I-124 Labeled Rhein-coated gold nanoparticles for targeted breast cancer

Abstract

Rhein, an anthraquinone derivative, and gold nanoparticles have demonstrated significant potential in facilitating diagnostic applications and for possible theranostic use in breast cancer research. Rhein is known to interact with estrogen-α (ERα), which is relevant in ERα-positive breast cancer models. In addition, gold nanoparticles, when used as nanocarriers, may support imaging performance and delivery, although therapeutic effects require further validation. This study outlines the preparation of [¹²⁴I]rhein-gold for position emission tomography (PET) imaging in an ERα-positive breast cancer mouse model. The radiochemical yield of [¹²⁴I]RCGs was 65% ± 8.2%, with a radiochemical purity of >98%. In the evaluation of cellular uptake, a high uptake rate exceeding 25% was observed within 15 minutes. Corresponding results indicated a significantly elevated initial uptake rate, with a standardized uptake value mean (SUV_mean) greater than 6 in small animal PET imaging. This study demonstrated the synthesis and biological evaluation of [¹²⁴I]RCGs. [¹²⁴I]RCGs showed high cellular uptake in a time-dependent manner. PET images of [¹²⁴I]RCGs confirmed preferential uptake in ERα-positive MCF-7 tumors from 15 min to 240 min. These results suggest that [¹²⁴I]RCGs are promising radiopharmaceuticals, demonstrating potential as experimental PET imaging agents associated with ERα-positive breast cancer, while targeting should be considered implicit without blocking or control data.

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1. Introduction

In recent years, the incidence of breast cancer patients has increased globally. Approximately one-third of individuals diagnosed with early-stage breast cancer will eventually develop metastatic disease.¹ Consequently, early diagnosis is crucial for preventing the metastasis of cancer to other organs. However, the early detection of breast cancer presents significant challenges owing to the asymptomatic nature of the disease.^2,3 To address this issue, prior research has focused on the identification of overexpressed receptors in breast cancer to aid early diagnosis.^4,5 Rhein, an anthraquinone derivative, has been noted to suppress various human cancer cell types, including those in the breast, colon, and lung.^6–8 The estrogen receptor (ER) is expressed in approximately 75% of breast cancer patients who are ER-positive and whose tumor growth is driven by endogenous estrogens.^9–11 The level of ER expression is a critical factor influencing the proliferation of breast cancer.¹² Studies have examined the expression of estrogen-α (ERα) in MCF-7 cells, as well as the absence of functional ERα in SK-BR-3 cells.^13,14 Rhein, derived from Rheum palmatum L., Cassia tora L., and Polygonum multiflorum Thunb, exhibits potent antioxidant and anticancer properties.¹⁵ Consequently, there is a growing need for further research on anthraquinone derivatives that can effectively target breast cancer. Gold nanoparticle (GNP)-based nanocarriers have been used in the treatment of various cancers. GNPs smaller than 100 nm are phagocytosed via scavenger receptor-mediated phagocytosis.^16,17 Gold, with its high atomic number (Z = 79), can enhance radiation effects when used with radioisotopes such as Iodine-125 (¹²⁵I) in biological systems.^18,19 Given these properties, numerous researchers have explored the application of GNPs in cancer treatment using radiation sources.²⁰ Gold nanoparticles with a diameter of 30 nm have been reported to enhance Na⁺, K⁺-ATPase activity in a concentration-dependent manner. Furthermore, gold nanoparticles of this size have demonstrated biosafety and biocompatibility in both in vitro and in vivo environments. It has also been reported that the liver toxicity of gold nanoparticles ranging from 10 nm to 60 nm is minimized at a diameter of 30 nm. These characteristics render gold nanoparticles particularly promising for applications in drug delivery, imaging, and therapeutic interventions.^21,22 Considering these aspects, this study was aimed at examining the feasibility of establishing a breast cancer treatment using anthraquinone-derivative-coated gold nanocomposites in conjunction with iodine-124 (¹²⁴I) for positron emission tomography (PET) imaging. The positron-emitting halogen ¹²⁴I has a long half-life (T_1/2 = 4.2 d), rendering it suitable for use as a PET imaging agent. The PET imaging capabilities of ¹²⁴I are particularly beneficial for this study, as it offers specific images with high spatial and contrast resolution.²¹ This report presents the evaluation of ¹²⁴I-labeled anthra-quinone-derivative-immobilized GNPs for ERα-targeted PET imaging.

