Pharmacokinetic, Biodistribution, and toxicity Study of Boronophenylalanine in hepatocellular carcinoma cells and Tumor- Bearing Mouse Model

Pharmacokinetic, Biodistribution, and toxicity Study of Boronophenylalanine in hepatocellular carcinoma cells and Tumor- Bearing Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pharmacokinetic, Biodistribution, and toxicity Study of Boronophenylalanine in hepatocellular carcinoma cells and Tumor- Bearing Mouse Model Tanglong Zhang, Pengcheng Zhang, Huanyu Zhang, Zhuoya Zhang, Xiaodong Jin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6184213/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Boron neutron capture therapy (BNCT) enables dual-targeted heavy-ion radiotherapy at the cellular level, with boronophenylalanine (BPA) as a key boron carrier. Although BPA shows clinical promise in liver cancer, there is relatively little basic research on its effect on hepatocellular carcinoma. Here, we systematically evaluated BPA uptake, safety, and pharmacokinetics. Boron uptake in HCC cells (Hepa1-6, HepG2) was quantified via ICP-AES, revealing concentration- and time-dependent accumulation (plateau at 6 h), while CCK-8 assays indicated significant cytotoxicity at 24 h. Hemolysis experiments confirmed BPA safety (0.1–1 mg/mL), and pharmacokinetic studies in Sprague-Dawley rats showed rapid boron distribution (peak at 25 ± 5.8 min) with a blood half-life of 74.71 ± 52.22 min. In cell-derived xenograft models, BPA achieved tumor-specific targeting, with tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) ratios exceeding 2 and 4, respectively, at 2 h post-injection, followed by rapid systemic clearance. These findings demonstrate BPA selective enrichment in liver tumors, favorable pharmacokinetics, and low toxicity, supporting its clinical utility in BNCT for HCC. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Oncology Boron neutron capture therapy Distribution of drugs HCC Hepa1-6 cells HepG2 BPA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Boron Neutron Capture Therapy (BNCT) represents a biologically targeted binary radiation therapy modality, characterized by its unique treatment mechanism. The therapeutic process involves the selective delivery of boron compounds to tumor cells, followed by irradiation with neutron beams. This interaction induces a nuclear reaction (10B + n → 7Li + 4He(α) + 2.792 MeV), generating high-linear energy transfer (LET) alpha particles and recoil lithium-7 nuclei, which are cytotoxic to tumor cells. Owing to the short penetration range (< 5–10 µm) of these alpha particles and 7Li nuclei, BNCT offers superior sparing of adjacent normal tissues (Fig. 1 ). In clinical practice, boronophenylalanine (BPA) has emerged as a predominant boron delivery agent, demonstrating therapeutic efficacy in various malignancies, including malignant melanoma ( 2 ), malignant brain tumors ( 3 ), recurrent head and neck cancers ( 4 ), and malignant mesothelioma ( 5 ). The therapeutic efficacy of BNCT is contingent upon the preferential accumulation of boron in tumor tissues, with minimal retention in normal tissues. To achieve optimal therapeutic outcomes, boron delivery agents must attain tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) concentration ratios exceeding 2 ( 6 ). Hepatocellular carcinoma (HCC), ranking as the third most prevalent malignancy worldwide, is associated with a dismal 5-year survival rate of merely 18% ( 7 ). The majority of HCC cases are diagnosed at advanced stages, where treatment options are predominantly palliative, encompassing targeted therapy, radiotherapy, and chemotherapy. However, the clinical utility of these modalities is limited by suboptimal efficacy and high recurrence rates ( 8 ). Consequently, the development of novel therapeutic strategies, such as BNCT, is imperative to enhance the clinical management and outcomes of HCC patients. Currently, boronophenylalanine (BPA) and sodium borocaptate (BSH) are the two boron delivery agents used in clinical practice, primarily employed in the treatment of high-grade gliomas, recurrent head and neck tumors, and melanoma. However, due to their limitations in water solubility and targeting efficiency, researchers have been actively developing novel boron delivery agents over the past four decades. These include boron-containing carboranes, porphyrins, amino acids, liposomes, boron-based compounds, and various nanoparticles ( 9 , 10 , 11 ). Despite these advancements, none of these new agents have yet been approved for clinical use. Most of the currently developed targeted boron drugs are modifications and innovations based on the structure of BPA. Therefore, in-depth research on BPA can significantly facilitate the development and clinical translation of next-generation agents. The aim of this study is to investigate the biodistribution of BPA in animal models, evaluate boron uptake and drug toxicity in liver cancer cells and tissues, and provide a foundation for the application of BNCT in liver cancer treatment. Carpano et al. ( 12 ) evaluated BPA uptake in MEL-J, A375, and M83 human melanoma cell lines and conducted biodistribution analysis in mice. Their results demonstrated a time-dependent increase in intracellular boron concentration, with the highest mean tumor boron concentration of 25.9 ± 2.6 µg/g observed 2 hours after BPA injection. Shinichi Terada et al. ( 13 ) explored the distribution of BPA in cervical cancer cells, revealing selective boron uptake in cervical cancer, with peak boron concentrations achieved 2.5 hours after intraperitoneal administration of BPA. Kulvik et al. ( 14 ) reported the biodistribution of boron following intravenous infusion of BPA-F complex in dogs. They found that blood boron concentration decreased over time after infusion initiation but was positively correlated with boron concentrations in the liver, lungs, and kidneys. The Tang team ( 15 ) investigated the distribution of BPA in gastric cancer, showing that blood boron concentrations in rats decreased rapidly within the first 30 minutes and then gradually declined. Boron distribution in various tissues was correlated with blood boron levels, except in the brain, kidneys, and bladder. Chen et al. ( 16 ) assessed the viability of B16 F10 cells co-cultured with BPA for 48 hours and found that BPA at various concentrations had minimal impact on cell viability (less than 10%), indicating its low toxicity. Zhang et al. ( 17 ) conducted a biodistribution study of BPA-F complex in three patients with extremity skin melanoma and reported no adverse reactions during or after BPA infusion. In our study, we observed that BPA exhibited negligible toxicity within the treatment time frame for hepatocellular carcinoma (HCC). Its uptake was concentration-dependent and demonstrated selective targeting in CDX models, providing a basis and dosimetric modeling for the application of BNCT in liver cancer treatment. These findings underscore the potential of BPA as a safe and effective boron delivery agent for BNCT in HCC. Results Drug uptake of BPA in Hepa1-6 and HepG2. The boron