Synthesis of heterodimer radionuclide nanoparticles for magnetic resonance and single-photon emission computed tomography dual-modality imaging

Abstract

We report a facile synthesis of bifunctional Fe₃O₄-Ag¹²⁵I heterodimers for use as dual-modality imaging agents in magnetic resonance (MR) and single-photon emission computed tomography (SPECT). We introduced ¹²⁵I, which is a clinically used radioisotope, as a SPECT reporter, into Fe₃O₄-Ag heterodimer nanoparticles to provide a new type of bifunctional contrast agent for MRI and SPECT imaging.

---

In recent years non-invasive imaging techniques, such as magnetic resonance imaging (MRI),¹ single-photon emission computed tomography (SPECT),² computed tomography (CT),³ photoacoustic tomography (PAT),⁴ and optical imaging,⁵ have become indispensible clinical methods for diagnosing various diseases. However, a single imaging modality is rarely sufficient to obtain all the demanded information.⁶ Using bimodal imaging probes and combining two techniques can synergize the advantages and/or mitigate the disadvantages of individual techniques. In the past few years, various nanocomposites have been developed with functional combinations including CT/MRI, MRI/PET, MRI/SPECT, CT/PAT, and SPECT/CT.^7–11 Our group has also developed heterostructured nanocomposites for CT/MRI.⁷ Compared with CT, MRI offers higher resolution of soft tissues without any associated radiation risks. Encouraged by the successful combination of SPECT and CT,¹¹ SPECT and MRI (SPECT/MRI) have been actively pursued by the imaging community in recent years.⁹ MRI has high spatial resolution with lower sensitivity, while SPECT has an outstanding sensitivity but lower resolution. MRI/SPECT dual-modal contrast agents can enable a hybrid imaging modality that takes advantage of these features. There is a demand for multimodal contrast agents for MRI/SPECT pre-clinically and clinically. One approach to achieve these nanomaterials is by labeling metal nanoparticles used in MRI, with radionuclides used in SPECT.

Magnetic iron oxide nanoparticles are known as T₂-relaxing contrast agents that decrease the MRI signal intensity by shortening the T₂ relaxation times of nearby protons, resulting in dark areas in images.¹ Iodine-125 (¹²⁵I) is a candidate single-photon emitting radionuclide with good potential in SPECT imaging application because of its suitable long half-life (t_1/2 = 61.14 days). Additionally, the production of this radioisotope is simple and cost-effective.

We hypothesized that ¹²⁵I could be inserted into a suitable nanocomposite functionalized by PEG to form a MRI/SPECT contrast agent. In this communication, Fe₃O₄-Ag heterostructured nanoparticles were selected for this purpose. Radiolabeling of the Fe₃O₄-Ag heterodimer nanoparticles was achieved by the rapid reaction of iodine with the Ag component of the nanoparticles. Scheme 1 illustrates the general synthetic strategy towards Fe₃O₄-Ag¹²⁵I nanoparticles. Firstly, the synthesis of Fe₃O₄-Ag nanoparticles was performed according to a reported procedure with some slight modifications.¹² In brief, the monodisperse Fe₃O₄ nanoparticles were synthesized through a thermal decomposition of an iron-oleate complex¹³ and then the Ag component was grown onto the Fe₃O₄ nanoparticles by adding the silver acetate into the reaction system. Secondly, the Fe₃O₄-Ag heterostructured nanoparticles were functionalized by a hydrophilic mPEG-LA polymer and transferred from hexane to water.¹⁴ The as-synthesized PEGylated Fe₃O₄-Ag heterostructured nanoparticles were found to be highly stable in water with no visible aggregation over one month. Finally, the Ag component of the heterostructured nanoparticles was reacted with sodium iodine-125, and stirred at room temperature to produce the radionuclide labeled Fe₃O₄-Ag¹²⁵I heterodimer nanoparticles (Scheme 1 and ESI†). The effectiveness of the heterodimer radionuclide nanoparticles as MRI contrast agents was evaluated via an in vitro MRI relaxivity measurement. The ¹²⁵I radiolabeled heterodimer nanoparticles were further tested as SPECT contrast agents via in vivo experiments.

