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Novel synthesis of superparamagnetic magnetite nanoclusters for biomedical applications†

Downloaded by National University of Singapore on 23 September 2011 Published on 15 August 2011 on http://pubs.rsc.org | doi:10.1039/C1JM11982F

Dipak Maity,‡a Prashant Chandrasekharan,b Pallab Pradhan,c Kai-Hsiang Chuang,de Jun-Min Xue,a Si-Shen Feng*b and Jun Ding*a Received 6th May 2011, Accepted 18th July 2011 DOI: 10.1039/c1jm11982f In this work, we report a novel method of single step facile synthesis of magnetite nanoclusters via thermal decomposition of iron(III) acetylacetonate in a liquid mixture of tri(ethylene glycol) (TREG) and triethanolamine (TREA). The optimized ratio of TREG : TREA has been found to be 1 : 4 (v/v) for the formation of well dispersed MNC-14 magnetite nanoclusters with high Ms values (75 emu g
1) as compared to MNC-10 magnetite nanoparticles (63 emu g
1). The MNC-14 nanoclusters were found to be nontoxic to MCF-7 cells up to an iron concentration of 10 mg ml
1. The MNC-14 nanoclusters yielded high specific absorption rate (SAR) values (500 Watt g
1 at 89 kA m
1 AC magnetic field and 240 kHz frequency) and thus qualified for their possible use in magnetic hyperthermia treatment, while MNC-10 nanoparticles possess a much lower SAR value of 135 Watt g
1. In vitro magnetic hyperthermia experiments (using the MNC-14 nanoclusters with the iron concentration of 0.5 mg ml
1) showed about 74% loss in viability of MCF-7 breast cancer cells indicating that they are a very suitable candidate for magnetic hyperthermia treatment of cancer. The r2 and r2* relaxivity values of MNC-14 nanoclusters (294.99 and 450.05 s
1 mM
1) as measured by a 9.4 T MRI scanner were higher than those for the MNC-10 nanoparticles (205.6 and 309.2 s
1 mM
1). The MNC-14 nanoclusters also showed very promising in vivo tumor imaging. Thus, the newly synthesized novel MNC-14 nanoclusters possess great potential in clinical MRI and magnetic hyperthermia applications and may be used simultaneously for cancer diagnosis and therapy.

1. Introduction Superparamagnetic iron oxide (particularly magnetite (Fe3O4) and maghemite (g-Fe2O3)) nanoparticles have been studied extensively for biomedical applications such as magnetic separation,1–3 drug delivery,4–6 hyperthermia treatment for tumors7–9 and contrast agents in magnetic resonance imaging (MRI).10–12 Recently, many groups have also found that the superparamagnetic iron oxide nanoclusters are very promising in biological applications.13–16 In all these studies, the iron oxide

a Department of Materials Science and Engineering, National University of Singapore, Singapore 117574. E-mail: [email protected] b Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117574. E-mail: [email protected] c Department of Biomedical Engineering, University of Texas at Austin, USA 78712 d Laboratory of Molecular Imaging, Singapore Bioimaging Consortium, A*STAR, Singapore 138667 e Clinical Imaging Research Center, A*STAR-National University of Singapore, Singapore 117456 † Electronic supplementary information (ESI) available. See DOI: 10.1039/c1jm11982f ‡ Now at the Department of Chemistry (E-mail: dipakmaity@gmail. com)

