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Water dispersible oleic acid-coated Fe3O4 nanoparticles for biomedical applications
Journal of Magnetism and Magnetic Materials ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Water dispersible oleic acid-coated Fe3O4 nanoparticles for biomedical applications P.B. Shete, R.M. Patil, B.M. Tiwale, S.H. Pawar n Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416006, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2014 Received in revised form 18 October 2014 Accepted 27 October 2014
Fe3O4 magnetic nanoparticles (MNPs) have proved their tremendous potential to be used for various biomedical applications. Oleic acid (OA) is widely used in ferrite nanoparticle synthesis because it can form a dense protective monolayer, thereby producing highly uniform and monodispersed particles. Capping agents such as oleic acid are often used because they form a protective monolayer, which is strongly bonded to the surface of nanoparticles. This is necessary for making monodisperse and highly uniform MNPs. Coating of Fe3O4 MNPs with OA makes the particles dispersible only in organic solvents and consequently limits their use for biomedical applications. Hence, in this work, the OA coated MNPs were again functionalized with chitosan (CS), in order to impart hydrophilicity on their surface. All the morphological, magnetic, colloidal and cytotoxic characteristics of the resulting core–shells were studied thoroughly. Their heating induction ability was studied to predict their possible use in hyperthermia therapy of cancer. Specific absorption rate was found to be increased than that of bare MNPs. & 2014 Published by Elsevier B.V.
Keywords: Alkaline precipitation Fe3O4 Chitosan Oleic acid Adsorption Hyperthermia
1. Introduction Nanosized magnetic iron oxide nanoparticles such as magnetite coated with hydrophilic water soluble polymers have received increasing attention due to their wide ranging biomedical applications in drug delivery [1], resonance imaging (MRI) contrast agents [2], in high-gradient magnetic field separations [3], treatment of retinal detachment [4], in bio-catalysis [5], as magnetic bio-separations [6] etc. Owing to their biocompatibility and low toxicity, magnetite (Fe3O4) nanoparticles (NPs) are the most attractive candidates for use in human body. Magnetic NPs (MNPs) for biomedical applications should fulfill a variety of requirements, including: (i) superparamagnetic behavior at room temperature, in order to avoid particle aggregation; (ii) large saturation magnetization, so as to show a large response under the application of a magnetic field; (iii) a limiting size in the order of 20 nm for in vivo applications, and (iv) bio-compatibility, such that nanoparticles are usually coated with either biological or biocompatible molecules. In order to make MNPs suitable for in vivo applications, they are coated with various polymers like chitosan (CS) [7], oleic acid (OA) [8], starch [9], pullulan [10], dextran [11] etc. Out of these, OA is probably the most common small molecule which is complexed with magnetite [12]. OA possesses a non-polar hydrocarbon tail n
Corresponding author. Fax: þ 91 231 2601595. E-mail address: [email protected] (S.H. Pawar).
and a polar carboxylic acid head group. Carboxylate anions are known to coordinate with the surface of magnetite, presumably through a coordination of iron atoms with both the carboxylate oxygens. The polar head group anchored on the magnetite surface, the non-polar tail extends into solution, causing the magnetite to be hydrophobic and dispersible in organic solvents. However coating of MNPs with OA makes the particles only dispersible in organic solvents and this limits their use for medical applications [13]. Fe3O4 NPs modified with OA decrease degree of agglomeration and favor the generation of hydrogen bonding between Fe3O4 and CS, thus improve their interfacial combination. Considering biodegradability and toxicity, much attention have been paid to chitosan, since it possesses interesting properties such as biocompatibility, biodegradability, film forming ability, gelation characteristics and bioadhesion. Another advantage of CS is that the amine groups can offer a variety of active sites for further biofunctionalization [14]. In the present investigation, Fe3O4 nanoparticles were prepared by alkaline precipitation method and were surface modified with OA. The precursor used for the synthesis of Fe3O4 was ferrous chloride only. A very little literature is available on the synthesis of Fe3O4 using FeCl2 only as the sole precursor for the nanoparticles [15,16]. The as-formed NPs were used for surface modification using OA which is hydrophobic in nature. Hence in order to make it suitable for biomedical applications, OA-Fe3O4 MNPs were further coated with CS, a biopolymer having hydrophilic nature. Bare (Fe3O4), OA coated (OA-Fe3O4) and CS-coated OA-Fe3O4 (CS-OA-Fe3O4) nanoparticles were characterized further for their
http://dx.doi.org/10.1016/j.jmmm.2014.10.137 0304-8853/& 2014 Published by Elsevier B.V.
