Engineering of Radiolabeled Iron Oxide Nanoparticles for Dual-Modality Imaging

Abstract

Over the last decade, radiolabeled iron oxide nanoparticles have been developed as promising contrast agents for dual-modality positron emission tomography/magnetic resonance imaging (PET/MRI) or single-photon emission computed tomography/magnetic resonance imaging (SPECT/MRI). The combination of PET (or SPECT) with MRI can offer synergistic advantages for non-invasive, sensitive, high-resolution, and quantitative imaging, which is suitable for early detection of various diseases such as cancer. Here, we summarize the recent advances on radiolabeled iron oxide nanoparticles for dual-modality imaging, through the use of a variety of PET (and SPECT) isotopes by using both chelator-based and chelator-free radiolabeling techniques.

INTRODUCTION

Molecular imaging, “the visualization, characterization and measurement of biological processes at the molecular and cellular levels in humans and other living systems”,¹ have enabled the visualization of specific molecular events in disease processes and have made great progress in modern diagnostics.^{2, 3} In general, molecular imaging modalities include optical bioluminescence (or optical fluorescence), ultrasound, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), single-photon emission computed tomography (SPECT), and positron emission tomography (PET).^4–6 MRI and PET are two of the most important imaging modalities that are used in daily clinical disease diagnosis. MRI is well-known for providing unmatched soft tissue details, however, suffers from relatively low sensitivity.⁷ The radionuclide-based SPECT and PET imaging are highly sensitive and quantitative nuclear imaging technologies, which share the same limitations of low spatial resolution. Modern PET and SPECT scanners all come with computed tomography (CT), where functional imaging obtained by PET and SPECT (which depicts the spatial distribution of metabolic or biochemical activity in the body) can be more precisely aligned or correlated with anatomic imaging obtained by CT scanning. PET/MRI is a raising hybrid imaging technology that incorporates MRI soft tissue morphological imaging and PET functional imaging (which can not be achieved by using PET or PET/CT alone) and is believed to play a vital role in clinical fields, such as oncology, cardiology, and neurology.^{8, 9} The first MRI-compatible PET system was reported in 2008 by using lutetium oxyorthosilicate scintillation crystals and avalanche photodiodes as PET detector.¹⁰ Unlike the conventional PET/CT where imaging information is acquired sequentially, PET/MRI can be performed simultaneous, leading to greatly improve the diagnostic potential.¹¹ Readers are referred to excellent reviewer articles in regards to PET/MRI system design.^12–15

Radiolabeled iron oxide nanoparticles have attracted great attention recently due to their ability to act both as MRI contrast agent and PET (or SPECT) imaging tracer, making them well-suited probes for dual-modality tumor imaging.¹⁶ Herein, we introduce recent advances in the engineering of radiolabeled iron oxide nanoparticles for PET/MRI and SPECT/MRI dual-modality imaging. A wide range of PET and SPECT isotopes and their radiolabeling techniques will be discussed.

CATEGORIES OF RADIOLABELED IRON OXIDE NANOPARTICLES

Iron oxide nanoparticle (IONP) is a T₂-weighted MRI contrast agent, which can shorten the T₂ relaxation time of water.¹⁷ The last decade has witnessed great advances of engineering of various kinds of magnetic iron oxide nanoparticles for MR imaging.^{18, 19} For example, cube-shaped iron oxide nanoparticles (IONPs) with a particle size of ~22 nm have been developed and showed an extremely high r₂ relaxivity (>700 mM⁻¹s⁻¹).²⁰ Besides, decorating of IONPs over other nanoplatforms, such as silica or polymers, has been considered to be an alternative method to improve the r₂ value.^{21, 22} Large-scale synthesis of uniform and extremely small-sized (<4 nm) iron oxide nanoparticles has also been reported for high-resolution T₁-weighted MR imaging.²³ Novel contrast agents for both nuclear and MR imaging can be achieved by labeling a variety of SPECT isotopes (such as ^99mTc, ¹²³I or ¹²⁵I, ¹¹¹In, etc.), and PET isotopes (such as ¹⁸F, ¹¹C, ⁶⁴Cu, ⁶⁸Ga, ⁶⁹Ge, ⁸⁹Zr, ⁷²As, etc.) to water-soluble iron oxide nanoparticles.

