64Cu2+ Complexes of Tripodal Amine Ligands on In Vivo Tumor and Liver Uptake and Intracellular Cu Distribution in the Extrahepatic Bile Duct Carcinoma Cell Line TFK-1: A Basic Comparative Study

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⁶⁴Cu²⁺Complexes of Tripodal Amine Ligands on In Vivo Tumor and Liver Uptake and Intracellular Cu Distribution in the Extrahepatic Bile Duct Carcinoma Cell Line TFK-1: A Basic Comparative Study

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Abstract

Copper (Cu) is a critical element for cancer cell proliferation and considerably accumulates in the nucleus. 64Cu2+ is an anticancer radiopharmaceuticals targeting the copper requirement of cancer cells. However, intravenously injected 64Cu2+ ions primarily accumulate in the liver. Ligand complexation of 64Cu2+ would be a promising method for increasing tumor delivery by reducing liver uptake. Herein, we used three tripodal amine ligands [tris(2-aminoethyl)amine (Tren), diethylenetriamine (Dien), and tris(2-pyridylmethyl)amine (TPMA)] to enclose 64Cu2+ ions and compared their in vivo tumor and liver uptake using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1. We examined intracellular Cu distribution using microparticle-induced X-ray emission (micro-PIXE) analysis for these compounds. 64Cu2+-Tren and 64Cu2+-Dien showed higher tumor uptake than 64Cu2+-TPMA and 64Cu2+ ions in TFK-1 tumors. Among the three 64Cu2+ complexes and 64Cu2+ ions, liver uptake was inversely correlated with tumor uptake. Micro-PIXE analysis showed that in vitro cellular uptake was similar to in vivo tumor uptake and nuclear delivery was the highest for 64Cu2+-Tren. Conclusively, an inverse correlation between tumor and liver uptake was observed using three 64Cu2+ complexes of tripodal amine ligands and 64Cu2+ ions. These results provide useful information for the future development of anticancer 64Cu radiopharmaceuticals.

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Medicine and Pharmacology - Medicine and Pharmacology

1. Introduction

Copper (Cu) is essential for cancer cell growth and proliferation [1,2,3]. The concentration of Cu is higher in tumor tissues and serum than in normal tissues in several types of cancers [4,5,6,7]. Cu accumulates in the nuclei of cancer cells and plays an important role in transcription-related tumor proliferation and metastasis [4,8]. Therefore, targeting the Cu requirement of cancer cells is considered a promising avenue for the development of anticancer drugs. Several studies have been conducted to investigate the potential of ⁶⁴Cu²⁺ ions as antitumor radiotherapeutic drugs [9,10,11,12,13]. ⁶⁴Cu is a useful radionuclide for theranostic purposes. ⁶⁴Cu decays via β⁺ (0.653 MeV, 17.4%), β⁻ (0.574 MeV, 40%), and electron capture (42.6%); γ-ray photons from electron-positron annihilation can be used for positron emission tomography (PET) imaging, whereas β⁻ particles and Auger electrons emitted from this nuclide can be used for therapeutic purposes to damage tumor cells [14]. In particular, Auger electrons emitted from ⁶⁴Cu demonstrate a strong capacity to damage cancer cell DNA [15]. ⁶⁴Cu²⁺ ion uptake is high in various cancers, which inhibits tumor growth [9,16,17]. However, preclinical and human ⁶⁴Cu²⁺ ions biodistribution studies reveal extensive liver distribution, particularly within the first 30 min after intravenous administration, with the ⁶⁴Cu²⁺ ion levels reaching a plateau in 60–90 min and being maintained for more than 20 h [18,19]. When ⁶⁴Cu²⁺ ions enter blood stream, ⁶⁴Cu²⁺ immediately binds to albumin in blood plasma, and the ⁶⁴Cu²⁺-albumin complex is subsequently trapped in the liver [20,21]. Therefore, further studies to improve the distribution of ⁶⁴Cu are essential for developing new anticancer drugs that utilize ⁶⁴Cu.

