Fe3O4 hybrids for cellular magnetic resonance imaging and fluorescence labeling

Materials Science and Engineering C 78 (2017) 817–825

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Functionalized graphene oxide/Fe3O4 hybrids for cellular magnetic resonance imaging and fluorescence labeling Chaohui Zhou a, Hui Wu a, Mingliang Wang c,⁎, Chusen Huang a, Dapeng Yang b,⁎, Nengqin Jia a,b,⁎⁎ a The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials and Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Department of Chemistry, Shanghai Normal University, Shanghai 200234, China b College of Chemical Engineering and Materials Science, Quanzhou Normal University, Quanzhou 362000, Fujian Province, China c Department of Radiology, Zhongshan Hospital, Fudan University, Shanghai 200032, China

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Article history: Received 29 November 2016 Received in revised form 10 April 2017 Accepted 13 April 2017 Available online 23 April 2017 Keywords: GO/Fe3O4 hybrids Contrast agents MRI Fluorescence labeling

a b s t r a c t In this work, we developed a T2-weighted contrast agent based on graphene oxide (GO)/Fe3O4 hybrids for efficient cellular magnetic resonance imaging (MRI). The GO/Fe3O4 hybrids were obtained by combining with coprecipitation method and pyrolysis method. The structural, surface and magnetic characteristics of the hybrids were systematically characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), AFM, Raman, FT-IR and XRD. The GO/Fe3O4 hybrids were functionalized by modifying with anionic and cationic polyelectrolyte through layer-by-layer assembling. The fluorescence probe fluorescein isothiocyanate (FITC) was further loaded on the surface of functionalized GO/Fe3O4 hybrids to trace the location of GO/Fe3O4 hybrids in cells. Functionalized GO/Fe3O4 hybrids possess good hydrophilicity, less cytotoxicity, high MRI enhancement with the relaxivity (r2) of 493 mM−1 s−1 as well as cellular MRI contrast effect. These obtained results indicated that the functionalized GO/Fe3O4 hybrids could have great potential to be utilized as cellular MRI contrast agents for tumor early diagnosis and monitoring. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Magnetic resonance imaging (MRI) is widely applied to visualize anatomical structures in biomedical research and clinical medicine for early tumor detection, diagnosis and monitor, which is capable of resolving physiological and anatomical details as its non-invasive nature, real-time monitoring and high spatial resolution [1–3]. In order to obtain excellent diagnosis quality, MRI contrast agents (CAs) were introduced to significantly enhance the imaging contrast between normal and pathological sites [4]. It is well known that paramagnetic Gd3 + complexes (e.g. Gd3+ chelate, Gd doped nanoparticles (NaYF4:Gd [5]) or lanthanide oxides (Gd2O3) [6,7]) are usually used as T1 contrast agents. Furthermore, Gd-based nanoparticles with fluorescent lanthanide elements of multifunctional systems have attracted great attention for MRI and fluorescence imaging due to superior optical and magnetic property. Some multifunctional systems have also been fabricated as

⁎ Corresponding authors. ⁎⁎ Correspondence to: N. Jia, The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials and Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Department of Chemistry, Shanghai Normal University, Shanghai 200234, China. E-mail addresses: [email protected] (M. Wang), [email protected] (D. Yang), [email protected] (N. Jia).

http://dx.doi.org/10.1016/j.msec.2017.04.139 0928-4931/© 2017 Elsevier B.V. All rights reserved.

mul-bimodal probes such as PEGylated Gd2O3:Tb3+ nanoparticles [8], Eu0.2Gd0.8PO4·H2O NPs [9]. Er3 +/Yb3 + and Tm3 +/Yb3 + doped GdF3 NPs [10] and Gd2O2S:Eu3 + [11] nanoparticles and so on. Nowadays, the Gd3 + chelate-based T1 MRI CAs are the most extensively used in clinic [12], however, they give rise to renal failure of patients in some case and are restricted by the US Food and Drug Administration (FDA) [13]. Thus, it is highly desired to develop new MRI CAs with high safety and efficacy. The majority of iron-based magnetic nanomaterials have been developed to T2 MRI CAs, which is considered safer. For example, iron oxide nanomaterials (Fe3O4,γ-Fe2O3,α-Fe2O3) [14–16], iron alloybased nanomaterials (FeCo, CoFe2O4 [17], NiFe2O4 [18], MnFe2O4 [19], FePt [20] and others). Furthermore, iron-based magnetic nanomaterials multifunctional systems have been extensively explored as mul-bimodal probes, Examples of such systems include magneto-fluorescent imaging agents [21–23], T1–T2 imaging agents [18], magneto-motive ultrasound imaging agents [24] and so on. Super-paramagnetic iron oxide (Fe3O4) NPs have been commonly used for T2-weighted CAs and could cause decrease in regional signal leading to darker images as its shortening spin–spin proton transverse relaxation times (T2) [25,26]. But Fe3O4 NPs tend to aggregate and often form precipitation in practical application, restricting their application both in vitro and vivo [27]. To address this issue, it is often desirable to modify Fe3O4 NPs with suitable functionalities or fabricate magnetic hybrids.

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C. Zhou et al. / Materials Science and Engineering C 78 (2017) 817–825

