Article pubs.acs.org/JPCC
Synthesis of Water-Dispersible Gd2O3/GO Nanocomposites with Enhanced MRI T1 Relaxivity Fenghe Wang,†,∥ Erwin Peng,†,∥ Bingwen Zheng,‡ Sam Fong Yau Li,‡,§ and Jun Min Xue*,† †
Department of Materials Science and Engineering, Faculty of Engineering, National University of Singapore (NUS), 7 Engineering Drive 1, Singapore 117574 ‡ Department of Chemistry, National University of Singapore (NUS), 3 Science Drive 3, Singapore 117543 § Department of Chemistry, NUS Environment Research Institute (NERI), 3 Science Drive 3, Singapore 117573 S Supporting Information *
ABSTRACT: Water-dispersible Gd2O3 (GDO) nanoparticles decorated graphene oxide (GO) nanocomposites (or GDO/ GO NCs) were successfully synthesized as novel magnetic resonance imaging contrast agents. The nanocomposites were prepared through a facile solvent evaporation method. The hydrodynamic size of the resultant nanocomposites could be adjusted easily by varying the sonication pretreatment time. When measured using 7 T MRI scanner, the relaxivity value (r1) of the GDO/GO NCs was as high as 34.48 mM−1 s−1, which is much higher than those of the typical commercial MRI T1 contrast agent materials such as Gd-DOTA and Gd-DTPA. The cytotoxicity study showed that the GDO/GO NCs exhibited better biocompatibility as compared to the previously reported Gd-based MRI contrast agents. It was demonstrated that GDO/ GO NCs were promising as magnetic resonance imaging (MRI) T1 contrast agents. none-free Gd3+ ion releasing, such as carbonate-based (e.g., Gd2(CO3)3), fluoride-based (e.g., GdF3 and NaGdF4), and gadolinium oxide (Gd2O3).14−17 The Gd2O3 NP is the most popular one among these inorganic Gd-compounds. The surface Gd3+ ion of Gd2O3 NPs offered all seven of its unpaired electrons for water hydration and cooperatively induced the longitudinal relaxation of the water proton.17,18 Such synergistic advantages offered by surface Gd3+ promised greater T1 relaxivity value (r1) than Gd-chelates. Small Gd2O3 NPs, due to its high surface to volume ratio (S/V), can concentrate huge number of magnetic ions in a small volume, thus offering a high signal-to-noise ratio.19 Furthermore, various targeting moieties or fluorescent dyes can be readily conjugated on surfaces of Gd2O3 NPs to develop multimodal CAs. To prepare high quality and monodisperse Gd2O3 NPs, high temperature thermal decomposition in nonpolar solvent was favored as compared to other hydrolytic/polar synthetic routes (e.g., hydrothermal, coprecipitation, and polyol thermal decomposition).19−23 However, Gd2O3 NPs prepared using the thermal decomposition method are coated with hydrophobic surface capping agents, which makes these NPs water insoluble. For meaningful biomedical application, further surface modification is essentially required. From literatures, various surface modification techniques have been employed to transfer hydrophobic NPs into aqueous solvent. These major
1. INTRODUCTION Magnetic resonance imaging (MRI) is an important noninvasive technique that has been widely used to diagnose diseases due to its high spatial resolution and good soft-tissue contrast.1−4 MRI works by either shortening the longitudinal relaxation time (T1) or the transverse relaxation time (T2) of water protons. The MRI signal tends to increase with T1 shortening, and it decreases with T2 shortening. Based on typical contrast mechanisms, MRI contrast agents (CAs) can be classified as either T1 CAs (positive CAs) or T2 CAs (negative CAs). At present, the negative T2 CAs are mainly used in passive targeting or blood pool imaging.5 However, the typical MRI signal obtained by negative T2 CAs is easily confused with other artifact signals, which include calcification, bleeding, metal deposits, etc.6,7 Positive T1 CAs have been widely used as extracellular agents, blood pool agents, and hepatobiliary agents in clinical application.5 With a brighter MR imaging, positive T1 CAs give an advantage in spatial resolution.8,9 Due to this, research on positive T1 CAs with high longitudinal relaxation rate, low intake dose, and low cytotoxicity is still attractive. Currently, the most commonly used T1 CAs for clinical MRI investigations is gadolinium chelates such as Gd-DOTA and Gd-DTPA, which were reported to have a T1 relaxivity value (r1) ranging from 3 to 5 mM−1 s−1.10,11 However, Gd-chelates could release a certain amount of free gadolinium ions, which may inhibit the calcium channels and have considerable cardiovascular and neurologic toxicity.12,13 Recently, various Gd-based inorganic nanoparticles (NPs) were investigated as effective T1 CAs due to their higher T1 relaxivity value (r1) and © 2015 American Chemical Society
Received: June 24, 2015 Revised: September 17, 2015 Published: September 21, 2015 23735
DOI: 10.1021/acs.jpcc.5b06037 J. Phys. Chem. C 2015, 119, 23735−23742
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the r1 values of Gd-chelates. It was demonstrated that the hydrodynamic size of GDO/GO NCs could be fine-tuned by adjusting the GO sonication pretreatment time. The particle loading amount on GO sheets could be controlled by tuning the particle to GO mass ratio, which influenced the T1 relaxivity value (r1) of GDO/GO NCs.
