5 0 ( 2 0 1 2 ) 2 1 6 2 –2 1 7 0
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/carbon
Water-dispersible multiwalled carbon nanotube/iron oxide hybrids as contrast agents for cellular magnetic resonance imaging Min Yin a, Mingliang Wang b, Fei Miao b, Yuxuan Ji a, Zhong Tian a, Hebei Shen a, Nengqin Jia a,* a
The Education Ministry Key Laboratory of Resource Chemistry, Department of Chemistry, Life and Environmental Science College, Shanghai Normal University, Shanghai 200234, China b Department of Radiology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China
A R T I C L E I N F O
A B S T R A C T
Novel magnetic resonance imaging (MRI) contrast agents composed of multiwalled carbon
Received 10 August 2011
nanotubes decorated with magnetic iron oxide nanoparticles were explored. They were
Accepted 6 January 2012
functionalized with multilayer polyelectrolytes through layer-by-layer assembling and
Available online 15 January 2012
were shown to be hydrophilic, biocompatible, and have a high MRI contrast. A targeted ligand folic acid was chemically bonded to the functionalized nanotubes for specific targeting and imaging of cancer cells in MRI. The results demonstrate that the material can be used as ideal targeted imaging agents and is sufficient to obtain strong MRI contrast. 2012 Elsevier Ltd. All rights reserved.
Molecular imaging is proving new approaches for early tumor detection, diagnosis and monitor, so it can lead to a wide scope of application not only in diagnostic radiology but also therapeutic medicine [1–3]. Various kinds of molecular imaging contrast agents have been studied for optical imaging, magnetic resonance imaging (MRI), computer tomography, ultrasound tomography, and positron emission tomography. Along with the advance of the nanotechnology more and more nanomaterials are utilized as versatile probes in different biomedical imaging. For example, semiconductor quantum dots and magnetic nanoparticles have been demonstrated as excellent imaging agents to detect and control biologically active processes at the molecular and cellular level [4–7]. MRI ranks among the best molecular imaging methodologies nowadays available in clinical medicine diagnostic as its non-invasive nature, real-time monitoring and multidimensional tomographic capabilities coupled with high spatial
resolution [8–11]. MRI has further advanced from the development of contrast agents that enabled more specific and clearer images and enlargements of detectable organs and systems. During the last decade, a great deal of attention has been paid to search for improved MRI ultrasensitive contrast agents. Current superparamagnetic iron oxide (SPIO) nanoparticles are prevalent in molecular imaging as MRI contrast agent because of their localized shortening of spin–spin proton transverse relaxation times (T2) and effective transverse relaxation times (T2 ), which permit negative contrast enhancement and darker images of the region of interest [12–15]. By functionalizing SPIO nanoparticles with targeting vectors (i.e. antibodies, small molecular, ligands, aptamers), known biomarkers for cancer (e.g. cell surface receptor) can be specifically bound to pathological tissue to induce the local imaging contrast between normal and tumor tissue [16–18]. In most applications, monodisperse spherical magnetic nanoparticles have been frequently used as contrast agents for MRI. However, due to their structural limitation, these
* Corresponding author: Fax: +86 21 64321833. E-mail address: [email protected]
(N. Jia). 0008-6223/$ - see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2012.01.026
5 0 ( 20 1 2 ) 2 1 6 2–21 7 0
spherical magnetic nanoparticles still need to be improved for making effective MRI contrast enhancement and developing the multifunctional nanoprobes. Recently, it is reported that several spherical magnetic nanoparticles-assembling elongated hybrids and nanoclusters could greatly improve the MRI property. The improvement is attributed to the synergistic effect of multiple magnetic nanoparticles and enhanced polyvalent binding to amplify particle affinity for cell surface receptors [19–23]. Therefore, it could be a feasible and promising strategy to develop novel magnetic nanoparticles-based hybrids as suitable MRI contrast agents. Carbon nanotubes present remarkable opportunities to meet a number of biological and biomedical applications including, but not limited to, protein and peptide transporter, drug and gene delivery, medical imaging, and cancer targeting/therapeutics [24–34]. The importance of generating carbon nanotube–nanoparticle heterostructures is that these hybrids are supposed to take advantage of both carbon nanotubes and nanoparticles in one discrete structure. Therefore, by combining the attractive tubular structure with magnetic property, SPIO-decorated carbon nanotubes can be an ideal candidate for the multifunctional nanomaterial application in a range of diverse fields. In our work, with the aim of exploring potentially excellent contrast agents for constructing intracellular molecular imaging agents for biomedical application, we prepared folic acid (FA)-conjugated functionalized multiwalled carbon nanotube (MWCNT)/magnetic nanoparticle hybrids via layer-by-layer assembling, which could be used as targeting contrast agent for cancer-cell specific imaging in MRI (Fig. 1). With the benefits of high specific surface area for high loading of nanoparticles and targeting biomolecules and the long tubular structure for providing a large number of interactions between the targeting ligands and their cell-surface receptors, the magnetic multiwalled carbon nanotubes (MAGCNTs) are therefore expected to improve their targeting magnetic relaxivity in MRI. To the authors’ knowledge, this is
the first study related to the functionalization of MAGCNTs with polyelectrolyte for cancer cell targeting and MR imaging. With this novel functionalization, the MAGCNTs exhibited high aqueous dispersion, low toxicity, and effective MRI cellular targeting imaging.