2. Experimental section

2.1. Materials and equipment

Rhein (4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid), sodium citrate di-hydrate, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES buffer solution), L-cysteine (Cys), chloramine T (N-chloro-p-toluenesulfonamide sodium salt), human serum, and hydrogen tetrachloroaurate(III) hydrate (HAuCl₄·3H₂O) were purchased from Sigma-Aldrich. All solvents were used without further purification. ¹²⁴I was obtained from the Korea Institute of Radiological and Medical Sciences (KIRAMS). All cancer cell lines were acquired from the American Type Culture Collection (ATCC).

Ultraviolet-visible (UV-vis) spectroscopy (Shimadzu UV-1800, Japan) was performed to measure the UV-vis absorbance of GNP, GNP-Cys, and GNP-Cys-Rhein systems. The transmission electron microscope (TEM, Hitachi H-7650) images were obtained at 12 kV for particle size and shape. Hydrodynamic size and ζ-potential measurements were conducted using a Zetasizer (Nano ZS, Malvern Instruments Ltd). Radioactive nanocomposites were characterized using the following equipment: radiochemical purity was assessed through radio-instant thin layer chromatography (Radio-iTLC, ZR-2000, Bioscan). The activity of the nanocomposite was measured using a dose calibrator (CRC-15R, Capintec). In vitro cellular uptake was determined using a gamma counter (1470 WIZARD2, PerkinElmer).

2.2. Preparation of gold nanoparticle (GNP)

30 nm GNPs were synthesized using a modified citrate reduction method. Prior to synthesis, all glassware was treated with aqua regia (3 : 1 HCl/HNO₃) and thoroughly dried. First, 10 mg of HAuCl₄·3H₂O was dissolved in 100 mL of distilled water. Next, 0.9 mL of a 3.5 mM sodium citrate dihydrate solution was rapidly added to the mixture. The solution was refluxed at 100 °C for 15 min, during which the color changed from yellow to red. Finally, the solution was allowed to cool to room temperature.

2.3. Preparation of Rhein-Cys-GNPs (RCGs)

The Rhein-Cys complex was synthesized using the carbodiimide coupling method. First, 0.04 mmol of Rhein was suspended in 700 μL of HEPES buffer solution. Next, 0.031 mmol of EDC and 0.038 mmol of NHS were added as coupling agents to form reactive NHS esters, and the reaction was allowed to proceed for 24 hours at room temperature. Subsequently, 100 μL of a 0.04 mmol L-cysteine solution was slowly added to the solution containing NHS ester-activated Rhein. Finally, the Rhein-Cys complex was introduced into a 30 nm gold colloidal solution, and Rhein-Cys-GNPs were formed through ultrasonic treatment in a bath for 30 minutes. The mixture was then purified using a 1 kDa centrifugal filter to remove unreacted byproducts and impurities. The resulting gold nanocomposites were washed with distilled water, filtered, and dried at 60 °C for 24 hours.

2.4. ¹²⁴I-labeled anthraquinone derivative

Before labeling the RCGs with ¹²⁴I, the labeling efficiency of ¹²⁴I on rhein was evaluated. First, 2 mg of rhein was dissolved in 200 μL of dimethylformamide (DMF), followed by the addition of Na[¹²⁴I] (37 MBq). Next, 5 mg of chloramine was added as an oxidizing agent, and the reaction was allowed to proceed at room temperature for 15 min. After the reaction, the ¹²⁴I-labeled rhein was purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system. A mobile phase consisting of 0.25 M ammonium acetate and methanol (MeOH) in a 3 : 7 ratio was employed, and a C18 reverse-phase chromatography column was used as the stationary phase.

