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shell nanoparticles as T1 and T2 dual mode MRI contrast agent
Talanta 131 (2015) 661–665
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Talanta journal homepage: www.elsevier.com/locate/talanta
Characterization of Fe3O4/SiO2/Gd2O(CO3)2 core/shell/shell nanoparticles as T1 and T2 dual mode MRI contrast agent Meicheng Yang a,b,1, Lipeng Gao c,1, Kai Liu c, Chunhua Luo c,n, Yiting Wang c, Lei Yu c, Hui Peng c,d,e, Wen Zhang a a
Chemistry Department, East China Normal University, Shanghai 200241, PR China Antibiotics Department, Shanghai Institute for Food and Drug Control, Shanghai 201203, PR China c Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai 200241, PR China d Polymer Electronic Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand e State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 July 2014 Received in revised form 13 August 2014 Accepted 15 August 2014 Available online 26 August 2014
Core/shell/shell structured Fe3O4/SiO2/Gd2O(CO3)2 nanoparticles were successfully synthesized. Their properties as a new type of T1–T2 dual model contrast agent for magnetic resonance imaging were investigated. Due to the introduce of a separating SiO2 layer, the magnetic coupling between Gd2O(CO3)2 and Fe3O4 could be modulated by the thickness of SiO2 layer and produce appropriate T1 and T2 signal. Additionally, the existence of Gd3 þ enhances the transverse relaxivity of Fe3O4 possibly because of the magnetic coupling between Gd3 þ and Fe3O4. The Fe3O4/SiO2/Gd2O(CO3)2 nanoparticles exhibit good biocompatibility, showing great potential for biomedical applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Fe3O4/SiO2/Gd2O(CO3)2 Nanoparticles Magnetic resonance imaging Contrast agents
1. Introduction Magnetic resonance imaging (MRI), one of noninvasive clinical diagnosis techniques with a high spatial resolution, is widely used for anatomical imaging of soft body tissues [1]. However, since normal tissues and lesions often show only small differences in relaxation time, the obtained images are not clear enough to make an accurate diagnosis. In order to improve the image contrast between normal and disease tissues, contrast agents are generally employed to change proton relaxation rates. Now, the most of available MRI contrast agents are paramagnetic complexes, such as gadolinium complexes [2,3], which facilitate the spin-lattice relaxation of protons and result in a positive MR image (T1-weighted images). The tremendous advances in nanotechnology have led to the development of new types of contrast agents based on inorganic nanoparticles (NPs), such as superparamagnetic iron oxide NPs which cause protons in their vicinity to undergo spin–spin relaxation and result in a negative MR image (T2-weighted images) [2,4,5]. Compared to conventional molecular contrast agents, inorganic NPs show the advantages of tunable size and n
Corresponding author. Tel.: þ 86 21 54342726; fax: þ 86 21 54345119. E-mail addresses: [email protected] (C. Luo), [email protected] (H. Peng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.talanta.2014.08.042 0039-9140/& 2014 Elsevier B.V. All rights reserved.
shape, as well as surface modification, which allow different biodistribution. Among inorganic nanoparticles, iron oxide NPs have been intensively investigated as a T2 contract agent in cell migration, apoptosis and cancer detection due to their relatively good biocompatibility [6–10]. Howerver, the signal enhancement caused by conventional iron oxide NPs is still unsatisfactory compared to other imaging modalities such as fluorescence and PET [11], due to their less saturation magnetization (Ms) resulting in a limited effect on r2 relaxivity. In order to enhence the saturation magnetization, magnetism-engineered iron oxide (MEIO) NPs have been reported with high MRI sensitivity for the detection of cancer markers [12]. Zn2 þ doped Mn–Fe–O ferrite NPs with a high Ms value have also been developed [13]. Huang et al. synthesized Gd3 þ -chelated Fe3O4@SiO2 magnetic nanoparticle with a Ms value of 94 emu g 1 [14]. Due to the existentce of Gd3 þ , the transverse relaxivity of the prepared NPs reached 681 (mmol/L) 1 s 1 [14]. Although the development of iron oxide NPs as T2 contrast agents has gained great successes, such single mode contrast agents are increasingly facing challenges arising from accurate imaging of small biological targets because of the negative contrast effect and magnetic susceptibility artifacts [11,15]. The obtained dark areas in MR images due to their negative contrast are often confused with a low-level MR signal arising from adjacent tissues such as bone or vasculature [11,16]. Therefore, the development of T1–T2 dual model contrast agents is highly
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attractive because two different T1 and T2 imaging modes can be selectively utilized to visualize different tissues and potentially give more accurate information [17,18]. For this aim, Bae et al. synthesized gadolinium-labeled Fe3O4 NPs and demonstrated the capability of these NPs as dual contrast agents for T1 and T2weighted magnetic resonance imaging [18]. Seo et al. reported that FeCo/graphitic-shell nanocrystals exhibit high T1 and T2 contrast effects [19]. In the present work, we report the synthesis of Fe3O4/SiO2/ Gd2O(CO3)2 core/shell/shell structured NPs (NPs) and their potential as a T1–T2 dual model contrast agent. In the designed structure of Fe3O4/SiO2/Gd2O(CO3)2 NPs, the SiO2 layer acts as a separating layer to modulate the magnetic coupling between T1 contrast material (Gd2O(CO3)2) and T2 contrast material (Fe3O4). The experimental results illustrates that the thickness of SiO2 layer has a significant effect on the values of r1 (longitudinal relaxivity) and r2 (transverse relaxivity).
