Synthesis and characterization of monodispersed water dispersible Fe3O4 nanoparticles and in vitro studies on human breast carcinoma cell line under hyperthermia condition

Monodispersed Fe3O4 magnetic nanoparticles (MNPs) having size of 7 nm have been prepared from iron oleate and made water dispersible by functionalization for biomedical applications. Three different reactions employing thioglycolic acid, aspartic acid and aminophosphonate were performed on oleic acid coated Fe3O4. In order to achieve a control on particle size, the pristine nanoparticles were heated in presence of ferric oleate which led to increase in size from 7 to 11 nm. Reaction parameters such as rate of heating, reaction temperature and duration of heating have been studied. Shape of particles was found to change from spherical to cuboid. The cuboid shape in turn enhances magneto-crystalline anisotropy (Ku). Heating efficacy of these nanoparticles for hyperthermia was also evaluated for different shapes and sizes. We demonstrate heat generation from these MNPs for hyperthermia application under alternating current (AC) magnetic field and optimized heating efficiency by controlling morphology of particles. We have also studied intra-cellular uptake and localization of nanoparticles and cytotoxicity under AC magnetic field in human breast carcinoma cell line.

. In case of oleic acid, peaks observed at 2890 and 2950 cm −1 correspond to CH 2 stretching vibration and peak at 3059 cm −1 corresponds to =C-H stretching vibration of unsaturated carbons (C 9 -C 10 ) of oleic acid 21 . The 1700 cm −1 peak corresponds to stretching vibration of C=O in oleic acid. The broad peak in 2200-3600 cm −1 corresponds to O-H stretching vibration of oleic acid in liquid phase. Ferric oleate has similar peaks of oleic acid, but the peak at 1700 cm −1 disappears and instead, the new peaks at 1557 and 1441 cm −1 appear and correspond to the anti-symmetric and symmetric stretching vibrations of carboxyl group (CO 2 − ) attached to Fe 3+ ions. The small peak at 3400 cm −1 corresponds to H 2 O present in oleate. In case of Fe 3 O 4 , extra peak at 607 cm −1 is observed as compared to oleate and this corresponds to Fe-O bond vibration. Detail studies are given in Supporting Information SI1.1 and 1.2.
Thermo-gravimetric analysis (TGA) curve of Fe 3 O 4 particles is shown in Fig. 1(iii). Total contribution of oleate or oleic acid molecules is 24%. Similar findings were reported elsewhere 21,22 . Differential thermal analysis (DTA) curve of Fe 3 O 4 shows three exothermic peaks at 350, 435 and 468 °C, which suggest the decomposition of oleate molecules.
TEM study. The size and morphology of Fe 3 O 4 particles are investigated by transmission electron microscopy (TEM). TEM images reveal quasi-spherical particles with size of 7-8 nm when rate of heating during synthesis is maintained at 4 °C/min. The particle size distribution was determined by the statistical evaluation of ~100 particles ( Fig. 2A). Selected area electron diffraction (SAED) patterns of the particles as well as the lattice fringes are observed in high resolution images. This is indicated that the particles are highly crystalline (Fig. 2B,C). From particles prepared by heating rate of 1.3 °C/min. Schematic diagram of conversion of spherical to cubic shape of a particle by core-shell model (Ostwald ripening phenomenon): (H1) slightly spherical particle coated by oleate ions along with magnetisation axes of face-centred cube (Fe 3 O 4 unit cell), (H2) particles in ferric oleate medium, (H3) formation of Fe 3 O 4 small paricles/cluster which surrounds a larger particle, (H4) Faster growth/ deposition along 〈111〉 direction as compared to 〈100〉 or 〈220〉 direction, (H5) formation of cubic shaped particle.
