shells for hyperthermia application: Improved colloidal stability and biocompatibility

Journal of Magnetism and Magnetic Materials 355 (2014) 22–30

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Superparamagnetic iron oxide/chitosan core/shells for hyperthermia application: Improved colloidal stability and biocompatibility R.M. Patil a, P.B. Shete a, N.D. Thorat a, S.V. Otari a, K.C. Barick b, A. Prasad b, R.S. Ningthoujam b, B.M. Tiwale a, S.H. Pawar a,n a b

Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416006, MS, India Chemistry Division, Bhabha Atomic Research Centre, Mumbai 400085, MS, India

art ic l e i nf o

a b s t r a c t

Article history: Received 25 September 2013 Received in revised form 9 November 2013 Available online 1 December 2013

Superparamagnetic magnetite nanoparticles are of great interest due to their potential biomedical applications. In the present investigation, Fe3O4 magnetic nanoparticles were prepared by alkaline precipitation using ferrous chloride as the sole source. An amphiphilic polyelectrolyte with the property of biocompatibility and functional carboxyl groups was used as a stabilizer to prepare a well-dispersed suspension of superparamagnetic Fe3O4 nanoparticles. The final material composed of Fe3O4 core and chitosan (CH) shell was produced. The amino groups of CH coated on Fe3O4 nanoparticles were further cross linked using glutaraldehyde (GLD) for stable coating. FTIR spectra, XPS and TGA confirmed the coating of CH/GLD on the surface of Fe3O4 nanoparticles. XRD patterns indicate the pure phase Fe3O4 with a spinel structure. The nanoparticles were superparamagnetic at room temperature with saturation magnetization values for bare and coated nanoparticles which were 51.68 emu/g and 48.60 emu/g, respectively. Zeta potential values showed higher colloidal stability of coated nanoparticles than the bare one. Cytotoxicity study up to 2 mg mL
1 concentration showed no drastic change in cell viability of nanoparticles after coating. Also, coated nanoparticles showed increased SAR value, making them suitable for hyperthermia therapy application. & 2013 Elsevier B.V. All rights reserved.

Keywords: Fe3O4 Magnetic nanoparticle Chitosan Hyperthermia Cross linking

1. Introduction Magnetic nanoparticles (MNPs) are receiving increasing interest in recent years. Generally nanocrystals exhibit distinct properties from those in bulk. The physico-chemical properties are influenced by the shape and size of the nanocrystals. Nanocrystaline magnetite has controllable size and is much smaller than a cell. The size is comparable to proteins and other biomolecules. The MNPs can be modified for interaction with these biological entities. MNPs are potentially very useful in the transport and immobilization of magnetically tagged biological entities using an external magnetic field. Many MNPs are reported to be having good biocompatibility and low toxicity [1,2]. Due to these properties, they are used for various biomedical applications such as targeted drug delivery [3], hyperthermia treatment [4], magnetic resonance imaging contrast enhancement [5], magnetic separation of biomolecules [6] and cancer diagnosis [7]. Hybrid organic–inorganic materials are of current interest because of n Corresponding author at: Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416006, MS, India. Tel.: þ 91 0231 2601202; fax: þ 91 0231 2601595. E-mail address: [email protected] (S.H. Pawar).

0304-8853/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jmmm.2013.11.033

their multifunctionality, ease of processibility and potential for large scale manufacturing [8]. Biocompatibility of MNPs is also an important issue for their suitability for biomedical applications. Key to the successful application of MNPs is the surface functionalization. There are two general surface modification approaches taken [9]: (1) Non-covalent adsorption: This is a reversible approach in which molecules with functional groups such as carboxylic acids or phosphoric acids are attached to MNPs. This approach is conceptually simple. (2) Covalent linkages: Ligands are attached to MNPs through a covalent linkage in this approach. There are various ways to prepare Fe3O4 nanoparticles, which have been reported earlier, such as arc discharge [10], mechanical grinding [11], laser ablation [12], microemulsions [13] and high temperature decomposition of organic precursors [14]. These methods may be able to prepare magnetite with controllable particle diameters. However, well-dispersed aqueous Fe3O4 nanoparticles have met with very limited success. Chemical co-precipitation [15] method is a convenient and cheap method having the potential to meet the increasing demand for the direct preparation of well dispersed (water-base) Fe3O4 nanoparticles. It also offers a low-temperature alternative to

