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Synthesis of functionalized Co0.5Zn0.5Fe2O4 nanoparticles for biomedical applications
Journal of Magnetism and Magnetic Materials ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials journal homepage: www.elsevier.com/locate/jmmm
Synthesis of functionalized Co0.5Zn0.5Fe2O4 nanoparticles for biomedical applications R.A. Bohara a, H.M. Yadav a, N.D. Throat b, S.S. Mali c, C.K. Hong c, S.G. Nanaware a, S.H. Pawar a,n a
Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416006, India Department of Molecular Cell Biology, Samsung Biomedical Research Center, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea c Advanced Chemical Engineering Department, Chonnam National University, Gwangju 500757, South Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 21 April 2014 Received in revised form 12 November 2014 Accepted 17 November 2014
In this paper, we report a simple one step method for the synthesis of uniform, water dispersible amine functionalized Co0.5Zn0.5Fe2O4 nanoparticles (AF-CZF) of size about 6 nm. The synthesis process was accomplished by refluxing Fe(acac)3, Co(acac)2 and Zn(acac)2 in diethylene glycol and ethanolamine. The magnetic nanoparticles were characterized by XRD, TGDTA, FTIR, SEM and TEM techniques. Their magnetic properties were also studied by using SQUID. The synthesized particles show superparamagnetism at room temperature. AF-CZF nanoparticles exhibit good cell viability, which is above 95% at a concentration of 80 mg mL 1 on MCF7 cell line. The AF-CZF can be a new versatile platform for many interesting biomedical applications. & 2014 Published by Elsevier B.V.
Keywords: Magnetic nanoparticles Amine functionalization Superparamagnetic Biocompatibility
1. Introduction Magnetic nanoparticles (MNPs) are attracting considerable interest as viable biomedical materials and research on them is growing due to their unique physical and chemical properties [1]. Biomedical applications of MNPs includes drug carriers, cellular labeling and tracking agents vectors for gene therapy, hyperthermia treatment and magnetic resonance imaging (MRI) contrast agent [2]. For successful application of MNPs for biomedical application they must satisfy certain criteria such as minimum cytotoxicity, improved colloidal stability in physiological media and ability to carry payloads, such as drug molecule or DNA for gene therapy [3]. For all this chemical modification of the MNPs surface is necessary for specific interactions. It is well known that proper modification of the nanoparticles surface through functionalization with –NH2 or –COOH groups assist conjugation of nanoparticles with different biomolecules using different linkages. Typically, in a multistep post-synthetic grafting process, phosphonates, organosilane, and other polymeric molecules are used to provide the desired functionality, also use of these costly organic chemicals in post-synthetic grafting makes the process inefficient for bulky synthesis. Also frequent exposure of magnetic particles n
Corresponding author. Fax: þ 91 231 2601595. E-mail addresses: [email protected] (R.A. Bohara), [email protected] (S.H. Pawar).
to harsh reaction conditions may affect magnetic properties [4]. Among different spinel ferrites cobalt ferrite (CoFe2O4) is well known and studied in detail because of its high coercivity and moderate saturation magnetization also magnetic properties of CoFe2O4 is greatly affected by the size of particles [5]. Substitution of nonmagnetic ions in cobalt ferrite exhibits improved properties such as excellent chemical stability, high magneto-crystalline anisotropy, magneto-optical properties as compared to other mixed ferrite [6]. To date many methods have been developed for synthesis of mixed ferrites such as co-precipitation [7] ceramic technique [8] hydrothermal synthesis [9] sonication [10] and low temperature combustion method [11]. However, research on thermal decomposition method of synthesis of Co0.5Zn0.5Fe2O4 (CZF) is comparatively limited. Typically this method includes decomposition of metal precursor in high boiling temperature solvents in the presence of stabilizing agents. However obtained MNPs are highly organic soluble, which restricts their use in most of biomedical applications [12]. Thermal decomposition is also been developed further for synthesis of water soluble magnetic nanoparticles [13]. Recently, Barick et al. [14] have developed highly water stable assembly of amine functionalized Fe3O4 using thermal decomposition method for MRI application, but other ferrites are less explored in this direction. In the present work we report for the first time one pot synthesis of amine functionalized CZF MNPs (AF-CZF) by thermal decomposition method which are water dispersible, monodisperse
http://dx.doi.org/10.1016/j.jmmm.2014.11.063 0304-8853/& 2014 Published by Elsevier B.V.
