Magnetic hyperthermia application of MnFe2O4 nanostructures processed through solvents with the varying boiling point

This article reports the synthesis of MnFe2O4 nanoparticles (NPs) by thermal decomposition method in different solvents such as diphenyl ether, benzyl ether and 1-octadecene with varying boiling point (bp). The physical and chemical properties of all the NPs were systematically studied. The NPs prepared using benzyl ether as solvent formed in cubic shape whereas spherical particles are formed in diphenyl ether and 1-octadecene. The solvent plays a significant role in the reaction and influence the morphology of the NPs. The hydrophobic particles are made hydrophilic by ligand exchange using tetramethyl-ammonium 11-aminoundecanote and the colloidal dispersion of the NPs are used for magnetic induction in varying alternating magnetic field (AMF) of frequency 314 kHz. The specific absorption rates that measure the heat generation in NPs are found to vary with the concentration of the NPs as well as the field strength. The cubic shaped NPs shows comparatively better SAR than the spherical NPs.


Introduction
The synthesis of inorganic NPs are of tremendous interest in the past two decades due to their distinct properties compared to the bulk analogue useful in a wide range of applications. Now a days many researches have been carried out to study the relation between variations in morphology to their physicochemical properties. In recent years, many studies are reported related to different size and shapes of inorganic metal, metal oxides and semiconductor NPs [1][2][3][4][5][6]. For biomedical applications, magnetic particles among inorganic NPs are highly interested particularly for drug delivery, magnetic resonance imaging and hyperthermia [7][8][9][10][11][12][13]. Magnetic ferrites have unique magnetic, optical, electronic and catalytic properties due to their spinel structure (AB 2 O 4 ) where A and B are tetrahedral and octahedral cation sites and O denotes the oxygen in anion sites. The spinel ferrites have face centred cubic structure with molecular formula MFe 2 O 4 (M = Fe, Mn, Ni, Zn, Co or Mg). Among ferrites, MnFe 2 O 4 can provide better biocompatibility and strong magnetisation for biomedical applications. Kim et al showed that MnFe 2 O 4 among other metal ion-doped ferrites provide better contrast agent for MRI [14]. In clinical studies for hepatic MRI, Mn 2+ containing contrast agent mangafodipir trisodium (MnDPDP, Teslascan) has been used [15]. By tuning the chemical composition, size and shapes of the magnetic NPs, the magnetic properties can be controlled. To get the desired physical and chemical properties of spinel ferrite, different chemical synthesis route has been the main focus of research. Different reported methods for the synthesis of magnetic NPs are ball milling, coprecipitation, sol-gel, hydrothermal, microwave-assisted route, microemulsion and thermal decomposition method. Different shapes of the NPs have been achieved by most of these methods but controlling the uniformity in large scale production is difficult. In this regard, the method involving decomposing of organometallic precursors in organic solvents shows better results in controlling the morphology of NPs when synthesized in large scale [16]. This fine-tune of sizes and shapes by thermal decomposition can be explained via the nucleation-growth theory proposed by Lamer [17]. The clear separation between nucleation and growth phase can be seen in the thermal decomposition method. The precursors generally used in thermal decomposition methods are oleates [16,18,19], acetates [20] and acetylacetonates [21,22]. The high-quality Fe 3 O 4 NPs can be produced by the thermal decomposition method with high magnetic saturation value [23]. In this method, oleic acid and oleylamine were used as surfactants which play a major role in the morphological formation. In general, surfactants are used to prevent aggregation by forming a coating on the particle results in the uniform growth size of the particles. For the nucleation process, these surfactants can also act as a reducing agent. The amine and carboxylic acid based surfactants can accelerate or delay the decomposition rate of precursors [24].
On the other hand, the solvent also plays an important role on the NP formation by thermal decomposition method. Lu et al [25] studied the effect of reducing agent and solvent on the synthesis of CoFe 2 O 4 NPs. They have presented the surfactant effect by changing the oleic acid and oleylamine in the equimolar concentration of 248 mM, 496 mM, and 620 mM keeping the same solvent and vice versa. The results show that the morphology CoFe 2 O 4 NPs varies from spherical to cubes to star shape with increase in surfactant to solvent ratio when synthesized using dioctyl ether (bp=287°C) as a solvent, whereas the NPs formed only in spherical shapes of different size when synthesized in 1-octadecene (bp=320°C) with different surfactant ratio. Sun and coworkers [22] have reported the synthesis of MFe 2 O 4 NPs by thermal decomposition method using different solvents. They reported the formation of two different sizes of NPs e.g. 4 and 6 nm by using phenyl and benzyl ether as solvents respectively keeping all other parameters same. The increase in NP size is concluded due to increase in the bp from 259°C for phenyl ether to 298°C for benzyl ether and inferred that the reaction temperature played significant role. Similarly, Zhou et al [26] demonstrated the controlled synthesis of anisotropic iron oxide nanoparticles in two different solvents 1-octadecene (bp=320°C) and tri-n-octylamine (bp=360°C) by changing the precursor ratios. From the series of synthesis, the one with precursor ratio of 1 mmol of iron oleate, 0.2 mmol of sodium oleate and 0.5 mmol of oleic acid in 1-octadecene as solvent produces iron oxide of truncated octahedron shape whereas in tri-n-octylamine solvent it formed in concave shapes. In the same report by Zhou et al cubic shaped iron oxide was synthesized in tri-n-octylamine as solvent without sodium oleate and reasoned that tri-n-octylamine act similar role as surfactant sodium oleate to deprotonate oleic acid from oleate (OL − ). Erik et al [27] reported synthesis of spherical and cubic shapes of iron oxide NPs using three different solvents 1-octadecene, 1-hexadecene (bp=287°C) and eicosane (bp= 343°C) where the the reaction temperature play important role for variation in morphology of NPs. Park et al [16] reported ultra-large scale synthesis of monodispersed iron oxide NPs by thermal decomposition method of iron oleate precursor in high bp solvents such as 1-hexadecene, octyl ether, 1-octadecene, 1-eicosene and trioctylamine. The nanocrystals are highly monodisperse in nature and the variation in particle-size are reported due to variaton in the reaction temperature. There are many literatures published on the formation magnetic NPs by thermal decomposition method where the temperature of the reaction and surfactant used play significant role in the morphology of NPs, however, the solvents role on the particle size and shape of NPs has not yet been clearly established [26,[28][29][30].
In this present work, MnFe 2 O 4 NPs are synthesized via a thermal decomposition method with three different solvents. The physico-chemical properties has been analysed through various characterisation techniques such as XRD, FESEM, XPS, TGA and VSM. The mechanism of morphological evolution in NPs have been explained and magneto-thermal induction capabilities MnFe 2 O 4 colloid has been assessed with the application of AMF.

