Assessing magnetic and inductive thermal properties of various surfactants functionalised Fe3O4 nanoparticles for hyperthermia

This work reports the fabrication of magnetite (Fe3O4) nanoparticles (NPs) coated with various biocompatible surfactants such as glutamic acid (GA), citric acid (CA), polyethylene glycol (PEG), polyvinylpyrrolidine (PVP), ethylene diamine (EDA) and cetyl-trimethyl ammonium bromide (CTAB) via co-precipitation method and their comparative inductive heating ability for hyperthermia (HT) applications. X-ray and electron diffraction analyses validated the formation of well crystallined inverse spinel structured Fe3O4 NPs (crystallite size of ~ 8–10 nm). Magnetic studies confirmed the superparamagnetic (SPM) behaviour for all the NPs with substantial magnetisation (63–68 emu/g) and enhanced magnetic susceptibility is attributed to the greater number of occupations of Fe2+ ions in the lattice as revealed by X-ray photoelectron spectroscopy (XPS). Moreover, distinctive heating response (specific absorption rate, SAR from 130 to 44 W/g) of NPs with similar size and magnetisation is observed. The present study was successful in establishing a direct correlation between relaxation time (~ 9.42–15.92 ns) and heating efficiency of each surface functionalised NPs. Moreover, heat dissipated in different surface grafted NPs is found to be dependent on magnetic susceptibility, magnetic anisotropy and magnetic relaxation time. These results open very promising avenues to design surface functionalised magnetite NPs for effective HT applications.


Results
Structural, microstructural and surface analysis. XRD 17 . From IR spectra of PEG-Fe 3 O 4, characteristic bands observed at ~ 2,924 cm −1 , ~ 1,406 cm −1 and ~ 1,060 cm −1 correspond to -CH 2 , -CH and C-O-C ether bond stretching vibrations respectively of the PEG chains 32 . As an ethylene glycol derivative, the OH groups of the PEG result in the major intense peak in the range of 3,000-3,500 cm −1 . Interaction via hydrogen bonding between the oxygen of PEG and the proton of protonated magnetite indicates PEG polymer chains were successfully grafted onto the Fe 3 O 4 surface 33 . In the FTIR spectra of PVP coated magnetite, peaks observed at ~ 1,082 cm −1 , ~ 1,420 cm −1 and ~ 1,645 cm −1 correspond to stretching vibrations of C-N stretching, CH 2 and C=O respectively of PVP. Interaction between oxygen in carbonyl group of PVP and the proton present in the protonated magnetite via hydrogen bonding plays the PVP grafting on Fe 3 O 4 . In case of EDA coated magnetite, bands at ~ 1,022 cm −1 and ~ 2,930 cm −1 can be ascribed to the asymmetrical axial deformations in the group [N(-CH 2 -) 3 ] of the tertiary amines and C-H axial deformities of the CH 2 group present in EDA 34 . Band occurred at ~ 1,638 cm −1 may be due to the overlap of C=N and C=C bonds and at ~ 1,429 cm −1 attribute to the amide band. This indicates the amine group of EDA makes interaction with magnetite. CTAB coated magnetite spectra exhibits characteristic band at ~ 1,627 cm − www.nature.com/scientificreports/ and bands at ~ 2,848 cm −1 and ~ 2,918 cm −1 are attributed to CH vibration 35 . Antisymmetric vibration of C-N and CH 3 stretching at ~ 960 cm −1 and ~ 1,470 cm −1 are also observed. These observed bands provide evidence for the interaction between the surface of magnetite and the chemisorbed CTAB molecules. Weight loss profile as observed in TGA plots ( Fig. 1iii) below 150 °C, between 150 and 380 °C and above 380 °C is correlated to the surface adsorbed water molecules/removal of hydroxyl group, the thermal decomposition of surfactants chemisorbed on the surface of NPs and thermally induced phase transformation of Fe 3 O 4 to γ-Fe 2 O 3 , respectively. A total weight loss of around 10, 12, 8, 10, 11 and 21% was observed for GA, CA, PEG, PVP, EDA and CTAB coated Fe 3 O 4 NPs respectively. Polymers exhibit an improved thermal stability compared to simple molecules or polysaccharides 36 which keeps PEG more thermally stable than others. A higher weight loss for CTAB coated Fe 3 O 4 compared to others indicates higher molecular mass of polymeric CTAB. Comparing the TGA plots, it is clear that the binding of surfactants on Fe 3 O 4 was effectively achieved.