Graphene is a novel one-atom-thick two-dimensional structure carbon materials, which has attracted a great deal of interest in material science because of their unique electronic, thermal, mechanical, and structural properties [28,29]. Graphene and grapheme derivatives have been explored applications in the nanomedicine filed, such as molecular imaging, drug delivery, cancer therapies, and biosensing [30– 32]. Graphene oxide (GO), one of the most extensively used grapheme derivatives in the biomedical filed owing to its excellent water solubility and low toxicity [33,34]. In addition, graphene oxide (GO) can confer enhancements in mass transport and higher effective surface area. The combination of graphene oxide (GO) with nanomaterials is able to offer numerous excellent physicochemical properties and functions for various applications [35]. A versatile nanocomposites based on GO have been built up by loading inorganic nanoparticles for cell imaging and drug delivery. Moreover, it has been reported graphene and graphene derivatives could not have obvious toxicity in vitro and in vivo experiments [36]. Several groups reported that the formation of aggregates of Fe3O4 nanoparticles could improve MRI contrast [35], but the uncontrolled aggregation of Fe3O4 nanoparticles can cause precipitation of the NPs and limited the application of Fe3O4 nanoparticles in vitro and vivo [37]. Therefore, the GO was employed to serve as a platform due to their highly effective surface area and controlled formation of MNP aggregates on the GO sheets, which could enhance MRI contrast of Fe3O4 nanoparticles [37]. Recently, the hybrids of Fe3O4 NPs and GO have attracted substantial attention due to both the superior properties of GO and magnetite nanoparticles for potential use as the contrast agent for MRI [38,39]. There are quite a few reports on synthetic methods of GO/Fe3O4 hybrids including in situ reduction of acetylacetone iron, chemical precipitation method, ion exchange and subsequent calcinations method, chemical deposition method and so on [40–48], but those methods still own some drawbacks such as uncontrolled loading amount of Fe3O4, easily leaching out from the GO in the process of application and having low saturation magnetization strength [48,49]. Therefore, it is believed that GO/Fe3O4 hybrids synthesized by suitable method may have great potential for applications in MRI. In this work, we constructed a potentially excellent contrast agent for cellular MR Imaging and fluorescence labeling. GO/Fe3O4 hybrids were synthesized by combining with co-precipitation method and pyrolysis method, followed by modification of anionic and cationic polyelectrolytes via layer-by-layer assembling to obtain functionalized GO/ Fe3O4 hybrids. Further, FITC as a fluorescence labeling was grafted to the surface of functionalized GO/Fe3O4 hybrids for monitoring their cellular internalization process. The MTT assay, prussian blue staining and fluorescence imaging experiments showed that the functionalized hybrids possess low toxicity, favorable water solubility and biocompatibility. Finally, magnetic resonance imaging study showed that the functionalized hybrids can be used as potential magnetic resonance imaging contrast agents for tumor early diagnosis and monitoring because of their excellent magnetic resonance imaging effect.

2.2. Synthesis of GO/Fe3O4 hybrids The GO was prepared by modified Hummer's method from purified natural graphite powder [50,51]. The GO/Fe3O4 hybrids were synthesized via combining with co-precipitation method and pyrolysis method with some modification following those reported literatures [52–54]. In a typical experimental procedure, FeCl3·6H2O (120 mg), and FeCl2·6H2O (450 mg) were dissolved in 10 mL of hot DEG at 90 °C in an oil bath, after 30 min of stirring, 2.5 mL of DEA was added. Meanwhile, the mixture of NaOH (6 mmol) and 5 mL of hot DEG were also introduced and kept stirring for 10 min again. Afterwards, the mixed solution of GO (15 mg) and 10 mL of DEG was homogenized under vigorous stirring. Finally, the mixture was brought to a 50 mL Teflon-lined autoclave, and the sealed autoclave was heated to 180 °C for 8 h. The products were collected and separated by centrifugation, washed with ethanol and water for three times, and then dried under vacuum. 2.3. Preparation of water-dispersed polymer- functionalized GO/Fe3O4 hybrids In order to endow the GO/Fe3O4 hybrids disperse in water with excellent solution stability, good biocompatible and further functionalization, the GO/Fe3O4 hybrids were grafted with functionalized polymer by layer-by-layer assembling method [55]. Briefly, 3 mg of GO/Fe3O4 were added into a 20 mL 1 wt% aqueous solution of an anionic polyelectrolyte PSS (polystyrene sulfonate sodium salt), the mixture solution was homogenized under vigorously stirring at 65 °C for 12 h. The large aggregates or contaminants were removed by filtering and excess of PSS was separated by a magnet. Then, the PSS wrapped GO/Fe3O4 were re-dispersed in pure water with brief sonication. We can make sure that the polymer were successful modified to the surface of GO/Fe3O4 hybrids by measuring of surface potential of GO/Fe3O4 hybrids. The GO/Fe3O4@PSS hybrids were further dispersed into a 20 mL solution containing 1 wt% of the cationic polyelectrolyte (PEI) and kept stirring for another 3 h. Finally, GO/Fe3O4@PSS@PEI was obtained by centrifuging, fully washing with three times. And the particles were re-dispersed in pure water for further applications. 2.4. Fluorescence labeling of functionalized GO/Fe3O4 hybrids with FITC FITC as fluorescence probe was further conjugated to the surface of functionalized GO/Fe3O4 by\\N_C_S to the amine groups of PEI coupling reaction to investigate the cellular uptake behavior of the hybrids [56]. Typically, 3 mg/mL functionalized GO/Fe3O4 hybrids dispersed in ethanol was firstly mixed with 1 mg/mL FITC solution, triethylamine was then added to the mixture for adjusting pH to slightly alkaline, and continuously shocked in the dark for 12 h. Finally, the mixture was centrifuged, washed three times with ethanol to remove excess FITC, and the FITC labeled-GO/Fe3O4 hybrids were re-dispersed in PBS solution.

2. Materials and methods

2.5. Characterization

2.1. Materials

The Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet Avatar 370 FT-IR spectrophotometer using KBr pellets. X-ray diffraction (XRD) patterns were determined by a Rigaku DMAX 2000 diffractometer equipped with Cu/Ka radiation (λ = 0.15405 nm) (40 kV, 40 mA). The ultraviolet–visible (UV–vis) absorption spectra were obtained with a UV-7502PC spectrophotometer. The morphology and composition of the hybrids were characterized by transmission electron microscopy (TEM) equipped with JEOL model JEM2100 high-resolution transmission electron microscope (HR-TEM) instrument. AFM images were obtained using a Multi-Mode V AFM (Veeco). Zeta potentials and size of the sample were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90). MTT assay could be determined by

Natural flake graphite used to prepare GO was purchased from Qingdao Dingding Graphite Products Factory. 1-Ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC), and N-hydroxysuccinnimide (NHS), polyethylenimine (PEI) (25 K), polystyrene sulfonate sodium salt (PSS) and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich and used as received. FeCl3·6H2O, FeCl2·4H2O, diethylene glycol (DEG), diethanolamine (DEA), sodium hydroxide, and ethanol were obtained from Sinopharm Chemical Reagent Co. Ltd. and used without further purification. All other reagents used were available commercially and were of the high purity grade.