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structural, morphological and magnetic properties in order to investigate the role of coating on particle. The resulting particles were well dispersed in water and studied for their heating induction effect so as to study their possible use in hyperthermia application.
analysis by XRD Philips PW-3710 diffractometer using CuKα radiation in the 2θ range from 0 to 100°. The XRD patterns were evaluated by X′pert high score software and compared with JCPDS card. The average crystallite size (t) was calculated from the diffraction line-width of XRD pattern, based on Scherrer′s relation:
t = 0.9λ /β cos θ 2. Materials and method 2.1. Materials Ferrous chloride (FeCl2 4H2O), HCl, acetic acid, acetone and NaOH were procured from HiMedia, Mumbai (Maharashtra), India. Methanol, chitosan and oleic acid were purchased from SigmaAldrich, USA. Double distilled water was used throughout the procedure. 2.2. 2 Experimental 2.2.1. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized via alkaline precipitation [17,18]. The brief procedure for preparation of MNPs is as follows: 2 g FeCl2 4H2O was dissolved in 50 mL 1 M HCl by heating upto 70 °C. 50 mL 3 M NaOH was added to it at 60 °C drop by drop with constant stirring. A black precipitate gets formed. This is nothing but magnetite (Fe3O4) NPs. The possible reaction taking place is showed below:
3 FeCl2 ·4H2 O + 6 NaOH + ½ O2 → Fe 3 O4 + 6 NaCl + 15 H2 O
(1)
The precipitate is allowed to settle down. The rate of settling is increased by applying external magnetic field. The precipitate is then separated, washed with D/W till the neutral pH, dried at RT and used further studies. The particles studied for various characters using XRD, SEM, TEM, FTIR, VSM and temperature susceptibility. 2.2.2. Surface modification of Fe3O4 nanoparticles with OA As-formed Fe3O4 MNPs were used for coating procedure. The particles were well dispersed in 200 ml methanol by ultrasonication. 50 ml OA was added while constant stirring at 80 °C. OA-Fe3O4 particles were filtered through Whatman filter paper no.1, washed three times with distilled water. The OA-Fe3O4 particles were separated from the filter paper using acetone. The particles were dried at room temperature to evaporate all the acetone. These particles were termed as OA-Fe3O4 NPs. 2.2.3. Coating of CS on OA-Fe3O4 NPs As-formed OA-Fe3O4 MNPs were used for coating procedure. The were dispersed in 1% CS solution in 2% acetic acid. The mixture was ultrasonicated for 30 min. After ultrasonication, the CS-coated Fe3O4 NPs (CS-OA-Fe3O4) allowed to settle, washed with distilled water 3 times to remove excess CS, separated and dried at 50 °C. These OA-particles were also thoroughly studied for their structural, morphological and magnetic characters. The coating of CS on Fe3O4 MNPs was confirmed by FTIR spectra and TG-DTA results. 2.3. Characterizations Bare, OA-Fe3O4 and CS-OA-Fe3O4 MNPs were studied for their structural, morphological and magnetic characters using XRD, FTIR, EDAX, SEM, TEM, VSM and tTemperature susceptibility. In order to use the particles for hyperthermia therapy, they were studied for their heating induction ability. The XRD pattern of MNPs drop coated and air-dried on the glass substrate was recorded to study the structural and phase
(2)
Where, β is the full width at half maximum (fwhm). TEM images were used to determine the morphology and size of the MNPs. For this purpose, the colloidal solution of the MNPs was transferred on to a carbon coated copper grid and allowed to air dry. The grid was then scanned using Philips CM200 model Transmission electron Microscope, operating voltage 20–200 kV with resolution 2.4 Å. The compositional analysis was done by Energy-dispersive analysis of X-ray spectroscopy (EDAX, JEOL JSM 6360). The Perkin-Elmer spectrometer, (Model no.783, USA) was used to get FT-IR spectra of MNPs in the range from 450 to 4000 cm 1 using KBr pellets to check the possible interaction of OA-Fe3O4 with chitosan. The magnetization measurements were performed on a superconducting quantum interference device (SQUID) magnetometer to investigate the saturation magnetization (Ms), blocking temperature (TB) and Curie temperature (TC). The measurements include field dependent hysteresis loops, (M–H), at two different temperatures 100 and 300 K with applied field range from 0 to 71 103 Oe. Induction heating of Fe3O4 nanoparticles for hyperthermia application was performed in a plastic micro centrifuge tube (1.5 ml) using an induction heating unit (Easy Heat 8310, Ambrell; UK) with a 6 cm diameter (4 turns) heating coil. To keep the temperature of the coil at ambient temperature, a provision of water circulation in coils was provided. MNPs suspended in 1 ml of distilled water was placed at the center of the coil and the applied frequency was 265 kHz. Particles are dispersed in water with a concentration ranging from 2, 5 and 10 mg ml 1 and ultrasonicated for 20 min to achieve a good dispersion of the NPs in carrier fluid. Samples were heated for 10 min with the desired current (200–400 A). For the conducted experiments, the magnetic field was calculated from the relationship:
H=
1.257ni in Oe L
(3)
where, n, i and L denote the number of turns, applied current and the diameter of the turn in centimeters, respectively. Calculated values of the magnetic field (H) at 200, 300 and 400 A were 167.6, 251.4 and 335.2 Oe (equivalent to 13.3, 20.0 and 26.7 kA m 1), respectively. Temperature was measured using an optical fiber probe with accuracy 0.1 °C.