R.M. Patil et al. / Journal of Magnetism and Magnetic Materials 355 (2014) 22–30

conventional powder synthesis techniques in the production of nanoparticles. The sizes of nanoparticles can be well controlled by apt surfactant in this method [16,17]. Chemical coprecipitation can produce fine, high-purity, stoichiometric particles of single and multicomponent metal oxides. Furthermore, if process conditions such as solution pH, reaction temperature, stirring rate, solute concentration and surfactant concentration are carefully controlled, oxide particles of the desired shape and sizes can be produced [18]. Highly stable aqueous dispersions of iron oxide nanoparticles have been obtained by using polymers as the stabilizer [19,20]. The surface of the nanoparticles has to be modified with nontoxic and biocompatible stabilizers for practical biomedical applications of superparamagnetic iron oxide nanoparticles. Several natural polymers, such as pullulan [21], dextran [22], starch [23] and BSA [24], have been used as protective layers on the superparamagnetic iron oxide nanoparticle surface which can provide biocompatibility, high solubility and hydrophilic properties to the MNPs. Chitosan is a polyaminosaccharide with significant biological (biodegradable, biocompatible, and bioactive) and chemical (polycationic, hydrogel, contains reactive groups such as –OH and –NH) properties. In addition, chitosan is an abundant, renewable, nontoxic, and biodegradable carbohydrate polymer and is obtained from the exoskeletons of shellfish and insects. Hence, chitosan has attracted a great deal of attention as a functional biopolymer for a wide variety of applications, especially in pharmaceutics, food and cosmetics [25]. Iron oxide nanoparticles due to good biocompatibility, superparamagnetism, low toxicity, high electron transfer capability and high adsorption ability have been used as an immobilizing matrix for application to biosensors. Further, Fe3O4 nanoparticles exhibit interesting properties like high surface area, lower mass transfer resistance and selective separation for immobilized biomolecules from a reaction mixture on the application of magnetic field. However, aggregation of Fe3O4 nanoparticles due to high surface area and magnetic dipole interaction between nanoparticles has limited their biomedical applications. This problem can perhaps be overcome by dispersing Fe3O4 nanoparticles in a biopolymer matrix of chitosan (CH) to prepare CH–Fe3O4 hybrid nanobiocomposite [26]. In the present paper, Fe3O4 nanoparticles were prepared using FeCl2 as the sole precursor [26a]. The as-prepared MNPs were further coated with chitosan (CH). Physically adsorbed CH was then cross-linked using glutaraldehyde (GLD). Fe3O4 nanoparticles and CH-coated Fe3O4 nanoparticles (Fe3O4–CH/GLD) were thoroughly studied for their structural, morphological and magnetic characterizations and the effect of CH coating on the Fe3O4 nanoparticles was observed. The particles were further studied for their capacity of heat generation by magnetic reversal losses to understand possible use in hyperthermia treatment as a biomedical application.

