Uniform Fe3O4/Gd2O3-DHCA nanocubes for dual- mode MR imaging

Multimodal imaging technology were extensively studied over past few years, because they offered complementary diagnosis information, which can increase the accuracy of diagnosis. The synthesis of contrast agents via simplified methods are desired for the development of multimodal imaging. Herein, uniformly distributed Fe3O4/Gd2O3 nanocubes for T1-T2 dual-mode contrast agents were rationally designed and successfully fabricated by our group. In this system, the Fe3O4/Gd2O3 nanocubes were coated with nontoxic 3,4-dihydroxyhydrocinnamic acid (DHCA) for better hydrophilia


Introduction
Magnetic Resonance (MR) imaging has been broadly used in clinical practice for diagnosing disease because of its excellent capability of differentiation of soft tissue, high space resolution, and non-invasive property [1][2][3][4][5]. However, with the increase of disease complexity and the low sensitivity of MR imaging, the diagnosis of diseases has become more and more challenging. Therefore, many researchers have committed to develop contrast agents [6][7][8][9]. MR imaging contrast agents can interact with surrounding hydrogen proton to shorten relaxation time and generate signal changes [10]. Generally, contrast agents can be divided into two kinds according to the effect of MR imaging, one is T1 contrast agents, which shorten longitudinal relaxation time and generate bright signals [11][12][13][14][15], the other is T2 contrast agents, which shorten transverse relaxation time and generate dark signals [16][17]. The advantage of T1 contrast agents is that they can generate bright signals, such as Gd-3 DTPA, which is broadly used for diagnostic imaging. Nevertheless, the renal toxicity of Gd-based contrast agents should not be ignored [18][19]. T2 contrast agents have lower toxicity compared to T1 contrast agents, such as Fe3O4 nanoparticle, but the exaggerated artifacts caused by contrast agents disturb the anatomical structure and then affect accuracy of diagnosis. Recent years, most of researchers pay more attention to develop multi-modal contrast agents. T1-T2 dual modal MR imaging contrast agents, which could effectively exploit their respective advantages and reduce the adverse impacts [20][21]. Moreover, it also offers complementary diagnostic information, which could improve the sensitivity and reliability for detecting lesions.
Fe3O4 nanoparticles have been extensively investigated as MRI contrast agents due to their good biocompatibility. Therefore, most of studies of T1-T2 dual modal contrast agents are based on Fe3O4 nanoparticles in recent years [22].
The present studies demonstrate that uniformly distributed Gd-embedded Fe3O4 nanoparticles possess an excellent MR imaging enhancement effect as T1-T2 dual modal contrast agent [23]. Moreover, some studies have suggested that nanocubes have a better MR imaging effect compared with nanoparticles. The reason is that cubic shape could induce irreversible dephasing in routine T2 sequences and a better T1 MR imaging [24][25][26]. Inspired by these illustrated studies, we report the fabrication of uniformly distributed cubic shape Fe3O4/Gd2O3, expecting to obtain a novel nanocubes with better MR imaging enhancement effect. In addition, in present work, the novel nanocubes were coated with 3,4-Dihydroxyhydrocinnamic (DHCA) which has higher exchange efficiency and a lower toxicity compared to frequently used modifications, which could make new nanocubes possess a better water-solubility for more stability in vivo applications [27][28].

