Study of ATP borate ester effects on cell sensitization to radiation emitted by a nuclear reactor

Study of ATP borate ester effects on cell sensitization to radiation emitted by a nuclear reactor 1 first-author 1 corresp Corresponding author, yongpeng@szu.edu.cn Corresponding author, yongpeng@szu.edu.cn yongpeng@szu.edu.cn https://orcid.org/0000-0002-7077-5835 1 1 College of Physics and Optoelectronic Engineering, Shenzhen University, NanHai Avenue 3688, Shenzhen 518060, China College of Physics and Optoelectronic Engineering, Shenzhen University, NanHai Avenue 3688, Shenzhen 518060, China ATP (adenosine triphosphate) borate ester as a new boron agent for boron neutron capture therapy was tested. It was synthesized via a dehydration reaction induced by heating adenosine triphosphate disodium with boric acid. Next, ATP borate ester pretreatments were assessed to study their effects on cell sensitization from exposure to thermal neutron irradiation emitted by a nuclear reactor. Using cell viability assays (CCK8), survival rates of A549 cells pretreated with or without boron-containing agents, including ATP borate ester and 4-dihydroxyborylphenylalanine (BPA), were measured. One week after feeding an ATP borate ester solution to tumor-bearing nude mice, elemental B content values of tumor muscle and blood were measured using ICP-MS (Inductively coupled plasma mass spectrometry). Meanwhile, other tumor tissue samples were placed in a culture medium, subjected to a 3-min neutron irradiation exposure, and then fixed in formalin 24 h later for dUTP nick end labeling (TUNEL) immunohistochemical staining analysis. Results showed that A549 cell irradiation sensitization (irradiation dose of 0.33Gy) varied with pretreatment. Sensitization values of the ATP borate ester pretreatment group were 1.3–14.1 with boron agent concentrations of 0.3–4.5 mM. Within 1.1–3.4 mM, ATP borate ester showed significantly higher sensitization values than BPA. Meanwhile, TUNEL results demonstrated that apoptosis rates of tumor tissue cells exposed to irradiation after ATP borate ester pretreatment significantly exceeded the corresponding rates for BPA-pretreated cells. In animal experiments, although the distribution ratio of ATP borate ester (tumor tissue/normal muscle, T/N) of 1.2 was not significantly different compared with that of BPA (1.3), the total ATP borate ester concentration in the tumor tissue (0.79 ± 0.05 μg/g) significantly exceeded that of BPA (0.58 ± 0.05 μg/g). Thus, compared with BPA, the greater enrichment of ATP borate ester in tumor tissues permits preferential targeting toward tumor cells for radiation sensitization. Therefore, ATP borate ester is superior to BPA for use in boron neutron capture therapy. ATP borate ester Boron agent Neutron radiation Sensitizing effect A549 cell lines 1 Introduction 1 Currently, radiotherapy is a routine treatment option for cancer. Its principle utilizes biological effects of ionizing radiation to directly or indirectly cause tumor cell damage. The direct effect is that under high-dose ionizing radiation, the biological body and cell macromolecules will be injured, leading to apoptosis or necrosis of tumor cells [ 1 1 ]. The indirect effect is that the ionizing radiation affects water molecules in organisms to produce free radicals [ 2 2 ] that comprise of unpaired electrons with high reactive activities. Furthermore, these free radicals can damage important components of DNA, proteins, and various biological membranes, thus resulting in the dysfunction of tumor cells [ 3 3 ]. In addition, tumor-associated antigens are released from dead tumor cells after radiotherapy, thereby producing an effect similar to that of an in situ tumor vaccine [ 4 4 ]. However, the prognosis of such treatments for certain tumors (e.g., gliomas or other tumors that develop deep in the body) is poor, while boron neutron capture therapy (BNCT) has demonstrated beneficial effects [ 5 5 ]. BNCT, a unique particle radiation therapy based on 10 B neutron capture response, kills tumor cells more effectively than