Zeta-potential (Fig. 1iv) showed that five out of six samples (GA- TEM micrographs (Fig. 2) exhibit spherical and well resolved monodispersed particles. Effective surface modification and reduced magnetic dipolar interaction between the particles lead to this monodispersity. The average particle size was estimated using log normal distribution function (considering 50 particles using Image J software). The particles exhibited a narrow size distribution ranging from an average of 7.8 nm to 10.5 nm (provided in Fig. S2) for the characterised samples which correlates with crystallite size estimated from XRD analysis. Since the particle size is approximately similar to crystallite size, it can be inferred that a thin layer of coating of surfactants is present surrounding the iron oxide core 37 42 . Also from C 1s XPS spectrum, two peaks located at 284.76 eV and 288.5 eV are assigned to the carbon atom (either bound to carbon or to hydrogen) and carbon atom in COOH or COOpeak respectively 38 . These results suggest that the surface modification with GA on Fe 3 O 4 NPs surfaces have accomplished successfully. Similarly, XPS analysis given in Fig. S3ii-vi for other samples inferred that the position of Fe 2+ and Fe 3+ peaks in Fe 2p region could vary based on the ligand species and also confirms the successful synthesis of corresponding surfactant coated Fe 3 O 4 NPs. Table S1 provides a summary of XPS binding energies and shifts for all coated samples.

Magnetic properties.
Magnetic measurements confirm the SPM nature for all samples, as can be seen in   is attributed to the non-collinear spin structure originated from the surface spin pinning and coatings at the interface of NPs. As the measured magnetic moment is less than theoretical value (4 µ B ) and the diameter of the particles obtained from TEM being much less than the single domain critical diameter (80 nm, reported for magnetite) 10,44 , the synthesized NPs are considered to be single domain. Moreover, from M-H curve it is observed that NPs exhibit maximum susceptibility (χ max = dM/dH) at low applied field. The magnetic susceptibility for all the NPs at room temperature is shown in Fig. 4 At room temperature, the maximum value of susceptibility (reported in Table 1) is found at fields of 136 Oe, 141 Oe, 155 Oe, 760 Oe, 140 Oe and 754 Oe respectively for the GA, CA, PEG, PVP, EDA and CTAB coated samples.
Zero field cooled (ZFC)/field cooled (FC) curves at an applied field of 450 Oe are depicted in Fig. 4i-vi. ZFC curve displays an increment in magnetisation with temperature and reached the maximum corresponding to the blocking temperature, T B . Below T B , the particles are in blocked state exhibiting an irreversible magnetisation with ferromagnetic nature whereas above T B , the particles are in a reversible SPM nature. Increment in magnetisation in ZFC curve originates from the contribution of blocked magnetic moments. Similarly, all the FC magnetisation decreases with rise in temperature which may be due to the randomisation of blocked magnetic moments upon thermal energy 45 . Such decrement in magnetisation below T B confirms the SPM nature. Similar magnetic behaviour reflects similar size and composition of the magnetic cores of all samples. Calculated T B values for the maximum magnetisation are tabulated in Table S2. T B values depend on the effective anisotropy and particle size according to 46,47 V where K is the effective anisotropy energy constant, k B is the Boltzmann constant, T B is the blocking temperature at which thermal energy becomes comparable to magnetic anisotropy energy barrier where the particle goes into the SPM regime and V is the volume of the MNP based on the particle size determined from HRTEM.  Table S2. It is found that GA-Fe 3 O 4 exhibits comparatively enhanced effective anisotropy of 2.05 × 10 5 erg/cm 3 than others which may contribute for higher heating efficiency under AC magnetic field. calorimetric heating performance. In order to assess the heating efficacy of the samples, temperaturetime calorimetric evaluations under magnetic field parameters of 450 Oe and 316 kHz were conducted. In biomedical applications, generally surface modified NPs are used to conjugate drug or biological molecules in order to target tumor specific surface receptors, however, for a comparative study, heating efficacy of uncoated NPs is also analysed and provided in Fig. S5. In view of clinical aspects