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nanoclusters have been synthesized using the traditional approach which relied on the aqueous coprecipitation of ferrous and ferric salts using a base such as ammonium hydroxide or sodium hydroxide and then stabilized with polymer,13 oleic acid14 and citrate ligands.15,16 The main problem in this approach is the uncontrolled aggregation of particles which eventually leads to the polydisperse size of the nanoclusters and secondly, the obtained nanoclusters are semi-amorphous in nature due to the lower reaction temperature (below 80  C) which eventually leads to very low saturation magnetization (Ms) (below 40 emu g
1) of the nanoclusters. Thus, the synthesis of stable colloidal magnetic nanoclusters with high magnetization is very challenging. To date, the thermal decomposition method is a very promising technique to fabricate high-quality monodisperse superparamagnetic iron oxide nanoparticles with high Ms (above 60 emu g
1).17–20 Typically, this method involves decomposition of iron(III) acetylacetonate, Fe(acac)3, in a high-boiling temperature solvent in the presence of stabilizing surfactants and reducing agents such as oleic acid and oleylamine. However, the obtained magnetite nanoparticles are organic soluble which makes them inappropriate for bio-medical applications. Therefore, many groups have further developed the thermal decomposition method to directly synthesize water-soluble magnetic J. Mater. Chem., 2011, 21, 14717–14724 | 14717

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nanoparticles.21,22 Recently, we have also reported the synthesis of water stable superparamagnetic magnetite nanoparticles by the high temperature decomposition of Fe(acac)3 in TREG medium in which the TREG has been used for a triple role as high-boiling solvent, reducing agent and stabilizer to efficiently control the particle growth and prevent interparticle aggregation.23,24 Here, we report the synthesis of the magnetite nanoclusters by the high temperature decomposition of Fe(acac)3 in a liquid mixture of TREG and TREA. To the best of our knowledge, this is the first report on the synthesis of magnetite nanoclusters by the thermal decomposition method. The idea behind the addition of TREA solvent with the TREG is to reduce the stabilizing effect of the TREG so that clustering of nanoparticles happens either to reduce their surface energy or due to the strong interparticle attractive magnetic force. The ratio of TREG and TREA has been systematically varied from 1 : 0 to 0 : 1 (v/v) to optimize the ratio to obtain well dispersed magnetite nanoclusters with higher Ms. We hypothesize that due to clustering of multiple magnetic nanoparticles into a single magnetic nanocluster, the saturation magnetization (Ms) would be increased and thereby their heating efficiency under alternating current (AC) magnetic field would be better as compared to individual magnetic nanoparticles. Thus, the magnetite nanoclusters (MNC-14) were further investigated for their suitability in magnetic hyperthermia applications. The efficacy of the MNC-14 nanoclusters for in vivo imaging application has also been evaluated.

2. Experimental Absolute ethanol, ethyl acetate, tri(ethylene glycol) (TREG, 99%), triethanolamine (TREA, 99%) and iron(III) acetylacetonate (Fe(acac)3, 97%) were purchased from Sigma-Aldrich and used without further purification. 2.1.

dispersed in water and the other half was dried overnight in an oven at 40  C to obtain dry nanoparticles for further characterization. All the samples prepared are listed in Table 1.

2.2.

Characterization of magnetite nanoclusters

The structure of the as prepared samples was identified by X-ray diffraction (XRD, Bruker D8 Advance) and their morphology was determined using transmission electron microscopy (TEM, JEOL 2010). Colloidal stability was determined by dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern Instruments). Magnetic properties were measured using a vibrating sample magnetometer (VSM, Lakeshore, Model 665) and a superconducting quantum interference device (SQUID, Quantum Design, MPMS XL). In vitro cell viability. Cell viability studies were performed on the MCF-7 human breast cancer cells using MTT assay. MCF-7 mammalian breast cancer cells were seeded in 96-well plates (Costar, IL, USA) at the density of 5000 viable cells/well and incubated 24 hours to allow cell attachment. The cells were incubated for 24 h with media containing MNC-14 nanoclusters at concentrations ranging from 0.655 to 10 mg of Fe per ml. Wells without the nanoclusters treatment were used as control. 10 ml of MTT was added into the 96 well plates five hours prior to the time point. The cells were incubated till the time point. After incubation, the media solution was removed from the wells, leaving the precipitate. Dimethylsulfoxide (100 ml) was added to the wells to dissolve the formazan crystals which were formed. Finally, the absorbance in each well was measured using a microplate reader (GENios, Tecan, Switzerland) at 550 nm. The cell viability was then calculated (n ¼ 8) by: % Cell viability ¼

