Anti-CEA-functionalized superparamagnetic iron oxide nanoparticles for examining colorectal tumors in vivo
© Huang et al.; licensee Springer. 2013
Received: 18 August 2013
Accepted: 26 September 2013
Published: 8 October 2013
Although the biomarker carcinoembryonic antigen (CEA) is expressed in colorectal tumors, the utility of an anti-CEA-functionalized image medium is powerful for in vivo positioning of colorectal tumors. With a risk of superparamagnetic iron oxide nanoparticles (SPIONPs) that is lower for animals than other material carriers, anti-CEA-functionalized SPIONPs were synthesized in this study for labeling colorectal tumors by conducting different preoperatively and intraoperatively in vivo examinations. In magnetic resonance imaging (MRI), the image variation of colorectal tumors reached the maximum at approximately 24 h. However, because MRI requires a nonmetal environment, it was limited to preoperative imaging. With the potentiality of in vivo screening and intraoperative positioning during surgery, the scanning superconducting-quantum-interference-device biosusceptometry (SSB) was adopted, showing the favorable agreement of time-varied intensity with MRI. Furthermore, biological methodologies of different tissue staining methods and inductively coupled plasma (ICP) yielded consistent results, proving that the obtained in vivo results occurred because of targeted anti-CEA SPIONPs. This indicates that developed anti-CEA SPIONPs owe the utilities as an image medium of these in vivo methodologies.
KeywordsCarcinoembryonic antigen Magnetic nanoparticles Scanning superconducting-quantum-interference-device biosusceptometry Magnetic resonance imaging
Colorectal tumors, which are caused by uncontrolled cell growth in the colon or rectum, have constituted the third most commonly diagnosed cancer in the world, especially in developed countries. In screening methods, a stool occult blood test is usually performed when the patient has experienced symptoms such as unusual bowel habits. When the result is positive, flexible sigmoidoscopy, barium enema, or colonoscopy is further applied. Because of discomfort and risks, such as the colonic perforation that can occur in these invasive methods, noninvasive methods, such as computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), are alternatively used to image not only the primary colorectal tumor but also metastatic tumors in other organs.
Two approaches can enhance the sensitivity and specificity of these medical imaging procedures. The first approach is the multimodality of structural imaging and functional imaging, such as the CT/PET and MRI/PET. The second is based on image contrast media using bioprobes. Here, the image contrast media are the radioactive materials for CT and PET and the superparamagnetic materials for MRI. It is well known that these radioactive media and methodologies entail a biological risk and that the clinically popular gadolinium medium of MRI superparamagnetic materials induces the side effect of kidney disease. Because iron oxide materials have a low risk of toxicity, superparamagnetic iron oxide nanoparticles (SPIONPs) coated with bioprobes have been developed for highly specific labeling of targeted tumors in examining and treating tumors.
Because carcinoembryonic antigen (CEA) is expressed in colorectal cancer, it is a useful indicator for treatment progress according to the decreasing CEA level in plasma. Therefore, anti-CEA SPIONPs were developed as the contrast medium of MRI for colorectal cancer. However, because MRI requires a no-metal and shielded environment, as well as the patient to lie inside a coil, the procedure is limited to preoperative examination rather than intraoperative examination. Therefore, the multimodality image contrast media of preoperative and intraoperative examinations have emerged recently, for example, nanoparticles used in preoperative MRI with an additional near-infrared fluorescent indicator for intraoperative optical imaging or an additional radioactive indicator for an intraoperative gamma imaging. However, synthesizing various functional indicators on nanoparticles increases not only the cost but also the toxicity risk.
To accommodate the needs of preoperative and intraoperative examinations using simple SPIONPs without additional indicators, the superior magnetic characteristics of SPIONPs should be examined for conducting different in vivo examinations. For example, the paramagnetic or superparamagnetic characteristics of SPIONPs have been used for performing the image contrast of MRI. Similarly, the nonlinear response of SPIONPs was developed to reveal SPIONP distributions by magnetic particle imaging (MPI). However, the field of view of MPI currently is quite small, for example, the beating heart of a mouse[14, 15]. Recently, a scanning superconducting-quantum-interference-device biosusceptometry (SSB) system, possessing the advantage of an ultrasonic-like operation, was developed to track SPIONPs noninvasively without using bioprobes in animals[16, 17]. The mechanism entails examining the in-phase component of the AC susceptibility of SPIONPs.
In this work, to validate the simple anti-CEA-functionalized SPIONPs demonstrating the ability to label colorectal tumors, anti-CEA-functionalized SPIONPs were synthesized and injected into mice implanted with colorectal tumors for MRI and SSB examinations in vivo.
The Animal Care and Use Committee of National Taiwan University approved all experimental protocols (No. 20110009), named 'Development of Core-technologies and Applications of Nano-targeting Low-field Magnetic Resonance Imaging.’ All experiments were conducted according to the animal care guidelines of the university.
For implanting the colorectal tumors, the injections of the CT-26 cell line were processed through the skin on the backs of 8-week-old mice. Three weeks later, 0.06 emu/g and 100 μl of anti-CEA SPIONPs in water were injected into the tail veins of five mice. Two mice, mouse 1 and mouse 2, were examined using SSB and MRI magnetic instruments. The SSB examination schedule was at the 0th, 14th, 26th, 40th, 68th, and 92nd hours for mouse 1 and at the 0th, 8th, 20th, and 42nd hours for mouse 2. The MRI examination schedule was 4 h later than each SSB examination time. Here, 0th represents the time before injection. Proving that the anti-CEA SPIONPs were bound to the tumor tissue required determining the Fe amount using inductively coupled plasma (ICP) and well-known tissue staining methods, such as hematoxylin and eosin (HE) staining, Prussian blue (PB) staining, anti-CEA staining, and cluster of differentiation 31 (CD 31) staining, to examine the tumor tissue of three mice, mouse 3, mouse 4, and mouse 5, which were euthanized at the 0th, 24th, and 98th hours, respectively.
