Non-aqueous to aqueous phase transfer of oleic acid coated iron oxide nanoparticles for hyperthermia application

Abstract

Iron oxide magnetic nanoparticles (MNPs) alone are suitable for a broad spectrum of applications, but the low stability and heterogeneous size distribution in aqueous medium represent major setbacks. These setbacks can however be reduced or diminished through functionalization of MNPs with various biocompatible surfactants. In this study, magnetite (Fe₃O₄) nanoparticles were modified using oleic acid (OA) to reduce their agglomeration. To render hydrophilicity and to increase the colloidal stability of the MNPs, they were further functionalized with betaine-HCl (BTH). The physiochemical properties were well characterized using X-ray diffraction, Fourier transform infrared, thermogravimetric analysis, transmission electron microscopy and superconducting quantum interference device of the OA-BTH coated Fe₃O₄ MNPs in order to use them for hyperthermia application. Zeta potential study and size distribution of nanoparticles showed increased stability of the nanoparticles. The coated MNPs show increase in specific absorption rate value of 91.03 W g⁻¹ at 335.2 Oe, making them more suitable for hyperthermia application. Cytotoxicity study was performed by MTT assay on L929 cell line for 24 h incubation period.

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Introduction

Hybrid inorganic–organic nanocomposite materials are of current interest because of their multifunctionality, ease of processibility, and potential for large-scale manufacturing. The inclusion of carbon nanostructures, metallic, magnetic, and semiconducting nanoparticles in polymer matrices has been investigated for a wide range of applications.¹ However, to date, effective coating of magnetic particles with natural or synthetic polymers is still a challenge, since the surfaces of magnetic particles are hydrophilic but polymers are hydrophobic. On the other hand, it is difficult to locate the dominant polymerization loci at the surface of the magnetic inorganic particles. Therefore, development of a versatile method to directly coat polymer onto magnetic particles is significant.

Core/shell structured particles, especially magnetic nanocomposite materials, have attracted increasing attention because they offer the possibility of new nanostructured materials with diverse applications for their unique magnetic responsivity, low cytotoxicity, and chemically liable surface.² Oil-soluble type functionalization employed in order to prevent or decrease the agglomeration of iron oxide MNPs and increase the stability give rise to the monodispersity, for instance, iron oxide MNPs frequently dispersed in long chain substance of hexadecane, the classic example being oleic acid (OA) [CH₃(CH₂)₇CH=CH (CH₂)₇COOH], which has a C18 tail with a cis-double-bond in the middle, forming a kink. Such kinks have been postulated to be necessary for effective stabilization. Additionally, oleic acid is widely used in ferrite nanoparticle synthesis because it can form a dense protective monolayer, thereby producing highly uniform and monodispersed particles.³

Capping agents such as oleic acid are often used because they form a protective monolayer, which is strongly bonded to the surface of nanoparticles. This is necessary for making monodisperse and highly uniform MNPs.^4,5 However, coating of MNPs with OA makes the particles dispersible only in organic solvents and consequently limits their use for biomedical applications.⁶ For biomedical applications in aqueous environments, this hydrophobic coating has to be replaced with a hydrophilic coating. Lattuada and Hatton⁷ reported that the oleic groups initially present on the above nanoparticle surfaces were replaced via ligand-exchange reaction with various capping agents bearing reactive hydroxyl moieties. This route was proposed a flexible methodology for the preparation of various types of monodisperse, water-soluble magnetic MNPs coated by different polymer brushes.

The shell can be generated by replacing the hydrophobic ligand with macromolecules such as peptides and hydrophilic polymers or other ligands such as DMSA (2,3-dimercaptosuccinic acid), betaine-HCl (BTH) and silanes.⁸ The richest natural source of biologically available methyl groups is betaine hydrochloride from beets, also known as trimethyl glycine.