other radiation therapies. By exploiting the high neutron capture cross-section of 10 B, BNCT relies on the interaction between boron and neutron irradiation, which results in the release of particles that can kill tumor cells [ 6 6 ]. After injecting an animal with a boron agent that accumulates preferentially at tumor sites, 10 B captures neutrons ( 10 B + 1n→7Li + α + γ) and emits α-rays (~2.3 MeV) [ 7 7 ]. Owing to the weak tissue penetration of α-rays, their tissue penetration distance is only 5–9 μm, a distance that is approximately the diameter of a biological cell. Therefore, the energy release of neutron radiation is primarily concentrated around 10 B-enriched tumor cells. However, currently used boron agents (such as BPA) yield only poor enrichment at target tumor sites [ 8 8 ], precluding their widespread use for antitumor BNCT treatment and highlighting the urgent need for better boron agents. Currently, the discovery of highly targeted and safe boron agents with low toxicity has been a bottleneck for the successful development of BNCT. A highly efficient boron agent must satisfy three requirements: First, the attainable concentration of 10 B in tumor cells must be the highest possible. Theoretical simulations, based on the Monte Carlo-based treatment planning system (INEL) program, show that the 10 B atomic dose required to effectively destroy cancer cells is ≥ 20 µg 10 B atoms per gram of tumor tissue, or 1 × 109 10 B atoms per cell [ 9 9 ]. Second, a given agent for tumor cells must exhibit strong selectivity to minimize damage to surrounding normal tissues, and the clearance of 10 B from blood and normal tissues must be more rapid than the time of clearance from tumor tissues. The synergy of these two effects results in a higher concentration of boron in tumor cells than in blood and normal tissues to specifically target the tumor cells. Third, boron drugs must exhibit low toxicity [ 10 10 ]. Hitherto, several generations of boron agents have been developed in rapid succession and yielded two types of agents that are currently in widespread use. One type is 4-dihydroxyborylphenylalanine (BPA), whose structure is related to amino-acid-like styrene-acrylic acid. It has been used in the treatment of glioblastoma, recurrent cancers of the head and neck, and melanoma [ 11 11 ]. In a prospective phase I/II trial of locally recurrent head and neck cancer, 17 BPA-treated patients received BNCT with radiation doses of 63–165 Gy. The complete response rate was 67%, and the overall survival and locoregional control were 47% and 28% at 2 years, respectively. Six patients exhibited acute toxicity [ 11 11 ]. The other is sulfhydryl borane (Na 2 B 12 H 11 SH, BSH), a super hydrophilic anionic boron cluster that has been tested for efficacy in the treatment of malignant glioma [ 12 12 ]. In an oral cancer model animal experiment, BSH (50 mg  10 B/kg) was administered to tumor-bearing mice. The boron concentration ratio tumor/normal tissue was approximately 1.1 [ 13 13 ]. However, these boron agents are not ideal; because the targeting intensity of BPA and BSH for tumor cells is generally low, they can be removed easily from the bloodstream post-administration owing to their low molecular weights and chemical instability [ 14 14 ]. In recent years, a third generation of boron carrier has been rapidly developed. These carriers can bind to antigens or receptors that are specifically expressed by tumors, resulting in a higher boron ratio in tumor cells relative to blood. More importantly, 10 B can be targeted to certain important sites (such as DNA and RNA) within the nuclei of tumor cells through drug uptake, thus reducing the required dose for BNCT [ 15 15 ]. Among these third-generation agents, epidermal growth factor receptor (EGFR)-targeted BPA that binds to epidermal growth factor (EGF) [ 16 16 ], folate receptor (FR)-targeted borate liposomes or boron-containing nanoparticles that bind to folic acid [ 17 17 ], carborane-conjugated polymers with loading of boron atoms [ 18 18 ], and borane-bonded porphyrin with