Method used
Manganese ferrite was synthesized using a modified thermal decomposition method [22]. In a typical synthesis, 1 mmol of Mn(acac) 2 , 2 mmol of Fe(acac) 3 , 10 mmol of oleylamine and oleic acid and 20 ml of solvent were magnetically stirred under a nitrogen atmosphere as shown in scheme-1. The solvents used were either diphenyl ether (PE) or 1-octadecene (ODC) or benzyl ether (BE) and the prepared MnFe 2 O 4 nanoparticles were named as MnF-PE, MnF-ODC, MnF-BE respectively. The solution was heated for 100°C under a nitrogen blanket for 30 min to remove moisture and impurities in the solvents used. Then, the temperature was increased to 200°C and maintained for another 30 min for nucleation. Followed by this, the temperature was increased to the boiling point of the solvent with the ramping rate of 3°per min and maintained for 60 min. After this, the reaction vessel containing the NPs are cooled down to room temperature under N 2 blanket. The dark black precipitate was collected using centrifugation method. Ethanol was added to the solvent to form precipitation and then centrifuged at 7000 rpm for 15 min. The collected precipitate was redispersed in 5 ml of hexane and centrifuged again by adding ethanol and it was repeated for 3 times. The final product was dispersed in hexane and stored. To make the particles water soluble, 2 ml of hexane dispersed NPs was mixed with 2 ml of 20 mg tetramethyl-ammonium 11-aminoundecanote in dichloromethane [22]. The mixture was sonicated for 2 min and the particles were isolated using a permanent magnet and redispersed in water. The thermal decomposition of Fe(acac) 3 and Mn(acac) 2 produce MnFe 2 O 4 in the presence of oleylamine and oleic acid as a surfactant in high boiling point solvent by releasing carbon dioxide and acetone byproducts as follows (1): It is well known that the reaction parameters like time, temperature and ratio of the solvent affect the morphology of the NPs. The reaction carried out is depicted in schematically in figure 1.