Absorbance of sample well  100 Absorbance of control well

Synthesis of magnetite nanoclusters

Typically, 2 mmol of Fe(acac)3 was dissolved in 20 ml of liquid mixture of TREG and TREA with 1 : 0, 1 : 1, 4 : 1, 1 : 1, 1 : 4 and 0 : 1 (v/v) ratios. The resulting mixture solution was magnetically stirred under a continuous flow of argon. The solution was dehydrated at 120  C for 1 h, and then quickly heated to reflux and maintained at the refluxing temperature (245–280  C) under the argon flow for 1 h. Thereafter, the black solution was cooled to room temperature and the nanoparticles were precipitated by addition of ethyl acetate. Finally, the obtained precipitates were washed several times with an ethanol and ethyl acetate (1 : 2 v/v) mixture followed by centrifugation at 8000 rpm. Finally, one half of the washed precipitates was

Magnetic hyperthermia study. The time-dependent calorimetric measurements of specific absorption rate (SAR) of the MNC nanoclusters were performed using a RF generator (EASYHEAT-5060, Ameritherm) operating at AC field. One millilitre of aqueous suspension of MNC-14 nanoclusters at concentrations ranging from 0.5 to 10 mg Fe per ml was subjected to 240 kHz frequency under 89 kA m
1 AC magnetic field and the time-dependent temperature rise was monitored for different times using an optical fibre based temperature probe (FLUOTEMP Series, FTP-LN2). The specific absorption rate (SAR) was calculated using the following equation:25 SAR ¼ C

Table 1 Different samples prepared using different TREG : TREA (v/v) ratios Samples

TREG : TREA (v/v)

Ms/emu g
1

MNC-10 MNC-41 MNC-11 MNC-14 MNC-01

1:0 4:1 1:1 1:4 0:1

63 66 70 75 80

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DT 1 Dt mFe

where C is the specific heat of solvent (here Cwater ¼ 4.18 J g
1  C), DT/Dt is the initial slope of the time-dependent temperature curve and mFe is the weight fraction of magnetic element (i.e. Fe) in the sample. For in vitro cellular hyperthermia, 1  106 exponentially growing MCF-7 cancer cells in DMEM cell culture medium containing 10% FBS and 1% antibiotic antimycotic solution were taken in 15 ml sterile polypropylene tubes and centrifuged This journal is ª The Royal Society of Chemistry 2011

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at 1000 rpm for 5 minutes to get cell pellets. The cells were re-suspended in 1 ml DMEM medium containing MNC-14 nanoclusters at 0.5 mg ml
1 Fe concentration and then subjected to an initial AC field of 89 kA m
1 using the RF generator operating at 240 kHz frequency. The temperature rise of the cell suspension was monitored using the optical fibre based temperature probe. After the temperature reached 45  C, the field was adjusted (by decreasing the field) to maintain the temperature of the cell suspension at about 45  C for a 1 h magnetic hyperthermia treatment. For comparison, same numbers of cells as above were treated using only the magnetic field (no MNC-14) and another with only the MNC-14 nanoclusters (no magnetic field) with 0.5 mg of Fe per ml concentration. Cells without any treatment were used as control. After the treatment, the cells were centrifuged and washed with PBS 2 times. Then, 2  104 cells were placed in a 96 well plate in 16 replicates followed by 48 h of incubation in 5% CO2 incubator. MTT assay was performed to evaluate the cell viability. For this purpose, the cell culture supernatants were replaced with 100 ml each of fresh medium containing 10 ml of 5 mg ml
1 MTT each. The plates were then incubated for 5 hours at 37  C. The purple formazan crystals were dissolved in 50% DMF in water solution containing 20% SDS (sodium dodecyl sulfate). Thereafter, the absorbance was measured at 550 nm wavelength in a plate reader. The percentage of cell viability was calculated using the following equation:

Cell viability ð%Þ ¼

SNR change at any time  100 SNR at time zero before contrast

3. Results and discussions 3.1.

Crystal structure

Fig. 1 shows the XRD patterns of the samples prepared at 1 : 0, 4 : 1, 1 : 1, 1 : 4 and 0 : 1 TREG : TREA (v/v) ratios. Positions of the diffraction peaks (220), (311), (400), (422), (511), (440) and (533) match well with the standard XRD patterns for bulk magnetite (JCPDS file no. 19-0629) indicating that the as prepared nanoclusters consist of pure magnetite (Fe3O4) phase. 3.2.

Morphology

Fig. 2 shows TEM images of the samples prepared at 1 : 0, 4 : 1, 1 : 1, 1 : 4 and 0 : 1 TREG : TREA (v/v) ratios. Insets show the corresponding HRTEM image of a single particle or cluster. It can be seen that the magnetite nanoparticles obtained up to the 4 : 1 ratio are relatively well dispersed and separated from each other due to the presence of TREG stabilizing coating on their surfaces23,24 (Fig. 2(a) and (b)). After this ratio, the single

Absorbance from the well treated with MNC-14 or magnetic filed or both  100 Absorbance from the well without any treatment

MRI characterizations and in vivo MRI. The longitudinal (T1) and transverse (T2 and T2*) relaxation times of aqueous suspensions of the MNC-14 nanoclusters were measured at 20  C using a Varian 9.4T MRI scanner. In vivo MRI studies were performed in SCID mice (female, weight, 20 g). All the mice experiments were approved by the Agency for Science, Technology and Research (A*STAR) Institutional Animal Care and Use Committee (IACUC). For this purpose, MCF-7 breast cancer cells were injected subcutaneously near the right flank of the SCID mice (female, 20 g). The tumor was allowed to develop to a volume of 150 mm3. The tumor-bearing SCID mice were imaged using a Bruker 7T Clinscan MRI. The MNC-14 nanoclusters were injected at the dose rate of 5 mg of Fe per kg body weight (100 ml, in 0.9% normal saline) through tail veins of the mice under 1% isoflurane anesthesia. T2-weighted images were acquired at various time points during 24 hours after injection using the fast spin-echo sequence (TR/TE ¼ 1500/36 ms, resolution ¼ 100 mm, thickness ¼ 1 mm). The SNR (signal to noise ratio) from the liver, kidney and tumor and the normalized contrast (%) were calculated and plotted against time. MRIcron 1.40 (Chris Rorden Copyright 1999–2005) was used for MRI image analysis. The MRI images were given color coding for easy understanding using MRIcron. SNR ¼

Normalized contrast change ð%Þ ¼

Mean signal from the region of interest Background standard deviation

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magnetite nanoparticles transformed into magnetite nanoclusters as shown in Scheme 1 (Fig. 2(c) and (d)). The HR-TEM images clearly indicate that each cluster consists of aggregates of

Fig. 1 XRD patterns of the samples prepared at (a) 1 : 0. (b) 4 : 1, (c) 1 : 1, (d) 1 : 4 and (e) 0 : 1 TREG : TREA (v/v) ratios, respectively.

