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Superparamagnetic iron oxide nanoparticles (SPION) stabilized by alginate

SPION-alginate
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: November 30, 2009.

Chemical name:Superparamagnetic iron oxide nanoparticles (SPION) stabilized by alginate
Abbreviated name:SPION-alginate
Synonym:SPIO-alginate
Agent Category:Nanoparticles
Target:Reticuloendothelial system (RES)
Target Category:Non-targeted (phagocytosis)
Method of detection:Magnetic resonance imaging (MRI)
Source of signal / contrast:Superparamagnetic iron oxide (SPIO)
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
No structure available

Background

[PubMed]

Superparamagnetic iron oxide nanoparticles (SPION) stabilized by alginate (SPION-alginate) have been developed as a contrast agent to improve the sensitivity of magnetic resonance imaging (MRI) in the detection of hepatocellular carcinoma (HCC) (1-3).

MRI is an imaging modality that is used to construct images of the nuclear magnetic resonance (NMR) signal, primarily from the hydrogen atoms in an object. The image contrast is achieved by the differences in the NMR signal intensity in different areas within the object, and the NMR signal intensity largely depends on the nuclear density (proton spins), the relaxation times (T1, T2, and T2*), and the magnetic environment of the tissues. Contrast agents serve to enhance the image contrast, thus improving the sensitivity and specificity of MRI in mapping information from tissues (4, 5). SPION comprise a class of novel MRI contrast agents that are composed of a ferric iron (Fe3+) and ferrous iron (Fe2+) core and a layer of dextran or other polysaccharide coating (4, 6). The iron nanoparticles have very large magnetic moment, which leads to local magnetic field inhomogeneity. Consequently, the NMR signal intensity is significantly decreased, appearing dark on T2- and T2*-weighted images. On the basis of size (i.e., diameter), SPION are commonly classified as oral SPIO (300 nm–3.5 µm), polydisperse SPIO (PSPIO, 50–150 nm), and ultrasmall SPIO (USPIO, <50 nm). In addition, USPIO nanoparticles with an iron oxide core that is monocrystalline in nature are referred to as monocrystalline iron oxide nanoparticles (MION), and MION with a chemically cross-linked and aminated polysaccharide shell are called cross-linked iron oxide nanoparticles (CLIO) (7).

SPION are predominantly used as a T2/T2* contrast agent in the clinic, though it could shorten both T1 and T2/T2* relaxation processes. Successful application of a SPION-based contrast agent is dependent on its size, size distribution, shape, magnetic susceptibility, and surface modification. In vivo, nonspecific SPION are mainly captured by the reticuloendothelial system, and they are more suitable for liver, spleen, and lymph node imaging (8). Because of the long plasma half-life (hours), they are also used as blood pool agents in magnetic resonance angiography (9). Specific SPION are developed by conjugating the respective targeting agents directly onto the SPION surface or onto its hydrophilic coating. Specific accumulation of the agents at disease-specific sites is achieved because of the target overexpression (often cell surface receptors) and receptor-mediated endocytosis and recycling (4, 5). The signal decrease is much more obvious in the lesions than in the surrounding normal tissues. An inverse strategy of the SPION-based molecular imaging is also applied in some studies by designing molecules that bind to targets expressing on normal tissues. This strategy has been proved to be valuable in imaging pancreatic ductal adenocarcinomas and HCC by targeting the receptors of bombesin, cholecystokinin, or asialoglycoprotein (10, 11). In this case, by decreasing the T2 signal of normal tissue surrounding a tumor more than that of the tumor, the contrast between healthy and tumor tissues is enhanced.

A SPION-based MRI contrast agent was developed by stabilizing the SPION with alginate (SPION-alginate) (1-3). In vivo application of this newly developed contrast agent improved the sensitivity of MRI in the detection of HCC in rat and rabbit HCC models.