3. Results and discussions Fig. 1(a) and (b) shows the powder XRD patterns for bare and OA-CS-coated Fe3O4 NPs respectively. The main characteristic peaks were obtained with the (hkl) values of (220), (311), (400), (422) and (511). These were then matched with the JCPDS file number 82-1533, which corresponds to Fe3O4 phase. Both the NPs show inverse spinel structure. The crystallite sizes of NPs were calculated from FWHM of the most intense peaks using the Debye–Scherrer formula. The crystallite sizes obtained were 20 nm and 10 nm for bare and OA-CS-coated MNPs respectively. However, the peaks became broad and intensity decreased after capping of Fe3O4 with OA-CS and hence it can be clearly stated that the particle size decreased after coating procedure. The coating of amorphous OA-CS polymer on crystalline Fe3O4 may induce
Please cite this article as: P.B. Shete, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.10.137i
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Fig. 1. XRD patterns obtained from bare Fe3O4 (a) and CS-OA-Fe3O4 (b) MNPs.
Fig. 3. Thermogravimetric spectra of bare Fe3O4 and CS-OA-Fe3O4 MNPs in nitrogen with a scanning rate of 10 °C min 1 up to 600 °C.
microstrain which resulted in broadening of peaks in case of coated sample [19–21]. The Selected Area Electron Diffraction (SAED) patterns for bare and coated NPs are shown in Fig. 2(a) and (b) respectively. It shows bright ring patterns indicating polycrystalline nature of the MNPs, as indicated by XRD patterns. The ring pattern corresponds to (220), (311), (400), (422) and (511) planes which can be clearly seen in XRD results. TGA provides a quantitative evidence of the coating of OA-CS on NPs. In this experiment, the MNPs are heated to 600 °C under flowing N2 and changes in mass loss of organic material were recorded. This is an extremely valuable technique for surface characterization of NPs. Typically, ligands that are bound more strongly desorb at higher temperatures. The weight losses of the bare Fe3O4, CS-OA-Fe3O4 MNPs were measured and shown in Fig. 3. Since the TG was performed under N2 atmosphere, the oxidation of coated and uncoated MNPs surface was greatly reduced. The weight loss of Fe3O4 NPs ( 5%) occurred upto temperature 200 °C was ascribed to the evaporation of water. For the CS-OA-Fe3O4 MNPs, after a gradual loss of water molecules in the polymer matrix, a great weight loss started after 2000 °C at which the decomposition of coating molecules occurs. CS decomposes at the temperature 200–300 °C [7]. Further degradation beyond 300 °C corresponds to decomposition of OA [13]. After the OA was decomposed completely, the residual substance mostly is magnetic particles. The percentage of CS in the coated MNPs was 8.3 wt% and that of OA was 27.38 wt% revealed in the TGA curve.
The size and shapes of the MNPs before and after surface modification were observed using TEM. The TEM images of Fe3O4 and CS-OA-Fe3O4 are shown in Fig.4. Bare Fe3O4 NPs were highly agglomerated with particle size 22.8 75.1 nm while OA-CS coated NPs were well dispersed with particle size 16.5 7 4.3 nm. Bare MNPs have strong magnetic dipole–dipole interaction and hence are attracted strongly and form big clusters causing bigger particle size. After efficient coating procedure, a non-magnetic layer is formed on surface of each particle which prevents increase in particle size. This may the reason why particle size reduces after coating procedure. These results are comparable with the XRD results. After surface modification, the particles maintained their original spherical shapes without any deformation or growth. The particles obtained were with less degree of aggregation and were well dispersed. Magnetic properties of the MNPs were studied using their M–H curves. M–H curves of bare and coated Fe3O4 MNPs at 300 K are shown in Fig. 5. The Saturation Magnetization (Ms), Coercivity (Ce) and Remenance (Mr) values calculated from the M–H curves for both bare and coated MNPs are given in Table 1. The graphs clearly show superparamagnetic nature of both the NPs at 300 K as Ce and Mr values are very negligible. Superparamagnetic behavior of MNPs at room temperature is very useful in in vivo applications as they do not retain magnetization before and after exposure to an external magnetic field, reducing the probability of particle aggregation due to magnetic dipole attraction [22]. This can be ascribed to the small size of NPs which were smaller than the
Fig. 2. Selected Area Electron Diffraction (SAED) patterns of bare Fe3O4 and CS-OA-Fe3O4 MNPs in (a) and (b) respectively.
Please cite this article as: P.B. Shete, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.10.137i
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Fig. 4. TEM images of (a) bare Fe3O4 and (b) CS-OA-Fe3O4 MNPs.
Fig. 5. M–H loops of bare Fe3O4, CS-OA-Fe3O4 MNPs at 300 K.