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(Scheme 1). The magnetite clusters are found to be self-assembled at the 0 : 1 ratio to form chain like structures (as shown in Fig. 2(e) and S1 in the ESI†) which could be either due to the absence of any stabilizing TREG agent or a strong attractive magnetic force between them. The well dispersed dumbbell shaped magnetite nanoclusters can also be formed by the thermolysis of Fe(acac)3 in a liquid mixture of TREG and diethanol amine (DEA) at the 1 : 1 (v/v) TREG : DEA ratio and the dumbbell shaped nanoclusters are also found to be self-assembled into chain like structures when the ratio is increased to a 0 : 1 (v/v) TREG : DEA ratio (as shown in Fig. S2 A and B in the ESI†, respectively). Thus, the TREG : TREA ratio is optimized at 1 : 4 to obtain well dispersed and bigger magnetite nanoclusters (MNC-14). The average size of the MNC-14 clusters is about 44 nm. Fig. 2(f) shows the selected area electron diffraction (SAED) of the MNC14 nanocluster prepared at the 1 : 4 TREG : TREA ratio. The SAED shows clear diffraction rings due to the (220), (311), (400), (422), (511) and (440) lattice diffractions of cubic Fe3O4, which corroborates well with the XRD result (Fig. 1). 3.3.

Fig. 2 TEM images of the samples prepared at (a) 1 : 0. (b) 4 : 1, (c) 1 : 1, (d) 1 : 4 and (e) 0 : 1 TREG : TREA (v/v) ratios, respectively (scale bars are 100 nm, 100 nm, 50 nm, 100 nm and 200 nm, respectively). Insets are the corresponding HRTEM image of single particle or cluster (scale bar is 5 nm for inset (b) and 10 nm for all other insets). (f) Selected area electron diffraction (SAED) pattern of the MNC-14 nanocluster.

many small particles and the number of clustering particles increases and thereby the cluster size increases with decreasing TREG : TREA ratio. This happens because of two main reasons: first of all, more and more particles come close to each other while the amount of stabilizing TREG agent decreases and secondly, while increasing the amount of base i.e. TREA concentration, the nucleation of the iron oxide particles becomes faster in the presence of more basic conditions so that the nucleated particles try to aggregate to reduce their surface energy. Thus, the initially nucleated nanoparticles grow into larger nanoclusters with decreased TREG : TREA ratio

Magnetic properties

Fig. 3A shows the VSM magnetization (M–H) curves of the samples prepared at 1 : 0, 4 : 1, 1 : 1, 1 : 4 and 0 : 1 TREG : TREA (v/v) ratios. It can be seen from the VSM curves that the saturation magnetization (Ms) value of the MNCs increases from 63 to 80 emu g
1 with decreasing TREG : TREA ratio from 1 : 0 to 0 : 1 (Table 1). This increase of Ms could be either due to the increase in the number of clustering particles or the increase in the cluster size as shown in the TEM studies (Section 3.2). The magnetic properties were also measured using SQUID measurements. Fig. 3B shows the temperature dependence of the

Fig. 3 (A) Magnetization (M–H) curves of the samples prepared at (a) 1 : 0, (b) 4 : 1, (c) 1 : 1, (d) 1 : 4 and (e) 0 : 1 TREG : TREA (v/v) ratios, respectively. (B) Zero-field cooled/field cooled (ZFC/FC) magnetization of the MNC-14 nanoclusters under an applied field of 50 Oe. The inset of (B) shows SQUID magnetization (M–H) curves of the MNC-14 nanoclusters at room temperature (300 K) and 10 K, respectively.

Scheme 1 Formation of magnetite nanoclusters by aggregation of magnetite nanoparticles.

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zero-field cooled/field cooled (ZFC–FC) SQUID magnetization curves of the MNC-14 nanoclusters under an applied field of 50 Oe. The feature of the ZFC–FC curves indicates that the MNC-14 nanoclusters are superparamagnetic in nature with the blocking temperature (TB) at around 210 K.27 The SQUID magnetization (M–H) curves of the MNC-14 nanoclusters at 300 K and 10 K are shown in the inset of Fig. 3B. The corresponding saturation magnetization (MS) is found to be 80 and 90 emu g
1, respectively. The MNC-14 nanoclusters are found to be ferromagnetic at 10 K while the zero coercivity and zero remanence on the M–H curve at 300 K again corroborate superparamagnetic behavior of the MNC-14 nanoclusters at room temperature.26 Thus, we have optimized the TREG : TREA ratio to 1 : 4 for the preparation of well dispersed superparamagnetic MNC-14 nanoclusters with high MS (75 emu g
1) and their potentialities for the magnetic hyperthermia and in vivo MRI imaging applications were further investigated. And the heating efficiency under an AC magnetic field and MRI relaxivities of MNC-14 nanoclusters are compared to those of the MNC-10 nanoparticles. 3.4.