Synthesis

[PubMed]

The contrast agent SPION-alginate was prepared with a two-step coprecipitation method as a formation of Fe3O4 by coprecipitation of ferric and ferrous ion with alkaline solution and stabilization of the SPION with alginate polymer (2). Briefly, ferric and ferrous chlorides were first dissolved in distilled water, followed by precipitation with NaOH solution at 60ºC. Sodium alginate solution was then added to the suspension containing the Fe3O4 nanoparticles with vigorous stirring for 30 min. The mixture was first heated for 1 h at 80ºC with slow stirring, and then it was sonicated for 20 min. The solution was dialyzed against deionized water to remove the FeCl2 and FeCl3 and then centrifuged to remove the solid materials. The supernatant containing SPION-alginate was collected (used as suspension sample), and a fraction of the supernatant was dried with a rotary evaporator at 50ºC to obtain a solid sample. Three kinds of sodium alginate were tested separately to stabilize the SPION, including Keltone LVCR (54 kDa, M/G ratio 1.5), Keltone HVCR (160 kDa, M/G ratio 1.5), and Manugel DMB (124 kDa, M/G ratio 0.6). All the above processes were run under N2 protection. The Fe3O4 contents in the solid samples varied with the types of alginate but changed little with different concentrations of alginate (LVCR, 1–4% (w/w)). With 1% LVCR, the Fe3O4 content reached up to 50% (w/w). In the suspension samples, the Fe3O4 concentrations were 1–4 mg/ml when coated or stabilized with LVCR alginate (1–4%, w/w), two-fold higher than concentrations when coated with HVCR or DMB.

X-ray diffraction analysis confirmed that the nanoparticles were Fe3O4 with an average diameter of 2.6 nm. Under electronic microscopy, the Fe3O4 nanoparticles were cube-shaped and monodispersed with a diameter of ~10 nm. The Fe3O4-alginate looked like a sponge with Fe3O4 nanoparticles embedded in the alginate substrate. The average hydrodynamic radius of the SPION-alginate was 193.8–483.2 nm, increasing with increased concentrations of alginate (1–4%, w/w). It has been assumed that the polydispersity of alginate is responsible for the large size of SPION-alginate because the Z-average hydrodynamic radius of the pure alginate solution was 200–500 nm. Under atomic force microscopy, the SPION-alginate appeared as a bright spot in liquid with an average diameter of 4.678 nm for the Fe3O4 core. In air, the Fe3O4 nanoparticles bound to the strands of alginate macromolecules with an average diameter of 7.530 nm, larger than in liquid. The binding of Fe3O4 with alginate was also confirmed with Fourier transform infrared spectroscopy, showing a specific shift of the frequencies in the spectra of SPION-alginate. The ξ-potentials of the SPION-alginate coated with LVCR alginate at 1–4% (w/w) ranged from −57.0 to −75.7 mV, similar to the ξ-potential of the alginate solution (−71.8 mV). No sedimentation was observed in bottles of the SPION-alginate filled with nitrogen gas even after 12 months of storage at 4ºC at pH 7.0, indicating that the SPION-alginate nanoparticles were highly stable.

The magnetic property of the SPION-alginate was investigated at 300 K. Magnetization did not reach saturation, even at the applied magnetic field strength of 10,000 oersted (Oe) and no hysteresis was found. These data indicated that the nanoparticles were superparamagnetic. The saturation magnetization (Ms) of the solid samples with 12.5% and 47.0% Fe3O4 was 55 and 52 emu/g, respectively, while the Ms of corresponding suspension samples with 1.09 and 3.02 mg/ml Fe3O4 was 40 and 38 emu/g, respectively. The Ms of the solution samples was 73% of the Ms of the solid samples. In T1-weighted images, the signal intensity of the SPION-alginate was stronger, and in T2-weighted images, the signal was weaker than that of the saline. With increased Fe3O4 concentrations of 0.074–0.300 mmol, T1 relaxation time decreased from 1,450 to 400 ms and T2 relaxation time decreased from 40 to 10 ms. The T1 and T2 relaxivities of the SPION-alginate in saline (1.5 T, 20ºC) were 7.9 ± 0.2 and 281.2 ± 26.4 mM−1 s−1, respectively. The relative magnitude of T1 and T2 relaxivities was 35.7, suggesting that the SPION-alginate nanoparticles had a moderate T1 shortening effect and a strong T2 shortening effect.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Ma et al. analyzed the potential adverse effect of the SPION-alginate on red blood cells and cytotoxicity to L929 cells (mouse fibroblasts) and RAW264.7 cells (mouse macrophages) (1, 3). L929 cells are widely used as the reference cells for polymer cytotoxicity analysis, and RAW264.7 cells are commonly used for cell uptake testing of SPION. Incubation of the rabbit erythrocytes with up to 0.57 mg Fe/ml SPION-alginate for 3 h at 37ºC did not result in evident hemolysis (<5%). No erythrocyte aggregation was observed after 1 h incubation at 37ºC. The cell growth of neither L929 nor RAW264.7 cells was inhibited upon exposure of 6.125–100 µg Fe/ml SPION-alginate for 24 h. After 24 h incubation with 12.5–50.0 µg Fe/ml SPION-alginate, almost all RAW264.7 cells were labeled with the SPION-alginate, presenting abundant Fe3O4 nanoparticles in the cytoplasm. The internalization was dependent on both Fe3O4 concentration and time.