2. Materials and methods Ferrous chloride (FeCl2  4H2O), hydrochloric acid, glacial acetic acid and sodium hydroxide (NaOH) were procured from HiMedia, India. Chitosan and glutaraldehyde (25%) were purchased from Sigma-Aldrich, USA. Double distilled water was used throughout the procedure. Magnetite nanocrystals were prepared using FeCl2  4H2O. In brief, 2 g FeCl2  4H2O was dissolved in 30 mL 1 M HCl. To this, 30 mL 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 was obtained. The precipitate was then separated out using external magnetic

23

field and dried at 100 1C. The as prepared nanoparticles were further used for the coating procedure. The possible reaction taking place is as shown below: 3FeCl2 4H2 O þ 6NaOH þ 12 O2 -Fe3 O4 þ 6NaCl þ15H2 O

ð1Þ

The obtained bare Fe3O4 nanoparticles were then coated with chitosan using the ultrasonication method. In brief, the obtained MNPs (1 g) were dispersed in 50 mL distilled water by ultrasonication for 20 min. This suspension was then added to 100 mL 1% chitosan (in 2% acetic acid) solution. Then the mixture was ultrasonicated for 30 min. After that, it was washed three times to remove excess CH and allowed to settle down using an external magnetic field. The chitosan coated MNPs were collected and dried at 50 1C. The CH coated nanoparticles (200 mg) were then dispersed in 25 mL of doubled distilled water and 10 mL of GLD was added to it and mechanically stirred for 8 h. The mechanical stirring was enough to crosslink CH. These resulting nanoparticles were washed with ethanol and water three times.

3. Characterizations The X-ray Diffraction (XRD) pattern of drop coated and airdried MNPs on the glass substrate was recorded to study the structural and phase analysis by an XRD Philips PW-3710 diffractometer using Cr Kα radiation (λ¼ 2.2897 Å) in the 2θ range from 201 to 1001. The XRD patterns were evaluated by the X′pert high score software and compared with the JCPDS card. The average crystallite size (t) was calculated from the diffraction line-width of XRD pattern, based on Scherrer's relation: t ¼ 0:9λ=β cos θ;

ð2Þ

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 a Perkin-Elmer spectrometer, (Model no.783, USA) in the range 450–4000 cm
1 using KBr pellets to check the possible interaction of Fe3O4 with chitosan. The compositional analysis was done by energy-dispersive analysis of X-ray spectroscopy (EDAX, JEOL JSM 6360). Scanning Electron Microscopic (SEM) and Transmission Electron Microscopic (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 a Philips CM200 model Transmission Electron Microscope, which had an operating voltage of 20–200 kV with resolution of 2.4 Å. The magnetization measurements were performed on a Superconducting Quantum Interference Device (SQUID) magnetometer to investigate the saturation magnetization (Ms), blocking temperature (TB) and the Curie temperature (TC). The M–H measurements were carried out at two different temperatures 100 and 300 K with applied field range from 0 to 72  104 Oe (2 T). Zeta potential and hydrodynamic diameter of particles were measured using a PSS/NICOMP 380 ZLS particle sizing system (Santa Barbara, CA, USA) with a red He–Ne laser diode at 632.8 Ǻe in a fixed 901 angle plastic cell. The zeta potential measurements were performed at 25 1C after a temperature homogenization time of 5 min. The measurements were carried out at different pHs 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 75 mV). Since the functionalized MNPs are to be used for biomedical applications, the issue of cytotoxicity has to be addressed. The

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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 [29–31]. The L929 cell line (mouse fibroblast) 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 the MTT assay. The L929 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) 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 1C in a 5% CO2 atmosphere. The cells were incubated with the concentration of 1  104 cells mL
1 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 1C in a 5% CO2 atmosphere for 24 h. After 24 h, 10 mL MTT solution was added into each well including control wells. The plates were incubated for 3 h at 37 1C 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 mL acidic isopropanol and finally the absorbance is read at 570 nm and from it the cell viability is calculated. The experiments were repeated three times and the data was graphically presented as the mean. Heating by magnetic reversal losses 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 an ambient temperature, a provision of water circulation in coils was provided. MNPs suspended in 1 ml of distilled water were placed at the centre of the coil and the applied frequency was 265 kHz. Particles were dispersed in water with a concentration ranging 2, 5 and 10 mg mL
1 and ultrasonicated for 20 min to achieve a good dispersion of the nanoparticles in carrier fluid. Samples were heated for 10 min with the desired current (200–400 Å). For the conducted experiments, the magnetic field was calculated from the relationship: H¼

1:257ni in Oe L

ð3Þ

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 Å were 167.6, 251.4 and 335.2 Oe (equivalent to 13.3, 20.0 and 26.7 kA m
1), respectively. Real field measurements were also taken using a digital guassmeter (Model: DGM-102, SES Instruments Pvt. Ltd., Roorkee) for the same set of current values and were recorded as 163.42, 251.4 and 326.85 Oe respectively. Temperature was measured using an optical fibre probe with an accuracy of 0.1 1C.

