All rights reserved.

Among all magnetic materials, iron oxide nanoparticles (IONPs) are known to be one of the best candidates for biomedical applications due to their biocompatibility, accessibility and facile synthesis [17]. IONPs have been synthesized by various techniques such as co-precipitation [18–20], thermal decomposition [21,22], microemulsion [23,24], thermal spraying [25], etc. The tendency of IONPs to form aggregates is a problem encountered in IONPs biomedical applications. Due to the high surface to the volume ratio of the IONPs, they form aggregates to reduce their surface energy levels leading to the destabilization of their colloidal stability in ferrofluids. One consequent problem of the reduced colloidal stability is the reduction of available specific surface area, an essential specification for drug loading capacity. One solution to enhance the colloidal stability is to use the coating agents [26,27]. Furthermore, the coating agents are able to enhance the biocompatibility of the IONPs [28–31]. Biosurfactants are biocompatible surface active agents produced by living organisms which enhance the colloidal nanoparticles stability. They are biodegradable, biocompatible and economically producible in large-scale [32,33]. Several biosurfactants and biopolymers including chitosan [34], dextran [35], hyaluronic acid [36], folic acid [14], etc. and synthetic polymers such as polyvinyl alcohol (PVA) [37], polyethylene glycol (PEG) [38], etc. have been widely used as the coating agents for the MNPs in many studies and are reviewed in [39,40]. Glycyrrhizic

M. Saeedi et al. / Materials Science and Engineering C 77 (2017) 1060–1067

acid (GA) is a biosurfactant extracted from the glycyrrhiza plant roots [41] and has not been used before as the coating agent for MNPs. GA has a seven aromatic rings with 894 g/mol molecular weight and is composed of two parts including one molecule of triterpenoid aglycone (hydrophobic portion) conjugated to two glycosyl groups (hydrophilic portion) (Fig. 1) [41,42]. The carboxyl groups are located in both hydrophilic and hydrophobic portions of the GA molecule. In addition to the high biocompatibility of GA, it has anti-inflammatory, anti-viral, anticancer and anti HIV properties [41–44]. In this paper, GA coated IONPs (GAIONPs) were synthesized by the in situ chemical co-precipitation method. The synthesized IONPs and GAIONPs were characterized by several analyses. The aim of this investigation is to study the hyperthermia process for potential cancer treatment using IONPs with a novel coating agent. In the following sections, the term MNPs refers to both IONPs and GAIONPs. 2. Materials and methods 2.1. Materials All chemicals and reagents used in this study were of analytical grade from mercantile companies without further purification. All chemicals required for nanoparticle synthesis and coating were purchased from Merck except for GA extracted from the roots of licorice plant in Surface Chemistry Laboratory, Iran University of Science and Technology, Tehran. Chemicals required for cell culture were purchased from Sigma-Aldrich. 2.2. Synthesis of MNPs MNPs were synthesized using in situ co-precipitation.

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in water and the impurities were separated by filtration and the filtered solution was dried in an oven at 70 °C overnight. 2.2.2. IONPs and GAIONPs preparation To prepare the IONPs, 18 g of NaNO3 and 0.15 g of NaOH were dissolved in 20 ml deionized water at 90 °C. Then, 0.9 g of FeSO4·7H2O was added to the solution and the solution was stirred at 90 °C for 1 h until its color turned into dark brown indicating the formation of IONPs [46,47]. The method used to prepare the GAIONPs was the same to the IONPs preparation with the difference that instead of using deionized water, 20 ml of 970 μM GA solution was used. 2.3. Characterization of MNPs The thermal analysis of the MNPs was performed using thermal gravimetric analysis and differential thermal analysis (TG/DTA; STA 504) in air atmosphere and the temperature was changed between 50 °C and 800 °C. The morphology and particle size of the MNPs were observed with a field emission scanning electron microscope (FE-SEM; Mira 3-XMU) and a transmission electron microscope (TEM; Philips CM120) at 120 kV. The crystal phase of the MNPs was characterized by X-ray diffraction (XRD) on a diffractometer (Philips PW-1730) in θ–2θ geometry using Cu-Kα radiation (λ ¼ 1:5418A). In order to validate the GA bonding on the surface of the MNPs, Fourier transform infrared spectroscopy (FT-IR; PE IR Spectrum ASCII PEDS 1.60) was performed in the range of 450–4400 cm−1. The specific surface area of the MNPs was measured using a Brunauer–Emmett–Teller test (BET; Nanosord Sensiran Co., Iran). Magnetic properties of the MNPs were measured by vibrating sample magnetometer (VSM; Meghnatis daghigh kavir Co., Iran). To assess size distribution of MNPs, a number of MNPs was selected in random from FE-SEM images and analyzed with digimizer software. 2.4. Hyperthermia efficiency of MNPs