Ligand complexation of ⁶⁴Cu²⁺ has been paid attention as a potential method for decreasing liver traps and increasing tumor delivery [21,22]. Therefore, we focused on tripodal amine ligands such as tris(2-aminoethyl)amine (Tren), diethylenetriamine (Dien), and tris(2-pyridylmethyl)amine (TPMA) and synthesized ⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, and ⁶⁴Cu²⁺-TPMA (Figure 1). As these tripodal amine ligands can enclose the ⁶⁴Cu ions to form stable ⁶⁴Cu²⁺ complexes, we hypothesized that they decrease liver uptake and increase tumor uptake compared with the liver and tumor uptake of only ⁶⁴Cu²⁺ ions.

In this study, we examined the in vivo tumor and liver uptake and investigated the intracellular Cu distribution of these complexes using a tumor-bearing xenograft mouse model of an extrahepatic bile duct carcinoma cell line TFK-1 with the above three ⁶⁴Cu²⁺ complexes. TFK-1 is a well-characterized and frequently used cell line to study extrahepatic bile duct cancer [23,24,25,26]. Bile duct carcinoma or cholangiocarcinoma is an aggressive tumor with a poor prognosis. The 5-year survival rate of extrahepatic bile duct cancer is 20–30% [27]. The only curative treatment for patients with extrahepatic bile duct carcinoma is surgical resection; however, the rate of resectability is low and the rate of recurrence is high [28]. Therefore, innovative drugs for this disease must be urgently developed. Clinical studies have reported that ceruloplasmin, which is related to active copper metabolism, is overexpressed and is a potential prognostic marker in bile duct cancer, similar to several other cancers [29,30,31].

Figure 1. Chemical structure of (a) ⁶⁴Cu²⁺-Tren, (b) ⁶⁴Cu²⁺-Dien, and (c) ⁶⁴Cu²⁺-TPMA.

2. Results

2.1. Determination of the Radiochemical Purity of ⁶⁴Cu²⁺ Complexes

The representative radio-chromatograms of ⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, ⁶⁴Cu²⁺-TPMA, and ⁶⁴Cu²⁺ ions are shown in Figure 2. ⁶⁴Cu²⁺-Tren (R_f = 0.83), ⁶⁴Cu²⁺-Dien (R_f = 0.78), and ⁶⁴Cu²⁺-TPMA (R_f = 0.44) were obtained with a radiochemical purity of > 95%. The R_f values of each ⁶⁴Cu²⁺ complex were similar to those of the corresponding complexes with non-radioactive Cu²⁺.

2.2. Tissue Distribution In Vivo

We compared the basic characteristics of the tissue distribution of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ in vivo. TFK-1 cells stably expressing red fluorescent protein (RFP) were used in this study (TFK-1-RFP). The tissue distribution and excretion of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ ions in mice bearing TFK-1-RFP cells 2.5 h after intravenous injections were examined (Figure 3). This time point was selected because a previous preclinical study showed that the liver and tumor distribution of Cu²⁺ ions plateaued 2 h after intravenous injection [18]. The biodistribution data are shown as the percentage of injected dose per gram for the organs (%ID/g) and blood and the percentage of injected dose (%ID) for the urine and feces. Cu²⁺-Tren and Cu²⁺-Dien showed higher tumor uptake than Cu²⁺-TPMA and Cu²⁺ ions (13.20 ± 2.65 %ID/g for Cu²⁺-Tren, 11.84 ± 2.47 %ID/g for Cu²⁺-Dien, 7.56 ± 0.41 %ID/g for Cu²⁺-TPMA, and 5.98 ± 1.09 %ID/g for Cu²⁺ ions) (P < 0.05). Cu²⁺-Tren showed a higher tumor uptake value than Cu²⁺-Dien; however, the difference was insignificant. There was no significant difference between Cu²⁺-TPMA and Cu²⁺ ions. Conversely, Cu²⁺-Tren and Cu²⁺-Dien showed lower liver uptake than Cu²⁺-TPMA and Cu²⁺ ions, respectively (26.38 ± 2.04 %ID/g for Cu²⁺-Tren, 26.33 ± 1.66 %ID/g for Cu²⁺-Dien, 34.58 ± 3.58 %ID/g for Cu²⁺-TPMA, and 34.96 ± 2.32 %ID/g for Cu²⁺ ions) (P < 0.05). There were no significant differences in liver uptake between Cu²⁺-Tren and Cu²⁺-Dien or between Cu²⁺-TPMA and Cu²⁺ ions. Figure 4 and Figure S1 show the correlation between tumor and tissue uptake. There was a significantly strong inverse correlation between tumor uptake and liver uptake (R = –0.9735, P < 0.05), whereas no correlation was observed in any of the other examined tissues (kidney, blood, heart, small intestine, large intestine, bone, muscle, parotid and submandibular, lung, spleen, pancreas, brain, and the remainder of the body). From two Cu²⁺ complexes (Cu²⁺-Tren and Cu²⁺-Dien) that showed high tumor uptake, Cu²⁺-Tren showed lower kidney uptake than Cu²⁺-Dien (10.87 ± 1.98 %ID/g for Cu²⁺-Tren and 24.23 ± 6.09 %ID/g for Cu²⁺-Dien) (P < 0.05) and higher urinary excretion than Cu²⁺-Dien (7.81 ± 2.54 %ID for Cu²⁺-Tren and 1.68 ± 0.46 %ID for Cu²⁺-Dien) (P < 0.05).