4. Results and discussion Fig. 1 shows the powder XRD patterns for the as-prepared and the CH-coated Fe3O4 MNPs, cross-linked with GLD. The XRD patterns revealed that the desired phase formation occurs in the samples. 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 the Fe3O4 phase. The particles were of inverse spinel structure. The Gaussian fit of the most intense peak (311) was used to calculate the full width at half maxima for determination of crystallite size (t) by the equation t¼0.9λ/β cos θ, where

Fig. 1. XRD patterns of (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs.

Fig. 2. FTIR spectra of (a) Fe3O4 MNPs, (b) Fe3O4–CH/GLD MNPs and (c) CH.

λ¼2.2897 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. From the XRD patterns of both samples, the reflection peaks are quite broad, suggesting their nanocrystallinity. The average crystallite size of bare Fe3O4 MNPs was found to be 14 nm and 11 nm for CH-coated MNPs. Fig. 2 shows the FTIR spectra of Fe3O4 and Fe3O4–CH/GLD MNPs along with CH over the range of 450–4000 cm
1. The band observed at 565 cm
1 corresponds to the intrinsic stretching vibration (Fetetra–O) of metal–oxygen at tetrahedral site, whereas the band observed at 455 cm
1 corresponds to the stretching vibration (Feocta–O) of metal–oxygen at octahedral site. The bands observed at 3376 and 1620 cm
1 corresponds to surface-adsorbed water molecules on Fe3O4. The bands observed at 634, 692 and 1403 cm
1 in the spectrum of CH corresponds to the bending vibration of C–H and the band observed at 1627 cm
1 corresponds to N–H bending vibration which showed efficient coating of CH on Fe3O4 MNPs. In the spectrum of Fe3O4–CH/GLD, the bands observed at 1020 and 1107 cm
1 relate to C–O stretching while 1377 and 1459 cm
1 relate to C–H bending. The bands observed at 2854 and 2924 cm
1 relate to C–H stretching vibrations. The broad band observed at around 3391 cm
1 corresponds to stretching vibration of N–H and O–H [27]. The band observed at 1624 cm
1 corresponds to imine (C ¼ N) confirmed the crosslinking of CH with GLD. No band is observed at 1720–1730 cm
1

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Fig. 3. Thermogravimetric spectra of (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs in nitrogen with a scanning rate of 10 1C min
1 up to 600 1C.