concentration in both Hepa1–6 and HepG2 cells was roughly positively correlated with the drug concentration and time. In the initial stage of co - culturing the two types of cells with the drug, the drug was taken up relatively quickly. After 6 hours, the upward trend of drug uptake became gentler, and the uptake reached its maximum at 24 hours. This indicates that the uptake of boron by Hepa1–6 and HepG2 cells is dependent on both concentration and time (Fig. 2 ). BPA is toxic to Hepa1-6 and HepG2 cells. To explore the toxicity of BPA on Hepa1-6 and HepG2 cells, we selected 5 concentration gradients from 0-1000 µg/mL for different times. The results showed that the cell viability decreased significantly at 24 h of co-culture. There is a positive correlation between concentration and time (Fig. 3 ). Analysis of the blood compatibility of BPA. Hemolysis experiments with BPA revealed its low hemolytic toxicity (Fig. 4 ). Erythrocyte hemolysis can predict the blood compatibility of drug carriers after administration of different concentrations. In high concentration (1 mg/mL), the hemolysis rate of BPA was only 1.4%, which showed a slight hemolytic effect but no obvious toxicity. In conclusion, the drug is relatively safe at concentrations below 1 mg/mL, with no erythrocyte agglutination and hemolysis. Thus, hemolysis is negligible at specific concentrations of boron, which makes further in vivo applications possible. Pharmacokinetics of BPA in rats. The analysis of drug distribution results showed that the blood boron concentration of rats injected intraperitoneally with BPA increased rapidly in the first 20 min, slowly increased from 20 to 30 min, reached its peak, and then declined with time (Fig. 5 ). As shown in Table 1 , analyses using non-compartmental models revealed the following boron PK parameters: MRT (0-last) :56.541 ± 1.161 min; MRT (0-∞) :55.798 ± 6.556 min; AUC (0-last) :319.281 ± 14.575 min × µg/mL; AUC (0-∞) :460.707 ± 170.724 min × µg/mL; T 1/2 z : 74.713 ± 52.22 min; Tmax : 25 ± 5.774 min; Cmax : 3.546 ± 0.303 µg/mL; The results showed that boron concentration reached a maximum within the first 30 min after intraperitoneal injection of BPA and then declined with time. Table 1 Pharmacokinetics of BPA in rats Items Unit Mean SD AUC (0-t) min × µg/mL 319.281 14.575 AUC (0-∞) min × µg/mL 460.707 170.724 MRT (0-t) min 56.541 1.161 MRT (0-∞) min 55.798 6.556 T 1/2z min 74.713 52.22 Tmax min 25 5.774 Cmax µg/mL 3.546 0.303 Note: MRT (0-last) : mean dwell time from the start of drug administration to the time point at which the last measurable drug concentration was measured; MRT (0-∞) : mean dwell time from the start of drug administration to complete elimination of drug; AUC (0-last) : the area under the curve from the start of dosing to the last measurable concentration point; AUC (0-∞) : the area under the curve from the beginning of administration to infinity; T 1/2z : elimination half-life; Tmax: the time required to reach the maximum plasma concentration in the body; Cmax: the maximum plasma concentration achieved in the body; Normal distribution of BPA in SD rat tissues. The concentration of BPA in heart, liver, spleen, lung, brain, and blood showed a consistent change with time. The concentration of BPA continued to rise and reached the maximum in the first 30 min after intraperitoneal administration, then showed a decreasing trend, and the drug was almost eliminated from the body at 2.5 hours. However, the kidney boron concentration increased to 14.1 µg/g at 20 min after administration, then decreased to 12.8 µg/g at 30 min, and then increased again after 30 min. From 10 minutes to 150 minutes, the maximum concentration of BPA in the brain was only 2.6 µg/g (Fig. 6 ). Pearson correlations were calculated by using all tissues as variables. There was a significant correlation between blood boron concentration and organs except kidney, heart (r = 0.846, p < 0.05), liver (r = 0.951, p < 0.03), spleen (r = 0.948, p < 0.04), lung (r = 0.949, p 0.05), and brain (r = 0.854, p < 0.03) (Table 2 ). These results suggest that BPA metabolism may be due to excretion through the kidney. These results also indicate that boron accumulation was not found in the tissues after BPA injection ( 2 ). Table 2 All tissues were used as variables for Person correlation calculation blood liver spleen lung kidney brain heart blood 1 liver 0.951 ** 1 spleen 0.948 ** 0.952 ** 1 lung 0.949 ** 0.969 ** 0.991 ** 1 kidney 0.680 0.571 0.664 0.704 1 brain 0.854 * 0.889 * 0.848 * 0.908 * 0.814 * 1 heart 0.846* 0.876 * 0.790 0.857 * 0.761 0.983 ** 1 *P < 0.05;**p < 0.01༛ The distribution of BPA in normal organs and tumors of CDX. To investigate whether BPA preferentially accumulates in tumor tissues over other normal organs, we examined the distribution of boron-containing drugs in tumor-bearing mice at 30 minutes, 1 hour, and 2 hours post-administration. The results demonstrated that boron-containing drugs were significantly enriched in tumor tissues 2 hours after tail vein injection. The boron concentration in tumors (7.52 µg/g) was higher than that in blood (1.87 µg/g), heart (3.31 µg/g), liver (2.28 µg/g), spleen (3.74 µg/g), lung (3.81 µg/g), and brain (3.11 µg/g). The tumor-to-blood or tumor-to-tissue (heart, liver, spleen, lung, brain) boron concentration ratios were 4.02, 2.28, 2.39, 2.13, 2.04, and 2.46, respectively (Fig. 7 ). However, the boron concentration in tumors was comparable to that in the kidneys, with a tumor-to-kidney boron concentration ratio of 0.96, which further supports the notion that boron is metabolized and excreted through the kidneys. These findings suggest that boron is more likely to distribute to tumor tissues than to blood or normal tissues. Tissue safety evaluation of BPA in CDX. Tissues were collected for safety assessment 2 h after the tail vein single injection of BPA (push injection). Histological findings revealed no abnormalities in the structure of the liver (a), brain (b), spleen (c), heart (d), kidney (e), and lung (f) (Fig. 8 ). Discussion BPA is a boronic acid derivative based on the phenylalanine skeleton. Phenylalanine is a high-affinity substrate for LAT1 (L-amino acid transporter). Due to the abnormal amino acid metabolism of tumor cells, LAT1 is highly expressed in tumor cells. Therefore, based on the transport mechanism of LAT1, BPA can be highly enriched in tumor cells with abnormal metabolism ( 18 , 19 ). BPA has achieved good efficacy in recurrent head and neck cancer, brain glioma, skin melanoma and mesothelioma ( 2 , 3 , 4 , 5 ). However, little attention has been paid to the treatment of liver cancer. Suzuki et al. ( 20 ) performed BNCT through boron accumulation in the arteries of liver cancer cells for the first time in clinical practice. They used a single dose of irradiation. After 1 month of treatment, a CT scan showed that the size of the right lobe tumor treated by BNCT was unchanged, and the left lobe tumor treated by chemoembolization was enlarged, which confirmed the feasibility of BNCT for tumor treatment. To improve the efficacy of BNCT in treating