2.3. In Silico Log P_o/w and Log S Studies of Cu²⁺ Complexes

Since the results in Figure 3 and Figure 4 indicate a tendency of liver uptake for these ⁶⁴Cu²⁺ complexes, a comparison of their physicochemical properties was performed. The octanol-water partition coefficient (log P_o/w) of the Cu²⁺ complexes was calculated using SwissADME software [32]. SwissADME is a free web tool run by the Swiss Institute of Bioinformatics (SIB), which provide predictive models for physicochemical properties, pharmacokinetics, drug compatibility, and medicinal chemistry compatibility [32]. The consensus log P_o/w (log P_o/w), which is the arithmetic mean of the values predicted by the five lipophilicity prediction models, was used in this study.

The calculated log P_o/w values for Cu²⁺-Tren, Cu²⁺-Dien, and Cu²⁺-TPMA were –0.98, –0.52, and 0.99, respectively (Table 1). The same software was also used to calculate water solubility log S (mol/L) using the estimated solubility model (ESOL) [33]. The calculated log S values for Cu²⁺-Tren, Cu²⁺-Dien, and Cu²⁺-TPMA were –1.91, –1.92, and –5.46, respectively (Table 1).

2.4. Intracellular Cu Distribution

Micro-particle-induced X-ray emission (Micro-PIXE) analysis was performed to investigate the in vitro whole cell, nuclear, and cytoplasmic uptake of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ ions at a 1 µm spatial resolution (Figure 5 and Figure 6). The in vitro whole-cell uptake showed a parallel tendency to the in vivo tumor uptake; Cu²⁺-Tren and Cu²⁺-Dien showed higher uptake than Cu²⁺-TPMA and Cu²⁺ ions, respectively (736.00 counts ± 156.03 for Cu²⁺-Tren, 530.60 counts ± 106.87 for Cu²⁺-Dien, 118.58 counts ± 59.33 for Cu²⁺-TPMA, and 49.43 counts ± 34.33 for Cu²⁺ ions) (P < 0.05). A positive correlation was observed between in vitro whole-cell uptake and in vivo tumor uptake (R = 0.992, P = 0.00772) (Figure S2). Similarly, we observed a consistent correlation between in vitro nuclear uptake and in vivo tumor uptake (417.6 counts ± 150.02 for Cu²⁺-Tren, 203.00 counts ± 25.13 for Cu²⁺-Dien, 94.82 counts ± 48.07 for Cu²⁺-TPMA, and 35.57 counts ± 25.02 for Cu²⁺ ions) (P < 0.05). Notably, among the three Cu²⁺ complexes and Cu²⁺ ions, Cu²⁺-Tren showed the highest average value in the percentage counts of nuclear uptake/whole-cell uptake, although there were no significant differences.