Chelator-based Synthesis of Radiolabeled Iron Oxide Nanoparticles

The most widely used radiolabeling strategy involves the use of exogenous chelators which could coordinate with certain radioisotopes to form stable complexes.^{24, 25} Different isotopes vary significantly in their coordination chemistry, making selection of the right chelator for a specific isotope vital. In this section, radiolabeled IONPs synthesized with the assistance of various kinds of chelators will be discussed.

^99mTc-labeled Iron Oxide Nanoparticles

The most commonly used radionuclide in SPECT is Technetium-99m (^99mTc, t_1/2=6 h).²⁰ Successful labeling of ^99mTc to nanoparticles is based on the fact that the reduced ^99mTcO₄⁻ (SnCl₂ is usually the reducing agent) can react with an electron donor group, such as the group –COO⁻ from diethylene triamine pentaacetic acid (DTPA) and 1,4,7-triazacyclononane-triacetic acid (NOTA), or the group –NH₂ from chitosan, to form a ^99mTc-chelate.²⁶ For example, Madru et al. prepared ^99mTc-labeled IONPs for multimodality SPECT/MRI imaging of sentinel lymph nodes.²⁷ The labeling method described in this work was simple and straightforward. When the oxidation state of ^99mTcO₄⁻ is reduced with a stannous chloride solution, ^99mTc binds to the functionalized polyethylene glycol (PEG) coating from the IONP surface. The radiolabeling yield was found to be 99% with high radiostability in both sterile water and human serum. ^99mTc-IONPs uptake in the SLN was found to be more than 200 %ID/g, whereas it was less than 2 %ID/g in liver and spleen. Results further indicated that ^99mTc-IONPs can be detected with both imaging techniques, and can act as multimodality contrast agents for sentinel lymph node mapping. ^99mTc-labeled NOTA-IONPs polymer-shelled microbubbles (MBs) and DTPA-IONPs MBs have also been developed for monitoring the distribution and clearance of nanoparticles in vivo.²⁶

A new class of dual-modality imaging agents based on the conjugation of radiolabeled bisphosphonates (BP) directly to the surface of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles have also been reported.^{28, 29} For example, researchers have prepared ^99mTc-PEG-BP-USPIO for T₁-weighted MRI-SPECT multimodal imaging (Figure 1A).²⁹ In vitro MRI studies showed that as-designed nanoparticles have a high r₁ with a low r₂/r₁ ratio of 9.5 mM⁻¹ s⁻¹ and 2.97, respectively. When compared with non-functionalized USPIO, the new contrast agent showed a similar signal enhancement by using four times lower dose. The nanoparticles also showed long blood circulation time (t_1/2=2.97 h), allowing the visualization of blood vessels and vascular organs with high spatial definition (Figure 1B). ^99mTc-labeled and Bevacizumab monoclonal antibody (mAb) conjugated USPIO (^99mTc-USPIO-bevacizumab) was also synthesized for targeted imaging of hepatocellular carcinoma.³⁰ Although therapeutic applications of radiolabeled iron oxide nanoparticle is not the main focus of this article, reports on engineering of these nanoparticles for combined magnetic hyperthermia (or radiation therapy) have shown their potential as novel theranostic nanoagents for both multimodality imaging and therapy.^31–33

Figure 1.

(A) An illustration of SPECT/MRI multimodality imaging using ^99mTc-PEG-BP-USPIO. (B) In vivo T₁-weighted MR imaging study on vessels (upper) and heart (down) of mice after injected with PEG(5)-BP-USPIO (Labels: H = heart, S = spleen, K = kidney, A =aorta, M = myocardium, LV = left ventricle. Reprinted with permission from [²⁹].