Scheme 1 Synthesis of Fe₃O₄-Ag¹²⁵I heterodimer nanoparticles for MRI and SPECT imaging.

Transmission electron microscope (TEM) was used to follow the synthesis procedure and to characterize the heterodimer nanoparticles. Fe₃O₄ nanoparticles with uniform morphology are shown in Fig. 1A. The average size of Fe₃O₄ nanoparticles was about 14 nm. The heterostructured Fe₃O₄-Ag nanoparticles are shown in Fig. 1B, which are well-dispersed in hexane. A well-established phase transfer process using the mPEG-LA polymer efficiently allowed the dispersion of the Fe₃O₄-Ag nanoparticles in an aqueous phase through metal–thiol bonds.¹⁴ A TEM image (Fig. S1†) confirmed that they still retained the heterodimer nanostructures in morphology after phase transfer. After coating with mPEG-LA, these Fe₃O₄-Ag nanoparticles (PEG-Fe₃O₄-Ag) were easily dispersed in water (Fig. S2†). No precipitates were observed even after one month. As shown in Fig. 1C, the size of the Ag¹²⁵I spherical component in heterodimer nanoparticles was about 9 nm. The addition of a radionuclide into the Fe₃O₄-Ag nanoparticles did not change the morphology of the samples. Furthermore, the energy dispersive X-ray spectroscopy (EDS) analysis (Fig. 1D) confirmed the presence of silver, iron, and iodine elements in the synthesized heterostructured nanoparticles.

Fig. 1 TEM images of (A) Fe₃O₄ nanoparticles, (B) Fe₃O₄-Ag heterodimer nanoparticles, (C) Fe₃O₄-Ag¹²⁵I heterodimer nanoparticles. (D) EDS data of Fe₃O₄-Ag¹²⁵I heterodimer nanoparticles. The specimens for TEM analysis were prepared by room temperature deposition on a carbon-coated copper grid.

Representative X-ray diffraction (XRD) patterns of the Fe₃O₄-Ag and Fe₃O₄-Ag¹²⁵I nanoparticles are shown in Fig. 2A, pointing out the occurrence of crystalline phases of both AgI and Fe₃O₄ in the heterostructures. Representative X-ray photoelectron spectroscopy (XPS) spectra of Fe₃O₄-Ag¹²⁵I nanoparticles (Fig. 2B and C) also showed the presence of Ag and I with the peak of binding energy occurring at 367.9 eV (for Ag 3d_5/2) and 618.4 eV (for I 3d_5/2), respectively.

Fig. 2 (A) XRD data of Fe₃O₄-Ag heterodimer nanoparticles (black) and Fe₃O₄-Ag¹²⁵I heterodimer nanoparticles (red). The samples were dispersed on Si substrates and dried under ambient conditions. XPS spectra of (B) Ag 3d and (C) I 3d for Fe₃O₄-Ag¹²⁵I heterodimer nanoparticles.

After characterization, we obtained T₂-MRI images of a phantom at various concentrations of Fe₃O₄-Ag¹²⁵I nanoparticles dispersed in deionized water using a GE 1.5 T Signa Excite MRI scanner. As the nanoparticle concentration increased, the signal intensity decreased significantly (Fig. 3, inset). The measured r₂ value was 139.8 mM⁻¹ s⁻¹ (Fig. 3) obtained by plotting 1/T₂ against the iron concentration ([Fe]) in the solution, which was close to that of a commercial MRI contrast agent.¹⁵ The T₂ contrast effect of the nanoparticles was saturated at a concentration of 0.34 mmol L⁻¹ and no signal attenuation was observed above this concentration. Since MRI is very sensitive, a sufficient T₂ contrast effect was observed even at low concentrations.