3. Discussion

⁶⁴Cu²⁺ ion is a promising anticancer therapeutic drug targeting the copper requirement of cancer cells; however, intravenously injected ⁶⁴Cu²⁺ ions primarily accumulate in the liver. Complexation of the ligand with ⁶⁴Cu²⁺ would be a potential way to increase delivery to the tumor by decreasing liver uptake.

In this study, we compared the in vivo tumor and liver uptake of three ⁶⁴Cu²⁺ complexes with tripodal amine ligands, ⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, and ⁶⁴Cu²⁺-TPMA, and with those of ⁶⁴Cu²⁺ ions using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1. In vivo tissue distribution showed that ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien showed higher tumor uptake and lower liver uptake than ⁶⁴Cu²⁺-TPMA and ⁶⁴Cu²⁺ ions, respectively. In addition, we observed a parallel trend between in vitro cellular uptake and in vivo tumor uptake, with ⁶⁴Cu²⁺-Tren exhibiting high whole-cell and nuclear uptake in TFK-1 cells.

Copper is an essential element for cancer cell growth and proliferation [1]; therefore, ⁶⁴Cu²⁺ ion has been extensively investigated as a potential antitumor radioactive therapeutic drug [9]. However, ⁶⁴Cu²⁺ ions were widely distributed in the liver immediately after intravenous administration [18,19]. Therefore, improving the distribution of ⁶⁴Cu²⁺ ion is important for the development of novel ⁶⁴Cu anti-cancer drugs. This study demonstrated the potential of ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien to improve ⁶⁴Cu²⁺ delivery to tumors by reducing liver traps. Consequently, these two ⁶⁴Cu²⁺ complexes emerge as promising candidates for further development. Cu is highly concentrated in tumor cells and tissues [4] and accumulates in the nuclei of cancer cells [4,8]. This study demonstrated that Cu²⁺-Tren and Cu²⁺-Dien showed higher whole-cell and nuclear uptakes than Cu²⁺-TPMA and the Cu²⁺ ions. Consequently, ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien are good candidates among the examined Cu²⁺ complexes and Cu²⁺ ions. Cu²⁺-Tren showed much higher in vitro nuclear uptake and (although there were no significant differences) a higher tendency of in vivo tumor uptake, in vitro whole cellular uptake, and percentage counts of nuclear uptake in the whole cell than ⁶⁴Cu²⁺-Dien. Cu²⁺-Tren showed rapid urinary excretion, whereas ⁶⁴Cu²⁺-Dien showed kidney retention at the examined time points. Based on these observations, Cu²⁺-Tren would be a better alternative to ⁶⁴Cu²⁺-Dien.

Notably, we found a significantly strong inverse correlation between tumor uptake and liver uptake; however, no correlation was observed in any of the other examined tissues (Figure 4 and Figure S1) for ⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, ⁶⁴Cu²⁺-TPMA, and ⁶⁴Cu²⁺ ions. Previous studies have shown that intravenously administered ⁶⁴Cu²⁺ ions immediately bind to albumin in the blood plasma, and the ⁶⁴Cu²⁺-albumin complex is subsequently trapped in the liver [20,21]. Ligand complexation of ⁶⁴Cu²⁺ can be a promising strategy to reduce liver traps and facilitate tumor delivery [21,22]. In addition, lipophilicity is an important factor in determining excretion [34,35,36]. Therefore, the present study focused on three ⁶⁴Cu²⁺ complexes of tripodal amine ligands with different lipophilicities, log P_o/w. Log P_o/w is an indicator of lipophilicity, and the calculated log P_o/w values for Cu²⁺-Tren, Cu²⁺-Dien, and Cu-TPMA were –0.98, –0.52, and 0.99, respectively. A previous study reported that if the log P_o/w values are less than 0, the compounds are classified as hydrophilic, and if the value is greater than 0, the compounds are classified as lipophilic [37,38]. In addition, hydrophilic compounds increase the portion of urinary excretion via the kidney, and lipophilic compounds are likely to show liver excretion [39,40]. Therefore, based on this knowledge and our results, it is considered reasonable that ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien with log P_o/w < 0 (hydrophilic) increased the proportion of urinary excretion via the kidney, whereas Cu²⁺-TPMA with a log P_o/w > 0 (lipophilic) showed liver excretion.