higher hydrophilicity and higher affinity [ 19 19 ] have been widely studied. In these abovementioned studies, for carborane PN150, the accumulation of boron in tumor tissues was 1.2–2.2 μg/g [ 18 18 ], and 0.25–0.275 μg B/ml 10 6 cells for boronated porphyrin (H2OCP) [ 19 19 ]. In this experiment, the ATP borate ester synthesized by heating nucleotide-containing substance ATP disodium with boric acid was tested as a superior third-generation BNCT boron agent. A few studies have been conducted regarding the use of boron-loaded ATP analogs for tumor-specific actions as BNCT agents, e.g., 3-carboranyl thymidine analogues (3CTAs) nucleoside prodrugs with a strong hydrophilicity and lower price [ 20 20 ]; aptamers transcribed with boranophosphate-substituted ribonucleoside analogs (guanosine 5′-(α-P-borano) or uridine 5′-(α-P-borano)) [ 21 21 ]. First, as a small molecule is required for RNA synthesis, nucleotide analogs are easily taken up by rapidly proliferating tumor cells [ 22 22 ], whereby 10 B atoms bound to them can enter tumor cells for effective tumor cell killing via BNCT. Simultaneously, the ATP borate ester, a molecule similar to the nucleotide that acts as an EGFR inhibitor, may competitively inhibit the autophosphorylation of EGFR tyrosine kinase [ 23 23 ] to block tumor growth [ 24 24 , 25 25 ] and bind to tumor cells [ 26 26 ] to enhance radiation sensitization in BNCT. Ester compounds are fully advantageous as boron carriers owing to their strong cell permeability and retention properties [ 27 27 ]. Furthermore, they can bind boron atoms to ATP molecules easily by forming ester bonds. Moreover, ATP borate ester contains three phosphate groups that endow it with high water solubility for enhanced cellular uptake. 2 Materials and methods 1 Adenosine triphosphate was purchased from BBI Life Science Co., Ltd. BPA was donated by Guangdong Dongyangguang Pharmaceutical Co., Ltd. Boric acid was purchased from Tianjin Damao Chemical Reagent Factory. Dimethyl sulfoxide (DMSO) was purchased from BBI Life Science Co., Ltd. Deuterated dimethyl sulfoxide was purchased from Beijing Jinyuxiang Science and Trade Co., Ltd. Ethanol was purchased from the Guangzhou Changjiang Fine Chemical Plant. RPMI-1640 medium was purchased from Sangon Biotech Co., Ltd. Cell Counting Kit-8 (CCK-8) was purchased from BBI Life Science Co., Ltd. Additionally, 30% H 2 O 2 was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Sodium dodecyl sulfate (SDS) lysis buffer was purchased from Sangon Biotech (Shanghai) Co., Ltd. EGFR/HER1/ErbB1 was purchased from Sino Biological Inc., China. A549 human non-small cell lung cancer cells were purchased from Shenzhen Top Biotechnology Co., Ltd. and human non-small cell lung cancer A549 subcutaneous tumor-bearing nude mice were purchased from the Guangdong Province Medical Laboratory Animal Center. A PPS-2510 organic synthesis device was purchased from Gongyi City Yuhua Instrument Co., Ltd. A speed-type mini centrifuge was purchased from Sangon Biotech (Shanghai) Co., Ltd. A Nicolet 6700 Fourier infrared spectrometer was purchased from Thermo Fisher Scientific, America. An AVANCE III 600 MHz superconducting Fourier NMR spectrometer was purchased from Bruker Technology Co., Germany. An Agilent 6210 Quadrupole LC/MS analyzer was obtained from Agilent Technologies Co., Ltd, China. A 754NPC UV-Vis spectrophotometer was obtained from Shanghai Instrument Co., Ltd. A NexION 300X inductively coupled plasma mass spectrometer was obtained from Perkin Elmer Instrument Co., Ltd. The Shenzhen University Microreactor is administered by the Institute of Nuclear Technology of Physics and Energy, Shenzhen University. 