for effective MHT applications, a minimal concentration range 48,49 (1 mg/ml, 2 mg/ml and 3 mg/ml) was selected for present study. The selected concentration can produce significant heating within 10 min without reaching the boiling point of water. An in vivo www.nature.com/scientificreports/   51 have chosen a concentration of 120 mg/ml. As evident from Fig. 5i(a,b) a gradual rise in temperature with time was observed for all the samples. Time required to attain the required HT temperature (42-46 °C) varies with different NPs under identical experimental conditions. Moreover, Fig. 5i(a,b) demonstrated that heating response is concentration dependent for all the samples. 1 mg/ml samples attained a peak value at ~ 43 °C and became saturated afterwards as generation of heat is balanced by dissipation of heat in MNPs. However, in the case of 2 mg/ml concentration, all samples reached beyond the HT limit (~ 46 °C). This rise in temperature can be a consequence of increased particle concentration in the solvent. In terms of time duration to attain clinically relevant HT response under magnetic field exposure, 1 mg/ml NPs is found to reach 43 °C after a prolonged exposure (~ 10 min) whereas in the case of 2 mg/ml NPs, HT limit (43 °C) was obtained within a comparatively lesser time span (~ 5 min). In the current context, it can be concluded that higher con-  Fig. 5ii illustrates the contribution of magnetic moment relaxation of NPs. SAR was calculated (in Table 2) from the initial slope of temperature-time curves as the temperature response with time is non-linear for non-adiabatic systems (due to thermal loss). The SAR of uncoated samples (depicted in Fig. S5) at NPs concentrations of 1 mg/ml, 2 mg/ml and 3 mg/ml are found to be higher than the coated samples due to the higher magnetisation obtained. An increment in SAR upon increasing the concentration can also be observed in the uncoated sample. In addition to the relaxation mechanisms, the magnetic susceptibility at HT temperature (42 °C) for the applied field (450 Oe) is also plotted (data obtained from M-T curve) and shown in Fig. 5iii. For elucidating the optimal concentration of all MNPs on heating efficiency, all the samples were further subjected to HT at a comparatively higher concentration of 3 mg/ml (Fig. 5i(c)) and SAR was determined ( Table 2). Highest SAR of 130 W/g was obtained for GA-Fe 3 O 4 whereas SAR values for the remaining samples exhibited a demoting trend with SAR values similar to 1 mg/ml concentrated samples. Since heat dissipation due to magnetic moment flip under AC magnetic field still persists leading to a higher magnitude of SAR value for 3 mg/ml GA-Fe 3 O 4 , optimum concentration for heating ability needs to be identified. Accordingly, further analysis of HT studies for 3 mg/ml and 5 mg/ml were performed (Fig. 6) and SAR values were determined. A substantial decrease in SAR of 42 W/g has been determined for 5 mg/ml concentration due to the presence of strong demagnetising interactions between the NPs.

Discussion
The present work has investigated the heating potentiality of Fe 3 O 4 NPs coated with various surfactants such as GA, CA, PEG, PVP, EDA and CTAB by elucidating the influence of surfactants on the magnetic as well as inductive thermal properties of Fe 3 O 4 NPs. Well defined broad peaks obtained from XRD revealed the nanocrystalline nature of Fe 3 O 4 and absence of any impurity peaks confirmed the single-phase nature. These various surfactant-coated Fe 3 O 4 NPs neither exhibited phase transitions nor phase shifts suggesting that introduction of surfactants during the reaction does not affect the crystal structure. This is evident from the XRD patterns of both coated and uncoated Fe 3 O 4 NPs exhibiting same peaks. Reduced crystallite sizes obtained (8-10 nm) after surface modification is attributed to the different nature and charge of surfactants used 32 . Hence it is clear that surfactants influence the crystallite size rather than its structure. From the FTIR data, metal-oxygen band commonly present at ~ 540 cm −1 confirms the presence of Fe 3+ ions which gets coordinated through the corresponding functional group. Though Fe 2+ ions are present in octahedral sites, Fe 3+ ions in the tetrahedral site at the periphery have a strong preference for the feasible coordination. This inference is supported by the results of TGA, XPS, zeta potential and the proposed reaction mechanism.