During the measurement process, the test mice were initially anesthetized, and their back tumors were then covered by a plastic plate with a tumor-fit hole to fix the relative orientation and distance of the tumor from the scanning probe unit. The scanning probe unit scanned the entire tumors in several scanning paths with a vertical interval of 0.1 mm. Thus, a magnetic image for the tumor could be constructed, as shown in Figure 2a. SPIONPs under AC field excitation generally expressed the characteristics of AC susceptibility. Therefore, the SSB signal from the in-phase component of the AC susceptibility of SPIONPs was in proportion to the SPIONP concentration.
The 3-T MRI (Bruker Biospec System, Karlsruhe, Germany) and a volume coil were used for T2-weighted images. In parallel with the arrangement of the anesthetized mouse, a long tube filled with deionized (DI) water was inserted as the intensity reference to dismiss the instrument drift at various times. Producing the coronal images of each entire mice body at 2-mm intervals required nearly 2 h.
In general, the uniformity of the static field and gradient field is distorted by SPIONPs, resulting in the dephasing of the proton nuclear spin and, subsequently, the reduction of nuclear magnetic resonance (NMR) intensity induced by the pulse field of MRI. Hence, the labeled tumor cells using bound SPIONPs expressed a darker image. Therefore, SPIONPs were the contrast agent of the MR images.
For ICP examination (EVISA Instruments, PE-SCIEX ELAN 6100 DRC, High Valued Instrument Center, National Science Council, Kaohsiung, Taiwan), two pieces of tumor tissue from one euthanized mouse were both weighted by a 0.1-g weight and then dissolved entirely in a HNO3 solution at a concentration of 65%; they were then diluted and examined. To evaluate the incorporation of an anti-CEA SPIONP quantity into the tumor tissue, the difference of Fe concentration between the varied post-injection and pre-injection times at the 0th hour was expressed as ΔCFe (ppm).
The tissue staining was processed (Laboratory Animal Center, National Taiwan University, Taipei, Taiwan), and the × 400 magnification of the optical images was observed using a light microscope. HE staining, PB staining, anti-CEA staining, and CD 31 staining were performed to identify the tumor tissue, Fe element distribution, and anti-CEA SPIONP distribution; and the degree of tumor angiogenesis was related to the transportation of anti-CEA SPIONPs.
Results and discussion
Figure 1b shows the curve of the magnetism-applied field (M-H) curve of anti-CEA SPIONPs. Based on the ultralow hysteresis in the M-H curve, the anti-CEA SPIONPs expressed superparamagnetic characteristics. Furthermore, the X-ray pattern of the anti-CEA SPIONPs in Figure 1c depicts the crystal structure of anti-CEA SPIONPs obtained by X-ray diffraction. The major peaks correspond with the standard X-ray pattern of Fe3O4 (JCPDS card no. 65–3107), verifying that the magnetic material was Fe3O4, a magnetic iron oxide (IO). In addition, the distribution of the hydrodynamic diameter, as shown in Figure 1d, indicates that anti-CEA SPIONPs have a mean diameter of 50 ± 2 nm. In other words, anti-CEA SPIONPs belong to the so-called 'ultrasmall superparamagnetic iron oxides (USPIOs)’.
Furthermore, regarding the mentioned favorable agreement between the SSB results and the MRI results of the upper region of a labeled tumor rather than the entire region, it was explained as follows. In tumor development, most of the scab tumors were possibly fiber tissue or dead tumor cells in the tumor center; however, the upper region, in which more distribution of live tumor cells occurred around the tumor center, constituted live cells for binding anti-CEA coating SPIONPs. Hence, for colorectal tumors labeled with developed anti-CEA SPIONPs, a two-dimensional (2D) magnetic image (Figure 2a) of SSB was in charge of in vivo screening initially and intraoperative positioning finally, and MRI worked for only preoperative imaging.
Figure 5b shows the variation of the average iron amounts in tumor tissues reaching the highest level at the 24th hour and recovering at the 98th hour to the initial level at the 0th hour. Therefore, the various amounts of both anti-CEA SPIONPs by tissue staining and Fe element distribution by ICP correspond with the magnetic results obtained by SSB and MRI.
In summary, anti-CEA SPIONPs with simple structures demonstrated superior magnetic characteristics for examining colorectal tumors in vivo. Because the dynamics of magnetic labeling was consistent with biological phenomena by tissue staining and ICP, the feasibility of examining targeted colorectal tumors by SSB and MRI was proved. This indicates that this type of anti-CEA SPIONP can be used in a complete series of medical applications, such as in vivo screening and intraoperative positioning, by SSB and conducting preoperative examination by MRI.
This work was supported by the National Science Council of Taiwan under grant numbers 102-2112-M-003-017, 102-2923-M-003-001, 102-2120-M-168-001, 102-2112-M-168-001, 102-2221-E-003-008-MY2, and 101–2221-E-003-005; the Department of Health under grant numbers DOH101-TD-N-111-004, DOH100-TD-N-111-008, and DOH100-TD-PB-111-TM022; and the National Taiwan Normal University.
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