In the present paper, Fe₃O₄ nanoparticles were prepared using FeCl₂ as the sole precursor. Very few data is available on this type of study to date. The as-prepared MNPs were coated with OA. The hydrophobic nature of these OA coated Fe₃O₄ nanoparticles were reversed by forming a shell of BTH. The bare and coated Fe₃O₄ nanoparticles (Fe₃O₄–OA-BTH) were thoroughly studied for their structural, morphological and magnetic characterizations and the effect of OA-BTH coating on the Fe₃O₄ nanoparticles was observed. The particles were again studied for induction heating ability for their possible use in hyperthermia treatment as a biomedical application.

Materials

Ferrous chloride (FeCl₂·4H₂O), hydrochloric acid (HCl), methanol, acetone and sodium hydroxide (NaOH) were procured from HiMedia, India. Oleic acid and betaine-HCl were purchased from Sigma-Aldrich, USA. Double distilled water was used throughout the procedure.

Method

MNPs were prepared using our recently reported method.⁹ In brief, 2 g of FeCl₂·4H₂O was dissolved in 20 mL, 2 M HCl by heating at 70 °C. To this, 25 mL of 3 M NaOH was added dropwise with continuous stirring till black precipitate was formed. The precipitate was then washed several times with distilled water till neutral pH. The precipitate was then separate out using external magnetic field and dried at 100 °C. The as prepared nanoparticles were further used for coating procedure. The possible reaction taking place is as shown below:

3FeCl₂·4H₂O + 6NaOH + 1/2O₂ → Fe₃O₄ + 6NaCl + 15H₂O (1)

The obtained bare Fe₃O₄ MNPs then coated with OA. 1 g of bare Fe₃O₄ MNPs were dispersed in 100 mL of methanol and then heated to 80 °C while continuous stirring on magnetic stirrer. To this 10 mL of OA was added dropwise. The mixture was kept for several hours at 80 °C till all methanol get evaporated. The remaining mixture was filtered to remove excess of OA and washed several times with double distilled water and finally with acetone. The OA coated MNPs were collected and dried at 50 °C.

The OA coated nanoparticles (200 mg) were then dispersed in 25 mL of 1% BTH with continuous stirring for 6 h. After that coated MNPs were separate out using external magnetic field and washed several times with double distilled water. The obtained MNPs were dried at 50 °C.

Characterization

The X-ray diffraction (XRD) pattern of drop coated and air-dried MNPs on the glass substrate was recorded to study the structural and phase analysis by XRD Philips PW-3710 diffractometer using Cr K_α (λ = 2.2897) radiation in the 2θ range from 0° to 100°. The XRD patterns were evaluated by X'pert high score software and compared with JCPDS card. The average crystallite size (t) was calculated from the diffraction line-width of XRD pattern, based on Scherrer's relation:

t = 0.9λ/β cos θ (2)

where, β is the full width at half maxima (FWHM).

The MNPs were used to get Fourier transform infrared (FTIR) spectra with the help of Perkin-Elmer spectrometer, (model no. 783, USA) in the range 450 to 4000 cm⁻¹ using KBr pellets to check the possible interaction of Fe₃O₄ with OA and BTH. The compositional analysis was done by energy-dispersive analysis of X-ray spectroscopy (EDAX, JEOL JSM 6360). Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) images were used to determine the morphology and size of the MNPs. For TEM, the colloidal solution of the MNPs was transferred on to a carbon coated copper grid and allowed to air dry. The grid was then scanned using Philips CM200 model transmission electron microscopy, operating voltage 20–200 kV with resolution 2.4 Å.

The magnetization measurements were performed on a Superconducting Quantum Interference Device (SQUID) magnetometer to investigate the saturation magnetization (M_s), blocking temperature (T_B) and Curie temperature (T_C). The measurements include field dependent hysteresis loops, (M–H), at two different temperatures 100 and 300 K with applied field range from 0 to ±2 × 10⁴ Oe (2 Tesla).