Adding ferric oleate (1 ml) to Fe 3 O 4 particles (7 nm) followed by heating at 320 °C for 1 hour changes the shape of the particles from spherical to cuboid (Fig. 2D). The particles are monodispersed and are found to be 11 nm (one side of cuboid). In case of addition of 5 mL of ferric oleate to the already synthesized particles (following the same heating conditions as mentioned above), formation of large number of smaller particles around the bigger particles is observed as seen in Fig. 2E. The particle size shows bimodal distribution. Small spherical shaped particles of ~6-8 nm are found along with bigger cuboid particles with size of 13-16 nm.
However, when 5 mL of ferric oleate is added followed by 8 hours heating, particles are found to be larger in size (16 nm) and they contain both spherical as well as cuboid shapes (Fig. 2F). Also it was noticed that the smaller particles were not present. Detail study about formation of bigger size particles is given in SI2. Interestingly, highly monodispersed spherical Fe 3 O 4 MNPs of bigger size (14 ± 1) are obtained when heated at a slower rate of 1.5 °C/min (i.e., instead of 4 °C/min) to reach 320 °C (Fig. 2G). As can be seen from the results, in order to prepare uniform spherical Fe 3 O 4 particles, a very slow heating rate of 1.5 °C per minute is essential and also, different shape and size of Fe 3 O 4 particles can be prepared using this method.
Increase in the size of the Fe 3 O 4 particles can be explained based on Ostwald ripening (Fig. 2H), in which the smaller particles get deposited on the surface of larger particles to gain stability by lowering surface energy 23,24 . When ferric oleate solution is added to the pristine particles followed by heated to the reaction temperature for longer times, small particles are formed which subsequently get deposited on the already present larger particles. The larger particles act as nucleation sites for newly formed cluster/molecules. Change of shape of particle from spherical to cuboid can't be explained purely on the basis of Ostwald ripening. It is obvious that some other factors also start playing important role in governing the shape of particles. A possible explanation is given below for transformation of spherical to cuboid shape. Upon heating the pristine spherical nanoparticles in presence of ferric oleate solution, newly formed smaller particles from ferric oleate preferably deposit on larger spherical nanoparticles on a specific plane that is easy to deposit and has less surface energy (γ). The sequence of planes in terms of surface energy is γ (111) < γ (100) < γ (110) < γ (220) in face-centered cube (fcc). Also, 〈111〉 direction has more magnetization than other directions in fcc Fe 3 O 4 unit cell 25 . In such a situation, newly formed nanoparticles prefer to deposit on the plane (111) (i.e., growth rate is faster along 〈111〉 direction) and thus, the cubic shaped particles are formed, when amount of ferric oleate added is 1 mL. It has been reported that a faster growth rate along 〈111〉 direction over 〈100〉 direction leads to cubic shaped particles 25 . The formation steps are shown in Fig. 2H(1-5). Upon increasing the amount of ferric oleate to 5 ml, formation of a mixture of spherical and cuboid shaped particles was observed. This may be due to the inhomogeneous deposition of smaller particles over larger particles in terms of direction (〈111〉 or 〈100〉) when amount of newly formed smaller particles from ferric oleate is comparable to that of the larger particles already present.

Functionalization of nanoparticles.