2.2.1. GA preparation Licorice, the root of the glycyrrhiza plant, is composed of GA, glabridin and other minor components. The extraction percentage of GA from the licorice in some solvents such as water, methanol and ethanol are 93.12%, 54.43% and 44.55%, respectively [45]. In the present study, water was used to extract GA from licorice. Licorice was dissolved

The thermal efficiency of the MNPs for hyperthermia application was evaluated by using an alternating magnetic field generator (HF-HST 45, Sadr Sanat Danesh Co., Iran). The magnetic field was generated by a solenoid coil with 52 mm inner diameter (5 turns). Induction heating was performed at the frequency of 300 kHz and the field

Fig. 1. Schematic representation of GAIONPs synthesis.

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intensity of 8.8 kA/m for 30 min. Concentration of the IONPs and GAIONPs in aqueous solution was 1 mg/ml and the temperature of two samples was measured by a digital thermometer with 0.1 °C resolution. The heating efficiency of MNPs is expressed as specific loss power (SLP) and is defined as follows [48]: SLP ¼ C P :

m f ∂T : mm ∂t t¼0

ð1Þ

where mm and mf are the mass of MNPs and aqueous solution, respecÞ is the initial slope of the solution temperature versus time tively. ð∂T ∂t curve in the hyperthermia process and CP is the specific heat capacity of the solution (CP ~ Cwater =4.18 J/g°C). 2.5. MNPs preparation for biological analysis The sterilization process was performed under a tissue culture hood. Firstly, MNPs were separated from the aqueous solution by neodymium magnet. Then, the MNPs were dispersed in 10 ml of 70% ethanol solution and after 30 min, the MNPs were separated from the ethanol solution by neodymium magnet. The separated MNPs were left under the hood until all of the residual liquid evaporated. The dried MNPs were washed twice with phosphate buffer saline (PBS). 2.5.1. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay L929 fibroblast cell line from the National Cell Bank of Iran (NCBI) Pasteur Institute was used for biological analysis. L929 cells were cultured in T75 cell culture flasks with Roswell Park Memorial Institute (RPMI-1640) culture medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were trypsinized from cell culture flask and seeded in a 96-well plate with the density of 104 cells/ml and incubated for 24 h at 37 °C in 5% CO2. After one day of incubation, the old medium was replaced by the fresh one containing dispersed MNPs with the concentrations of 1, 10, 100, 200 μg/ml. One control group with zero MNPs concentration was also considered. Afterwards, the cells were incubated for another 24 h. After 24 h, the medium was removed and the wells were washed twice with PBS. 200 μl of MTT solution was added into each well and the plate was incubated for 4 h. Then, the MTT solution was removed and 100 μl of isopropanol was added to each well. The formazan was dissolved into isopropanol and after 30 min the well plate color changed to purple. The absorbance was quantified by a microplate reader (BioTek, Elx800 Absorbance Reader) at the wavelength of 545 nm. 2.5.2. In vitro hyperthermia We investigated the cell viability in induction heating of GAIONPs by trypan blue method. 150 μg of GAIONPs dispersed in 1.5 ml culture medium was added to micro-tubes containing 1.5 ml of culture medium with (4 × 105) cells (hence, the GAIONPs concentration is 50 μg/ml). A control group with identical cell density containing no GAIONPs was also prepared. Both micro-tubes were placed in the center of the induction coil and exposed to a magnetic field with 11.7 kA/m intensity and 300 kHz frequency. The experiment was performed in a 37 °C laboratory water bath. Samples were taken from each micro-tube and the cells were counted by hemocytometer under a light microscope. The viability percentage was calculated as follows: Viability ð%Þ ¼