¹¹¹In- and ¹²⁵I-labeled Iron Oxide Nanoparticles

Indium-111 (¹¹¹In, t_1/2=2.8 d) is another radionuclide in clinical nuclear medicine for its reasonably long half-life. Misri et al. developed a dual-modality molecular imaging probe by conjugating ¹¹¹In-labeled antimesothelin antibody mAbMB (i.e.¹¹¹In-mAbMB) to IONPs.³⁴ DTPA was used the chelator for ¹¹¹In labeling. IONPs were coated with carboxy methyl dextran, providing carboxylic acid groups for the ¹¹¹In-mAbMB antibody conjugation. In vivo SPECT and MRI dual-modality imaging was carried out on A431K5 tumor-bearing mice. Specific uptake of ¹¹¹In-mAbMB-IONPs in A431K5 tumor (mesothelin-positive tumor model, 4.8 %ID/g) was found significantly higher than the non-specific accumulation in A431 tumor (mesothelin-negative tumor model, 2.7 %ID/g). Although a much higher uptake in spleen (up to 68 %ID/g) was observed in the study, the combination of SPECT with MRI holds the potential to obtain both functional and anatomical imaging information with high signal sensitivity and contrast, thereby providing a powerful diagnostic tool for early diagnosis and treatment planning of mesothelin-expressing cancers in the future.

In another study, ¹¹¹In-labeled, and chimeric L6 (ChL6), a human–mouse mAb chimera, conjugated IONPs were developed for pharmacokinetics, tumor active targeting, and alternating magnetic frequency (AMF) therapy studies.³⁵ Tumor uptake was detected to be about 14 %ID/g at 48 h post injection. External AFM was applied on the injected mice and magnetic hyperthermia tumor toxicity studies were carried out. Results showed tumor growth delay in all groups, except for the group with the lowest heat dose. Electron microscopy further confirmed the presence of necrosis after AMF treatment. This was one of the few reports that showed the potential of using radiolabeled INOPs for tumor targeted thermal therapy. IONPs labeled with ¹¹¹In, iron-59 (⁵⁹Fe, t_1/2=49.5 d) and carbon-14 (¹⁴C, t_1/2=5700 y) have also been reported for evaluating the in vivo integrity of radiolabeled IONPs.³⁶

Radioisotopes of iodine have been extensively used in clinical nuclear medicine imaging and radiation therapy. There are 37 known isotopes of iodine from ¹⁰⁸I to ¹⁴⁴I, and four of those, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I, are suitable for SPECT or PET imaging. Tang and co-workers synthesized a SPECT/MRI/optical trimodality probe by labeling fluorescent silica coated IONPs with iodine-125.^{37 125}I-labeling was achieved by the Iodogen oxidation method. This novel probe was used to label mesenchymal stem cells (MSCs) and quantitatively track their migration and biodistribution in ischemic rats. As-developed nanoprobes showed high labeling efficiency and could allow in vivo tracking of labeled MSC with high spatial resolution and anatomical localization through SPECT and MRI imaging. The long half-life (59 d) of ¹²⁵I also enabled a long-term tracking and imaging of the labeled cells. No detection limitation was reported in this study.

⁶⁴Cu- and ⁶⁸Ga-labeled Iron Oxide Nanoparticles

When compared with SPECT imaging, PET imaging may offer increased accuracy, higher sensitivity and better resolution.³⁸ Hybrid imaging of high-resolution anatomical MRI and PET might offer an even better solution for future early cancer diagnosis. Copper-64 (⁶⁴Cu, t_1/2=12.7 h) is a positron emitter with a reasonably long half-life and well-established radiolabeling techniques. Chelators, such as DTPA, NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and bis-dithiocarbamatebisphosphonate (DTCPB) have been used for the radiolabeling of ⁶⁴Cu to INOPs for PET/MRI dual-modality imaging.^{39, 40}