approaches include (i) ligand exchange, (ii) amphiphilic polymeric encapsulation, and (iii) silica coating. One of the simplest surface modification techniques, ligand exchange, generally suffered from the potential of uncontrolled aggregation due to weak binding between the ligand and NPs’ surface.24,25 Moreover, ligand exchange is usually surfacesensitive and is limited to certain type of ligands, which depends on the NPs’ surface. To obtain better aqueous solubility, hydrophobic NPs were encapsulated using either amphiphilic polymeric encapsulation or silica coating processes. Amphiphilic polymer encapsulation method offered a wide applicability and better colloidal stability. However, it is hard to obtain monodisperse NPs with individual coating using this method. Hydrophobic NPs are generally aggregated to form nanoclusters.26,27 In such structure, the distance between the magnetic core and water protons is increased, leading to a decrease in longitudinal relaxation rate (1/T1 ∝ d6, where d is the distance between paramagnetic center and water proton). An even more devastating impact is the significant reduction in the effective total surface area and surface to volume ratio (S/ V) of the magnetic cores in such nanocluster structure, which will further decrease r1 value (r1 ∝ S/V). Similar to the amphiphilic polymer encapsulation, silica coating method normally results in thick silica coating layer or aggregation of the magnetic cores,28−30 which leads to an ineffective interaction between water protons and magnetic cores. Due to the current limitations of the surface modification techniques, properly designed phase transfer methods with large effective particle surface area and limited increment in the distance between the magnetic cores and the water proton remain to be great challenges. To date, graphene oxide (GO) has attracted a lot of attention due to its hydrophilicity, unique physical structure, and chemical properties.31−33 GO was reported to be highly soluble in aqueous solvent due to the presence of various hydrophilic oxygenated functional groups at its edges and basal planes including epoxy, hydroxyl, and carboxyl.34 Besides promoting hydrophilicity, these functional groups also favor the modification of GO with other molecules via covalent or noncovalent bondings.35 Recently, some nanoparticles/GO nanocomposites (NCs) were reported as effective T1 contrast agents for MR imaging (summarized in Table S1). Most papers reported that Fe3O4 NP loaded GO NCs could offer effective T2 contrast effect.36−40 Lui’s group reported the functionalized Fe3O4/GO NCs, which could used for targeted drug delivery, photothermal therapy, and MR imaging.41,42 These studies indicated that GO sheets were popular nanoparticle carriers, which aid in transferring hydrophobic NPs into aqueous solution. At present, there is no report on Gd2O3/GO NC based positive CAs. In this work, a facile method was proposed to load hydrophobic gadolinium oxide (GDO) NPs on hydrophilic GO sheets to obtain water-soluble MRI T1 CAs with high T1 relaxivity value (r1). There was no prior surface modification on the hydrophobic GDO NPs during the formation of the water-soluble Gd2O3/GO (GDO/GO) NCs. GDO NPs were adsorbed onto the hydrophobic basal planes of GO through hydrophobic−hydrophobic interactions or π−π stacking.43,44 Change in the distance between paramagnetic core and water proton was very subtle. Meanwhile, the sheetlike GDO/GO NCs offered large effective surface area; thus, the obtained GDO/GO NCs was able to exhibit high T1 relaxivity value (r1). By using this method, the GDO/GO NCs exhibited effective T1 enhancement effect, which was 7 times