Table 1 The Saturation magnetization (Ms), Coercivity (Ce) and Remenance (Mr) values calculated from the M–H loops for both bare and CS-OA-Fe3O4 MNPs.
Ms (emu/g) Mr (emu/g) Ce (Oe) Mr/Ms
Fe3O4
CS-OA-Fe3O4
36.98 3.13 0.05 0.08
29.71 1.95 0.05 0.06
superparamagnetic critical size (20 nm) [23]. Ms value of bare NPs is observed to be 36.98 emu/g which is small compared to that of theoretical value of bulk Fe3O4 (Ms ¼92 emu/g) since Ms generally decreases with a decrease in magnetic particle size [26]. Ms has been reported to decrease as the particles size of Fe3O4 decreases below 30 or 20 nm, due to finite size effect [22,24]. The reduced magnetization in coated MNPs could also result from the small particle surface effect [25] which refers to the disordered alignment of surface atomic spins induced by reduced coordination and broken exchange between surface spins [26]. This surface effect is more prominent in small particles as the ratio of surface atoms to the interior atoms increases with a decrease in particle size. The zeta potential values and hydrodynamic diameters of bare and coated NPs suspensions in water with respect to pH 2 to 10 are shown in Fig. 6. The zeta potentials of bare NPs at pH 2 to 10 were 17.35, 12.02, 11.00, 10.33, 5.92, 12.00 and 25.6, 31.6 and 32.87 mV and that of coated NPs were 40.13, 32.69, 18.90, 12.49, 10.23, 1.2, 47.87, 54.43 and 58.52 mV, respectively. The
zeta potential of coated particles is more positive in the range of pH 2–6, as compared to bare indicating that the positive charges on the coated NPs increase with a decrease in pH. At higher pH, again, zeta potential of coated NPs is more negative than the bare ones. The isoelectric points (pI) for bare and coated nanoparticles were found to be around 6.7 and 7 respectively. The bare nanoparticles possess negative charge at physiological pH which was in agreement with the literature [27]. Naturally, at their respective pI values, both MNPs show highest hydrodynamic diameters due to highest degree of agglomeration. The bare MNPs have larger size due to higher degree of agglomeration and consequently show lager hydrodynamic diameter than the coated MNPs. Though coated MNPs are said to be more hydrophilic, they show smaller hydrodynamic diameter due to their much smaller size, as shown by TEM images. MTT assay was performed in order to check cytotoxic effect of MNPs. The cytotoxicity study of both, bare and coated nanoparticles was done on L929 and cell line with different concentrations of nanoparticles and the obtained data is shown in Fig. 7. The L929 cell line was incubated with nanoparticles for 48 h with the concentrations of 0.1, 0.5, 1.0, 1.5 and 2.0 mg mL 1 at 37 °C in 5% CO2 atmosphere. The relative cell viability (%) compared with control well containing cells without nanoparticles are calculated by the equation: [A]tested / [A]control 100. Fig. 7 shows the cell viability after incubation with different concentrations of both bare and coated Fe3O4 nanoparticles. It clearly reveals that after 48 h, bare MNPs started to exhibit their cytotoxicity while CS-OA-coated MNPs still showed almost 100% viability. Therefore coated MNPs are more suitable for in vivo applications than the bare MNPs, owing to their lower cytotoxicity.
4. Conclusion This study demonstrates the effect of capping of OA-CS on the surface behavior of Fe3O4 MNPs. XRD and Zeta potential studies confirmed coating of Fe3O4 MNPs with OA-CS. XRD pattern proved inverse spinel structure of the particles. TEM images showed that the particles are monodispersed, spherical-shaped having diameter of 22.8 75.1 nm and 16.5 7 4.3 nm in case of bare and coated particles respectively. SAED and XRD proved polycrystalline nature of particles. VSM showed that coating procedure decreased magnetization of the Fe3O4 NPs. Both the MNPs are superparamagnetic at room temperature with negligible Ce and Mr. Zeta potential studies showed a high colloidal stability of both the suspensions in water as base media at extreme pH values. Cell
Please cite this article as: P.B. Shete, et al., Journal of Magnetism and Magnetic Materials (2014), http://dx.doi.org/10.1016/j. jmmm.2014.10.137i
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Fig. 6. Hydrodynamic diameter and zeta potential as a function of pH for (a) Fe3O4 and (b) CS-OA-Fe3O4 MNPs dispersed in water.
References
Fig. 7. Cytotoxicity profiles of MNPs for 48 h on L929 cell line at different concentrations (0.1, 0.5, 1.0, 1.5 and 2.0 mg mL 1).
viability assay of both the MNPs showed very low cytotoxic effect on L929 cell line even after 48 h incubation period. Thus hydrophilicity can be rendered to hydrophobic surface of OA-Fe3O4 NPs using CS. Moreover, synthesized MNPs are suitable for hyperthermia therapy applications owing to their smaller size, superparamagnetic behavior at room temperature, higher magnetization values, high colloidal stability and low cytotoxicity.