All reagents used were available commercially and were of high purity grade. MWCNTs (95% purity, diameter 10–20 nm, length 1–2 lm) were obtained from Shenzhen Nanotech Port Co. Ferrocene, polyethylenimine (PEI) (25 K), polystyrene sulfonate sodium salt (PSS), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and folic acid (FA) were purchased from Sigma–Aldrich and used as received. Human cervical carcinoma HeLa cells were obtained from Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS, China).
Synthesis of magnetic multiwalled carbon nanotubes
The shortened MWCNTs were first acquired according to our previously reported method . In a typical procedure: the MWCNTs were added in a concentrated sulfuric acid/nitric acid mixture (3:1 v/v) and sonicated in a sonic bath for 16 h. After this treatment, the shortened MWCNTs were obtained after filtration and were then thoroughly rinsed. Then, superparamagnetic nanoparticles were strongly attached to the shortened MWCNTs through the decomposition of ferrocene at high temperatures according to a reported method with slight modification . In brief, 50 mg of MWCNTs and 50 mg of ferrocene were placed in a 30 mL crucible and sealed in a stainless autoclave with air. The autoclave was heated to 425 C for 2 h to transform ferrocene into magnetic nanoparticles and then cooled to room temperature naturally. The
Fig. 1 – Synthesis and targeting of MAGCNTs–PSS–PEI–FA conjugates for magnetic resonance imaging (MRI) of cancer cells.
5 0 ( 2 0 1 2 ) 2 1 6 2 –2 1 7 0
dark product was collected and washed with absolute ethanol repeatedly to remove excess unreacted ferrocene. Finally, it was vacuum-dried at 60 C for 6 h. The sizes of magnetic nanoparticles were examined at room temperature using transmission electron microscopy (TEM). The chemical states of MAGCNTs were studied by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. The magnetic properties of the MAGCNTs were measured using a vibrating sample magnetometer (VSM).
2.3. Preparation MAGCNTs
Cell labeling by Prussian blue staining
After incubation with MAGCNTs–PSS–PEI particles at the iron concentration of 0.08 mM for 6 h, cells were washed four times to remove excess MAGCNTs–PSS–PEI. The cells were then fixed for 40 min using 4% paraformaldehyde. Cultures were then washed three times with PBS and incubated with Perls’ reagent (4% potassium ferrocyanide/12% HCl, 50:50, v/ v) for 1 h. The cells were washed with PBS and observed using optical microscopy.
To make the MAGCNTs disperse in water with satisfying stability and further functionalize, polymer wrapping was performed by the following method. Briefly, 3 mg of MAGCNTs were dispersed into a 20 mL 1 wt.% aqueous solution of an anionic polyelectrolyte, PSS (polystyrene sulfonate sodium salt) and vigorously stirred to ensure that well dispersion of individual MAGCNTs, the reaction continued at 65 C for 12 h. The solution was filtered to remove large aggregates or contaminants, and excess unbound PSS was removed by a magnet. The resulting MAGCNTs–PSS product was redispersed in pure water by brief sonication and a stable colloid of PSS wrapped MAGCNTs in water was obtained. The surface zeta-potential of MAGCNTs–PSS was measured by a Malvern Zetasizer Nano 90. After polymer wrapping with the anionic polyelectrolyte, the MAGCNTs–PSS were further mixed with a 20 mL 1 wt.% aqueous solution of the cationic polyelectrolyte PEI with shaking. After adsorption of PEI for 3 h, the suspension was centrifuged at 12,000 rpm for 15 min. The obtained MAGCNTs–PSS–PEI were washed by three alternate cycles of centrifuging and re-suspending the particles in pure water. The surface zeta-potential of MAGCNTs–PSS–PEI was measured as above. The iron concentration on the MAGCNTs was quantitatively determined by using inductively coupled plasma optical emission spectroscopy (ICP-OES).
