Introduction

Magnetic resonance imaging (MRI) has an important role in diagnosis of different diseases involving body soft tissues [ 1 ]. To obtain better diagnosis and differentiation of the lesions, using of MRI contrast media is essential in many cases [ 2 , 3 ]. Contrast agents in MRI influence image contrast by changing longitudinal (T1) and transverse (T2) relaxation times of the protons. Gadolinium (Gd³⁺) based contrast materials are positive contrast agents due to their obvious impact on longitudinal relaxation time of water protons [ 2 ]. Gadolinium is a choice positive contrast agent in MRI because of the seven unpaired electrons which make it one of the elements with a high magnetic moment [ 4 ]. In addition, gadolinium based nanomaterials have potential for anticancer drug loading and tumor targeting [ 5 , 6 ]. Size, shape and surface properties of gadolinium based nanomaterials are the important factors that influence correlation of gadolinium ions with water protons [ 7 ]. Unlike positive contrast media of MRI, negative contrast enhanced materials show a higher effect on transverse relaxation rate of water protons [ 8 ].

Recently, gadolinium oxide nanoparticles (Gd₂O₃) have been developed as a high-performance positive contrast material for MR imaging [ 9 , 10 ]. Due to the high surface-to-volume ratio of Gd₂O₃ nanoparticles, their effect on longitudinal relaxation rate of the protons is higher than that of gadolinium chelates [ 11 ]. Gd₂O₃ nanoparticles can be doped into the other lanthanides. In doping process, some impurities of the lanthanide is added to Gd₂O₃ nanoparticles, leading to changes in the nanoparticles properties [ 12 ]. Since some of the lanthanides such as Tb³⁺ and Eu³⁺ have optical properties, by introducing these elements into Gd₂O₃ nanoparticles, optical properties are added to the nanomaterials. Therefore, the terbium doped Gd₂O₃ nanoparticles [ 13 ], and the europium doped nanoparticles [ 14 , 15 ] have been investigated for dual mode MRI/fluorescence imaging in other studies. In addition to applications of doped Gd₂O₃ nanoparticles in the imaging methods, the rare earth or lanthanide doped Gd₂O₃ nanoparticles along with other materials have been studied for other biomedical applications such as drug delivery [ 16 , 17 ], tumor targeting [ 18 ], and photodynamic therapy [ 19 , 20 ].

Neodymium (Nd) is one of the rare elements. The optical properties of the neodymium doped Gd₂O₃ nanorods have been investigated [ 21 ]. However, to the best of our knowledge, the effect of neodymium doping into Gd₂O₃ nanoparticles (Nd³⁺:Gd₂O₃) on the MRI r1 and r2 relaxivities of the particles has been not considered. Therefore, the purpose of the current study was to investigate the effect of Nd³⁺ doping on MRI relaxivity of the gadolinium oxide nanoparticles.

Material and Methods

Preparation of the oleic acid coated Nd3+:Gd2O3 nanoparticles

In this experimental study, preparation of the Gd₂O₃ nanoparticles was carried out based on Katamian et al. study [ 22 ]. For this purpose, 0.9 mmol GdCl₃.6H₂O was dissolved in 2 ml dilute nitric acid (2 mol L⁻¹) and 2.7 mmol citric acid was added to the solution. The mixture was stirred during 3 h. By using dilute ammonia, the final pH was controlled to be 6-7 (Solution A). As the next step, the acrylamide monomers (0.5 g) were added into the clear solution A and was stirred for 20 min (Solution B). Then, the resulted solution was heated in a water bath. The system was stirred continuously during the whole process. As the temperature was increased, the solution was became gradually transparent. For the synthesis of Nd³⁺:Gd₂O₃ nanoparticles, the proper amount of Nd2O3 added into the transparent solution of Nd:Gd was 3:100. At the temperature about 80 °C, a little amount of compound initiator AIBN (C₈H₁₂N₄) was added into the solution which led to a quick polymerization. Subsequently, the transparent polymeric resin was obtained with no precipitation. At the last step, to achieve a xerogel, the synthesized gel was dried at 100 °C for 24 h. Then, in order to burn out the organic residues, the xerogel was heated at 300 °C in a laboratory furnace for 10 h and calcined at 550 °C for 5 h.

For preparation of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles [ 11 ], 100 mmol oleic acid was added to 1 mL of Nd³⁺:Gd₂O₃ nanoparticles suspension which was dispersed in ethyl ether 3000 mg/L of Gd³⁺ concentration. After 24 h stirring, sodium bicarbonate solution (0.1 M) with a pH of 9 was added and stirring was performed for 2 h. Then, for dispersion of oleic acid coated Gd₂O₃ in water, a probe solicitor with amplitude of 60% was used for 10 minutes. In order to complete evaporation of ethyl ether, the suspension was stirred for 1 day at room temperature. For purification of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles, syringe filtration with 90 °K was used.