2.1 Nuclear reactor experimental device 2 For neutron irradiation, the Shenzhen University micro nuclear reactor and its reaction device, as shown in Fig. 1 Fig. 1 , were used; position 1 is the inner irradiation tube, while the bottom of the tube that is located 23 cm from the core serves as an irradiation point. The sample cell tube shown at position 4 must reach the bottom of the irradiation tube to receive neutron irradiation. The maximum power of the microreactor is 30 kW, and the maximum neutron flux is 1 × 1012 n·cm-2s-1. Glass sheets supporting live adherent cancer cells were soaked in boron agent solutions (untreated cancer cells were used as a blank control), and then placed in test tubes. The culture solution was added to tubes that were then sealed and inserted (one at a time) into the reactor inlet. Each tube was pushed to the bottom of the reactor by the external airflow provided by the pneumatic system. BNCT was initiated by neutron bombardment; subsequently, the sample tubes were blown out through the export tube after a predetermined time. In this experiment, background irradiation was performed when the reactor was shut down to deliver a thermal neutron flux of 1 × 108·cm-2s-1 at the irradiation site. The neutron irradiation process is shown in Fig. 3 Fig. 3 . The water equivalent absorption dose rate at the irradiation site, as measured using a dosimeter, was 0.11 Sv·min-1. 2.2 Synthesis of ATP borate ester 2 2.2.1 Synthesis 3 The ATP borate ester was synthesized in a DMSO solvent by heating to induce dehydration, as shown in Fig. 2 Fig. 2 . After mixing 100 mg of adenosine triphosphate disodium salt with 100 mg of boric acid (at a molar ratio of approximately 1:10), 2000 μl of DMSO was added to form a premixed solution that was then heated at 130 °C for 1.5 h in an organic synthesis apparatus. Next, the mixture was centrifuged, filtered, recrystallized, and then suspended in ethanol to obtain a precipitate. The final product was obtained after drying the precipitate at 80 °C after separation and purification. To increase the integration of boron atoms in the boron ester product, care was taken to avoid the formation of a high molecular weight polymer composed of adenosine triphosphate during heating by maintaining boric acid in excess during the reaction. However, because excess boric acid renders the solution acidic, the solution was recrystallized after several rounds of centrifugal filtration during synthesis, thus resulting in the carryover of residual boric acid into the final product. Therefore, prior to performing cytotoxicity experiments, the pH of the boron solution was adjusted to within a physiological range as necessary, although the cell culture medium buffers used in the experiments would likely minimize any pH-related adverse effects of boric acid on cells. 2.2.2 Identification of ATP borate ester product 3 At room temperature (25℃), infrared spectra of samples were obtained using a Nicolet 6700 Fourier transform infrared spectrometer. Nuclear magnetic resonance 1H and 13 C spectra were obtained using an AVANCE III 600-MHz-type superconducting Fourier nuclear magnetic resonance spectrometer at room temperature with the DMSO solvent, with the chemical shift of the element shown in ppm. The samples were analyzed using mass spectrometry at 30 °C with 0.1% formic acid aqueous solution and methanol eluent at a flow rate of 0.35 ml/min using an Agilent 6210 Quadrupole LC/MS liquid chromatography-mass spectrometer. Structures of the synthesized ATP borate ester products were characterized by a comprehensive analysis of the three types of spectra. 2.3 Study of inhibition of EGFR autophosphorylation by ATP borate ester 2 The ability of the ATP borate ester to inhibit the autophosphorylation of EGFR (p-EGFR) was analyzed using western blot. An autoradiography study was performed on polyacrylamide gel electrophoresis. Samples contained 2 µl of 0.52 mg/ml human EGFR/HER1/ErbB1 (Sino Biological Inc., China) and various volumes of inhibitors, including (5 µl of 4.5 mM ATP borate ester) or (0, 1, 2, or 4 µl of 4.5 mM ATP borate ester with 5 µl of 4.5 mM ATP (BBI Life Science Co., Ltd)) or 5 µl of 4.5 mM BPA (Guangdong Dongyangguang Pharmaceutical Co., Ltd.). Cell proteins from different agent-treated A549 cells were extracted after adding an SDS lysis buffer to the cells, and heating proteins at 100 °C. The proteins were separated by electrophoresis at 100 V for 2 h, transferred into a PVDF membrane at 120 V, and then blocked in BSA for 1 h. EGFR, P-EGFR, and β-actin protein antibodies were added and incubated overnight at 4 °C. After washing with PBST, the membranes were incubated with corresponding secondary antibodies at room temperature for 1 h. Finally, the membranes were developed and scanned to obtain gel images. 