Supplementary Information
In vivo Immunotoxicity Evaluation of Gd2O3
Nanoprobe Prepared by Laser Ablation in Liquid for
MRI Preclinical Application
Journal of Nanoparticle Research
†
†
Xiumei Tian . Xiaoying Guan . Ningqi Luo. Fanwen Yang. Dihu Chen. Ye Peng. Jixiang Zhu.
Li Li*. Xiaoming Chen*
Department of Biomedical Engineering, Guangzhou Medical University, Guangzhou Guangdong
Province ,195, Dongfeng Xi Road, China.
E-mail: xmchenw@126.com (X.C.)
L.Li. ()
State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center, Sun Yat-sen
University Cancer Center, Guangzhou, Guangdong Province ,651, Dongfeng Dong Road, China.
E-mail: li2@mail.sysu.edu.cn (L.L.)
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1. Synthesis of the Gd2O3 nanoprobe
The LAL apparatus has been described in our previous work (Luo et al. 2013). In brief, a thin
sheet of Gd was initially ultrasonically cleaned with ethanol and deionized water, then placed into
a container and covered by 5 mm deionized water. A microsecond Nd:YAG laser (wavelength, λ
= 1,064 nm) at a repetition of 100 Hz with a 6 μs pulse duration was focused onto the Gd target.
The laser fluence was 70 mJ/pulse with an ablation time of 15 min. The upper colloid of the
ablated samples was collected by centrifugation at 12,000 rpm for 15 min and filtered (pore size
of 2.2 µm) to exclude large particles. The nanoprobe demonstrated high colloidal stability.
2. Characterization of the Gd2O3 nanoprobe
Transmission electron microscopy (TEM, JEOL, Inc., Japan) and X-ray photoelectron
spectrometry (ESCALab250, Thermo Fisher Scientific, USA) were used to identify the morphology,
structure and components of the products. A 3.0 T clinical MRI scanner (Trio, Siemens,
Erlangen, Germany) was used to measure the relaxivity (r1) of the in vitro and in vivo T1weighted MR images. To determine the contrast enhancement of the Gd2O3 nanoprobe in vitro,
various concentrations (0.025 mM to 0.3 mM) of Gd3+ were subject to T1-weighted phantom
MRI.
3. Results and Discussion
3.1 Characterization of the Gd2O3 nanoprobe
Morphology of the novel Gd2O3 nanoprobe was characterized by TEM. Typical TEM images
are shown in Figure 1(A, B) and suggest our spherical particles obtained good dispersibility with
an interplanar distance of 0.3268 nm. X-ray photoelectron spectroscopy (XPS) determined the
chemical state of the Gd elements. As shown in Figure 1C, the binding energy of 148.0 eV and
142.7 eV were assigned to Gd4d3/2 and Gd4d5/2, respectively. The peak positions are consistent
with the energy level for Gd in Gd2O3 (Raiser et al. 1991), thus verifying the valence state of the
elements in the sample. Furthermore, energy dispersive X-ray spectroscopy (EDS) revealed the
atomic percentage of Gd and O in Gd2O3 (Figure 1D). In our previous works, the data of XRD
showed that no obvious impurity phases are detected; And T1-weighted MR images from in vitro
experiments demonstrated an increase in signal intensity of the Gd2O3 nanoprobe with increasing
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of Gd3+ concentration, suggesting that these nanoparticles act as T1-weighted contrast agents (Luo
et al. 2013).
Figure 1. Characterization of the Gd2O3 nanoprobe. (A) Typical TEM micrograph and (B)
HRTEM image of Gd2O3 nanocrystals (interplanar distance 0.3268 nm). (C) Gd4d XPS spectrum
of the Gd2O3 nanoprobe. (D) EDS of Gd2O3 nanoprobe.
Figure 2. Apoptosis rate of Raw264.7 cells were measured by flow cytometry 48 h after
incubation of PBS, LPS, Gd-DTPA (10 μM) and Gd2O3 nanoprobe (10 μM). Samples were
stained by Annexin V and PI.
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3.2 the stability of the Gd2O3 nanoprobe in PBS
10 mM the Gd2O3 nanoprobe were added into two tubes with 10 mL and 20mL PBS (pH7.4),
respectively , then divided into 5 tubes, and covered and placed at 37℃ for 48h and 72h. The
samples were centrifuged at 15000rpm for 30min. The supernatants were transferred into the
new eppendorf tubes, and the concentration of Gd3+ ions were measured by the Thermo Electron
X7, ICP-MS (Thermo Instrument System Inc.America). The concentrations of Gd3+ ions in the
supernatants are 0.008±0.0001 nM and 0.017±0.0003 nM at 48h and 72h, respectively. The data
indicated that there was no free toxic Gd3+ ions dissociated from the Gd2O3 nanoprobe in PBS at
48h and 72h.
Figure 3. Histogram plot of BL CD69 and LN CD69 expression and the ROS level.
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Figure 4. Histogram plot of CD11b, CD206, CD40 and CD80 in monocytes/macrophages.
Reference
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