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Composites of Aminodextran-Coated Fe3O4 Nanoparticles and Graphene Oxide for Cellular Magnetic Resonance Imaging Weihong Chen,†,‡,§ Peiwei Yi,† Yi Zhang,† Liming Zhang,† Zongwu Deng,† and Zhijun Zhang*,† †
Division of Nanobiomedicine, and Division of Nanobionics, Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou 215123, China ‡ Graduate University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China § Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 2 Beiyitiao Street, Zhongguancun, Haidian District, Beijing 100190, China ABSTRACT: Formation of composites of dextran-coated Fe3O4 nanoparticles (NPs) and graphene oxide (Fe3O4-GO) and their application as T2-weighted contrast agent for efficient cellular magnetic resonance imaging (MRI) are reported. Aminodextran (AMD) was first synthesized by coupling reaction of carboxymethyldextran with butanediamine, which was then chemically conjugated to meso-2,3-dimercaptosuccinnic acid-modified Fe3O4 NPs. Next, the AMD-coated Fe3O4 NPs were anchored onto GO sheets via formation of amide bond in the presence of 1-ethyl-3-(3dimethyaminopropyl) carbodiimide (EDC). It is found that the Fe3O4-GO composites possess good physiological stability and low cytotoxicity. Prussian Blue staining analysis indicates that the Fe3O4-GO nanocomposites can be internalized efficiently by HeLa cells, depending on the concentration of the composites incubated with the cells. Furthermore, compared with the isolated Fe3O4 NPs, the Fe3O4-GO composites show significantly enhanced cellular MRI, being capable of detecting cells at the iron concentration of 5 μg mL 1 with cell density of 2 105 cells mL 1, and at the iron concentration of 20 μg mL 1 with cell density of 1000 cells mL 1. KEYWORDS: graphene oxide, Fe3O4 nanoparticles, composites, MRI, cell labeling
’ INTRODUCTION Superparamagnetic Fe3O4 nanoparticles (NPs) are widely used in biomedical fields such as magnetic resonance imaging (MRI), biological separation, and hyperthermia therapy.1 3 MRI labeling of cells by Fe3O4 NPs is a powerful technique for evaluation of the location and distribution of cells in a noninvasive manner.4 6 Fe3O4 NPs induce localized inhomogeneity of magnetic field, and therefore cause decrease in regional signal intensity due to shortening of T2 relaxation time. To achieve enhanced MRI contrast effect and better biocompatibility and physiological stability, it is often desirable to modify Fe3O4 NPs with suitable functionalities.7 It is also reported that formation of aggregates of Fe3O4 NPs leads to enhanced relaxation rate (r2).8 12 Ai and colleagues9 found that the T2 relaxivities of Fe3O4-loaded polymeric micelles (r2 = 169 Fe mM 1 s 1) are significantly larger than that of the corresponding dextran-coated Fe3O4 NPs (30 50 Fe mM 1 s 1). Liu et al.10 reported that under a magnetic field of 3 T, multiple Fe3O4 NPs containing micelles have an r2 up to 345 Fe mM 1 s 1, 4-fold of single Fe3O4 NP-containing micelles (84 Fe mM 1 s 1). However, when used in vivo, the aggregation of Fe3O4 NPs often leads to precipitation, causing shortening of circulation time in blood.13,14 To address this issue, we have developed a GO-based platform r 2011 American Chemical Society
for formation of aggregates of dextran-coated Fe3O4 NPs to induce more efficient T2 shortening. We demonstrate that the dextran-coated Fe3O4 NPs can anchor onto graphene sheet to form clusters or aggregates, affording enhanced MRI contrast compared to the isolated Fe3O4 NPs. In the mean time, the oxygen-containing functionalities such as carboxylic acid, epoxide, and hydroxyl groups of the GO sheets improves, to a large extent, the biocompatibility and physiological stability of the aggregated dextran-modified Fe3O4 NPs.15,16 There are some reports on formation of composites of GO and magnetic nanoparticles and their applications in MRI, magnetically targeted drug delivery, removal of contaminants from wastewater.17 24 Cong et al.17 reported for the first time the synthesis and MRI effect of composites of Fe3O4 NPs and chemically reduced GO. However, there is still no report on cellular MRI labeling using Fe3O4-GO nanocomposites as contrast agents. In the present work, the Fe3O4 NPs were prepared via thermal decomposition approach developed by Woo et al.,25 followed by surface modification with meso-2,3-dimercaptosuccinnic acid Received: July 23, 2011 Accepted: September 1, 2011 Published: September 01, 2011 4085