2.4 Study of irradiation sensitizing effect of boron neutron radiation 2 2.4.1 Cell lines 3 A549 (EGFR wild type, K-ras mutant) lung cancer cells were purchased from Shenzhen Tuopu Biotechnology Co., Ltd. The cells used for experimentation were cultured in DMEM-HG complete medium (containing 10% FBS and 1% antibiotics). 2.4.2 Changes in viability of cells cultured in boron agent solutions before irradiation 3 The CCK-8 method was used to detect the effects of boron agents on A549 cell viability. The cultured cells adhered to glass sheets of dimensions 40 mm × 10 mm at a density of 106 cells per sheet in RPMI 1640 cell culture medium. Next, the cells were incubated in test tubes containing 4 ml of culture medium for 5 h. Each of the three types of borate agents (ATP borate ester, BPA, and boric acid) was added to the respective test tubes to achieve a final boron agent concentration of 4.5 mM (pH = 7 adjusted with NaOH). Additionally, a positive control group (addition of 50 μl 30% H 2 O 2 ) and a negative control group were set up and cultured at 37 °C. After incubation for 4 h, the CCK-8 reagent was dripped into each test tube to a final proportion of 10% of the total volume; subsequently, the contents were completely mixed and cultured at 37 °C for 4 h. The absorbance of the mixed solution was detected using a 754NPC UV-Vis spectrophotometer (450 nm). Results were compared to assess cell activity changes. 2.4.3 Cell neutron irradiation sensitizing experiment 3 After culturing the cells in various concentrations of the borate agent solutions for 5 h, the sheets of cancer cells were washed sequentially by a step-wise transfer to three test tubes containing the clean culture medium to remove residual agents adhering to cell surfaces. Subsequently, the sheets were placed into new test tubes containing fresh medium (4 ml) and sealed using an electric soldering iron. Samples cultured without borate agents served as the blank irradiation control group. The micro nuclear reactor of Shenzhen University served as a radiation source to deliver a thermal neutron flux of 1 × 108·cm-2s-1 at the irradiation site. The sample tubes were fed individually into the irradiation site of the reactor for low-energy neutron irradiation for 3 min to deliver approximately 0.33 Gy of irradiation per sample. After irradiation, the sample tubes were blown out of the irradiation site chamber. Next, the irradiation sensitizing effects of the three boron agent pretreatments on A549 cell viability were studied using the CCK-8 kit. The process of cell neutron radiation is shown in Fig. 3 Fig. 3 . 2.5 Distribution of boron agents in tumor-bearing mice and sensitizing effect of neutron irradiation on tumor tissues 2 A tumor-bearing nude mice model that incorporates non-small cell lung cancer A549 cells was developed at the Guangdong Medical Laboratory Animal Center (15 g/mouse). The tumors of the mice were cultured until they each grew to a diameter of approximately 10 mm. All the nude mice were females and were randomly divided into three treatment groups: deionized water as the negative control group (a), deionized water + BPA borate solution as group (b), and deionized water + ATP borate ester solution as group (c). The mice were fed 0.1 ml of each boron agent at the same concentration (9 mM, pH = 7), once in the morning and once again in the evening for 4 days. Biological distributions of the tested drugs were determined 3 days after feeding was ceased. The mice were then killed; 0.2 g of tumor tissue, 0.2 g of blood, and 0.2 g of peripheral normal muscle tissue were used for measuring boron content by ICP-MS. Next, 0.5 g of the tumor was placed into 3 ml of culture solution and then sealed and irradiated for 3 min followed by culturing for 24 h. Next, the tumor tissue was removed from the culture and fixed with formalin. The apoptosis of tumor cells was detected by immunohistochemical analysis using the terminal-deoxynucleotidyl transferase (TDT)-mediated nick end labeling (TUNEL) method. All animal experiments were performed in accordance with the relevant international regulations [ 28 28 ]-[ 29 29 ]. 