Water solubility is one of the critical factors in achieving the desired drug concentration in systemic circulation to achieve the required pharmacological response [41]. To successfully develop intravenous formulations, the water solubility must be high to deliver sufficient quantities of the active ingredient through a limited drug dosage [42]. The calculated log S (mol/L) values for Cu²⁺-Tren, Cu²⁺-Dien, and Cu²⁺-TPMA were –1.91, –1.92, and –5.46, respectively, suggesting that ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien are highly soluble and have potential as intravenous preparations.

This study has several limitations. First, this study aimed to perform a basic comparative study of ⁶⁴Cu complexes with tripodal amine ligands and used only one cell line of extrahepatic bile duct carcinoma. In future studies, it will be beneficial to use other cell lines of bile duct carcinoma and of different types of cancer. Second, this study demonstrated that ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien showed higher in vivo and in vitro tumor uptake in TFK-1 cells. Further studies on the detailed biodistribution over time and the in vitro and in vivo therapeutic efficacy are warranted for the future development of ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien. Finally, we did not focus on the transport mechanisms of the Cu²⁺ complexes in this study. Previous studies on the cytotoxicity of several copper(II) complexes for cancer treatment have demonstrated that copper transporter 1 plays critical roles in cancer cell uptake of copper(II) complexes [43]. Therefore, copper transporter 1 may also contribute to the transportation of ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien. The elucidation of the transport mechanism of these Cu²⁺ complexes is critical for future developmental studies of these compounds.

4. Materials and Methods

4.1. Reagents and Materials

Ligands Tren, Dien, and TPMA were obtained from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Copper(II) chloride dihydrate and ammonium acetate were of guaranteed reagent grade and were obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Copper(II) perchlorate was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Ultrapure water was purchased from Kanto Chemical Co. (Tokyo, Japan). All reagents and solvents were used without further purification. Cu²⁺-Tren, Cu²⁺-Dien, and Cu²⁺-TPMA used for the TLC analysis were prepared by previous literature [44,45] and the complexes used in the micro-PIXE analysis were synthesized from previous literature [46,47,48].

4.2. Preparation of ⁶⁴Cu²⁺ Complexes

⁶⁴CuCl₂ was obtained from PDR Pharma (Tokyo, Japan). ⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, and ⁶⁴Cu²⁺-TPMA (10 MBq/21 nmol) were synthesized by adding a ⁶⁴Cu²⁺ aqueous solution (10 MBq/50 µL) to each ligand aqueous solution (21 nmoL/50 µL). To determine the radiochemical purity of the prepared ⁶⁴Cu²⁺ complexes, 1 µL of each sample solution was spotted on an HPTLC NH₂ Silica Gel 60 F₂₅₄ glass plate (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and developed using aqueous ammonium acetate solution (0.5 M). Radioactivity on the HPTLC plates was measured using a radio-TLC system (Raytest PET MiniGITA Star; Elysia s.a, Liège, Belgium).

4.3. Cell Line and Culture

The human extrahepatic bile duct carcinoma cell line TFK-1 was obtained from the RIKEN Bioresource Research Center (Ibaraki, Japan) and immediately expanded and frozen in our laboratory. For animal experiments, TFK-1 cells stably expressing red fluorescent protein (RFP) (TFK-1-RFP) were used. To establish the TFK-1-RFP cell line, TFK-1 cells were transfected with RFP lentivirus (Lenti-Labeler Cell Labeling System, System Biosciences, CA, USA) following the manufacturer’s protocol. A clone strongly expressing RFP was selected by limiting dilution and was denoted as TFK-1-RFP. Early passage TFK-1 and TFK-1-RFP cells, with < 2–3 months of cumulative subculture, were used for all experiments. Cells were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco Corp, NY, USA) and incubated at 37 °C in a humidified atmosphere of 5% CO₂. Exponentially growing cells were detached from the plates using trypsin (0.5 w/v% Trypsin-5.3 mmol/L EDTA·4Na Solution without Phenol Red (×10), Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and the number of viable cells was determined using the trypan blue dye (Bio-Rad, California, USA) exclusion method.