Three different protocols are used here for making water dispersible, monodispersed Fe 3 O 4 nanoparticles, which may have potential for application in drug delivery, hyperthermia treatment, etc. [26][27][28][29][30][31][32] . These protocols open an easy way to convert particles to another functional group so that it can be utilized for different applications (SI3). Amount of oleic acid on the surface of the particles and iron were calculated theoretically (see SI4) using the cubical or spherical model of the particles. The theoretically calculated value is in close agreement with the experimentally measured thermogravimetric analysis (TGA) value. The contribution of oleic acid or oleate in oleic acid coated Fe 3 O 4 is about 24 wt.% in TGA. Exothermic reaction in differential thermal analysis (DTA) suggests evolution of different gases (e. g., CO 2 , CO, C x H y ) which results into simultaneous oxidation and reduction reactions during formation of Fe 3   Hyperthermia study. For typical demonstration of heating efficacy, the Fe 3 O 4 particles coated with aspartic acid molecules are dispersed in water; and the dispersed particles (5 or 10 mg per 1 mL) are kept in induction coil (frequency = 265 kHz, current = 300 or 400A). 10 mg/mL of pristine Fe 3 O 4 particles (7 nm) produce 31-33 °C within 600 seconds at 400 A (335 Oe) ( Fig. 4(a)), but could not reach hyperthermia temperature (HT = 42 °C). Similarly, 5 mg/mL of bigger particles (cuboid shaped nanoparticles, 1 ml for 1 h) at 400 A could not reach HT Scientific REPORTS | (2018) 8:14766 | DOI:10.1038/s41598-018-32934-w ( Fig. 4(b)). But, 10 mg/mL at 300 and 400A could reach HT in 1137 and 255 s, respectively. In another case (bigger particles, 5 mL for 1 h, 5 and 10 mg/ml could reach HT at 400 A ( Fig. 4(c)) within 337 and 270 s, respectively. 5 and 10 mg/ml of the bigger particles (5 ml for 8 h case) reach HT at 400A at 245 and 170 s, respectively ( Fig. 4(d)). These results suggest that the time required for reaching HT decreases with increasing particle size and magnetic field. Specific absorption rates (SAR) of pristine particles (0 mL, 1 h), bigger particles synthesized by (1 mL, 1 h), (5 mL, 1 h) and (5 mL, 8 h) are found to be 4.1, 42.7, 30.9 and 46.5 Wg −1 , respectively and here, SAR is expressed in term of Watts per 1 g of Fe 3 O 4 . In our earlier studies 6,21 , SAR values of 30-40 Wg −1 for agglomerated Fe 3 O 4 particles (size 10-12 nm) were found under similar conditions of magnetic field and AC frequency. Thus, change of morphologies has made a large change in magnetic properties. Considering nanoparticles of same volume, there is difference in magnetic anisotropy between the cuboid and spherical shaped nanoparticles. Coercivity of cuboid shaped particles is higher than that of spherical particles (see SI5). Cuboid shaped magnetic nanoparticles have large magnetic anisotropy constant (K) than the spherical shaped magnetic nanoparticles. The increase in heating in cuboid shaped particles is due to increase in magnetic anisotropy. SAR value increases by increase of particle size or change of shape from spherical to cuboid. Thus, magnetic nanoparticles will be useful in heat generation for hyperthermia based cancer therapy in economic ways (lower amount of materials, easy processing/dispersion, reduced current, time, etc.). The heat generation from magnetic fluid under AC magnetic field comes from relaxation phenomena and hysteresis loop 5,6,20,33 . Relaxation phenomena include Brownian motion of particles with liquid medium and single domain relaxation (10 −5 -10 −10 s −1 ) and hysteresis loss arises during AC frequency (265 kHz) and applied magnetic field (see SI6). Relaxation phenomena are dominating factor in case of smaller particles sizes, whereas hysteresis loss is dominating factor in case of bigger particles. For comparison, heating behaviors of other particles coated with thioglycolic acid as well as aminophosphonate are shown in Fig. 4(a). Variation in heating is due to the different amounts of capping agents present over the surface of particles.
Zeta-potential study. Zeta-potential possessed by the particles dispersed in water is measured at pH 5, 6, 7 and 8. The zeta-potential values of thioglycolic acid, aspartic acid and aminophosphonate functionalized particles (core: 7 nm) at pH = 5 are −28.1 ± 1, −5.1 ± 0.6 and −25.5 ± 0.1 mV, respectively. With increase of pH up to 8, The zeta-potential measurements for the Dulbecco's Modified Eagle Medium (DMEM) and the DMEM with serum are performed and found to be value of −8.1 ± 0.2 and −5.9 ± 0.5 mV, respectively. The zeta-potential measurements of the nanoparticles functionalized with thioglycolic acid, aspartic acid and aminophosphonate functional groups are also performed in these two media. The zeta-potential value of the thioglycolic acid functionalized particles in the DMEM is found to be −13.6 mV. But, its value in the DMEM containing serum is +2.5 mV. For MNPs functionalized with aspartic acid, corresponding zeta-potential values in DMEM and DMEM with serum are −11.5 and −0.03 mV, respectively. Zeta-potential values of MNPs functionalized with aminophosphonate in the DMEM and the DMEM with serum are −3.0 and −9.2 mV, respectively.
