Targeted Radionuclide Therapy (TRT) has shown to be a successful strategy against cancer (1–3). Its success relies on the localized delivery of large amounts of radiation which cause irreversible damage to cancer cells while minimizing the damage to healthy tissue (4). The radiopharmaceuticals used in TRT (Figure 1) consist of a compound (e.g., hormones, peptides, nucleotides, oligonucleotides, and antibodies) and a high-LET emitting radionuclide that specifically binds to a cell site (3).

The most sensitive region to ionizing radiation in the cell is genomic DNA (5). Radiation energy can be deposited in the DNA through direct action -by ionizing charged particles- or indirect action -by interacting with water radiolysis products like hydroxyl radicals, solvated electrons, and hydrogen atoms (6). These interaction processes can lead to two types of DNA damage: a single-strand break (SSB) or a double-strand break (DSB), and in the absence of a DNA repair process, derives in cell death through mitotic catastrophe or apoptosis (7).

Auger emitters (AE) are radionuclides that have aroused a high clinical interest due to their extremely short range, localized dose deposition, and low toxicity when decaying outside the cell nucleus, such as in the cytoplasm (8); examples of AE include ⁶⁷Ga, ^99mTc, ¹¹¹In, ¹²³I, ¹²⁵I, and ⁶⁴Cu. The AE’s have been shown to have a high relative biological effectiveness, similar to the alpha particles at distances shorter than 11 nm, which is comparable to the DNA molecule’s diameter (8). Auger electrons are ejected from electron orbitals due to nuclear decay modes such as electron capture or internal conversion (9). The energy of those electrons can be greater than 25 keV, but the yield per decay is very low (~ 0.1). Most electrons have energies less than 5 keV and deposit all their energy within a nanometer-micrometer range (9). Furthermore, many of the parent radionuclides also emit β-particles or photons that could be suitable for combined therapy and diagnosis (10).

We can understand the TRT status with AE by analyzing preclinical studies, clinical trials and other novel approaches. In preclinical studies compounds labeled with AE such as [¹¹¹In]In-BnDTPA-F3, [¹²³I]MST-312, [¹²⁵I]C5, and [^99mTc]C3 have been demonstrated to have a potent cytotoxic effect, intracellular uptake, and DSB induction (11–13). In clinical studies the [¹²⁵I]IUdR and the ¹²⁵I-labeled murine anti-EGFR mAb showed a biological relapse as well as safe and well-tolerated treatments (14). A novel approach using [¹¹¹In]In-DTPA showed no clinical side effects in patients, disease stabilization, and tumor size reduction (14, 15). In addition, over the past decade a new class of radiopharmaceuticals called theranostics have revolutionized nuclear medicine applications. This option opens the possibility of treatment and medical imaging, heralding a new era in the field.

⁶⁴Cu is a radionuclide with theranostics potential that has recently generated broad interest (16), and numerous preclinical reports have explored the therapeutic use of ⁶⁴Cu in experimental mouse models of cancers. For example, Ferrari et al. (17) studied [⁶⁴Cu]CuCl₂ for glioblastoma 2 (U87MG) in mice, reporting a good response and size reduction in tumors; in some cases, the tumors completely disappeared. Jin et al. (18) evaluated the therapeutic potential of [⁶⁴Cu]Cu-cyclam-RAFT-c(-RGDfK-)4 in glioblastoma cells in mice. Meanwhile, a new type of therapy that combines ⁶⁴Cu -based TRT with immunotherapy has been reached, in order to enhance the therapeutic efficacy of a radiopharmaceutical targeting α_vβ₃ integrin ([⁶⁴Cu]Cu-DOTA-EB-cRGDfK) (19). On the other hand, Qin et al. (20) demonstrated the therapeutic potential of [⁶⁴Cu]CuCl₂ for malignant melanoma in mice; the tumor growth was found to be reduced in models that received [⁶⁴Cu]CuCl₂ treatment. Until recently, only a limited number of clinical studies in humans have been reported using [⁶⁴Cu]CuCl₂ as radiopharmaceutical, mainly to evaluate the biodistribution and radiation dosimetry in healthy subjects and patients (21, 22).