4.4. Animal Model

Animal experimental procedures were approved by the Animal Ethics Committee of the National Institutes for Quantum Science and Technology (QST, Chiba, Japan) and conducted following institutional guidelines. SCID beige mice (7-week-old females) were obtained from Charles River Laboratories Japan (Yokohama, Japan) and used for in vivo biodistribution experiments. TFK-1-RFP cells (5 × 10⁶ cells) suspended in RPMI-1640 were pre-mixed with Matrigel at a ratio of 50:50 (v/v) and subcutaneously injected into the flanks of the mice. Mice bearing tumors of approximately 5 mm in diameter were used to examine tissue distribution in vivo.

4.5. Tissue Distribution In Vivo

To compare the tumor and liver uptake of ⁶⁴Cu²⁺ complexes (⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, and ⁶⁴Cu²⁺-TPMA) and ⁶⁴Cu ion, the in vivo tissue distribution was investigated in mice bearing TFK-1-RFP cells. Mice were administered 1.85 MBq of ⁶⁴Cu²⁺-Tren, ⁶⁴Cu²⁺-Dien, ⁶⁴Cu²⁺-TPMA, and ⁶⁴Cu²⁺ ions intravenously (n = 3–4/group) and sacrificed at 2.5 h to collect tissues (parotid, submandibular, heart, lung, liver, kidney, spleen, pancreas, small intestine, large intestine, brain, muscle, bone, tumor, and the remainder of the body) and blood. Urine and feces were collected during the 2.5 h post-administration using polyethylene-laminated filter paper. Radioactivity levels were counted using a γ-counter (1480 Automatic gamma counter Wizard 3; PerkinElmer Inc., Waltham, MA, USA). The %ID/g for organs and blood and the %ID for urine and feces were calculated.

4.6. In Silico Log P_o/w and Log S Studies of ⁶⁴Cu²⁺ Tripodal Amine Complexes

The log P_o/w values of the Cu²⁺ complexes were calculated using SwissADME software [32]. Swiss ADME-free web tools provide predictive models for physicochemical properties, pharmacokinetics, drug compatibility, and medicinal chemistry compatibility. Water solubility (log S), one of the most important physicochemical properties for drugs, was also calculated using the same software.

4.7. Micro-PIXE Analysis

A micro-PIXE analysis was carried out at the QST Electrostatic Accelerator Facility [49]. The system consists of a 3.0 MeV proton microbeam (ϕ = 1 μm) combined with a 1.7 MV tandem accelerator and an ion source. The cells were detached from the culture plates using trypsin to make cell suspensions of 8.0 × 10⁴ cells/mL in RPMI-1640 medium. The cell suspension was dropped on a 5 μm Mylar film (Chemplex Inc, FL, USA) in a culture dish and incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 2 d. The cells were incubated in a medium with 1 mM non-radioactive compounds (Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ ions) for 4 h. Thereafter, cells were washed twice with phosphate-buffered saline, fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and rinsed three times with 150 mM ammonium acetate buffer. Cells were subsequently stained with DAPI (4’,6-diamidino-2-phenylindole; Bio-Rad, California, USA) for nuclear localization and observed under a fluorescence microscope (BZ-X810, Keyence, Osaka, Japan). The samples were air-dried at room temperature for more than 24 h and subjected to micro-PIXE analysis [49]. The sample preparation procedure was based on previous literature with modifications [50]. The distributions of Cu, P, K, Ca, Al, Fe, and S in each group were determined using the Kα lines. Each micro-PIXE image was obtained for five cells per group. The cell morphology in micro-PIXE analysis was determined by matching micro-PIXE images of phosphorus and potassium, which are distributed uniformly throughout the cells. Nuclei localization was determined via fluorescence microscope images of DAPI staining. Cu signals in the nucleus, cytoplasm, and whole cells were counted.