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
R.A. Bohara et al. / Journal of Magnetism and Magnetic Materials ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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in nature. Our synthesis method involves heating acetonates precursor in polyol medium (diethylene glycol) and ethanolamine. Cytotoxicity studies of the prepared MNPs have also been performed to evaluate their potentiality in biomedical application.
temperature on magnetic properties. Hydrodynamic diameter (HDD) of particle was measured by PSS/NICOMP 380 ZLS particles sizing system (Santa Barbara, CA, USA) with a red diode laser at 632.8 Å in a fixed angle 90° plastic cell in water. 2.4. Cell culture
2. Experimental 2.1. Materials Fe(acac)3, Co(acac)2, and Zn(acac)2 were purchased from Sigma–Aldrich. Diethylene glycol and ethanolamine were purchased from Molychem India. All chemicals used here were of analytical grade and used without further purification. 2.2. Synthesis of AF-CZF MNPs The water dispersible AF-CZF were prepared by the high thermal decomposition method. A mixture of Co(acac)2, (1.5 mmol), Zn(acac)2 (1.5 mmol) and Fe(acac)3 (3.00 mmol) dissolved in diethylene glycol (40 mL) was continuously heated at 110 °C under vigorous magnetic stirring. After heating for 1 h, 15 mL ethanolamine was added. The reaction mixture was refluxed at 160 °C for 6 h during which fine black colored colloidal particles appeared in the reaction mixture and cooled down to room temperature. The particles were washed several times with ethanol and separated by magnetically and dried in hot air oven at 80 °C for 2 h.
Since the functionalized MNPs are to be used for biomedical application, the issue of cytotoxicity has to be addressed. The viability of MCF7 cell line in the presence of MNPs was assessed relative to the cells in the control experiment (without MNPs) using sulforhodamine assay (SRB) [15]. The cell were seeded into 96-well plate at densities of 1 104 cells per well for 48 h. Then the MNPs of four different concentrations ranging from 20, 40, 60 and 80 mg mL 1 were added to cells and incubated for 48 h. Then the cells were washed with phosphate buffer saline (PBS) and processed for SRB assay to determine the cell viability. To carry this, cell were fixed with a solution of 10% trichloroacetic acid and stained with 0.4% SRB dissolved in 1% acetic acid. Cell bound dye was extracted with 10 mM unbuffered Tris Buffer solution (pH 10.5) and then absorbance was measured at 560 nm using a plate reader. The cell viability was calculated using following formulae:
% Viability = absorbance of treated cells /absorbance of control cells × 100.