which showed glutaraldehyde crosslinks chitosan by the Schiff base formation [28]. Fig. 3 shows thermogravimetric (TG) curves of uncoated and CH/GLD coated Fe3O4 nanoparticles measured by a thermogravimetric analyzer. As TG was performed under N2 atmosphere, the oxidation of MNPs was greatly reduced. It provides additional quantitative evidence on the structure of coating on surface of nanoparticles. The 3.5% weight loss due to evaporation of physically adsorbed water in the temperature range below 140 1C was observed for both samples. Three major weight loss stages are observed in thermogram for Fe3O4–CH/GLD, one below 140 1C which can be ascribed to evaporation of water, while the other one beginning at about 170 1C due to the decomposition of CH. This pattern is in good agreement with patterns reported in literature for CH coating [32]. The third stage begins at about 300 1C was due to the decomposition of GLD. The differences in total weight loss for both samples help us to calculate the percentage CH/GLD molecules attached to the surface of nanoparticles. Thus it is assumed that about 70% of CH/GLD was adsorbed on to the surface of nanoparticles. Above the temperature of 500 1C both samples attain stability in terms of weight loss. For further confirmation, X-ray photoelectron spectroscopy (XPS) of coated MNPs was used. XPS spectra of the sample are shown in Fig. 4. C 1s peak at 286.26 eV (Fig. 4(a)) was due to the spectrometer rotary pump oil that inevitably contaminates the samples at working pressure (1  10
5 Pa). The survey scan spectra also confirmed O 1s peak (Fig. 4(b)), which is at about 529.12 eV, corresponding to oxide oxygen. Fig. 4(c) shows Fe 2p core-level spectrum of the coating. The intensity of the main-peak shoulder of Fe 2p3/2 at 709.16 eV, is characteristic of Fe2 þ ions. The binding energy at 710.5 eV for the Fe 2p3/2 main peak is consistent with typical values for ferric oxides [33]. The double peaks are broadened due to the appearance of Fe2 þ (2p3/2) and Fe2 þ (2p1/2), in agreement with the literature that peaks broaden for Fe3O4 on the appearance of Fe2 þ (2p3/2) and Fe2 þ (2p1/2). This phenomenon confirms the product is Fe3O4 rather than γ-Fe2O3 [34]. Presence of N 1s peak at 399.58 eV confirms the presence of CH on the surface of MNPs. Fig. 5 shows SEM images of Fe3O4 (a) and Fe3O4–CH/GLD (b) MNPs. The images clearly show higher degree of agglomeration in bare Fe3O4 MNPs compared to Fe3O4–CH/GLD MNPs. This is because of reduction in dipole–dipole interaction in coated nanoparticles due to the presence of organic layer. The particles are spherical in shape as shown by the images. The EDAX spectra were used for quantitative elemental analysis of bare and CH-coated Fe3O4 nanoparticles, which are shown in

25

Fig. 6(a) and (b) respectively. The corresponding peaks in bare nanoparticles are due to Fe and O only, while CH-coated nanoparticles 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. The details of elements analysis is given in Table 2. Fig. 7 represents the TEM images for both uncoated and CH/ GLD coated Fe3O4 nanoparticles. Fig. 7(a) clearly shows the formation of spherical nanoparticulates with sizes 20.5 73.8 nm and 22.2 73.4 nm respectively. From Fig. 7(b), one can see that the CH/GLD coated Fe3O4 nanoparticles are spherical and have good dispersibility (non-agglomerated) as compared to uncoated nanoparticles. The average particulate diameter is found to increase after functionalization with CH/GLD molecules. Improvement in dispersibility after coating with CH/GLD may be attributed to the presence of the non-magnetic surface layer of CH/GLD 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. 7 (insets) show bright ring patterns indicating polycrystalline nature of the MNPs, as indicated by XRD patterns. The ring patterns correspond to (220), (311), (400), (422), (511) and (440) planes which can be clearly seen in XRD results. In order to use these nanoparticles for in vivo biomedical applications, they must be in the form of aqueous colloidal suspensions. It is important to study surface chemistry and stability of the particles in water as a function of pH to accomplish this requisite. Electrophoretic mobility data was transformed to zeta potential values, which were related to the surface charge density and depend on the oxide composition, crystalline form, size and surface characteristics [35]. Suspensions of superparamagnetic Fe3O4 nanoparticles have van der Waals forces and magnetic dipole–dipole interactions generated from residual magnetic moments, which tend to agglomerate the particles. Therefore repulsive forces are required to keep each particle discrete and prevent it from amassing as larger and faster setting agglomerates. Steric hindrance plays an important role in stabilizing suspensions, which is accomplished by the protective shields on the oxide surface produced by molecules or polymers [36]. The zeta potential values and hydrodynamic diameters of bare and coated nanoparticle suspensions in water with respect to pH are shown in Fig. 8. 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 40.13, 27.69, 6.90,
20.99 and
28.52 mV, respectively, the zeta potential of coated particles is more positive in the range of pH 2–4, as compared to bare indicating that the positive charges on the Fe3O4–CH/GLD nanoparticles increase with a decrease in pH, may be due to the protonation of free amino groups at low pH. The protonated amino groups provide enough charge to stabilize coated nanoparticles at pH 2 and 4. At pH 6, zeta potential for coated nanoparticles is lower because the isoelectric point of chitosan is 6.3 [25], and the suspension at pH 6 is stabilized by the steric stabilization mechanism. At higher pH, zeta potential of bare nanoparticles is more negative than coated. The isoelectric points (pIs) for bare and coated nanoparticles were found to be around 6.7 and 6.4 respectively. The bare nanoparticles possess negative charge at physiological pH which was in agreement with the literature [37]. Dynamic Light Scattering (DLS) measurements were carried out to investigate the hydrodynamic size of bare and CH/GLD–Fe3O4 nanoparticles. The hydrodynamic diameter (number-weighted) distribution of unmodified and CH/GLD stabilized Fe3O4 nanoparticles at a scattering angle of 901 is shown in Fig. 8. The bare nanoparticles showed higher particle size as compared to coated ones due to the formation of agglomerates by the virtue of dipole– dipole interaction. The particle size of coated nanoparticles was in