number of living cells 100 initial number of living cells

ð2Þ

2.5.3. Hyperthermia evaluation in a tissue mimicking phantom To precisely investigate the heating ability of MNPs in a pseudophysiological environment, we constructed an agar gel tissue mimicking phantom to simulate hyperthermia process in a human tissue. The

tissue phantom model comprised two parts, a 3 cm diameter spherical region where the MNPs are homogenously distributed and a 4.2 cm diameter and 6 cm height cylindrical region with no MNPs surrounding the sphere. The spherical part of the phantom model can represent a tissue portion into which the MNPs are injected and distributed and the surrounding part can represent the tissue portion throughout which the MNPs are not distributed yet. The phantom model was exposed to an alternating magnetic field with the frequency of 300 kHz and intensity of 8.8 kA/m for 30 min. The temperature profiles were measured at 4 points indicated in Fig. 2c. The thermo-physical properties such as thermal conductivity, density and specific heat of a typical tissue (brain [49]) are 0.527 W/m·K, 1050 kg/m3 and 3305 J/kg·K and for the agar gel (%3) [50] are 0.566 W/m·K, 1000 kg/m3 and 3900 J/kg·K, respectively. We assumed that the thermo-physical properties of the gel and gel-IONPs mixture were approximately identical [50]. To prepare the MNPs mixed agar gel, 20 ml of water was mixed with 20 mg of IONPs and 0.6 g of agar powder at 60 °C. To form it into a sphere, the mixture was injected into a 3 cm diameter plastic sphere shell and after solidifying at the room temperature, the shell was removed. To prepare the MNPs free agar gel, 60 ml of water was mixed with 1.8 g of agar powder at 60 °C. Then, half of the mixture was poured into a 100 ml beaker and when the gel started to solidify, the spherical gel was placed into the baker and then, the rest of the MNPs free agar gel was added to the beaker. 2.5.4. Cells and IONPs interaction study by Prussian blue staining The Prussian blue staining is a qualitative method to investigate the cells and IONPs physical interactions. The cells were seeded with the density of 105 cells/ml in a 6-well plate and incubated for 24 h at 37 °C in 5% CO2. After 24 h of incubation, the cells were incubated for another 24 h in the presence of MNPs at 100 μg/ml concentration. A control group with no MNPs was also considered. After the second 24 h of incubation, the supernatant of all wells was removed and the wells were washed twice with PBS. Then, the cells were fixed with 500 μl/ well of 5% glutaraldehyde solution. After 45 min, the solution was removed and the wells were washed twice with PBS. Each well was treated with 100 μl of 20% potassium ferrocyanide and 100 μl of 20% HCl. After 20 min, the solution was removed and the wells were washed with PBS. To improve a contrasting background between cells and MNPs, the cells were counterstained with Safranin 1% for 5 min and the wells were washed with PBS again. The images were captured with 100 μm magnification. 3. Result and discussion 3.1. MNPs characterization Fig. 3a indicates the XRD pattern of synthesized GAIONPs and hkl values of magnetite. XRD pattern shows peaks at 30.38o, 35.76o, 43.50o, 54.12o, 57.58o, 63.08o, 74.40o and 90.44o which match well with the standard pattern of magnetite crystalline cubic structure (JCPDS card No. 01-075-0449). The average crystallite size of MNPs was theoretically determined with Debye-Scherrer equation defined as below: D¼