Zeta potential and hydrodynamic diameter of particles was measured using a PSS/NICOMP 380 ZLS particle sizing system (Santa Barbara, CA, USA) with a red He–Ne laser diode at 632.8 Å in fixed angle 90° plastic cell. The zeta potential measurements were performed at 25 °C after a temperature homogenization time of 5 min. The measurements were carried out at different pH from 2 to 10. For reproducibility, at least three measurements were conducted for each pH value. The instrument calibration was checked before each experiment using a latex suspension of known zeta potential (i.e., −55 ± 5 mV).

Since the functionalized MNPs are to be used for biomedical applications, the issue of cytotoxicity has to be addressed. The viability of the L929 cell line in the presence of MNPs was assessed relative to cells in the control experiment (no MNP present) using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay which has been described as a very suitable method for the detection of biomaterial toxicity.^10,11 L929 cell line was obtained from the National Centre for Cell Sciences, Pune, India and the detailed toxicity study was done in the National Toxicology Centre Pune, India (ISO 10993/USP 32 NF 27) by MTT assay. The L929 cells were grown in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% v/v fetal bovine serum, kanamycin (0.1 mg mL⁻¹), penicillin G (100 U mL⁻¹), and sodium bicarbonate (1.5 mg mL⁻¹) at 37 °C in a 5% CO₂ atmosphere. The cells were incubated with the concentration of 1 × 10⁴ cells per mL in the medium for 24 h in a 96-well microtitre plate. After 24 h, the old media was replaced by fresh media and different proportions of sterile magnetic particles of MNPs (0.1, 0.5, 1.0, 1.5 and 2.0 mg mL⁻¹ of cultured media) were added. Then the total medium was incubated at 37 °C in a 5% CO₂ atmosphere for 24 h. After 24 h, 10 μL MTT solution was added into each well including control wells. The plates were incubated for 3 h at 37 °C in a 5% CO₂ atmosphere for metabolization of MTT with the nanoparticles and cell media. Then, the total medium was removed by flicking the plates and only anchored cells remained in the wells. The cells were then washed with PBS and any formazan formed was extracted in 200 μL acidic isopropanol and finally the absorbance is read at 570 nm and from it the cell viability is calculated. The experiments were replicated three times and the data was graphically presented as the mean.

Induction heating of Fe₃O₄ nanoparticles for hyperthermia application was performed in a plastic micro centrifuge tube (1.5 mL) using an induction heating unit (Easy Heat 8310, Ambrell; UK) with a 6 cm diameter (4 turns) heating coil. To keep the temperature of the coil at ambient temperature, a provision of water circulation in coils was provided. MNPs (2, 5 and 10 mg) suspended in 1 mL of distilled water was placed at the centre of the coil and the applied frequency was 265 kHz. Particles were dispersed in water with ultrasonication for 20 min to achieve a good dispersion of the MNPs in carrier fluid. Samples were heated for 10 min with the desired current (200–400 A). For the conducted experiments, the magnetic field was calculated from the relationship:

where n, i and L denote the number of turns, applied current and the diameter of the turn in centimeters, respectively. Calculated values of the magnetic field (H) at 200, 300 and 400 A were 167.6, 251.4 and 335.2 Oe (equivalent to 13.3, 20.0 and 26.7 kA m⁻¹), respectively. Temperature was measured using an optical fibre probe with accuracy 0.1 °C.

Results and discussion

For biomedical application of nanoparticles, effective coating of nanoparticles is an important task. As mentioned above the nanoparticles were first modified with OA, these MNPs were only form stable suspension in organic solvents. To change the polarity of these MNPs they were again functionalized with BTH. The crystal structure of as-prepared and the OA-BTH coated Fe₃O₄ MNPs was shown in Fig. 1. The XRD patterns reveal that both samples consist of highly crystalline particles with an inverse spinel structure and lattice parameters similar to those of magnetite.¹² The main characteristic peaks were obtained with the (hkl) values of (220), (311), (400), (422), (511) and (440). These were then matched with the JCPDS file number 82-1533, which corresponds to Fe₃O₄ phase. The Gaussian fit of the most intense peak (311) was used to calculate the full width at half maxima for determination of crystallite size (D) by the equation D = 0.9λ/β cos θ, where λ = 2.2897 Å, the wavelength of incident X-ray, θ is the corresponding Bragg's diffraction angle and β is full width at half maxima of the (311) peak. The average crystallite size of bare Fe₃O₄ MNPs found to be 16 nm and 10 nm for OA-BTH coated MNPs. Reports also support the fact that post-synthesis coating procedure also may result in decreasing the crystallite size.^20,21