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Thermo Multiskan Spectrum (Varian Cary 100). The cells images were observed by inverted fluorescent microscope (Olympus XI 71). The T2weighted MRI imaging and relaxation time was carried out on GE signa 3.0-T HDX. 2.6. Cellular experiments 2.6.1. Cell culture The human pancreatic cancer BxPC-3 cell line was obtained from Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS, China). The cells were cultured in Dulbecc's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were maintained at 37 °C in a humidified and 5% CO2 incubator. They were routinely harvested by treatment with a trypsin-ethylene diamine tetraacetic acid (EDTA) solution (0.25%). 2.6.2. Cell cytotoxicity A standard MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, Sigma–Aldrich) assay was performed to evaluate the cytotoxicity of the GO/Fe3O4@ PSS @PEI hybrids. BxPC-3 cells were seeded in 96-well plates at a density of 1 × 104 cells per well in 100 μL culture medium at 37 °C with 5% CO2 for 24 h. And after incubation with various concentrations of GO/Fe3O4@PSS@PEI for 24 h or 48 h, the relative cellular viability was examined by MTT assay. The data were presented as mean ± SD. 2.6.3. Prussian blue staining The BxPC-3 cells were treated with GO/Fe3O4@PSS@PEI particles at different concentrations for 6 h, and cells were washed four times with PBS to remove excess of GO/Fe3O4@PSS@PEI. The cells were treated with 4% paraformaldehyde for 40 min, washed three times with PBS and treated with Perls' reagent (4% potassium ferrocyanide/12% HCl, 50:50, v/v) for 1 h. Finally, the cells were rinsed with PBS for three times to remove of regents and observed using optical microscopy. 2.6.4. Cellular iron content The inductively coupled plasma optical emission spectroscopy (ICPOES) instrument (Varian) analysis was carried out to determined iron concentrations in the GO/Fe3O4 hybrids and uptaken by the cells. The samples were lysed after the addition of 30% HNO3 and heated to 60 °C for 1 h. And likewise, digestion of the cells was performed in concentrated nitric acid at 90 °C for 6 h. Finally, all those samples were diluted to 25 mL before measurement. The measurement was repeated for three times. 2.6.5. Intracellular fluorescence imaging observation The BxPC-3 cells were incubated with 25 μg/mL GO/Fe3O4@PSS@PEIFITC hybrids for 6 h. Then the cells were subsequently washed with PBS three times to remove GO/Fe3O4@PSS@PEI-FITC hybrids that were not uptaken by cells. The imaging of cells was performed using inverted fluorescence microscope with a 488 nm laser as the excitation source. 2.7. In vitro MR imaging MRI experiments were carried out on a 3.0-T clinical MRI instrument (GE signa3.0-T HDx). The T2-weighted images were obtained using spin-echo imaging sequence, the relevant parameters as following: repetition rate (TR) = 1000 ms, various echo times (TE) = 12.8, 25.6, 38.4, 51.2, 64.0, 76.8 ms, field of view (FOV) = 12.0 cm, slice thickness = 2 mm, the T2 value was measured by analyzing of corresponding software. The T2 values of GO/Fe3O4@PSS@PEI hybrids with varying iron concentrations ranging from 0.004 to 0.08 g/L were performed on a 3.0-T clinical MRI scanner at room temperature. After acquiring the T2weighted MR images, the signal intensity was measured within a

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manually drawn region-of-interest for each sample. Relaxation rates R2 (R2 = 1/T2) were calculated from T2 values at different iron concentrations. BxPC-3 cells (2 × 106) were incubated with different concentrations of GO/Fe3O4@ PSS@PEI hybrids for 6 h at 37 °C in cell culture medium. The cells were washed three times with PBS, harvested and processed for MR imaging as following, the cells were dispersed in 2.5% glutaraldehyde PBS solution inside Eppendorf tubes for 1 h, then, discarded the supernatant carefully. Each cell tube was respectively dispersed in 0.5% agarose gel solution with gentle shaking to keep suspending. MR imaging of cells was performed with a clinical 3.0-T MR imager. 3. Results and discussion 3.1. Synthesis and characterization of the GO/Fe3O4 hybrids The synthesis process of the GO/Fe3O4@PSS@PEI-FITC hybrids is schematically illustrated in Scheme 1. Starting from GO prepared by a modified Hummer's method, the GO/Fe3O4 hybrids were synthesized by combining with co-precipitation method and pyrolysis method [57]. Then, anionic polyelectrolyte polystyrene sulfonate sodium salt (PSS) used as a wrapping polymer were firstly modified to the surface of GO/Fe3O4 hybrids. GO/Fe3O4 @PSS was further functionalized with polyethylenimine (PEI) to offer amine groups on the surface of GO/ Fe3O4 hybrids for further conjugating with a fluorescence labeling (FITC). As can be seen from TEM images, the as prepared GO illustrates the flake-like shape and layer-layer structure (Fig. 1(a)). Fe3O4 nanoparticles with size of 5–10 nm were homogeneous deposited on GO (Fig. 1(b)), it is proved that GO/Fe3O4 hybrids were successful synthesized. AFM images (Fig. 1(c–d)) displayed that the average diameter of GO and GO/Fe3O4 hybrids were about 200–600 nm. The crystalline structures of the GO/Fe3O4 hybrids were identified with XRD. The XRD pattern of the as-prepared GO/Fe3O4 hybrids (Fig. 2(a)) reveals that the as-prepared Fe3O4 nanoparticles are cubic phase, which was corresponding well with the standard cubic phase of Fe3O4 (JCPDS Card No. 19-0629). In the meantime, the diffraction peaks at 26.0° is ascribed to characteristic (0 0 2) reflections of graphite [58]. All those results indicated that GO/Fe3O4 hybrids were successfully constructed. Raman spectroscopy is a widely used technique to characterize the disorder and defect structures of carbon [58]. Fig. 2(b) shows the Raman spectra of GO (black line) and GO/Fe3O4 (red line) hybrids. As can be seen in Fig. 2(b), the Raman spectrum of GO exhibited a Dband peak at ~1331 cm−1 and G-band peak at ~1571 cm−1 corresponding to the breathing mode of -point phonons of A1g symmetry and the first-order scattering of the E2g phonons, respectively [59]. Generally, the D band assigned to the sp3 carbons in graphene sheets is associated with the disorder degree of carbon. While G band assigned to the E2g phonon of C sp2 atoms is associated with order carbon atoms [60], [61]. In contrast, the D band of GO/Fe3O4 hybrids becomes higher and broader. This can be due to the fact that more detects exists in GO/ Fe3O4 hybrids during the preparation process. In the Raman spectrum of GO/Fe3O4 hybrids, the D band and G bang are shifted to ~1290 cm−1 and ~1571 cm−1, respectively, which was mainly caused by strong interacting between the GO sheet and the Fe3O4. Moreover, the intensity ratio (ID/IG) of GO/Fe3O4 hybrids (0.96) was found to be higher than that of GO (0.67), suggesting that functionalized GO was more disordered [60,62]. All the above results prove that Fe3O4 nanoparticles have been coated on the GO surface. FT-IR spectra of the GO and GO/Fe3O4 hybrids are also shown in Fig. 2(c). Via analyzing the IR spectra of GO and GO/Fe3O4 hybrids, it can be seen that the peak at 1620 cm−1 is attributed to the skeletal vibrations of unoxidized graphitic domains while the peak at 1400 cm−1 corresponds to the bending absorption of carboxyl group O_C\\O, and the band at 1080 cm−1 is due to the deformation of the C\\O [63]. As can be seen, the broad band appearing at around 3427 cm−1 and around 3427 cm−1 of GO are weakened much in comparison with GO/Fe3O4