All the coated samples are highly stable due to significant interparticle electrostatic or steric repulsive force and consequently good dispersion stability as indicated by zeta potential results. Existence of negative charge on magnetite surface is ascribed to the surface deprotonation and presence of hydroxyl, carbonyl or carboxylic groups attachment 53 whereas positive charge is owing to the cationic surfactant used. GA (with two carboxyl and one amino group) and CA (with three carboxyl and one OH group) are polysaccharides possessing strong binding affinity to Fe 3  www.nature.com/scientificreports/  www.nature.com/scientificreports/ a bi-dentate ligand with two nitrogen atoms at the centre having lone pair serve as Lewis base which coordinates with the surface of Fe 3 O 4 56 . CTAB provides a structure of 16-carbon and an ammonium group which acts as a long tail and head which is attached to three methyl groups 57 . Hence, the presence of quaternary amine at the outermost layer in CTAB is responsible for the binding interaction and positive charge 58 . More suitably, electrostatic stabilisation is obtained in ionic surfactant like CA, GA and EDA whereas steric stabilisation dominates in polymeric surfactants like PEG, PVP and CTAB.
The possible reaction mechanism leading to the formation of magnetite (Fe 3 O 4 ) from the Fe-precursors such as ferrous (FeCl 2 ) and ferric chloride (FeCl 3 ) with alkali (NH 4 OH) precipitation may be given as 59 : Alkaline NH 4 OH plays a crucial role for co-precipitation of Fe 2+ and Fe 3+ ions present in the mixed solution by providing hydroxyl (OH − ) ions to precipitate unstable metallic hydroxides. During heating, water (H 2 O) molecules get eliminated from the metal mixed hydroxides leading to the formation of Fe 3 O 4 . Inert atmosphere (nitrogen) used throughout the experiment prevented oxidation and helped in size reduction of NPs when compared with other methods without removing oxygen 43,60 . The proposed mechanisms are illustrated in Fig. S6. It is inferred that -COOH groups present in GA and CA linked with the -OH groups available on the surface of Fe 3 O 4 as COOH groups possess a higher affinity towards metallic oxides. It was reported that MNPs synthesized via co-precipitation method comprised of a number of OH groups on the surface 61,62 which can bind surface active agents. In the present study, Fe 3 O 4 NPs in aqueous medium resulted in water dissociation to produce OH groups on the surface. Hence, stabilisation of Fe 3 O 4 NPs has been achieved by the direct attachment of the respective surfactants (containing carboxyl, hydroxyl or amino groups) to the OH group present on the surface of the precipitated NPs. Here, Fe-OH bond on magnetite NPs surface reacts with COOH group present in both GA and CA molecule through an acid-base reaction resulting Fe-O-C species by eliminating H 2 O. It is reported that COOH or OH groups act as hydrogen donor or acceptor with the Fe 3 O 4 NPs via hydrogen bond 60 . OH and C=O groups present in PEG 63 and PVP respectively are responsible for interaction via hydrogen-bonding interactions. Positively charged hydrogen ion (H + ) from NH 2 groups in EDA reacts with OHleaving NH − which coordinates with Fe 2+ ions. Similarly, positively charged nitrogen (N + ) group present in CTAB is responsible for interaction. These conclusions strongly support the surface modification of Fe 3 O 4 NPs with varying surfactants.
