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Journal of Nanoscience and Nanotechnology Vol. 11, 2730–2734, 2011

Synthesis of Hydrophilic Superparamagnetic Magnetite Nanoparticles via Thermal Decomposition of Fe(acac)3 in 80 Vol% TREG + 20 Vol% TREM Dipak Maity1 , Pallab Pradhan2 , Prashant Chandrasekharan3 , S. N. Kale4 , Borys Shuter5 , Dhirendra Bahadur2 , Si-Shen Feng3 , Jun-Min Xue1
∗ , and Jun Ding1
∗ RESEARCH ARTICLE

1

2

Department of Materials Science and Engineering, NUS 117574, Singapore Department of Metallurgical Engineering and Materials Science, IIT Bombay 400076, India 3 Department of Chemical and Biomolecular Engineering, NUS 117574, Singapore 4 Department of Electronic-Science, Fergusson College, Pune 411004, India 5 Department ofDelivered Diagnostic Radiology, NUS 119260, by Ingenta to: Singapore

Institute of Molecular and Cell Biology In this paper, we report single step synthesis of hydrophilic superparamagnetic magnetite nanoparIP : 137.132.123.69 and their characterization of the properties relevant to biomedical ticles by thermolysis of Fe(acac)3 Sat, 11 Jun 2011 11:52:53 applications like hyperthermia and magnetic resonance imaging (MRI). Size and morphology of the particles were determined by Transmission electron microscopy (TEM) while phase purity and structure of the particles were identified by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Magnetic properties were evaluated using vibrating sample magnetometer (VSM) and superconducting quantum interference device (SQUID) measurements. The as prepared nanoparticles were found to be superparamagnetic with the blocking temperature of 136 K and were easily suspendable in water. Cytotoxicity studies on human cervical (SiHa), mouse melanoma (B16F10) and mouse primary fibroblast cells demonstrated that up to a dose of 0.1 mg/ml, the magnetite nanoparticles were nontoxic to the cells. To evaluate the feasibility of their uses in hyperthermia and MRI applications, specific absorption rate (SAR) and spin–spin relaxation time (T2) were measured respectively. SAR has been calculated to be above 80 Watt/g for samples with the iron concentration of 5–20 mg/ml at 10 kA/m AC magnetic field and 425 kHz frequency. r2 relaxivity value was measured as 358.4 mM−1 S−1 which is almost double as compared to that of the Resovist®, a commercially available MRI contrast agent. Thus the as-prepared magnetite nanoparticles may be used for hyperthermia and MRI applications due to their promising SAR and r2 values.

Keywords: Magnetite, Superparamagnetic, Cytotoxicity, MRI, Hyperthermia.

1. INTRODUCTION Superparamagnetic magnetite nanoparticles have attracted much attention in the bio-medical field such as in hyperthermia treatment of cancer and as magnetic resonance imaging (MRI) contrast enhancement agent.1–4 Thermal decomposition of Iron(III) acetylacetonate, Fe(acac)3 in presence of oleic acid and oleylamine is very promising for the synthesis of superparamagnetic magnetite (Fe3 O4
nanoparticles.5 6 However, the obtained nanoparticles are only suspendable in organic solvent which makes them inappropriate for biomedical applications. ∗

Authors to whom correspondence should be addressed.

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Therefore, several groups have tried to developed the thermal decomposition method to synthesize hydrophilic magnetite nanoparticles,7 8 which can be suspended in aqueous medium and thereby can be used in biomedical applications. Here, we have prepared hydrophilic magnetite nanoparticles by the thermolysis of Fe(acac)3 in a mixture of Tri(ethylene glycol) (TREG) and Triethanol amine (TREM). The TREM has been used along with the TREG to efficiently control the particle growth and thereby improving the monodispersity of the nanoparticles. In vitro biocompatibility, MRI and hyperthermia studies of the as prepared nanoparticles have been performed to evaluate the feasibility of their use in biomedical applications. 1533-4880/2011/11/2730/005

doi:10.1166/jnn.2011.2693

Maity et al.