2. EXPERIMENTAL SECTION 2.1. Materials. Graphene oxide (GO) aqueous solution (5 mg/mL) was purchased from Graphene Supermarket (New York); oleic acid (99%), gadolinium nitrate hexahydrate (Gd(NO) 3 ·6H 2 O, 99.99%), pluronic F-127, methanol (99.8%), acetone (99.5%), 1-octadecene (ODE), and tetrahydrofuran (THF; 99.9%) were purchased from Sigma-Aldrich. Chloroform (Fisher Scientific; 99.99%) and hydrochloric acid (37 wt %) were used as received. 2.2. Preparation of GDO Nanoparticles. The synthesis of GDO NPs followed the procedures as reported papers with some modification.19 Typically, GDO NPs (average particle size = 3 nm) were synthesized in a one-pot reaction using the basic formulation: gadolinium nitrate hexahydrate (2 mmol), oleic acid (4 mmol), and 1-octadecene (5 g) were charged into a three-neck round-bottom flask. The mixture was stirred for 2 h at 110 °C until the gadolinium nitrate hexahydrate was completely dissolved in the solvent as a Gd-oleate precursor. The temperature was then raised to 290 °C and held for 3 h. After cooling down to room temperature, the resulting colloidal solution was washed 5 times with methanol (20 mL) and acetone (20 mL). Finally, the GDO NPs were dispersed in chloroform (50 mg/mL). By varying the amount of reactants, the 17 nm GDO NPs were obtained. These particles were abbreviated as GDO1 for 3 nm nanoparticles and GDO2 for 17 nm nanoplates. 2.3. Preparation of GDO/GO Water-Soluble Nanocomposites. Hydrophobic GDO nanoparticles in chloroform were first collected by centrifugation and redispersed into THF solvent (5 mg/mL). Typically, 2 mL of GDO THF solution and 20 mL of DI water were added to a certain amount of GO solution. The mixture was homogenized for 2 min and subsequently stirred at 70 °C for 30 min to allow THF evaporation. After cooling down to room temperature, the obtained solution was stored in a sealed glass bottle. In this article, we first used GDO1 NPs as particle source to prepare GDO/GO NCs. The particle to GO mass ratio was 1:5 and GO was used as received without any treatment. The asobtained sample was labeled as GDO1/GO-NCs. In order to study the effective of sonication time on reducing GDO/GO hydrodynamic size, GDO/GO NCs with different GO sonication pretreatment time were prepared. Samples prepared with GDO1 NPs and GO pretreated 0, 30, and 60 min were labeled as GDO1/GO-0, GDO1/GO-30, and GDO1/GO-60, respectively. Similarly, samples prepared with GDO2 NPs and GO presonicated 0, 30, and 60 min were labeled as GDO2/ GO-0, GDO2/GO-30, and GDO2/GO-60, respectively. Several GDO/GO NCs were prepared with different particle to GO mass ratio. The GDO1/GO-1, GDO1/GO-2, and GDO1/GO-3 samples were prepared using GDO1 NPs and GO solution with 1 h sonication pretreatment; the particles to GO mass ratio were 1:2, 1:3, and 1:5, respectively. The GDO2/ GO-1, GDO2/GO-2, and GDO2/GO-3 samples were prepared using GDO2 NP and GO solution with 1 h sonication pretreatment; the particles to GO mass ratio were 1:2, 1:3, and 1:5, respectively. 23736
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The Journal of Physical Chemistry C 2.4. Preparation of GDO/F127 Nanocomposites. The magnetic GDO/F127 nanocomposite aqueous solution was prepared by the combination of mini-emulsion and solvent evaporation processes. The procedure of encapsulation was listed as follows: F127 solution in chloroform and a solution of oleic acid capped GDO nanoparticles in chloroform were mixed and sonicated in bath ultrasonic for 10 min. Then the mixed solution was emulsified with water (10 × volume excess, 1 wt % PVA as stabilizer) using an ultrasonic homogenizer for 5 min. The ultrasonication was performed in an ice bath to minimize the evaporation of chloroform before the stable emulsion be formed. Finally, the produced solution was transferred to a preheated baker at 70 °C under rapid stirring for 30 min to allow the chloroform to evaporate. The colloid was washed with water and methanol (Vwater/Vmethanol = 1:1) mixture for two times and then dispersed in DI water for further characterization. 