For example, a novel amine-activated chelator (amine-Bz-DOTA) was developed and conjugated to the surface of dextran sulfate coated IONPs for ⁶⁴Cu radiolabeling.⁴⁰ The new labeling procedure was able to avoid the cross-link of IONPs (which caused nanoparticles aggregation) and enabled a higher labelling yield. By using NOTA as the chelator, we also developed a ⁶⁴Cu-labeled, cRGD-functionalized, and therapeutic drug doxorubicin (DOX)-conjugated IONPs for drug delivery and PET/MRI imaging (Figure 2A, B).⁴¹ Enhanced and specific accumulation of cRGD-conjugated IONPs in U87MG tumor-bearing mice was demonstrated by using PET imaging (Figure 2C). In a similar study, Lee et al. developed a RGD-conjugated and ⁶⁴Cu-labeled iron oxide nanoparticles for PET/MRI dual-modality tumor imaging.⁴² Both small-animal PET and T₂-weighted MR imaging show integrin-specific delivery of RGD-conjugated IONPs, together with prominent reticuloendothelial system uptake due to the large particle size (>40 nm). Quantitative PET imaging and region of interest analysis showed about 10.1 %ID/g in mice injected with ⁶⁴Cu-DOTA-IONPs-RGD conjugates at 4 h post injection, while tumor uptakes of the non-targeted and blocking groups were only 4 %ID/g and 3 %ID/g, respectively.

Figure 2.

(A) A schematic illustration of the ⁶⁴Cu-NOTA-IONP(DOX)-cRGD nanoconjugates for combined tumor-targeted drug delivery and PET/MRI imaging. (B) A TEM image of IONP(DOX)-cRGD. (C) In vivo PET images of U87MG tumor-bearing mice 24 h after injection of different nanoconjugates. From left to right: targeted group (⁶⁴Cu-NOTA-IONP-cRGD), non-targeted group (⁶⁴Cu-NOTA-IONP) and blocking group. Tumors were marked by yellow arrow-heads. Reprinted with permission from [⁴¹].

A more complex hetero-nanostructure with two different functional nanomaterials (i.e. gold and iron oxide) within one structure has been used as the platform for radiolabeling and targeted trimodality (PET/MRI/optical) imaging (Figure 3A).⁴³ As-synthesized hybrid nanoparticles have a dumbbell shape (Figure 3B, C), and can be further modified with PEG and chelators for prolonged blood circulation time and radiolabeling. Both in vivo PET and MRI demonstrated the specific targeting of anti-EGFR Affibody protein conjugated and ⁶⁴Cu-labeled hybrid nanoparticle, denoted as ⁶⁴Cu-NOTA-Au-IONP-Affibody (Figure 3D, E).

Figure 3.

(A) A schematic illustration of Au-IONP-Affibody. (B) A TEM image of Au-IONP. (C) A high-angle annular dark field image of Au-IONP. The Au nanoparticles were shown as the bright dots. (D) In vivo PET imaging of A431 tumor-bearing mice acquired 24 h after the injection of ⁶⁴Cu-NOTA-Au-IONP-Affibody. From left to right: targeted group and blocking group. (E) In vivo MR imaging of A431 tumor-bearing mice acquired 24 h after the injection of ⁶⁴Cu-NOTA-Au-IONP-Affibody. Tumors were marked by yellow arrow-heads. Reprinted with permission from [⁴³].

Gallium-68 (⁶⁸Ga, t_1/2= 68 min) can be easily synthesized using ⁶⁸Ge/⁶⁸Ga generators. Kim et al. reported a ⁶⁸Ga radiolabeled tumor-targeting IONPs, using NOTA as the radiolabeling chelator.⁴⁴ The authors used oleanolic acid (OA), a tumor-targeting molecule, to modify the IONPs, and then coupled it with NOTA for ⁶⁸Ga radiolabeling. The ⁶⁸Ga-NOTA-OA-IONPs were intravenously injected into HT29 tumor-bearing mice for in vivo PET/MRI imaging. The tumor uptake of ⁶⁸Ga-NOTA-OA-IONPs was found to be about 3 %ID/g. No non-targeted and blocking studies were provided to demonstrate the targeting specificity of ⁶⁸Ga-NOTA-OA-IONPs.