The cytotoxicity assay by flow cytometry
HeLa cells were maintained in MEM (Minimum Essential Medium) supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL penicillin G and 100 mg/mL streptomycin in an incubator at 37 C in a humidified 5% CO2 atmosphere. Cells at a logarithmic phase were detached and seeded in 25-mL flasks at a density of 2–3 · 105 cells per flask in a 2-mL volume. After culturing overnight at 37 C to allow exponential growth, 200 ll of MAGCNTs–PSS–PEI hybrids in 0.9% NaCl solution at different concentrations based on iron nanoparticles concentration (0.05, 0.1, 0.15, and 0.2 mM) were added to each flask. After being incubated, the cells were collected from the corresponding groups, washed twice with cool PBS and re-suspended in PBS. Cell viability was evaluated by a Becton–Dickinson FACS Calibur after incubation in various concentrations of nanotube solutions. For the cell cytometry, the cells were observed immediately after the initial incubation and the level of cell death was compared with control cells by propidium iodide (PI) staining.
Biofunctionalization of MAGCNTs–PSS–PEI with FA
FA (3.7 mg, 0.0084 mmol) and EDC (9.3 mg, 0.021 mmol) were dissolved in 3 mL of DMSO and the mixture was stirred at room temperature for 3 h to activate the c-carboxylic acid of FA. The PSS–PEI-coated MAGCNTs were dispersed in 20 mL of distilled water. Two milliliters of the FA solution was added drop-wise into the aqueous dispersion of MAGCNTs–PSS–PEI under stirring at room temperature. After reaction for 6 h, the MAGCNTs–PSS–PEI–FA conjugates were magnetically separated from the free FA using magnets. The conjugates were washed three times with distilled water and redispersed in 0.9% NaCl by brief sonication. The MAGCNTs–PSS–PEI–FA conjugates were characterized by UV–vis absorption spectroscopy.
2.7. Quantitative intracellular iron content assessment by inductively coupled plasma-optical emission spectroscopy The iron concentration of labeled cells was assessed using inductively coupled plasma optical emission spectroscopy (ICP-OES). A predetermined number of MAGCNTs–PSS–PEI– FA-labeled, MAGCNTs–PSS–PEI-labeled and unlabeled control HeLa cells (1 · 106 cells) were pelleted and 200 mL of 65% (v/ v) nitric acid were added for 2 h. The obtained cell extracts were then diluted in water to reach an iron concentration compatible for reference to a standard curve. The total iron content (pg) was measured at 238.2 nm by means of a VistaMPX ICP-OES device (Varian, USA).
Magnetic resonance imaging (MRI)
MRI experiments were carried out on a 3.0-T clinical MRI instrument (GE signa3.0-T HDx). MR images were acquired by T2-weighted with the imaging parameters as follow: 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. In order to obtain relaxivity value, we also used T2 -weighted with the following set parameters: repetition rate (TR) = 300 ms, various echo times (TE) from 3.2 to 26.6 ms, field of view (FOV) = 12.0 cm, slice thickness = 2 mm. The software was used for data acquisition, reconstruction and visualization/analysis of the images. After acquiring the images, the T2 value was measured by region of interest (ROI) analysis of T2 -weighted image using the manuallydrawn circular ROI for each of the samples. To estimate the transverse relaxivities r2 value, we measured the effective transverse time T2 values of the MAGCNTs–PSS–PEI with varying iron concentrations (0–0.2 mM, iron concentration in
5 0 ( 20 1 2 ) 2 1 6 2–21 7 0
hybrids was determined using inductively coupled plasma) in water. Furthermore, T2 -weighted images were acquired. The MR contrast effects of MAGCNTs–PSS–PEI and MAGCNTs–PSS–PEI–FA in vitro were obtained as follow. MAGCNTs–PSS–PEI and MAGCNTs–PSS–PEI–FA were dispersed in 0.9% NaCl solution (in each case maintaining an equivalent iron concentration for respective comparisons). 1 · 106 HeLa cells were incubated with MAGCNTs–PSS–PEI or MAGCNTs– PSS–PEI–FA with Fe concentrations of 0.08 mM at 37 C with 5% CO2 for 6 h in a humid incubator. The MAGCNTs–PSS– PEI–FA labeled, MAGCNTs–PSS–PEI labeled and unlabeled cells were washed three times with PBS, trypsinized and centrifuged to prepare the pellets. The pellets were fixed with 2.5% glutaraldehyde in PBS for 1 h, then discarded the supernatant carefully. Five hundred microliters of 0.5% agarose gel was added to each cell pellets in 1.5 mL Eppendorf tubes, and the cells were suspended by gentle shaking. MRI of these tubes was performed with a clinical 3.0-T MR imager.