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Scheme 1. Schematic Diagram Showing Preparation of (A) AMD-Coated Fe3O4 and (B) Fe3O4-GO Composites
(DMSA) and then aminodextran (AMD). The AMD-coated Fe3O4 NPs were finally conjugated to the GO via formation of amide bond between amine groups of the AMD-functionalized Fe3O4 NPs and carboxylic acid groups of the GO sheets. Thusprepared Fe3O4-GO nanocomposites exhibit good physiological stability and low cytotoxicity, and, more importantly, efficient cellular labeling. To the best of our knowledge, this is the first report on the efficient cellular MRI using Fe3O4-GO composites.
’ EXPERIMENTAL SECTION Materials. Native graphite flake was purchased from Alfa Aesar. Dextran 70K, pentacarbonyl-iron (Fe(CO)5), bromoacetic acid, butanediamine, DMSA, dimethylsulfoxide (DMSO), 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC), and N-hydroxysuccinnimide (NHS), and oleic acid were purchased from Sigma-Aldrich. RPMI 1640 culture medium and fetal bovine serum were purchased from Invitrogen. WST-1 was purchased from Biyuntian Biotechnology Institute. All other reagents were analytical grade and used as received. Ultrapure water (18.2 MΩ cm 1) was used in all experiments. Instrumentation. 13C nuclear magnetic resonance (NMR) spectra were recorded on a Varian 400 MHz spectrometer. FT-IR spectra of the samples (KBr pellet) were collected at a resolution of 4 cm 1 with a Thermo Nicolet 6700 FTIR spectrometer. The morphology and composition of NPs were characterized by Tecnai G2 F20 S-Twin transmission electron microscopy (TEM) equipped with an Energy Dispersive Spectrometry. The average size of NPs was measured by a particle size analyzer (ZEN3600-nanoZS, Malvern). Magnetic measurement was carried out at room temperature on a Physical Property Measurement System (PPMS, Quantum Design Inc.). The iron concentration was measured using atomic absorption spectrometry (SpectrAA-Duo 220 FS, Varian). WST assay was performed with a Biotek Elx 800 Microplate Reader. Cell lines were cultured in a waterjacketed CO2 incubator (Thermo 3111). The MRI was carried out on a 11.7 T Bruker micro 2.5 micro-MRI system with a conventional spin echo acquisition. Relaxivity (r2) with unit mM 1 s 1 was calculated through the curve-fitting of the reciprocal of the relaxation time versus the iron concentration (mM Fe). Synthesis of Carboxymethyldextran (CMD) and Aminodextran (AMD). The schematic diagram showing preparation of AMD-coated Fe3O4 was presented in Scheme 1. First, CMD was and Johnsson.26 Briefly, 1 g prepared following the approach by L€ofas of dextran was dissolved in 5 mL of 2 M NaOH containing 0.25 M bromoacetic acid. The solution was stirred overnight, dialyzed against
water for 24 h, against 0.1 M HCl for 24 h, and finally against water for 24 h. The solution was then lyophilized and stored at 4 °C until required for use. AMD was obtained by the coupling reaction of CMD and butanediamine in the presence of EDC and NHS according to published protocol.27 In brief, CMD (200 mg) was dissolved in ultrapure water, to which butanediamine (0.07 mL) was added and then the pH of the solution was adjusted to 8.0 with HCl. The resulting solution was mixed with EDC (480 mg) and NHS (480 mg), the total volume was adjusted to 4 mL with ultrapure water. The reaction was allowed to take place at room temperature overnight. The AMD was obtained by dialysis for 48 h and then freeze-drying. Preparation of DMSA-Coated Fe3O4 NPs. Five nm Fe3O4 NPs were synthesized by thermal decomposition of Fe(CO)5 according to the literature.25 Thus-prepared Fe3O4 NPs were then coated with DMSA following the approach by Chen et