Acknowledgements The authors are thankful to Dr. S. D. Sartale, Pune University, Pune (Maharashtra), India for providing SAED patterns. The authors are grateful to DST, India (DST no: SR/NM/NS-126/2010(G) for its financial support.
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Water dispersible oleic acid-coated Fe3O4 nanoparticles for biomedical applications P.B. Shete, R.M. Patil, B.M. Tiwale, S.H. Pawar n Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416006, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2014 Received in revised form 18 October 2014 Accepted 27 October 2014
Fe3O4 magnetic nanoparticles (MNPs) have proved their tremendous potential to be used for various biomedical applications. Oleic acid (OA) is widely used in ferrite nanoparticle synthesis because it can form a dense protective monolayer, thereby producing highly uniform and monodispersed particles. Capping agents such as oleic acid are often used because they form a protective monolayer, which is strongly bonded to the surface of nanoparticles. This is necessary for making monodisperse and highly uniform MNPs. Coating of Fe3O4 MNPs with OA makes the particles dispersible only in organic solvents and consequently limits their use for biomedical applications. Hence, in this work, the OA coated MNPs were again functionalized with chitosan (CS), in order to impart hydrophilicity on their surface. All the morphological, magnetic, colloidal and cytotoxic characteristics of the resulting core–shells were studied thoroughly. Their heating induction ability was studied to predict their possible use in hyperthermia therapy of cancer. Specific absorption rate was found to be increased than that of bare MNPs. & 2014 Published by Elsevier B.V.
Keywords: Alkaline precipitation Fe3O4 Chitosan Oleic acid Adsorption Hyperthermia
1. Introduction Nanosized magnetic iron oxide nanoparticles such as magnetite coated with hydrophilic water soluble polymers have received increasing attention due to their wide ranging biomedical applications in drug delivery [1], resonance imaging (MRI) contrast agents [2], in high-gradient magnetic field separations [3], treatment of retinal detachment [4], in bio-catalysis [5], as magnetic bio-separations [6] etc. Owing to their biocompatibility and low toxicity, magnetite (Fe3O4) nanoparticles (NPs) are the most attractive candidates for use in human body. Magnetic NPs (MNPs) for biomedical applications should fulfill a variety of requirements, including: (i) superparamagnetic behavior at room temperature, in order to avoid particle aggregation; (ii) large saturation magnetization, so as to show a large response under the application of a magnetic field; (iii) a limiting size in the order of 20 nm for in vivo applications, and (iv) bio-compatibility, such that nanoparticles are usually coated with either biological or biocompatible molecules. In order to make MNPs suitable for in vivo applications, they are coated with various polymers like chitosan (CS) [7], oleic acid (OA) [8], starch [9], pullulan [10], dextran [11] etc. Out of these, OA is probably the most common small molecule which is complexed with magnetite [12]. OA possesses a non-polar hydrocarbon tail n
Corresponding author. Fax: þ 91 231 2601595. E-mail address: [email protected] (S.H. Pawar).
and a polar carboxylic acid head group. Carboxylate anions are known to coordinate with the surface of magnetite, presumably through a coordination of iron atoms with both the carboxylate oxygens. The polar head group anchored on the magnetite surface, the non-polar tail extends into solution, causing the magnetite to be hydrophobic and dispersible in organic solvents. However coating of MNPs with OA makes the particles only dispersible in organic solvents and this limits their use for medical applications [13]. Fe3O4 NPs modified with OA decrease degree of agglomeration and favor the generation of hydrogen bonding between Fe3O4 and CS, thus improve their interfacial combination. Considering biodegradability and toxicity, much attention have been paid to chitosan, since it possesses interesting properties such as biocompatibility, biodegradability, film forming ability, gelation characteristics and bioadhesion. Another advantage of CS is that the amine groups can offer a variety of active sites for further biofunctionalization [14]. In the present investigation, Fe3O4 nanoparticles were prepared by alkaline precipitation method and were surface modified with OA. The precursor used for the synthesis of Fe3O4 was ferrous chloride only. A very little literature is available on the synthesis of Fe3O4 using FeCl2 only as the sole precursor for the nanoparticles [15,16]. The as-formed NPs were used for surface modification using OA which is hydrophobic in nature. Hence in order to make it suitable for biomedical applications, OA-Fe3O4 MNPs were further coated with CS, a biopolymer having hydrophilic nature. Bare (Fe3O4), OA coated (OA-Fe3O4) and CS-coated OA-Fe3O4 (CS-OA-Fe3O4) nanoparticles were characterized further for their
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structural, morphological and magnetic properties in order to investigate the role of coating on particle. The resulting particles were well dispersed in water and studied for their heating induction effect so as to study their possible use in hyperthermia application.