2.3. Characterizations
3. Results and discussion
The structural, morphological and magnetic properties of functionalized MNPs were studied using X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), thremogravimetric analysis (TGA), transmission electron microscopy (TEM) and magnetization measurements were performed on Quantum Design SQUID-VSM. The particles were also studied for cytotoxicity study in order to use them for further biomedical applications. Phase identification and structural analysis of amine functionalized MNPs were studied using XRD (Philip-3710) with Cr-Kα radiation (λ ¼1.5418 Å) in the 2θ range from 20° to 80°. The pattern were evaluated by X-Pert high score software and compared with the Joint Committee on Powder Diffraction Standards (JCPDS) (card numbers 00-22-1086 and 22-1012). The crystallite size was calculated from the full width at half maximum (FWHM) of the highest intensity diffraction peak, which is based on Debye– Scherrer's relation:
3.1. Structural and morphological studies
t = 0.9λ /β cos θ
(2)
X-ray diffraction pattern of AF-CZFMNPs is shown in Fig. 1. XRD analysis showed that the synthesized material structure corresponds with the cubic spinel structure. All XRD peaks are well matched with JCPDS card no. 22-1086 of the (CoFe2O4) and JCPDS card no. 22-1012 of the (ZnFe2O4). The crystallite size was estimated by Scherrer formula, which is about of 5 nm. The lattice parameters were calculated from the reflection of (311) plane using standard formula is found to be 0.842 nm. It was found that lattice parameter of AF-CZF MNPs was higher than lattice parameter of CoFe2O4. This may be due to the diffusion of zinc ions into tetrahedral sites which leads to increase in lattice parameter of AF-
(1)
where t is the crystallite size, λ is the wave length of Cu-Kα radiation, β is the FWHM and θ is the diffraction angle of strongest peak. The morphology and size of the MNPs were determined from TEM micrographs. For this purpose, the colloidal solution of the MNPs was transferred onto a carbon coated carbon grid and allowed to air dry. The grid was then scanned using a Philips CM 200 model TEM, with an operating voltage of 20–200 kV and resolution of 2.4 Å. A Perkin–Elmer Spectrometer (Model no. 783 USA) was used to get FTIR spectra of MNPs in the range of 450–4000 cm 1 using KBr pellets to confirm the amine functionalization on the surface and to check the possible interaction between them. M–H curves were recorded to calculate the important magnetic properties like saturation magnetization and coercivity by VSM at room temperature. M–T measurement was performed with SQUID at filed 100 Oe to study the effect of
Fig. 1. XRD pattern for amine functionalized CZF MNPs.
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
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Fig. 4. SEM micrographs of amine functionalized CZF MNPs.
Fig. 2. FTIR spectra of amine functionalized CZF MNPs.
16.8%, which is between 200 and 300 °C is due to the surface adsorbed molecules of diethylene glycol. The weight loss of 9.5%, above 400 °C is ascribed to removal of surface bonded ethanolamine molecules. Fig. 4 shows the representative SEM images of AF-CZF MNPs. The morphology reveals that particles are agglomerated in nature and most of them are spherical in shape. The agglomeration may be due to the oriented attachment growth mechanism which leads to the minimization of surface energy and it is dependent on the collective behavior of nanoparticles and intermolecular force existing between them [4]. Fig. 5(a) shows TEM micrograph of AFCZF MNPs, it can be observed that particle shows uniform size with a spherical shape. The average particle size is about 6 nm and matches with the crystallite size calculated from XRD pattern. The inset in Fig. 5(b) is the selected area electron diffraction (SAED) of a AF-CZF MNPs, which shows bright ring patterns and reveals the crystalline nature of sample.
CZF MNPs. XRD peaks broading suggest fine nanocrystalline nature of the sample. The presence of ethanolamine on the particle surface MNPs were confirmed by using FTIR spectroscopy technique. Fig. 2 shows FTIR spectra of AF-CZF MNPs, the appearance of peak at 580 cm 1 attributes metal oxygen bonding. The appearances of the peaks at 883 cm 1, 1353 cm 1, 1630 cm 1 and 3350 cm 1 corresponds to N–H wagging, C–N stretching, NH2 scissoring and N–H stretching respectively. This observation confirms the existence of ethanolamine molecules on surface [16]. Thremogravimetric analysis (TGA) of the AF-CZF MNPs was carried out and presented in Fig. 3. In this experiment, the MNPs are heated to 700 °C under flowing N2 and change in mass loss of organic material were recorded. TGA allows us to determine the bonding strength of ligand to the nanoparticles surface and its thermal stability. As the TGA was performed under an N2 atmosphere, the oxidation of the functionalized MNPs surface was greatly reduced. A three stage decomposition profile is apparent as the initial weight loss of near about 3.6% is related to the removal of surface moisture from ferrite surface. The second weight loss of
The magnetic properties of AF-CZF MNPs were obtained from VSM at room temperature with an applied field up to 740 kOe. Field dependent magnetic measurements show (Fig. 6) very
Fig. 3. Thremogravimetry curve of amine functionalized CZF MNPs.
Fig. 5. (a) TEM micrograph and (b) SAED pattern of amine functionalized CZF MNPs.