al.28 In brief, dioctyl ether (20 mL) and oleic acid (1.92 mL) was added to a three-necked flask and then heated to 110 °C for 2 h. Fe(CO)5 0.4 mL (3.04 mmol) was dissolved into the solution then heated to reflux (300 °C) for another 1 h, 5 nm Fe3O4 NPs was obtained. Twenty mg of the Fe3O4 NPs was dissolved in 2 mL toluene, then DMSA (20 mg) and DMSO (2 mL) was added to the solution and stirred at 25 °C for 12 h. This precipitant was then washed with ethyl acetate. Finally, the DMSA-coated Fe3O4 NPs were transferred into 2 mL of ultrapure water. Synthesis of Fe3O4-GO Composites. Scheme 1 illustrates synthesis route of the Fe3O4-GO composites. Water-soluble GO was prepared according to the procedure described in our previous work.29 Five milliliter aqueous solution of the DMSA-coated Fe3O4 NPs (∼0.018 M) was adjusted to pH 8.0 with triethylamine, to which 120 mg of EDC, 120 mg of NHS, and 0.34 mL of AMD (∼0.02 M) were added. The reaction solution was stirred at room temperature for 48 h, and the obtained raw product, AMD-coated Fe3O4 NPs, was transferred to Millipore centricon (120, 000 molecular mass cutoff), then washed three times by ultrapure water. For preparation of Fe3O4-GO composites, 1.5 mL of the AMD-coated Fe3O4 NPs (∼0.015 M), 80 mg of EDC, 80 mg of NHS, and 4 mL of GO (∼0.5 mg mL 1) were mixed and stirred overnight. The Fe3O4-GO composites were obtained after centrifugation and through washing with ultrapure water. Cell Line and Cell Culture. The HeLa cell line (human cervical carcinoma cell) was kindly provided by Professor Haiyan Liu, Soochow University. Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and incubated in a humidified atmosphere at 37 °C with 5% CO2. WST Assay. WST assay was performed to evaluate the cytotoxicity of the Fe3O4-GO composites and AMD-Fe3O4 NPs. The cells were 4086
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Figure 1.
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13
C NMR spectra of (A) CMD and (B) AMD.
seeded in 96-well plates at a density of 1 104 cells in 100 μL culture medium and maintained for 24 h. Then, cells were incubated for 24 h with the Fe3O4-GO composites and AMD-coated Fe3O4 NPs (as a control) at different iron concentration of 10 μg mL 1, 20 μg mL 1, 40 μg mL 1, and 80 μg mL 1 respectively, then washed with PBS buffer for three times and added fresh RPMI 1640 medium supplemented with 10% fetal bovine serum. The relative cellular viability was examined by the WST assay. The data were presented as mean ( SD. Prussian Blue Staining. The HeLa cells were seeded in 96-well plates then incubated with Fe3O4-GO composites at different concentration for 24 h. The cells were then fixed with 4% paraformaldehyde for 15 min and incubated with Pearls reagent (4% potassium ferrocyanide and 12% HCl, 50:50 vol vol 1) for 30 min at room temperature under agitation. The cells were then rinsed well with PBS, counterstained with 0.5% neutral red for 5 min and rinsed with PBS for three times. Cellular Iron Content. The iron content in the cells was determined by means of atomic absorption spectrophotometry after mineralization. Two millilter samples containing 2 105 cells mL 1 were lysed after the addition of 5 mL of 30% HCl and heated to 70 °C for 1 h. The measurements were repeated three times. Cell Labeling. To determine the minimum number of labeled cells detectable by MRI, we incubated different numbers of HeLa cells per mL (1 103, 1 104, 1 105, 1 106) with the Fe3O4-GO composites at iron concentration of 20 μg mL 1 for 24 h, and then injected into the NMR tube, respectively. Likewise, the minimum detectable iron concentration was obtained by incubation of separate batches of cells (2 105 cells mL 1) at different iron concentration of 5, 10, 20, and 40 μg mL 1 for 24 h, respectively. Imaging parameters of T2-weighted images (T2WI) by multi slice multi echo (MSME) experiments on 11.7 T Bruker micro 2.5 micro-MRI system with repetition time (TR) = 2500 ms, echo time (TE) = 90 ms, imaging matrix = 128 128, slice thickness =1 mm, and file of vision (FOV) = 2.0 cm 2.0 cm.