The MNPs are found to be stable in the cell-culture medium (DMEM) with serum for more than three hours. The stability charts of the MNPs aspartate dispersed in water and DMEM with serum are shown in the supporting information (see Figs S39 and S40). The possible reason for this stability is due to steric stabilization 34 . In general, the MNPs having positive charge on the surface are more prone to internalize the cell than MNPs having negative charge on the surface. However, there are reports of internalization of negatively charged nanoparticles also in literature 35 . The internalization of negatively charged nanoparticles is believed to occur through nonspecific binding and clustering of the particles on cationic sites on the plasma membrane (that are relatively scarcer than negatively charged domains) and their subsequent endocytosis. Our fluorescence imaging results showed significant uptake of MNP-aspartate-FITC in the MCF-7 cells. MNP-aspartate-FITC has a zeta potential of −0.03 mV (close to net zero charge) in presence of DMEM with serum (pH = 7.4), as compared to its zeta potential of −26 mV in distilled water at pH = 8. This significant reduction in negative zeta potential may be due to the interaction of these MNPs with the serum proteins. Probably, such reduced negative charge on MNPs may help in internalization of MNPs on the MCF-7 cells. MCF-7 cells have the zeta potential of −20 ± 0.4 mV 36 . However, the detailed mechanism of internalization needs to be further studied.
Internalization study of magnetic nanoparticles in human breast adenocarcinoma cells. MCF-7 cells were treated with magnetic nanoparticles functionalized with different groups (viz., aspartic acid, thioglycolic acid and aminophosphonate). Using Prussian blue staining technique, blue coloured spots corresponding to presence of magnetic nanoparticles could be observed on the surface and inside the tumors cells, which again varied depending on nature of functionalized groups. These images indicate the internalization of magnetic nanoparticles in the tumor cells. It is interesting to observe that magnetic nanopaprticles functionalized with thioglycolate show faint staining in most of the tumor cells. Compared to this formulation, nanoparticles functionalized with aminophosphonate show more clustered localization in cell culture and the corresponding images are shown in Fig. S41. However, nanoparticles functionalized with aspartic acid show higher interaction and uptake in tumor cells resulting in darker blue spots. The differential staining and localization of nanoparticles functionalized with different groups may be associated with low Fe content or more homogenous distribution of MNPs in tumor cells. To further validate the higher intracellular uptake of MNP-aspartate in MCF-7 cells by Prussian blue studies, MNP-aspartate was labeled with a fluorescent dye (fluorescein iso-thiocyanate, FITC), followed by treatment of MCF-7 cells with 400 µl of MNP-aspartate-FITC. The cells were visualized by fluorescence microscopy. Green fluorescence is observed in the cytoplasm and near the surface of the cells. The nucleus stained with DAPI didn't show co-localization with MNP-aspartate-FITC as observed in the merged image (Fig. 5) suggesting that the MNPs are localized mainly in the cytoplasm and cell surface.

Cell cytotoxicity study of magnetic nanoparticles in human breast adenocarcinoma cells with or without AC magnetic field.