3.2. Magnetic study
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
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Fig. 6. M–H curves and zero filed cooling and filed cooling of amine functionalized CZF MNPs.
negligible hysteresis parameter (coercivity) and remenance magnetization at 300 K, which reveals the superparamagnetic nature of MNPs. The saturation magnetization (Ms) of the sample was 46.03 emu/g which is smaller than the bulk ferrite. These difference in magnetization value between the nanosize ferrites and bulk ferrites can be attributed to finite size effect [5]. The temperature dependent magnetization (FC-ZFC) of the AF-CZF is measured in the applied magnetic field of 100 Oe and the obtained results are shown in the Fig. 6. The M–T measurements show blocking temperature of the sample. The feature of the FC-ZFC curve indicating nanoparticles are superparamagnetic in nature [17]. The ZFC curve reached the maximum at about 100 K which corresponds to the blocking temperature (TB) of the sample. Above TB the sample shows superparamagnetic behavior. However, the superimposition of the ZFC and FC curves take place at a certain temperature (TSEP) and which is 160 K. The superimposition of ZFC and FC curves is one of the characteristic features of superparamagnetic system [18]. So, the present synthesis technique is enable to produce superparamagnetic AF-CZF MNPs. 3.3. Colloidal stability study
magnetic field, the magnetic dipole–dipole interactions between particles can cause their agglomeration. Therefore, the hydrodynamic size distributions are larger than those observed by TEM [19]. Thus lower value of HDD suggest the enhance stability of particle in aqueous media which may be due to surface bonded ethanolamine [16]. 3.4. Cytotoxicity study The biocompatibility of MNPs is a key factor in view of their biological applications. For biomedical application MNPs are intentionally engineered to interact with cells, it is important to ensure that these enhancements are not causing any adverse effect. The cytotoxicity were done on MCF7 (human breast cancer cell line) with different concentration of nanoparticles and the obtained data is shown in the figure. The cell lines were incubated with nanoparticles for 48 h with concentration of 10, 20, 40, 80 m g mL 1 in the 5% CO2 atmosphere. Fig. 8 shows the cell viability, as it can be seen from the graph cell viability remains unchanged for 48 h up to 60 mg/mL concentration, for the MCF7 cell line. While at
Dynamic Light Scattering (DLS) measurements were carried out to investigate the hydrodynamic size of MNPs shown in Fig. 7. The average maxima of DLS size for AF-CZF MNPs give the average hydrodynamic diameter of about 51.3 mm (710 nm), with polydispersity of 0.2 which suggest the monodisperse nature of the samples. The discrepancy in size measurements from TEM and DLS techniques is observed readily as even in the absence of external
Fig. 7. HDD at pH ∼7 in water for amine functionalized CZF MNPs.
Fig. 8. Cytotoxicity profile of amine functionalized CZFMNPs for 48 h on MCF7 cell line at different concentrations (10, 20, 40 and 80 mg/mL).
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
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80 mg/mL concentration the obtained viability was 95%. The results indicate that the viability of MCF7 cell line is not affected by the presence of amine functionalized CZF, suggesting that nanoparticles are highly biocompatible and does not possess toxic effect. These nanoparticles stand out to potential material for biomedical applications.
4. Conclusion In the present investigation, a simple one step method has been developed for the synthesis of water dispersible, AF-CZF nanoparticles. The structural, magnetic and cytotoxic properties of MNPs were studied. The prepared magnetic nanoparticles possess high aqueous stability. The FC and ZFC measurement strongly supports the superparamagnetic behavior at room temperature. The size of MNPs is about 6 nm. SRB assay reveals that prepared MNPs are biocompatible up to the 80 mg/mL concentration on MCF7 cell line. We demonstrated that AF-CZF MNPs preserved the option of superparamagnetism and biocompatibility for their applicability in biomedical filed such as drug delivery and hyperthermia application.
Acknowledgment
5
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Authors are very grateful to Prof. S.K. Dhar, TIFR, Mumbai for M–T measurement, SAIF-NEHU, Shillong for TEM facility. The authors also acknowledge SAIF Kochi for SEM facility.