Colloidal stability

The colloid stability of the MNC-14 nanoclusters suspended in fetal bovine serum (FBS) was determined by DLS measurements. The particle size histogram at 0 h and particle size variation up to 24 h are shown in Fig. S3 A and B in the ESI†, respectively. The average hydrodynamic diameter of the MNC-14 nanoclusters suspended in the serum was estimated to be 187 nm (polydispersity index, PDI ¼ 0.127). The hydrodynamic diameter remained around 182 nm up to 24 h (Fig. S3 B†), indicating the good colloidal stability of the MNC-14 nanoclusters in the serum. 3.5.

Cytotoxicity studies

Fig. 4 shows the percentage cell viability of MCF-7 breast cancer cells following treatment of the MNC-14 nanoclusters in vitro. It can be seen from the graph that the MNC-14 nanoclusters do not show any cytotoxicity in MCF-7 cells within the range of 0.625–

Fig. 4 Cytotoxicity profile of the MNC-14 nanoclusters on MCF-7 breast cancer cells.

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10 mg Fe per ml concentrations. Thus, the MNC-14 nanoclusters are cytocompatible up to the iron concentration of 10 mg ml
1 with MCF-7 cancer cells. 3.6.

Magnetic hyperthermia studies

Fig. 5A shows the time-dependent temperature rise of 1 ml aqueous suspension of the MNC-14 nanoclusters with iron concentrations of 0.5, 1, 2, 4 and 8 mg ml
1 on exposure to 89 kA m
1 AC magnetic field at 240 kHz frequency. It can be seen that the temperature rises with the increased concentration of the MNC-14 nanoclusters and the required time to raise the temperature up to 42  C is about 7.5, 5.6, 3.8, 2.6 and 1.6 minutes, respectively. This heating could be due to Neel and Brownian loss, which arises from rotation of the magnetization vector and the MNC-14 nanoclusters itself, respectively.9 The inset of Fig. 5A depicts the field dependent SAR values indicating that the SAR values vary nearly with H2 which is very similar to that reported previously.28,29 The SAR value under 89 kA m
1 AC magnetic field at 240 kHz frequency is about 500 Watt g
1 for the MNC-14 nanoclusters with the 0.5 mg ml
1 iron concentration. However, the SAR of the MNC-10 nanoparticles is measured to be about 135 Watt g
1. Therefore, the larger SAR value indicates that the heating rate of the MNC-14 nanoclusters under the AC magnetic field is much faster than that for the MNC-10 nanoparticles. This could be either due to the bigger particle size or higher Ms of MNC-14 nanoclusters over the MNC-10 nanoparticles (Table 2). Fig. 5B shows the in vitro cytotoxic effect on MCF-7 breast cancer cells treated with magnetic hyperthermia using the MNC14 nanoclusters (0.5 mg ml
1 of Fe concentration) upon exposure to 89 kA m
1 AC field at 240 kHz frequency. As a comparison, the cancer cells were also exposed to only magnetic field (89 kA m
1 AC, without adding any MNC-14 nanoclusters) and only MNC-14 nanoclusters with 0.5 mg ml
1 of Fe concentration (without exposing to the magnetic field). Results (Fig. 5B) show that the magnetic hyperthermia at 45  C for 1 h caused 74% of cell death, while cell viability in the case of exposure to only the magnetic field or only MNC-14 nanoclusters was comparable to that of the control. Thus, it was confirmed that the loss of MCF-7 cancer cell viability was due to the magnetic hyperthermia treatment. Fig. 5C and D show optical microscope images of MCF-7 breast cancer cells treated with only AC magnetic field (without MNC-14) and with magnetic hyperthermia (using both MNC-14 and AC magnetic field). It can be seen that the cells treated with magnetic hyperthermia are round in shape, have lost their ability to attach to the surface and fail to grow (as shown in Fig. 5D) while the cells treated with only magnetic field (as shown in Fig. 5C) are attached to the surface having normal shape and are viable. Thus, it was confirmed that the loss of cell viability (as also shown by the MTT assay, Fig. 5B) is due to the magnetic hyperthermia treatment only. The percentage loss of MCF-7 cancer cell viability due to the MNC-14 nanoclusters as well as the MNC-10 nanoparticles are compared in Table 2. It can be seen that the percentage cell death is greater for the MNC-14 nanoclusters (74%) than for the MNC10 nanoparticles (62%). This could be due to the larger Ms and SAR values and thereby the faster heating rate of MNC-14 nanoclusters under the AC magnetic field as compared to J. Mater. Chem., 2011, 21, 14717–14724 | 14721