2.6 Statistical analysis 2 The SPSS software (version 21.0) was used for statistical analysis. All values are expressed as mean ± standard deviation (SD). Differences between groups with p < 0.05 are considered to be significant. 3 Results and discussion 1 3.1 Identification of ATP borate ester product 2 3.1.1 Fourier transform infrared analysis (FTIR) 3 The FTIR spectrum of the synthetic product of ATP borate ester synthesis is shown in Fig. 4 Fig. 4 . Figure 4 Figure 4 shows the FTIR spectra of adenosine triphosphate disodium (shown in orange) and synthetic ATP borate ester products (shown in black). Within the area above the curve of the spectrum(shown in black), the characteristic absorption peak of borate ester is visible at 1076 cm -1 , and the symmetrically telescopic vibration peaks of B-O are visible at 949 cm -1 . The stretching vibration peak of O-H is visible at 3431 cm -1 , and is slightly weaker than the corresponding peak of adenosine triphosphate, indicating the involvement of pentose hydroxyl groups. The result indicates that the synthesized product is a borate ester. 3.1.2 Nuclear magnetic resonance spectroscopy 3 The NMR spectra of the synthesized ATP borate ester is shown in Fig. 5 Fig. 5 . Figure 5 (a) Figure 5 (a) shows that δ of 4.55 and 4.24 ppm are the hydroxyl peaks of pentose in ATP. Figure 5 (b) Figure 5 (b) shows that the peaks disappeared, indicating that the hydroxyl group of pentose reacted to form the borate ester. 3.1.3 LC/MS analysis 3 The mass spectrum of the synthetic ATP borate ester is shown in Fig. 6 Fig. 6 . As shown in Fig. 6 (a) Fig. 6 (a) , the solute contains a substance of relative molecular weight 507, identified as ATP. As shown in Fig. 6 (b) Fig. 6 (b) , a substance of molecular weight 631 appears in the product. It is estimated that its structure represents one ATP molecule combined with two boric acid molecules, as shown in Fig. 2 Fig. 2 . As shown in Fig. 6 (c), (d) Fig. 6 (c), (d) , the substance with a molecular weight of 631 was not found at a higher reaction temperature, indicating that the synthesized borate ester may oxidize and decompose when the reaction temperature is higher than 140 °C. 3.2 Results of inhibition of EGFR autophosphorylation by ATP borate ester 2 The ATP borate ester inhibition of EGFR autophosphorylation is shown in Fig. 7 Fig. 7 . The Western blot results seen in Fig. 7 Fig. 7 indicate that ATP does not present any inhibitory effect on the autophosphorylation of EGFR (p-EGFR). In the mixed samples of ATP and the ATP borate ester, the inhibition effects enhanced with the increase in the ATP borate ester proportion, and the ATP borate ester alone exhibited the strongest inhibition. Furthermore, BPA was observed as a weak suppression of p-EGFR. EGFR is a type of receptor tyrosine kinase. As an EGFR inhibitor, the ATP borate ester inhibits phosphorylation by connecting to tyrosine kinase, which competes with ATP. The results indicated that the ATP borate ester could inhibit the autophosphorylation of EGFR. 3.3 Study of irradiation sensitizing effect of boron neutron radiation 2 3.3.1 Activity changes of tumor cells cultured in borate ester solution before irradiation 3 Viability changes of tumor cells cultured in borate ester solution are shown in Table 1 Table 1 . 1 1   1 1 A (H 3 BO 3 ( n = 5)) 1 1 B (BPA  ( n = 5))  1 1 C (ATP borate ester ( n = 5)) 1 1 P 1 1 Cell viability 1 1 92%±5.0% 1 1 90%±6.0% 1 1 88%±6.0% 1 1 > 0.05 The results show that boric acid and borate esters have a marked inhibitory effect on tumor cell activity, compared with the control, with no significant tumor cell activity differences observed between the boric acid and borate ester agents at 5 h of culture. 