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
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Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials journal homepage: www.elsevier.com/locate/jmmm
Synthesis of functionalized Co0.5Zn0.5Fe2O4 nanoparticles for biomedical applications R.A. Bohara a, H.M. Yadav a, N.D. Throat b, S.S. Mali c, C.K. Hong c, S.G. Nanaware a, S.H. Pawar a,n a
Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416006, India Department of Molecular Cell Biology, Samsung Biomedical Research Center, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea c Advanced Chemical Engineering Department, Chonnam National University, Gwangju 500757, South Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 21 April 2014 Received in revised form 12 November 2014 Accepted 17 November 2014
In this paper, we report a simple one step method for the synthesis of uniform, water dispersible amine functionalized Co0.5Zn0.5Fe2O4 nanoparticles (AF-CZF) of size about 6 nm. The synthesis process was accomplished by refluxing Fe(acac)3, Co(acac)2 and Zn(acac)2 in diethylene glycol and ethanolamine. The magnetic nanoparticles were characterized by XRD, TGDTA, FTIR, SEM and TEM techniques. Their magnetic properties were also studied by using SQUID. The synthesized particles show superparamagnetism at room temperature. AF-CZF nanoparticles exhibit good cell viability, which is above 95% at a concentration of 80 mg mL 1 on MCF7 cell line. The AF-CZF can be a new versatile platform for many interesting biomedical applications. & 2014 Published by Elsevier B.V.
Keywords: Magnetic nanoparticles Amine functionalization Superparamagnetic Biocompatibility
1. Introduction Magnetic nanoparticles (MNPs) are attracting considerable interest as viable biomedical materials and research on them is growing due to their unique physical and chemical properties [1]. Biomedical applications of MNPs includes drug carriers, cellular labeling and tracking agents vectors for gene therapy, hyperthermia treatment and magnetic resonance imaging (MRI) contrast agent [2]. For successful application of MNPs for biomedical application they must satisfy certain criteria such as minimum cytotoxicity, improved colloidal stability in physiological media and ability to carry payloads, such as drug molecule or DNA for gene therapy [3]. For all this chemical modification of the MNPs surface is necessary for specific interactions. It is well known that proper modification of the nanoparticles surface through functionalization with –NH2 or –COOH groups assist conjugation of nanoparticles with different biomolecules using different linkages. Typically, in a multistep post-synthetic grafting process, phosphonates, organosilane, and other polymeric molecules are used to provide the desired functionality, also use of these costly organic chemicals in post-synthetic grafting makes the process inefficient for bulky synthesis. Also frequent exposure of magnetic particles n
Corresponding author. Fax: þ 91 231 2601595. E-mail addresses: [email protected] (R.A. Bohara), [email protected] (S.H. Pawar).
to harsh reaction conditions may affect magnetic properties [4]. Among different spinel ferrites cobalt ferrite (CoFe2O4) is well known and studied in detail because of its high coercivity and moderate saturation magnetization also magnetic properties of CoFe2O4 is greatly affected by the size of particles [5]. Substitution of nonmagnetic ions in cobalt ferrite exhibits improved properties such as excellent chemical stability, high magneto-crystalline anisotropy, magneto-optical properties as compared to other mixed ferrite [6]. To date many methods have been developed for synthesis of mixed ferrites such as co-precipitation [7] ceramic technique [8] hydrothermal synthesis [9] sonication [10] and low temperature combustion method [11]. However, research on thermal decomposition method of synthesis of Co0.5Zn0.5Fe2O4 (CZF) is comparatively limited. Typically this method includes decomposition of metal precursor in high boiling temperature solvents in the presence of stabilizing agents. However obtained MNPs are highly organic soluble, which restricts their use in most of biomedical applications [12]. Thermal decomposition is also been developed further for synthesis of water soluble magnetic nanoparticles [13]. Recently, Barick et al. [14] have developed highly water stable assembly of amine functionalized Fe3O4 using thermal decomposition method for MRI application, but other ferrites are less explored in this direction. In the present work we report for the first time one pot synthesis of amine functionalized CZF MNPs (AF-CZF) by thermal decomposition method which are water dispersible, monodisperse
http://dx.doi.org/10.1016/j.jmmm.2014.11.063 0304-8853/& 2014 Published by Elsevier B.V.