Various in vitro studies have described the DNA-damage inflicted by ⁶⁴Cu. Fernandes-Guerreiro et al. (23) evaluated the radiobiological effects of the [⁶⁴Cu]CuCl₂ uptake in a panel of PCa cell lines. This study revealed that PCa cells exhibited a higher Cu uptake than non-tumoral cells. Also, they demonstrated that [⁶⁴Cu]CuCl₂ was able to reach the nuclear cell compartment producing significant genotoxicity and cytotoxicity in PC3, which were less efficient than normal cells in repairing the DNA-damage induced by [⁶⁴Cu]CuCl₂. McMillan et al. (24) on the other hand performed survival fraction studies with Chinese hamster ovary (CHO) wild type and DNA repair–deficient xrs5 cells exposed to [⁶⁴Cu]Cu-ATSM under hypoxic conditions, and by γH2AX foci assays confirmed DSBs and other complex types of chromosomal aberrations, both typical of high-LET radiation, providing strong evidence that [⁶⁴Cu]Cu-ATSM damages DNA via Auger electrons. More recently, Serban et al. (25) analyzed the DNA-damage and stress responses inflicted in various human normal and tumor cell lines after the exposure to [⁶⁴Cu]CuCl₂. All investigated cells, regardless of their tumoral or normal status, incorporate ⁶⁴Cu ions similarly, but their fate after exposure was cell-dependent. They found that an activity concentration of 40 MBq/mL of [⁶⁴Cu]CuCl₂ delivers a therapeutic effect in human colon carcinoma cells, but also caused harm to normal fibroblasts, yet lower than tumoral cells. An activity concentration of 20 MBq/mL was also able to induce DNA-damage and oxidative stress in colon cancer cells, and even when the therapeutic effect on tumor cells might be partial, the radiotoxicity to normal cells is expected to be lower.

Using computational modeling and experiments, researchers have observed and reported DSB caused by AE like ¹²³I, ¹²⁵I, ¹¹¹In, and ^99mTc when incorporated into the DNA (26–33). We have previously estimated the damage that ⁶⁴Cu, ¹²⁵I and ¹¹¹In caused to the DNA through the use of Geant4-DNA and the DBSCAN algorithm, considering the AE radionuclides randomly distributed in the cellular compartments (such as nucleus, cytoplasm and cell surface); the DNA content was also randomly distributed (no geometrical model) within the nucleus (34). Thus, ⁶⁴Cu has not been studied as a source of DSB damage when it is incorporated into the DNA structure. In the present work, we used a DNA geometry model, incorporated the AE ⁶⁴Cu in two positions within the DNA genome, and calculated the DSB damage as well as the total number of atoms incorporated that would cause a therapeutic effect. The motivation for this research comes from the continuous interest in new radiopharmaceuticals with AE such as the ⁶⁴Cu. Is our hope these data help estimate the total radioactivity needed for treatments against diseases, such as cancer.

⁶⁴Cu is a very promising AE for TRT due to its favorable nuclear and chemical properties and high cytotoxicity, which is attributed to the generation of low-energy electrons during its decay. Nevertheless, the use of ⁶⁴Cu remains scarce, as there is little evidence of its use on humans to assess its therapeutic potential. Guerreiro et al. (23) reported using a panel of Prostate Cancer (PCa) cell lines exhibiting a deficient DNA damage repair upon exposure to [⁶⁴Cu]CuCl₂. While Rigui et al. reported a dosimetry study in patients with prostate cancer, showing that the absorbed dose per administered activity was low, they also suggested that clinical trials are needed to evaluate the therapeutic effectiveness of ⁶⁴Cu. Thus, computational modeling is a more convenient for to estimating the therapeutic effectiveness in the short term. To the best of our knowledge, this is the first paper that reports on the DSB yields per decay incorporated in DNA for ⁶⁴Cu.

A comparison study using monoenergetic electrons was performed for model verification purposes. Figure 5 shows the DSB yields for monoenergetic electrons as a function of energy. The DSB yields follow an increasing behavior starting at an electron energy of 100 eV (1.10 ± 0.02 DSB/Gy/Da). The curve reaches a maximum value of 1.85 ± 0.03 DSB/Gy/Da at 500 eV. This behavior is expected because electrons with energy in the range of 100–200 eV have been reported to be two to four times more effective on inducing a DSB than electrons with energy between 100 and 1,000 keV (55–57). Finally, the DSB yield decreases monotonically until it reaches 0.83 ± 0.01 DSB/Gy/Da at 100 keV. While our calculated data fell within the available measured data, the comparison with calculated values reported by Nikjoo et al. (45) show that our results are lower for the energy range from 0.1 to 4.5 keV, but higher for the interval from 4.5 to100 keV. The discrepancies are attributed to the different DNA damage models and cross-section data used from different Monte Carlo engines.