Results and discussion
Magnetic multiwalled carbon nanotubes were synthesized through thermal decomposition of ferrocene. Firstly, the pristine MWCNTs were shortened to a desired size (about 100– 300 nm of nanotube length) by treating with strong acids and constant sonication, followed by washing with distilled water several times. Then, the MAGCNTs were synthesized by employing a one-step approach, in which ferrocene decomposed at high temperature of 425 C, and transformed into iron oxides to deposit on MWCNTs. Fig. 2A shows the TEM image of MAGCNTs. It can be clearly seen that iron oxide nanoparticles adhered to the walls of MWCNTs and the size of the particles was estimated to be 7–8 nm, indicating their superparamagnetic properties [36,37]. As shown in Fig. 2B, the magnetic properties of the MAGCNTs exhibited typical superparamagnetic characteristics owing to the existence of nano-sized magnetic particles on the nanotubes, and the MAGCNTs suspended in distilled water were easily attracted to magnets (inset in Fig. 2B). The obtained MAGCNTs were characterized by XRD, XPS and Raman spectroscopy (see Figs.
S1–S3 inSupplementary material), revealing that the magnetic nanoparticles in the hybrids are mainly Fe2O3. Furthermore, it could be investigated that these superparamagnetic nanoparticles were firmly fixed on the CNTs to keep it from breaking off while the hybrids were treated with repetitive washing, severe agitation, and prolonged sonication. However, the obtained MAGCNTs were relatively poorly dispersed in aqueous media, which limited their applications in biological fields. Well dispersing MAGCNTs with satisfying stability in aqueous media is a key prerequisite with respect to in vitro and in vivo applications of MAGCNTs as MRI contrast agents. Dissolution of CNT in water have been facilitated by surfactants, polymers, proteins, phospholipids, and other chemical modifications [38–41]. In our work, anionic polyelectrolyte polystyrene sulfonate sodium salt (PSS) was firstly used as a wrapping polymer providing remarkably stable aqueous dispersions of MAGCNTs. The surface zeta-potential of MAGCNTs–PSS was determined by a Malvern Zetasizer Nano ZS 90. As shown in Fig. 3A, the zeta-potential of MAGCNTs–PSS suspended in water was found to be 73.5 mV, which is a key factor in affecting the stability of MAGCNTs. The high negative surface charges could be ascribed to the high density of sulfonate groups on the negatively charged polyelectrolyte PSS, acting as a primer onto the MAGCNTs surface for the subsequent, homogeneous adsorption. In order to improve the efficiency of water-dispersible MAGCNTs transfer in vitro and offer amine groups on the surface of MAGCNTs for further conjugating with a targeting ligand, the MAGCNTs–PSS were further modified with a cationic polyelectrolytes polyethylenimine (PEI) through electrostatic interactions. Zeta potential measurements show that after the electrostatic interactions, the surface zeta-potentials value of MAGCNTS–PSS–PEI suspended in water dramatically increased to +45.0 mV (Fig. 3A). The results suggest that the negatively charged surface of MAGCNTs–PSS was fully covered with PEI, which in turn provides a homogeneous distribution of positive charges. The assynthesized MAGCNTs–PSS–PEI have abundant amine groups on their surface which provide convenient sites for covalent
Fig. 2 – (A) TEM image of pristine MAGCNTs showing the heterostructured complexes of the MWCNT/iron oxide nanoparticle hybrids. (B) Magnetic hysteresis curves of MAGCNTs (black line) and MWCNTs (red line). Inset: photograph of MAGCNTs and MWCNTs suspended in distilled water under an applied magnetic field. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
5 0 ( 2 0 1 2 ) 2 1 6 2 –2 1 7 0
Fig. 3 – (A) Zeta potential distributions of MAGCNTs (red line), MAGCNTs–PSS (green line), MAGCNTs–PSS–PEI (blue line). (B) TEM image of MAGCNTs wrapped by PSS and PEI, the marked thickness profile along the nanotube (indicate by arrow) reveals that PEI and PSS coat on MWCNTs surface. (C) Photograph of (a) pristine MAGCNTs dispersed in water (0.15 mg/mL), (b–e) MAGCNTs–PSS–PEI well dispersed in different solvents (0.15 mg/mL). (b) Water, (c) 0.9% NaCl, (d) PBS, and (e) MEM cell culture medium. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Fig. 4 – The cytotoxicity effects of the MAGCNTs–PSS–PEI hybrids. HeLa cells treated with MAGCNTs–PSS–PEI hybrids ranging from (a–e) 0, 0.05, 0.1, 0.15, 0.2 mM iron concentration in culture medium at 37 C for 6 h. After incubation and washings, the cells were stained with propidium iodide (PI) and analyzed by flow cytometry using 20,000 events. The histograms were plotted on a log scale. The bar represents the percentage of dead HeLa cells.