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Scheme 1. Schematic illustration of fabrication of the GO/Fe3O4@PSS@PEI-FITC hybrids.

hybrids. Besides, the new absorption band at 573 cm−1 is attributed to the Fe\\O stretching vibrations [64], further confirming the presence of magnetite nanoparticles in the hybrids. The magnetic properties of GO/Fe3O4 hybrids were measured by a SQUID magnetometer at room temperature (Fig. 2(d)). The magnetization hysteresis loop of near zero coercivity indicated the superparamagnetic property of GO/Fe3O4 hybrids owing to the existence of Fe3O4 nanoparticles on the GO sheets. Furthermore, the GO/Fe3O4 hybrids dispersed in aqueous solutions could be completely separated from water by a magnet (inset of Fig.1d), clearly demonstrating its excellent magnetic properties. 3.2. Surface modification of GO/Fe3O4 hybrids It is noteworthy that the properties of nanoparticles such as good biocompatibility, good dispersion and excellent stability in biological

environment with water at neutral pH and high ionic strength are key prerequisites with respect to in vitro and in vivo applications. To render the as-prepared GO/Fe3O4 hybrids biocompatible and functional groups on their surfaces, surface functionalization with hydrophilic polymer PSS and PEI was carried out following a literature protocol [55]. As shown in Fig. 3(a), the surface zeta potential of the hybrids changed from +38.1 mV (GO/Fe3O4 hybrids) to −60.3 mV (GO/Fe3O4@PSS), indicating the successful coating of PSS on the hybrids through electrostatic interactions. The negatively charged sulfonate groups on polyelectrolyte PSS were coated on the surface of GO/Fe3O4 hybrids, making the surface zeta potential of GO/Fe3O4 hybrids change to negative. Then, a cationic polyelectrolytes polyethylenimine (PEI) was introduced to the surface of GO/Fe3O4@PSS through electrostatic interactions which makes the GO/Fe3O4 hybrids more stable in biological environment and also benefit for the further functionalization to improve the

Fig. 1. TEM images of GO (a) and GO/Fe3O4 hybrids (b), AFM images of the (a) GO and (b) GO/Fe3O4 hybrids.

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Fig. 2. (a) XRD pattern of the Fe3O4 and GO/Fe3O4 hybrids, (b) Raman spectra of the GO (blank) and GO/Fe3O4 hybrids (red), (c) FT-IR spectra of the GO (blank) and GO/Fe3O4 hybrids (red), (d) magnetic hysteresis curves of GO (blank) and GO/Fe3O4 hybrids (red). Inset is photograph of GO and GO/Fe3O4 dispersed in distilled water under an applied magnetic field. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

efficiency of water dispersibility and offer amine groups for further conjugating with FITC. Zeta potential measurements (Fig. 3(a)) showed that the surface zeta potential value of GO/Fe3O4@PSS@PEI dramatically increased to +55.1 mV after surface modification by PEI polymer (Fig. 3(a)), which could be ascribed to the abundant amine groups of PEI on their surface and make the the zeta potential change to positive. Thus, GO/Fe3O4 hybrids were successful functioned through this layerby-layer assembling of multilayer polyelectrolytes. The resulting GO/ Fe3O4@PSS@PEI also exhibited good dispersity and stability in water, PBS buffer solution, salt solution and cell culture medium (Fig. 3(b)) and without precipitation.

MTT was carried out to determine the relative viabilities of cells after incubated with different concentrations of GO/Fe3O4@PSS@PEI hybrids. It can be observed from Fig. 4 that BxPC-3 cells still maintained greater than 75% cell viability after 24 h of treatment with the hybrids at the iron concentration as high as 100 μg mL−1. Furthermore, the cytotoxicity of GO/Fe3O4@PSS@PEI hybrids after incubation for 48 h only changed slightly compared to that of 24 h. These results suggest that the GO/Fe3O4@PSS@PEI hybrids employed in our work own less cytotoxicity over a wide range of iron concentration (0–100 μg mL− 1), which can be used for biomedical applications such as cellular MRI contrast agents.

3.3. In vitro cytotoxicity of the GO/Fe3O4 hybrids

3.4. Internalization of GO/Fe3O4@PSS@PEI-FITC hybrids in vitro

The biocompatibility of materials is of great importance for their further biomedical applications. Herein, standard cell viability assay using

Prussian blue staining experiments are widely used to examine iron distribution in cells [65,66]. In order to investigate intracellular GO/

Fig. 3. (a) Zeta potential values of GO, GO/Fe3O4, GO/Fe3O4@PSS and GO/Fe3O4@PSS@PEI; (b) photograph of pristine GO/Fe3O4 dispersed in water (a), GO/Fe3O4@PSS@PEI hybrids dispersed in different solvents (b) water, (c) 0.9% NaCl, (d) PBS, (e) DMEM cell culture medium.

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Fig. 4. In vitro cell viability of BxPC-3 cells incubated with GO/Fe3O4@PSS@PEI hybrids at different iron concentrations at 37 °C for 24 h and 48 h as compared to untreated cells.

Fe3O4 @PSS@PEI hybrids incorporation and their labeling efficiency, prussian blue staining experiment was performed to evaluate the BxPC-3 cells treated with GO/Fe3O4@PSS@PEI hybrids for 24 h. As shown in Fig. 5, comparing with the untreated cells, it was clearly investigated that amounts of dense blue granules were found in the labeled cells, which indicated GO/Fe3O4@PSS@PEI hybrids can be effectively uptaken by cells [4]. Furthermore, the uptake behavior of cells was also investigated by fluorescence microscopy. BxPC-3 cells were incubated with GO/Fe3O4@PSS@PEI-FITC hybrids for 6 h, followed by inverted fluorescence microscope observation to trace the location of the GO/Fe3O4@PSS@PEI-FITC hybrids. The live cells were imaged in bright filed (Fig. 5(e)) with 488 nm excitation (Fig. 5(f)). The strong green fluorescence signal from the nanoparticles was observed inside the cells and distributed throughout the cell, suggesting that GO/ Fe3O4@PSS@PEI-FITC hybrids can be efficiently uptaken by BxPC-3 cells. Therefore, the FITC-labeled GO/Fe3O4@PSS@PEI hybrids could be used for cellular labeling through fluorescence imaging, which could provide direct investigation in cellular uptake of the hybrids complementary to cellular MR imaging.