Even though all MNPs have exhibited SPM nature experimentally, negligible coercivity occurred due to difference in proportion of factors such as particle volume, magnetic anisotropy and thermal energy. These factors concomitantly influence the magnetic moments to randomly fluctuate resulting in SPM behaviour. Though significant size effect on magnetic saturation values for various surfactant coated NPs was not observed, observed M s values may be attributed to the varying surface state of NPs. Surfactants co-ordinately anchored to the Fe 3 O 4 surface may alter the surface state thereby varying the magnetic properties 64 . Each surfactant has interacted with the magnetic core differently exhibiting varying magnetic response. Coatings affect the canting angles of magnetic moments of Fe core in its magnetic sublattice generating magnetic spin disorder 65 which results in lower magnetisation than the bulk magnetite value 35 . This is more evident from the higher M s value obtained for the uncoated Fe 3 O 4 NPs i.e., magnetisation value of Fe 3 O 4 NPs reduces after surface functionalization. Consequently, www.nature.com/scientificreports/ the obtained saturation magnetization values arise as a result of both the volumes of respective diamagnetic coating and total iron oxide. Similarly, substantial number of Fe 2+ ions present in GA, CA, PEG, and EDA coated samples are responsible for enhanced magnetic susceptibility compared to PVP and CTAB coated ones which is evident from the XPS spectra (details given in Table S1). Moreover, different anisotropy (K) values obtained (Table S2) imply the different amount of energy required to orient the entire magnetic moment of corresponding grains (iron oxide core and coatings). Since all the samples have approximately similar size and magnetisation values, variation in hyperthermic ability can be attributed to the distinctive Neel-Brownian relaxation mechanisms induced by different coatings 18 . This eventually affects the heat dissipation upon an external magnetic field. Magnetic anisotropy is a predominant factor affecting the Neel-Brownian relaxation of MNPs subjected to an AC magnetic field 66 . When magnetic spin reversal and particle rotation occurs in parallel, the effective relaxation time is calculated using the equation: In the present study τ N and τ B are calculated using the modified Neel relaxation and Brownian equations 18 , as can be seen in Table 2. Though Brownian mechanism is not directly linked to magnetic behaviour associated with MNPs, it holds a noteworthy impact to intensify the magnetic behaviour in terms of increment in NP size within the SPM limit. In this study, Brownian contribution is negligible compared to Neel contribution which is evident from the calculated values. Therefore, utmost contribution to heat dissipation is attributable to the Neel losses and its relaxation time, τ N is found to be effectively controlled by the anisotropy constant K.
Magnetic field reversal time of 5.03 × 10 -7 s (τ m = 1 2πυ ) is found to be shorter than the magnetic relaxation time (τ) (obtained in Table 2) of NPs on exposure to magnetic field of 316 kHz frequency which strongly confirms the heat dissipation is strictly via magnetic moment relaxation mechanisms. Faster relaxation time, which denotes maximum number of sufficient attempts to overcome the energy barrier for magnetisation reversal, occurs for GA-Fe 3 O 4 NPs. Even though M s value obtained for CA-Fe 3 O 4 is highest among all, faster relaxation time for magnetic flipping obtained for GA-Fe 3 O 4 has led to the maximum SAR value. Even though PVP coated NPs possess a lower M s than PEG, EDA and CTAB, it results in a higher SAR due to a comparatively higher anisotropy and faster relaxation time. A maximum magnetic susceptibility is observed for GA-Fe 3 O 4 at 42 °C among all samples. In our investigation, anisotropy, magnetic susceptibility and effective relaxation time dominantly contributed to SAR value. In addition to this, SAR is found to be reduced for higher concentrated samples due to increased magnetostatic interactions (lowers the anisotropy energy barrier). Such interactions consequently lead to the reduction in Neel-Brownian relaxation (no mobility between NPs) 21 i.e., NPs not at all rotate under the applied magnetic field as they are considered as large entities possessing weak remnant magnetization so the torque undergone is trivial. It can also be concluded that magnetic field lines closure configuration reduces the interaction between each NP thereby causing for reduced magnetic moment of aggregates.