To determine the hyperthermia efficacy of MNP-aspartate in MCF-7 cells, they were treated with increasing concentrations of nano-formulation (1.5 and 2.5 mg/ml), followed by induction heating for 20 min and determination of cell cyto-toxicity by PI cell cycle analysis (Fig. 6). Results showed that as compared to only nanoparticles (8.5 ± 1.3% and 10.3 ± 0.6% cells in sub-G1 for 1.5 and 2.5 mg/ml MNP-aspartate treatments, respectively), MCF-7 cells treated with MNP-aspartate and hyperthermia therapy showed significant increase in % cells in sub-G1 phase of cell cycle (17.2 ± 1.2% and 23.02 ± 2.1% cells in sub-G1 for 1.5 mg/ ml + hyperthermia and 2.5 mg/ml + hyperthermia treatments, respectively). The histogram analysis of flow cytometry is provided in Fig. S42. These results indicate that MN-aspartate in combination with AC magnetic field induces significant cell cyto-toxicity by apoptosis, suggesting its potential for cancer hyperthermia therapy applications.

Cell culture experiments. Human Breast Adenocarcinoma cell line (MCF-7) was obtained from National
Centre for Cell Sciences, Pune, India. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; GIBCO, Invitrogen, Carlbad, CA, USA) supplemented with 10% fetal calf serum (FCS: Himedia Laboratories, Mumbai, India) and antibiotics (100 U ml −1 penicillin and 100 µg ml −1 streptomycin) in a humidified atmosphere of 5% CO 2 at 37 °C. For studying the intracellular uptake, MNP-aspartate were labeled with FITC, followed by testing its internalization efficacy in MCF-7 cells (protocol for FITC labeling is given in SI7.8). Briefly, MCF-7 cells (1 × 10 6 ) were seeded on glass coverslips for overnight at culture conditions, followed by treatment with MNP-aspartate-FITC for 3 h. The cells were then washed with PBS, followed by fixing in 4% paraformaldehyde for 20 min. at room temperature (RT). The cells were further washed with PBS and mounted on slide using Prolong Gold mounting media containing DAPI (Molecular Probes, USA). The cells were visualized by fluorescence microscopy under 40 X magnification.
For determining the hyperthermia efficacy of MNP-aspartate, 0.5 × 10 6 cells were seeded in 35 mm Petri-dishes for overnight at culture conditions, followed by treatment with different concentrations of MNP-aspartate (viz., 1.5 and 2.5 mg/ml) for 3 h. Further, the cells were subjected to hyperthermia treatment for 20 min, followed by further incubation at culture conditions for 24 h. The cell viability was determined by PI cell cycle analysis by flow cytometry. Briefly, the cells were harvested by trypsinization followed by washing with PBS. The cell pellet was fixed with ice cold absolute methanol and stored at −20 °C till further use. For flow cytometry analysis, the cells were first permeabilized (0.1% Triton X-100 and 1 mg/ml sodium citrate in PBS), followed by staining with PI [containing freshly added RNAse solution (50 µg/tube)]. The flow cytometry (Partec, Germany) analysis was carried out at 488 nm excitation and 585 nm emission wavelengths. 20,000 cells were analyzed and % cells in sub-G1 phase of cell cycle were determined by using Cyflogic software.

Synthesis of Fe 3 O 4 particles.
Reaction of ferric chloride with oleic acid at room temperature was used to prepare iron oleate. Iron oleate solution was slowly heated at the rate of 4 °C per minute to reach 320 °C in presence of 1-octadecene in inert atmosphere. Once the desired temperature was reached, it was maintained for 1 h. Change of brownish to black coloration was observed after about 30 minutes of heating suggesting formation of Fe 3 O 4 particles. The particles were characterized by Fourier transform infrared (FT-IR) spectroscopy, thermo-gravimetric analysis (TGA) and differential thermal analysis (DTA). The detailed procedures for synthesis have been given in SI1. Thus, oleic acid coated Fe 3 O 4 particles were prepared. These particles were found to be dispersible in chloroform and hexane etc., but not dispersible in water which can be ascribed to hydrophobic nature of the oleic acid capping. Amount of oleic acid present on the surface of the particles and iron were calculated theoretically (SI4) using the spherical and cuboid models of the particles. Different possible arrangements of COO − groups of oleate molecules are also given in detail in SI4. From TGA data, we found oleic acid contributed a minimum of ~24% of the particle's weight, thus functionalization reactions on the particles itself saved lots of reagent.