Fig. 1 XRD patterns of the as prepared Fe₃O₄ (a) and Fe₃O₄–OA-BTH (b) MNPs.

Surface functionalization of MNPs was confirmed by FTIR and thermogravimetric (TG) analysis. Fig. 2 shows the FTIR spectra of Fe₃O₄ and Fe₃O₄–OA-BTH MNPs over the range of 450 to 4000 cm⁻¹. The band observed at 565 cm⁻¹ corresponds to the intrinsic stretching vibration (Fe_tetra–O) of metal–oxygen at tetrahedral site.¹³ The peak observed at 3434 cm⁻¹ correspond to surface-adsorbed water molecules on Fe₃O₄.

Fig. 2 FTIR spectra of Fe₃O₄, Fe₃O₄–OA and Fe₃O₄–OA-BTH MNPs.

However, the presence of oleic acid was confirmed by two CH₃ stretching at 2930 cm⁻¹ and 2860 cm⁻¹. According to the literature, C=O stretch band of the carboxyl group shows a peak at 1710 cm⁻¹ in the spectrum of the pure liquid oleic acid,¹⁴ which was absent in the spectrum of the coated nanoparticles and there appeared two new bands at 1541 and 1639 cm⁻¹, which were characteristic of the asymmetric ν_as(COO⁻) and the symmetric ν_s(COO⁻) stretch. A strong adsorption at 1030 cm⁻¹ arises from C–O single bond stretching. Carboxylate and CH₃ stretching confirmed the presence of OA in coated samples. This proved oleic acid was chemisorbed onto the MNPs surface via its carboxylate group. The peak observed at 1350 cm⁻¹ in OA-BTH coating corresponds to C–N stretching which confirmed the successful coating of BTH.^15,16 BTH may form coronary layer over OA layer.

Fig. 3 shows TGA curves of uncoated, OA coated and OA-BTH coated Fe₃O₄ nanoparticles measured by thermogravimetric analyzer. As TG was performed under N₂ atmosphere, the oxidation of magnetic nanoparticles was greatly reduced. It provides additional quantitative evidence on the structure of coating on surface of nanoparticles. The 1.5% weight loss due to evaporation of physically adsorbed water in the temperature range below 120 °C was observed for both bare and coated samples. Three major weight loss stages were observed in thermogram for Fe₃O₄–OA-BTH, one below 120 °C which can be ascribed to evaporation of water, while the other one beginning at about 120 °C was attributed to the decomposition of BTH.¹⁶ The third stage begins at about 240 °C was attributed to the decomposition of OA. This pattern is in good agreement with the patterns reported in literature for OA coating and can be explained by chemisorption of OA molecules on to the surface of nanoparticles. The differences in total weight loss for both samples help us to calculate the percentage OA-BTH molecules attached to surface of nanoparticles. Thus it is implicit that about 17% of the OA-BTH was adsorbed on to the surface of nanoparticles. In that about 7% of BTH was present and about 10% of OA was present (TG of OA coating shows about 10% of loss). Above the temperature 500 °C both samples attains the stability in terms of weight loss.

Fig. 3 Thermogravimetric spectra of bare Fe₃O₄, Fe₃O₄–OA and Fe₃O₄–OA-BTH MNPs in nitrogen with a scanning rate of 10 °C min⁻¹ up to 600 °C.