analysis by XRD Philips PW-3710 diffractometer using CuKα radiation in the 2θ range from 0 to 100°. The XRD patterns were evaluated by X′pert high score software and compared with JCPDS card. The average crystallite size (t) was calculated from the diffraction line-width of XRD pattern, based on Scherrer′s relation:
t = 0.9λ /β cos θ 2. Materials and method 2.1. Materials Ferrous chloride (FeCl2 4H2O), HCl, acetic acid, acetone and NaOH were procured from HiMedia, Mumbai (Maharashtra), India. Methanol, chitosan and oleic acid were purchased from SigmaAldrich, USA. Double distilled water was used throughout the procedure. 2.2. 2 Experimental 2.2.1. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized via alkaline precipitation [17,18]. The brief procedure for preparation of MNPs is as follows: 2 g FeCl2 4H2O was dissolved in 50 mL 1 M HCl by heating upto 70 °C. 50 mL 3 M NaOH was added to it at 60 °C drop by drop with constant stirring. A black precipitate gets formed. This is nothing but magnetite (Fe3O4) NPs. The possible reaction taking place is showed below:
3 FeCl2 ·4H2 O + 6 NaOH + ½ O2 → Fe 3 O4 + 6 NaCl + 15 H2 O
(1)
The precipitate is allowed to settle down. The rate of settling is increased by applying external magnetic field. The precipitate is then separated, washed with D/W till the neutral pH, dried at RT and used further studies. The particles studied for various characters using XRD, SEM, TEM, FTIR, VSM and temperature susceptibility. 2.2.2. Surface modification of Fe3O4 nanoparticles with OA As-formed Fe3O4 MNPs were used for coating procedure. The particles were well dispersed in 200 ml methanol by ultrasonication. 50 ml OA was added while constant stirring at 80 °C. OA-Fe3O4 particles were filtered through Whatman filter paper no.1, washed three times with distilled water. The OA-Fe3O4 particles were separated from the filter paper using acetone. The particles were dried at room temperature to evaporate all the acetone. These particles were termed as OA-Fe3O4 NPs. 2.2.3. Coating of CS on OA-Fe3O4 NPs As-formed OA-Fe3O4 MNPs were used for coating procedure. The were dispersed in 1% CS solution in 2% acetic acid. The mixture was ultrasonicated for 30 min. After ultrasonication, the CS-coated Fe3O4 NPs (CS-OA-Fe3O4) allowed to settle, washed with distilled water 3 times to remove excess CS, separated and dried at 50 °C. These OA-particles were also thoroughly studied for their structural, morphological and magnetic characters. The coating of CS on Fe3O4 MNPs was confirmed by FTIR spectra and TG-DTA results. 2.3. Characterizations Bare, OA-Fe3O4 and CS-OA-Fe3O4 MNPs were studied for their structural, morphological and magnetic characters using XRD, FTIR, EDAX, SEM, TEM, VSM and tTemperature susceptibility. In order to use the particles for hyperthermia therapy, they were studied for their heating induction ability. The XRD pattern of MNPs drop coated and air-dried on the glass substrate was recorded to study the structural and phase
(2)
Where, β is the full width at half maximum (fwhm). TEM images were used to determine the morphology and size of the MNPs. For this purpose, the colloidal solution of the MNPs was transferred on to a carbon coated copper grid and allowed to air dry. The grid was then scanned using Philips CM200 model Transmission electron Microscope, operating voltage 20–200 kV with resolution 2.4 Å. The compositional analysis was done by Energy-dispersive analysis of X-ray spectroscopy (EDAX, JEOL JSM 6360). The Perkin-Elmer spectrometer, (Model no.783, USA) was used to get FT-IR spectra of MNPs in the range from 450 to 4000 cm 1 using KBr pellets to check the possible interaction of OA-Fe3O4 with chitosan. The magnetization measurements were performed on a superconducting quantum interference device (SQUID) magnetometer to investigate the saturation magnetization (Ms), blocking temperature (TB) and Curie temperature (TC). The measurements include field dependent hysteresis loops, (M–H), at two different temperatures 100 and 300 K with applied field range from 0 to 71 103 Oe. Induction heating of Fe3O4 nanoparticles for hyperthermia application was performed in a plastic micro centrifuge tube (1.5 ml) using an induction heating unit (Easy Heat 8310, Ambrell; UK) with a 6 cm diameter (4 turns) heating coil. To keep the temperature of the coil at ambient temperature, a provision of water circulation in coils was provided. MNPs suspended in 1 ml of distilled water was placed at the center of the coil and the applied frequency was 265 kHz. Particles are dispersed in water with a concentration ranging from 2, 5 and 10 mg ml 1 and ultrasonicated for 20 min to achieve a good dispersion of the NPs in carrier fluid. Samples were heated for 10 min with the desired current (200–400 A). For the conducted experiments, the magnetic field was calculated from the relationship:
H=
1.257ni in Oe L
(3)
where, n, i and L denote the number of turns, applied current and the diameter of the turn in centimeters, respectively. Calculated values of the magnetic field (H) at 200, 300 and 400 A were 167.6, 251.4 and 335.2 Oe (equivalent to 13.3, 20.0 and 26.7 kA m 1), respectively. Temperature was measured using an optical fiber probe with accuracy 0.1 °C.