Characterization techniques used
The crystal structure of the samples were examined using powder x-ray diffractometer (D8 Advance Bruker diffractometer) which employ Cu-Kα radiation with λ=1.5406 Å. Debye-Scherrer equation was used to calculate the crystallite size of samples. The microstructural images were taken in a field emission scanning electron microscope (FESEM, FEI Quanta 200). The magnetic parameters were measured in the vibrating sample magnetometer (VSM, LakeShore). Fourier transform infrared (FTIR) spectrum were recorded in Shimadzu IR Prestige spectrometer. Magnetic induction heating experiments for all the samples were performed using Amberal EASYHEAT-8310, 4.2 kW system. For heating efficiency, different concentration of 0.5, 1 and 2 mg ml −1 of the NPs were dispersed in water and taken in vials, sonicated and placed in the AMF. The difference in temperature was measured using a optical fibre temperature sensor. The time versus temperature graphs were plotted and the initial slopes were calculated. SAR for the samples were estimated using the following equation (2) =´´D Where C water is the specific heat of water with value of 4.2 J g −1°C−1 , m is the mass of the NPs in aqueous suspension and ΔT/Δt is the initial slope of the temperature curve.  Generally, the size of the NPs linearly increases with the boiling temperature of the solvents showing the dependency of growth rate to the reaction temperature [16,33]. However, in the present study, in case of BE, the NPs size are larger than the particles synthesized from ODC. This deviation in size shows the functional group of the solvent alters the stability and decomposition kinetics of iron complexes which affects the nucleation and growth rate of the NPs. By considering the LaMer model [17], the nucleation dependency on the solvent is shown in figure 4(d). The synthesis of NPs takes place by two steps nucleation and growth process. From figure 4(d), the barrier of the activation energy for nucleation is higher than the growth step. The burst nucleation takes place when concentration (C) is greater than C min , where C min is the minimum monomer concentration required for nucleation. The higher monomer concentration occurs with the decomposition of precursors at higher temperatures. The radius of the stable nuclei formation is inversely proportional to the supersaturation. Thus at C<C max , where C max is the supersaturation of the monomer, a lesser amount of nuclei with larger radius are formed but with C>C max , a larger amount of nuclei with smaller radius are formed. The use of 1-octadecene at the higher temperature would be in agreement with higher C max value and hence particles are formed with smaller radius [24]. The NPs synthesized with BE solvent show perfect cubic shape with the side length of 13 nm. It is well known that the shape of the particle can be varied by changing the oleic acid and oleylamine ratio in the reaction [25]. In the present study, all the synthesis parameters and reagents are kept fixed except for the use of different solvents. Thus, morphology also relies on the reductive environment in the thermal decomposition reaction. In general, BE acts as an antioxidant and hence, in the presence of oxygen it turns into benzaldehyde and benzoic acid that give a strong reductive environment when compared to other solvents. So, the monomer and nucleation formation in BE were much faster and the formed nucleation entered into kinetic growth phase earlier. In general, face-centred cubic (fcc) structure of spinel ferrite nanocrystal has three low-energy facets e.g. (100), (110), and (111). The highest surface energy possessed by the (111) planes and the growth along this leads to the formation of cubical nanostructures [34]. Thus bigger size cubic shaped NPs are formed in benzyl ether whereas for phenyl ether and 1-octadecene, the nucleation was dominant over the growth process resulting in smaller size spherical shaped NPs.

Magnetic properties
The magnetisation the samples at 300 K as the function of the applied magnetic field are shown in figure 5(a) with the inset graph showing the coercivity of the samples. The hysteresis behaviour of the NPs are consistent with nearly superparamagnetic behaviour. The samples MnF-PE, MnF-BE and MnF-ODC possessed M s value of 42.0, 51.3 and 48.9 emu g −1 respectively. In agreement with previous studies [35][36][37], the Ms value are related to the size of the NPs. The lower Ms in smaller sized particles are associated with the surface spin disorder by excess long chain capping ligand attached directly on the surface or by the absence of some ions from the spinel lattice [38,39]. The Ms values of the NPs are quite lower than the bulk MnFe 2 O 4 which possesses Ms value of 80 emu g −1 [40]. Additionally, the coercivity Hc is decreasing with the increase in size. This can be supported with single domain particle theory proposed by Stoner and Wohlfarth [41] which shows Hc depends on both the Ms and anisotropy constant (K) given by the equation (3)

Thermogravimetry analysis
Weight loss profile measurements were recorded from room temperature to 800°C ( figure 5(b)). TGA of MnFe 2 O 4 NPs show weight loss of 2.5% to 3.3% when size varied from 9 nm to 13 nm. The materials are almost stable up to 200°C without any loss. Weight loss observed from 200 to 450°C is due to the decomposition of the oleic acid and oleylamine surfactants [44]. Since all the samples were prepared with the same weight of the precursors and volume of the surfactants, the ratio of surfactant coated on the NPs surface is proportional to the size of the NPs. The surface-to-volume ratio increases as the particle size decreases, and the surfactant amount on the surface decreases [45]. The weight loss profiles indicate the surface functionalization of the NPs which are highly stable in hydrophobic solvents such as hexane and toluene.