Fig. 3 Plots of 1/T₂versus iron concentration. Transverse (r₂) relaxivity was determined by linear flitting of the plots (inset: T₂-weighted MR images of the same phantom).

We assessed the biocompatibility of the Fe₃O₄-Ag¹²⁵I nanoparticles toward mammalian cells by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a HUVEC (normal) and HeLa cell line, respectively. The PEG-Fe₃O₄-Ag¹²⁵I nanoparticles were tested over a dosage range of 0–1.5 mM of Fe₃O₄-Ag¹²⁵I nanoparticles at 37 °C. The MTT assay (Fig. 4E) showed no appreciable cytotoxicity (cell viability > 85%) detected up to a concentration of 1.5 mM after 24 h of exposure. This high biocompatibility is a great advantage of Fe₃O₄-Ag¹²⁵I nanoparticles for further clinical use. We further demonstrated the cytocompatibility of Fe₃O₄-Ag¹²⁵I nanoparticles through optical microscopy. The HUVEC and HeLa cells were incubated with the Fe₃O₄-Ag¹²⁵I nanoparticles (1.5 mM) at 37 °C for 24 h, respectively. As shown in Fig. 4A–D, there was no obvious change in the cellular morphology after treated with the Fe₃O₄-Ag¹²⁵I nanoparticles, which agreed with the MTT data.

Fig. 4 The HUVEC (A, B) and HeLa (C, D) cell morphology before (0 h) and after (24 h) treatment with heterodimer radionuclide nanoparticles imaged with phase contrast microscopy. (E) The cytotoxicity evaluated by MTT assay in the range of 0–1.5 mM iron concentration of heterodimer radionuclide nanoparticles.

To confirm the ¹²⁵I labeling yield, various concentrations of Na¹²⁵I were added into PEG-Fe₃O₄-Ag heterostructured nanoparticles (2 mL, 1 mmol Fe L⁻¹). Free radioisotopes were removed from the radiolabeled nanoparticles by filtering through a sterile membrane. The content of the free iodine was less than 1%, as estimated by analysis of the activity of the supernatant. The radiolabeling yield of Fe₃O₄-Ag¹²⁵I heterostructured nanoparticles was found to be above 80% after 30 min of stirring (Fig. 5A). A mixture of free ¹²⁵I and water showed a nearly zero labeling yield, confirming the successful labeling of ¹²⁵I to Fe₃O₄-Ag heterostructured nanoparticles.

Fig. 5 (A) Radioactivity quantization of heterodimer radionuclide nanoparticles. (B) In vivo representative SPECT images of Kunming mice acquired 1 h after injection of Fe₃O₄-Ag¹²⁵I (left) and free ¹²⁵I (right) (n = 5/group).

To demonstrate the feasibility of Fe₃O₄-Ag¹²⁵I heterostructured radionuclide nanoparticles for in vivo SPECT imaging, a heterostructured radionuclide nanoparticle solution (100 μL, 120 μCi) was intravenously injected into Kunming mice (n = 5) via the tail vein. A solution of Na¹²⁵I with the same radioactivity was used as a comparison. In the whole-body SPECT imaging of mice, liver and spleen were all clearly visualized at 1 h post-injection (Fig. 5B left), but little or no accumulation in kidney, muscle or other major organs. Upon injection of Na¹²⁵I at 1 h (Fig. 5B, right), nanoparticles showed darkening of the kidney and the bladder, but nearly no liver and spleen uptake. Dominant liver (31.98 ± 2.44% ID g⁻¹) and spleen (41.87 ± 4.41% ID g⁻¹) uptake of Fe₃O₄-Ag¹²⁵I nanoparticles was observed at 1 h post-injection (Table S1†). These results suggest that such heterostructured radionuclide nanoparticles show potential capability in tumor imaging of mice. Besides, the heterostructured nanoparticles demonstrate a new type of dual-modality probe for MRI/SPECT imaging through compromising their capabilities in high spatially-resolved MRI and sensitive SPECT imaging from Fe₃O₄ nanoparticles and ¹²⁵I-radiolabelled nanoparticles, respectively. More specific research studies on targeting and combination of other therapies are under investigation.