2. Experimental 2.1. Materials Anhydrous iron chloride (Z98%), sodium oleate (Z 95%, capillary GC), tetraethyl orthosilicate (TEOS, 98%) and Gd(NO3)3 (99%) were purchased from Sigma-Aldrich Ltd. Oleic acid (90%, technical grade), 1-octadecene (90%, technical grade) and Igepal CO-520 (containing 50 mol% hydrophilic group) were obtained from AlfaAesar Ltd. Other chemicals were purchased from the China National Medicine Company (Shanghai, China) and were analytical grade or better. All chemicals were used without further purification. HL-7702 (Hepatic cells), Bel-7404 and SMMC-7721 (Hepatocarcinoma cells) were purchased from Shanghai Cellular Institute of China Scientific Academy. These cells were cultured in PRMI1640 medium (GIBCO, USA), supplemented with 10% FBS (BioInd, Israel), 100 units mL 1 penicillin G, and 100 μg mL 1 streptomycin (GIBCO, USA). In all experiments, the cells were maintained in 100-mm culture dishes (Corning, USA) at 37 1C in a humidified atmosphere of 5% CO2 and 95% air (FORMA Series 2 Water Jacket, Thermo Scientific, USA). 2.2. Synthesis of Fe3O4/SiO2/Gd2O(CO3)2 core/shell/shell NPs Fe3O4 NPs were first synthesized according to Ref. [20]. In brief, 2.78 g (3 mmol) of iron-oleate which was treated at 30 1C in a vacuum oven for 24 h before use and 0.48 mL of oleic acid (1.5 mmol) were dissolved in 10 mL of 1-octadecene. The reaction mixture was heated to 320 1C at a constant heating rate of 3.3 1C min 1, then kept at that temperature for 60 min. After the reaction, the solution was cooled to room temperature and a mixture of 10 mL of hexane and 40 mL of acetone were added to precipitate Fe3O4 NPs. Coating Fe3O4 NPs with a SiO2 shell was done by using a known method [21] with a slight modification. Polyoxyethylene(5)nonylphenyl ether (Igepal CO-520, 1.68 mmol) and Fe3O4 NPs (1.2 mg) were dispersed in cyclohexane and vortexed. Then, ammonium hydroxide (30%, 105 μL) was added to form a brown reverse microemulsion, followed by the addition of tetraethyl orthosilicate
(92 μL, TEOS). The reaction was kept for 72 h at room temperature. The formed Fe3O4/SiO2 NPs were precipitated by the addition of methanol and collected by centrifugation. To control the SiO2 thicknesses from 8 to 20 nm, the amount of TEOS were changed from 24 to 149 μL, respectively. The growth of Gd2O(CO3)2 shell followed the previous reported procedure [22]. The obtained Fe3O4/SiO2 NPs were dispersed in a diethylglycol solution containing 38.0 mmol L 1 Gd(NO3)3 and 2.0 mol L 1 (NH2)2CO. The reaction mixture was heated to 80 1C under N2 atmosphere with continuous stirring. After the reaction was finished, the NPs were precipitated by adding acetone and collected by centrifugation.
2.3. Characterization The micrographs of NPs were taken by using transmission electron microscope (TEM, JEM-2100F, JEOL, Japan). X-ray diffraction (XRD) spectra were collected from 101 to 701 (2θ), with a step of 0.021 by using a Ni filtered Cu Kα (λ ¼1.5418 Å) radiation source (D/Max-2550V, Rigaku Co.). The JCPDS PDF database was utilized for phase identification. Magnetic measurements were performed on a superconducting Quantum Interference Device (SQUID, PPMS-9, Quantum Design Inc., USA). The samples were filled in a small container made of polyvinyl chloride, whose diamagnetic moment was subtracted from the measured magnetization values. The magnetic hysteresis loops were recorded at room temperature in a field sweep from 5000 Oe to 5000 Oe at a rate of 50 Oe s 1.
2.4. MRI measurements MR imaging of Fe3O4/SiO2/Gd2O(CO3)2 solutions were performed by using a 3T MRI (SIEMENS MAGNETOM Trio I-class, Germany). T1 images were measured with a slice thickness of 2 mm, the acquisition number of 1, 60 60 mm field of view (FOV), 256 256 matrices and an echo time (TE) of 12 ms. The T2 images were obtained by using in a fast spin-echo sequence (TR¼4000 ms, TE¼75 ms, FOV¼60 mm, matrix¼256 256, slice thickness¼ 2 mm, acquisition number¼ 1). r1 was measured by using various TI in an inverse recovery (IR) pulse sequence (TI¼ 24, 100, 200, 400, 600, 900, 1200, 2000, 3000, and 5000 ms, TR¼7000 ms, TE¼13 ms, FOV¼89 179 mm, matrix¼350 704 mm, slice thickness¼ 5 mm, average¼1). r2 was measured by using Spin Echo (SE) pulse sequence (TR¼ 3000 ms, TE¼14.3 ms, FOV¼87 280 mm, matrix¼240 768 mm, slice thickness¼5 mm, average¼1).
2.5. MTT assay The cytotoxicity of Fe3O4/SiO2/Gd2O(CO3)2 NPs was evaluated by MTT viability assay. HL-7702, Bel-7404 and SMMC-7721 cells were cultured at 1 105 cells on 96-well plates for 24 h, and then incubated with the different concentrations of NPs for 24, 48 and 72 h, respectively. After that, the cells were cultured with MTT reagents for 4 h. The products were dissolved with DMSO on the plate, and the absorbance at 490 nm was measured with a microplate reader (Multiskan MK3, Thermo Scientific, USA).