3.3.2 Cell neutron irradiation sensitizing experiment 3 A549 tumor cells were cultured in a borate ester solution and then washed and placed in a fresh culture medium. The sensitizing results after 3 min of neutron irradiation are shown in Fig. 8 Fig. 8 . The radiation dose rate at the site was 0.11 Gy /min for a total irradiation dose of 0.33 Gy per sample. After cell irradiation experiments were performed using the micro nuclear reactor of Shenzhen University as the neutron source, cell viability was measured using the CCK-8 method and absorbance readings were normalized to the negative and positive control absorbance readings. Fig. 8(a) Fig. 8(a) shows the survival rate of A549 cells exposed to the same dose of 0.33 Gy of neutron irradiation based on various boron agent concentrations. It can be concluded from the diagram that compared with the negative control group (non-boron agent and non-irradiation), neutron irradiation results in cell death, whereby cell viability decreases as boron concentration increases. However, the observed effects are different in several agents. For example, as the boron agent concentration reached a certain value (2.3 mM), the sensitizing effect of the ATP borate ester increased much more rapidly and was significantly higher compared with the corresponding effects of BPA and boric acid (H 3 BO 3 ). Alternatively, as the concentration approached 4.5 mM, the cell mortality of A549 cells in ATP borate ester and BPA culture groups increased significantly. This indicates that the sensitizing effects of the ATP borate ester and BPA significantly exceeded that of H 3 BO 3 . * designates P < 0.05 and indicates a significant difference among groups. Tyrosine is a type of amino acid that is highly enriched in cancer cells, exhibits a high expression of tyrosinase, and can be metabolized by cells. BPA is a tyrosine analog; its structure is shown in Fig. 9 Fig. 9 . At low and medium concentrations, a mediocre irradiation sensitizing effect of BPA was observed, which approximates that of H 3 BO 3 , while high-concentration BPA (4.5 mM) exhibited strong irradiation sensitization power. For ATP borate ester, the agent entered cancer cells more effectively with increasing concentration and may participate in RNA synthesis, resulting in the integration of B atoms in RNA. It is known that radiation will cause genic damage of cells [ 30 30 ]-[ 31 31 ], especially when irradiation is performed specifically on genetic sensitive targets. Consequently, upon irradiation with neutrons, the RNA of cancer cells treated by ATP borate ester may be rapidly broken down to induce significant gene damage and viability loss of tumor cells. Furthermore, in the presence of ATP, EGFR itself becomes more powerful for inducing DNA or RNA decomposition [ 32 32 ], thus inhibiting the genic repair of tumors. Additionally, ATP borate ester is known to compete with ATP to target EGFR tyrosine kinase receptors on the surface of tumor cells [ 23 23 ] and inhibit the autophosphorylation of EGFR ( Fig. 7 Fig. 7 ) such that it can further synergistically suppress tumor. Figure 8(b) Figure 8(b) shows that the sensitizing value (control survival rate/sample survival rate) of the ATP borate ester (2.8–11.0) is significantly higher than that of BPA (1.5–5.1) at low culture concentrations (1–3.5 mM). Within the concentration range of 1.1 to 3.4 mM, the sensitizing value of ATP borate ester exceeded that of BPA by 1.8 fold, while at a higher concentration (4.5 mM), the sensitizing values of ATP borate ester and BPA were both higher, with no significant differences between the two agents. The results indicated that the ATP borate ester exhibited superior radiation sensitization and superior targeting of cancer cell binding than the other two boron agents at low treatment concentrations, owing to the ATP borate ester containing two B atoms per unit molecule and its greater water solubility. Although the sensitization effects may not be optimal at lower concentrations, using a lower concentration of boron agent may help prevent adverse biological side effects. In summary, ATP borate ester appears to be a promising candidate for BNCT. 3.4 Distribution of boron agents in tumor-bearing mice and sensitizing effect of neutron irradiation on tumor tissues 2 Elemental B content values in tumor samples detected by ICP-MS are shown in Table 2 Table 2 . Results of apoptosis detection in tumor cells via the TUNEL immunohistochemical method are shown in Fig. 9 Fig. 9 . 