3. Results and discussions Fig. 1(a) and (b) shows the powder XRD patterns for bare and OA-CS-coated Fe3O4 NPs respectively. The main characteristic peaks were obtained with the (hkl) values of (220), (311), (400), (422) and (511). These were then matched with the JCPDS file number 82-1533, which corresponds to Fe3O4 phase. Both the NPs show inverse spinel structure. The crystallite sizes of NPs were calculated from FWHM of the most intense peaks using the Debye–Scherrer formula. The crystallite sizes obtained were 20 nm and 10 nm for bare and OA-CS-coated MNPs respectively. However, the peaks became broad and intensity decreased after capping of Fe3O4 with OA-CS and hence it can be clearly stated that the particle size decreased after coating procedure. The coating of amorphous OA-CS polymer on crystalline Fe3O4 may induce
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Fig. 1. XRD patterns obtained from bare Fe3O4 (a) and CS-OA-Fe3O4 (b) MNPs.
Fig. 3. Thermogravimetric spectra of bare Fe3O4 and CS-OA-Fe3O4 MNPs in nitrogen with a scanning rate of 10 °C min 1 up to 600 °C.
microstrain which resulted in broadening of peaks in case of coated sample [19–21]. The Selected Area Electron Diffraction (SAED) patterns for bare and coated NPs are shown in Fig. 2(a) and (b) respectively. It shows bright ring patterns indicating polycrystalline nature of the MNPs, as indicated by XRD patterns. The ring pattern corresponds to (220), (311), (400), (422) and (511) planes which can be clearly seen in XRD results. TGA provides a quantitative evidence of the coating of OA-CS on NPs. In this experiment, the MNPs are heated to 600 °C under flowing N2 and changes in mass loss of organic material were recorded. This is an extremely valuable technique for surface characterization of NPs. Typically, ligands that are bound more strongly desorb at higher temperatures. The weight losses of the bare Fe3O4, CS-OA-Fe3O4 MNPs were measured and shown in Fig. 3. Since the TG was performed under N2 atmosphere, the oxidation of coated and uncoated MNPs surface was greatly reduced. The weight loss of Fe3O4 NPs ( 5%) occurred upto temperature 200 °C was ascribed to the evaporation of water. For the CS-OA-Fe3O4 MNPs, after a gradual loss of water molecules in the polymer matrix, a great weight loss started after 2000 °C at which the decomposition of coating molecules occurs. CS decomposes at the temperature 200–300 °C [7]. Further degradation beyond 300 °C corresponds to decomposition of OA [13]. After the OA was decomposed completely, the residual substance mostly is magnetic particles. The percentage of CS in the coated MNPs was 8.3 wt% and that of OA was 27.38 wt% revealed in the TGA curve.
The size and shapes of the MNPs before and after surface modification were observed using TEM. The TEM images of Fe3O4 and CS-OA-Fe3O4 are shown in Fig.4. Bare Fe3O4 NPs were highly agglomerated with particle size 22.8 75.1 nm while OA-CS coated NPs were well dispersed with particle size 16.5 7 4.3 nm. Bare MNPs have strong magnetic dipole–dipole interaction and hence are attracted strongly and form big clusters causing bigger particle size. After efficient coating procedure, a non-magnetic layer is formed on surface of each particle which prevents increase in particle size. This may the reason why particle size reduces after coating procedure. These results are comparable with the XRD results. After surface modification, the particles maintained their original spherical shapes without any deformation or growth. The particles obtained were with less degree of aggregation and were well dispersed. Magnetic properties of the MNPs were studied using their M–H curves. M–H curves of bare and coated Fe3O4 MNPs at 300 K are shown in Fig. 5. The Saturation Magnetization (Ms), Coercivity (Ce) and Remenance (Mr) values calculated from the M–H curves for both bare and coated MNPs are given in Table 1. The graphs clearly show superparamagnetic nature of both the NPs at 300 K as Ce and Mr values are very negligible. Superparamagnetic behavior of MNPs at room temperature is very useful in in vivo applications as they do not retain magnetization before and after exposure to an external magnetic field, reducing the probability of particle aggregation due to magnetic dipole attraction [22]. This can be ascribed to the small size of NPs which were smaller than the
Fig. 2. Selected Area Electron Diffraction (SAED) patterns of bare Fe3O4 and CS-OA-Fe3O4 MNPs in (a) and (b) respectively.
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Fig. 4. TEM images of (a) bare Fe3O4 and (b) CS-OA-Fe3O4 MNPs.
Fig. 5. M–H loops of bare Fe3O4, CS-OA-Fe3O4 MNPs at 300 K.
Table 1 The Saturation magnetization (Ms), Coercivity (Ce) and Remenance (Mr) values calculated from the M–H loops for both bare and CS-OA-Fe3O4 MNPs.