Although chelator-based radiolabeling techniques have been used for decades, concerns about the complexity of coordination chemistry, possible altering of pharmacokinetics of carriers, and potential detachment of radioisotopes during imaging have driven the need for developing a simpler yet better technique for future radiolabeling. There is an emerging concept of intrinsically radiolabeled nanoparticles, which can be synthesized using methods such as hot-plus-cold precursors, specific trapping, cation exchange and proton beam activation.^{45, 46} In the next section, we will discuss radiolabeled IONPs using chelator-free method.

Chelator-free Synthesis of Radiolabeled Iron Oxide Nanoparticles

¹⁸F- and ¹¹C-labeled Iron Oxide Nanoparticles

Fluorine-18 (¹⁸F, t_1/2=109 min) is a widely available PET isotope. Devaraj et al. reported an ¹⁸F-radiolabeled Vivotag-680 functionalized IONPs for multimodality imaging.⁴⁷ IONPs were coated with aminated cross-linked dextran, which was functionalized first with near-infrared fluorochrome Vivotag-680. After that, ¹⁸F-PEG was conjugated using copper-catalyzed azide-alkyne click chemistry, forming a trimodal nanoparticle (¹⁸F-CLIO) that is suitable for multimodality imaging (PET, fluorescence and MRI). The radiochemical purity of ¹⁸F-CLIO was detected to be >99% according to high-performance liquid chromatography analysis. Results also showed that the detection threshold of ¹⁸F-CLIO for PET imaging was 200 times more sensitive than MRI. In vivo dynamic PET imaging showed high signal-to-noise ratios. Furthermore, ¹⁸F-CLIO presented a vascular half-life of 5.8 h in mice and subsequent internalization into liver, spleen and phagocytic cells of other lymphatic organs. Another interesting chelator-free labeling method was reported by Cui and co-workers, who labeled ¹⁸F to Fe₃O₄@Al(OH)₃ by taking advantages of the high affinity between Al(OH)₃ and fluoride anions.⁴⁸

Carbon-11(¹¹C, t_1/2=20.3 min) is another non-metal isotope with a relatively short half-life that can be used for making ¹¹C-labeled IONPs.⁴⁹ In a study reported by Ramesh Sharma and co-workers, [¹¹C]CH₃I was used to react with carboxylic acid (–COOH) or amine (–NH₂) functional groups modified IONPs. Although the radiolabeling yield was lower than 3%, ¹¹C-labeled IONPs had sufficient radioactivity to perform PET imaging for short-term dynamics and biodistribution studies. The low radiolabeling yield was primarily due to INOPs agglomeration and low carboxylic acid or amine functional ligand density on the surface of nanoparticles. In vivo dual-modality PET/MRI of mouse showed an excellent correlation between PET and MRI data for the distributions of ¹¹C-labeled IONPs.

*As- and ⁶⁹Ge-labeled Iron Oxide Nanoparticles

Arsenic (As) has 4 positron emitting (^70/71/72/74As) and 3 electron emitting (^74/76/77As) radioisotopes with half-lives ranging from 52.6 min to 17.8 days, which could be useful for both PET and internal radiotherapy applications.⁵⁰ However, few techniques are currently available for incorporation of these radionuclides into biologically relevant targeting vectors. Inspired by an ancient groundwater decontamination process, where both As^III and As^V can be incorporated by magnetite or IONPs,^51–54 we demonstrated a simple but highly efficient strategy for the synthesis of radioarsenic-labeled IONPs (i.e. *As-IONP, *=71, 72, 74, 76) without the use of any chelators (Figure 4A). The underlying mechanism of arsenic trapping by IONP involves the formation of highly stable arsenic complexes, where As^IIIO₃ trigonal pyramids or As^VO₄ tetrahedra occupy vacant FeO₄ tetrahedral sites on the octahedrally terminated (111) surface of the magnetite nanoparticles.⁵⁵ Oleic acid capped IONPs were first synthesized and transfer to water phase by modifying with poly(acrylic acid) (PAA) (Figure 4B, C). The labeling of *As (*= 71, 72, 74, 76) to IONPs was later demonstrated to be fast, iron concentration dependent, and highly specific. Although the in vivo stability of *As-IONPs still needs to be improved, the PEGylated *As-IONPs showed improved serum stability and less bladder uptake in vivo. PET/MRI dual-modality lymph node mapping using *As-IONPs@PEG was also demonstrated in vivo (Figure 4D, E). Germanium-69, (⁶⁹Ge, t_1/2= 39.05 h) is another novel potential PET radioisotope, whose in vivo applications are hampered by its complex coordination chemistry in aqueous medium. To circumvent this challenge, we also exploited the high affinity of germanium for metal oxides to develop the first chelator-free ⁶⁹Ge-labeled IONPs based agent for PET/MRI lymph node mapping.⁵⁶