Synthesis of Hydrophilic Superparamagnetic Magnetite Nanoparticles via Thermal Decomposition of Fe(acac)3

2. EXPERIMENTAL DETAILS

optical fibre based temperature probe (Luxtron, USA). The SAR was calculated using the following equation:

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Typically, 2 mmol of Fe(acac)3 was dissolved in a mixT 1 ture of 5 ml of TREM and 20 ml of TREG (1:4 v/v). The SAR = C (1) resulting solution was magnetically stirred under a flow of t mFe  argon. The solution was dehydrated at 120 C for 1 h, and where C is the specific heat of solvent (here Cwater = then quickly heated to reflux and kept at this temperature 418 J/g  C), T /t is the initial slope of the timefor 2 h. The black solution was cooled to room temperdependent temperature curve and mFe is weight fraction of ature and the nanoparticles were precipitated by addition magnetic element (i.e., Fe) in the sample.9 10 of ethyl acetate. Then, the nanoparticles were washed sevT2 relaxation time of aqueous suspension of magnetite eral times using 1:2 (v/v) ethanol and ethyl acetate mixnanoparticles with the Fe concentration of 0.0125–0.4 mM ture followed by centrifugation. Finally, aqueous magnetic was measured at 25  C using a Siemens Symphony 1.5T fluid was obtained suspending the as-prepared nanopartiMRI scanner with a head coil. The T2 relaxation time was cles in water medium. TEM was used to characterize size computed using in-house software (MATLAB V7) by fitand morphology of the nanoparticles. Structure and phase ting appropriate exponential functions. Based on the T2 purity of the nanoparticles were identified by XRD and values, the specific relaxivity (r2
, which are a measure of FTIR. Magnetic properties of the nanoparticles were studthe induced change of the spin–spin relaxation rate (T2−1
ied by VSM and SQUID measurements. per unit concentration, were calculated. In vitro biocompatibility of the nanoparticles were evalDelivered by Ingenta to: uated by cytocompatilibility studies with SiHa (human Institute of Molecular and Cell Biology cervical), B16F10 (mouse melanoma) and mouse primary IP : 137.132.123.69 3. RESULTS AND DISCUSSION fibroblasts isolated from skin epithelium of Swiss albino Sat, 11 Jun 2011 11:52:53 mice using MTT assay. The cells were seeded at den3.1. Structural Characterization sity of 1 × 105 cells/ml in 96-well plates. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) Our XRD study showed that all X-ray diffraction peaks supplemented with 10% Fetal bovine serum, 2 mM Lcould be identified with the magnetite spinel structure. glutamine, 100 u/ml of penicillin/streptomycin followed Figure 1 depicts the FTIR spectra for the as-prepared magnetite nanoparticles. The peaks at about 2962–2809, by incubation at 37  C in 5% CO2 for overnight. Next 1632–1600, 1485–1340, 1060, and 910 cm−1 are attributed day, the cells were treated with fresh medium containto C–H stretching, O–H stretching, C–H bending, C–O ing the magnetite nanoparticles at different concentrations stretching and O–H bending vibrations, respectively arise ranging from 0.00625–0.4 mg Fe/ml of culture medium from TREG and/or TREM adsorbed to the particle in triplicates and the plates were further incubated for surface.11 12 The broad band between 3600 and 3000 cm−1 24 h. Thereafter, the culture medium containing magcentered at about 3300 cm−1 is due to the O–H stretchnetite nanoparticles was removed and 5 mg/ml MTT soluing vibration attributed also for the TREG and/or TREM tion was added and the cells were incubated for 4 h at and water adsorbed to the particle surface. In addition, the 37  C in 5% CO2 incubator. Then, dimethyl sulfoxide strong absorption band at about 580 cm−1 is due to Fe–O was added and stirred to dissolve insoluble purple colstretching vibration of the Fe3 O4 nanoparticles.13 ored formazan crystal. The optical density (OD) of colored formazan solution was determined by measuring the absorbance at 570 nm in ELISA microplate reader (BioRad, Hercules, CA). The mean optical density (OD) value of three wells (for each concentration of nanoparticles dosed to the cells) was used for assessing the cell viability that was expressed as percentage of control [% Cell viability = OD of nanoparticle-treated cells/OD of control cells × 100]. AC field induced heating ability of the as prepared magnetite nanoparticles was evaluated by specific absorption rate (SAR) determined from the time-dependent calorimetric measurements using a RF generator. One milliliter of aqueous suspension of magnetite nanoparticles with the Fe concentration of 5–20 mg/ml were subjected to 4–10 kA/m AC field using a RF generator (CLF-5000, Comdel, USA) operating at 425 kHz frequency and time dependent temperature rise was monitored for different times using an Fig. 1. FT-IR spectra of the magnetite nanoparticles.

RESEARCH ARTICLE

Synthesis of Hydrophilic Superparamagnetic Magnetite Nanoparticles via Thermal Decomposition of Fe(acac)3

Maity et al.