Magnetic hyperthermia
Magnetic hyperthermia heating application is based on Neel and Brownian motion of the magnetic NPs. In the presence of an applied AMF, the moment of magnetic particle rotates (Neel relaxation) as well as the particle itself rotates (Brownian relaxation) and relaxes back to normal orientation with the phase lag between the direction of magnetic moment and applied field. This phase lag relaxation process releases heat energy. The particle heating efficiency is calculated as specific loss power (SLP) or specific absorption rate (SAR). The SAR value of the particles should be higher for the safe application of magnetic hyperthermia in clinical uses. This is because the NPs with high SAR provide efficient heat generation at low concentration. By Rosenweig's theory, the heat generation depends on the parameters like frequency of the applied magnetic field, particle size, magnetization, magnetic anisotropy and viscosity of the solvent [46].
Clinically available NanoActivator(MagForceAG, Germany) operates at the applied field of 18 kA m −1 with a frequency of 100 kHz [47]. This low frequency is used to minimize the effect of generation of eddy current in the human body by Faraday's law. Till date, on maximum tolerable frequency and field, there is limited research and clinical data. Atkinson et al in 1984 proposed that the frequency and strength of the field applied cannot be increased beyond a certain limit (maximum frequency=13.56 MHz or the product of frequency and field strength=4.85×10 8 A ms −1 ) in clinical trials for safety reasons [48]. However, in certain research, it is shown that this limit can be exceeded. Even in phase 1 and 2 trial of MagForce, patients with glioblastoma tolerate up to 13.5 kA m −1 whereas patients with prostate cancer tolerate till 5 kA m −1 [49,50]. It is due to the difference in tissue conductivity and dielectric constant. Considering this, further research is required on the maximum tolerable limit of applied magnetic field strength and frequency.
Another major factor on which the magnetic hyperthermia highly depends is the concentration of the administered material in the tumour site. In a clinical trial, patients with glioblastoma cancer have been administered by magnetite NPs up to 31.36 mg of Fe/cm 3 [51]. The recommended dosage of FDA approved Feraheme (AMAG Pharmaceuticals, USA) is 510 mg [52]. Thus, heat generation by NPs in magnetic hyperthermia varied by some of the parameters such as magnetic field parameters (strength and frequency) and the concentration of the NPs in the colloid. Here, we have discussed and analysed the effect of two parameters field strength and concentration.
For studying the heating efficacy of the NPs, surface modified MnFe 2 O 4 with Tetramethyl-ammonium 11-aminoundecanote is dispersed in water. The sample concentrations used for hyperthermia studies were 0.5, 1 and 2 mg ml −1 . For induction heating, 9 turns of 6 cm diameter coil is used with the applied field (Frequency=314 kHz, Field strength=375, 425, 475, 525 and 575 Oe). The hyperthermia temperature of 42°C is reached by all the samples under different parameters were recorded by the fibre optic sensor. Figure 6 shows the linear increase in temperature with time under different field strength for all the samples at a concentration of 2 mg ml −1 . It is seen that with the rise in the applied field, the time required to reach the hyperthermic temperature decreases. The hyperthermic behaviour is directly proportional to the magnetic parameters of the NPs. MnF-BE NPs requires less time to reach the temperature under different field and different concentration when compared with MnF-ODC and MnF-PE. This may be due to the high Ms value and cubic shape of the particles since the cubic shaped particle poses high anisotropy value as compared to the spherical shape. The SAR values calculated from the initial slope obtained from the time versus temperature graph for all concentrations at different applied fields are given in figure 7. The high SAR value was possessed by all the NPs from different solvents. The maximum SAR value was obtained by MnF-BE is 438 W g −1 at a concentration of 2 mg ml −1 and with an applied field of 575 Oe compared to other NPs might be due to the cubic shape. This is a high SAR value than the reported literature [42,53]. These results prove that the degree of induced heat by the NPs can be controlled by the concentration and applied field and indicates the suitability of these NPs in magnetic hyperthermia application.

Conclusion
In summary, the simple thermal decomposition method with different high boiling solvent can be used to synthesis high quality MnFe 2 O 4 with different morphology. The dual functioning role of solvents as reducing agents also proved from the different shapes of the NPs obtained. By keeping the reagents and surfactants ratio as constant, the heating rate or boiling point of the solvents determine the size and shape of the particles. The synthesized particle MnF-PE and MnF-ODC show nearly spherical shape whereas the particle synthesized in BE MnF-BE shows cubic shape. The ligand exchange process by Tetramethyl-ammonium 11-aminoundecanote produces highly stable hydrophilic NPs. The potential of these synthesized NPs as magnetic hyperthermia agent shows better SAR values when compared with most of the research data and commercial agents. The SAR efficiency of NPs is size and morphology dependent. These stable NPs with superior magnetic properties can be utilized as hyperthermia agent for cancer therapy.