Scheme 1. The schematic synthesis of the Fe3O4/SiO2/Gd2O(CO3)2 NPs.
M. Yang et al. / Talanta 131 (2015) 661–665
3. Results and discussion The synthesis procedure of Fe3O4/SiO2/Gd2O(CO3)2 NPs is represented in Scheme 1 and described in detail in the experimental section. Fig. 1a shows a typical TEM image of prepared Fe3O4/SiO2/ Gd2O(CO3)2 core/shell/shell structured NPs. The diameter of Fe3O4 core is about 12 nm. The thicknesses of SiO2 shell and Gd2O(CO3)2 shell are about 16 nm and 1.5 nm, respectively. To confirm the crystal structure of prepared Fe3O4/SiO2/Gd2O(CO3)2 NPs, X-ray diffraction (XRD) was employed to analyze the samples obtained in each synthetic step. The results are given in Fig. 2. Curve I in Fig. 2 is the XRD spectrum of prepared Fe3O4 NPs. There are six characteristic peaks at 30.501 (2 2 0), 35.841 (3 1 1), 43.461 (4 0 0), 53.901 (4 2 2), 57.381 (5 1 1) and 62.901 (4 4 0), which are in good agreement with the standard XRD data of Fe3O4 (JCPDS No. 19-06290). After coating with SiO2, the characteristic peaks of Fe3O4 NPs can still be seen (Fig. 2, curve II), although the intensities of the diffraction peaks were greatly weakened. This result illustrates that the coating of SiO2 did not change the crystal structure of Fe3O4 NPs. The hump centered at 2θ E221 is a typical feature of amorphous SiO2. While, after the growth of very thin Gd2O(CO3)2 layer (1.5 nm), the XRD signals of Gd2O(CO3)2 are very weak because of very thin layer of Gd2O(CO3)2. So the Gd2O(CO3)2 shell was over grown and the measured XRD spectrum is shown in Fig. 2 (curve III) which is matched well with the literature values of Gd2O(CO3)2 (JCPDS ♯: 430604). The magnetic properties of prepared NPs were characterized at 300 K and are shown in Fig. 3. It can be seen from Fig. 3a that the sample of Fe3O4 NPs has a saturation magnetization (Msat) value of 56 emu g 1. This value is lower than that of bulk Fe3O4 (about
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85–100 emu g 1) [23], because the magnetization of samples decreases with decreasing particle size. The magnetic hysteresis loop of Fe3O4 NPs also illustrates that the Fe3O4 NPs exhibit superparamagnetism at room temperature, since no hysteresis is observed and both remanence and coercivity are approximately zero [24]. After the growth of the separating SiO2 layer, the Msat value dramatically decreases to 1.4 emu g 1 (Fig. 3b, curve II) mainly due to a decrease of the content of Fe3O4 in one gram of the sample. However, the Msat value increases to 2.0 emu g 1 after the further growth of Gd2O(CO3)2 shell. This could be explained by the fact that Gd2O(CO3)2 is paramagnetic and shows magnetization in the presence of an externally applied magnetic field.
Fig. 2. X-ray diffraction patterns of (I) Fe3O4 NPs, (II) Fe3O4/SiO2 and (III) Fe3O4/ SiO2/Gd2O(CO3)2 NPs.
Fig. 1. (a) Schematic and TEM image of core–shell type Fe3O4/SiO2/Gd2O(CO3)2. (b) TEM images of Fe3O4/SiO2/Gd2O(CO3)2 with variable separating layer thickness (8, 16 and 20 nm), having a fixed Fe3O4 core (12 nm in diameter) and a Gd2O(CO3)2 shell (1.5 nm).
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Fig. 3. The magnetic hysteresis loops of (I) Fe3O4, (II) Fe3O4@SiO2 and (III) Fe3O4@SiO2@Gd2O(CO3)2 NPs.