Synthesis and characterization of FGDA
The schematic illustration of the fabrication of FGDA nanocubes presented in Scheme 1. The oleate metal mixtures as precursors were used to produce Fe3O4/Gd2O3-Oleic Acid (FGOA) nanocubes by thermal decomposition. In the procedure, reaction temperature and time both play important roles in the size of nanocubes. The FGOA were obtained by refluxing at 310 o C for 30 min. From TEM pictures (Fig 1a, d), it can be seen that the size of cubic FGOA nanocubes are 7.44 ± 0.10 nm and they possess good monodispersity. After the treatment of ligand-exchange, the nanocubes still present good monodispersity, and the size of FGDA nanocubes (about 6.33 ± 0.09 nm), which looks a bit decrease compared to FGOA nanocubes (Fig 1b, e). It may be caused by the change in the surface of nanocubes when FGOA nanocubes were converted to FGDA nanocubes. EDS was performed to study the main element contributions and element distributions of the FGDA nanocubes. EDS spectrum shows mainly ferrum, gadolinium and oxygen elements in the nanocubes. No other impurity elements can be detected except carbon element, which contributes from carbon film on copper mesh. EDS mapping indicates that ferrum and gadolinium elements distribute uniformly in the FGDA nanocubes (Fig 1f, g). In addition, the HRTEM (Fig   1c) shows that the interplanar spacing of nanocubes is 0.296±0.02 nm, which corresponds to (220) crystal plane in Fe3O4 [29]. The FGOA nanocubes exhibit hydrophobic property because of alkyl groups, which limits its application as contrast agent for in vivo use. Therefore, oleic acid on surface of FGOA was considered to be exchanged by DHCA to achieve hydrophilic surface using ligand-exchange method. FT-IR was conducted to verify the surface modifier of the nanocubes with or without DHCA exchange. By comparing the FT-IR spectra of FGOA nanocubes and FGDA nanocubes (Fig 1i), it can be observed that two samples display totally different characteristic absorption peaks. The characteristic absorption peaks of FGOA nanocubes at 2937cm -1 and 2857cm -1 correspond to the stretching vibration of -CH3 and -CH2, which indicates oleic acid indeed forms on FGOA nanocubes. The characteristic absorption peak of FGDA nanocubes at 1617cm -1 corresponds to the stretching vibration of benzene ring skeleton, which indicates that DHCA successfully modifies FGDA nanocubes.
As shown in Fig 1j, the field-dependent magnetization (M-H) curves were conducted at 300 K to estimate the magnetic properties of FGDA nanocubes. Fe3O4 nanocubes served as control. It shows that Fe3O4 nanocubes and FGDA nanocubes are both superparamagnetic. The saturation magnetization of Fe3O4 nanocubes and FGDA nanocubes were 0.2132 emu g -1 and 0.7612 emu g -1 , respectively. The latter exhibits much higher saturation magnetization than the former, which is probably due to the change of compound structure induced by the addition of gadolinium. The values of saturation magnetization are both low, which may be caused by the small size of nanocubes [31][32].

Relaxation rate measurement
To estimate the MR imaging contrast enhancement of FGDA nanocubes as T1-T2 dualmode contrast agent, MR imaging of FGDA nanocubes sample at different concentrations were conducted to measure r1 and r2. Fe3O4 nanocubes and Gd2O3 8 nanoparticles were scanned as control. Figure 2 a, and c, show that the MR enhancement of FGDA is better than that of Gd2O3 nanoparticles and Fe3O4 nanocubes. To investigate the ability of FGDA nanocubes accurately, the r1 value and r2 value were calculated to be 67.57 ± 6.2 mM -1 s -1 and 24.2 ± 1.46 mM -1 s -1 , respectively. Both values present much higher than their control groups. Furthermore ，the r1 value are also higher than the former study [20], it may be caused by the special structure. The investigation confirms that the MR imaging enhancement of FGDA nanocubes containing Fe3O4 and Gd2O3 is significantly improved compared to pure Gd2O3 nanoparticles (r1 = 11.75 ± 0.62 mM -1 s -1 ) and pure Fe3O4 nanocubes (r2 = 2.36 ± 0.59 mM -1 s -1 ). It demonstrates that the FGDA nanocubes can be applied in MR imaging as sensitive T1-T2 dual modal contrast agent.

Biocompatibility
The toxicity of FGDA nanocubes to cells plays an important role in its application [33].
The CCK-8 assay was conducted to detect the viability of L929 cells. Figure 3a indicates that the cell viability of all FGDA nanocubes groups at 12 h have no significant difference compared to control group. The reason may be that L929 cells have to adapt to the medium containing FGDA nanocubes at the initial culturing stage. After culturing   (fig 4d). The above results prove that the FGDA nanocubes can be applied to diagnosis as T1-T2 dual modal contrast agent.