The EDAX spectra were used for quantitative elemental analysis of bare and OA-BTH coated Fe₃O₄ MNPs, which are shown in Fig. 4(a) and (b) respectively. The corresponding peaks in bare MNPs are due to Fe and O only, while OA-BTH-coated MNPs show additional peaks corresponding to C and N as expected. Both the spectra do not show any additional impurity peak implying purity of the samples.

Fig. 4 EDAX spectra of bare Fe₃O₄ (a) and Fe₃O₄–OA-BTH (b) MNPs.

Fig. 5 represents the TEM images for both uncoated and OA-BTH coated Fe₃O₄ nanoparticles. Fig. 5(a) clearly shows the formation of spherical nanoparticulates with size 20.7 ± 4.8 nm in an agglomerated form. From Fig. 5(b), one can see that the OA-BTH coated Fe₃O₄ nanoparticles were spherical in shape with size 19.3 ± 3.9 nm and have good dispersibility (non-agglomerated) as compared to uncoated nanoparticles. Improvement in dispersibility after coating with OA-BTH may be attributed to presence of the non-magnetic surface layer of OA-BTH which readily decreases the interparticle interaction i.e. dipole–dipole interaction and thus enhances the dispersibility. The corresponding Selected Area Electron Diffraction (SAED) patterns in Fig. 5 (insets) show bright ring patterns indicating polycrystalline nature of the MNPs, as indicated by XRD patterns. The ring pattern corresponds to (220), (311), (400), (422), (511) and (440) planes which can be clearly seen in XRD pattern.

Fig. 5 TEM images of bare Fe₃O₄ (a) and Fe₃O₄–OA-BTH (b) MNPs.

In general, zeta potential is used to express the colloidal stability of MNPs dispersed in aqueous media. Colloidal stability of MNPs depends upon electrostatic as well as steric repulsion. The zeta potential values and hydrodynamic diameters of bare and coated nanoparticles suspensions in water with respect to pH are shown in Fig. 6. The zeta potentials of bare nanoparticles at pH 2, 4, 6, 8, and 10 were 20.05, 17.02, 15.00, −27.92 and −35.00 mV and that of coated nanoparticles were 00.00, 04.91, −12.21, −19.11 and −40.03 mV, respectively, the values of coated particles is more negative in the range of pH 6–10, as compared to bare indicating that the negative charges on the Fe₃O₄–OA-BTH nanoparticles increase with a increase in pH, may be due to the protonation of free carboxylate groups at high pH. The isoelectric point of bare and coated nanoparticles were determined as about pH 6.6 and 4.5 respectively. These observations suggest that the surface of the bare nanoparticles were coated efficiently with OA-BTH. Hence the coating of bare nanoparticles with OA-BTH allows obtaining a stable dispersion at neutral pH values required to work with biological samples.¹⁷

Fig. 6 Zeta potential and hydrodynamic diameters of bare and coated nanoparticles suspensions in water as a function of pH.

Dynamic Light Scattering (DLS) measurements were carried out to investigate the hydrodynamic size of bare and Fe₃O₄–OA-BTH nanoparticles. The hydrodynamic diameter distributions of bare and Fe₃O₄–OA-BTH nanoparticles at a scattering angle of 90° are shown in Fig. 6. The bare nanoparticles showed higher particle size as compared to coated one due to formation of agglomerates by the virtue of dipole–dipole interaction. The particle size of coated nanoparticles was in good agreement with the TEM and zeta potential results; the size was reduced by the repulsive forces acted on the particles due to formation of electrostatic and steric interactions of the coated material.

The particle size distributions of both the nanoparticles when dispersed in water were measured by DLS technique and obtained results are shown in Fig. 6. The average hydrodynamic diameters of bare MNPs at pH 2–10 was about 60–75 nm indicating higher degree of agglomeration. The OA-BTH-coated MNPs showed higher particle size distribution about 67 nm and 58 nm at pH 2 and 4 respectively. At higher pH, 6, 8 and 10 the particle size was greatly reduced which is in agreement with zeta potential value at respective pH. Moreover, one should observe the absence of large aggregates in the coated sample which indicates that the bare MNPs are polydispersive while OA-BTH-coated MNPs are monodispersive. The SEM, TEM and DLS results confirms that the OA-BTH coating on Fe₃O₄ prevents the agglomeration of Fe₃O₄ MNPs.