Characterization

The crystal structure of the nanoparticles was detected by powder X-ray diffraction (XRD) using a Siemens diffractometer (D500). A Philips scanning electron microscope (SEM: ES 30 kW) was used to determine the size and shape of the nanoparticles.

MTT assay

In vitro MTT assay was performed on A549 cell line for the three groups in triplicate including the blank, the oleic acid coated Nd³⁺:Gd₂O₃ and bare nanoparticles according to the Zeini et al. study [ 23 ].

MRI experiments

Similar concentrations of the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles were prepared and fixed in agarose gel to provide uniform dispersion. Both series of the samples were put in the 3 mL Eppendorf plastic tubes to prevent any imaging artifacts. The Eppendorf tubes were placed in a Perspex water filled phantom. The T1 and T2 weighted images of the nanoparticles were prepared with a 1.5 Tesla MRI system (Philips, Ingenia).

The spin echo T1 weighted MR images were acquired using the variable TRs (100 to 2500 ms) and a fix TE (8 ms), flip angle = 90 degree, field of view (FOV) = 190 mm², slice thickness = 6 mm² and acquisition pixel size = 0.95 × 0.75 mm². For preparing the T2 weighted MR images, a multi spin echo sequence was utilized with a fix TR (4000 msec) and 16 alternate TEs (16 to 256 ms). Other imaging parameters were similar to the T1 weighted images.

Signal intensity of each image was measured by DICOM software of the MRI machine. Plotting the curves of the T1 and T2 relaxation times was done by MATLAB software. The relaxivity graphs which show changes of the 1/T1 and 1/T2 versus Gd³⁺ concentrations of the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles were plotted by excel software.

Results

Characterization tests and MTT assey

Figure 1 shows the XRD pattern of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles. The Gd₂O₃ nanoparticles peaks are seen in the figure while there is no peak related to neodymium.

Figure 1. X-ray diffraction (XRD) pattern of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles.

The SEM image of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles is seen in Figure 2. According to the figure, the spherical-like nanoparticles were uniform in the shape and the average size of them was 40 nm.

Figure 2. Scanning electron microscope (SEM) image of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles.

The cell toxicity of the Nd³⁺:Gd₂O₃ nanoparticles with and without oleic acid coating is illustrated in Figure 3. Reduction of A549 cells viability with increasing of the concentrations of both nanoparticles is seen in the figure. The coated Nd³⁺:Gd₂O₃ (light brown columns) and the bare nanoparticles (blue columns) showed a cell viability more than 80% up to concentrations of 0.18 and 0.10 mM, respectively.

Figure 3.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results for the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles (light brown columns) and the bare nanoparticles (blue columns).

MRI

The T1 and T2 weighted images of the various concentrations of the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles are shown in the insets of Figures 4A and B, respectively. In the T1 weighted images (inset of Figure 4A), the signal intensity of the Gd₂O₃ nanoparticles was much higher than that of Nd³⁺:Gd₂O₃ at similar concentrations. Inset of Figure 4B illustrates the concentration dependent signal intensity reduction for both the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles with a higher effect on the neodymium doped nanoparticles.

The r1 relaxivity of the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles were calculated as 24.051 and 2.974 mM⁻¹s⁻¹, respectively (Figure 4A). On the other hand, the r2 relaxivity of the coated Nd³⁺:Gd₂O₃ nanoparticles was calculated as 61.163 mM⁻¹s⁻¹ while it was 35.282 mM⁻¹s⁻¹ for the Gd₂O₃nanoparticles (Figure 4B).

Figure 4. A) r1 and B) r2 relaxivity graphs of the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles. Inset of Figure A and B show the T1 and T2 weighted images of the nanoparticles, respectively. In each inset, the first row demonstrates the zero concentration (at left) and from the low to the high concentrations of the coated Gd₂O₃ nanoparticles. The second row is related to the same concentrations of the coated Nd³⁺:Gd₂O₃ nanoparticles.

Discussion

Characterization tests

According to Figure 1 which shows the XRD pattern of the Nd³⁺:Gd₂O₃ nanoparticles, the main characteristic peaks were matched with the Gd₂O₃ in cubic phase (JCPDS: 12-0797) with space group Ia3. The XRD peaks were related only to the Gd₂O₃ nanoparticles and there were no peaks corresponding to neodymium. The finding indicates successful doping of Nd³⁺ into the Gd₂O₃ nanoparticles.

As it is seen in Figure 3, the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles showed cytocompatibility at the higher concentrations compared to the bare nanoparticles. The cell viability for the coated nanoparticles was slightly lower than 80% only at the highest concentration (0.22 mM). On the other hand, the bare Nd³⁺:Gd₂O₃ nanoparticles were cytocompatible only at the lower concentrations (up to 0.10 mM), showing significant effect of the oleic acid coating on the cytocompatibility of the nanoparticles.