4.8. Statistical Analysis

Data were expressed as the mean ± SD. Multiple comparisons were conducted using parametric one-way analysis of variance with the Tukey−Kramer post-hoc test. All statistical analyses were conducted at a significance level of P < 0.05. Data analyses were performed using JMP 13.2.0 (SAS Institute, Cary, NC, USA).

5. Conclusions

This study demonstrated that ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien showed higher in vivo tumor uptake and in vitro cellular and nuclear uptake than ⁶⁴Cu²⁺-TPMA and ⁶⁴Cu²⁺ ions in TFK-1 cells. In the in vivo study, an inverse correlation was observed between tumor and liver uptake among the three examined ⁶⁴Cu²⁺ complexes and ⁶⁴Cu²⁺ ions. ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien are promising candidates to develop anticancer ⁶⁴Cu drugs. This study provides useful information for the future development of ⁶⁴Cu radiopharmaceuticals.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org. Figure S1: Correlation between tumor uptake and tissue uptake. Figure S2: Comparison of in vivo tumor uptake and in vitro whole-cell uptake.

Author Contributions

Conceptualization, M.S., H.M., and Y.Y.; methodology, M.S., M.T., M.H., and Y.Y.; formal analysis, M.S., M.T., C.I., H.M., F.H., T.T., M.O., H.S., M.-R.Z., H.K., and Y.Y.; investigation, M.S., M.T., C.I., H.M., F.H., T.T., and Y.Y.; writing—original draft preparation, M.S., M.T., H.M., and Y.Y.; writing—review and editing, M.S., M.T., C.I., H.M., F.H., T.T., M.O., H.S., M.-R.Z., T.H., H.K., Y.Y., and Y.D.; supervision, Y.Y., and Y.D.; funding acquisition, Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the JST FOREST Program (grant number JPMJFR2116) and JSPS KAKENHI (grant number JP22H03031).

Institutional Review Board Statement

The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Committee of the National Institutes for Quantum Science and Technology (QST, Chiba, Japan) in 2018 (approval no. 13-1022-7).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used in this study are available from the corresponding author upon request.

Conflicts of Interest

H.M., H.K., and Y.Y. are co-founders and board members of LinqMed Inc. C.I., F.H., and T.T. are employees of LinqMed Inc.

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Figure 2. Radio-TLC (thin-layer chromatography) analysis. Representative chromatogram of (a) ⁶⁴Cu²⁺-ion, (b) ⁶⁴Cu²⁺-Tren, (c) ⁶⁴Cu²⁺-Dien, and (d) ⁶⁴Cu²⁺-TPMA.

Figure 3. Tissue distribution in vivo. Tissue distribution and excretion of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and the Cu²⁺ ions in TFK-1-RFP-xenografted mice 2.5 h after injections.

Figure 4. Correlation between tumor and tissue uptake. Values of tumor and tissue uptake and excretion of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ ions in TFK-1-RFP-xenografted mice 2.5 h after injections, shown in Figure 3, are used. This figure illustrates correlations among liver, kidney, blood, heart, small intestine, large intestine, bone, and muscle (those among parotid and submandibular gland, lung, spleen, pancreas, brain, and the remainder of the body are shown in Figure S1).

Figure 5. Micro-PIXE analysis. Representative images of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ ions are shown. Bright field (left column), fluorescence from DAPI (middle column), and micro-PIXE (right column) images.

Figure 6. The quantitative analysis of micro-PIXE. In vitro whole cell (a) and nuclear (b) and cytoplasmic (c) uptake of Cu²⁺-Tren, Cu²⁺-Dien, Cu²⁺-TPMA, and Cu²⁺ ion are shown, respectively. The percentage of nuclear uptake to whole cell uptake is also shown (d). * indicates significant differences (P < 0.05).

Table 1. Basic physicochemical properties of Cu²⁺ complexes in this study.

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