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
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in nature. Our synthesis method involves heating acetonates precursor in polyol medium (diethylene glycol) and ethanolamine. Cytotoxicity studies of the prepared MNPs have also been performed to evaluate their potentiality in biomedical application.
temperature on magnetic properties. Hydrodynamic diameter (HDD) of particle was measured by PSS/NICOMP 380 ZLS particles sizing system (Santa Barbara, CA, USA) with a red diode laser at 632.8 Å in a fixed angle 90° plastic cell in water. 2.4. Cell culture
2. Experimental 2.1. Materials Fe(acac)3, Co(acac)2, and Zn(acac)2 were purchased from Sigma–Aldrich. Diethylene glycol and ethanolamine were purchased from Molychem India. All chemicals used here were of analytical grade and used without further purification. 2.2. Synthesis of AF-CZF MNPs The water dispersible AF-CZF were prepared by the high thermal decomposition method. A mixture of Co(acac)2, (1.5 mmol), Zn(acac)2 (1.5 mmol) and Fe(acac)3 (3.00 mmol) dissolved in diethylene glycol (40 mL) was continuously heated at 110 °C under vigorous magnetic stirring. After heating for 1 h, 15 mL ethanolamine was added. The reaction mixture was refluxed at 160 °C for 6 h during which fine black colored colloidal particles appeared in the reaction mixture and cooled down to room temperature. The particles were washed several times with ethanol and separated by magnetically and dried in hot air oven at 80 °C for 2 h.
Since the functionalized MNPs are to be used for biomedical application, the issue of cytotoxicity has to be addressed. The viability of MCF7 cell line in the presence of MNPs was assessed relative to the cells in the control experiment (without MNPs) using sulforhodamine assay (SRB) [15]. The cell were seeded into 96-well plate at densities of 1 104 cells per well for 48 h. Then the MNPs of four different concentrations ranging from 20, 40, 60 and 80 mg mL 1 were added to cells and incubated for 48 h. Then the cells were washed with phosphate buffer saline (PBS) and processed for SRB assay to determine the cell viability. To carry this, cell were fixed with a solution of 10% trichloroacetic acid and stained with 0.4% SRB dissolved in 1% acetic acid. Cell bound dye was extracted with 10 mM unbuffered Tris Buffer solution (pH 10.5) and then absorbance was measured at 560 nm using a plate reader. The cell viability was calculated using following formulae:
% Viability = absorbance of treated cells /absorbance of control cells × 100.
2.3. Characterizations
3. Results and discussion
The structural, morphological and magnetic properties of functionalized MNPs were studied using X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), thremogravimetric analysis (TGA), transmission electron microscopy (TEM) and magnetization measurements were performed on Quantum Design SQUID-VSM. The particles were also studied for cytotoxicity study in order to use them for further biomedical applications. Phase identification and structural analysis of amine functionalized MNPs were studied using XRD (Philip-3710) with Cr-Kα radiation (λ ¼1.5418 Å) in the 2θ range from 20° to 80°. The pattern were evaluated by X-Pert high score software and compared with the Joint Committee on Powder Diffraction Standards (JCPDS) (card numbers 00-22-1086 and 22-1012). The crystallite size was calculated from the full width at half maximum (FWHM) of the highest intensity diffraction peak, which is based on Debye– Scherrer's relation:
3.1. Structural and morphological studies
t = 0.9λ /β cos θ
(2)
X-ray diffraction pattern of AF-CZFMNPs is shown in Fig. 1. XRD analysis showed that the synthesized material structure corresponds with the cubic spinel structure. All XRD peaks are well matched with JCPDS card no. 22-1086 of the (CoFe2O4) and JCPDS card no. 22-1012 of the (ZnFe2O4). The crystallite size was estimated by Scherrer formula, which is about of 5 nm. The lattice parameters were calculated from the reflection of (311) plane using standard formula is found to be 0.842 nm. It was found that lattice parameter of AF-CZF MNPs was higher than lattice parameter of CoFe2O4. This may be due to the diffusion of zinc ions into tetrahedral sites which leads to increase in lattice parameter of AF-