In the designed structure of Fe3O4/SiO2/Gd2O(CO3)2 NPs, SiO2 acts as a separating layer to modulate the magnetic coupling between T1 contrast material (Gd2O(CO3)2) and T2 contrast material (Fe3O4). So another two NPs samples were prepared with a SiO2 layer of 8 nm and 20 nm. Their TEM images are shown in Fig. 1b. The performances of Fe3O4/SiO2/Gd2O(CO3)2 NPs with different SiO2 layer thickness as T1–T2 dual model contrast agent were investigated and the results are shown in Fig. 4. From the images produced by T1-weighted MR sequences (Fig. 4a), it can be seen that the T1 contrast effect is dramatically changed with the thickness of the separating SiO2 layer. When the SiO2 layer was 8 nm thick, T1 contract effect is very weak (Fig. 4a, left) due to the closely located T1 and T2 contrast agents. The magnetic field generated by the superparamagnetic Fe3O4 NPs perturbs the dipolar interactions between electron spins of gadolinium and nuclear spins of water, resulting in the quenching of T1 signal. Increasing the thickness of the SiO2 layer to 16 nm and 20 nm causes the change of T1 contract from dark grey to bright, which illustrates greatly improved T1 contract effect. Such change can also be clearly seen in the color coded MR image (Fig. 4a, bottom). This result indicates that the perturbation to T1 signals caused by the Fe3O4 NPs could be tuned by the separating SiO2 layer. The relaxivity coefficient r1, which is the value of MR contrast effects, changes in the order 3.7, 32.9, and 32.2 (mmol/L) 1 s 1 as the thickness of the SiO2 layer increases in the respective order 8, 16, and 20 nm. The T2 contrast gradually changed from dark to dark grey with the increase of SiO2 thickness, as shown in Fig. 4b. The T2 relaxivity coefficient (r2) of the Fe3O4/SiO2/Gd2O(CO3)2 changes in the order 312, 269, and 208 (mmol/L) 1 s 1 as the SiO2 thickness increases in the respective order 8, 16, and 20 nm
Fig. 4. (a) T1- and (b) T2-weighted MR images and their color coded images of Fe3O4/SiO2/Gd2O(CO3)2 with varying SiO2 thickness by using 3T MRI. Contrast agents: 200 μM (Gd) for T1 image, 100 μM (Fe) for T2 images. The image of H2O was taken together for the purpose of comparison. In the color coded image, positive and negative contrasts were indicated by the red and blue color, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 r1 and r2 of Fe3O4/SiO2/Gd2O(CO3)2 NPs with different thickness of SiO2 layer. SiO2 thickness
8 nm
16 nm
20 nm
r1 ((mmol/L) 1 s 1) r2 ((mmol/L) 1 s 1)
3.7 312.0
32.9 269.4
32.2 208.0
(shown in Table 1). It is known that the local magnetic field generated by the Fe3O4 core prompts the transverse T2 relaxation rate of water nuclear spins in the vicinity [25]. However, this magnetic field is distance-dependent with 1/d3 (d is the distance from the T2 agent) [23]. So the increase of the thickness of SiO2 layer reduces this magnetic field to the surrounding water molecules, resulting in reduced T2 contrast effects. Even so, the r2 value of 208 (mmol/L) 1 s 1 in the case of 20 nm of SiO2 layer is still higher than that of commercial MR imaging contrast agents (Ferumoxide: 190.5 (mmol/L) 1 s 1) [26]. The enhanced r2 value
M. Yang et al. / Talanta 131 (2015) 661–665
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hepatocarcinoma cells after 24 h incubation. Fig. 6 give the half maximal inhibitory concentrations (IC50) of Fe3O4/SiO2/Gd2O (CO3)2 NPs to these three cell lines. It is obvious that Fe3O4/SiO2/ Gd2O(CO3)2 NPs show the highest biocompatibility to Bel-7404 cell lines.
4. Conclusions
Fig. 5. Biocompatibility of Fe3O4/SiO2/Gd2O(CO3)2 NPs on (a) HL-7702, (b) Bel-7404 and (c) SMMC-7721 cell lines by MTT viability assay relative to untreated controls.
We have successfully synthesized a new type of magnetic NPs with a core/shell/shell structure. The SiO2 layer was used as a separating layer to modulate the magnetic coupling between T1 contrast material (Gd2O(CO3)2) and T2 contrast material (Fe3O4). T1- and T2-weighted MR images illustrate that the prepared Fe3O4/SiO2/Gd2O(CO3)2 NPs could be used as T1 and T2 dual model contrast agent. The r2 value of Fe3O4/SiO2/Gd2O(CO3)2 NPs with 20 nm of SiO2 layer is 208 (mmol/L) 1 s 1. The enhanced r2 value is possibly due to the magnetic coupling between Gd3 þ and Fe3O4. The cytotoxicity studies show that the prepared NPs exhibit good biocompatibility to the tested cell lines.
Acknowledgements We appreciate the financial support of Innovation Program of Shanghai Municipal Education Commission (12ZZ041), NSFC (61177011), Shanghai Municipal Commission for Science and Technology (11JC1403800), PCSIRT and NCET. References
Fig. 6. Comparison of the IC50 values of Fe3O4/SiO2/Gd2O(CO3)2 NPs on HL-7702, Bel-7404 and SMMC-7721 cell lines after the incubation of 48 h.