’ RESULTS AND DISCUSSION Synthesis and Characterization of the Fe3O4-GO Composites. Scheme 1 shows schematic diagram illustrating preparation
of the Fe3O4-GO composites. First, carboxymethyldextran (CMD) was synthesized by the reaction of dextran with bromoacetic acid. Next, aminodextran (AMD) was obtained by reaction of CMD with butanediamine, which was then covalently conjugated to the DMSA-coated Fe3O4 NPs to endow the Fe3O4
Figure 2. FT-IR spectra of (A) AMD, (B) DMSA-Coated Fe3O4 NPs, and (C) AMD-Coated Fe3O4 NPs.
NPs with good biocompatibility and physiological stability. We chose AMD, a dextran derivative with high bicompatibility and biodegradability, to modify the Fe3O4 NPs because dextrancoated Fe3O4 NPs are widely used MRI contrast agent approved by US Food and Drug Administration (FDA).30 Next, AMDmodified Fe3O4 NPs were conjugated to the GO sheets via formation of amide bond between carboxylic groups of GO and amine groups of AMD. CMD and AMD were characterized by 13C NMR spectra (Figure 1). CMD: 13C NMR (D2O): δ 98.9 (C-1), 72.6 (C-2), 74.6 (C-3), 71.3 (C-4), 70.8(C-5), 66.5 (C-6), 73.6 (CH2 7), 178.8 (C=0 8). AMD: 13C NMR (D2O): δ 97.6 (C-1), 71.3 (C-2), 73.2 (C-3), 70.1 (C-4), 69.5(C-5), 65.3 (C-6), 72.2 (CH2 7), 173.6 (C=0 8), 35.8 (CH2 9), 23.7 (CH2 10), 35.1 (CH2 11), 38.7 (CH2 12). For CMD, the resonance of the carboxylate carbon at 178.8 ppm and the resonance of the anomeric carbon atom C-1 at 98.9 ppm are observed. The intense peak at 70.8 ppm results from the overlap of the resonances due to C-2, C-3, C-4, and C-5. The resonance due to the unsubstituted methylene C-6 is observed at 66.5 ppm. The NMR result of the synthesized CMD in this work is consistent with that of the compound previously reported.31,32 Figure 1B presents 13C NMR spectrum of AMD. Clearly, covalent attachment of butanediamine to the CMD resulted in 4087
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Figure 3. (A) Photographs of the Fe3O4-GO composites dispersed in water (left) and in the RPMI 1640 medium with 10% fetal bovine serum (right), respectively. Photos were taken after the samples were prepared and stored at ambient condition for 24 h. (B) TEM images of the oleic acid coated Fe3O4 NPs (left), AMD-Fe3O4 NPs (central), and Fe3O4-GO composites (right).