Fig. 5. Photomicrographs of Prussian blue staining of BxPC-3 cells incubated with the Fe3O4/GO @PSS@PEI hybrids at different iron concentrations of (a) 0 μg mL−1 (control), (b) 10 μg mL−1, (c) 20 μg mL−1, (d) 40 μg mL−1 for 6 h, respectively. (e) Bright-filed image and (f) fluorescence image of BxPC-3 cells after incubated with Fe3O4/GO@PSS@PEI-FITC hybrids for 6 h.

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Fig. 6. (a) T2-weighted MRI images of GO/Fe3O4@PSS@PEI hybrids suspended in water at different iron concentrations; (b) a plot of T2 relaxation rate r2 (1/T2) against iron concentration of GO/Fe3O4@PSS@PEI hybrids; (c) T2-weighted MRI images of BxPC-3 cells treated with different concentrations of GO/Fe3O4@PSS@PEI hybrids for 6 h; (d) the signal intensity percentage of the labeled BxPC-3 cells, as compared with the untreated cells in PBS, as a function of the GO/Fe3O4@PSS@PEI hybrids concentration; (inset of panel (d)): the iron uptake content in BxPC-3 cells after incubation with different concentrations of GO/Fe3O4@PSS@PEI hybrids.

3.5. In vitro MR imaging To investigate the MRI contrast enhancement of GO/Fe3O4@PSS@PEI hybrids, T2-weighted MR images of the GO/Fe3O4@PSS@PEI hybrids solutions at different iron concentration were acquired on a clinical 3.0-T MR system at room temperature. From Fig. 6(a, b), T2-weighted images revealed the concentration-dependent darkening effect and the transverse relativity (r2) of GO/Fe3O4@PSS@PEI hybrids was calculated to be 493 mM−1 s− 1, suggesting that the GO/Fe3O4@PSS@PEI hybrids could be utilized as potential MRI contrast agents. We further investigated the diagnostic potential of the GO/Fe3O4@ PSS@PEI hybrids as cellular MRI contrast agent. The contrast effect of the GO/Fe3O4@PSS@PEI hybrids labeled BxPC-3 cells were also conducted at a clinical 3.0-T MR system with the same parameters. As indicated in Fig. 6(c), GO/Fe3O4@PSS@PEI hybrids labeled cells showed the significant negative contrast enhancement depending concentration of GO/ Fe3O4@PSS@PEI hybrids. Meanwhile, it is found that MRI signal intensity of BxPC-3 cells (Fig. 6(d)) decreased with the increase of the incubation concentration of GO/Fe3O4@PSS@PEI hybrids. The amounts of GO/ Fe3O4@PSS@PEI hybrids taken up by cells increased with the increase of sample concentration, which is confirmed by the quantitative ICPOES analysis results (the inset in Fig. 6(d)). Therefore, all these results suggested that the functionalized GO/Fe3O4 hybrids have great potential as intracellular MRI contrast agents.

4. Conclusions In summary, we have successful prepared novel water-dispersible GO/Fe3O4 hybrids as efficient MRI contrast agents as well as fluorescence labeling. The obtained functionalized GO/Fe3O4 hybrids displayed good biocompatible, low cytotoxicity and significant MRI negative contrast enhancement effect in solution. Moreover, the T2-weighted MRI signal on BxPC-3 cancer cells decreased significantly after treatment with the GO/Fe3O4@PSS@PEI hybrids, suggesting the potential application of the hybrids as efficient cellular MRI contrast agents. In addition, the excellent fluorescence imaging could provide additional information complementary to cellular MR imaging. This preliminary research

may highlight the great benefit of graphene-based hybrids for cancer diagnosis and imaging applications. Acknowledgements This work was supported by Shanghai Education Committee (15ZZ070), the National Natural Science Foundation of China (21373138), Program for Changjiang Scholars and Innovative Research Team in University (IRT_16R49) and International Joint Laboratory on Resource Chemistry (IJLRC). References [1] H.R. Herschman, Molecular imaging: looking at problems, seeing solutions, Science 302 (5645) (2003) 605–608, http://dx.doi.org/10.1126/science.1090585. [2] T. Schroeder, Imaging stem-cell-driven regeneration in mammals, Nature 453 (7193) (2008) 345–351, http://dx.doi.org/10.1038/nature07043. [3] R. Weissleder, U. Mahmood, Molecular imaging, Radiology 219 (2) (2001) 316–333, http://dx.doi.org/10.1148/radiology.219.2.r01ma19316. [4] H. Shokrollahi, Contrast agents for MRI, Mater. Sci. Eng. C 33 (8) (2013) 4485–4497, http://dx.doi.org/10.1016/j.msec.2013.07.012. [5] R. Kumar, M. Nyk, T.Y. Ohulchanskyy, C.A. Flask, P.N. Prasad, Combined optical and MR bioimaging using rare earth ion doped NaYF4 nanocrystals, Adv. Funct. Mater. 19 (6) (2009) 853–859, http://dx.doi.org/10.1002/adfm.200800765. [6] J.Y. Park, M.J. Baek, E.S. Choi, S. Woo, J.H. Kim, T.J. Kim, J.C. Jung, K.S. Chae, Y. Chang, G.H. Lee, Paramagnetic ultrasmall gadolinium oxide nanoparticles as advanced T1 MRI contrast agent: account for large longitudinal relaxivity, optimal particle diameter, and in vivo T1 MR images, ACS Nano 3 (11) (2009) 3663–3669, http://dx.doi. org/10.1021/nn900761s. [7] C. Zhou, H. Wu, C. Huang, M. Wang, N. Jia, Facile synthesis of single-phase mesoporous Gd2O3:Eu nanorods and their application for drug delivery and multimodal imaging, Part. Part. Syst. Charact. 31 (6) (2014) 675–684, http://dx.doi.org/10.1002/ ppsc.201300342. [8] R.M. Petoral, F. Söderlind, A. Klasson, A. Suska, M.A. Fortin, N. Abrikossova, L. Selegård, P.-O. Käll, M. Engström, K. Uvdal, Synthesis and characterization of Tb3+-doped Gd2O3 nanocrystals: a bifunctional material with combined fluorescent labeling and MRI contrast agent properties, J. Phys. Chem. C 113 (17) (2009) 6913–6920, http://dx.doi.org/10.1021/jp808708m. [9] Y. Li, T. Chen, W. Tan, D.R. Talham, Size-dependent MRI relaxivity and dual imaging with Eu0.2Gd0.8PO4·H2O nanoparticles, Langmuir 30 (20) (2014) 5873–5879http:// dx.doi.org/10.1021/la500602x. [10] T. Passuello, M. Pedroni, F. Piccinelli, S. Polizzi, P. Marzola, S. Tambalo, G. Conti, D. Benati, F. Vetrone, M. Bettinelli, A. Speghini, PEG-capped, lanthanide doped GdF3 nanoparticles: luminescent and T2 contrast agents for optical and MRI multimodal imaging, Nano 4 (24) (2012) 7682–7689http://dx.doi.org/10.1039/c2nr31796f.