liver cancer, we evaluated the concentration and time parameters of BPA in cell and animal models, aiming to prove the possibility of applying BNCT. The biodistribution of BPA in rats indicates that BPA does not stay in the body for a long time after injection. Its average clearance half-life is only 74.7 minutes. Pearson correlation analysis shows that blood boron concentration is positively correlated with boron concentration in other tissues except kidney, suggesting that increasing blood boron concentration may increase boron in tumors. This further suggests that BPA may be cleared in the form of urine. Kulvik et al. ( 21 ) also found that blood boron concentration decreased with time after boron drug infusion, and boron concentration in the brain changed little, which is consistent with our study. Therefore, during BNCT treatment, long- term stable drug administration may further increase tumor boron concentration. We studied the cytotoxicity and drug uptake of different concentrations and different times of BPA co-cultured with Hepa1-6 and HepG2. After co-culturing BPA with Hepa1-6 and HepG2 cells for 24 hours, it exerted obvious toxic effects on both types of cells, and these effects showed a concentration- and time-dependent relationship. The intracellular boron concentration is positively correlated with drug concentration. This provides the best conditions for cell irradiation experiments. This is the first report on the absorption and toxicity of boron in liver cancer cells. Hermawan et al. ( 22 ) also showed preferential enrichment of boron concentration in breast cancer cells. Yoshida et al. ( 23 ) found that in glioma cells, the uptake of BPA by cancer cells was significantly higher than that of normal cells without obvious toxicity. On this basis, Couto et al. ( 24 ) has developed a bimodal therapeutic agent for glioblastoma, whose uptake is 1.7 times that of BPA and which significantly improves the survival rate. When the drug concentration is lower than 1 mg/mL, as the drug concentration increases, the degree of hemolysis gradually increases. The maximum hemolysis rate is 1.4%, but no obvious hemolysis is observed in vitro. This indicates that the drug has little effect on the stability of red blood cells at low concentrations and has a certain degree of safety. For BNCT treatment, a high boron content in tumors is the key to treatment. The boron concentration ratio of tumor/blood or tumor/normal tissue should be at least greater than 2 times. In the CDX model, we compared single injection for 30 minutes, 1 hour and 2 hours. The results showed that the boron concentration in tumors with single injection for 2 hours was significantly higher than that in blood and other tissues (heart, liver, spleen, lung, brain). The ratio of the tumor to the tissue is greater than 2. Wang et al. ( 25 ) reported that the uptake of BPA in F98 glioma reached the maximum value 1 hour after administration. Huang et al. ( 26 ) indicated that BNCT mainly eliminates radiation-resistant liver cancer cells by targeting DNA damage and repair responses. Alamón et al. ( 27 ) synthesized the boron-rich selective epidermal growth factor receptor (EGFR)- inhibitor hybrid 1. In U87 cells and tumor-bearing rats, it exhibited an enrichment ability superior to that of BPA, and significantly prolonged the survival period of the rats after BNCT treatment. The possibility of boron distribution in tumor tissues is higher than that in blood and normal tissues. BNCT treatment of HCC is feasible. At the same time, a large number of clinical and experimental studies have proved that BPA is safe ( 28 ). At the same time, we did not observe any abnormal structures such as brain, heart, lung, liver, kidney, and spleen on the paraffin sections of normal tissues of mice after administration, which once again proves its safety at the histological level. Conclusions In summary, the results indicate that BPA is preferentially enriched in HCC cells without significant toxicity, providing a protocol and rationale for BNCT treatment of HCC. In the future, detailed molecular experiments are needed to deeply explore the molecular mechanism by which Boron Neutron Capture Therapy (BNCT) kills liver cancer cells. Methods Materials Hepa1-6 and HepG-2 cells were purchased from Shanghai Beyotime Biotechnology Co., Ltd, which were cultured in DMEM (Dalian Meilun Biotechnology Co., Ltd) medium containing 10% fetal bovine serum (Wuhan Servicebio Technology Co., Ltd) at 37℃ and 5% CO 2 . The Ethics committee of the First Hospital of Lanzhou University approved the animal experiment plan in this paper. Eight-week-old female SD rats and 6-week-old female nude rats were purchased from Lanzhou Institute of Animal Research in Gansu Province, and the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were conducted in accordance with the ARRIVE guidelines. Measurements of the Boron Concentration. The tissue samples containing BPA were mixed with the appropriate concentrated nitric acid at 55°C for 1 hour and subsequently placed in a 55°C water bath for 4 hours. Once the tissue was completely dissolved, the solution was calibrated with ultra-pure water. The boron concentration in each sample was determined by using ICP-AES. The culture dishes were collected at different time points during the co-culture of cells and BPA. The cells were gently rinsed with physiological saline three times. An appropriate amount of concentrated nitric acid was added, and then the dishes were placed in an oven at 55°C and shaken for 40 minutes. The cells were collected with ultra-pure water and transferred into a 15 mL centrifuge tube. The volume was uniformly calibrated to 10 mL. Finally, the boron content was detected by ICP-AES. Preparation of BPA-fructose complex solution. The 10 B-labeled BPA ( 10 B-BPA) was provided by the Key Laboratory of Heavy Ion Radia-tion Biomedicine, Institute of Modern Physics, Lanzhou Academy of Sciences, China (Fig. 9 ). BPA and fructose (Shanghai Beyotime Biotechnology Co., Ltd) were first mixed in ultra-pure water at a molar ratio of 1:1.5, and then 1N NaOH was added to pH 10.5 under continuous stirring, followed by titration to pH 7.6 with 1N HCl. Finally, the solution was filtered and sterilized using a 0.22 µm filter. The concentration of boron prepared was 1 mg/mL. All the BPA appearing in the subsequent experiments was prepared by the fructose-complex method. Uptake and toxicity of BPA on Hepa1-6 and HepG2. Hepa1-6 is a murine hepatoma cell line derived from BW7756 hepatoma tumor, HepG2 is derived from human hepatocellular carcinoma tissue, and boron intake was determined during exponential growth phase. The cells in logarithmic growth phase were digested, and spread on 6-well plate. Co-cultured with BPA (at concentrations of 1, 5, 10, 25, 50, 100, 200 µg/mL) for 1, 3, 6, and 12 hours respectively. At the end of co-culture, the cells were washed three times with precooled saline, 500 µL concentrated nitric acid was added and placed in an oven at 55°C for 40 min with shaking, and then the volume was fixed with ultra-pure water. Boron concentration