Volume 13 Issue 9 - May 7, 2010 PDF
Bifunctional Gd2O3/C Nanoshells for MR Imaging and NIR-Therapeutic Applications
Chih-Chia Huang,1 Chia- Hao Su,2 Wei-Ming Li,1 Tzu-Yu Liu,1 and Chen-Sheng Yeh*1
  1. Department of Chemistry, National Cheng Kung University
  2. Center for Translational Research in Biomedicial Science, Chang Gung Memorial Hospital
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Nanosize materials have practical applications in early detection and destruction of cancer cells, which would be useful for local therapeutic treatments of tumor sites without affecting neighboring healthy tissues, and for in vivo detection of therapeutic efficiency. Multifunctional nanomaterials incorporated therapeutics, molecular targeting, and diagnostic imaging capabilities have been considered as the next generation of multifunctional nanomedicine. In this study, a Gd2O3/C nanoshells (with 6 wt% graphite carbon) was synthesized by modifying our previous method (Chem. Mater. 2008, 20, 3840–3848) and applied for in vivo MR imaging and photothermal therapy. Scheme 1 illustrates the material synthesis via a gelatin templating process (~ 384 nm in diameter) and the studies of in vivo MR imaging and selective photothermolysis of cancer cells. The as-prepared gelatin/inorganic composites were initially thermal treated at 440 ˚C in air, followed by an anneal process at 600 ˚C under N2 to obtain crystalline Gd2O3/C nanoshells.
Scheme 1. Synthesis of Gd2O3/C@PSMA nanoshells and their biomedical applications.

Figure 1. (a) SEM image of Gd2O3/C nanoshells prepared via a two-step thermal treatment: calcination at 440 ˚C in air, followed by annealing at 600 ˚C under N2. (b) XRD patterns for the nanoshells prepared at 440 ˚C in air and at 600 ˚C in N2. (c) Raman spectrum of Gd2O3/C nanoshells prepared after annealing at 600 ˚C under N2, showing G and D bands due to the presence of carbon. (d) MTT assays of Gd2O3/C and Gd2O3/C@PSMA nanoshells cultured with A549 malignant lung cancer cells.
Fig. 1 shows a SEM image of Gd2O3/C nanoshells with an average particle size of ~ 138 nm and a shell thickness of ~ 19.2 nm (obtained by counting 150 particles). Fig. 1b shows X-ray diffraction (XRD) analysis of nanoshell structures. The crystalline nanoshells were obtained after annealing treatment at 600 ˚C, which could be designated as a cubic Gd2O3. Fig. 1c shows the Raman signals of Gd2O3/C nanoshells, labeling as G- and D- peaks appeared at ~1570 cm-1 and ~1340 cm-1, respectively. The G band is related to sp2-hybridized carbon atoms in a graphite layer. The D band originates from disordered graphite.

To improve the hydrophilic and biocompatible nature of Gd2O3/C nanoshells, PSMA surfactant was introduced to modify the nanoshells, leading to a water-dispersible suspension of Gd2O3/C@PSMA nanoshells (the inset in Fig. 1c). The cytotoxicity of Gd2O3/C and Gd2O3/C@PSMA nanoshells was estimated using cell viability (A549 malignant lung cancer cells) with MTT assay for 1 day cell culture (Fig. 1d). Based on these sample doses (0-500 μg/mL), the Gd2O3/C@PSMA nanoshells exhibit good cell viability, where a cell viability of 90.4 % at high dose concentration of 500 μg/mL was reached. The better biocompatibility of Gd2O3/C@PSMA nanoshells than that of Gd2O3/C nanoshells (76.2 % at 500 μg/mL) could be due to their better hydrophilic property.

A series of different Gd3+ concentrations was calculated for the r1 and r2 relaxivities. Based on the the plots of the longtitudinal and transverse relaxation rate versus the Gd ion concentrations, the proton longitudinal (r1) and transverse (r2) relaxivities were determined as 10.3 s-1mM-1 (r1) and 11.0 s-1mM-1 (r2) for Gd2O3/C nanoshells, r2/r1 = 1.1, and 14.0 s-1mM-1 (r1) and 26.2 s-1mM-1 (r2) for Gd2O3/C@PSMA nanoshells, r2/r1 = 1.9. The ratio of r2/r1 is the reference value for MR contrast agent enhancement, where the r2/r1 value is close to 1.0 for favor positive contrast enhancement and the ratio of r2/r1 is larger than 1.5 for viewing as negative contrast agents. Based on the r2/r1 ratio, the Gd2O3/C nanoshells act as positive MR contrast agents (short T1 relaxation time) and the Gd2O3/C@PSMA nanoshells are more likely to serve as negative MR contrast agents (short T2 relaxation time).