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Fig. 5 (A) Time dependent temperature rise of 1 ml aqueous suspension of MNC-14 nanoclusters with different iron concentrations on exposure to 89 kA m
1 AC field at 240 kHz frequency. Inset shows field dependent SAR values of 1 ml sample with 0.5 mg ml
1 iron concentration. (B) Cell viability plot shows the cytotoxic effect on MCF-7 breast cancer cells treated with magnetic hyperthermia (45  C), treated with MNC-14 nanoclusters only, and treated with magnetic field only in comparison with the control cells. (C and D) Optical microscope images of control MCF-7 breast cancer cells treated with magnetic field only and magnetic hyperthermia using MNC-14 nanoclusters (scale bar 100 mm).

unclustered MNC-10 nanoparticles. Thus, the MNC-14 nanoclusters are much better for magnetic hyperthermia applications than the MNC-10 nanoparticles as the amount of magnetic materials required for the treatment would be less and undesired toxicity which may arise due to high dose in vivo can be avoided.

3.7.

MRI studies

Fig. 6A and B show the transverse relaxation rates (1/T2 and 1/T2*) for various Fe concentrations of MNC-10 nanoparticles and MNC-14 nanoclusters measured at 9.4 T, respectively. The corresponding relaxivities (r2, and r2*) were calculated (Table 3). It is clear that the r2 and r2* relaxivities of MNC-14 (294.99 and 450.05 s
1 mM
1) are higher than those of the MNC-10 nanoparticles (205.6 and 309.2 s
1 mM
1) which could be either due to the clustering of magnetic nanoparticles or the higher Ms of MNC-14 nanoclusters over the MNC-10 nanoparticles (Table 1). Thus, the MNC-14 nanoclusters can also be used as a promising MRI T2 contrast agent. Table 2 Average particle size, saturation magnetization (Ms), SAR and percentage cell death under the AC magnetic field for MNC-10 nanoparticles and MNC-14 nanoclusters

Samples

Average particle size/nm

Ms/emu g
1

SAR/Watt g
1

Cell death (%)