To understand the effect of coating on the magnetization behavior of Fe₃O₄ system the M verses H measurements were carried out as a function of applied field and temperature. Fig. 7(a) and (b) show the M–H curves of both samples at 100 and 300 K respectively. On the basis of SQUID measurements, it can be seen from the hysteresis curves for bare and functionalized Fe₃O₄ MNPs at 100 and 300 K that almost negligible coercivity or remanence existed, indicating the superparamagnetic behavior of Fe₃O₄ MNPs before and after coating. The values of magnetization, coercivity and remanence observed from the experiment are given in Table 1. It can be seen from Fig. 7 that magnetization decreased with coating of OA-BTH. This is because magnetization is proportional to the amount of weight for the same magnetic material. Organic coating (OA-BTH) layers on magnetic material increases the amount of non magnetic substance which reduces the overall magnetization of the material.

Fig. 7 MH curves of Fe₃O₄ and Fe₃O₄–OA-BTH MNPs at 100 K (a) and 300 K (b).

Table 1 Saturation magnetization (M_s), coercivity (C_e) and remanence (M_r) values calculated from the MH curves for both bare and OA-BTH coated Fe₃O₄ MNPs

	Fe3O4		Fe3O4–OA-BTH
	100 K	300 K	100 K	300 K
Ms (emu g^−1)	74.30	74.24	55.74	51.68
Mr (emu g^−1)	17.60	2.04	13.91	2.23
Hc (Oe)	145.66	12.41	159.72	16.85
Mr/Ms	0.24	0.03	0.25	0.04

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Fig. 8(a–c) represents the temperature kinetic curves obtained after application of an alternating magnetic field on both samples which dispersed in water with concentration of 2 mg mL⁻¹ and (d) SAR vs applied AC magnetic field for both samples. Temperature kinetic curves represents, rise in temperature is dependent on the applied magnetic field for both the samples. For superparamagnetic nanoparticles the greatest relaxation losses are due to Brownian modes (heat due to friction arising from total particle oscillations) and Neel modes (heat due to rotation of the magnetic moment with each field oscillation).¹⁸ The heat dissipation by superparamagnetic nanoparticles is given by the following equation

P = μ₀πχ′′fH² (4)

where P is the heat dissipation value, μ₀ the magnetic field constant, χ′′ the AC magnetic susceptibility (imaginary part), f the frequency of the applied AC magnetic field, and H the strength of the applied AC magnetic field. If an assembly of MNPs is put into an alternating magnetic field of frequency f and amplitude μ₀H_max, the amount of heat A released by the magnetic nanoparticles during one cycle of the magnetic field simply equals the area of their hysteresis loop, which can be expressed as.

Fig. 8 (a–c) Temperature kinetic curves obtained after application of an alternating magnetic field on both the samples dispersed in water with a concentration of 2 mg mL⁻¹, (d) SAR vs applied AC magnetic field for both samples.

Then the SAR is

SAR = Af (6)

where f is the frequency of the AC magnetic field and it is expressed as f = ω/2π, M magnetization and H applied magnetic field. The heat loss due to eddy currents (ED) is given by the following equation,where μ is the permeability of a material, d, the diameter of the particle and ρ, resistivity of the material.

The specific absorption rate (SAR) is calculated by using following relation.

where c is the sample-specific heat capacity, which was calculated as a mass weighted mean value of MNPs and water. The heat-capacity of both samples is negligible because of its low concentration, and thus a heat capacity for water (4.18 J g⁻¹ K⁻¹) was taken as the sample's heat capacity. ΔT/Δt is the initial slope of the time-dependent temperature curve. Here, we considered time up 1–5 min to calculate the slope. The value of m_magn is considered as the amount of MNPs per total amount of MNPs and water. The estimated SAR values from Fig. 8(a–c) and by using eqn (8) for both the samples are graphically represented in Fig. 8(d).