is possibly due to the magnetic coupling between Gd3 þ and Fe3O4. The similar phenomenon has also been found in Gd3 þ -chelated Fe3O4@SiO2 magnetic nanoparticle [14]. Due to the aim of biomedical application, it is necessary to evaluate the biocompatibility of Fe3O4/SiO2/Gd2O(CO3)2 NPs. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay was employed to measure the cytotoxicity of the NPs on hepatic cell line (HL-7702) and hepatocarcinoma cell lines (Bel-7404 and SMMC-7721). Fig. 5 shows the viability of each cell line exposed to Fe3O4/SiO2/Gd2O (CO3)2 NPs in a range of concentration from 50 μg mL 1 to 500 μg mL 1 for 24 h, 48 h and 72 h, respectively. As to the viability at 24 h, only 5% of cell viability loss has been seen for HL-7702 and Bel-7404 up to a concentration of 300 μg mL 1 (Fig. 5a and b). Though exposed up to 500 μg mL 1 Fe3O4/SiO2/ Gd2O(CO3)2 NPs for 24 h, the cytotoxicity was under 30% on all three cells. There is no obvious dose-effect relationship on HL7702 and Bel-7404 cells observed. After 48 h incubation, the cytotoxicity on HL-7702 and SMMC-7721 cells was still under 50% when the NPs concentration was under 100 μg mL 1. The cytotoxicity of the Fe3O4/SiO2/Gd2O(CO3)2 NPs was obviously higher after 72 h incubation on all three cell lines. Interestingly, at the concentration of 50 and 100 μg mL 1, the cytotoxicity on HL-7702 is almost the same at three designed time points. From these results, we could conclude that the cytotoxicity of Fe3O4/ SiO2/Gd2O(CO3)2 NPs is nearly the same between hepatic and
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Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Characterization of Fe3O4/SiO2/Gd2O(CO3)2 core/shell/shell nanoparticles as T1 and T2 dual mode MRI contrast agent Meicheng Yang a,b,1, Lipeng Gao c,1, Kai Liu c, Chunhua Luo c,n, Yiting Wang c, Lei Yu c, Hui Peng c,d,e, Wen Zhang a a
Chemistry Department, East China Normal University, Shanghai 200241, PR China Antibiotics Department, Shanghai Institute for Food and Drug Control, Shanghai 201203, PR China c Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai 200241, PR China d Polymer Electronic Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand e State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 July 2014 Received in revised form 13 August 2014 Accepted 15 August 2014 Available online 26 August 2014
Core/shell/shell structured Fe3O4/SiO2/Gd2O(CO3)2 nanoparticles were successfully synthesized. Their properties as a new type of T1–T2 dual model contrast agent for magnetic resonance imaging were investigated. Due to the introduce of a separating SiO2 layer, the magnetic coupling between Gd2O(CO3)2 and Fe3O4 could be modulated by the thickness of SiO2 layer and produce appropriate T1 and T2 signal. Additionally, the existence of Gd3 þ enhances the transverse relaxivity of Fe3O4 possibly because of the magnetic coupling between Gd3 þ and Fe3O4. The Fe3O4/SiO2/Gd2O(CO3)2 nanoparticles exhibit good biocompatibility, showing great potential for biomedical applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Fe3O4/SiO2/Gd2O(CO3)2 Nanoparticles Magnetic resonance imaging Contrast agents
1. Introduction Magnetic resonance imaging (MRI), one of noninvasive clinical diagnosis techniques with a high spatial resolution, is widely used for anatomical imaging of soft body tissues [1]. However, since normal tissues and lesions often show only small differences in relaxation time, the obtained images are not clear enough to make an accurate diagnosis. In order to improve the image contrast between normal and disease tissues, contrast agents are generally employed to change proton relaxation rates. Now, the most of available MRI contrast agents are paramagnetic complexes, such as gadolinium complexes [2,3], which facilitate the spin-lattice relaxation of protons and result in a positive MR image (T1-weighted images). The tremendous advances in nanotechnology have led to the development of new types of contrast agents based on inorganic nanoparticles (NPs), such as superparamagnetic iron oxide NPs which cause protons in their vicinity to undergo spin–spin relaxation and result in a negative MR image (T2-weighted images) [2,4,5]. Compared to conventional molecular contrast agents, inorganic NPs show the advantages of tunable size and n
Corresponding author. Tel.: þ 86 21 54342726; fax: þ 86 21 54345119. E-mail addresses: [email protected] (C. Luo), [email protected] (H. Peng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.talanta.2014.08.042 0039-9140/& 2014 Elsevier B.V. All rights reserved.
shape, as well as surface modification, which allow different biodistribution. Among inorganic nanoparticles, iron oxide NPs have been intensively investigated as a T2 contract agent in cell migration, apoptosis and cancer detection due to their relatively good biocompatibility [6–10]. Howerver, the signal enhancement caused by conventional iron oxide NPs is still unsatisfactory compared to other imaging modalities such as fluorescence and PET [11], due to their less saturation magnetization (Ms) resulting in a limited effect on r2 relaxivity. In order to enhence the saturation magnetization, magnetism-engineered iron oxide (MEIO) NPs have been reported with high MRI sensitivity for the detection of cancer markers [12]. Zn2 þ doped Mn–Fe–O ferrite NPs with a high Ms value have also been developed [13]. Huang et al. synthesized Gd3 þ -chelated Fe3O4@SiO2 magnetic nanoparticle with a Ms value of 94 emu g 1 [14]. Due to the existentce of Gd3 þ , the transverse relaxivity of the prepared NPs reached 681 (mmol/L) 1 s 1 [14]. Although the development of iron oxide NPs as T2 contrast agents has gained great successes, such single mode contrast agents are increasingly facing challenges arising from accurate imaging of small biological targets because of the negative contrast effect and magnetic susceptibility artifacts [11,15]. The obtained dark areas in MR images due to their negative contrast are often confused with a low-level MR signal arising from adjacent tissues such as bone or vasculature [11,16]. Therefore, the development of T1–T2 dual model contrast agents is highly
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attractive because two different T1 and T2 imaging modes can be selectively utilized to visualize different tissues and potentially give more accurate information [17,18]. For this aim, Bae et al. synthesized gadolinium-labeled Fe3O4 NPs and demonstrated the capability of these NPs as dual contrast agents for T1 and T2weighted magnetic resonance imaging [18]. Seo et al. reported that FeCo/graphitic-shell nanocrystals exhibit high T1 and T2 contrast effects [19]. In the present work, we report the synthesis of Fe3O4/SiO2/ Gd2O(CO3)2 core/shell/shell structured NPs (NPs) and their potential as a T1–T2 dual model contrast agent. In the designed structure of Fe3O4/SiO2/Gd2O(CO3)2 NPs, the SiO2 layer acts as a separating layer to modulate the magnetic coupling between T1 contrast material (Gd2O(CO3)2) and T2 contrast material (Fe3O4). The experimental results illustrates that the thickness of SiO2 layer has a significant effect on the values of r1 (longitudinal relaxivity) and r2 (transverse relaxivity).