824

C. Zhou et al. / Materials Science and Engineering C 78 (2017) 817–825

[11] S.A. Osseni, S. Lechevallier, M. Verelst, P. Perriat, J. Dexpert-Ghys, D. Neumeyer, R. Garcia, F. Mayer, K. Djanashvili, J.A. Peters, E. Magdeleine, H. Gros-Dagnac, P. Celsis, R. Mauricot, Gadolinium oxysulfide nanoparticles as multimodal imaging agents for T2-weighted MR, X-ray tomography and photoluminescence, Nano 6 (1) (2014) 555–564, http://dx.doi.org/10.1039/c3nr03982j. [12] R. Jenjob, N. Kun, J.Y. Ghee, Z. Shen, X. Wu, S.K. Cho, D.H. Lee, S.-G. Yang, Enhanced conjugation stability and blood circulation time of macromolecular gadoliniumDTPA contrast agent, Mater. Sci. Eng. C 61 (2016) 659–664, http://dx.doi.org/10. 1016/j.msec.2016.01.008. [13] D.R. Broome, Nephrogenic systemic fibrosis associated with gadolinium based contrast agents: a summary of the medical literature reporting, Eur. J. Radiol. 66 (2) (2008) 230–234, http://dx.doi.org/10.1016/j.ejrad.2008.02.011. [14] A.J. Nijdam, T.R. Nicholson Iii, J.P. Shapiro, B.R. Smith, J.T. Heverhagen, P. Schmalbrock, M.V. Knopp, A. Kebbel, D. Wang, S.C. Lee, Nanoparticulate iron oxide contrast agents for untargeted and targeted cardiovascular magnetic resonance imaging, Curr. Nanosci. 5 (5) (2009) 88–102, http://dx.doi.org/10.2174/157341309787314601. [15] R. Brooks, T(2)-shortening by strongly magnetized spheres: a chemical exchange model, Magn. Reson. Med. 47 (2) (2002) 388–391, http://dx.doi.org/10.1002/ mrm.10059. [16] B.R. Smith, J. Heverhagen, M. Knopp, P. Schmalbrock, J. Shapiro, M. Shiomi, N.I. Moldovan, M. Ferrari, S.C. Lee, Localization to atherosclerotic plaque and biodistribution of biochemically derivatized superparamagnetic iron oxide nanoparticles (SPIONs) contrast particles for magnetic resonance imaging (MRI), Biomed. Microdevices 9 (5) (2007) 719–727, http://dx.doi.org/10.1007/s10544-007-9081-3. [17] B. Ruprecht, M. Wilkening, A. Feldhoff, S. Steuernagel, P. Heitjans, High temperature polyol synthesis of superparamagnetic CoFe2O4 nanoparticles for magnetic resonance imaging contrast agents, J. Inorg. Mater. 24 (5) (2009) 967–972, http://dx. doi.org/10.1039/B901293A. [18] T.H. Shin, J.S. Choi, S. Yun, I.S. Kim, H.T. Song, Y. Kim, K.I. Park, J. Cheon, T1and T2 dualmode MRI contrast agent for enhancing accuracy by engineered nanomaterials, ACS Nano 8 (4) (2014) 3393–3401, http://dx.doi.org/10.1021/nn405977t. [19] X. Cui, S. Belo, D. Krüger, Y. Yan, R.T. de Rosales, M. Jaureguiosoro, H. Ye, S. Su, D. Mathe, N. Kovács, Aluminium hydroxide stabilised MnFe2O4 and Fe3O4 nanoparticles as dual-modality contrasts agent for MRI and PET imaging, Biomaterials 35 (22) (2014) 5840–5846, http://dx.doi.org/10.1016/j.biomaterials.2014.04.004. [20] D.K. Jha, K.S. Varadarajan, A.B. Patel, P. Deb, Direct synthesis of water dispersible superparamagnetic TGA capped FePt nanoparticles: one pot, one shot, Mater. Chem. Phys. 156 (2015) 247–253, http://dx.doi.org/10.1016/j.matchemphys.2015.03.016. [21] D.K. Jha, K. Saikia, S. Chakrabarti, K. Bhattacharya, K.S. Varadarajan, A.B. Patel, D. Goyary, P. Chattopadhyay, P. Deb, Direct one-pot synthesis of glutathione capped hydrophilic FePt-CdS nanoprobe for efficient bimodal imaging application, Mater. Sci. Eng. C 72 (2017) 415–424, http://dx.doi.org/10.1016/j.msec. 2016.11.077. [22] M.M. Ali, M.P. Bhuiyan, B. Janic, N.R. Varma, T. Mikkelsen, J.R. Ewing, R.A. Knight, M.D. Pagel, A.S. Arbab, A nano-sized PARACEST-fluorescence imaging contrast agent facilitates & validates in vivo CEST MRI detection of glioma, Nanomedicine 7 (12) (2012) 1827–1837, http://dx.doi.org/10.2217/nnm.12.92. [23] L. Cheng, K. Yang, Y. Li, J. Chen, C. Wang, M. Shao, S.T. Lee, Z. Liu, Facile preparation of multifunctional upconversion nanoprobes for multimodal imaging and dualtargeted photothermal therapy, Angew. Chem. 50 (32) (2011) 7385, http://dx.doi. org/10.1002/anie.201101447. [24] M. Mehrmohammadi, T.H. Shin, M. Qu, P. Kruizinga, R.L. Truby, J.H. Lee, J. Cheon, S.Y. Emelianov, In vivo pulsed magneto-motive ultrasound imaging using high-performance magnetoactive contrast nanoagents, Nano 5 (22) (2013) 11179–11186, http://dx.doi.org/10.1039/c3nr03669c. [25] Y.-X.J. Wang, S.M. Hussain, G.P. Krestin, Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging, Eur. Radiol. 11 (11) (2001) 2319–2331, http://dx.doi.org/10.1007/s003300100908. [26] D.L.J. Thorek, A.K. Chen, J. Czupryna, A. Tsourkas, Superparamagnetic iron oxide nanoparticle probes for molecular imaging, Ann. Biomed. Eng. 34 (1) (2006) 23–38, http://dx.doi.org/10.1007/s10439-005-9002-7. [27] Y. Zhang, X. Sun, L. Zhu, H. Shen, N. Jia, Electrochemical sensing based on graphene oxide/Prussian blue hybrid film modified electrode, Electrochim. Acta 56 (3) (2011) 1239–1245. [28] A.K. Geim, A.H. Macdonald, Graphene: exploring carbon flatland, Phys. Today 60 (8) (2007) 35–41, http://dx.doi.org/10.1063/1.2774096. [29] K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I.V. Grigorieva, A.A. Firsov, Electric field effect in atomically thin carbon films, Science (New York, N.Y.) 306 (5696) (2004) 666–669, http://dx.doi.org/10.1126/science.1102896. [30] L. Feng, Z. Liu, Graphene in biomedicine: opportunities and challenges, Nanomedicine 6 (2) (2011) 317–324, http://dx.doi.org/10.2217/nnm.10.158. [31] Z. Liu, X.-J. Liang, Nano-carbons as theranostics, Theranostics 2 (3) (2012) 235–237, http://dx.doi.org/10.7150/thno.4156. [32] F. Schedin, A.K. Geim, S.V. Morozov, E.W. Hill, P. Blake, M.I. Katsnelson, K.S. Novoselov, Detection of individual gas molecules adsorbed on graphene, Nat. Mater. 6 (9) (2007) 652–655, http://dx.doi.org/10.1038/nmat1967. [33] Z. Liu, J.T. Robinson, X. Sun, H. Dai, PEGylated nanographene oxide for delivery of water-insoluble cancer drugs, J. Am. Chem. Soc. 130 (33) (2008) 10876–10877, http://dx.doi.org/10.1021/ja803688x. [34] H. Zhang, T. Yan, S. Xu, S. Feng, D. Huang, M. Fujita, X.D. Gao, Graphene oxidechitosan nanocomposites for intracellular delivery of immunostimulatory CpG oligodeoxynucleotides (Mater. Sci. Eng.), C 73, 2017 144–151, http://dx.doi. org/10.1016/j.msec.2016.12.072. [35] D. Chen, H. Feng, J. Li, Graphene oxide: preparation, functionalization, and electrochemical applications, Chem. Rev. 112 (11) (2012) 6027–6053, http://dx.doi.org/ 10.1021/cr300115g.