MRI

The higher signal intensity of the Gd₂O₃ nanoparticles compared to the Nd³⁺:Gd₂O₃ particles at similar concentrations (inset of Figure 4A) indicates stronger effect of the Gd₂O₃ nanoparticles on the T1 shortening of the water protons.

An appropriate Gd-based T1 contrast agent enhances the signal intensity and contrast in the T1 weighted MR images proportional to the gadolinium ions concentration. Therefore, the samples with the higher concentrations of gadolinium ions show the higher signal intensity. In the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles, some of the gadolinium ions are replaced with Nd³⁺, leading to the changes in the magnetic domains which affect the T1 relaxation time, and consequently, r1 relaxivity. The value of r1 relaxivity is an important factor to determine the efficacy of Gd-based nanoparticles. Based on the r1 relaxivity graphs of the oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles, the r1 values were calculated as 24.051 and 2.974 mM^-1s^-1, respectively (Figure 4A). Consequently, the r1 value of the Gd₂O₃ nanoparticles was obtained about 8 times higher compared to the Nd³⁺:Gd₂O₃ nanoparticles.

Despite oleic acid coating of the Gd₂O₃ nanoparticles which is a hydrophobic coating material, the r1 relaxivity of the nanoparticles in this study is also higher than those of the nanostructures in the other studies such as the ultrasmall Gd₂O₃ nanoparticles with coating of polyethylene glycol diacid (PEGD)-250 and PEGD-600 [ 24 ], and the polyethylenimine (PEI) coated ultrasmall Gd₂O₃ nanoparticles (PEI-1300) [ 25 ]. Although the physicochemical properties of the Gd₂O₃ nanoparticles are different among the studies, the main reason for the higher r1 relaxivity of the Gd₂O₃ nanoparticles in this work is their hollow sphere structure. Preparation of the nanoparticles in the present study was carried out by polymer pyrolysis method, leading to production of the hollow sphere Gd₂O₃ nanoparticles. This situation provides a high access of the gadolinium ions to the water protons, and therefore, a high T1 shortening and r1 relaxivity is resulted.

The oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles showed the higher T2 effect and image darkness in the T2 weighted images compared to the Gd₂O₃ nanoparticles (inset of Figure 4B). As it is seen in Figure 4B, the r2 relaxivity of the coated Nd³⁺:Gd₂O₃ nanoparticles (61.163 mM^-1s^-1) was about 1.7 times higher than that of the Gd₂O₃ nanoparticles (35.282 mM^-1s^-1). This finding implies a dominant T2 shortening effect in the Nd³⁺:Gd₂O₃ nanoparticles due to the doping of the Nd³⁺ into the nanoparticles which accelerates the spin-spin interactions.

The r2/r1 ratio for the Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles were calculated to be 1.467 and 20.566, respectively. The r2/r1 ratio is an indicator to determine whether a magnetic material enhances T1 or T2 contrast of MR images. Since the value of r2/r1 for the Gd₂O₃ nanoparticles is close to unity, the material is considered as a T1 contrast agent. On the other hand, the Nd³⁺:Gd₂O₃ nanoparticles with a high amount of the r2/r1 ratio have potential to enhance T2 contrast of the images.

Comparing the r1 relaxivity of the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles with other lanthanide doped Gd₂O₃ nanoparticles in the similar magnetic field intensity showed the lower r1 for the Nd³⁺ doped nanoparticles compared to the terbium doped [ 13 ] and the europium doped [ 26 ] nanoparticles. It can be due to the different effect of Nd³⁺ doping on the magnetic domains of the Gd₂O₃ nanoparticles compared to terbium and europium. Since the r2 values of the terbium doped and europium doped Gd₂O₃ nanoparticles were not reported, comparing the r2 amounts was not possible.

Conclusion

In the current study, we investigated the effect of Nd³⁺ doping on r1 and r2 relaxivities of the Gd₂O₃ nanoparticles. The oleic acid coated Gd₂O₃ and Nd³⁺:Gd₂O₃ nanoparticles were prepared. The successful preparation of the nanoparticles and their compatibility with the A549 cells for 24 h incubation were confirmed by XRD and MTT assay results, respectively. The average size of the spherical-like shape nanoparticles was 40 nm. A considerable drop in the r1 relaxivity and an improvement in the r2 relaxivity were observed for the oleic acid coated Nd³⁺:Gd₂O₃ nanoparticles compared to the Gd₂O₃ particles. The findings revealed that Nd³⁺ doping into the Gd₂O₃ nanoparticles leads to the changes in the MRI properties of the Gd₂O₃ nanoparticles.

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