2.5. ¹²⁴I-labeled RCGs

To prepare [¹²⁴I]RCGs, 2 mg of RCGs was resuspended in 200 μL of DMF and allowed to react with Na[¹²⁴I] (37 MBq) and 5 mg of chloramine T as an oxidizing agent. The reaction was allowed to proceed for 15 min at room temperature (27 °C). The radioactivity of free ¹²⁴I and ¹²⁴I-labeled RCGs was measured using a radioisotope dose calibrator. To eliminate free ¹²⁴I, the mixture was purified by centrifugation at 10 000 rpm for 10 min.

2.6. Serum stability of [¹²⁴I]RCGs

To assess the biodegradation of [¹²⁴I]RCGs, their stability in human serum was evaluated at various time points. Specifically, 3.7 MBq of [¹²⁴I]RCGs was incubated with 1.0 mL of human serum at 37 °C for durations ranging from 0.25 to 24 h. The samples were subsequently analyzed using radio-TLC, with the mobile phase consisting of a 3 : 1 mixture of chloroform and methanol. In this study, stability was operationally defined as the percentage of intact [¹²⁴I]RCGs, determined by radio-TLC. The stability (%) for human serum was determined according to the formula:
where the numerator represents the radioactivity corresponding to the intact [¹²⁴I]RCGs band on the TLC plate, and the denominator is the total radioactivity of both intact and free [¹²⁴I]. This study was performed at various time points to evaluate the serum stability of the [¹²⁴I]RCGs.

2.7. Partition coefficient of [¹²⁴I]RCGs

The 1-octanol/phosphate-buffered saline (PBS) partition coefficients (log P) of [¹²⁴I]RCGs were determined using the shake-flask method. A total of 3.7 MBq of [¹²⁴I]RCGs was added to a conical tube containing 500 μL of PBS (pH 7.4) and 500 μL of 1-octanol. The mixture was shaken for 5 min at a constant temperature to allow sufficient distribution between the two phases, and then centrifuged at 10 000 rpm for an additional 5 min. After centrifugation, the radioactivity in 100 μL of each phase was measured using a γ-counter.

2.8. In vitro cell culture and cellular uptake of [¹²⁴I]RCGs

The in vitro binding affinity of [¹²⁴I]RCGs to breast cancer cells was evaluated using human breast cancer cell lines. Human breast cancer cells (MCF-7 and SK-BR-3) were routinely cultured in 100 mm culture plates at 37 °C with 5% CO₂ in a humidified atmosphere. The cells were grown in the RPMI 1640 medium supplemented with 10% fetal bovine serum and 100-fold diluted 1% penicillin (10 000 U mL⁻¹) and streptomycin (10 000 μg mL⁻¹). Cancer cells were detached and dissociated using a 0.05% trypsin/0.02% EDTA solution for cellular uptake studies. Subsequently, the cancer cells were seeded (1 × 10⁵ cells/well) into 24-well culture plates and incubated under standard conditions. [¹²⁴I]RCGs (185 kBq/μg per well) were added to each well to assess the cellular uptake at various time points (15, 30, 60, and 120 min).

2.9. In vivo establishment of MCF-7 tumor model and PET Imaging

Before injecting the MCF-7 cells, an estradiol pellet (0.36 mg, 60 d release) was subcutaneously implanted into the dorsal region of each female athymic nude mouse (6 weeks old, weighing 20–22 g). This procedure provided the necessary estrogen supplementation needed for the growth of ER-positive MCF-7 tumors. After implantation, we waited 1 to 2 d for the pellet to begin releasing estrogen. Then, 100 μL of the prepared cell suspension containing approximately 1 × 10⁶ MCF-7 cells was subcutaneously injected into the right thigh of each mouse for PET imaging. [¹²⁴I]RCGs were injected intravenously at a dose of 3.7 MBq/20 μg per mouse. This experiment was conducted when the tumor size was approximately 100 mm³.

3. Results

3.1. Synthesis and characterization

To achieve a synergistic combination of GNPs, we synthesized ¹²⁴I-labeled anthraquinone-coated gold nanocomposites. The preparation of 30 nm GNPs was accomplished through nucleation and growth processes. The strong interaction between thiol groups and gold served as the foundation for the fabrication of robust self-assembled monolayers for coating the anthraquinone derivatives. In general, ¹²⁴I in the −1 oxidation state occurs through its reaction with chloramine T, an electrophilic species in the +1 oxidation state. This reaction results in the substitution of an activated proton from the aromatic ring of tyrosine at the ortho position relative to the phenol group. The synthesis of the nanocomposites is illustrated in Fig. 1. Various analytical methods, including UV spectroscopy, high-performance liquid chromatography (HPLC), dose calibrator measurements, zeta potential analysis, and TEM, were used to characterize the GNPs, rhein, RCGs, and [¹²⁴I]RCGs.