linking of biological molecules. Combining with the TEM result (Fig. 3B), direct evidence for the presence of polyelectrolytes on MAGCNTs surface can be found. The thickness of
the polymer coating was about 5–6 nm. Thus, through this layer-by-layer assembling of multilayer ployelectrolytes, the MAGCNTs–PSS–PEI were found to be well dispersed and stable
5 0 ( 20 1 2 ) 2 1 6 2–21 7 0
Fig. 5 – (A) T2 -weighted MR images (top: color maps, bottom: black and white images) of the PSS-PEI–coated MAGCNTs at various concentrations in water, a discrete contrast is observed according to the concentration of the iron oxide nanoparticles. The color bar changing from blue to red indicates the gradual decrease in MR signal intensity. (B) Plot of spin–spin effective transverse relaxation rate (R2 ) against Fe concentration for MAGCNTs–PSS–PEI hybrids, from which the relaxivity (r2 ) is determined. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
in water, salt solution, PBS buffer solution and cell culture medium (Fig. 3C). To assess the biocompatibility of the polymer-coated MAGCNTs–PSS–PEI, we initially studied the cytotoxicity effects of the MAGCNTs–PSS–PEI on Human cervical cancer HeLa cells using flow cytometry. As shown in Fig. 4, the HeLa cells treated with MAGCNTs–PSS–PEI displayed a low toxicity at different concentrations of the hybrids based on iron concentrations ranging from 0.05 to 0.2 mM. Especially, there is a minimum level of cell death similar to the untreated cells upon treatment with MAGCNTs–PSS–PEI at iron concentrations below 0.1 mM, at which concentration was sufficient to produce a significant MR contrast effect in vitro. These data
indicate that the as-prepared water-dispersible MAGCNTs– PSS–PEI hybrids are biocompatible that can be used in biomedical fields. To investigate the MAGCNTs–PSS–PEI hybrids as improved contrast agents in MRI, we studied the differences in the image contrasts created by the MAGCNTs–PSS–PEI samples with different iron content. The T2 -weighted images of the MAGCNTs–PSS–PEI with varying iron concentrations (0–0.2 mM) in water were obtained using a clinical 3.0-T MRI system ( Fig. 5A). The signal intensity of the MR image is related to the adhered iron oxide nanoparticles on the nanotubes. In color maps based on effective transverse time T2 values, it is clear that as the iron concentration of the MAGCNTs–PSS– PEI increases, the image color change from blue to red indicates the gradual decrease of MR signal intensity, which is consistent with those observations based on the intensity of black color. The water-dispersible magnetic nanoparticles decorating the MWCNTs exhibited a high effective transverse relaxivity r2 value of 263.04 mM 1 S 1 (Fig. 5B). Here, the reciprocal relaxation time, 1/T2 = R2 , defined as the relaxation rate, is plotted versus the concentration of superparamagnetic irons. The slope of the curve yields the concentration independent relaxivity, r2 . These results clearly show that the MAGCNTs–PSS–PEI could be very promising serving as an effective MRI contrast agent. We further investigated the availability on cell internalization of the MAGCNTs–PSS–PEI and their MR contrast effect in the cells. It is important to know intracellular MAGCNTs–PSS– PEI incorporation and their labeling efficiency. For this purpose, we performed Prussian blue staining investigation of the HeLa cells treated with MAGCNTs–PSS–PEI for 6 h. As shown in Fig. 6A, a large amount of blue color staining could be seen in almost all of treated cells, which can be ascribed that the iron nanoparticles fixed on the MWCNTs reacted to potassium ferrocyanide and hydrochloric acid formed Prussian blue. The results indicate that the MAGCNTs–PSS–PEI hybrids could effectively transport into the cells with high labeled rate. Furthermore, we evaluated the MR contrast effect of MAGCNTs–PSS–PEI hybrids uptaking into the cells. HeLa cells were treated with the MAGCNTs–PSS–PEI for 6 h and immobilized in agar (0.5%, w/v) in 1.5 mL centrifuge tubes for T2-weighted scan at a clinical 3.0-T MR system. The control samples consisted of the untreated cells and the cells
Fig. 6 – (A) Prussian blue-stained cell image of MAGCNTs–PSS–PEI hybrids treated Human cervical cancer HeLa cells. Intracytoplasmic blue particles are clearly visible with Prussian blue staining. (B) MRI effective transverse relaxation times (T2 ) of untreated control HeLa cells and HeLa cells treated with MAGCNTs–PSS–PEI, plain MWCNTs-PSS–PEI.