To make these particles water dispersible, we planned three different strategies. Here, methyl oleate was used as a model before trying it on oleic acid coated nanoparticles. As methyl oleate has no acidic functional group (-COOH), addition and cleavage reactions to the C=C of oleate can be carried out. Otherwise, this functional group will affect other mechanism and also, this model is almost the same with the particle system. The -COO − group of oleic acid is used to cap Fe 3 O 4 particles. Synthetic procedure for methyl oleate is given in SI7.1.
Addition of thioglycolic acid to the double bond of methyl oleate. About 1 g of methyl oleate was dissolved in 10 mL of hexane and was mixed with 62 mg (2 equivalents) of thioglycolic acid in isopropanol. It was exposed to the ultraviolet lamp of 15 W having emission maxima at 254 nm. The reaction was monitored with the help of thin layer chromatography (TLC). The products (P1 & P2) were purified using column chromatography and characterized by nuclear magnetic resonance (NMR) and FT-IR spectroscopy (SI7.2A). The yield of the reaction was ~90% and the typical reaction scheme is shown in Fig. 7a.
We performed similar reaction on the nanoparticles in which methyl oleate was substituted with oleic acid coated Fe 3 O 4 nanoparticles. The thioglycolic acid functionalized particles were separated by centrifugation and were washed with methanol. These particles were characterized by FT-IR spectroscopy (SI7.2B), and found to be water dispersible (Fig. 1iv-A).
Addition of aspartic acid to oxirane derivatives of methyl oleate. About 20 mg of the methyl oleate was dissolved in 20 mL of dichloromethane and this solution was cooled to 0 °C. Dried 3-chloroperbenzoic acid in dichloromethane was added drop-wise to the cooled solution with stirring. The reaction mixture was kept for 3 h stirring at the room temperature. The product was purified and characterized by FT-IR and NMR spectroscopy (SI7.3A). The yield of the reaction was ~80%. The obtained oxirane and aspartic acid (2 equivalents) were dissolved in dimethyl formamide (DMF) and heated to 60 °C with the addition of catalytic amount of boron trifluoride. The reaction was monitored using TLC (thin layer Chromatography) and it was found to be complete in 4 h. The product (Q) was purified and characterized by FT-IR and NMR spectroscopy. The yield of the reaction was ~70% and the typical reaction scheme is shown in Fig. 7b. Similar reaction was performed on the nanoparticles. The aspartic acid functionalized particles were washed with methanol to remove excess aspartic acid and separated by centrifugation. The particles were characterized by FT-IR spectroscopy (SI7.3B). These particles were found to be water dispersible (Fig. 1iv-B).
Synthesis of aminophosphonate from aldehyde obtained from methyl oleate. About 10 mg of methyl oleate was dissolved in 30 mL of dichloromethane and cooled to −50 °C to prevent explosion due to molozonide that is formed by the reaction of ozone on C-C double bond of methyl oleate. Maintaining this temperature, ozone gas was passed. After passing for one and half hour, blue colouration of the reaction mixture was observed. It indicated excess of ozone gas present in the reaction medium. Triphenyl phosphine (3 equivalents) was added to the reaction mixture along with passing of oxygen gas. The reaction mixture was kept stirring for overnight at room temperature. The product was purified using column chromatography and characterized by FT-IR and NMR spectroscopy (SI7.4A.1). The yield of the reaction was ~90%.
Using the aldehyde having ester group only, α-aminophosphonate of tertiary butyl amine and diethyl phosphite was synthesized by Kabachnik -Fields reaction. The product (R) was purified and characterized by FT-IR and NMR spectroscopy (SI7.4A.2). Detailed procedure is given in the supporting information. The typical reaction scheme is shown in Fig. 7c.