Fig. 1 Schematic diagram of gold nanocomposites with ¹²⁴I (Created in BioRender. Lee, J. (2024) https://BioRender.com/p54g744).

3.2. UV-vis spectroscopy

The UV-Vis absorption spectra of rhein, 30 nm GNPs, GNP-cysteine, and GNP-Cys-Rhein complexes are presented in Fig. 2. The spectrum of rhein alone exhibits a characteristic absorption peak at approximately 430 nm, corresponding to its intrinsic electronic transitions. In contrast, the 30 nm GNPs show a distinct surface plasmon resonance (SPR) peak centered around 520 nm, which is typical for GNPs of 30 nm size.²²

Fig. 2 UV-Vis absorption spectra and adsorption shift of gold nanocomposites.

Upon conjugation with L-cysteine (GNP-Cys), the SPR peak of GNPs exhibits a slight broadening and shift, indicating successful surface modification with L-cysteine molecules. The spectrum of the GNP-Cys-Rhein complex reveals additional features, including a slight redshift in the SPR peak and an increase in absorbance intensity near 430 nm. These observations suggest that rhein molecules are effectively conjugated to the GNP-Cys surface, resulting in alterations to the optical properties of the nanoparticles.

These results confirm the successful functionalization and conjugation of Rhein onto cysteine-modified gold nanoparticles. The observed spectral changes provide evidence of strong interactions between Rhein and the GNP-Cys surface, likely through coordination or covalent bonding mechanisms.

3.3. Particle size and shape

Cysteine exhibits a notably high affinity for gold due to its sulfhydryl(thiol) side chain and was utilized as a cross-linking agent to attach rhein to gold nanoparticles, As demonstrated in the TEM images, aggregation occurred following the introduction of cysteine to the gold nanoparticles (Fig. 3). Furthermore, it was confirmed that a layer formed on the surface of the gold nanoparticles after the addition of rhein.

Fig. 3 Representative TEM images of (a) 30 nm gold nanoparticles, (b) GNP-Cys and (c) GNP-Cys-rhein.

3.4. Gold nanoparticle functionalization

The functionalization of GNPs was achieved using L-cysteine as an interface. First, rhein was conjugated to L-cysteine through an EDC/NHS coupling reaction. This conjugate was then attached to citrate-stabilized GNPs. As indicated in Table 1, the thickness of the anthraquinone shell was determined to be between 3 and 4 nm through hydrodynamic size measurements of GNPs, GNP-Cys complexes, and GNP-Cys-rhein complexes. Additionally, the surface charge of these entities was found to be negative.

Table 1 Hydrodynamic size and charge of GNPs, GNP-Cys, GNP-Cys-rhein

	Hydrodynamic size (nm)	ζ-potential (mV)
GNPs	35.0	−42.8 ± 4.19
GNP-Cys	37.2	−78.7 ± 11.8
GNP-Cys-rhein	38.4	−48.0 ± 9.14

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3.5. ¹²⁴I-labeled rhein

To improve the labeling technique of ¹²⁴I before its application to rhein coated with GNPs, a synthesis study was conducted involving ¹²⁴I and rhein, employing chloramine T as an oxidizing agent. The labeling yield of ¹²⁴I introduced into rhein was calculated based on the radioactivity of the [¹²⁴I]Rhein fraction obtained after HPLC purification (Fig. 4). The radiochemical yield (RCY) of [¹²⁴I]Rhein was 25–35%, with a radiochemical purity (RCP) exceeding 95%.

Fig. 4 Analysis by HPLC of the iodinated product [¹²⁴I]Rhein.

