AuNP-DG: Deoxyglucose-Labeled Gold Nanoparticles as X-ray Computed Tomography Contrast Agents for Cancer Imaging
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- Aydogan, B., Li, J., Rajh, T. et al. Mol Imaging Biol (2010) 12: 463. doi:10.1007/s11307-010-0299-8
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To study the feasibility of using 2-deoxy-d-glucose (2-DG)-labeled gold nanoparticle (AuNP-DG) as a computed tomography (CT) contrast agent with tumor targeting capability through in vitro experiments.
Gold nanoparticles (AuNP) were fabricated and were conjugated with 2-deoxy-d-glucose. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Two groups of cell samples were incubated with the AuNP-DG and the unlabeled AuNP, respectively. Following the incubation, the cells were washed with sterile PBS to remove the excess gold nanoparticles and spun to cell pellets using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. The reconstructed CT images were analyzed using a commercial software package.
Significant contrast enhancement in the cell samples incubated with the AuNP-DG with respect to the cell samples incubated with the unlabeled AuNP was observed in multiple CT slices.
Results from this study demonstrate enhanced uptake of 2-DG-labeled gold nanoparticle by cancer cells in vitro and warrant further experiments to study the exact molecular mechanism by which the AuNP-DG is internalized and retained in the tumor cells.
Key wordsGold nanoparticleContrast-enhanced CT2-Deoxy-d-glucoseCancer imagingTumor targeting
In order to accurately stage and treat malignancies, precise knowledge of tumor location, size, and lymphatic or distant spread is essential. While imaging with computed tomography (CT), magnetic resonance imaging, and ultrasound imaging provides valuable anatomical information, they lack the high sensitivity and specificity offered by a functional imaging modality such as positron emission tomography (PET). However, despite its ability to detect functional processes in the body, PET imaging has several drawbacks including the production, transportation, and cost of the radiopharmaceuticals which limit a wider spread of this technology. Also, PET images do not provide anatomical information, and are therefore inadequate for radiation or surgical treatment planning. Recent development of hybrid PET/CT scanners and sophisticated image registration algorithms allows for combined image sets from CT and PET to be used in the diagnosis and staging of malignant diseases. However, despite the benefits of combined PET/CT, the full potential of CT imaging cannot be realized because the superb spatial resolution provided by CT scans is not shared by the PET images. Current PET technology has reported limitations in detecting tumors of fewer than 109 cells (∼1 cm in diameter) . This has significance for the early diagnosis of cancer, where small malignant lesions can be missed by PET scans.
In the last decade, studies have demonstrated that CT imaging can, when combined with an X-ray contrast agent, offer both anatomical and functional data . However, this technique, termed functional CT, has not gained widespread clinical use because of the limitations of current contrast agents. To date, the most commonly used X-ray contrast agents are iodine-based compounds. Despite their clinical use, iodine-based contrast agents have several drawbacks including a high osmolality and a short blood half-life (<10 min) that requires imaging immediately after administration. Also, iodine has a moderate atomic number (Z) that limits the level of achievable CT contrast, decreasing its usefulness in radiation therapy planning, which relies almost exclusively on these values. More importantly, commercially available iodine-based X-ray contrast agents lack tumor-specific targeting ability. Conjugates with targeting moieties, such as antibodies, have thus far failed to deliver iodine to disease sites at a concentration detectable by current CT scanners. In addition to iodine-based agents, several other experimental X-ray contrast materials have been tested with varying degrees of success [3–6]. Nonetheless, the development of intravascular X-ray contrast agents based on other mid-Z to high-Z material, especially those agents with tumor-specific targeting capability, has not been successful due to performance, cost, and toxicity issues [7–9].
Recent advancements in nanotechnology have allowed for the development of novel contrast agents, including gold nanoparticles (AuNP), to improve functional CT imaging. Several investigators have demonstrated the feasibility of using various materials at the nanometer scale as X-ray contrast agents [10–15]. Due to its size, atomic number, and surface area to volume ratio, AuNP offers advantages over iodine-based compounds. Gold, because of its higher atomic number, attenuates X-rays more effectively than iodine and therefore produces superior contrast. Furthermore, the large molecular weight of the AuNPs, ranging from tens to hundreds kilodaltons, provides longer biological half-life than iodine-based compounds. Lastly, a unique property of the AuNPs that remains to be explored is their large surface-to-volume ratio. This large ratio differs distinctively from that of molecular or bulk materials and allows more freedom for surface modification.