2.5. CCK-8 Cytotoxicity Assay. Human breast cancer cells (MCF-7 cells) were seeded into a 96-well plate at 7500 cells per well and were incubated in Dulbecco’s Modified Eagle Medium (DMEM, include 10% FBS) for 24 h at 37 °C, 5% CO2 in air. The growth medium was removed before the MCF-7 cells were treated with GDO/GO NCs in DMEM/10%FBS. The cell lines were incubated with the particles for 24 h. Then CCK-8 solution (10 μL) was added to each well and shaken slightly to mix well. After a further incubation time of 4 h, the plate was analyzed using a microplate reader by measuring the absorbance at 450 nm. 2.6. MRI Measurement. MRI experiments were conducted in a 7 T MRI scanner (Varian, Agilent Inc., USA). The MRI samples were prepared by drawing GDO/GO NCs solutions at different gadolinium concentration into 1 mL of disposable poly(propylene) syringes. For the T1 measurement, inversion− recovery fast spin−echo sequence with 8 inversion times (TI; ranging from 10 ms to 4 s) was employed. Meanwhile for the T2 measurement, multislice multiecho sequence was employed [others parameters: TR = 6 s (for T1) and 2 s (for T2); matrix size = 128 × 128; field of view = 40 × 40 mm; slice thickness = 4 mm]. 2.7. Characterization. The morphologies of the obtained samples were examined by using transmission electron microscopy (TEM, JEOL JEM-3010). The TEM samples of GDO and GDO/GO were prepared by dropping a drop of the magnetic samples solution (in their respective solvent, either CHCl3 or water) to the TEM copper grid, followed by airdrying at ambient conditions. For the GDO/GO morphology analysis, lacey Formvar stabilized with carbon grid (with completely open holes) was used instead of the conventional copper grid with specimen support film. The selected area electron diffraction (SAED) pattern of GDO and GDO/GO samples were also recorded by using the TEM. The crystal phases of magnetic particles were verified by X-ray powder diffraction (XRD) patterns recorded on a powder diffractometer (Bruker D8 Advanced Diffractometer System) with Cu Kα (1.5418 Å). The hydrodynamic diameters of the magnetic NCs were measured using a Malvern Zetasizer Nano ZS. Fourier transform infrared spectroscopy (FT-IR) spectra of powder samples were recorded on a Varian 3100 Excalibur Series FT-IR Spectrophotometer. For MRI measurement and cell cytotoxicity assay, the Gd concentration of GDO/NPs were determined by using inductively coupled plasma (ICP) analysis, which was performed using a PerkinElmer Dualview Optima 5300 DV ICP-OES system (PerkinElmer, Waltham, MA). The
inductively coupled plasma (ICP) samples were prepared by dissolving GDO/NPs in 37% HCl, followed by centrifugation (10 000 rpm, 10 min) to remove GO aggregates. Magnetic properties of NPs were characterized by a LakeShore Model 7407 Vibrating Sample Magnetometer (VSM) at 300 K (room temperature).
3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of GDO NPs. By varying the amount of the precursors used during the thermal decomposition synthesis, two different hydrophobic GDO NPs, namely, GDO1 (3 nm) and GDO2 (17 nm), were obtained. The morphologies and microstructures of GDO1 and GDO2 were analyzed with transmission electron microscopy (TEM) images shown in Figure 1a,b. From the TEM image given in
Figure 1. TEM image of (a) 3 nm Gd2O3 nanoparticles (GDO1) and (b) 17 nm Gd2O3 nanoparticles (GDO2) dispersed in CHCl3. TEM size distribution of (c) GDO1 and (d) GDO2, and (e) their respective XRD patterns.