Similar reaction was performed on the particles. The amino-phosphonate functionalised particles were washed with ethyl acetate and collected with centrifugation. The particles were characterized with FT-IR spectroscopy (SI7.4B) and found to be water dispersible ( Fig. 1iv-C).

Synthesis of bigger Fe 3 O 4 particles.
To study the heating effect of different sizes and shapes of Fe 3 O 4 particles under AC magnetic field, bigger particles were prepared. A stock solution of 500 mL of ferric oleate in 1 -octadecene (0.16 g/mL) was prepared. About 1 mL of ferric oleate was added to the already synthesized particles that were synthesized starting from 10 mL of stock solution. Following the same procedure of particles synthesis, reaction mixture was heated for 1 h at 320 °C (heating rate of 4 °C per minute to reach 320 °C). In the second set, 5 mL of ferric oleate was added to the already synthesized particles and reaction mixture was heated for 1 h at 320 °C. The particles formed after addition of 1 and 5 mL of ferric oleate are designated as 1 mL and 5 mL, respectively. In the third set, 5 mL of ferric oleate was added to the already synthesized particles and reaction mixture was heated for 8 h at 320 °C. The size and morphology of the particles were investigated with TEM ( Fig. 2D-F).
Characterization. FT-IR spectra were recorded using Bomem spectrometer (Hartmann and Braun, MB -100 series). NMR data were recorded on a Bruker 400 MHz spectrometer using tetramethylsilane or H 3 PO 4 as the standard reference. X-ray diffraction (XRD) patterns were recorded using Rigaku Miniflex 600 machine and crystallite size (t) was calculated using the Scherrer equation t = (0.9 λ)/(B cosθ), where λ is the wavelength of Cu Kα, B the half width at maximum intensity and θ the Bragg's angle. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) data were recorded with TG -DTA -EGA Setaram (Setsys evolution) in an argon atmosphere in 20-600 °C. Transmission electron microscopic images of the particles were taken with 200 keV JEOL (HRTEM) (SI1).
The magnetization and magnetic hysteresis measurements were performed using a Physical Property Measurement System (PPMS) (Model: 6000, Quantum Design) equipped with vibrating sample magnetometer (VSM) option and a superconducting magnet producing fields up to ±9 Tesla. To carry out this measurement, the samples were packed in a pocket (capsule) made from polytetrafluoroethylene (PTFE) tape. The mass of the sample was chosen in the range of 8-12 mg in order to obtain a good signal-to-noise ratio. For zero field cooled (ZFC) measurement, the samples were at first cooled in zero magnetic field down to 5 K and then magnetizations were recorded by increasing the temperature in an applied magnetic field (i. e. 50 Oe). For field cooled measurement on warming (FC or FCW), samples were cooled down to 5 K in the presence of magnetic field and then magnetizations were recorded by increasing temperature in the presence of the same field (i.e. 50Oe). To obtain the field dependence of the magnetization (i.e. hysteresis loop), the samples were brought to a specific temperature and then the sample's magnetic moment, as a function of the magnetic field with field stabilization time 90 s, was recorded in the magnetic field range of ±9 Tesla.
For FT-IR, TGA-DTA and XRD experiments, pristine Fe 3 O 4 particles (prepared with a heating rate of 4 °C per minute to reach 320 °C) were used.

Conclusions
Monodispersed Fe 3 O 4 MNPs have been prepared from iron oleate. These particles have been made water dispersible by functionalization with different functional groups. Shape of particles was found to change from spherical to cuboid. The cuboid shape in turn enhances magneto-crystalline anisotropy (K u ). Particle size can be increased by heating pristine particles in presence of ferric oleate solution for different periods of time. The increase in size of particle has been explained by Oswald ripening mechanism. The time required to reach hyperthermia temperature changes with shape and size of particles. The MNPs functionalized with aspartic acid showed better interaction or internalization in human breast cancer cells and showed enhanced cell killing under AC magnetic field. These results suggest potential of these nano-formulations for cancer hyperthermia therapy applications. However, it needs to be further studied in detail.