1 1   1 1  B in   blood (µg/g wt) 1 1 B in normal muscle (µg/g wt) 1 1 B   in tumor tissue  (µg/g wt) 1 1 T/N  1 1 Group A 1 1 0.021±0.016 1 1 0.019±0.010 1 1 0.020±0.012 1 1 1.0 1 1 Group B 1 1 2.7±0.06 1 1 0.43±0.03 1 1 0.58±0.05 1 1 1.3 1 1 Group C 1 1 1.6±0.05 1 1 0.68±0.003 1 1 0.79±0.05 1 1 1.2 1 1 P AB , P AC 1 1 <0.05 1 1 <0.05 1 1 <0.05 1 1 1 1 P BC 1 1 <0.05 1 1 <0.05 1 1 <0.05 1 1 T/N, the distribution ratio of boron in tumor tissues and normal muscles, is used to describe the tumor enrichment of boron agents. As shown in Table 2 Table 2 , the relative value of B tumor enrichment (T/N) in mice pretreated with the ATP borate ester is not significantly different from that of mice pretreated with BPA (1.2 and 1.3, respectively). However, for the absolute amount of B uptake by tumor cells (µg/g wt), the ATP borate ester (0.79±0.05µg/g) exhibited a higher value than BPA (0.58±0.05µg/g). Therefore, the ATP borate ester exhibits better irradiation sensitization than BPA. As shown in Fig. 10 Fig. 10 , the percentage of TUNEL-positive tumor cells in group C (ATP borate ester) is significantly higher than the corresponding percentages for group A (control) and group B (BPA), indicating that the irradiation sensitizing effect of the ATP borate ester is stronger than that of BPA owing to the possibility of greater B incorporation in sensitive target molecules (RNA, DNA, etc.). In addition, ATP borate ester exhibits a strong p-EGFR inhibitory effect that results in enhanced radiation damage and apoptosis. The ATP borate ester, synthesized by heating ATP in the presence of boric acid, is an ideal boron carrier for targeting cancer cells. Similar to ATP, ATP borate ester is a binding substrate for EGFR that is highly expressed on cancer cells as a tyrosine kinase receptor. Thus, the ATP borate ester can selectively bind to tumor cells [ 26 26 ]-[ 33 33 ], transporting elemental B to the targets to significantly improve the irradiation sensitizing effect of BNCT. Furthermore, as a p-EGFR inhibitor, this boron agent can effectively inhibit tumor growth and metastasis while exhibiting an irradiation sensitizing effect by inhibiting the repair of radiation-damaged DNA. Additionally, this agent may enter the nucleus and participate in RNA synthesis as a nucleotide analog that results in B incorporation in the RNA of tumor cells. Nevertheless, the specific mechanism is not confirmed in this study. Finally, in addition to its affordability, biocompatibility, water solubility, low toxicity, and degradability, the ATP borate ester can be produced by a simple batch process. In summary, the ATP borate ester can be regarded as a practical and effective next-generation boron-delivery agent for BNCT. 4 Conclusion 1 A new boron-delivery agent for BNCT, i.e., ATP borate ester, was synthesized from boric acid and adenosine triphosphate disodium. For cell irradiation sensitization using a radiation dose of 0.33 Gy, the ATP borate ester sensitization values (control cell survival rate/cell survival rate with boron agent) increased with the concentration of boron agents. In the range of 1.1–3.4 mM, the sensitization value of the ATP borate ester was more than 1.8-fold higher than that of BPA. Furthermore, TUNEL experiments showed that the apoptosis rates of ATP borate-ester-treated tumor cells were significantly higher than the corresponding rates obtained using BPA. 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The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-019-0713-0 http://dx.doi.org/10.1007/s41365-019-0713-0 . 2020 Nuclear Science and Techniques 1 31