5 0 ( 2 0 1 2 ) 2 1 6 2 –2 1 7 0
FA MAGCNTs-PSS-PEI MAGCNTs-PSS-PEI-FA
Wavelength(nm) Fig. 7 – UV–vis absorption spectra of the FA (black curve), and the MAGCNTs–PSS–PEI (red curve) and the MAGCNTs– PSS–PEI–FA (green curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
treated with plain MWCNTs–PSS–PEI. The significant decreasing in the T2 relaxation time arising from MAGCNTs–PSS–PEItreated cells was observed as compared to those from the control samples (Fig. 6B), confirming the MAGCNTs–PSS–PEI is a potential candidate for improved MRI contrast agents both in solution and inside cells. To validate the diagnostic potential of the MAGCNTs–PSS– PEI as a platform for cancer cell targeting and imaging, we further investigated their utility in vitro for MRI cancer targeted detection. As the attachment of tumor-specific targeting ligands to nanomaterials has the promise to enhance tumor targeting for improved detection and therapy. In this work, the water-dispersible MAGCNTs–PSS–PEI hybrids were conjugated with the cancer-targeting ligand folic acid (FA)  via amide reaction between the carboxyl group of the FA and the amine group of PEI segment to obtain the MAGCNTs–PSS–PEI–FA conjugates, which were verified by UV–vis absorption spectroscopy ( Fig. 7). The non-targeted MAGCNTs–PSS–PEI only displayed a weak peak at 265 nm (red curve) while an obvious absorption peak of MAGCNTs– PSS–PEI–FA at 282 nm (green curve) can be attributed to the FA characteristic absorption peak (black curve). It is quite documented that the FA molecules had successfully anchored to the MAGCNTs–PSS–PEI as targeting ligand. As it is known that Human cervical cancer HeLa cells line exhibited a high level of folate receptor (FR) over-expression [43,44], the HeLa cells were chosen as model cell lines in our study to evaluate the specific targeting MRI capability of the MAGCNTs–PSS–PEI–FA conjugates. The cells were treated with either folate-targeted MAGCNTs–PSS–PEI or non-targeted MAGCNTs–PSS–PEI at a concentration based on 0.08 mM iron concentration for 6 h, and subjected to MRI experiments based on a T2 -weighted scan. As expected, the T2 -weighted MR image of the MAGCNTs–PSS–PEI–FA treated cells was much darker than those of the MAGCNTs–PSS–PEI treated cells and the control nontreated HeLa cells (Fig. 8A). Furthermore, this effect is clearly seen in color maps based
Fig. 8 – In vitro T2 -weighted MR images (A) black and white images; (B) color maps) of control nontreated HeLa cells, MAGCNTs–PSS–PEI treated HeLa cells and MAGCNTs–PSS– PEI–FA treated HeLa cells. The corresponding T2 relaxation times are shown. (C) Inductively coupled plasma-optical emission spectroscopy analysis of the targeting labeling efficiency for MAGCNTs–PSS–PEI–FA to HeLa cells compared to MAGCNTs–PSS–PEI to HeLa cells. MAGCNTs–PSS–PEI–FA showed improved cancer cell targeting.
on T2 values (Fig. 8B). The T2 relaxation time decreased from 69.6 ms of control nontreated cells to 38.3 ms for the MAGCNTs–PSS–PEI treated cells and 25.1 ms for the MAGCNTs–PSS–PEI–FA treated cells. This corresponds to a T2 MAGCNTs–PSS–PEI reduction to 44.97% of the initial value (control HeLa cells) whereas MAGCNTs–PSS–PEI–FA reduced the T2 to 63.94% of the initial value, exhibiting strong targeting specificity MRI contrast effect of the FA-targeted conjugates. Furthermore, the quantitative analysis of ICP-OES confirmed that the HeLa cells treated with MAGCNTs– PSS–PEI–FA had a higher iron content than the those treated with non-targeted MAGCNTs–PSS–PEI and nontreated control cells (Fig. 8C), indicating the MAGCNTs–PSS–PEI with the cancer-targeting moiety (FA) could be taken up to a greater degree into the targeted cell through possible receptor-mediated endocytosis. Therefore, the MAGCNTs–PSS–PEI–FA conjugates could be utilized as effective MRI contrast agents for the targeted detection of cancer cells.
We have developed novel water-dispersible MWCNT/iron oxide nanoparticle hybrids as specific targeting MRI contrast
5 0 ( 20 1 2 ) 2 1 6 2–21 7 0
agents. The obtained functionalized MAGCNTs were hydrophilic, biocompatible and exhibited a high contrast effect in MRI. Cancer cells targeted with the MAGCNTs–PSS–PEI–FA in vitro were detectable by a clinical MRI system, showing the specific targeting MRI capability of the MAGCNTs–PSS– PEI–FA conjugates. The MAGCNTs–PSS–PEI hybrids may be further functionalized with fluorescence molecular and anticancer drugs, and thus be very useful for future development of MAGCNT-based platforms for the real-time monitoring of drug distribution to the target tissue as well as simultaneous magnetic and optical dual imaging diagnostics.