the decrease by 1.3 ppm in the chemical shift of glucose unit. The presence of peaks at 23.7, 35.1, 35.8, and 38.7 ppm due to methylene C-10, C-11, C-9, and C-12, respectively, confirms successful covalent linking of butanediamine to CMD. Figure 2 depicts FTIR spectra of (A) AMD, (B) DMSA-Fe3O4 NPs, and (C) AMD-coated Fe3O4 NPs, respectively. Two characteristic peaks at 2855 and 2921 cm 1 in all curves are assignable to symmetric and asymmetric stretching modes of CH2. The peak at 1637 cm 1 is attributed to the CdO stretching mode of COOH groups of the three samples. In addition, the IR peak at 478 cm 1 (Figure 2B) corresponds to the stretching vibration of Fe O bond for the Fe3O4 NPs, which shifts to 481 cm 1 for the AMD-coated Fe3O4 NPs (Figure 2C). The two peaks at 1151 and 1109 cm 1 assignable to C O C of the pyranose of dextran, appear in spectra A and B of Figure 2, suggesting successful conjugation of AMD onto the surface of the Fe3O4 NPs. Thus-prepared Fe3O4-GO composites are well-dispersed in water (Figure 3 A (left)), and remain stable for two months without precipitation. The average size of the Fe3O4-GO composites was determined to be 174.4 nm measured by the dynamic light scattering (DLS), similar to that of the GO sheet, indicating no agglomeration of the composites formed in aqueous solution. Moreover, the composites are well-dispersed and stable in RPMI 1640 medium with 10% fetal bovine serum, a physiological solution after stored for 24 h (Figure 3A (right)). The morphology and size of the oleic acid-coated Fe3O4 NPs, AMD-Fe3O4 NPs, and Fe3O4-GO composites were characterized
Figure 4. Magnetization curves of (A) DMSA-coated Fe3O4 NPs, (B) AMD-Fe3O4 NPs, and (C) Fe3O4-GO composites.
by TEM (Figure 3B). The TEM data indicate that the as-prepared oleic acid coated Fe3O4 NPs are uniform spherical NPs with size of 5 nm, and replacement of the oleic acid ligand by AMD renders the Fe3O4 NPs well dispersed in water (Figure 3B(central)). It is clearly seen from Figure 3B (right) that the Fe3O4 NPs were assembled and formed aggregates onto the GO surface. The aggregation of the magnetic NPs benefits their MRI sensitivity, which will be discussed in detail below. Figure 4 gives magnetic hysteresis loops of the DMSA-coated Fe3O4 NPs, AMD-Fe3O4 NPs, and Fe3O4-GO composites. The 4088
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Figure 5. T2 relaxation rates (1/T2 s 1) of the Fe3O4-GO composites, the DMSA-Fe3O4 NPs, and the AMD-Fe3O4 NPs as a function of iron concentration (mM) at 11.7 T.
Figure 6. Relative viability of HeLa cells incubated with AMD-Fe3O4 (red) and Fe3O4-GO (black) for 24 h, respectively. Error bars were based on quartet samples.