3.6. Synthesis of ¹²⁴I-labeled RCGs

The RCY achieved using the established procedure was 65% ± 8.2% (n = 3), with an RCP of 98%, as determined by radio-TLC (mobile phase: 85% MeOH). Using the chloramine T method, the successful synthesis of ¹²⁴I-labeled RCGs was accomplished, resulting in an efficient labeling reaction.

3.7. Partition coefficient and in vitro serum stability

The partition coefficient (log P) of the ¹²⁴I-labeled RCGs was assessed in a mixture of 1-octanol and phosphate-buffered saline (pH 7.4). Subsequently, the radioactivity was quantified using a dosimeter, and log P values were calculated. The measured log P value was −0.738 ± 0.098, indicating the hydrophilic nature of the agent (Fig. 5a). The stability of the ¹²⁴I labeled RCGs exceeded 98% at 15 min and 88% after 24 h. Additionally, the ¹²⁴I labeled RCGs demonstrated stability exceeding 90% for up to 4 h (Fig. 5b).

Fig. 5 (a) Determination of lipophilicity of ¹²⁴I labeled RCGs and (b) stability of ¹²⁴I-labeled RCGs in Human Serum.

3.8. In vitro cellular uptake

The cellular uptake of human breast cancer cells specifically, the hormone-dependent MCF-7 and hormone-independent SK-BR-3 cell lines was evaluated using [¹²⁴I]RCGs. The results demonstrated that [¹²⁴I]RCGs exhibited higher uptake in ERα-positive, estrogen-responsive MCF-7 cells after a 60-minute incubation period, with injected dose (ID%) values of 28.6% for MCF-7 and 24.6% for SK-BR-3. Binding affinity in both cell lines decreased following a 120-minute incubation period (Fig. 6). These findings align with previous reports indicating that MCF-7 cells express high levels of ERα, whereas SK-BR-3 cells are ERα-negative.^23,24 Although blocking assays were not conducted in this study due to experimental limitations, the observed trends support the rationale for ERα-targeted imaging. Further validation through blocking experiments or competitive binding assays would strengthen the evidence for ERα-mediated specificity.

Fig. 6 Cellular uptake studies of ¹²⁴I labeled RCGs. Values are mean ± standard deviation (n = 3).

3.9. Small-animal PET imaging

The whole-body distribution of [¹²⁴I]RCGs in an MCF-7 bearing mouse model is illustrated in Fig. 7. The MCF-7 tumors could be distinctly identified, with a high tumor-to-background contrast recorded at various time points (15 min, 30 min, 60 min, 120 min and 240 min). The PET images demonstrated significant tumor uptake shortly after intravenous administration. The in vivo specificity of [¹²⁴I]RCGs was confirmed in an estrogen-α positive tumor, with a high uptake of 6.11 ± 1.20 SUV_mean (Fig. 7a). Furthermore, the tumor-to-thyroid ratios were calculated to be 5.31 (Fig. 7b). The biodistribution studies conducted on the MCF-7 bearing mouse model for breast cancer suggest that [¹²⁴I]RCGs may serve as an effective tracer for ERα imaging.

Fig. 7 Micro-PET image of ¹²⁴I-RCGs. (a) Images at different time points post-injection; (b) the metabolic behavior of ¹²⁴I-RCGs in the tumor and major organs.