Acknowledgments This work was supported by Program for New Century Excellent Talents in University (NCET-08-0897), National 973 Project (No. 2010CB933901), Shanghai Sci. & Tech. and Education Committee (09SG43, S30406), Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University (DZL806).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.carbon.2012.01.026.
R E F E R E N C E S
 Weissleder R, Ntziachristos V. Shedding light onto live molecular targets. Nat Med 2003;9:123–8.  Lee JH, Huh YM, Jun YW, Seo JW, Jang JT, Song HT, et al. Artificially engineered magnetic nanoparticles for ultrasensitive molecular imaging. Nat Med 2007;13:95–9.  Weissleder R, Pittet MJ. Imaging in the Era of molecular oncology. Nature 2008;452:580–9.  Wu XG, Liu HJ, Liu JQ, Haley KN, Treadway JA, Larson JP, et al. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol 2003;21:41–6.  Bruchez Jr M, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science 1998;281:2013–6.  Hu FQ, MacRenaris KW, Waters EA, Liang TY, Schultz-Sikma EA, Eckermann AL, et al. Water-soluble magnetite nanoparticles with high relaxivity for magnetic resonance imaging. J Phys Chem C 2009;113:20855–60.  Lu J, Ma SL, Sun JY, Xia CC, Liu C, Wang ZY, et al. Manganese ferrite nanoparticle micellar composites as MRI contrast agent for liver imaging. Biomaterials 2009;30:2919–28.  Gao MY, Liu SJ, Jia B, Qiao RR, Yang Z, Yu ZL, et al. A novel type of dual-modality molecular probe for MR and nuclear imaging of tumor: preparation, characterization and in vivo application. Mol Pharm 2009;6:1074–82.  Na HB, Lee JH, An K, Park Y, Park M, Lee IS, et al. Development of a T1 contrast agent for magnetic resonance imaging using MnO nanoparticles. Angew Chem Int Ed 2007;46:5397–401.  Schroeder T. Imaging stem-cell-driven regeneration in mammals. Nature 2008;453:345–51.  Weissleder R, Mahmood U. Molecular imaging. Radiology 2001;219:316–33.
 Wang YXJ, Hussain SM, Krestin GP. Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging. Eur Radiol 2001;11:2319–31.  Thorek DLJ, Chen AK, Czupryna J, Tsourkas A. Superparamagnetic iron oxide nanoparticle probes for molecular imaging. Ann Biomed Eng 2006;34:23–38.  Bulte JWM, Kraitchman DL. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed 2004;17:484–99.  Mowat P, Franconi F, Chapon C, Lemaire L, Dorat J, Hindre F, et al. Evaluating SPIO-labelled cell MR efficiency by threedimensional quantitative T2 MRI. NMR Biomed 2007;20:21–7.  Artemov D, Mori N, Okollie B, Bhujwalla ZM. MR molecular imaging of the Her-2/neu receptor in breast cancer cells using targeted iron oxide nanoparticles. Magn Reson Med 2003;49:403–8.  Wu PC, Su CH, Cheng FY, Weng JC, Chen JH, Tsai TL, et al. Modularly assembled magnetite nanoparticles enhance in vivo targeting for magnetic resonance cancer imaging. Bioconjugate Chem 2008;19:1972–9.  Jun YW, Huh YM, Choi J, Lee JH, Song HT, Kim S, et al. Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging. J Am Chem Soc 2005;127:5732–3.  Park JH, Maltzahn GA, Zhang LL, Schwartz MP, Ruoslahti E, Bhatia SN, et al. Magnetic iron oxide nanoworms for tumor targeting and imaging. Adv Mater 2008;20:1630–5.  Park JH, Maltzahn G, Zhang LL, Derfus AM, Simberg D, Harris TJ, et al. Systematic surface engineering of magnetic nanoworms for in vivo tumor targeting. Small 2009;5:694–700.  Wang CG, Chen JJ, Talavage T, Irudayaraj J. Gold nanorod/ Fe3O4 nanoparticle ‘‘Nano-Pearl-Necklaces’’ for simultaneous targeting, dual-mode imaging, and photothermal ablation of cancer cells. Angew Chem Int Ed 2009;48:2759–63.  Lee JH, Jun YW, Yeon SI, Shin JS, Cheon J. Dual-mode nanoparticle probes for high-performance magnetic resonance and fluorescence imaging of neuroblastoma. Angew Chem Int Ed 2006;45:8160–2.  