2. Experimental 2.1. Materials Anhydrous iron chloride (Z98%), sodium oleate (Z 95%, capillary GC), tetraethyl orthosilicate (TEOS, 98%) and Gd(NO3)3 (99%) were purchased from Sigma-Aldrich Ltd. Oleic acid (90%, technical grade), 1-octadecene (90%, technical grade) and Igepal CO-520 (containing 50 mol% hydrophilic group) were obtained from AlfaAesar Ltd. Other chemicals were purchased from the China National Medicine Company (Shanghai, China) and were analytical grade or better. All chemicals were used without further purification. HL-7702 (Hepatic cells), Bel-7404 and SMMC-7721 (Hepatocarcinoma cells) were purchased from Shanghai Cellular Institute of China Scientific Academy. These cells were cultured in PRMI1640 medium (GIBCO, USA), supplemented with 10% FBS (BioInd, Israel), 100 units mL 1 penicillin G, and 100 μg mL 1 streptomycin (GIBCO, USA). In all experiments, the cells were maintained in 100-mm culture dishes (Corning, USA) at 37 1C in a humidified atmosphere of 5% CO2 and 95% air (FORMA Series 2 Water Jacket, Thermo Scientific, USA). 2.2. Synthesis of Fe3O4/SiO2/Gd2O(CO3)2 core/shell/shell NPs Fe3O4 NPs were first synthesized according to Ref. [20]. In brief, 2.78 g (3 mmol) of iron-oleate which was treated at 30 1C in a vacuum oven for 24 h before use and 0.48 mL of oleic acid (1.5 mmol) were dissolved in 10 mL of 1-octadecene. The reaction mixture was heated to 320 1C at a constant heating rate of 3.3 1C min 1, then kept at that temperature for 60 min. After the reaction, the solution was cooled to room temperature and a mixture of 10 mL of hexane and 40 mL of acetone were added to precipitate Fe3O4 NPs. Coating Fe3O4 NPs with a SiO2 shell was done by using a known method [21] with a slight modification. Polyoxyethylene(5)nonylphenyl ether (Igepal CO-520, 1.68 mmol) and Fe3O4 NPs (1.2 mg) were dispersed in cyclohexane and vortexed. Then, ammonium hydroxide (30%, 105 μL) was added to form a brown reverse microemulsion, followed by the addition of tetraethyl orthosilicate
(92 μL, TEOS). The reaction was kept for 72 h at room temperature. The formed Fe3O4/SiO2 NPs were precipitated by the addition of methanol and collected by centrifugation. To control the SiO2 thicknesses from 8 to 20 nm, the amount of TEOS were changed from 24 to 149 μL, respectively. The growth of Gd2O(CO3)2 shell followed the previous reported procedure [22]. The obtained Fe3O4/SiO2 NPs were dispersed in a diethylglycol solution containing 38.0 mmol L 1 Gd(NO3)3 and 2.0 mol L 1 (NH2)2CO. The reaction mixture was heated to 80 1C under N2 atmosphere with continuous stirring. After the reaction was finished, the NPs were precipitated by adding acetone and collected by centrifugation.
2.3. Characterization The micrographs of NPs were taken by using transmission electron microscope (TEM, JEM-2100F, JEOL, Japan). X-ray diffraction (XRD) spectra were collected from 101 to 701 (2θ), with a step of 0.021 by using a Ni filtered Cu Kα (λ ¼1.5418 Å) radiation source (D/Max-2550V, Rigaku Co.). The JCPDS PDF database was utilized for phase identification. Magnetic measurements were performed on a superconducting Quantum Interference Device (SQUID, PPMS-9, Quantum Design Inc., USA). The samples were filled in a small container made of polyvinyl chloride, whose diamagnetic moment was subtracted from the measured magnetization values. The magnetic hysteresis loops were recorded at room temperature in a field sweep from 5000 Oe to 5000 Oe at a rate of 50 Oe s 1.
2.4. MRI measurements MR imaging of Fe3O4/SiO2/Gd2O(CO3)2 solutions were performed by using a 3T MRI (SIEMENS MAGNETOM Trio I-class, Germany). T1 images were measured with a slice thickness of 2 mm, the acquisition number of 1, 60 60 mm field of view (FOV), 256 256 matrices and an echo time (TE) of 12 ms. The T2 images were obtained by using in a fast spin-echo sequence (TR¼4000 ms, TE¼75 ms, FOV¼60 mm, matrix¼256 256, slice thickness¼ 2 mm, acquisition number¼ 1). r1 was measured by using various TI in an inverse recovery (IR) pulse sequence (TI¼ 24, 100, 200, 400, 600, 900, 1200, 2000, 3000, and 5000 ms, TR¼7000 ms, TE¼13 ms, FOV¼89 179 mm, matrix¼350 704 mm, slice thickness¼ 5 mm, average¼1). r2 was measured by using Spin Echo (SE) pulse sequence (TR¼ 3000 ms, TE¼14.3 ms, FOV¼87 280 mm, matrix¼240 768 mm, slice thickness¼5 mm, average¼1).
2.5. MTT assay The cytotoxicity of Fe3O4/SiO2/Gd2O(CO3)2 NPs was evaluated by MTT viability assay. HL-7702, Bel-7404 and SMMC-7721 cells were cultured at 1 105 cells on 96-well plates for 24 h, and then incubated with the different concentrations of NPs for 24, 48 and 72 h, respectively. After that, the cells were cultured with MTT reagents for 4 h. The products were dissolved with DMSO on the plate, and the absorbance at 490 nm was measured with a microplate reader (Multiskan MK3, Thermo Scientific, USA).