[36] K. Yang, Y. Li, X. Tan, R. Peng, Z. Liu, Behavior and toxicity of graphene and its functionalized derivatives in biological systems, Small 9 (9–10) (2013) 1492–1503, http://dx.doi.org/10.1002/smll.201201417. [37] I. Raynal, P. Prigent, S. Peyramaure, A. Najid, C. Rebuzzi, C. Corot, Macrophage endocytosis of superparamagnetic iron oxide nanoparticles: mechanisms and comparison of ferumoxides and ferumoxtran-10, Investig. Radiol. 39 (1) (2004) 56–63, http://dx.doi.org/10.1097/01.rli.0000101027.57021.28. [38] W. Chen, P. Yi, Y. Zhang, L. Zhang, Z. Deng, Z. Zhang, Composites of aminodextrancoated Fe3O4 nanoparticles and graphene oxide for cellular magnetic resonance imaging, ACS Appl. Mater. Interfaces 3 (10) (2011) 4085–4091, http://dx.doi.org/10. 1021/am2009647. [39] X. Shi, H. Gong, Y. Li, C. Wang, L. Cheng, Z. Liu, Graphene-based magnetic plasmonic nanocomposite for dual bioimaging and photothermal therapy, Biomaterials 34 (20) (2013) 4786–4793, http://dx.doi.org/10.1016/j.biomaterials. 2013.03.023. [40] J. Shen, Y. Hu, M. Shi, N. Li, H. Ma, M. Ye, One step synthesis of graphene oxide− magnetic nanoparticle composite, J. Phys. Chem. C 114 (3) (2010) 1498–1503, http://dx.doi.org/10.1021/jp909756r. [41] H.-P. Cong, J.-J. He, Y. Lu, S.-H. Yu, Magnetic graphene: Water-Soluble MagneticFunctionalized Reduced Graphene Oxide Sheets: In situ Synthesis and Magnetic Resonance Imaging Applications, Small 6 (2) (2010)http://dx.doi.org/10.1002/ smll.201090003 (n/a-n/a). [42] V.K. Singh, M.K. Patra, M. Manoth, G.S. Gowd, S.R. Vadera, N. Kumar, In situ synthesis of graphene oxide and its composites with iron oxide, New Carbon Mater. 24 (2) (2009) 147–152, http://dx.doi.org/10.1016/S1872-5805(08)60044-X. [43] T. Szabó, A. Bakandritsos, V. Tzitzios, E. Devlin, D. Petridis, I. Dékány, Magnetically modified single and turbostratic stacked graphenes from tris(2,2′-bipyridyl) iron(II) ion-exchanged graphite oxide, J. Phys. Chem. B 112 (46) (2008) 14461–14469, http://dx.doi.org/10.1021/jp802879a. [44] F. He, J. Fan, D. Ma, L. Zhang, C. Leung, H.L. Chan, The attachment of Fe3O4 nanoparticles to graphene oxide by covalent bonding, Carbon 48 (11) (2010) 3139–3144, http://dx.doi.org/10.1016/j.carbon.2010.04.052. [45] H. He, C. Gao, Supraparamagnetic, conductive, and processable multifunctional graphene nanosheets coated with high-density Fe3O4 nanoparticles, ACS Appl. Mater. Interfaces 2 (11) (2010) 3201–3210, http://dx.doi.org/10.1021/ am100673g. [46] P. Lian, X. Zhu, H. Xiang, Z. Li, W. Yang, H. Wang, Enhanced cycling performance of Fe3O4–graphene nanocomposite as an anode material for lithium-ion batteries, Electrochim. Acta 56 (2) (2010) 834–840, http://dx.doi.org/10.1016/j.electacta. 2010.09.086. [47] M. Zhang, D. Lei, X. Yin, L. Chen, Q. Li, Y. Wang, T. Wang, Magnetite/graphene composites: microwave irradiation synthesis and enhanced cycling and rate performances for lithium ion batteries, J. Mater. Chem. 20 (26) (2010) 5538–5543, http://dx.doi.org/10.1039/c0jm00638f. [48] X. Yang, X. Zhang, Y. Ma, Y. Huang, Y. Wang, Y. Chen, Superparamagnetic graphene oxide-Fe3O4 nanoparticles hybrid for controlled targeted drug carriers, J. Mater. Chem. 19 (18) (2009) 2710–2714, http://dx.doi.org/10.1039/b821416f. [49] Q. Liu, W. Ren, Z.-G. Chen, B. Liu, B. Yu, F. Li, H. Cong, H.-M. Cheng, Direct synthesis of carbon nanotubes decorated with size-controllable Fe nanoparticles encapsulated by graphitic layers, Carbon 46 (11) (2008) 1417–1423, http://dx.doi.org/10.1016/j. carbon.2008.06.021. [50] X. Sun, Z. Liu, K. Welsher, J.T. Robinson, A. Goodwin, S. Zaric, H. Dai, Nano-graphene oxide for cellular imaging and drug delivery, Nano Res. 1 (3) (2008) 203–212, http://dx.doi.org/10.1007/s12274-008-8021-8. [51] W.S. Hummers, R.E. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80 (6) (1958) 1339–1399, http://dx.doi.org/10.1021/ja01539a017. [52] L. Yu, H. Wu, B. Wu, Z. Wang, H. Cao, C. Fu, N. Jia, Magnetic Fe3O4-reduced graphene oxide nanocomposites-based electrochemical biosensing, Nano-Micro Letters 6 (3) (2014) 258–267, http://dx.doi.org/10.1007/bf03353790. [53] H. Wu, G. Liu, Y. Zhuang, D. Wu, H. Zhang, H. Yang, H. Hu, S. Yang, The behavior after intravenous injection in mice of multiwalled carbon nanotube/Fe3O4 hybrid MRI contrast agents, Biomaterials 32 (21) (2011) 4867–4876, http://dx.doi.org/10. 1016/j.biomaterials.2011.03.024. [54] M. Chen, Y. Nam Kim, A. Cuncheng Li, S.O. Cho, Preparation and characterization of magnetic nanoparticles and their silica egg-yolk-like nanostructures: a prospective multifunctional nanostructure platform, J. Phys. Chem. C 112 (17) (2008) 6710–6716, http://dx.doi.org/10.1021/jp710775j. [55] M. Yin, M. Wang, F. Miao, Y. Ji, Z. Tian, H. Shen, N. Jia, Water-dispersible multiwalled carbon nanotube/iron oxide hybrids as contrast agents for cellular magnetic resonance imaging, Carbon 50 (6) (2012) 2162–2170, http://dx.doi.org/10.1016/j. carbon.2012.01.026. [56] L. Wan, Q. Zhao, P. Zhao, B. He, T. Jiang, Q. Zhang, S. Wang, Versatile hybrid polyethyleneimine–mesoporous carbon nanoparticles for targeted delivery, Carbon 79 (2014) 123–134, http://dx.doi.org/10.1016/j.carbon.2014.07.050. [57] Q. Wu, G. Zhao, C. Feng, C. Wang, Z. Wang, Preparation of a graphene-based magnetic nanocomposite for the extraction of carbamate pesticides from environmental water samples, J. Chromatogr. A 1218 (44) (2011) 7936–7942, http://dx.doi.org/ 10.1016/j.chroma.2011.09.027. [58] H.-L. Guo, X.-F. Wang, Q.-Y. Qian, F.-B. Wang, X.-H. Xia, A green approach to the synthesis of graphene nanosheets, ACS Nano 3 (9) (2009) 2653–2659, http://dx.doi. org/10.1021/nn900227d. [59] S. Alwarappan, A. Erdem, C. Liu, C.-Z. Li, Probing the electrochemical properties of graphene nanosheets for biosensing applications, J. Phys. Chem. C 113 (20) (2009) 8853–8857, http://dx.doi.org/10.1021/jp9010313. [60] S. Li, Y. Hu, Q. Xu, J. Sun, B. Hou, Y. Zhang, Iron- and nitrogen-functionalized graphene as a non-precious metal catalyst for enhanced oxygen reduction in an