The DSB yield for ⁶⁴Cu incorporated in the genome was simulated by modeling the AE incorporation to 0.25 and 1.15 nm off the DNA central axis and obtaining 0.171 ± 0.003 and 0.190 ± 0.003 per decay, respectively (Table 1).

As a means of verification of the calculation method, DSB yield calculations were performed for the ¹²⁵I incorporated in a DNA base pair, and the results were compared with experimental and calculated data available in the literature for this AE, which is considered the gold standard. Our calculation for ¹²⁵I incorporated in a DNA base pair was 1.94 ± 0.01 (0.25 off the central DNA axis); this value was compared with the data measured by Krisch and Ley (53), who performed studies with ¹²⁵I incorporated into the DNA of bacteriophage in the form of 5-iododeoxyuridine ([¹²⁵I]IUdR) and obtained 1.01 ± 0.13 per decay of ¹²⁵I. Humm and Charlton (29) also obtained a similar value using Monte Carlo simulations. The differences between our result and the reported by Krisch and Ley (53) might be due to the highly packed DNA structure in the cell nucleus model used in this work, which has been shown to be an important factor in the production of DSB’s in comparison with oligonucleotide or plasmid DNA fragments (58). Another study elaborated through Monte Carlo simulation, which was carried out by Ftániková and Böhm (28), obtained a value of 2.41 ± 0.8 DSB per decay for the ¹²⁵I. This calculation is also consistent with Walika’s result, and the differences between our results and the reported by Ftániková and Böhm (28) could be explained because they ignored the protecting character of histones that act as scavengers for chemical species.

The lethality analysis was performed by applying Eq. 1 and the yield of DSB/decay presented in Table 1. The results indicated that the lethality produced by 3,107 ± 28 initial atoms of ⁶⁴Cu incorporated into DNA is equivalent to that of 17,416 ± 46 initial atoms of the gold standard ¹²⁵I in a complete cell cycle of 24 h. This result corresponds to about 0.18 times fewer initial atoms of ⁶⁴Cu to achieve the same lethal damage as ¹²⁵I. The difference in initial atoms to reach the same lethality is mainly due to the longer half-life of ¹²⁵I (60 days) compared with the half-life of ⁶⁴Cu (12.7 h). When we compared with other AE such as ¹²³I, ¹¹¹In, and ^99mTc we observed that the number of initial atoms was less than the required for ⁶⁴Cu: 451 ± 2, 1,625 ± 8, and 1,095 ± 4, respectively, for the three AE. The differences observed are mainly due to the energies and electron yield per decay of each EA in addition to their half-lives. Table 2 compares our calculation of initial atoms and activities and those reported by Humm and Charlton (29) for the AE ¹²⁵I, ¹²³I and ^99mTc. The lower values obtained in this work are due to differences in the nuclear cell model, and the DSB yield results from calculations as seen in Table 1.

Regarding the production of ¹²⁵I, this process is carried out mainly in nuclear reactors; however, this technique presents serious disadvantages due to the long hours of irradiation required, and the production of other radionuclides considered contaminants, such as ¹²⁶I with a half-life of 13.1 days. There are other techniques, such as batch production and continuous systems, however, they also present challenges, such as the low amount of useful ¹²⁵I or the need for two irradiation systems in the nuclear reactor’s core (59). On the other hand, many studies have demonstrated the feasibility of the ⁶⁴Cu production through standardized methods in compact cyclotrons and radiosynthesis modules. The impurities produced after the irradiation of the ⁶⁴Cu are extremely low; additionally, they can produce an appropriate quantity and high quality of ⁶⁴Cu, which is suitable for labeling different ligands to be used in therapy and diagnosis (60, 61).