Figure 1a, the resultant GDO1 NPs were nearly spherical in shape with average TEM particle size of 2.89 ± 0.35 nm (see Figure 1c). The SAED pattern of GDO1 given in the Figure 1a inset showed the typical ring-type patterns for a polycrystalline material indexed to the Gd2O3. This indicated that the asprepared GDO1 NP sample was well-crystallized. Meanwhile, morphology of the GDO2 NPs (Figure 1b) was determined to be plate-like, as observed from the side-view TEM images (see Figure S1). The average lateral diameter of the GDO2 nanoplates sample was 16.96 ± 1.72 nm (see Figure 1d). The intense SAED pattern in the Figure 1b inset verified the crystalline phase of GDO2 NPs. In both SAED patterns, the diffraction rings were able to match with the more intense reflections from Gd2O3 powder, namely, the (222), (440), (622), (662), and (844) crystal planes. The crystalline phase of both GDO NPs samples was also reinforced from the XRD patterns of GDO1 and GDO2 NPs given in Figure 1e. The main characteristic peaks could be indexed readily to the cubic phase of Gd2O3 (JCPDS pdf #12-0797). Within the detection limit of XRD, no impurity was present. The magnetic properties of both GDO1 and GDO2 NPs were shown in 23737
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combining GDO1 with GO, the FT-IR spectrum of GDO1/ GO-NCs showed all the characteristic peaks of both in GO and GDO1 spectrum (see Figure S3). The thermal gravimetric analysis (TGA) study revealed that GDO1 NPs was composed of only 13.06% of the GDO1/GO-NCs mass (see Figure S4). This explained the relatively low abundance of oleic acid characteristic peaks in GDO1/GO-NCs. 3.3. Effect of Sonication Time on GDO/GO Size. In order to understand the sonication time effect on GDO/GO NCs hydrodynamic size, the pretreatment sonication time of GO prior to the nanocomposites fabrication was varied from 0 to 60 min. As shown in Table 1, when the amount of NPs and GO were fixed, the hydrodynamic size of GDO/GO NCs decreased with the increase in the sonication time. When GDO/GO NCs were prepared using the 1 h sonication time, the overall average hydrodynamic sizes of resultant for GDO1/ GO and GDO2/GO NCs were 283.4 and 162.1 nm, respectively. The smaller hydrophobic GDO1 nanoparticles (3 nm) with high surface-to-volume ratio tended to lower the system total surface energy when GDO1/GO was formed by forming a more aggregated structure. Thus, all GDO1/GO samples have lager hydrodynamic size than GDO2/GO samples. Due to the presence of GO host, the GDO/GO NCs were inherently stable in water (see the time-dependent average hydrodynamic size plot in Figure S5). 3.4. Particle Loading Amount on GO Sheets. Moreover, by varying the amount of GDO in the homogenization process, the amount of GDO NPs on GO sheets could be adjusted. A series of GDO1/GO NCs were synthesized in the order of decreasing particle loading amount, denoted as GDO1/GO-1, GDO1/GO-2 , and GDO1/GO-3. The GDO1/GO-1, GDO1/ GO-2, and GDO1/GO-3 samples were prepared using GDO1 NPs and GO solution with 1 h sonication pretreatment; the particles to GO mass ratio were 1:2, 1:3, and 1:5, respectively. Similarly, GDO2/GO series were prepared using GDO2 NPs, the particles to GO mass ratio were 1:2, 1:3, and 1:5 for GDO2/GO-1, GDO2/GO-2, and GDO2/GO-3, respectively. At high particle loading amount (GDO1/GO-1), the GDO1 NPs were observed to be densely distributed onto GO sheets (see Figure 4a,b) in an evenly manner as the whole image exhibited a dark contrast. At this loading, multiple layers of GDO1 NPs were expected from the GDO1/GO-1 structure due to the interdigitation of the oleic-acid surface capping agent between the adjacent hydrophobic GDO1 NPs on GO surface. Such configuration was favored, particularly in aqueous phase, for the small 3 nm GDO1 NPs in order to minimize the interfacial energy. When GDO1 NPs loading amount was gradually decreased, GDO1 NPs distribution was no longer uniform. This resulted in several segments of the GO sheets in GDO1/GO-2 being covered with less GDO1 NPs as compared to GDO1/GO-1. This was clearly observed in TEM images (Figure 4c,d), as dark contrast area corresponded to high
Figure S2. It was found that both GDO1 and GDO2 NPs exhibited paramagnetic behavior at 300 K. To verify the chemical composition of the as-prepared hydrophobic GDO1 NPs, the powder sample of GDO1 was investigated by X-ray photoelectron spectroscopy (XPS). Figure 2a showed high-resolution XPS spectrum of Gd (3d)
Figure 2. High-resolution XPS spectrum of Gd 4d (a) and O 1s (b) energy levels.