Ms (emu/g) Mr (emu/g) Ce (Oe) Mr/Ms
Fe3O4
CS-OA-Fe3O4
36.98 3.13 0.05 0.08
29.71 1.95 0.05 0.06
superparamagnetic critical size (20 nm) [23]. Ms value of bare NPs is observed to be 36.98 emu/g which is small compared to that of theoretical value of bulk Fe3O4 (Ms ¼92 emu/g) since Ms generally decreases with a decrease in magnetic particle size [26]. Ms has been reported to decrease as the particles size of Fe3O4 decreases below 30 or 20 nm, due to finite size effect [22,24]. The reduced magnetization in coated MNPs could also result from the small particle surface effect [25] which refers to the disordered alignment of surface atomic spins induced by reduced coordination and broken exchange between surface spins [26]. This surface effect is more prominent in small particles as the ratio of surface atoms to the interior atoms increases with a decrease in particle size. The zeta potential values and hydrodynamic diameters of bare and coated NPs suspensions in water with respect to pH 2 to 10 are shown in Fig. 6. The zeta potentials of bare NPs at pH 2 to 10 were 17.35, 12.02, 11.00, 10.33, 5.92, 12.00 and 25.6, 31.6 and 32.87 mV and that of coated NPs were 40.13, 32.69, 18.90, 12.49, 10.23, 1.2, 47.87, 54.43 and 58.52 mV, respectively. The
zeta potential of coated particles is more positive in the range of pH 2–6, as compared to bare indicating that the positive charges on the coated NPs increase with a decrease in pH. At higher pH, again, zeta potential of coated NPs is more negative than the bare ones. The isoelectric points (pI) for bare and coated nanoparticles were found to be around 6.7 and 7 respectively. The bare nanoparticles possess negative charge at physiological pH which was in agreement with the literature [27]. Naturally, at their respective pI values, both MNPs show highest hydrodynamic diameters due to highest degree of agglomeration. The bare MNPs have larger size due to higher degree of agglomeration and consequently show lager hydrodynamic diameter than the coated MNPs. Though coated MNPs are said to be more hydrophilic, they show smaller hydrodynamic diameter due to their much smaller size, as shown by TEM images. MTT assay was performed in order to check cytotoxic effect of MNPs. The cytotoxicity study of both, bare and coated nanoparticles was done on L929 and cell line with different concentrations of nanoparticles and the obtained data is shown in Fig. 7. The L929 cell line was incubated with nanoparticles for 48 h with the concentrations of 0.1, 0.5, 1.0, 1.5 and 2.0 mg mL 1 at 37 °C in 5% CO2 atmosphere. The relative cell viability (%) compared with control well containing cells without nanoparticles are calculated by the equation: [A]tested / [A]control 100. Fig. 7 shows the cell viability after incubation with different concentrations of both bare and coated Fe3O4 nanoparticles. It clearly reveals that after 48 h, bare MNPs started to exhibit their cytotoxicity while CS-OA-coated MNPs still showed almost 100% viability. Therefore coated MNPs are more suitable for in vivo applications than the bare MNPs, owing to their lower cytotoxicity.
4. Conclusion This study demonstrates the effect of capping of OA-CS on the surface behavior of Fe3O4 MNPs. XRD and Zeta potential studies confirmed coating of Fe3O4 MNPs with OA-CS. XRD pattern proved inverse spinel structure of the particles. TEM images showed that the particles are monodispersed, spherical-shaped having diameter of 22.8 75.1 nm and 16.5 7 4.3 nm in case of bare and coated particles respectively. SAED and XRD proved polycrystalline nature of particles. VSM showed that coating procedure decreased magnetization of the Fe3O4 NPs. Both the MNPs are superparamagnetic at room temperature with negligible Ce and Mr. Zeta potential studies showed a high colloidal stability of both the suspensions in water as base media at extreme pH values. Cell
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Fig. 6. Hydrodynamic diameter and zeta potential as a function of pH for (a) Fe3O4 and (b) CS-OA-Fe3O4 MNPs dispersed in water.
References
Fig. 7. Cytotoxicity profiles of MNPs for 48 h on L929 cell line at different concentrations (0.1, 0.5, 1.0, 1.5 and 2.0 mg mL 1).
viability assay of both the MNPs showed very low cytotoxic effect on L929 cell line even after 48 h incubation period. Thus hydrophilicity can be rendered to hydrophobic surface of OA-Fe3O4 NPs using CS. Moreover, synthesized MNPs are suitable for hyperthermia therapy applications owing to their smaller size, superparamagnetic behavior at room temperature, higher magnetization values, high colloidal stability and low cytotoxicity.
Acknowledgements The authors are thankful to Dr. S. D. Sartale, Pune University, Pune (Maharashtra), India for providing SAED patterns. The authors are grateful to DST, India (DST no: SR/NM/NS-126/2010(G) for its financial support.
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