Larsen BA, Haag MA, Serkova NJ, Shroyer KR, Stoldt CR. Controlled aggregation of superparamagnetic iron oxide nanoparticles for the development of molecular magnetic resonance imaging probes. Nanotechnology 2008;19:265102–6.  Choi JH, Nguyen FT, Barone PW, Heller DA, Moll AE, Patel D, et al. Multimodal biomedical imaging with asymmetric single-walled carbon nanotube/iron oxide nanoparticle complexes. Nano Lett 2007;7:861–7.  Wong N, Kam S, Dai HJ. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J Am Chem Soc 2005;127:6021–6.  Liu Z, Tabakman SM, Chen Z, Dai HJ. Preparation of carbon nanotube bioconjugates for biomedical applications. Nat Prot 2009;4:1372–82.  Prato M, Kostarelos K, Bianco A. Functionalized carbon nanotubes in drug design and discovery. Acc Chem Res 2008;41:60–8.  Bhirde AA, Patel V, Gavard JL, Zhang GF, Sousa AA, Masedunskas A, et al. Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery. ACS Nano 2009;3:307–16.  Jia NQ, Lian Q, Shen HB, Wang C, Li XY, Yang ZN. Intracellular delivery of quantum dots tagged antisense oligodeoxynucleotides by functionalized multiwalled carbon nanotubes. Nano Lett 2007;7:2976–80.  Miyawaki J, Yudasaka M, Imai H, Yorimitsu H, Isobe H, Nakamura E, et al. In vivo magnetic resonance imaging of single-walled carbon nanohorns by labeling with magnetite nanoparticles. Adv Mater 2006;18:1010–4.
5 0 ( 2 0 1 2 ) 2 1 6 2 –2 1 7 0
 Achraf AF, Katarzyna C, Ghislaine L, Sophie G, Emmanuelle CS, Yannick C. In vivo imaging of carbon nanotube biodistribution using magnetic resonance imaging. Nano Lett 2009;9:1023–7.  Jeyarama SA, Michael LM, Annie MT, Trinanjana M, Stephen L, Kelvin W, et al. Single-walled carbon nanotube materials as T2-weighted MRI contrast agents. J Phys Chem C 2009;113:19369–72.  Sitharaman B, Kissell KR, Hartman KB, Tran LA, Baikalov A, Rusakova I, et al. Superparamagnetic gadonanotubes are high-performance MRI contrast agents. Chem Commun 2005;31:3915–7.  Richard C, Doan BT, Beloeil JC, Bessodes M, To´th E´, Scherman D. Noncovalent functionalization of carbon nanotubes with amphiphilic Gd3+ chelates: toward powerful T1 and T2 MRI contrast agents. Nano Lett 2008;8:232–6.  Sun ZY, Liu ZM, Wang Y, Han BX, Du JM, Zhang JL. Fabrication and characterization of magnetic carbon nanotube composites. J Mater Chem 2005;15:4497–501.  Pankhurstt QA, Pollard RJ. Fine-particle magnetic oxides. J Phys Condens Matter 1993;5:8487–508.  Alivisatos P. The use of nanocrystals in biological detection. Nat Biotechnol 2004;22:47–52.  Strano MS, Miller MK, Allen MJ, Moore VC, O’Connell MJ, Kittrell C, et al. The role of surfactant adsorption during
ultrasonication in the dispersion of single-walled carbon nanotubes. J Nanosci Nanotechnol 2003;3:81–6. Moore VC, Strano MS, Haroz EH, Hauge RH, Smalley RE. Individually suspended single-walled carbon nanotubes in various surfactants. Nano Lett 2003;3:1379–82. Chen X, Tam UC, Czlapinski JL, Lee GS, Rabuka D, Zettl A, et al. Interfacing carbon nanotubes with living cells. J Am Chem Soc 2006;128:6292–3. Qin SH, Qin DQ, Ford WT, Herrera JE, Resasco DE, Bachilo SM, et al. Solubilization and purification of single-wall carbon nanotubes in water by in situ radical polymerization of sodium 4-styrenesulfonate. Macromolecules 2004;37:3965–7. Low PS, Kularatne SA. Folate-targeted therapeutic and imaging agents for cancer. Curr Opin Chem Biol 2009;13:256–62. Kamaly N, Kalber T, Thanou M, Bell JD, Miller AD. Folate receptor targeted bimodal liposomes for tumor magnetic resonance imaging. Bioconjugate Chem 2009;20:648–55. Sonvico F, Mornet S, Vasseur S, Dubernet C, Jaillard D, Degrouard J, et al. Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments. Bioconjugate Chem 2006;16:1181–8.