MNC-10 MNC-14

10 44

63 75

135 500

62 74

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We have further evaluated the efficacy of the MNC-14 nanoclusters for in vivo MRI imaging applications. The MNC-14 nanoclusters were injected into SCID mice having the subcutaneous xenograft tumor of MCF-7 cancer cells. Fig. 7 shows the signal intensity normalized by the signal in the background at different time points in the tumor (blue line), liver (red line) and kidney (green line). The inset of Fig. 7 shows the normalized contrast change in the tumor up to 24 h. Left images in Fig. 8 show the corresponding axial image of the tumor (top left) and liver and kidney (bottom left) before injection while right images in Fig. 8 show the axial image of the tumor (top right) and liver and kidney (bottom right) after 6 hours injection of the MNC-14 nanoclusters. It can be seen from Fig. 7 that the percentage of signal reduction for the liver (80%) is higher as compared to the kidney (20% up to 4 h) and tumor (10%) and this is also obvious from the large signal intensity reduction in the postcontrast image of liver (Fig. 8) which is due to massive accumulation of the MNC-14 nanoclusters in the liver. The initial contrast change with 20% signal reduction up to 4 h in the kidney suggests that the MNC-14 nanoclusters are accumulated in the kidney. However, as they are excreted quickly the T2 contrast of the kidney is abolished thereafter. It can also be observed that there is 10% contrast changes in the tumor which is relatively less as compared to that of the liver (Fig. 7). However, there is a significant reduction of signal intensity in the post-contrast tumor image which is obvious from the colored intensity scale (Fig. 8). The tumor color changes from white-to-yellow (high signal intensity) to a red-to-orange shade indicating low signal intensity. The reduction in signal intensity of the tumor tissue is This journal is ª The Royal Society of Chemistry 2011

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Fig. 6 Transverse relaxation rates (1/T2 and 1/T2*) vs. Fe concentration measured at 9.4 T for (A) MNC-10 nanoparticles and (B) MNC-14 nanoclusters.

Table 3 Relaxivity of MNC-10 nanoparticles and MNC-14 nanoclusters measured at 9.4 T Relaxivity/s
1 mM
1 Samples

r2

r2*

MNC-10 MNC-14

205.6 294.99

309.2 450.05

Fig. 8 Top images show the axial image of tumor (marked by arrow) and bottom images show the axial image of liver and kidney (marked by arrow) before injection (left) and after 6 hours of injection (right) of the MNC-14 nanoclusters. The change in signal intensity can be noticeable and can be compared to the scale given.

Fig. 7 The normalized contrast change at different time points in tumor (blue line), liver (red line) and kidney (green line). Inset highlights the normalized contrast change in tumor up to 24 h.

due to the accumulation of the MNC-14 nanoclusters in the tumor region. Thus it can be concluded that the MNC-14 nanoclusters could be used as a potential MRI contrast agent and have a tendency to accumulate at tumor sites by means of passive targeting in which the nanoclusters penetrate through the leaky vasculature of the tumor site by an enhanced permeation and retention (EPR) mechanism. However, tumor accumulation of the MNC-14 nanoclusters can be further increased by using a permanent magnetic field gradient near the tumor area following the principle of magnetic drug targeting (MDT).30 This journal is ª The Royal Society of Chemistry 2011

Taken together, the MNC-14 nanoclusters as synthesized by a single step facile method are able to heat up efficiently under AC magnetic field and can be detected by MRI due to their high T2 contrast enhancement properties. Thus, these nanoclusters are very promising for both cancer diagnostic and therapeutic (theranostic) purposes. However, further in vivo investigations are warranted before their clinical use.

4. Conclusions Magnetite nanoclusters have been successfully synthesized by a facile one-step thermal decomposition of Fe(acac)3 in a liquid mixture of TREG and TREA. The well dispersed superparamagnetic MNC-14 nanoclusters obtained at an optimized 1 : 4 TREG : TREA (v/v) ratio resulted in higher Ms (75 emu g
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(MNC-10). The MNC-14 nanoclusters are cytocompatible. The heating rate of MNC-14 nanoclusters under an AC magnetic field are faster and thus cause more percentage loss of MCF-7 cancer cell viability (74%) than that for the MNC-10 nanoparticles (62%). Moreover, MNC-14 nanoclusters show higher r2 and r2* relaxivities (294.99 and 450.05 s
1 mM
1) than those for the MNC-10 nanoparticles (205.6 and 309.2 s
1 mM
1). The MNC-14 nanoclusters are suitable for in vivo MRI imaging too. Thus, the novel MNC-14 nanoclusters are a very promising candidate for clinical MRI imaging as well as for magnetic hyperthermia treatment of cancer.

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