Scheme 1. The schematic synthesis of the Fe3O4/SiO2/Gd2O(CO3)2 NPs.
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3. Results and discussion The synthesis procedure of Fe3O4/SiO2/Gd2O(CO3)2 NPs is represented in Scheme 1 and described in detail in the experimental section. Fig. 1a shows a typical TEM image of prepared Fe3O4/SiO2/ Gd2O(CO3)2 core/shell/shell structured NPs. The diameter of Fe3O4 core is about 12 nm. The thicknesses of SiO2 shell and Gd2O(CO3)2 shell are about 16 nm and 1.5 nm, respectively. To confirm the crystal structure of prepared Fe3O4/SiO2/Gd2O(CO3)2 NPs, X-ray diffraction (XRD) was employed to analyze the samples obtained in each synthetic step. The results are given in Fig. 2. Curve I in Fig. 2 is the XRD spectrum of prepared Fe3O4 NPs. There are six characteristic peaks at 30.501 (2 2 0), 35.841 (3 1 1), 43.461 (4 0 0), 53.901 (4 2 2), 57.381 (5 1 1) and 62.901 (4 4 0), which are in good agreement with the standard XRD data of Fe3O4 (JCPDS No. 19-06290). After coating with SiO2, the characteristic peaks of Fe3O4 NPs can still be seen (Fig. 2, curve II), although the intensities of the diffraction peaks were greatly weakened. This result illustrates that the coating of SiO2 did not change the crystal structure of Fe3O4 NPs. The hump centered at 2θ E221 is a typical feature of amorphous SiO2. While, after the growth of very thin Gd2O(CO3)2 layer (1.5 nm), the XRD signals of Gd2O(CO3)2 are very weak because of very thin layer of Gd2O(CO3)2. So the Gd2O(CO3)2 shell was over grown and the measured XRD spectrum is shown in Fig. 2 (curve III) which is matched well with the literature values of Gd2O(CO3)2 (JCPDS ♯: 430604). The magnetic properties of prepared NPs were characterized at 300 K and are shown in Fig. 3. It can be seen from Fig. 3a that the sample of Fe3O4 NPs has a saturation magnetization (Msat) value of 56 emu g 1. This value is lower than that of bulk Fe3O4 (about
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85–100 emu g 1) [23], because the magnetization of samples decreases with decreasing particle size. The magnetic hysteresis loop of Fe3O4 NPs also illustrates that the Fe3O4 NPs exhibit superparamagnetism at room temperature, since no hysteresis is observed and both remanence and coercivity are approximately zero [24]. After the growth of the separating SiO2 layer, the Msat value dramatically decreases to 1.4 emu g 1 (Fig. 3b, curve II) mainly due to a decrease of the content of Fe3O4 in one gram of the sample. However, the Msat value increases to 2.0 emu g 1 after the further growth of Gd2O(CO3)2 shell. This could be explained by the fact that Gd2O(CO3)2 is paramagnetic and shows magnetization in the presence of an externally applied magnetic field.
Fig. 2. X-ray diffraction patterns of (I) Fe3O4 NPs, (II) Fe3O4/SiO2 and (III) Fe3O4/ SiO2/Gd2O(CO3)2 NPs.
Fig. 1. (a) Schematic and TEM image of core–shell type Fe3O4/SiO2/Gd2O(CO3)2. (b) TEM images of Fe3O4/SiO2/Gd2O(CO3)2 with variable separating layer thickness (8, 16 and 20 nm), having a fixed Fe3O4 core (12 nm in diameter) and a Gd2O(CO3)2 shell (1.5 nm).
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Fig. 3. The magnetic hysteresis loops of (I) Fe3O4, (II) Fe3O4@SiO2 and (III) Fe3O4@SiO2@Gd2O(CO3)2 NPs.