profiles of all the NPs are characteristic of superparamagnetic nanoparticles. The saturation magnetization of the as-prepared oleic acid-coated Fe3O4 NPs is determined to be 27 emu g 1, much lower than the bulk Fe3O4, due to size-dependent magnetization and presence of organic ligands on the surface of the Fe3O4 NPs.25 Surface modification of the as-prepared Fe3O4 NPs by organic ligands DMSA and then AMD, and formation of Fe3O4-GO composites further reduced their saturation magnetization, being 14, 11.5, and 7.3 emu g 1, respectively, due to decrease of magnetic component in the nanoparticles and the composites. The saturation magnetization of the Fe3O4-GO composites is still high compared with that of the Fe3O4-GO reported by others.18,20 To investigate the effect of aggregation of Fe3O4 NPs on the MRI property, we compared the MRI intensities of the Fe3O4GO composites and the corresponding DMSA- and AMDcoated Fe3O4 NPs (Figure 5). At the magnetic field of 11.7 T, T2 relaxivity of the Fe3O4-GO composites is determined to be 76 Fe mM 1 s 1, much higher than that of the DMSA-Fe3O4 NPs (r2 = 24 Fe mM 1 s 1) and the AMD-Fe3O4 NPs (r2 = 21 Fe mM 1 s 1) (Figure 5), indicative of enhanced T2 relaxivity caused by formation of Fe3O4 aggregates on the GO sheets. T2 relaxivity of the AMD-Fe3O4 NPs is lower than that of the DMSA-Fe3O4 NPs, due to binding of AMD to the DMSAFe3O4 NPs. Cytotoxicity of the Fe3O4-GO Composites. The viability of HeLa cells incubated with the AMD-coated Fe3O4 NPs and Fe3O4-GO composites was examined by WST assay. It was found that the AMD-Fe3O4 NPs showed very low cytotoxicity even at iron concentration up to 80 μg mL 1 (Figure 6). The Fe3O4-GO composites with different iron concentrations, of 10, 20, 40, and 80 μg mL 1 show very low cytotoxicity, with relative cellular viability of 100, 96, 92, and 91%, respectively. The cytotoxicity of Fe3O4-GO composites only increased slightly compared to the corresponding AMD- Fe3O4 NPs, most likely due to aggregation and anchoring of the Fe3O4 NPs onto GO.33 All these results suggest that the Fe3O4-GO composites employed in our work are practically noncytotoxic, which is ideal for their application in biomedical applications such as cellular MRI contrast agent, cell separation, and magnetically targeted drug delivery. Cellular Iron Content. The iron content of the cells reflects efficiency of the cellular uptake of the Fe3O4 NPs, and therefore is closely related to the MRI detection limit. The iron content per cell was obtained by atomic absorption spectrophotometry after
mineralization. In our experiment, the iron content of HeLa cells (2 105 cells mL 1) incubated with Fe3O4-GO composites at different iron concentrations (10, 20, 40, and 80 μg mL 1) for 24 h was determined to be 9.2, 15.1, 16.4, and 17.1 pg per cell, respectively, whereas unlabeled cells contain about 0.338 pg per cell. The iron content obtained in this study is consistent with that reported by others.34,35 Arbab et al.36 reported 6 pg of iron per cell (HeLa) and 13 pg of iron per cell (human mesenchymal stem cell, msc), respectively, when the HeLa and MSC cells were incubated with ferumoxides-PLL complexes at a concentration of 25 μg mL 1. Bhattarai et al.37 obtained iron content of 9 pg per cell for RAW cells labeled with chitosan-coated Fe3O4 NPs (20 μg mL 1). Interestingly, the cellular iron content in case of Fe3O4-GO composites are much higher than that for dextranand chitosan-coated Fe3O4 NPs,36,37 suggesting more efficient cellular uptake of the Fe3O4-GO composites than the Fe3O4 NPs, which will benefit the contrast enhancement to be discussed below. Prussian Blue Staining. Iron inside the cells (Figure 7) was visualized by Prussian Blue staining versus untreated control, a standard protocol widely used to examine cellular uptake of iron oxide NPs.36,38 We found that the Fe3O4-GO composites labeled cells are unaffected in their viability and proliferating capacity with nearly 99% labeling efficiency.39 In Figure 7, the regions highlighted in the Prussian Blue section were magnified (20 ). The labeled cells showed clusters of dense blue granules in the cytoplasm, which indicates the location of the Fe3O4-GO composites. When incubated with the Fe3O4-GO composites at iron concentration of 20 μg mL 1 for 24 h, the cells contains more iron than the cells incubated with the composites at lower iron concentration (10 μg mL 1). This result suggests more efficient cellular uptake of the composites with higher iron concentration. The result from Prussian Blue staining is in good agreement with the iron content per cell obtained by atomic absorption spectrophotometry. In Vitro MR Imaging. Labeling HeLa cells with the Fe3O4 NPs enhances the cell-to-background contrast and makes them visible in MR images. The Fe3O4-GO composites labeled HeLa (Figure 8) showed relaxation rate enhancement, depending on the dose and cell density. We could detect as few as 1000 cells mL 1 on the 11.7 T magnet when HeLa cells were incubated with the Fe3O4-GO composites at iron concentration of 20 μg mL 1 for 24 h. Jendelova et al.35 reported detection of 625 cells μL 1 using dextran-coated Fe3O4 NPs (Endorem) in 4089
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Figure 7. Photomicrographs of Prussian Blue staining of HeLa cells: (A) unlabeled cells as a control, (B) cells incubated with the Fe3O4-GO composites at iron concentration of 10 μg mL 1, and (C) cells labeled with the Fe3O4-GO composites at iron concentration of 20 μg mL 1 for 24 h, respectively.