with two strong binding peaks. The binding energy of 1187.6 eV corresponded to the Gd 3d5/2 energy level, and 1219.6 eV corresponded to the Gd 3d3/2 energy level. The O (1s) binding energy peak was shown in Figure 2b. The O 1s binding energy was dominated by one major peak located at 531.4 eV, corresponding to the bind between O2− and Gd3+; confirming the Gd2O3 phase. These observations were in good agreement with reports in the literature for Gd2O3 nanoparticles.18 3.2. Formation of Water-Soluble GDO/GO NCs. In the first attempt to prepare GDO/GO NCs, GDO1 NPs was selected as the core NPs. In the typical synthesis process, the particle to GO mass ratio was 1:5. GO was used as received without any treatment. The TEM images of the obtained GDO1/GO-NCs were shown in Figure 3a,b. The high
Figure 3. TEM image of GDO/GO-NCs dispersed in H2O.
magnification TEM image showed that GDO1 NPs were uniformly loaded onto the GO sheets. The SAED pattern (inset of Figure 3b) indicated that the NP loading onto GO sheets had no influence toward the crystal structure of the GDO1 NPs. FT-IR measurement was also carried out to verify the chemical bonding of the resultant nanocomposites. After
Table 1. Effect of GO Sonication Time on Reducing GDO/GO Hydrodynamic Size sample
Gd2O3 (mg)
GO (mg)
sonication pretreatment time (min)
hydrodynamic size (d·nm)
GDO1/GO-0 GDO1/GO-30 GDO1/GO-60 GDO2/GO-0 GDO2/GO-30 GDO2/GO-60
5 5 5 5 5 5
15 15 15 15 15 15
0 30 60 0 30 60
633.1 407.9 283.4 417.6 221.7 162.1
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The hydrodynamic sizes and the dynamic light scattering (DLS) size distributions of the resultant water-dispersible GDO/GO NCs with different core NPs and different NPs to GO mass ratio, measured at 300 K, were summarized in Figure 5. Typically, the overall hydrodynamic sizes of GDO/GO NCs
Figure 4. TEM images of GDO1/GO nanocomposites dispersed in H2O, with particle/GO mass ratio of (a,b) 1:2 (GDO1/GO-1), (c,d) 1:3 (GDO1/GO-2), and (e,f) 1:5(GDO1/GO-3).
Figure 5. DLS distribution of (a) GDO1/GO nanocomposites and (b) GDO2/GO nanocomposites (insets: size vs particle to GO mass ratio).
decreased with decrease in the GDO NPs loading amount. The average hydrodynamic sizes were 1000.0, 283.4, and 268.9 nm for GDO1/GO-1, GDO1/GO-2, and GDO1/GO-3, respectively. Meanwhile, the average hydrodynamic sizes for GDO2/ GO composites were 246.1, 163.5, and 156.2 nm for GDO2/ GO-1, GDO2/GO-2, and GDO2/GO-3, respectively. 3.5. Relaxivity Studies. The magnetic relaxometric properties of GDO/GO samples were assessed using a 7 T MRI scanner. The concentration-dependent T 1 and T 2 relaxation times of all the GDO/GO NCs samples were summarized in Figure 6a,b (as well as Figure S7). The calculated longitudinal and transversal relaxivity values (r1 and r2, respectively) for different GDO/GO NCs (at different core size and loading amount) were tabulated in Table 2. All GDO/ GO NCs samples that have been prepared, exhibited rather high r1 values (from 19.17 to 34.48 mM−1 s−1) with relatively low r2/r1 ratios (less than 10). These data emphasized that the fabricated GDO/GO samples could produce effective T1 contrast enhancement effects, which were 4 to 7 times the r1 values of various Gd-chelates reported in the literatures. Overall, there are two major factors influencing T1 relaxivity value (r1) of Gd based T1 CAs. The first factor is the number of surface Gd3+ ions, which is commonly perceived as the surfaceto-volume ratio (S/V). Small GDO NPs possess larger S/V and generally the r1 values increased with decreasing particle size. The second factor is the distance between the paramagnetic center and the surrounding water proton. It is critical to