0:94λ β cosθ

ð3Þ

where λ is the X-ray wave length (1.548 Å), θ is the Bragg angle and β (radians) is the FWHM (full-width at half maximum) of the maximum intensity peak (311). Using Eq. (3), the average size of GAIONPs was calculated at 18.24 nm. To demonstrate the presence of GA on IONPs, FT-IR analysis was performed on GA, IONPs and GAIONPs (Fig. 3b). The peaks at 3420 and 1624 cm−1 observed in all three samples are related to the stretching and bending vibrations of the O\\H bond, respectively. These peaks


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Fig. 2. Top (a) and side (b) view of tissue mimicking phantom model; (c) four points at which the temperature is measured versus time during the hyperthermia process.

are due to the presence of water absorbed on the surface of all samples [48]. The peaks at 1430 and 2928 cm−1 are related to the bending and stretching vibrations of the C\\H bond in the GA, respectively [35] and the peak at 1052 cm− 1 is attributed to the C\\O stretching vibration of the GA primary alcoholic functional group [51]. A peak at 1364 cm−1 in GA and GAIONPs confirms the presence of carboxyl groups (−COOH) [52]. The carboxyl groups are located in both hydrophilic and hydrophobic portions of GA molecule and the ones on the hydrophobic portion are believed to make hydrogen bonds with the oxygen of Fe3O4 [53] and the hydrophilic portion is in contact with the surrounding aqueous solution (Fig. 1). The peaks at 580 and 638 cm−1

are related to the Fe\\O bond vibration of MNPs. GAIONPs show a larger peak at 580 cm−1 and the larger peak for IONPs is at 638 cm−1. The shift might be due to the interaction between Fe\\O of IONPs and carboxyl group of GA [54]. The percentage amount of GA coating agent on the IONPs was measured by TG/DTA analysis shown in Fig. 3c. The TG results indicate a comparative weight loss between the IONPs and GAIONPs as the temperature rises which proves the presence of GA on IONPs. The total weight loss difference between the IONPs and GAIONPs was 5.6%. The DTA results for GAIONPs show an endothermic dip below 200 °C and three exothermic peaks at 311 °C, 506 °C and 662 °C. The endothermic

Fig. 3. (a) XRD pattern of GAIONPs, (b) Fourier transform infrared spectra of (i) IONPs,(ii) GAIONPs and (iii) GA, (c) TG/DTA analysis for GAIONPs. Inset: TG analysis comparative curves between IONPs and GAIONPs, (d) VSM curves of the MNPs. Inset: magnetization behavior near zero magnetic field.

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Fig. 4. (a) TEM and (b) FESEM images of GAIONPs (c) size distribution of GAIONPs from 100 random particles in FE-SEM image.

peak below 200 °C can be attributed to the water vaporization [55,56]. The decomposition of GA with herbal structure should also occur below 200 °C. The exothermic peak at 311 °C can be attributed to the oxidation of iron and formation of amorphous phase, the second exothermic peak at 506 °C can be related to the oxidation of Fe3O4 to α-Fe2O3 and the last exothermic peak at 662 °C can be assigned to the full oxidation of remaining magnetite and iron to α-Fe2O3 [57].

The magnetization curves of IONPs and GAIONPs measured by VSM are indicated in Fig. 3d. The saturation magnetization (ms) values of IONPs and GAIONPs were 56.8 and 52.2 emu/g, respectively. The lowered value of GAIONPs ms with respect to that of the IONPs is due to the presence of non-magnetic GA on the surface of IONPs. Also, the very low coercivity (Hc) and remanent magnetization (mr) values of both IONPs and GAIONPs indicates superparamagnetic behavior of the

Fig. 5. (a) Hyperthermia process for 1 mg/ml GAIONPs and IONPs solutions exposed to an alternating magnetic field of 8.8 kA/m intensity and 300 kHz frequency. (b) Hyperthermia process for IONPs in agar gel phantom with 1 mg/ml concentration and magnetic field intensity of 8.8 kA/m and 300 kHz frequency. Error bars show standard deviation, n = 3. (c) Schematic representation of the magnetic field lines in the coil.