in cells using ICP-AES and expressed as µg/10 7 cells. The number of cells was counted with Neubauer’s chamber. The cells in logarithmic growth phase were digested, and 2.5×10 4 /mL cell suspension was spread on a 96-well plate, and 200 µL cell suspension was added to each well. After the cells were attached to the well, the supernatant was discarded, and the prepared BPA (0.1, 1, 5, 10, 25, 50, 100, 200, 500 µg/mL) was added to the 96-well plate, and 150 µL was added to each well. After 3, 6, 12, and 24 hours of co-culture, 100 µL of 10% CCK8 (a reagent for cell proliferation and cytotoxicity detection) was added. The samples were incubated in a 37°C incubator in the dark for 1–3 hours. Then, the absorbance was measured at 450 nm using an enzyme-labeled instrument. Graphpad Prism was used for plotting. In vitro hemolysis of BPA. Blood was collected from the orbital venous plexus of four nude rats respectively. The blood was centrifuged at 3000 rpm for 10 min, rinsed, and centrifuged repeatedly with normal saline, and red blood cells were collected and diluted to 4% red blood cell suspension with normal saline. Saline solutions containing 0.1, 0.2, 0.5, and 1mg/mL BPA were prepared. An equal volume of red blood cell suspension was mixed with various concentrations of BPA solutions. Co-cultured for 4 hours at 37°C. Then the red blood cell suspension with distilled water was set as the positive control group, and the red blood cell suspension with normal saline was set as the negative control. Then, the mixture was centrifuged at 3000 rpm for 10 min, measure the absorbance of the supernatant at 540 nm. According to the measured absorbance value, the hemolysis rate of each concentration of drug was calculated by the formula: Hemolysis rate (%) = (A sample-A negative) / (A positive-A negative) ×100%. The pharmacokinetic characteristics of boron in SD rats following tail vein injection of BPA. Thirty 8-week-old female SD rats were intravenously injected with BPA at a dose of 500 mg/kg using a tail vein injection method. The injection was administered via intravenous push. Under sodium pentobarbital anesthesia, blood was collected from the apex of the heart, and organs such as the heart, liver, spleen, lung, kidney, and brain were harvested at 10, 20, 30, 60, 90, and 150 minutes after administration. ICP-AES was used for detection. One milliliter of blood was taken to determine the boron concentration, which was expressed as µg/g of blood or tissue. Pharmacokinetic parameters were calculated using Winnonlin software with a non-compartment model, and a curve of boron concentration as a function of time was plotted. Pearson correlation calculations were performed using all tissues as variables. Biodistribution of BPA in normal tissues and tumors of CDX. To establish a subcutaneous animal model, Hepa1-6 cells (2×10 7 /mL) in the exponential growth phase were subcutaneously injected (push injection) into 6week-old nude rats (Fig. 10 ). To evaluate boron uptake, BPA (500 mg/kg) was injected via the tail vein and sacrificed 0.5, 1 and 2 h after injection. Boron concentrations in 100 µl blood and tumor or normal tissue were measured using ICP-AES and expressed as µg/g tissue. Safety of BPA in CDX. In order to verify the safety of BPA at the dose used in this study, we selected 9 nude rats bearing tumors and conducted 3 replicate experiments. The nude rats were injected with BPA (500 mg/kg) via the tail vein at 2 h after injection, and different organs were extracted and paraffin sections were prepared to observe histological damage. Statistical analysis. Data are expressed as mean ± SD. Two-way analysis of variance (ANOVA) was performed to evaluate the significance of the differences. Winnonlin software was used to analyze the pharmacokinetic parameters. Statistical analysis included Graphpad Prism 8, SPSS26.0 statistical software, and p values < 0.05 were considered significant. Declarations Author Contributions Conceptualization, Tanglong Zhang, Huanyu Zhang and Juntao Ran; Formal analysis, Pengcheng Zhang; Investigation, Tanglong Zhang and Huanyu Zhang; Meth-odology, Tanglong Zhang and Zhuoya Zhang; Project administration, Juntao Ran and Xiaodong Jing; Resources, Pengcheng Zhang and Ting Zhao; Software, Pengcheng Zhang; Supervision, Juntao Ran; Writing – original draft, Tanglong Zhang and Zhuoya Zhang; Writing – review & editing, Juntao Ran. Funding This research was funded in part by and the National Natural Science Foundation of China (NSFC), grant number 82360583, the Lanzhou University Medical Research Innovation Capacity Enhancement Program, grant number lzuyxcx-2022-159, Natural Science Foundation of Gansu Province of China (Grant No. 23JRRA0952) and The First Hospital of Lanzhou University Intramural Fund, Excellent Doctoral Start-up Fund (Grant No. ldyyyn2022-91）. Ethics declarations Competing interests The authors declare no competing interests. Institutional Review Board Statement: The animal study protocol was approved by The Committee on the Ethics of Animal Experiments of Lanzhou University First Hospital (LDYYLL2024-684). We used isoflurane inhalation anesthetic to euthanize the experimental mice. D ata a vailability The data supporting the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments The authors would like to thank Boyi Yu for his expert technical assistance in the animal modeling experiments. References Yura, Y. & Fujita, Y. Boron neutron capture therapy as a novel modality of radiotherapy for oral cancer: Principle and antitumor effect. Oral Sci. Int. 10 , 9–14 (2013). Morita, T. et al. Dynamic changes in 18F-borono-L-phenylalanine uptake in unresectable, advanced, or recurrent squamous cell carcinoma of the head and neck and malignant melanoma during boron neutron capture therapy patient selection. Radiat. Oncol. 13 , 4 (2018). Lan, T. L. et al. Using salvage Boron Neutron Capture Therapy (BNCT) for recurrent malignant brain tumors in Taiwan. Appl. Radiat. Isot. 160 , 109105 (2020). Hirose, K. et al. Boron neutron capture therapy using cyclotron-based epithermal neutron source and borofalan (10B) for recurrent or locally advanced head and neck cancer (JHN002): An open-label phase II trial. Radiother Oncol. 155 , 182–187 (2021). Suzuki, M. et al. Feasibility of boron neutron capture therapy (BNCT) for malignant pleural mesothelioma from a viewpoint of dose distribution analysis. *Int J. Radiat. Oncol. Biol. Phys. * **66** , 1584–1589 (2006). Beck-Sickinger, A. G. et al. New Boron Delivery Agents. Cancer Biother Radiopharm . 38 , 160–172 (2023). Ferlay, J. et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int. J. Cancer . 144 , 1941–1953 (2019). Chen, S., Cao, Q., Wen, W. & Wang, H. Targeted therapy for hepatocellular carcinoma: Challenges and opportunities. Cancer Lett. 460 , 1–9 (2019). Barth, R. F., Mi, P. & Yang, W. Boron delivery agents for neutron capture therapy of cancer. Cancer Commun. 