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Fig. 4. The XPS of Fe3O4–CH/GLD nanoparticles: (a) peak for C, 1s, (b) peak for O, 1s, (c) details of the Fe 2p1/2 and Fe 2p3/2 peaks and (d) peak for N, 1s that implied the presence of coating on the surface of Fe3O4 nanoparticles.

Fig. 5. SEM images of (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs.

Fig. 6. EDAX spectra of (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs.

R.M. Patil et al. / Journal of Magnetism and Magnetic Materials 355 (2014) 22–30

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Fig. 7. TEM images of the (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs with respective SAED patterns in insets.

Fig. 8. Hydrodynamic diameter and zeta potential as a function of pH for (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs dispersed in water.

Fig. 9. M–H curves of Fe3O4 and Fe3O4–CH/GLD MNPs at (a) 100 K and (b) 300 K.

good agreement with the TEM and zeta potential results; the size was reduced by the repulsive forces acted on the particles due to the formation of electrostatic and steric interactions of the coated material. To understand the effect of coating on the magnetization behavior of Fe3O4 system we have carried out the M vs H measurements as a function of applied field and temperature. Fig. 9(a) and (b) shows the M–H curves of both samples (in powder form) at 100 and 300 K. 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. 9 that magnetization decreased with coating of CH/GLD. This is because magnetization is proportional to the amount of weight for the same magnetic material. Organic coating (CH/GLD) layers on magnetic material increases the amount of non-magnetic substance which reduces the overall magnetization of the material . Fig. 10 shows the temperature susceptibility spectra of Fe3O4 nanoparticles and Fe3O4–CH/GLD nanoparticles. The sudden drop

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R.M. Patil et al. / Journal of Magnetism and Magnetic Materials 355 (2014) 22–30

in magnetic susceptibility at  580 1C confirms the presence of Fe3O4 in both samples. It is the Curie temperature of Fe3O4. The Curie temperature of maghemite is higher than 600 1C [38,39] which confirms the presence of magnetite in the final product of the reaction. Fig. 11(a)–(c) represents the temperature kinetic curves obtained after application of an alternating magnetic field on both samples which are dispersed in water with concentration of 2 mg mL
1 and (d) SAR vs applied magnetic field for both samples. Temperature kinetic curves represent that rise in temperature is dependent on the applied magnetic field for both samples. For superparamagnetic nanoparticles the greatest relaxation losses are due to the Brownian modes (heat due to friction arising from total particle oscillations) and the Neel modes (heat due to rotation of the magnetic moment with each field oscillation). The heat dissipation by superparamagnetic nanoparticles is given by the following equation: P ¼ μ0 πχ″f H 2