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Table 1 SLP values calculated from the fitted quadratic function. sample IONPs GAIONPs Point 1 Point 2 Point 3 Point 4

F(t) 2

ΔT = −0.0052x + 0.3336x + 0.2262 ΔT = −0.0047x2 + 0.2571x + 0.1738 ΔT = −0.0013x2 + 0.0921x − 0.0071 ΔT = −0.0053x2 + 0.3100x + 0.2262 ΔT = −0.0065x2 + 0.3764x + 0.3452 ΔT = −0.0066x2 + 0.3600x + 0.3357

synthesized MNPs. Furthermore, both Hc and mr of the GAIONPs were smaller than that of the IONPs indicating their lower susceptibility against agglomeration. The average particle size (n = 100) obtained from FE-SEM image was found to be 25.3 nm (Fig. 4c) while the calculated crystallite size from Eq. (3) was 18.24 nm. The difference is due to the presence of GA on the surface of IONPs and also the formation of agglomerates. TEM image of GAIONPs shown in Fig. 4b unveils the core-shell structure of GAIONPs. The specific surface areas of IONPs and GAIONPs measured by BET test were obtained at 83.569 and 103.936 m2/g, respectively. It confirms the expansion of IONPs specific surface area by GA coating agent making it a more appropriate carrier for drug loading purposes. 3.2. Hyperthermia process evaluation The temperature profiles of the GAIONPs and IONPs in aqueous solutions during a hyperthermia process were measured and are shown in Fig. 5a. According to Fig. 3d, the saturation magnetization of IONPs is greater than that of the GAIONPs and therefore, for the solutions with equal concentrations of IONPs and GAIONPs (1 mg/ml) exposed to an identical alternating magnetic field frequency and intensity, the heat amount dissipated from IONPs should be greater than that of the GAIONPs. The experimental results indicate such an expectation (Fig. 5a). To calculate the SLP, the temperature profiles of both GAIONPs and IONPs were fitted with a quadratic function. Using Eq. (1), the calculated values of SLP for both MNPs are indicated in Table 1. The results for four indicated points in Fig. 2c of tissue mimicking phantom exposed to an alternating magnetic field of 300 kHz and 8.8 kA/m are shown in Fig. 5b. The temperature of point 3 located at the center of the phantom has the maximum increase of approximately 6 °C after 30 min of hyperthermia process. Since the radial positions of points 2 and 4 are equal, identical temperature profiles are expected for both points. However, as the results show, there is a discrepancy

Þ (0C/min) ð∂T ∂t t¼0

SLP (W/g)

R-squared value

0.3336 0.257 0.092 0.310 0.376 0.360

23.26 17.92 5.98 20.15 24.47 22.58

0.9908 0.9872 0.9993 0.9855 0.9790 0.9699

between their temperature profiles. The difference is due to the presence of non-uniform induced magnetic field inside the coil. As the schematic representation of the magnetic field in the coil shows (Fig. 5c), the magnetic field lines at the coil center are parallel while they diverge at the coil ends which results in weaker and less dense magnetic field at the coil ends with respect to that of the coil center. Therefore, the resulted induced temperature raise at point 4 is more than that of the point 2. To calculate the SLP, the temperature profiles were fitted with a quadratic function (trendlines are not indicated in Fig. 5b) and the calculation results are shown in Table 1. As the results show, the SLP of the IONPs in agar gel at the center of the phantom is larger and at the outer portions is less than that of the IONPs in aqueous solution. In the aqueous solution, since the MNPs are free to move, the generated heat is distributed throughout the solution and a homogenous temperature is obtained, however, the MNPs in the agar gel are not able to move and therefore, the heat accumulation at the gel center dominates its thermal diffusion into the outer portions resulting in higher temperature raise and consequently higher SLP at the gel center in comparison to the SLP of MNPs in aqueous solution. In the outer portions of the gel, the generated heat diffuses out into the MNPs free gel portion resulting in less temperature raise and consequently less SLP with respect to the SLP of MNPs in the aqueous solution. 3.3. Cytotoxicity assay and hyperthermia induced cell death evaluation Based on the MTT assay results, the calculated IC50 values for IONPs and GAIONPs were 85.47 and 228.32 μg/ml, respectively. The results show low cytotoxicity for GAIONPs in L929 cells within the range of 1–100 μg/ml concentration. The significant reduction in the percentage of cell viability for GAIONPs occurs in 200 μg/ml in spite of the high biocompatibility of GA coating [58,59]. Comparing the GAIONPs and IONPs cytotoxicities, GAIONPs are more biocompatible with respect to the IONPs due to the presence of GA coating on the IONPs surface which enhances their biocompatibility.