Animal Studies

Rodents

[PubMed]

The pharmacokinetics of the SPION-alginate was investigated in healthy male Sprague-Dawley rats after intravenous administration (1, 3). Twenty-nine rats were used for whole-blood studies by dividing the rats into a low-dose group (109.5 µmol Fe/kg, n = 18 rats), a high-dose group (218.9 µmol Fe/kg, n = 3 rats), an alginate-alone group (n = 3 rats), and a physiological saline group (n = 5 rats). An additional 20 rats were set for serum studies (n = 5/group). Ex vivo whole-blood studies showed that the blood iron reached the maximum level at 5 min, followed by a gradual decrease to the lowest level at 48 h after injection (from 515.5 to 266.3 µg/ml for the low-dose group, and from 647.7 to 333.6 µg/ml for the high-dose group). The blood iron level returned to the baseline (~471.9 µg/ml before injection) at 96 h after injection. In the rats given alginate alone or saline, the patterns of blood iron level changes were similar, mildly decreased at first and recovered later. However, the blood iron levels in all rats were lower than the baseline up to 48 h after injection of alginate alone or saline. The baseline serum iron level fluctuated between 6.06 and 15.22 µg/mmol. After injection of the SPION-alginate, the serum iron level increased rapidly and then decreased to the baseline at 3 h for the low-dose group and at 6 h for the high-dose group. The half-life of the SPION-alginate was 0.27 ± 0.06 h at the low-dose and 0.65 ± 0.22 h at the high-dose. The serum iron concentration change over time after injection of the SPION-alginate were provided in table 1 by Ma et al. (3), however the relationship between the injected does and the iron amount in serum were not described.

Tissue distribution studies showed that the iron levels in the liver and spleen increased markedly after intravenous injection of low-dose SPION-alginate. More than 80% of the injected iron accumulated in the liver, and ~10% accumulated in the spleen. The iron accumulation peaked at 30 min in the liver (235.05 ± 19.62 µg Fe/kg) and at 24 h in the spleen (402.28 ± 187.31 µg Fe/kg). The iron elimination was slower from the liver than from the spleen. Less than 2% of the injected iron accumulated in the lungs, heart, and kidneys. Staining of the liver and spleen tissues demonstrated that the iron accumulated in the Kupffer cells of the hepatic sinusoid and in the cord and red pulp of the spleen at 30 min to 14 days after injection. At 30 min, the iron particles were found to be mainly distributed in the peripheral region of the hepatic lobules, and in the center region at 96 h. The iron particles were still observable at 14 days after injection.