(1)
where t is the crystallite size, λ is the wave length of Cu-Kα radiation, β is the FWHM and θ is the diffraction angle of strongest peak. The morphology and size of the MNPs were determined from TEM micrographs. For this purpose, the colloidal solution of the MNPs was transferred onto a carbon coated carbon grid and allowed to air dry. The grid was then scanned using a Philips CM 200 model TEM, with an operating voltage of 20–200 kV and resolution of 2.4 Å. A Perkin–Elmer Spectrometer (Model no. 783 USA) was used to get FTIR spectra of MNPs in the range of 450–4000 cm 1 using KBr pellets to confirm the amine functionalization on the surface and to check the possible interaction between them. M–H curves were recorded to calculate the important magnetic properties like saturation magnetization and coercivity by VSM at room temperature. M–T measurement was performed with SQUID at filed 100 Oe to study the effect of
Fig. 1. XRD pattern for amine functionalized CZF MNPs.
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Fig. 4. SEM micrographs of amine functionalized CZF MNPs.
Fig. 2. FTIR spectra of amine functionalized CZF MNPs.
16.8%, which is between 200 and 300 °C is due to the surface adsorbed molecules of diethylene glycol. The weight loss of 9.5%, above 400 °C is ascribed to removal of surface bonded ethanolamine molecules. Fig. 4 shows the representative SEM images of AF-CZF MNPs. The morphology reveals that particles are agglomerated in nature and most of them are spherical in shape. The agglomeration may be due to the oriented attachment growth mechanism which leads to the minimization of surface energy and it is dependent on the collective behavior of nanoparticles and intermolecular force existing between them [4]. Fig. 5(a) shows TEM micrograph of AFCZF MNPs, it can be observed that particle shows uniform size with a spherical shape. The average particle size is about 6 nm and matches with the crystallite size calculated from XRD pattern. The inset in Fig. 5(b) is the selected area electron diffraction (SAED) of a AF-CZF MNPs, which shows bright ring patterns and reveals the crystalline nature of sample.
CZF MNPs. XRD peaks broading suggest fine nanocrystalline nature of the sample. The presence of ethanolamine on the particle surface MNPs were confirmed by using FTIR spectroscopy technique. Fig. 2 shows FTIR spectra of AF-CZF MNPs, the appearance of peak at 580 cm 1 attributes metal oxygen bonding. The appearances of the peaks at 883 cm 1, 1353 cm 1, 1630 cm 1 and 3350 cm 1 corresponds to N–H wagging, C–N stretching, NH2 scissoring and N–H stretching respectively. This observation confirms the existence of ethanolamine molecules on surface [16]. Thremogravimetric analysis (TGA) of the AF-CZF MNPs was carried out and presented in Fig. 3. In this experiment, the MNPs are heated to 700 °C under flowing N2 and change in mass loss of organic material were recorded. TGA allows us to determine the bonding strength of ligand to the nanoparticles surface and its thermal stability. As the TGA was performed under an N2 atmosphere, the oxidation of the functionalized MNPs surface was greatly reduced. A three stage decomposition profile is apparent as the initial weight loss of near about 3.6% is related to the removal of surface moisture from ferrite surface. The second weight loss of
The magnetic properties of AF-CZF MNPs were obtained from VSM at room temperature with an applied field up to 740 kOe. Field dependent magnetic measurements show (Fig. 6) very
Fig. 3. Thremogravimetry curve of amine functionalized CZF MNPs.
Fig. 5. (a) TEM micrograph and (b) SAED pattern of amine functionalized CZF MNPs.