loading rate parts and lighter contrast area corresponded to the GO sheets segment with either less or no GDO1 NPs. Lastly, by further decreasing particle loading amount, GDO1 NPs distribution on GO sheets in GDO1/GO-3 sample became uniform, but much less densely packed (see Figure 4e,f). This resulted in the creation of void area in between the hydrophobic oleic-acid coated GDO1 NPs. Unlike the previous GDO1 NPs, the larger plate-like GDO2 NPs (17 nm) tended to aggregate on the GO sheets surface, especially at low loading amount. At high loading amount, GDO2 NPs were densely distributed onto GO sheets, in a uniform manner (see Figure S6a,b). Meanwhile, decreasing GDO2 NPs loading amount inadvertently caused uneven GDO2 NP distribution (as shown in Figure S6c,d). Further decreasing particle loading amount would no longer result in the uniform GDO2 NPs distribution on GO sheets, in contrast to the previously observed GDO1/GO NCs sample. This could be attributed to the fact that GDO2 NPs were larger and heavier than the smaller GDO1 NPs. As such, there was limited GDO NPs available to sufficiently decorate and cover the GO sheets uniformly at low GDO NPs loading amount. The similar trend observed was that the separation distance between the aggregated GDO NPs increased with decrease in the GDO NPs loading amount. The SAED patterns in Figure 4 and Figure S5 insets indicated that the loading of different GDO NPs core sizes onto the GO sheets had little influence on the GDO NPs morphology and crystal structure, even after sonication. 23739
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increase in r1 value for both GDO1/GO and GDO2/GO samples. As the loading amount decreased, there was more space for water diffusion along the surface of GO sheets due to the increase in the GDO NPs separation distance. The possible water diffusion pathway on GDO/GO NCs was illustrated in Figure S8. Such increase in the separation distance helps the water molecule to diffuse faster on GO surface, and therefore contributes positively toward the shortening of the water protons longitudinal relaxation process. The concentrationdependent darkening and brightening contrasting effect of the GDO/GO NPs could be clearly observed from the T1-weighted and T2-weighted MR images of GDO1/GO-3 sample given in Figure 6c. In a control experiment, pluronic F127 polymer encapsulated GDO1 nanoclusters were prepared through a mini-emulsion and solvent evaporation process (MESE), following established protocols.27 The hydrodynamic size of the as-obtained GDO1/ F127 nanoclusters decreased with decreasing particle loading amount (Table S2). The r1 value of the obtained GDO1/F127 was 9.33 mM−1 s−1 (Figure S9), which was only one-third of that of GDO1/GO NCs (Figure 6 and Figure S7). This could be ascribed to the two aforementioned reasons. First, as compared to the GDO/GO samples, the polymer encapsulation of the nanoclusters resulted in an increase in the distance between paramagnetic cores and water protons; thus, the effective interaction between magnetic cores and water protons was inhibited. Second, the whole nanoclusters behaved as a large particle; thus, the effective total surface area or effective S/ V ratio decreased, leading to a lower r1 value. 3.6. Cytotoxicity Study. Cellular cytotoxicity of NCs is an important aspect for biological application. To assess the cellular cytotoxicity of the GDO/GO samples, breast cancer cells (MCF-7) were cultured and incubated with GDO/GO NCs at different metal (Gd) concentrations for 24 h at 37 °C. Subsequently, the cell viability was evaluated using Cell Counting Kit-8 (CCK-8) assays. Figure 7 showed the cell
Figure 6. (a) T1 relaxation rate of GDO/GO nanocomposites, (b) T2 relaxation rate of GDO/GO nanocomposites, and (c) T1-weighted phantom MR images of GDO1/GO-3.