Conclusions

In conclusion, we have developed Fe₃O₄-Ag¹²⁵I heterostructured radionuclide nanoparticles as novel dual-modality imaging agents for MRI and SPECT. Combining Fe₃O₄-Ag nanoparticles with radioisotopes (¹²⁵I) may provide new synthetic routes for dual-modality contrast agents. The Fe₃O₄-Ag¹²⁵I heterostructured radionuclide nanoparticles showed low cell toxicity, clearly reduced T₂-MRI signal intensity and high radiolabeling efficiency. Furthermore in vivo SPECT images of mice showed strong uptake of this nanomaterial by liver and spleen. We anticipate that these heterodimer radionuclide nanoparticles will have great potential in biological and medical imaging.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 21373006, 51402203, 21207164), the National Basic Research Program of China (973 Program 2014CB931900), the Natural Science Foundation of Jiangsu Province for Young Scholars (BK20140326), the Natural Science Foundation of Jiangsu Higher Education Institutions (14KJB430021), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Application Fundamental Research Program of Suzhou City (SYS201224). We thank Prof. Chunying Chen at the National Center for Nanoscience and Technology (NCNST) China for helpful discussions. We also thank Prof. Kai Yang and Ms Xiaoyan Zhong for radionuclide assistance.

Notes and references

1. C. Xu and S. Sun, Adv. Drug Delivery Rev., 2013, 65, 732; G. Hadjipanayis, J. Bonder, S. Balakrishnan, X. Wang, H. Mao and C. Hadjipanayis, Small, 2008, 4, 1925; J. Zeng, L. Jing, Y. Hou, M. Jiao, R. Qiao, Q. Jia, C. Liu, F. Fang, H. Lei and M. Gao, Adv. Mater., 2014, 26, 2694; Y. Li, Y. Lu, H. Hong, Y. Chen, X. Ma, L. Guo, Z. Wang, J. Chen, M. Zhu, J. Ni, H. Gu, J. Lu and J. Ying, Chem. Commun., 2011, 47, 6320; Y. Pan, X. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012, 41, 2912; Z. Zhao, Z. Zhou, J. Bao, Z. Wang, J. Hu, X. Chi, K. Ni, R. Wang, X. Chen, Z. Chen and J. Gao, Nat. Commun., 2013, 4, 2266; A. Chrastina and J. Schnitzer, Int. J. Nanomed., 2010, 5, 653.
2. M. Perrier, M. Busson, G. Massasso, J. Long, V. Boudousq, J. Pouget, S. Peyrottes, C. Perigaud, C. Porredon-Guarch, J. de Lapuente, M. Borras, J. Larionova and Y. Guari, Nanoscale, 2014, 6, 13425.
3. D. Huo, J. He, H. Li, H. Yu, T. Shi, Y. Feng, Z. Zhou and Y. Hu, Colloids Surf., B, 2014, 117, 29; K. Ai, Y. Liu, J. Liu, Q. Yuan, Y. He and L. Lu, Adv. Mater., 2011, 23, 4886.
4. L. Cheng, J. Liu, X. Gu, H. Gong, X. Shi, T. Liu, C. Wang, X. Wang, G. Liu, H. Xing, W. Bu, B. Sun and Z. Liu, Adv. Mater., 2014, 26, 1886; Z. Li, S. Yin, L. Cheng, K. Yang, Y. Li and Z. Liu, Adv. Funct. Mater., 2014, 24, 2312.