In the designed structure of Fe3O4/SiO2/Gd2O(CO3)2 NPs, SiO2 acts as a separating layer to modulate the magnetic coupling between T1 contrast material (Gd2O(CO3)2) and T2 contrast material (Fe3O4). So another two NPs samples were prepared with a SiO2 layer of 8 nm and 20 nm. Their TEM images are shown in Fig. 1b. The performances of Fe3O4/SiO2/Gd2O(CO3)2 NPs with different SiO2 layer thickness as T1–T2 dual model contrast agent were investigated and the results are shown in Fig. 4. From the images produced by T1-weighted MR sequences (Fig. 4a), it can be seen that the T1 contrast effect is dramatically changed with the thickness of the separating SiO2 layer. When the SiO2 layer was 8 nm thick, T1 contract effect is very weak (Fig. 4a, left) due to the closely located T1 and T2 contrast agents. The magnetic field generated by the superparamagnetic Fe3O4 NPs perturbs the dipolar interactions between electron spins of gadolinium and nuclear spins of water, resulting in the quenching of T1 signal. Increasing the thickness of the SiO2 layer to 16 nm and 20 nm causes the change of T1 contract from dark grey to bright, which illustrates greatly improved T1 contract effect. Such change can also be clearly seen in the color coded MR image (Fig. 4a, bottom). This result indicates that the perturbation to T1 signals caused by the Fe3O4 NPs could be tuned by the separating SiO2 layer. The relaxivity coefficient r1, which is the value of MR contrast effects, changes in the order 3.7, 32.9, and 32.2 (mmol/L) 1 s 1 as the thickness of the SiO2 layer increases in the respective order 8, 16, and 20 nm. The T2 contrast gradually changed from dark to dark grey with the increase of SiO2 thickness, as shown in Fig. 4b. The T2 relaxivity coefficient (r2) of the Fe3O4/SiO2/Gd2O(CO3)2 changes in the order 312, 269, and 208 (mmol/L) 1 s 1 as the SiO2 thickness increases in the respective order 8, 16, and 20 nm
Fig. 4. (a) T1- and (b) T2-weighted MR images and their color coded images of Fe3O4/SiO2/Gd2O(CO3)2 with varying SiO2 thickness by using 3T MRI. Contrast agents: 200 μM (Gd) for T1 image, 100 μM (Fe) for T2 images. The image of H2O was taken together for the purpose of comparison. In the color coded image, positive and negative contrasts were indicated by the red and blue color, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 r1 and r2 of Fe3O4/SiO2/Gd2O(CO3)2 NPs with different thickness of SiO2 layer. SiO2 thickness
8 nm
16 nm
20 nm
r1 ((mmol/L) 1 s 1) r2 ((mmol/L) 1 s 1)
3.7 312.0
32.9 269.4
32.2 208.0
(shown in Table 1). It is known that the local magnetic field generated by the Fe3O4 core prompts the transverse T2 relaxation rate of water nuclear spins in the vicinity [25]. However, this magnetic field is distance-dependent with 1/d3 (d is the distance from the T2 agent) [23]. So the increase of the thickness of SiO2 layer reduces this magnetic field to the surrounding water molecules, resulting in reduced T2 contrast effects. Even so, the r2 value of 208 (mmol/L) 1 s 1 in the case of 20 nm of SiO2 layer is still higher than that of commercial MR imaging contrast agents (Ferumoxide: 190.5 (mmol/L) 1 s 1) [26]. The enhanced r2 value
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hepatocarcinoma cells after 24 h incubation. Fig. 6 give the half maximal inhibitory concentrations (IC50) of Fe3O4/SiO2/Gd2O (CO3)2 NPs to these three cell lines. It is obvious that Fe3O4/SiO2/ Gd2O(CO3)2 NPs show the highest biocompatibility to Bel-7404 cell lines.
4. Conclusions
Fig. 5. Biocompatibility of Fe3O4/SiO2/Gd2O(CO3)2 NPs on (a) HL-7702, (b) Bel-7404 and (c) SMMC-7721 cell lines by MTT viability assay relative to untreated controls.
We have successfully synthesized a new type of magnetic NPs with a core/shell/shell structure. The SiO2 layer was used as a separating layer to modulate the magnetic coupling between T1 contrast material (Gd2O(CO3)2) and T2 contrast material (Fe3O4). T1- and T2-weighted MR images illustrate that the prepared Fe3O4/SiO2/Gd2O(CO3)2 NPs could be used as T1 and T2 dual model contrast agent. The r2 value of Fe3O4/SiO2/Gd2O(CO3)2 NPs with 20 nm of SiO2 layer is 208 (mmol/L) 1 s 1. The enhanced r2 value is possibly due to the magnetic coupling between Gd3 þ and Fe3O4. The cytotoxicity studies show that the prepared NPs exhibit good biocompatibility to the tested cell lines.
Acknowledgements We appreciate the financial support of Innovation Program of Shanghai Municipal Education Commission (12ZZ041), NSFC (61177011), Shanghai Municipal Commission for Science and Technology (11JC1403800), PCSIRT and NCET. References
Fig. 6. Comparison of the IC50 values of Fe3O4/SiO2/Gd2O(CO3)2 NPs on HL-7702, Bel-7404 and SMMC-7721 cell lines after the incubation of 48 h.
is possibly due to the magnetic coupling between Gd3 þ and Fe3O4. The similar phenomenon has also been found in Gd3 þ -chelated Fe3O4@SiO2 magnetic nanoparticle [14]. Due to the aim of biomedical application, it is necessary to evaluate the biocompatibility of Fe3O4/SiO2/Gd2O(CO3)2 NPs. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay was employed to measure the cytotoxicity of the NPs on hepatic cell line (HL-7702) and hepatocarcinoma cell lines (Bel-7404 and SMMC-7721). Fig. 5 shows the viability of each cell line exposed to Fe3O4/SiO2/Gd2O (CO3)2 NPs in a range of concentration from 50 μg mL 1 to 500 μg mL 1 for 24 h, 48 h and 72 h, respectively. As to the viability at 24 h, only 5% of cell viability loss has been seen for HL-7702 and Bel-7404 up to a concentration of 300 μg mL 1 (Fig. 5a and b). Though exposed up to 500 μg mL 1 Fe3O4/SiO2/ Gd2O(CO3)2 NPs for 24 h, the cytotoxicity was under 30% on all three cells. There is no obvious dose-effect relationship on HL7702 and Bel-7404 cells observed. After 48 h incubation, the cytotoxicity on HL-7702 and SMMC-7721 cells was still under 50% when the NPs concentration was under 100 μg mL 1. The cytotoxicity of the Fe3O4/SiO2/Gd2O(CO3)2 NPs was obviously higher after 72 h incubation on all three cell lines. Interestingly, at the concentration of 50 and 100 μg mL 1, the cytotoxicity on HL-7702 is almost the same at three designed time points. From these results, we could conclude that the cytotoxicity of Fe3O4/ SiO2/Gd2O(CO3)2 NPs is nearly the same between hepatic and
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