Specifically, the study revealed that GA coated Fe 3 O 4 NPs with enhanced anisotropy, enhanced magnetic susceptibility, faster relaxation time and with a higher SAR release a larger amount of heat which is potentially stable for clinical MHT applications compared to other samples. Moreover, the intrinsic loss power (ILP) values of the corresponding surfactant coated Fe 3 O 4 NPs were calculated and summarised in Table 2. Though the calculated ILP values are lower, these MNPs can still be used as nano heaters for MHT therapy since these values are similar to certain commercially available functionalised superparamagnetic ferrofluids such as Nanomag-D-spio (0.23 nHm 2 /kg), Micromode ferrofluids (0.15-0.35 nHm 2 /kg) etc 67 . Further the functionalised NPs are biocompatible in nature and can be conjugated with other drug molecules for chemothermal therapy of cancer as evident from our earlier published results 26,27,68 . Figure 7 shows the schematic illustration of as synthesized surface modified SPIONs and their testing towards MHT.

conclusion
The heating potentiality of aqueous stable biocompatible Fe 3 O 4 NPs coated with various surfactants such as GA, CA, PEG, PVP, EDA and CTAB with well-defined particle size, shape, magnetic anisotropy and susceptibility was successfully investigated. XPS asserted the presence of functional groups on the iron oxide core. MHT studies found that heating efficacy varied with different surface coatings due to effective anisotropy, relaxation time and magnetic susceptibility. Maximum magnetic susceptibility of 0.075 emu/gOe exhibited at HT temperature under magnetic field of 450 Oe leads to the highest SAR of 130 W/g in GA coated Fe 3 O 4 NPs. In addition, shorter relaxation time and enhanced anisotropy also cause for maximum heat dissipation within short period. Moreover, this study pinpointed the fact that SAR is concentration dependent as exhibited by all functionalized NPs. The as synthesized NPs possessing enhanced heating efficacy proved its potential candidacy for future clinical MHT applications. Physical and chemical characterization. Crystalline structure was identified using X-ray diffraction (XRD) pattern using a diffractometer (D8 Advanced, Bruker) with a Cu-Kα radiation source (λ = 1.5406 Å, 40 kV). Debye-Scherrer method was used for the measurement of average crystallite size. Surface functionalization was characterized with the aid of Fourier transform infrared (FTIR) spectra recorded on a Shimadzu IR Prestige ranging from 400 to 4000 cm −1 on a Shimadzu IR Prestige. Thermogravimetric-differential thermal analysis (TGA) was performed by an SDT Q60 V20.9 thermal analyzer. For TGA, samples were heated from room temperature to 800 °C in a nitrogen atmosphere under a flow rate of 100 ml/min and a temperature ramp of 20 °C/min. Colloidal stability of MNPs in water was investigated by Horiba zeta potential analyzer and average of three values was calculated. High-resolution transmission electron microscopy (HRTEM) images and selected area diffraction (SAED) patterns were obtained using transmission electron microscope (TEM, Philips, Model: CM 200) operated under an accelerating voltage of 200 kV. Elemental composition and oxidation states of the surface species were analyzed by X-ray photoelectron spectroscopy (XPS) spectra obtained from Kratos AXIS ULTRA-DLD utilising Al Kα excitation source (14 kV). Magnetic measurements were conducted with the aid of a magnetic properties measurement system (MPMS-XL, Quantum Design).

Materials and methods
Evaluation of heating efficacy in terms of SAR. MHT measurements were carried out for determining the SAR using calorimetric method. Calorimetric HT was performed with the aid of a 4.2 kW Ambrell Easy heat 8310 system. Colloidal suspension of 1 mg/ml, 2 mg/ml and 3 mg/ml for all the prepared samples in water was used and the heating response was measured upon external magnetic field parameters of 450 Oe and 316 kHz. In order to prevent agglomeration between NPs, thorough sonication was performed for all samples prior to HT measurements. The temperature rise with respect to the exposure time for corresponding magnetic field parameters for all the samples was recorded. The heat dissipated in terms of SAR, expressed in W/g, was examined by the following formula: where, C s is the specific heat capacity of solvent (C water = 4.187 J/g℃); M SOL and M MNPs are masses of the solvent and MNPs used for measurement, and T t is the temperature-time dependent slope. An intrinsic parameter, intrinsic loss power (ILP), for power dissipation with respect to applied frequency (f) and amplitude (H) is also considered in case of calorimetric HT measurements i.e., www.nature.com/scientificreports/