38 , 35 (2018). Couto, M. & Cerecetto, H. Advancements in the Synthesis and Biological Properties of Carboranes and HighBoron Related Compounds: A Comprehensive Exploration with Emphasis on BNCT Applications. J. Braz Chem. Soc. 10.21577/0103-5053.20240109 (2024). Boron-Based Compounds: Potential and Emerging Applications in Medicine . (Wiley, Hoboken, (2018). Carpano, M. et al. Experimental Studies of Boronophenylalanine ((10)BPA) Biodistribution for the Individual Application of Boron Neutron Capture Therapy (BNCT) for Malignant Melanoma Treatment. Int. J. Radiat. Oncol. Biol. Phys. 93 , 344–352 (2015). Terada, S. et al. Boron uptake of boronophenylalanine and the effect of boron neutron capture therapy in cervical cancer cells. Appl. Radiat. Isot. 197 , 110792 (2023). Kulvik, M. et al. Biodistribution of boron after intravenous 4-dihydroxyborylphenylalanine-fructose (BPA-F) infusion in meningioma and schwannoma patients: A feasibility study for boron neutron capture therapy. Appl. Radiat. Isot. 106 , 207–212 (2015). Tang, F. et al. Evaluation of Pharmacokinetics of Boronophenylalanine and Its Uptakes in Gastric Cancer. Front. Oncol. 12 , 925671 (2022). Chen, J. et al. Unlocking the potential: phenylboronic acid as a nuclear-targeting boron agent for neutron capture therapy. Med. Oncol. 41 , 104 (2024). Zhang, Z. et al. Biodistribution studies of boronophenylalanine in different types of skin melanoma. Appl. Radiat. Isot. 163 , 109215 (2020). Liu, Z. et al. Boramino acid as a marker for amino acid transporters. Sci. Adv. 1 , e1500694 (2015). Nomoto, T. et al. Poly(vinyl alcohol) boosting therapeutic potential of p-boronophenylalanine in neutron capture therapy by modulating metabolism. Sci. Adv. 6 , eaaz1722 (2020). Suzuki, M. et al. First attempt of boron neutron capture therapy (BNCT) for hepatocellular carcinoma. Jpn J. Clin. Oncol. 37 , 376–381 (2007). Kulvik, M. E. et al. Boron biodistribution in beagles after intravenous infusion of 4-dihydroxyborylphenylalanine-fructose complex. Appl. Radiat. Isot. 61 , 975–979 (2004). Hermawan, A. et al. Cellular uptake evaluation of pentagamaboronon-0 (PGB-0) for boron neutron capture therapy (BNCT) against breast cancer cells. Invest. New. Drugs . 37 , 1292–1299 (2019). Yoshida, F. et al. Difference in BPA uptake between glioma stem-like cells and their cancerous cells. Appl. Radiat. Isot. 164 , 109234 (2020). Couto, M. et al. Bimodal Therapeutic Agents Against Glioblastoma, One of the Most Lethal Forms of Cancer. Chemistry 26 , 14335–14340 (2020). Wang, H. E. et al. Evaluation of 4-borono-2-18F-fluoro-L-phenylalanine-fructose as a probe for boron neutron capture therapy in a glioma-bearing rat model. J. Nucl. Med. 45 , 302–308 (2004). Huang, C. Y. et al. Boron Neutron Capture Therapy Eliminates Radioresistant Liver Cancer Cells by Targeting DNA Damage and Repair Responses. J. Hepatocell Carcinoma . 9 , 1385–1401 (2022). Alamón, C. et al. A Potential Boron Neutron Capture Therapy Agent Selectively Suppresses High-Grade Glioma: In Vitro and in Vivo Exploration. Mol. Pharm. 20 , 2702–2713 (2023). Malouff, T. D. et al. Boron Neutron Capture Therapy: A Review of Clinical Applications. Front. Oncol. 11 , 601820 (2021). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6184213","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449842220,"identity":"c664eb2c-7252-4ef2-876d-8b2a5a623a2e","order_by":0,"name":"Tanglong Zhang","email":"","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Tanglong","middleName":"","lastName":"Zhang","suffix":""},{"id":449842221,"identity":"cd1f65f4-a4d7-482e-a3ea-cde98cbe2acf","order_by":1,"name":"Pengcheng Zhang","email":"","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Pengcheng","middleName":"","lastName":"Zhang","suffix":""},{"id":449842222,"identity":"d3cd9b6d-b1de-4273-bf6d-b239606ecb77","order_by":2,"name":"Huanyu Zhang","email":"","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Huanyu","middleName":"","lastName":"Zhang","suffix":""},{"id":449842223,"identity":"8d7cdc72-7b6f-4843-a8d6-b5e1ce2b7f2a","order_by":3,"name":"Zhuoya Zhang","email":"","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhuoya","middleName":"","lastName":"Zhang","suffix":""},{"id":449842224,"identity":"a980e67f-6e3d-4c46-ad71-0b5cc6a4a3b5","order_by":4,"name":"Xiaodong Jin","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Jin","suffix":""},{"id":449842225,"identity":"658a1544-f04a-446c-a913-6ceac20c5ba1","order_by":5,"name":"Ting Zhao","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Zhao","suffix":""},{"id":449842226,"identity":"b193f99c-d036-4d7d-af74-9818ba0de144","order_by":6,"name":"Juntao Ran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYJACA4YKCzBDgngtB85IkKiF4WAbKVrM288eKP44T8Le4ADzwds8DHZ5BLXInMlLMDi4TYLZ4ABbsjUPQ3IxQS0SDDkGIC1sBgd4zKR5GA4kNhDUwv8GqGWOBI/BAf5vRGqRANnSICEBtIWNWC1AW84ckzCQPMxmbDnHIJkYh+WYGVTU2NjzHW9+eONNhR1hLUDAZgCmmEGEARHqQWofEKduFIyCUTAKRiwAAAGKM4ybEUEUAAAAAElFTkSuQmCC","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Juntao","middleName":"","lastName":"Ran","suffix":""}],"badges":[],"createdAt":"2025-03-08 12:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6184213/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6184213/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82001203,"identity":"4ab560e6-a459-45e3-a695-1046495b5160","added_by":"auto","created_at":"2025-05-05 19:47:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1958527,"visible":true,"origin":"","legend":"\u003cp\u003eNeutrons combine with boron-bearing tumor cells to undergo nuclear fission.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/ec043e7516a04685fb221f8d.jpg"},{"id":82002285,"identity":"b988b054-cba6-45d8-8d88-701bbbf064d9","added_by":"auto","created_at":"2025-05-05 20:11:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74603,"visible":true,"origin":"","legend":"\u003cp\u003eDrug uptake of BPA at different concentrations and different times in Hepa1-6 and HepG2 cells.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/69dd4c4890b3729b811db5dd.jpg"},{"id":82001206,"identity":"cb11896f-0413-4e35-91d8-509a78d4e6cf","added_by":"auto","created_at":"2025-05-05 19:47:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74708,"visible":true,"origin":"","legend":"\u003cp\u003eThe comparison of cell viability after co-culturing BPA with Hepa1-6 and HepG2 cells; the figure displays the cell viability values measured after 3 hours, 6 hours, 12 hours, and 24 hours of co-culture.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/febc9e3ca2dae2dfa4faf0c6.jpg"},{"id":82001884,"identity":"a15908f4-d7f5-4e12-a79a-bfb9f948fce8","added_by":"auto","created_at":"2025-05-05 20:03:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112376,"visible":true,"origin":"","legend":"\u003cp\u003eA: a, b, c, d were 0.1, 0.2, 0.5, 1 mg/mL BPA; e is the negative control added with normal saline. f represents the positive control added with distilled water. B: degree of hemolysis at different concentrations of BPA; The BPA concentration (0.1-1 mg/mL); the data (n = 4) are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/9c2c1119ea5f24b229d693ae.jpg"},{"id":82001886,"identity":"10ffe916-3645-4476-91f8-dacd907c0208","added_by":"auto","created_at":"2025-05-05 20:03:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54148,"visible":true,"origin":"","legend":"\u003cp\u003eCurves of boron concentration in rats' blood as a function of time\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/7017b373427a87463418a75a.jpg"},{"id":82001586,"identity":"40b5711a-143d-4e61-8dfb-97cb5c489762","added_by":"auto","created_at":"2025-05-05 19:55:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112301,"visible":true,"origin":"","legend":"\u003cp\u003eThe boron concentration-time curves of BPA in the heart, liver, spleen, lung, kidney, brain, and blood of rats.