3.2. Magnetic study
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Fig. 6. M–H curves and zero filed cooling and filed cooling of amine functionalized CZF MNPs.
negligible hysteresis parameter (coercivity) and remenance magnetization at 300 K, which reveals the superparamagnetic nature of MNPs. The saturation magnetization (Ms) of the sample was 46.03 emu/g which is smaller than the bulk ferrite. These difference in magnetization value between the nanosize ferrites and bulk ferrites can be attributed to finite size effect [5]. The temperature dependent magnetization (FC-ZFC) of the AF-CZF is measured in the applied magnetic field of 100 Oe and the obtained results are shown in the Fig. 6. The M–T measurements show blocking temperature of the sample. The feature of the FC-ZFC curve indicating nanoparticles are superparamagnetic in nature [17]. The ZFC curve reached the maximum at about 100 K which corresponds to the blocking temperature (TB) of the sample. Above TB the sample shows superparamagnetic behavior. However, the superimposition of the ZFC and FC curves take place at a certain temperature (TSEP) and which is 160 K. The superimposition of ZFC and FC curves is one of the characteristic features of superparamagnetic system [18]. So, the present synthesis technique is enable to produce superparamagnetic AF-CZF MNPs. 3.3. Colloidal stability study
magnetic field, the magnetic dipole–dipole interactions between particles can cause their agglomeration. Therefore, the hydrodynamic size distributions are larger than those observed by TEM [19]. Thus lower value of HDD suggest the enhance stability of particle in aqueous media which may be due to surface bonded ethanolamine [16]. 3.4. Cytotoxicity study The biocompatibility of MNPs is a key factor in view of their biological applications. For biomedical application MNPs are intentionally engineered to interact with cells, it is important to ensure that these enhancements are not causing any adverse effect. The cytotoxicity were done on MCF7 (human breast cancer cell line) with different concentration of nanoparticles and the obtained data is shown in the figure. The cell lines were incubated with nanoparticles for 48 h with concentration of 10, 20, 40, 80 m g mL 1 in the 5% CO2 atmosphere. Fig. 8 shows the cell viability, as it can be seen from the graph cell viability remains unchanged for 48 h up to 60 mg/mL concentration, for the MCF7 cell line. While at
Dynamic Light Scattering (DLS) measurements were carried out to investigate the hydrodynamic size of MNPs shown in Fig. 7. The average maxima of DLS size for AF-CZF MNPs give the average hydrodynamic diameter of about 51.3 mm (710 nm), with polydispersity of 0.2 which suggest the monodisperse nature of the samples. The discrepancy in size measurements from TEM and DLS techniques is observed readily as even in the absence of external
Fig. 7. HDD at pH ∼7 in water for amine functionalized CZF MNPs.
Fig. 8. Cytotoxicity profile of amine functionalized CZFMNPs for 48 h on MCF7 cell line at different concentrations (10, 20, 40 and 80 mg/mL).
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80 mg/mL concentration the obtained viability was 95%. The results indicate that the viability of MCF7 cell line is not affected by the presence of amine functionalized CZF, suggesting that nanoparticles are highly biocompatible and does not possess toxic effect. These nanoparticles stand out to potential material for biomedical applications.
4. Conclusion In the present investigation, a simple one step method has been developed for the synthesis of water dispersible, AF-CZF nanoparticles. The structural, magnetic and cytotoxic properties of MNPs were studied. The prepared magnetic nanoparticles possess high aqueous stability. The FC and ZFC measurement strongly supports the superparamagnetic behavior at room temperature. The size of MNPs is about 6 nm. SRB assay reveals that prepared MNPs are biocompatible up to the 80 mg/mL concentration on MCF7 cell line. We demonstrated that AF-CZF MNPs preserved the option of superparamagnetism and biocompatibility for their applicability in biomedical filed such as drug delivery and hyperthermia application.
Acknowledgment
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Authors are very grateful to Prof. S.K. Dhar, TIFR, Mumbai for M–T measurement, SAIF-NEHU, Shillong for TEM facility. The authors also acknowledge SAIF Kochi for SEM facility.
Please cite this article as: R.A. Bohara, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.11.063i
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