Figure 8. T2 weighted MR images: (A) HeLa cells (2 105 cells mL 1) incubated with the Fe3O4-GO composites at different concentrations for 24 h. (B) Fe3O4-GO composites (20 μg Fe mL 1) incubated with HeLa cells at different cell density for 24 h. The imaging parameters with 11.7 T magnet system: TR = 2500 ms,TE = 90 ms, imaging matrix =128 128, slice thickness = 1 mm, FOV = 2.0 cm 2.0 cm.
gelatin on a 4.7 T clinical scanner. Magnitsky and colleagues40 detected 100 cells (C17.2 cells) labeled with dextran-coated Fe3O4 NPs (Feridex) at a concentration of 25 μg mL 1. It should be mentioned that the detection sensitivity of the magnetically labeled cells depends on many factors, including magnetic field intensity, resolution of acquired images, and the nature of the magnetic NPs used. In our study, MR relaxation rate (1/ T2) of HeLa cells (2 105 cells mL 1) incubated with the Fe3O4-GO composites at different concentrations for 24 h is still very high (31 mM 1 s 1), though significantly lower than that of the Fe3O4-GO composites without incubation with cells (76 mM 1 s 1). The decrease of longitudinal relaxivity is due to the slowering of water diffusion inside the cell, when the Fe3O4GO composites are strongly confined in the endosomes.41
’ CONCLUSIONS We have developed a strategy for preparation of Fe3O4-GO composites as efficient MRI contrast agent. The nanocomposites were synthesized by coupling reaction of the AMD-coated Fe3O4 NPs with GO via EDC chemistry. Thus-prepared magnetic composites exhibit good physiological stability and low cytotoxicity. Further in vivo study is desired to investigate if the Fe3O4-GO composites have longer blood circulation time than the widely used dextran-coated Fe3O4 NPs. We showed that the internalization of the Fe3O4-GO composites did not affect the cellular viability and proliferation. More importantly, compared to the corresponding Fe3O4 NPs, the Fe3O4-GO composites
exhibit significantly improved T2 weighted MRI contrast, which is explained by that the Fe3O4 NPs formed aggregates on the GO sheets, resulting in a considerable enhanced T2 relaxivity. This work demonstrated potential application of Fe3O4-GO composites as biocompatible and efficient cellular MRI contrast agent. Further work on in vivo MRI cellurlar labeling by the Fe3O4-GO composites is desired for their clinical application.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Tel: 86-512-62872556. Fax: 86-512-62603079.
’ ACKNOWLEDGMENT Financial support of this work by Natural Science Foundation of China (20873090, 21073224), National Basic Research Program of China (2010CB933504), and CAS (KJCX2.YW.M12) are gratefully acknowledged. The authors are grateful to Platform for Characterization and Test at SINANO for their assistance in TEM measurement. ’ REFERENCES (1) Huh, Y. M.; Jun, Y. W.; Song, H. T.; Kim, S.; Choi, J. S.; Lee, J. H.; Yoon, S.; Kim, K. S.; Shin, J. S.; Suh, J. S.; Cheon, J. J. Am. Chem. Soc. 2005, 127, 12387–12391. 4090
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