Figure 4.

(A) A schematic illustration of chelator-free synthesis of *As (or ⁶⁹Ge)-IONPPAA-PEG. (B) A TEM image of oleic acid capped IONPs. (C) A TEM image of PAA modified IONPs. (D) In vivo PET imaging of lymph nodes after the injection of *As-IONPPAA-PEG. (D) In vivo MR imaging of lymph nodes after the injection of *As-IONPPAA-PEG. Reprinted with permission from [⁶⁷].

64Cu- and ⁸⁹Zr-labeled Iron Oxide Nanoparticles

Besides labeling *As and ⁶⁹Ge to IONPs using specific trapping strategy, intrinsically ¹¹¹In-, ⁶⁴Cu-, iron-59 (⁵⁹Fe, t_1/2=44.5 d) labeled IONPs could also be synthesized by using hot-plus-cold precursors technique.^57–59 Recently, we further developed MoS₂-IONP nanosheets for ⁶⁴Cu chelator-free radiolabeling and multimodality image-guided photothermal therapy (PTT) (Figure 5A).⁶⁰ MoS₂-IONPs were prepared by self-assembling of IONPs on the surface of MoS₂ nanosheets via sulfur chemistry, and were PEGylated for improved in vivo stability (Figure 5B). ⁶⁴Cu could be easily labeled to the MoS₂-IONPs by taking advantages of the high affinity between ⁶⁴Cu²⁺ ions and sulfur atoms. Labeling yield was measured to be 85% at optimal experimental condition. PET imaging of as-developed ⁶⁴Cu-MoS₂-IONPs in 4T1 tumor-bearing mice showed about 6 %ID/g passive targeting efficacy (Figure 5C). In vivo MR imaging further confirmed the accumulation of nanoparticles in the tumor site (Figure 5D). We also demonstrated effective image-guided PTT by exposing the MoS₂-IONPs injected mice to an 808 nm laser. Enhanced PTT effect is also expected by further conjugating ⁶⁴Cu-MoS₂-IONPs with targeting ligands in follow-up studies.

Figure 5.

(A) A schematic illustration of using ⁶⁴Cu-MoS₂-IONPs for multimodality image-guided photothermal therapy. (B) A TEM image of PEGylated MoS₂-IONPs. (C) PET imaging of 4T1 tumor-bearing mice after the injection of ⁶⁴Cu-MoS₂-IONPs. (D) MR imaging of 4T1 tumor-bearing mice after the injection of ⁶⁴Cu-MoS₂-IONPs. Reprinted with permission from [⁶⁰].