Figure 2. (a) In vivo progressive MRI events. T1-weighted images of male BALB/c mice administrated with Gd2O3/C nanoshells at the indicated temporal points (pre-injection, immediate post, 1 h and 2h) (The white and black arrows indicate the kidneys and liver, respectively). (b) The signal intensities of liver and kidney in T1-weighted imaging at the indicated temporal points. In vivo progressive MR images of Gd2O3/C@PSMA nanoshells. (c) T2-weighted and (d) T2*-weighted images of male BALB/c mice at the temporal points (preinjection, immediate post, 1h, and 2 h). (e). The signal intensities of liver in T2- and T2*-weighted imaging at the temporal points. The white arrows indicate the liver.
The mice were administrated with Gd2O3/C nanoshells (3 mg/Kg) via the jugular vein. At post immediately, the imaging of kidneys (white arrow in Fig. 2a) became slightly brighter. Both the kidney and liver (black arrow in Fig. 2a) areas appeared brighter than pre-contrast images after 2 h of circulation. After post 2h by the injection of Gd2O3/C nanoshells, both liver and kidney were enhanced about 10% and 16%, respectively (Fig. 2b). Fig. 2c and 2d shows T2- and T2*-weighted imaging for Gd2O3/C@PSMA nanoshells. Figure 2c shows in vivo T2-weighted imaging, pre- and post-contrast images, of Gd2O3/C@PSMA nanoshells that decreased signal intensity in the liver area (white arrow) with time. The anatomic T2*-weighted imaging of the mice also exhibits the imaging intensity of the liver region darkened as the time increased (Fig. 2d). The T2- and T2*-weighted imaging of liver were enhanced about 36% and 15%, respectively, at post 1h by injection of Gd2O3/C@PSMA nanoshells, as shown in Figure 2e. These animal MR assay experiments demonstrate that Gd2O3/C nanoshells would serve as a positive contrast agent. The enhancement property could be switched from positive to negative contrast agent by coating PSMA on Gd2O3/C nanoshells.

The biodistribution was measured as a function of time and analyzed by ICP analysis. We administrated dosages of Gd2O3/C and Gd2O3/C@PSMA nanoshells at 3.0 mg/Kg. The uptake of Gd2O3/C and Gd2O3/C@PSMA nanoshells in the heart and kidney was low (0.5-24h) and shows apparent accumulation in the liver, lung and spleen and peaked at 3 h after injection. We also found the particles could circulate in the vessels and are cleared out gradually from organs after 24 h.

Figure 3. (a) Anti-EGFR conjugated with Gd2O3/C@PSMA nanoshells (500 μg/mL) treated with A549 cancer cells were irradiated by laser dosages of 20 W/cm2 (top row), 15 W/cm2 (middle row), and 10 W/cm2 (bottom row) for 7 min. (b) The cell viability versus NIR laser power for A549 cells treated with anti-EGFR conjugated with Gd2O3/C@PSMA nanoshells, Au nanorods and silica@Au nanoshells. The samples dosage with fixed at 500 μg/mL.
The 808 nm laser irradiation power densities were varied from 10 to 20 W/cm2 with 7 min of irradiation to evaluate the cancer cell killing efficiency by 500 μg/mL of Gd2O3/C@PSMA nanoshells (ε ~ 1563 M-1cm-1 at 808 nm). The Gd2O3/C@PSMA nanoshells conjugated with anti-EGFR antibodies (electrostatic interaction) were used for targeting and destroying A549 lung cancer cells. After NIR laser treatment, the cells were incubated with fluorescent labels of calcein AM and EthD-1 dyes, producing green emission in viable cells and red emission in dead cells, respectively. It was found that a significant loss of viability, at which a clear void in calcein AM staining and a red fluorescence spot in EthD-1 staining, began at 15 W/cm2, as shown in Fig. 3a (middle row). However, no damage to A549 cells was observed when the laser dosage was turned down to 10 W/cm2, as shown in Fig. 3a (bottom row). The cell viability versus NIR laser power for A549 cells treated with anti-EGFR conjugated Gd2O3/C@PSMA nanoshells, Au nanorods (40.1 ± 3.4 nm in length and 10.3 ± 1.8 nm in diameter) and silica@Au nanoshells (a diameter of 125.4 ± 13.6 nm with Au shell thickness of ~12.1 nm) was conducted to understand the efficiency of photothermolysis in cancer cells, as seen in Fig.3b. The sample dosage was fixed at 500 μg/mL for each nanomaterial. The results indicated suggest that Gd2O3/C@PSMA nanoshells (a loss of viability beginning in the power range of 10–15 W/cm2) act as efficient photothermal absorbers for destroying cancer cells as compared with Au nanorods and silica@Au nanoshells (both above 25 W/cm2).

In conclusion, we have presented the first report of Gd2O3/C nanoshells showing MR anatomic imaging enhancement and photothermolysis of killing cancer cells. The MR assays showed that we could switch the imaging contrast effect from positive Gd2O3/C contrast agents to negative Gd2O3/C@PSMA contrast agents. The graphite carbon coated on Gd2O3 nanoshells has a good absorbance in the near-infrared (NIR) region, acting as photothermal therapeutic agents. As compared with Au nanorods and silica@Au nanoshells, the Gd2O3/C@PSMA nanoshells performed more efficient photothermal absorbers to destroy cancer cells.
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