Synthesis of metal oleic precursor
The synthesis methods were described in previous study [34]. 10 mmol Sodium oleate was dissolved in 60 ml ultrapure water and 20 ml ethanol, 5.0 mmol ferric trichloride, 1.0 mmol gadolinium chloride hexahydrate were dissolved in 20 ml ultrapure water in a beaker. Then, the mixture was added to a 250 ml three-necked flask drop by drop.
The reaction mixture was heated and refluxed at 75 o C for 4 h. The reaction was cooled to room temperature and added 20 ml hexane after accomplished. Then the mixture was transferred to a separating funnel, the organic phase in top was collected in a beaker and the aqueous phase in bottom was discarded. The collected organic phase was washed with water in a separating funnel. The obtained metal oleate complex was dried at 55 o C for 24 h to form a ceraceous product. The ferric oleate was synthesized in the similar way.

Synthesis of Fe3O4/Gd2O3-OA (FGOA) nanocubes
The uniformly distributed FGOA nanocubes were synthesized by one-step thermal decomposition. 1.12 g metal oleate precursor, 0.17 ml oleic acid and 15 ml 1octadecene were added in a 250 ml three-necked flask. The reaction system was heated up to 200 o C and kept the temperature for 30 min and then heated up to 310 o C at a rate of 4 o C/min and kept refluxing for 30 min. All procedures mentioned above were under nitrogen atmosphere. When the reaction mixture was cooled down to room temperature, 80 ml ethanol was added into the mixture to precipitate the nanocubes. 15 And the nanocubes were collected by centrifuging at 8000 rotate per min (rpm) for 5 min. Then the obtained hydrophobic nanocubes were washed several times using ethanol and hexane. Finally, the obtained product, FGOA nanocubes, was resuspended in 3 ml hexane. To intuitively observe the cytotoxicity of FGDA nanocubes, live-dead staining was operated using Calcein AM (C1430, Thermo Fisher, USA) and Ethidium Homodimer 1 (E1169, Thermo Fisher, USA). Briefly, L929 cells were seeded in a 24-well plate at a density of 3×10 4 cell per well. After 24 h incubation, the medium was exchanged with 1 ml cell medium containing 60 μg/ml FGDA nanocubes. The live-dead staining was performed after incubation of 12 h, 24 h and 48 h. Cells were observed by inverted phase contrast microscope (Nikon, TiS, Japan).

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The animal protocol was complied with the regulation of experimental animal management of Shanxi Medical University. Three Sprague Dawley (SD) rats were employed to test the MR imaging effect in vivo of FGDA nanocubes. Under the 2% isoflurane inhalation, MR imaging were performed on rats. T1WI and T2WI were acquired by the turbo spin echo (TSE) sequence with the parameter of TR/TE = 550/14 ms and TR/TE = 2510/101 ms, respectively. After FGDA nanocubes were injected through tail vein at a dose of 0.8mg Fe/kg and MR imaging was performed at 10 min, 30 min, and 60 min post-injection.

Histological staining
Immediately after the MR imaging, the rats were euthanized by intravenous injection of overdosed chloral hydrate and the lumber muscles were harvested and placed in 4% paraformaldehyde. Then Prussian blue staining was performed.

In vivo toxicity evaluation of FGDA nanocubes
FGDA nanocubes were injected intravenously into rats at a dose of 2mg Fe/kg. After 2 weeks, the rats were euthanized by intravenous injection of overdosed chloral hydrate and the heart, liver, spleen, lung, kidney were harvested and placed in 4% paraformaldehyde. Then the H&E staining were performed, SD rats of control were injected with normal saline intravenously.

Data analysis
The data were expressed as the means ± standard deviations. Statistical significance was assessed using the Student's t-test, and the values were considered significant at P < 0.05.