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/5cc89114f05bb6e524b431de.jpg"},{"id":82001207,"identity":"71b6aae7-f27b-4c0c-ada0-c3b1f45a5835","added_by":"auto","created_at":"2025-05-05 19:47:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":90989,"visible":true,"origin":"","legend":"\u003cp\u003eObserve the boron content in normal tissues and tumors at 30 minutes, 1 hour and 2 hours after tail-vein injection of BPA in tumor-bearing mice; and observe the ratios of tumor to normal tissue (T/N) and tumor to blood (T/B).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/5f9bbec6c97343cd472eb6c8.jpg"},{"id":82001221,"identity":"b21fad1c-c997-4031-9de6-63ccb8b53535","added_by":"auto","created_at":"2025-05-05 19:47:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":720504,"visible":true,"origin":"","legend":"\u003cp\u003eParaffin sections of normal tissues from rats injected with BPA 2h after tail vein injection.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/e3966bb2d47c5f233e8ff0db.jpg"},{"id":82001888,"identity":"5c615563-bd7f-4714-b82a-90b09fd66e14","added_by":"auto","created_at":"2025-05-05 20:03:30","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":62461,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of BPA.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/6a141def37d9c3e72ef79e8a.jpg"},{"id":82001231,"identity":"98b408be-3954-45e5-b8da-7262f7724d4b","added_by":"auto","created_at":"2025-05-05 19:47:30","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":230347,"visible":true,"origin":"","legend":"\u003cp\u003eSubcutaneous tumor model of liver cancer in rats (CDX). We selected 27 nude rats and carried out three independent replicate experiments. The injection method adopted was intravenous push injection. Each time, 9 rats were divided into 3 groups, namely the 30-minutes post-BPA injection group, the 1-hour post-BPA injection group, and the 2-hours post-BPA injection group. minutes.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/91d9a95bfc16515df77a0eed.jpg"},{"id":82002497,"identity":"82a72a1d-8f9c-4494-a0f1-7025c919ec77","added_by":"auto","created_at":"2025-05-05 20:19:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3932508,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/9e55dc10-da21-4e65-8d7e-cc188be5bec8.pdf"},{"id":82001588,"identity":"ba71c282-5ba1-4941-ab90-4a85c5ffd2d3","added_by":"auto","created_at":"2025-05-05 19:55:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":237694,"visible":true,"origin":"","legend":"","description":"","filename":"ApprovalbyEthicsCommitteeofLZUNo.1Hospital.docx","url":"https://assets-eu.researchsquare.com/files/rs-6184213/v1/9beb08f68d50f6926f147d42.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pharmacokinetic, Biodistribution, and toxicity Study of Boronophenylalanine in hepatocellular carcinoma cells and Tumor- Bearing Mouse Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBoron Neutron Capture Therapy (BNCT) represents a biologically targeted binary radiation therapy modality, characterized by its unique treatment mechanism. The therapeutic process involves the selective delivery of boron compounds to tumor cells, followed by irradiation with neutron beams. This interaction induces a nuclear reaction (10B\u0026thinsp;+\u0026thinsp;n \u0026rarr; 7Li\u0026thinsp;+\u0026thinsp;4He(α)\u0026thinsp;+\u0026thinsp;2.792 MeV), generating high-linear energy transfer (LET) alpha particles and recoil lithium-7 nuclei, which are cytotoxic to tumor cells. Owing to the short penetration range (\u0026lt;\u0026thinsp;5\u0026ndash;10 \u0026micro;m) of these alpha particles and 7Li nuclei, BNCT offers superior sparing of adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn clinical practice, boronophenylalanine (BPA) has emerged as a predominant boron delivery agent, demonstrating therapeutic efficacy in various malignancies, including malignant melanoma (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), malignant brain tumors (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), recurrent head and neck cancers (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), and malignant mesothelioma (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The therapeutic efficacy of BNCT is contingent upon the preferential accumulation of boron in tumor tissues, with minimal retention in normal tissues. To achieve optimal therapeutic outcomes, boron delivery agents must attain tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) concentration ratios exceeding 2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHepatocellular carcinoma (HCC), ranking as the third most prevalent malignancy worldwide, is associated with a dismal 5-year survival rate of merely 18% (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The majority of HCC cases are diagnosed at advanced stages, where treatment options are predominantly palliative, encompassing targeted therapy, radiotherapy, and chemotherapy. However, the clinical utility of these modalities is limited by suboptimal efficacy and high recurrence rates (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Consequently, the development of novel therapeutic strategies, such as BNCT, is imperative to enhance the clinical management and outcomes of HCC patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCurrently, boronophenylalanine (BPA) and sodium borocaptate (BSH) are the two boron delivery agents used in clinical practice, primarily employed in the treatment of high-grade gliomas, recurrent head and neck tumors, and melanoma. However, due to their limitations in water solubility and targeting efficiency, researchers have been actively developing novel boron delivery agents over the past four decades. These include boron-containing carboranes, porphyrins, amino acids, liposomes, boron-based compounds, and various nanoparticles (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Despite these advancements, none of these new agents have yet been approved for clinical use. Most of the currently developed targeted boron drugs are modifications and innovations based on the structure of BPA. Therefore, in-depth research on BPA can significantly facilitate the development and clinical translation of next-generation agents. The aim of this study is to investigate the biodistribution of BPA in animal models, evaluate boron uptake and drug toxicity in liver cancer cells and tissues, and provide a foundation for the application of BNCT in liver cancer treatment. Carpano et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) evaluated