C. Zhou et al. / Materials Science and Engineering C 78 (2017) 817–825 air-cathode microbial fuel cell, J. Power Sources 213 (2012) 265–269, http://dx.doi. org/10.1016/j.jpowsour.2012.04.002. [61] A.C. Ferrari, J. Robertson, Interpretation of Raman spectra of disordered and amorphous carbon, Phys. Rev. B 61 (20) (2000) 14095–14107, http://dx.doi.org/10. 1103/PhysRevB.61.14095. [62] K.N. Kudin, B. Ozbas, H.C. Schniepp, R.K. Prud'homme, I.A. Aksay, R. Car, Raman spectra of graphite oxide and functionalized graphene sheets, Nano Lett. 8 (1) (2008) 36–41, http://dx.doi.org/10.1021/nl071822y. [63] S. Stankovich, R.D. Piner, S.T. Nguyen, R.S. Ruoff, Synthesis and exfoliation of isocyanate-treated graphene oxide nanoplatelets, Carbon 44 (15) (2006) 3342–3347, http://dx.doi.org/10.1016/j.carbon.2006.06.004.

825

[64] X. Wang, Z. Zhao, J. Qu, Z. Wang, J. Qiu, Fabrication and characterization of magnetic Fe3O4–CNT composites, J. Phys. Chem. Solids 71 (4) (2010) 673–676, http://dx.doi. org/10.1016/j.jpcs.2009.12.063. [65] S. Ju, G. Teng, Y. Zhang, M. Ma, F. Chen, Y. Ni, In vitro labeling and MRI of mesenchymal stem cells from human umbilical cord blood, Magn. Reson. Imaging 24 (5) (2006) 611–617, http://dx.doi.org/10.1016/j.mri.2005.12.017. [66] S.-L. Hu, J.-Q. Zhang, X. Hu, R. Hu, H.-S. Luo, F. Li, Y.-Z. Xia, J.-T. Li, J.-K. Lin, G. Zhu, H. Feng, In vitro labeling of human umbilical cord mesenchymal stem cells with superparamagnetic iron oxide nanoparticles, J. Cell. Biochem. 108 (2) (2009) 529–535, http://dx.doi.org/10.1002/jcb.22283.