To our knowledge, the lethality of the ⁶⁴Cu upon localization in DNA has not been reported previously. The electron yield (~0.18/decay) during the decay of ⁶⁴Cu is lower in comparison to the other radionuclides evaluated in this work, mainly the ¹²⁵I (~24/decay), which is consistent with the lower number of DSBs produced when it is incorporated in DNA genome. This apparent disadvantage can be offset by the lower number of initial atoms of ⁶⁴Cu needed to produce the same lethality compared to ¹²⁵I (Table 2). On the other hand, most AE must be conjugated to a proper molecule to be incorporated into the vicinity of the DNA and produce cellular toxicity. ⁶⁴Cu has been extensively studied due to its favorable physical and chemical properties with radiolabeled complexes; it has also been successfully evaluated with a wide variety of biomolecules conjugated with suitable chelators with this positron emitter. However, in recent years, ⁶⁴Cu in the simple chemical form of copper dichloride [⁶⁴Cu]CuCl₂ (without any radiolabeling process) has been identified as a potential agent for TRT because this element is highly regulated naturally at the cellular level through complex molecularly regulated processes that bind and transport copper to different compartments of the cell and cell nuclei, Beaino et al. (62). This fact avoids the need to attach this AE to a specific molecule, thus avoiding the complex process of radiolabeling and the need for expensive target-specific ligands, such as peptides and antibodies, and it would be easy to implement and produce the radiotracer in a suitable form for therapeutic applications. In addition, among the AEs evaluated in this work, apart from ^99mTc, ⁶⁴Cu is the only radionuclide that, due to its radioactive emissions during its decay (including positrons), makes it possible to obtain high-quality Positron Emission Tomography (PET) images while producing lethal effects on cells, which provides a considerable advantage.

The main limitation of this study is the estimation of biological effects of the Auger electron emitters incorporated in the DNA structure, without considering the probability of getting inside there. The number of radioactive nuclei reaching a specific molecular target depends on many factors including the affinity of the vector molecule (or radionuclide itself) for the molecular target, the density of the molecular target or specific receptor, and the amount of radioactivity administered. In the specific case of ⁶⁴Cu, experimental studies performed by Fernandes-Guerreiro et al. (23) showed that [⁶⁴Cu]CuCl₂ is able to reach the nuclear compartment of various PCa cell lines and non-tumoral cells. The percentage of nuclear uptake was cell-dependent and was in the range of 10 to 40%, however it is unknown which percentage of this activity, if any, is incorporated in the DNA structure. Nevertheless, it is possible to consider other approaches to target the DNA structure using molecular vectors such as oligonucleotides, so our assumption of the radioactive nuclei reaching the DNA structure, or its vicinity, is feasible. The next challenge after being capable of binding AEs to DNA in sufficient quantity to cause lethal damage will be to develop suitable methods to estimate the radiation absorbed dose which is an imperative for targeted radionuclide therapy.

DNA damage caused by ⁶⁴Cu incorporated in the genome was quantified in this work through a nuclear mammalian cell model with Monte Carlo track structure simulations. The therapeutic effect of ⁶⁴Cu, based on the D₃₇ value, suggests the ability of this AE to have a lethal effect when incorporated into the DNA genome. The initial activity per cell calculated to cause lethal damage can be used to estimate the total activity necessary to administer in a group of cells or tissue for TRT. On the other hand, although the initial activity of ⁶⁴Cu required to obtain lethality is higher than the required by other AEs analyzed in this work, the number of initial atoms to cause lethal damage is 1/5 times less than the required by ¹²⁵I due to its shorter half- life of 12.7 h for ⁶⁴Cu. Unlike other AE, ⁶⁴Cu emits positrons which allows for PET imaging and provides lethality for cancer cells, making it an excellent candidate for TRT.

Moreover, it is known that copper, due to its chemical properties, is capable of being internalized in cells and nuclei close to DNA without the need to be attached to a vector molecule, unlike other AE radionuclides that need to be attached to molecules to be able to bind to DNA. This characteristic provides a notable advantage, making its production for TRT simpler than other radiopharmaceuticals. However, more studies are needed to understand the molecular processes responsible for its interaction with the DNA molecule and to verify if ⁶⁴Cu, in this form, can bind to DNA in a sufficient quantity to cause lethal damage.

Further studies are required to optimize the subsequent application of ⁶⁴Cu as part of the Targeted Radionuclide Therapy in humans.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

JC-H: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. JR-M: Conceptualization, Formal analysis, Writing – review & editing. EP-R: Investigation, Methodology, Supervision, Writing – review & editing. MA-R: Conceptualization, Funding acquisition, Resources, Writing – review & editing.