4. Discussion

Prior studies have reported that rhein inhibits glucose uptake in tumor cells,^25,26 leading to membrane-associated functions that induce cell death. To validate the selective accumulation of the estrogen-α receptor,^6,27 we selected rhein, an anthraquinone derivative, and introduced GNPs labeled with positron emitter ¹²⁴I for targeted breast cancer imaging. GNPs are being increasingly recognized for their effectiveness in drug delivery owing to their enhanced cellular uptake, biocompatibility, hydrophilicity, non-immunogenicity, and reduced toxicity. Before labeling the rhein-gold nanocomposites with ¹²⁴I, the labeling procedure utilizing chloramine oxidizer for [¹²⁴I]rhein was optimized. The results of the synthesis of [¹²⁴I]Rhein were evaluated to have a labeling yield of 25–35% within 15 min, and a high radiochemical purity of more than 95% was confirmed. After confirming its high reactivity, the synthesized rhein-gold nanocomposite was labeled with ¹²⁴I in a single-step reaction. Gold nanoparticles with a diameter of 30 nm are regarded as biologically safe and biocompatible. Previous research indicates that the liver toxicity of gold nanoparticles, which ranges from 10 nm to 60 nm in diameter, is minimized at 30 nm. These biocompatible gold nanoparticles were synthesized by meticulously controlling the amount of citrate, a reducing agent, to achieve the desired spherical morphology of 30 nm. To assess the accumulation of the RCGs in breast cancer cells, we evaluated its uptake in the MCF-7 and SKBR-3 breast cancer cell lines. The uptake was significantly higher in the MCF-7 cell line, which overexpresses the estrogen alpha hormone, demonstrating an uptake rate exceeding 25% within the first 15 minutes and gradually increasing over the course of 60 minutes. Based on these results, PET imaging studies were conducted to confirm the rapid uptake and release of the nanocomposite in tumors. Consistent with the cellular uptake findings, the [¹²⁴I]Rhein-coated GNPs showed high tumor accumulation efficiency within 15 minutes following intravenous injection into MCF-7 tumor-implanted mice, and were rapidly excreted after 60 minutes. This was validated as a potential targeting imaging agent by conducting an active targeting assay on the MCF-7 breast cancer cell line, which overexpresses the estrogen-α receptor (Table 2).

Table 2 Biodistribution of [¹²⁴I]RCGs in mice at different time points after injection (Mean ± SD, SUV). Values represent radioactivity concentrations in selected organs (n = 3)

Time (min)	Lung	Liver	Thyroid	Tumor
15	0.30 ± 0.09	1.17 ± 0.16	0.18 ± 0.04	2.82 ± 0.98
30	0.42 ± 0.13	1.28 ± 0.17	0.44 ± 0.06	2.54 ± 0.53
60	0.29 ± 0.07	0.46 ± 0.11	0.45 ± 0.04	1.78 ± 0.61
120	0.53 ± 0.10	0.40 ± 0.14	1.72 ± 0.38	0.39 ± 0.07
240	0.23 ± 0.07	0.26 ± 0.09	0.65 ± 0.11	0.41 ± 0.06

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5. Conclusion

The study successfully synthesized ¹²⁴I-labeled rhein-coated gold nanoparticles and demonstrated their potential as targeted PET imaging agents for breast cancer, particularly for estrogen receptor-alpha (ERα) positive tumors. The nanoparticles exhibited significant cellular uptake in ERα-positive MCF-7 cells, confirming their targeting efficiency. Furthermore, high tumor uptake and clear imaging were confirmed in PET images of a breast cancer xenograft mouse model. These results suggest that [¹²⁴I]RCG has promise as an early diagnostic tool for breast cancer. While this study focused on the diagnostic potential of [¹²⁴I]RCG, its therapeutic efficacy was not evaluated. Future studies evaluating the therapeutic efficacy of [¹²⁴I]RCG are essential for establishing a comprehensive theranostic platform. In terms of clinical applicability, [¹²⁴I]RCG demonstrates significant potential due to its gold nanoparticle-based structure and strong tumor selectivity, and has the potential to replace existing ERα-targeted tracers. Compared with existing ERα PET tracers such as ¹⁸F or ¹¹C labeled estrogen derivatives, [¹²⁴I]RCG, although still in the early preclinical stage, is expected to be a promising candidate for ERα-targeted breast cancer diagnosis and treatment due to its high tumor-targeting efficiency and rapid clearance from major organs.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Korea Atomic Energy Research Institute and approved by the Institutional Animal Care and Use Committee (KAERI-IACUC-2025-001).

Author contributions

Jun Young Lee designed and organized the research, developed the methodology, conducted the investigation, curated and analyzed the data, created the visualizations, and was the primary contributor to writing the original draft. Jeong Hoon Park contributed to the conceptualization and methodology, provided resources, supervised and administered the project, secured funding, and was responsible for reviewing and editing the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5nj02035b.

Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00210260 and RS-2023-00237149). We would like to acknowledge the researchers at the cyclotron facility of the Korea Atomic Energy Research Institute (Jeongeup, Korea) and the Korea Institute of Radiological & Medical Science (Seoul, Korea) for their contributions to the application of the radioisotope.

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