Fig. 6. (a) Fibroblast L929 cells viability incubated with IONPs and GAIONPs. The cells viability percentage was obtained with optical density (OD) values for the samples distributed in 6 replicates. (b) Hyperthermia induced cell death on fibroblast L929 cells with and without the presence of GAIONPs exposed to an alternating magnetic field with the intensity of 11.7 kA/m and the frequency of 300 kHz.

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Fig. 7. Prussian blue staining assay (a) control group cells, (b) cells with IONPs.

To investigate the hyperthermia induced cell death, hyperthermia process was performed and the results are shown in Fig. 6b. With the implemented magnetic field, the temperature increase of 4 °C was achieved in 30 min (the results are not shown). The in vitro hyperthermia study results show that the solution containing GAIONPs exposed to an alternating magnetic field reduces the cell viability significantly while the alternating magnetic field itself has no lethal effect on the cells as indicated by the control group (Fig. 6b). It should be also noted that the GAIONPs without induced magnetic field at the concentration of 50 μg/ml does not reveal cytotoxic effect on the L929 cells as the MTT assay results indicated (Fig. 6a) which means that the only factor for the induced cell death is the elevated culture medium solution temperature caused by the heat generated by the GAIONPs exposed to the alternating magnetic field. As studies on other cell lines indicated that early signs of cell death through apoptosis mechanism triggered by temperature elevation require time to be detectable [60,61], the immediate induced cell death in the short term hyperthermia process can be possibly attributed to the cell necrosis mechanism, although precise determination of cell death mechanism requires detailed and complex analyses. 3.4. MNPs-cells interactions The Prussian blue staining assay was used to investigate how the MNPs affect the cells morphology. The concentration used for the assay was around the IC50 of the IONPs. Comparing the cells morphology with and without the presence of MNPs indicates that the presence of MNPs surrounding the cells alters the cells morphology. As Fig. 7a shows, the cells without the presence of MNPs have spindle shaped morphology while the cells morphology surrounded by the MNPs is changed and the cells are not able to keep their spindle shaped morphology (Fig. 7b). The assay also indicates the cell internalization of MNPs (Fig. 7b). 4. Conclusion In the present study, GA coated IONPs were prepared by the oxidative precipitation method. The XRD analysis confirmed the formation of magnetite crystal phase and the crystal size of MNPs was calculated by Debye-Scherrer equation at 18.24 nm. From the FE-SEM images, the GAIONPs average size was measured at 25.3 nm. The presence of GA coating agent on the surface of IONPs was confirmed by FT-IR, TG/ DTA, VSM, BET measurements. The saturation magnetization (ms) values of IONPs and GAIONPs were measured at 56.8 emu/g and 52.2 emu/g, respectively, indicating high enough value for the saturation magnetization of the synthesized MNPs making them suitable candidates for magnetic hyperthermia applications. The BET measurements indicated that GA coating agent increases the available specific surface area of the IONPs by approximate 25%. The increased specific surface