Fig. 4. ZFC and FC curves of the magnetite nanoparticles under an Fig. 2. TEM image of the magnetite nanoparticles (scale bar −20 nm). applied field of 100 Oe. Inset is the HRTEM image of a single magnetite nanocrystal (scale bar − 5 nm). Delivered by Ingenta to:

cooled (ZFC–FC) magnetization of the magnetite nanopar-

Institute of Molecularticles and Cell Biology measured by SQUID under an applied field of IP : 137.132.123.69 3.2. Size and Morphology Study 100 Oe. The feature of the ZFC–FC curves is also indiSat, 11 Jun 2011 11:52:53 cating that the nanoparticles are of SPM in nature.15 The Figure 2 shows the TEM image of well dispersed asprepared magnetite nanoparticles in an aqueous media. It can be seen that the particles are relatively monodisperse with an average size of about 16 nm. Inset of Figure 2 is showing the HRTEM image of a single magnetite nanocrystal.

ZFC curve reached the maximum at about 136 K, which corresponded to the blocking temperature (TB
of the sample. Above TB the sample is superparamagnetic and below is ferromagnetic. 3.4. Cytotoxicity Studies

3.3. Magnetic Properties Figure 3 shows the magnetization (M–H) curve of the magnetite nanoparticles at room temperature. The saturation magnetization (MS ) of the magnetite nanoparticles is found to be 68 emu/g. Zero coercivity and zero remanence on the M–H curve indicate that the nanoparticles are superparamagnetic (SPM) in nature.14 Figure 4 shows the temperature dependence of the zero-field cooled/field

Fig. 3.

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Magnetization (M–H ) curves of the magnetite nanoparticles.

The cytotoxicity profile of the magnetite nanoparticle on the SiHa, B16F10 and mouse primary fibroblast cells have been shown in Figure 5. The observation reveals that B16F10 and mouse primary fibroblast cells do not show any cytotoxicity with the magnetite nanoparticles in the range of 0.00625–0.4 mg Fe/ml doses. However, in the range of 0.1–0.4 mg Fe/ml concentration, the cell viability is decreased slightly for SiHa cell lines (∼90% viability as

Fig. 5. Cytotoxicity profile of the magnetite nanoparticles on SiHa (human cervical), B16F10 (mouse melanoma) and mouse primary fibroblasts cells.

J. Nanosci. Nanotechnol. 11, 2730–2734, 2011

Maity et al.

Synthesis of Hydrophilic Superparamagnetic Magnetite Nanoparticles via Thermal Decomposition of Fe(acac)3

SAR values vary nearly with H2 as reported in literature.16 The heating effects of the superparamagnetic nanoparticles under alternating fields are due to Neel and Brownian loss which arise from rotation of the magnetization vector and the nanoparticles itself under AC magnetic field, respectively.3 SAR values have been calculated to be 84.3, 83.2, and 87.6 Watt/g of Fe for the samples with iron concentration of 5, 10, and 20 mg/ml, respectively while that have been found to be increased from 16.8 to 83.2 Watt/g with the increase of the AC field from 4.1 to 10 kA/m. Clearly, the use of higher Fe concentration and much higher AC field generates the high SAR value, suggesting that as-prepared magnetite nanoparticles may be used for hyperthermia treatment. 3.6. MRI Studies

Figure 8 shows the T2 relaxation rate (1/T2
of the samples compared to control), which could be due to mild cytotoxic by Ingenta containingto: as-prepared magnetic nanoparticles for different Delivered effect at higher concentrations in this cell line. it iron Institute of Thus, Molecular andconcentrations. Cell BiologyIt can be seen that the relaxation rates is evident from the cytotoxicity study that the as-prepared varied linearly with the iron concentration according to the IP : 137.132.123.69 magnetite nanoparticles are cytocompatible up to 11 the Jun iron 2011 following equation:7 Sat, 11:52:53 concentration of 0.1 mg/ml with SiHa, B16F10 and mouse primary fibroblast cells. 1/T2 = 1/T20 + r2 F e (2) 3.5. Hyperthermia Studies Figure 6 shows the time dependent temperature rise of aqueous suspension of the magnetite nanoparticles with the iron concentration of 5–20 mg/ml on exposure to 10 kA/m AC field at 425 kHz frequency. A systematic increase in the temperature with increase in the concentration of the dispersed nanoparticles is observed as a function of time and AC field. Figure 7 depicts the field dependent SAR values of the aqueous magnetic fluid with 10mg/ml Fe concentration at 4–10 kA/m AC field and 425 kHz frequency. It can be observed from the graph that the

Fig. 7. Field dependent SAR values of 1 ml sample with 10 mg/ml iron concentration on exposure to 4–10 kA/m AC field.

J. Nanosci. Nanotechnol. 11, 2730–2734, 2011

where 1/T2 is the observed relaxation rate in the presence of magnetite nanoparticles, 1/T20 is the relaxation rate of pure water, [Fe] is the concentration of magnetite nanoparticles, and r2 is the transverse relaxivity, which represent the efficiency of the magnetite nanoparticles as a contrast agent shortens the proton relaxation times. The r2 value of 358.4 mM−1 S−1 of the as-prepared magnetite nanoparticles is almost double as compared to that (189 mM−1 S−1
of the commercial iron oxide (Resovist®, Schering).17 18 This difference may be due to the higher crystallinity or bigger size and thereby higher magnetization of the nanoparticles as they are prepared by high temperature decomposition

Fig. 8. T2 relaxation rate (1/T2
plotted against the Fe concentration of aqueous suspension of the magnetite nanoparticles.