Zirconium-89 (⁸⁹Zr, t_1/2=78.4 h) is a radioisotope with a relatively low positron energy (β⁺_avg=395.5 keV), making it highly suitable for long blood circulating monoclonal antibody-based PET imaging.⁶¹ Desferrioxamine (DFO), a hexadentate ligand with three hydroxamate groups that provide six oxygen donors for metal binding, is currently the preferred chelator for labeling of ⁸⁹Zr.⁶² For example, ⁸⁹Zr-DFO-ferumoxytol was developed for PET/MRI mapping of deep-tissue lymph nodes in live animals.⁶³ Recently, a chelater-free iron bonding and heat-induced radiolabeling of IONPs was also developed.⁶⁴ Holland and co-workers demonstrated that ferumoxytol could be labeled with the ⁸⁹Zr, ⁶⁴Cu and ¹¹¹In under the similar general reaction conditions (i.e. 120 °C under pH 8) without using any chelates.⁶⁴ In vivo pharmacokinetics and distribution of ⁸⁹Zr-ferumoxytol nanoparticles were preformed using PET/CT imaging, and showed the circulating of radiolabeled nanoparticles in the blood during as well as high liver and spleen uptake in the mice. As-developed labeling strategy might also apply to other metal or non-metal oxide nanoparticles.

SUMMARY AND OUTLOOK

In conclusion, radiolabeled IONPs have emerged as a novel dual-modality contrast agents which already shown their potential for providing non-invasive, high-resolution and quantitative imaging results. Table 1 further provides a collection of representative radiolabeled IONPs via different radiolabeling methods. Despite the progress that has been made in the last decade, challenges still exist for engineering of radiolabeled IONPs for future dual-modality imaging and clinical translation.

Table 1.

Representative examples of radiolabeled iron oxide nanoparticles

Radioisotopes	Half-life	Nanoparticles	Radiolabeling methods	Chelators	Applications	References
99mTc	6 h	IONPs-PEG	Chelator-free	N/A	SPECT/MRI	27
		IONPs-Poly(vinyl alcohol)	Chelator-based	DTPA, NOTA	SPECT/MRI	26
		USPIO	Chelator-based	Bisphosphonates	SPECT/MRI	28, 29
111In	67.2 h	IONPs-dextran	Chelator-based	DTPA	SPECT/MRI	34
		IONPs-PEG	Chelator-free	N/A	N/A	57
		Ferumoxytol	Chelator-free	N/A	N/A	64
125I	59 d	IONPs@SiO2	Iodogen oxidization method	N/A	SPECT/MRI/Optical	37
68Ga	68 min	INOPs	Chelator-based	NOTA	PET/MRI	44
18F	109.8 min	Aminated cross-linked dextran IONPs	Click chemistry	N/A	PET/MRI	47
11C	20.3 min	IONPs-NH2 or IONPs-COOH	Methylation reactions	N/A	PET/MRI	49
64Cu	12.7 h	IONPs-PEG	Chelator-based	DOTA	PET/MRI	39
		IONPs-dextran	Chelator-based	DOTA	N/A	40
		IONPs-PEG	Chelator-based	NOTA	PET/MRI	41
		MoS2-IONPs	Chelator-free	N/A	PET/MRI	60
		Ferumoxytol	Chelator-free	N/A	N/A	64
89Zr	3.3 d	Ferumoxytol	Chelator-based	DFO	PET/MRI	63
		Ferumoxytol	Chelator-free	N/A	PET/CT	64
69Ge	69 h	IONPs@PAA	Chelator-free	N/A	PET/MRI	68
72As	26 h	IONPs@PAA	Chelator-free	N/A	PET/MRI	67

Firstly, most of radiolabeled IONPs reviewed in this article have hydrodynamic size range of 10 to 200 nm, which caused high and retained accumulation in the reticuloendothelial system (RES) organs. Considering the fact that the Food and Drug Administration requires all injected contrast agents to be cleared completely within a reasonable period,^{65, 66} engineering of radiolabeled IONPs that can be cleared by the renal system will be one of the next major focuses. Secondly, specific delivery of the radiolabeled IONPs to tumor site is critical for dual-modality imaging. Engineering of tumor actively targeted radiolabeled IONPs is still in its early stage with only a few examples being reported. Thirdly, due to the lack of accessibility to the PET/MRI scanners, most of current PET and MRI images were acquired separately. The true advantages of simultaneously PET/MRI in early cancer diagnosis using radiolabeled IONPs are believed to be further revealed in the near future.