MRI was performed with a 3.0-T MR scanner in healthy male Sprague-Dawley rats (n = 6), rats with diethylnitrosoamine-induced primary liver cancer (n = 29), and rabbits transplanted with VX2 tumor. The signal/noise ratio (SNR) was calculated by dividing the signal intensity by the standard deviation of noise. The contrast/noise ratio (CNR) was calculated with the following formula: (signal intensity of the HCC – signal intensity of the cirrhotic liver tissue)/standard deviation of noise. In healthy rats, the SNR in the liver decreased at 10 min and remained stable up to 180 min after injection. For the rat primary tumor model, 15 rats survived at week 18 of experiments and were used for MRI studies. All 15 rats were accompanied by severe hepatic cirrhosis, and no HCC was found with MRI in any rat before injection of the SPION-alginate. Interestingly, 22 HCCs were found in 11 rats after injection of the SPION-alginate, and the other 4 rats were diagnosed with simple cirrhosis. The SNR in the healthy liver tissue decreased from 48.95 ± 4.87 before injection to 6.43 ± 3.37 after injection, and in the cirrhotic tissue it decreased from 45.51 ± 11.71 to 23.47 ± 7.52. There was no marked change for the SNR in the HCC (45.51 ± 11.71 before injection and 40.53 ± 12.98 after injection). Are they looking simply at SPION? The HCC should be much lower if they are looking at SPION; it looks like they are looking at other sources of iron? The CNR of the HCC increased from 0 before injection to 17.69 ± 3.69 after injection. The contrast between HCC and liver parenchyma was significantly increased. With Perls staining of the tissues after MRI, the SPION were observed in the normal liver parenchyma, hyperplastic nodules, and hemangiomas, but not in the HCC. MRI with SPION alone was not performed for comparison of the contrast enhancement between SPION-alginate and SPION alone.

Similar results were obtained with the MRI studies on the rabbit VX2 tumor model. The MRI signal intensity in the liver parenchyma decreased, but did not decrease in the VX2 tumor. The borderline of the tumor was more obvious in comparison with that of unenhanced images. Perls staining again showed that the SPION were present in the normal part of the liver but not in the tumor.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
Ma H.L., Qi X.R., Ding W.X., Maitani Y., Nagai T. Magnetic targeting after femoral artery administration and biocompatibility assessment of superparamagnetic iron oxide nanoparticles. J Biomed Mater Res A. 2008;84(3):598–606. [PubMed: 17618488]
2.
Ma H.L., Qi X.R., Maitani Y., Nagai T. Preparation and characterization of superparamagnetic iron oxide nanoparticles stabilized by alginate. Int J Pharm. 2007;333(1-2):177–86. [PubMed: 17074454]
3.
Ma H.L., Xu Y.F., Qi X.R., Maitani Y., Nagai T. Superparamagnetic iron oxide nanoparticles stabilized by alginate: pharmacokinetics, tissue distribution, and applications in detecting liver cancers. Int J Pharm. 2008;354(1-2):217–26. [PubMed: 18191350]
4.
Islam T., Josephson L. Current state and future applications of active targeting in malignancies using superparamagnetic iron oxide nanoparticles. Cancer Biomark. 2009;5(2):99–107. [PubMed: 19414927]
5.
Corot C., Robert P., Idee J.M., Port M. Recent advances in iron oxide nanocrystal technology for medical imaging. Adv Drug Deliv Rev. 2006;58(14):1471–504. [PubMed: 17116343]
6.
Bulte J.W., Kraitchman D.L. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 2004;17(7):484–99. [PubMed: 15526347]
7.
Di Marco M., Sadun C., Port M., Guilbert I., Couvreur P., Dubernet C. Physicochemical characterization of ultrasmall superparamagnetic iron oxide particles (USPIO) for biomedical application as MRI contrast agents. Int J Nanomedicine. 2007;2(4):609–22. [PMC free article: PMC2676801] [PubMed: 18203428]
8.
Tanimoto A., Kuribayashi S. Application of superparamagnetic iron oxide to imaging of hepatocellular carcinoma. Eur J Radiol. 2006;58(2):200–16. [PubMed: 16414230]
9.
Maes R.M., Lewin J.S., Duerk J.L., Misselwitz B., Kiewiet C.J., Wacker F.K. A new type of susceptibility-artefact-based magnetic resonance angiography: intra-arterial injection of superparamagnetic iron oxide particles (SPIO) A Resovist in combination with TrueFisp imaging: a feasibility study. Contrast Media Mol Imaging. 2006;1(5):189–95. [PubMed: 17193696]
10.
Tanimoto A., Kuribayashi S. Hepatocyte-targeted MR contrast agents: contrast enhanced detection of liver cancer in diffusely damaged liver. Magn Reson Med Sci. 2005;4(2):53–60. [PubMed: 16340158]
11.
Montet X., Weissleder R., Josephson L. Imaging pancreatic cancer with a peptide-nanoparticle conjugate targeted to normal pancreas. Bioconjug Chem. 2006;17(4):905–11. [PubMed: 16848396]

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