The contrast enhancement in the AuNP sample is clearly seen in this picture. The measured CT intensity of the AuNP sample is 900 ± 50 HU, consistent with the result shown by Kim et al. . The voxel intensity histograms for both the water and the AuNP sample are shown in Fig. 2b.
The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay because of its positive response to fluorodeoxyglucose (FDG) . Prior to the imaging experiments, trypan blue staining was performed on the A-549 cells after incubation with varying concentrations of AuNP-DG suspended in PBS and Dulbecco's Modified Eagle Medium (DMEM) media in order to assess cell membrane integrity as a marker for cell viability. These data were used to find non-toxic levels of AuNP-DG concentration in the growth media that would not disrupt the cell membrane, but still be high enough to achieve a suitable contrast level.
The cell samples were imaged using the microCT scanner immediately after the centrifugation. For image acquisition, the vials containing the cell pellets were stacked horizontally with their long axes parallel to the bed of the microCT scanner. The scanner settings used were identical to those used to image the pure AuNP suspension.
The A-549 cells used in these experiments are derived from human lung cancers where FDG/PET is commonly employed. Since the A-549 cells contain wild-type p53 and undergo cell cycle arrest following serum withdrawal, we chose to use complete cell growth media in our experiments that may have led to some competition with the glucose receptors. However, translation of these results to in vivo environment awaits direct experimental verification in the future. While the exact mechanism by which the unlabeled AuNP enhances the contrast in these in vitro assays remains to be investigated in future experiments, the cell membrane appeared intact in a majority of the cells after the AuNP-DG incubation in our cell viability assays.
In this study, we demonstrated, through in vitro experiments, that the addition of 2-DG to the AuNP enhances tumor cell uptake. This enhanced cellular uptake suggests that AuNP-DG could serve as a CT contrast agent with tumor-specific targeting capability. Validation of whether AuNP-DG is similar to 2-DG in its metabolism is currently being investigated in our laboratory. Uptake of 2-DG, similar to glucose, appears to depend on both the glucose transport proteins and the hexokinase enzyme. While the former allows intracellular uptake of glucose and its analogs, the latter ensures the trapping of these substances inside the cell by phosphorylating their 6th carbon. Both events lead to the intracellular accumulation of 2-DG-6-phosphate which is limited in its further metabolism resulting in accumulation of this metabolite. The upregulation of either glucose transporters or hexokinases in cancer cells may independently play a role in the greater accumulation of 2-DG . Thus, it is important to investigate whether addition of AuNP to 2-DG will interfere with either of these enzymes. A priori, we hypothesize that AuNP-DG should be an active substrate for glucose transporters since previously it was demonstrated that replacement of the hydrogen group on the second carbon of 2-DG with larger moieties results in minimal steric hindrance . On the other hand, the glucose binding pocket of hexokinase appears to be more conservative to the size of the molecule on the second carbon of 2-DG . Therefore, the nanoparticle may interfere with the binding of AuNP-DG to hexokinase. However, glucose analogs that cannot be phosphorylated by hexokinase, for instance, 3-O-methyl-glucose and 6-fluoro-deoxy-d-glucose, have been shown to be a tracer of greater glucose uptake in cells that over-express glucose transporters . Thus, even if the AuNP-DG is a substrate of only the glucose transporter but not hexokinase, it may still be sufficient to employ this agent for functionally targeted CT imaging since it may exploit high glucose uptake of tumor cells. Overall, our results in which the cellular AuNP-DG signal is found to be superior to AuNP alone suggest that the 2-DG moiety on the nanoparticle preserves some of its activity in facilitating the uptake of this molecule into tumor cells.
Functional CT technique offers a new set of capabilities in cancer imaging by providing unmatched high-resolution anatomical and functional images in a single CT scan. Higher-resolution functional and molecular image information can be expected to be one of the most important contributors in improving cancer therapy, since early detection of cancer still offers the best prognosis . Since X-ray CT and AuNP-DG technologies could be widespread and readily available, successful clinical implementation of the AuNP-DG will offer an effective functional contrast agent for high-resolution X-ray and CT imaging that could have a large impact on cancer research, diagnosis, and treatment planning. Results from these experiments strongly suggest enhanced uptake of the AuNP-DG over the unlabeled AuNP by the highly glycolytic cancer cells in vitro and indicate that AuNP-DG could serve as a CT contrast agent with tumor targeting capability. The exact mechanism by which the AuNP-DG is internalized and retained in the tumor cells remains to be studied in future experiments.
This work was partially supported by Research Training in Medical Physics 5 T32-EB002103-19. Use of the Center for Nanoscale Materials at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Science, under Contract No. DE-AC02-06CH11357.