ð4Þ

where P is the heat dissipation value, μ0 is the magnetic field constant, χ″ is the AC magnetic susceptibility (imaginary part), f is the frequency of the applied AC magnetic field, and H is 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 μ0Hmax, the amount of heat A released by the MNPs Table 1 The saturated magnetization (Ms), coercivity (Ce) and remanence (Mr) values calculated from the M–H curves for both bare and CH/GLD coated Fe3O4 MNPs. Fe3O4–CH/GLD

Fe3O4

Ms (emu/g) Mr (emu/g) Hc (Oe) Mr/Ms

100 K

300 K

100 K

300 K

55.74 13.91 160.82 0.25

51.68 2.23 16.85 0.04

52.30 13.46 151.55 0.26

48.60 2.69 17.28 0.05

Table 2 Elemental analysis for both bare and CH/GLD coated Fe3O4 MNPs. Element

Fe O C N

At% Fe3O4

Fe3O4–CH/GLD

85.43 14.67 0.00 0.00

53.26 23.12 19.88 4.74

during one cycle of the magnetic field simply equals the area of their hysteresis loop, which can be expressed as Z A¼

þ H max
H max

μ0 MðHÞdH

ð5Þ

Then the SAR is SAR ¼ Af

ð6Þ

where f is the frequency of the AC magnetic field and it is expressed as f¼ω/2π, M is the magnetization and H is the applied magnetic field. The heat loss due to eddy currents (ED) is given by the following equation: ED ¼

ðμπdf HÞ2 20ρ

ð7Þ

where μ is the permeability of a material, d is the diameter of the particle and ρ is resistivity of the material. The specific absorption rate (SAR) is calculated by using following relation: SAR ¼ c

ΔT 1 Δt mmagn

ð8Þ

where c is the sample-specific heat capacity, which was calculated as a mass weighted mean value of MNPs and water. The heatcapacity of both samples is negligible because of its low concentration, and thus the heat capacity of water (4.18 Jg
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 to 1–5 min to calculate the slope. The value of mmagn is considered as the amount of MNPs per total amount of MNPs and water. The estimated SAR values from Fig. 11(a)–(c) and by using Eq. (8) for both the samples are graphically represented in Fig. 11(d). In this paper, it is demonstrated experimentally that the hyperthermia effect of Fe3O4 nanoparticles enhances dramatically after functionalization with CH/GLD. The first possible reason for the enhanced hyperthermic effect after coating is that the ability of the CH/GLD coating to retain the superparamagnetic fraction of Fe3O4 is much better as compared to Fe3O4 alone. The second is that coating layer prevents the formation of larger aggregates of Fe3O4 (also confirmed by DLS results) which makes better suspension of CH/GLD functionalized nanoparticles in 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 [29]. Thus the hyperthermia study also strongly supports the coating of CH/ GLD on Fe3O4 nanoparticles which prevents particle agglomeration.

Fig. 10. The temperature susceptibility graphs of (a) Fe3O4 and (b) Fe3O4–CH/GLD MNPs.

R.M. Patil et al. / Journal of Magnetism and Magnetic Materials 355 (2014) 22–30

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Fig. 11. (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 and (d) SAR vs applied magnetic field for both samples.

coated Fe3O4 nanoparticles. Over 93% and 94% cell viability was still obtained after 24 h incubation with 2 mg mL
1 concentration of bare Fe3O4 and coated nanoparticles respectively. This shows that the coating of nanoparticles did not affect cytotoxicity much even up to 2 mg mL
1 concentration after 24 h. In the present paper this much concentration for both cytotoxicity and heating induction ability is studied in order to use it for hyperthermia therapy application. We have mentioned possible mechanisms of interactions of nanoparticles with the cells in our earlier report [29].

5. Conclusions

Fig. 12. Cytotoxicity profiles of MNPs for 24 h on the L929 cell line at different concentrations (0.1, 0.5, 1.0, 1.5 and 2.0 mg mL
1).