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Fig. 6. Time dependent temperature rise of 1 ml sample with different iron concentration on exposure to 10 kA/m AC field at 425 kHz frequency.

Synthesis of Hydrophilic Superparamagnetic Magnetite Nanoparticles via Thermal Decomposition of Fe(acac)3

Maity et al.

RESEARCH ARTICLE

technique. The higher relaxivity value suggests that asprepared magnetite nanoparticles can act as MRI T2 contrast agents.

5. S. Sun and H. Zeng, J. Am. Chem. Soc. 124, 8204 (2002). 6. D. Maity, S.-G. Choo, Ji. Yi, J. Ding, and J.-M. Xue, J. Magn. Magn. Mater. 321, 1256 (2009). 7. Z. Li, H. Chen, H. Bao, and M. Gao, Chem. Mater. 16, 1391 (2004). 8. J. Wan, W. Cai, X. Meng, and E. Liu, Chem. Commun. 47, 5004 4. CONCLUSIONS (2007). 9. S. Vasseur, E. Duguet, J. Portier, G. Goglio, S. Mornet, E. Hadová, The as-prepared hydrophilic magnetite nanoparticles are K. Knížek, M. Maryško, P. Veverka, and E. Pollert, J. Magn. Magn. single crystalline, monodisperse and superparamagnetic in Mater. 302, 315 (2006). nature. Cytotoxicity studies show that the nanoparticles are 10. I. Hilger, K. Frühauf, W. Andrä, R. Hiergeist, R. Hergt, and W. A. Keiser, Acad Radio 9, 198 (2002). biocompatible up to the iron concentration of 0.1 mg/ml. 11. D. Maity and D. C. Agrawal, J. Magn. Magn. Mater. 308, 46 The significant temperature rise of the magnetite nanopar(2007). ticles upon exposure of AC magnetic field upto 10 kA/m 12. J. R. Dyer, Applications of Absorption Spectroscopy of Organic at 425 kHz frequency and larger r2 value confirms their Compounds, Prentice-Hall, Inc., Englewood Cliffs, New Jersey potential applicability for magnetic hyperthermia and MRI (1965). 13. R. M. Cornell and U. Schwertmann, The Iron Oxides: Structure, applications. Properties, Reactions, Occurrence and Uses, 2nd edn., Wiley–VCH, Weinheim (2003). 14. F. Montagne, O. Mondain-Monval, C. Pichot, H. Mozzanega, and References and Notes A. Elaissari, J. Magn. Magn. Mater. 250, 302 (2002). 15. C. Liu,to: B. Zou, A. J. Rondinone, and Z. J. Zhang, J. Am. Chem. 1. P. Tartaj, M. P. Morales, T. Gonzalez-Carreno, S.Delivered Veintemillas-by Ingenta Soc. 122, 6263 (2000). Verdaguer, and C. J. Serna, J. Magn. Magn. Mat. 290–291, 28 Institute of Molecular16. andR. Cell Biology Hergt, R. Hiergeist, M. Zeisberger, G. Glöckl, W. Weitschies, (2005). IP : 137.132.123.69 L. P. Ramirez, I. Hilger, and W. A. Kaiser, J. Magn. Magn. Mater. 2. P. Pradhan, J. Giri, G. Samanta, H. D. Sarma, K. P. Mishra, 280, 358 (2004). J. Bellare, R. Banerjee, and D. Bahadur, J. Biomed.Sat, Mater.11 Res.Jun Part 2011 11:52:53 17. C. Corot, P. Robert, J. Idée, and M. Port, Adv. Drug Deliv. Rev. B: Appl. Biomater. 81B, 12 (2007). 58, 1471 (2006). 3. S. Mornet, S. Vasseur, F. Grasset, and E. Duguet, J. Mater. Chem. 18. P. Reimer, C. Marx, E. J. Rummeny, M. Müller, M. Lentschig, 14, 2161 (2004). T. Balzer, K. H. Dietl, U. Sulkowski, T. Berns, K. Shamsi, and P. E. 4. A. Jordan, R. Scholz, P. Wust, H. Fahling, and R. Felix, J. Magn. Peters, J. Magn. Reson. Imaging 7, 945 (1997). Magn. Mater. 201, 413 (1999).

Received: 26 June 2009. Accepted: 6 October 2009.

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