BPA uptake in MEL-J, A375, and M83 human melanoma cell lines and conducted biodistribution analysis in mice. Their results demonstrated a time-dependent increase in intracellular boron concentration, with the highest mean tumor boron concentration of 25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 \u0026micro;g/g observed 2 hours after BPA injection. Shinichi Terada et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) explored the distribution of BPA in cervical cancer cells, revealing selective boron uptake in cervical cancer, with peak boron concentrations achieved 2.5 hours after intraperitoneal administration of BPA. Kulvik et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) reported the biodistribution of boron following intravenous infusion of BPA-F complex in dogs. They found that blood boron concentration decreased over time after infusion initiation but was positively correlated with boron concentrations in the liver, lungs, and kidneys. The Tang team (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) investigated the distribution of BPA in gastric cancer, showing that blood boron concentrations in rats decreased rapidly within the first 30 minutes and then gradually declined. Boron distribution in various tissues was correlated with blood boron levels, except in the brain, kidneys, and bladder. Chen et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) assessed the viability of B16 F10 cells co-cultured with BPA for 48 hours and found that BPA at various concentrations had minimal impact on cell viability (less than 10%), indicating its low toxicity. Zhang et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) conducted a biodistribution study of BPA-F complex in three patients with extremity skin melanoma and reported no adverse reactions during or after BPA infusion.\u003c/p\u003e \u003cp\u003eIn our study, we observed that BPA exhibited negligible toxicity within the treatment time frame for hepatocellular carcinoma (HCC). Its uptake was concentration-dependent and demonstrated selective targeting in CDX models, providing a basis and dosimetric modeling for the application of BNCT in liver cancer treatment. These findings underscore the potential of BPA as a safe and effective boron delivery agent for BNCT in HCC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDrug uptake of BPA in Hepa1-6 and HepG2.\u003c/p\u003e \u003cp\u003eThe boron concentration in both Hepa1\u0026ndash;6 and HepG2 cells was roughly positively correlated with the drug concentration and time. In the initial stage of co - culturing the two types of cells with the drug, the drug was taken up relatively quickly. After 6 hours, the upward trend of drug uptake became gentler, and the uptake reached its maximum at 24 hours. This indicates that the uptake of boron by Hepa1\u0026ndash;6 and HepG2 cells is dependent on both concentration and time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBPA is toxic to Hepa1-6 and HepG2 cells.\u003c/p\u003e \u003cp\u003eTo explore the toxicity of BPA on Hepa1-6 and HepG2 cells, we selected 5 concentration gradients from 0-1000 \u0026micro;g/mL for different times. The results showed that the cell viability decreased significantly at 24 h of co-culture. There is a positive correlation between concentration and time (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the blood compatibility of BPA.\u003c/p\u003e \u003cp\u003eHemolysis experiments with BPA revealed its low hemolytic toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Erythrocyte hemolysis can predict the blood compatibility of drug carriers after administration of different concentrations. In high concentration (1 mg/mL), the hemolysis rate of BPA was only 1.4%, which showed a slight hemolytic effect but no obvious toxicity. In conclusion, the drug is relatively safe at concentrations below 1 mg/mL, with no erythrocyte agglutination and hemolysis. Thus, hemolysis is negligible at specific concentrations of boron, which makes further in vivo applications possible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePharmacokinetics of BPA in rats.\u003c/p\u003e \u003cp\u003eThe analysis of drug distribution results showed that the blood boron concentration of rats injected intraperitoneally with BPA increased rapidly in the first 20 min, slowly increased from 20 to 30 min, reached its peak, and then declined with time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, analyses using non-compartmental models revealed the following boron PK parameters: MRT (0-last) :56.541\u0026thinsp;\u0026plusmn;\u0026thinsp;1.161 min; MRT (0-\u0026infin;) :55.798\u0026thinsp;\u0026plusmn;\u0026thinsp;6.556 min; AUC (0-last) :319.281\u0026thinsp;\u0026plusmn;\u0026thinsp;14.575 min\u0026thinsp;\u0026times;\u0026thinsp;\u0026micro;g/mL; AUC (0-\u0026infin;) :460.707\u0026thinsp;\u0026plusmn;\u0026thinsp;170.724 min\u0026thinsp;\u0026times;\u0026thinsp;\u0026micro;g/mL; T\u003csub\u003e1/2\u003c/sub\u003ez : 74.713\u0026thinsp;\u0026plusmn;\u0026thinsp;52.22 min; Tmax : 25\u0026thinsp;\u0026plusmn;\u0026thinsp;5.774 min; Cmax : 3.546\u0026thinsp;\u0026plusmn;\u0026thinsp;0.303 \u0026micro;g/mL; The results showed that boron concentration reached a maximum within the first 30 min after intraperitoneal injection of BPA and then declined with time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePharmacokinetics of BPA in rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC (0-t)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emin\u0026thinsp;\u0026times;\u0026thinsp;\u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e319.281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.575\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC (0-\u0026infin;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emin\u0026thinsp;\u0026times;\u0026thinsp;\u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e460.707\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e170.724\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRT (0-t)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.161\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRT (0-\u0026infin;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003e1/2z\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTmax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.774\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCmax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.303\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: MRT (0-last) : mean dwell time from the start of drug administration to the time point at which the last measurable drug concentration was measured; MRT (0-\u0026infin;) : mean dwell time from the start of drug administration to complete elimination of drug; AUC (0-last) : the area under the curve from the start of dosing to the last measurable concentration point; AUC (0-\u0026infin;) : the area under the curve from the beginning of administration to infinity; T\u003csub\u003e1/2z\u003c/sub\u003e: elimination half-life; Tmax: the time required to reach the maximum plasma concentration in the body; Cmax: the maximum plasma concentration achieved in the body;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNormal distribution of BPA in SD rat tissues.