The cytotoxicity study of both, bare and coated nanoparticles, was done on L929 cell line with different concentrations of nanoparticles and the obtained data is shown in Fig. 12. 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 1C in a 5% CO2 atmosphere. The relative cell viability (%) compared with the control well containing cells without nanoparticles is calculated by the equation: [A]tested/[A]control  100. Fig. 12 shows the cell viability after incubation with different concentrations of both bare and

The focus of this work is to develop a simple technique for the preparation of Fe3O4 nanoparticles and demonstrating their applicability in the biomedical field. From the present study, it is concluded that pure and stable phase of Fe3O4 nanoparticles can be obtained using FeCl2 as the sole source and without using any other oxidant. Hence the conventional method is more simplified and made cost-effective in the present work. The obtained particles are of polycrystalline nature. TEM images showed that the particles are round-shaped having a size 20.5 73.8 nm and 22.2 73.4 nm in cases of bare and coated particles respectively. The resulting Fe3O4 nanoparticles were superparamagnetic at room temperature with a high saturation magnetization value. CH can be efficiently coated on the surface of Fe3O4 nanoparticles using a simple technique of ultrasonication. Cross-linking of amino groups in CH using GLD increases the stability of coating. Zeta

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potential and DLS measurements of both the particles prove to have 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 and cytotoxicity studies are highly encouraging due to monodispersivity, high saturation magnetization values, high SAR values and low cytotoxicity. Acknowledgemnts The authors are thankful to Dr. S.D. Sartale, Pune University, Pune (MS), India for providing SEM images and EDAX patterns. They are also thankful to Dr. N. Basavaiah and their students, IIG, Mumbai (MS), India for temperature susceptibility measurements. The authors are also grateful to Dr. T.R. Waghmode, Shivaji University, Kolhapur (MS), India for providing TGA results of the samples. They are also thankful to Dr. D. Das and Dr. R.K. Vatsa, Chemistry Division, BARC for providing induction heating facilities. The M–H measurements were performed at magnetism laboratory under the supervision of Dr. Alok Banergee and Dr. R.J. Choudhary in the UGC-DAE Consortium for Scientific Research, Indore. TEM images were obtained from SAIF, IIT, Mumbai. The authors are thankful to them. The authors are grateful to DST, DAE-BRNS, India for their financial support. The authors are thankful to the National Toxicology Center, Pune and the National Center for Cell Science, Pune (MS), India for cytotoxicity study. References [1] J.M. Perez, T. O′Loughin, F.J. Simeone, R. Weissleder, L. Josephson, J. Am. Chem. Soc. 124 (2002) 2856–2857. [2] A. Dyal, K. Loos, M. Noto, S.W. Chang, C. Spagnoli, K.V. Shafi, A. Ulman, M. Cowman, R.A. Gross, J. Am. Chem. Soc. 125 (2003) 1684–1685. [3] M. Ferrari, Nat. Rev. Cancer 5 (2005) 161–171. [4] A. Ito, H. Honda, T. Kobayashi, Cancer Immunol. Immunother. 55 (2007) 320–328. [5] Y.M. Huh, Y.W. Jun, H.T. Song, S.W. Kim, J.S. Choi, J.H. Lee, S. Yoon, K.S. Kim, J.S. Shin, J.S. Suh, J. Cheon, J. Am. Chem. Soc. 127 (2005) 12387–12391. [6] J.M. Perez, F.J. Simeone, Y. Saeki, L. Josephson, R. Weissleder, J. Am. Chem. Soc. 125 (2003) 10192–10193. [7] Y.W. Jun, Y.M. Huh, J.S. Choi, J.H. Lee, H.T. Song, S. Kim, S. Yoon, K.S. Kim, J.S. Shin, J.S. Suh, J. Cheon, J. Am. Chem. Soc. 127 (2005) 5732–5733.

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