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

Copper (Cu) is a critical element for cancer cell proliferation and considerably accumulates in the nucleus. 64Cu2+ is an anticancer radiopharmaceutical that targets the copper requirement of cancer cells. However, intravenously injected 64Cu2+ ions primarily accumulate in the liver. Ligand complexation of 64Cu2+ may be a promising method for increasing tumor delivery by reducing liver uptake. In this study, 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 uptakes 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 of 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.


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 cancer [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 64 Cu 2+ ions as antitumor radiotherapeutic drugs [9][10][11][12][13]. 64 Cu 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 Pharmaceuticals 2024, 17, 820 2 of 12 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 64 Cu demonstrate a strong capacity to damage cancer cell DNA [15]. 64Cu 2+ ions uptake is high in various cancers, which inhibits tumor growth [9,16,17].However, preclinical and human 64 Cu 2+ ions biodistribution studies have revealed extensive liver distribution, particularly within the first 30 min after intravenous administration, with 64 Cu 2+ ion levels reaching a plateau in 60-90 min and being maintained for more than 20 h [18,19].When 64 Cu 2+ ions enter the blood stream, 64 Cu 2+ immediately binds to albumin in blood plasma, and the 64 Cu 2+ -albumin complex is subsequently trapped in the liver [20,21].Therefore, further studies to improve the distribution of 64 Cu are essential for developing new anticancer drugs that utilize 64 Cu.
hibits tumor growth [9,16,17].However, preclinical and human Cu ions biodistribution studies have revealed extensive liver distribution, particularly within the first 30 min after intravenous administration, with 64 Cu 2+ ion levels reaching a plateau in 60-90 min and being maintained for more than 20 h [18,19].When 64 Cu 2+ ions enter the blood stream, 64 Cu 2+ immediately binds to albumin in blood plasma, and the 64 Cu 2+ -albumin complex is subsequently trapped in the liver [20,21].Therefore, further studies to improve the distribution of 64 Cu are essential for developing new anticancer drugs that utilize 64 Cu.
In this study, we examined 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 64 Cu 2+ 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 for 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 to treat this disease must be urgently developed.Clinical studies have reported that when ceruloplasmin, which is related to active copper metabolism, is overexpressed it is a potential prognostic marker for bile duct cancer, similar to several other cancers [29][30][31].

Determination of the Radiochemical Purity of 64 Cu 2+ Complexes
The representative radio-chromatograms of 64 Cu 2+ -Tren, 64 Cu 2+ -Dien, 64 Cu 2+ -TPMA, and 64 Cu 2+ ions are shown in Figure 2. 64 Cu 2+ -Tren (Rf = 0.83), 64 Cu 2+ -Dien (Rf = 0.78), and 64 Cu 2+ -TPMA (Rf = 0.44) were obtained with a radiochemical purity of >95%.The Rf values of each 64 Cu 2+ complex were similar to those of the corresponding complexes with nonradioactive Cu 2+ .In this study, we examined 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 64 Cu 2+ 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 for 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 to treat this disease must be urgently developed.Clinical studies have reported that when ceruloplasmin, which is related to active copper metabolism, is overexpressed it is a potential prognostic marker for bile duct cancer, similar to several other cancers [29][30][31].

In Silico Log P o/w and Log S Studies of Cu 2+ Complexes
As results in Figures 3 and 4 indicate a tendency of liver uptake for the studied 64 Cu 2+ complexes, a comparison of their physicochemical properties was performed.The octanol-water partition coefficients (log P o/w ) of the Cu 2+ complexes were calculated using SwissADME software (http://www.swissadme.ch)[32].SwissADME is a free web tool operated by the Swiss Institute of Bioinformatics (SIB), which provides 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 values predicted by five lipophilicity prediction models, was used in this study.