Table 2. Summary of MR Relaxivities of Various Nanocomposites, as Measured Using MRI 7 T Scanner sample
core size (nm)
particle to GO ratio
r1 (mM−1 s−1)
r2 (mM−1 s−1)
r2/r1 ratio
GDO1/GO-1 GDO1/GO-2 GDO1/GO-3 GDO2/GO-1 GDO2/GO-2 GDO2/GO-3
3 3 3 17 17 17
1:2 1:3 1:5 1:2 1:3 1:5
33.13 33.89 34.48 19.17 19.87 21.60
173.82 191.19 193.85 104.00 107.73 135.88
5.25 5.64 5.62 5.43 5.42 6.29
optimize this distance in order to improve the relaxivity transfer. In additional to this, the water diffusion rate is also strongly influenced by the 1/T1 relaxivity rate. In this article, two kinds of NPs were used as magnetic core for T1 CAs: spherical GDO1 NPs with average size of 3 nm, and plate-like GDO2 NPs with 17 nm in diameter and 3 nm in thickness. GDO1 particles had larger S/V ratio than the platelike GDO2 NPs. Thus, the GDO1/GO NCs should have exhibited shorter 1/T1 relaxivity rate as compared to the GDO2/GO NCs. Such trend was observed from the results presented in Figure 6a,b whereby the r1 values for GDO1/GO1, GDO1/GO-2, and GDO1/GO-3 (33.13, 33.89, and 34.48 mM−1 s−1, respectively) were almost more than ca. 60% higher than the r1 values for GDO2/GO-1, GDO2/GO-2, and GDO2/GO-3 (19.17, 19.87, and 20.60 mM−1 s−1, respectively). In addition, it was also observed in Figure 6 that reduction in GDO NPs loading amount on GO sheets caused a slight
Figure 7. Cell viability of MCF-7 cell lines incubated with different concentrations of GDO/GO NCs.
viability of MCF-7 cells incubated with GDO/GO NCs at concentration range of 6.25 to 150 μM. There was no significant decrease in the viability of the MCF-7 cells within the Gd concentration of 50 μM for both GDO1/GO-3 and GDO2/GO-3 NCs. When the Gd ions concentrations were above 50 μM, the cell viability started to decline proportionally. The cell viability was still greater than 90% upon incubation with GDO/GO NCs when Gd concentration was 50 μM (or 7.864 mg/L). This result showed a better biocompatibility than 23740
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The Journal of Physical Chemistry C
(SPORE, COY-15-EWI-RCFSA/N197-1), Ministry of Education (R-143-000-582-112).
some published gadolinium based contrast agents, such as commercial T1 contrast agent Gd-DTPA, other modified Gdcomplexes, and some Gd2O3 based CAs (Table S3). Meanwhile, Gd2O3/GO NCs showed an excellent positive contrast effect (34.48 mM−1 s−1) compared to reported CAs as listed in the table. In summary, GDO/GO NCs had good compatibility and high relaxivity rate, which made them promising positive CAs for MR imaging. In order to improve the GDO/GO NCs biocompatibility in physiological solutions, several well-established surface modifications can be implemented, such as pluronic F127, mPEGNH2, and 4-arm PEG amine. The PEG functional group can act like a spacer, preventing the direct interaction of the hydrophobic NPs as well as the GO sheets with the surrounding ions. Conjugation GDO/GO NCs with 4-armPEG amine was one of the simplest and effective methods that can improve the biocompatibility of GDO/GO in various physiological solutions (Figures S10 and S11) and pH levels (Figure S12).
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4. CONCLUSIONS In summary, hydrophilic GDO/GO nanocomposites were synthesized directly by sonicating hydrophobic GDO NPs and hydrophilic GO mixture, followed by organic solvent evaporation. All the as-prepared GDO/GO NCs exhibited significant T1 contrast enhancement, which were 4−7 times greater than commercial Gd-chelates. The hydrodynamic size of GDO/GO NCs can be simply controlled by varying GO pretreatment time. Particle loading amount can influences the T1 relaxivity value (r1) due to (i) the change of particle separation distance on GO sheets, and (ii) the enhancement of water diffusion on GO sheets. Due to the high surface to volume ratio, the water-soluble NCs prepared by small NPs exhibited better T1 contrast enhancement than the NCs prepared by large NCs. The cytotoxicity study showed that GDO/GO NCs exhibited an acceptable biocompatibility. The obtained water-soluble GDO/GO NCs were determined to be suitable for MRI applications as effective T1 contrast agents.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.5b06037. Additional characterization data (inclusive of the TEM, VSM, FT-IR, TGA, and MR relaxometric analysis) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +65 65164655. Fax: +65 67763604. E-mail: msexuejm@ nus.edu.sg. Author Contributions ∥
REFERENCES
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by Ministry of Education Singapore FRC Grant WBS R-284-000-102-112. Sam Fong Yau Li would like to acknowledge financial support from National Research Foundation and Economic Development Board 23741
DOI: 10.1021/acs.jpcc.5b06037 J. Phys. Chem. C 2015, 119, 23735−23742
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DOI: 10.1021/acs.jpcc.5b06037 J. Phys. Chem. C 2015, 119, 23735−23742