In this paper, it is demonstrated experimentally that the hyperthermia effect of Fe₃O₄ MNPs enhances dramatically after functionalized with OA-BTH. First possible reason for the enhanced hyperthermic effect after coating is that the ability of the OA-BTH coating to retain the superparamagnetic fraction of the Fe₃O₄ much better as compared to Fe₃O₄ alone. The second is that coating layer prevents the formation of larger aggregates of Fe₃O₄ (also confirmed from DLS results) which make the better suspension of OA-BTH functionalized MNPs in the water as compared to naked Fe₃O₄. It is reported in our recent publication that the well dispersed superparamagnetic particles enhances the hyperthermic effect through Brownian and Neel's spin relaxations.¹⁹ Thus the hyperthermia study also strongly supports the coating of OA-BTH on Fe₃O₄ MNPs and prevents particle agglomeration.

The cytotoxicity study of both, bare and coated nanoparticles was done on L929 and cell line with different concentrations of nanoparticles and the obtained data is shown in Fig. 9. The L929 cell line was incubated with nanoparticles for 24 h with the concentrations of 0.1, 0.5, 1.0, 1.5 and 2.0 mg mL⁻¹ at 37 °C in a 5% CO₂ atmosphere. The relative cell viability (%) compared with control well containing cells without nanoparticles are calculated by the equation: [A]_tested/[A]_control × 100.²⁰ Fig. 9 shows the cell viability after incubation with different concentrations of both bare and coated Fe₃O₄ nanoparticles for 24 h. It clearly reveals that even after 24 h, coated MNPs give 80% cell viability and exhibit negligible cytotoxicity at 2 mg mL⁻¹ concentration. Therefore coated MNPs are suitable for in vivo applications owing to their lower cytotoxicity. In the present paper this much concentration for both the cytotoxicity and heating induction ability is studied in order to use them for hyperthermia therapy application.

Fig. 9 Cytotoxicity profiles of MNPs for 24 h on L929 cell line at different concentrations (0.1, 0.5, 1.0, 1.5 and 2.0 mg mL⁻¹).

Conclusions

From the present study, it was concluded that pure and stable phase Fe₃O₄ nanoparticles can be obtained using FeCl₂ as the sole source and without using any other oxidant. The resulting Fe₃O₄ nanoparticles were superparamagnetic at room temperature with high saturation magnetization value. The successful functionalization OA and BTH was achieved and the interaction of it at the interface is discussed in detail. As OA coated MNPs were hydrophobic in nature, BTH gave the OA coated MNPs a high degree of dispersibility in polar solvents. The resulting MNPs formed highly stable colloid suspensions. The MNPs showed negligible cytotoxicity on L929 cell line. Both, bare and OA-BTH Fe₃O₄ nanoparticles were proved to be very efficient for their use in hyperthermia treatment on the basis of their high SAR values. Zeta potential and DLS measurements of both the particles prove higher colloidal stability for the coated nanoparticles which is important in order to use them for in vivo applications. The preliminary results obtained from magnetic, hyperthermia, zeta potential and cytotoxicity study were highly encouraging due to monodispersivity, high saturation magnetization values, high SAR values and low cytotoxicity which makes the functionalized nanoparticles an important candidate for biomedical applications.

Acknowledgements

The authors are thankful to Dr S. D. Saratale, Pune University, Pune (M S), India for providing SAED patterns. The authors are also grateful to Dr T. R. Waghmode for giving TG-DTA results of the samples. They are also thankful to Dr D. Das, Dr R. K. Vatsa, Chemistry Division, BARC for providing Induction heating facilities. The M–H measurements were performed at the magnetism laboratory under the supervision of Drs Alok Banergee and R. J Choudhary in the UGC-DAE Consortium for Scientific Research, Indore. The authors are grateful to DST, DAE-BRNS, India for their financial support.

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