Discussion
64 Cu 2+ ions are promising as an anticancer therapeutic drug targeting the copper requirement of cancer cells; however, intravenously injected 64 Cu 2+ ions primarily accumulate in the liver.Complexation of the ligand with 64 Cu 2+ may be a potential way to increase delivery to the tumor by decreasing liver uptake.
In this study, we compared in vivo tumor and liver uptakes of three 64 Cu 2+ complexes with tripodal amine ligands 64 Cu 2+ -Tren, 64 Cu 2+ -Dien, and 64 Cu 2+ -TPMA with those of 64 Cu 2+ ions using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1.In vivo tissue distribution showed that 64 Cu 2+ -Tren and 64 Cu 2+ -Dien showed higher tumor uptake and lower liver uptake than 64 Cu 2+ -TPMA and 64 Cu 2+ ions.In addition, we observed a parallel trend between in vitro cellular uptake and in vivo tumor uptake, with 64 Cu 2+ -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, 64 Cu 2+ ions have been extensively investigated as a potential antitumor radioactive therapeutic drug [9].However, 64 Cu 2+ ions were widely distributed in the liver immediately after intravenous administration [18,19].Therefore, improving the distribution of 64 Cu 2+ ions is important for the development of novel 64 Cu anti-cancer drugs.This study demonstrated the potential of 64 Cu 2+ -Tren and 64 Cu 2+ -Dien to improve 64 Cu 2+ delivery to tumors by reducing liver traps.Consequently, these two 64 Cu 2+ complexes emerged 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 Cu 2+ -Tren and Cu 2+ -Dien showed higher whole-cell and nuclear uptakes than Cu 2+ -TPMA and Cu 2+ ions did.Consequently, 64 Cu 2+ -Tren and 64 Cu 2+ -Dien are good candidates among the examined Cu 2+ complexes and Cu 2+ ions.Cu 2+ -Tren showed much higher in vitro nuclear uptake and (although there were no significant differences) a higher tendency for in vivo tumor uptake, in vitro whole-cell uptake, and percentage counts of nuclear uptake in the whole cell than 64 Cu 2+ -Dien did.Cu 2+ -Tren showed rapid urinary excretion, whereas 64 Cu 2+ -Dien showed kidney retention at the examined time points.Based on these observations, Cu 2+ -Tren is a better alternative than 64 Cu 2+ -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 (Figures 4 and S1) for 64 Cu 2+ -Tren, 64 Cu 2+ -Dien, 64 Cu 2+ -TPMA, and 64 Cu 2+ ions.Previous studies have shown that intravenously administered 64 Cu 2+ ions immediately bind to albumin in the blood plasma, and the 64 Cu 2+ -albumin complex is subsequently trapped in the liver [20,21].Ligand complexation of 64 Cu 2+ 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 64 Cu 2+ 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 Cu 2+ -Tren, Cu 2+ -Dien, and Cu-TPMA were −0.98, −0.52, and 0.99, respectively.A previous study reported that if a log P o/w value is less than 0, the compound is classified as hydrophilic, and if the value is greater than 0, the compound is classified as lipophilic [37,38].In addition, hydrophilic compounds increase the proportion 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 64 Cu 2+ -Tren and 64 Cu 2+ -Dien with log P o/w < 0 (hydrophilic) increased the proportion of urinary excretion via the kidney, whereas Cu 2+ -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, 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 Cu 2+ -Tren, Cu 2+ -Dien, and Cu 2+ -TPMA were −1.91, −1.92, and −5.46, respectively, suggesting that 64 Cu 2+ -Tren and 64 Cu 2+ -Dien are highly soluble and have potential as intravenous preparations.
This study had several limitations.First, this study aimed to perform a basic comparative study of 64 Cu 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 different types of cancer.Second, this study demonstrated that 64 Cu 2+ -Tren and 64 Cu 2+ -Dien showed higher in vivo and in vitro tumor uptake in TFK-1 cells.Further studies of their detailed biodistribution over time and in vitro and in vivo therapeutic efficacy are warranted for the future development of 64 Cu 2+ -Tren and 64 Cu 2+ -Dien.Finally, we did not focus on the transport mechanisms of Cu 2+ complexes in this study.Previous studies of 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 64 Cu 2+ -Tren and 64 Cu 2+ -Dien.Elucidation of the transport mechanism of these Cu 2+ complexes is critical for future developmental studies of these compounds.

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.The Cu 2+ -Tren, Cu 2+ -Dien, and Cu 2+ -TPMA used for TLC analysis were prepared as previously described in the literature [44,45], and complexes used in the micro-PIXE analysis were also synthesized as previously described [46][47][48].

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, Palo Alto, 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, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and incubated at 37 • C in a humidified atmosphere of 5% CO 2 .Exponentially growing cells were detached from plates using trypsin (0.5 w/v% Trypsin-5.3mmol/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, Hercules CA, USA) exclusion method.

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 × 10 6 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.

In Silico Log P o/w and Log S Studies of 64 Cu 2+ Tripodal Amine Complexes
The log P o/w values of the Cu 2+ complexes were calculated using SwissADME software [32].SwissADME 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 of drugs, was also calculated using the same software.

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.Cells were detached from culture plates using trypsin to make cell suspensions of 8.0 × 10 4 cells/mL in RPMI-1640 medium.Cell suspensions were dropped on a 5 µm Mylar film (Chemplex Inc., Palm City, FL, USA) in a culture dish and incubated at 37 • C in a humidified atmosphere containing 5% CO 2 for 2 d.Cells were incubated in a medium with 1 mM non-radioactive compounds (Cu 2+ -Tren, Cu 2+ -Dien, Cu 2+ -TPMA, and Cu 2+ 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, Hercules, CA, USA) for nuclear localization and observed under a fluorescence microscope (BZ-X810, Keyence, Osaka, Japan).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 the previous literature with modifications [50].Distributions of Cu, P, K, Ca, Al, Fe, and S in each group were determined using 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 cells.Nuclei localization was determined via fluorescence microscope images of DAPI staining.Cu signals in the nucleus, cytoplasm, and whole cells were counted.

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).

Conclusions
This study demonstrated that 64 Cu 2+ -Tren and 64 Cu 2+ -Dien showed higher in vivo tumor uptake and in vitro cellular and nuclear uptake than 64 Cu 2+ -TPMA and 64 Cu 2+ ions in TFK-1 cells.In the in vivo study, an inverse correlation was observed between tumor and liver uptake among the three examined 64 Cu 2+ complexes and 64 Cu 2+ ions. 64Cu 2+ -Tren and 64 Cu 2+ -Dien are promising candidates for the development of anticancer 64 Cu drugs.This study provides useful information for the future development of 64 Cu radiopharmaceuticals.

Table 1 .
Basic physicochemical properties of Cu 2+ complexes in this study.