5. Z. Zhang, J. Wang and C. Chen, Adv. Mater., 2013, 25, 3869.
6. A. Louie, Chem. Rev., 2010, 110, 3146; N. Bertrand, J. Wu, X. Xu, N. Kamaly and C. Farokhzad, Adv. Drug Delivery Rev., 2014, 66, 2; N. A. Frey, S. Peng, K. Cheng and S. Sun, Chem. Soc. Rev., 2009, 38, 2532; D. Ho, X. Sun and S. Sun, Acc. Chem. Res., 2011, 44, 875.
7. J. Zhu, Y. Lu, Y. Li, J. Jiang, L. Cheng, Z. Liu, L. Guo, Y. Pan and H. Gu, Nanoscale, 2014, 6, 199.
8. L. Zhou, X. Zheng, Z. Gu, W. Yin, X. Zhang, L. Ruan, Y. Yang, Z. Hu and Y. Zhao, Biomaterials, 2014, 35, 7666; X. Cui, S. Belo, D. Krüger, Y. Yan, R. Rosales, M. Jauregui-Osoro, H. Ye, S. Su, D. Mathe, N. Kovács, I. Horváth, M. Semjeni, K. Sunassee, K. Szigeti, M. Green and P. Blower, Biomaterials, 2014, 35, 5840; R. Sharma, Y. Xu, S. Kim, M. Schueller, D. Alexoff, S. Smith, W. Wang and D. Schlyer, Nanoscale, 2013, 5, 7476; L. Wang, H. Xing, S. Zhang, Q. Ren, L. Pan, K. Zhang, W. Bu, X. Zheng, L. Zhou, W. Peng, Y. Hua and J. Shi, Biomaterials, 2013, 34, 3390; J. Gao, G. Liang, B. Zhang, Y. Kuang, X. Zhang and B. Xu, J. Am. Chem. Soc., 2007, 129, 142; K. Dong, Z. Liu, J. Liu, S. Huang, Z. Li, Q. Yuan, J. Ren and X. Qu, Nanoscale, 2014, 6, 2211.
9. M. Suchy, R. Bartha and R. Hudson, RSC Adv., 2013, 3, 3249.
10. C. Hadjipanayis, M. Bonder, S. Balakrishnan, X. Wang, H. Mao, S. Gambhir, A. Ahmad, I. Uddin, V. Dube and S. Kumar, J. Nucl. Med., 2011, 52, 1613.
11. Y. Shao, S. Cherry, K. Farahani, K. Meadors, S. Siegel, R. Silverman and P. Marsden, Phys. Med. Biol., 1997, 42, 1965; M. Judenhofer, H. Wehrl, D. Newport, C. Catana, S. Siegel, M. Becker, A. Thielscher, M. Kneilling, M. Lichy, M. Eichner, K. Klingel, G. Reischl, S. Widmaier, M. Rocken, R. Nutt, H. Machulla, K. Uludag, S. Cherry, C. Claussen and B. Pichler, Nat. Med., 2008, 14, 459; H. Herzog, Z. Med. Phys., 2012, 22, 281.
12. J. Jiang, H. Gu, H. Shao, E. Devlin, G. Papaefthymiou and J. Ying, Adv. Mater., 2008, 20, 4403.
13. J. Park, K. An, Y. Hwang, J. Park, H. Noh, J. Kim, J. Park, N. Hwang and T. Hyeon, Nat. Mater., 2004, 3, 891.
14. L. Cheng, K. Yang, Y. Li, J. Chen, C. Wang, M. Shao, S. Lee and Z. Liu, Angew. Chem., Int. Ed., 2011, 50, 7385.
15. M. Wang, C. Wang, K. Young, L. Hao, M. Medved, T. Rajh, H. Fry, L. Zhu, G. Karczmar, C. Watson, J. Jiang, N. Markovic and V. Stamenkovic, Chem. Mater., 2012, 24, 2423.

---

Footnotes

† Electronic supplementary information (ESI) available: Details of general experimental procedures, TEM image. See DOI: 10.1039/c4nr07255c

‡ These authors contributed equally.

---