area enhances the drug loading efficiency which is necessary for drug delivery applications. The heat generation capability of MNPs in aqueous solution and in a tissue mimicking phantom model made up of agar gel was investigated by exposing them to an alternating magnetic field. The measurements in the aqueous solution indicated that the IONPs and GAIONPs raised the solution temperature by approximately 5.5 °C and 4 °C after 30 min of exposure to a 8.8 kA/m and 300 kHz magnetic field. Similar results were obtained in the tissue mimicking phantom model. The MTT assay results on L929 cells indicated that the synthesized MNPs are biocompatible. Comparing the cytotoxicity of IONPs and GAIONPs indicated that GA coating agent increased the biocompatibility of the IONPs and reduced their cytotoxicity. Cell internalization of MNPs was investigated by Prussian blue staining assay and it was indicated that the MNPs are internalized by the cells. The assay images also showed that the cells lost their spindle shaped morphology due to the MNPs presence. The in vitro hyperthermia results indicated that the hyperthermia induced cells death was significant with respect to the control group. Due to the high biocompatibility of the synthesized MNPs, their significant capability to induce cell death in the hyperthermia process and their remarkable available active surface area for drugs carriage, they are appropriate to be considered for the thermotherapy combined with chemotherapy for cancer treatment. References [1] J. Estelrich, M.J. Sánchez-Martín, M.A. Busquets, Int. J. Nanomedicine 10 (2015) 1727–1741. [2] A. Ray Chowdhuri, D. Bhattacharya, S.K. Sahu, Dalton Trans. 45 (2016) 2963–2973. [3] C. Felton, A. Karmakar, Y. Gartia, P. Ramidi, A.S. Biris, A. Ghosh, Drug Metab. Rev. 46 (2014) 142–154. [4] M. Tajabadi, M.E. Khosroshahi, S. Bonakdar, Opt. Photon. J. 5 (2015) 212. [5] V.M. Khot, A.B. Salunkhe, N.D. Thorat, R.S. Ningthoujam, S.H. Pawar, Dalton Trans. (Cambridge, England: 2003) 42 (2013) 1249–1258. [6] P.I.P. Soares, C.A.T. Laia, A. Carvalho, L.C.J. Pereira, J.T. Coutinho, I.M.M. Ferreira, C.M.M. Novo, J.P. Borges, Appl. Surf. Sci. 383 (2016) 240–247. [7] S. Rana, N.G. Shetake, K.C. Barick, B.N. Pandey, H.G. Salunke, P.A. Hassan, Dalton Trans. 45 (2016) 17401–17408. [8] V.V. Mody, A. Cox, S. Shah, A. Singh, W. Bevins, H. Parihar, Appl. Nanosci. 4 (2014) 385–392. [9] M. Lewin, N. Carlesso, C.-H. Tung, X.-W. Tang, D. Cory, D.T. Scadden, R. Weissleder, Nat. Biotechnol. 18 (2000) 410–414. [10] E.A. Lee, H. Yim, J. Heo, H. Kim, G. Jung, N.S. Hwang, Arch. Pharm. Res. 37 (2014) 120–128. [11] A. Makridis, K. Topouridou, M. Tziomaki, D. Sakellari, K. Simeonidis, M. Angelakeris, M.P. Yavropoulou, J.G. Yovos, O. Kalogirou, J. Mater. Chem. B 2 (2014) 8390–8398. [12] H. Mamiya, B. Jeyadevan, Sci. Rep. 1 (2011) 157. [13] S. Dutz, R. Hergt, J. Mürbe, R. Müller, M. Zeisberger, W. Andrä, J. Töpfer, M.E. Bellemann, J. Magn. Magn. Mater. 308 (2007) 305–312. [14] Q.L. Jiang, S.W. Zheng, R.Y. Hong, S.M. Deng, L. Guo, R.L. Hu, B. Gao, M. Huang, L.F. Cheng, G.H. Liu, Y.Q. Wang, Appl. Surf. Sci. 307 (2014) 224–233. [15] P. Guardia, R. Di Corato, L. Lartigue, C. Wilhelm, A. Espinosa, M. Garcia-Hernandez, F. Gazeau, L. Manna, T. Pellegrino, ACS Nano 6 (2012) 3080–3091. [16] C. Xu, S. Sun, Dalton Trans. (Cambridge, England: 2003) (2009) 5583–5591. [17] A. Ruiz, P.C. Morais, R. Bentes de Azevedo, Z.G.M. Lacava, A. Villanueva, M. del Puerto Morales, J. Nanopart. Res. 16 (2014) 2589. [18] M.I. Khalil, Arab. J. Chem. 8 (2015) 279–284.

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