\u003c/p\u003e \u003cp\u003eThe concentration of BPA in heart, liver, spleen, lung, brain, and blood showed a consistent change with time. The concentration of BPA continued to rise and reached the maximum in the first 30 min after intraperitoneal administration, then showed a decreasing trend, and the drug was almost eliminated from the body at 2.5 hours. However, the kidney boron concentration increased to 14.1 \u0026micro;g/g at 20 min after administration, then decreased to 12.8 \u0026micro;g/g at 30 min, and then increased again after 30 min. From 10 minutes to 150 minutes, the maximum concentration of BPA in the brain was only 2.6 \u0026micro;g/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Pearson correlations were calculated by using all tissues as variables. There was a significant correlation between blood boron concentration and organs except kidney, heart (r\u0026thinsp;=\u0026thinsp;0.846, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), liver (r\u0026thinsp;=\u0026thinsp;0.951, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03), spleen (r\u0026thinsp;=\u0026thinsp;0.948, p\u0026thinsp;\u0026lt;\u0026thinsp;0.04), lung (r\u0026thinsp;=\u0026thinsp;0.949, p\u0026thinsp;\u0026lt;\u0026thinsp;0.04), kidney (r\u0026thinsp;=\u0026thinsp;0.680, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and brain (r\u0026thinsp;=\u0026thinsp;0.854, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results suggest that BPA metabolism may be due to excretion through the kidney. These results also indicate that boron accumulation was not found in the tissues after BPA injection (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAll tissues were used as variables for Person correlation calculation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblood\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eliver\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003espleen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ekidney\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ebrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eheart\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eblood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eliver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.951\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003espleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.948\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.952\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.949\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.969\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.991\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ekidney\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.664\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebrain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.854\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.889\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.848\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.908\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.814\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eheart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.846*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.876\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.857\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.983\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05;**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01༛\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe distribution of BPA in normal organs and tumors of CDX.\u003c/p\u003e \u003cp\u003eTo investigate whether BPA preferentially accumulates in tumor tissues over other normal organs, we examined the distribution of boron-containing drugs in tumor-bearing mice at 30 minutes, 1 hour, and 2 hours post-administration. The results demonstrated that boron-containing drugs were significantly enriched in tumor tissues 2 hours after tail vein injection. The boron concentration in tumors (7.52 \u0026micro;g/g) was higher than that in blood (1.87 \u0026micro;g/g), heart (3.31 \u0026micro;g/g), liver (2.28 \u0026micro;g/g), spleen (3.74 \u0026micro;g/g), lung (3.81 \u0026micro;g/g), and brain (3.11 \u0026micro;g/g). The tumor-to-blood or tumor-to-tissue (heart, liver, spleen, lung, brain) boron concentration ratios were 4.02, 2.28, 2.39, 2.13, 2.04, and 2.46, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, the boron concentration in tumors was comparable to that in the kidneys, with a tumor-to-kidney boron concentration ratio of 0.96, which further supports the notion that boron is metabolized and excreted through the kidneys. These findings suggest that boron is more likely to distribute to tumor tissues than to blood or normal tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTissue safety evaluation of BPA in CDX.\u003c/p\u003e \u003cp\u003eTissues were collected for safety assessment 2 h after the tail vein single injection of BPA (push injection). Histological findings revealed no abnormalities in the structure of the liver (a), brain (b), spleen (c), heart (d), kidney (e), and lung (f) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBPA is a boronic acid derivative based on the phenylalanine skeleton. Phenylalanine is a high-affinity substrate for LAT1 (L-amino acid transporter). Due to the abnormal amino acid metabolism of tumor cells, LAT1 is highly expressed in tumor cells. Therefore, based on the transport mechanism of LAT1, BPA can be highly enriched in tumor cells with abnormal metabolism (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). BPA has achieved good efficacy in recurrent head and neck cancer, brain glioma, skin melanoma and mesothelioma (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, little attention has been paid to the treatment of liver cancer. Suzuki et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) performed BNCT through boron accumulation in the arteries of liver cancer cells for the first time in clinical practice. They used a single dose of irradiation. After 1 month of treatment, a CT scan showed that the size of the right lobe tumor treated by BNCT was unchanged, and the left lobe tumor treated by chemoembolization was enlarged, which confirmed the feasibility of BNCT for tumor treatment. To improve the efficacy of BNCT in treating liver cancer, we evaluated the concentration and time parameters of BPA in cell and animal models, aiming to prove the possibility of applying BNCT.\u003c/p\u003e \u003cp\u003eThe biodistribution of BPA in rats indicates that BPA does not stay in the body for a long time after injection. Its average clearance half-life is only 74.7 minutes. Pearson correlation analysis shows that blood boron concentration is positively correlated with boron concentration in other tissues except kidney, suggesting that increasing blood boron concentration may increase boron in tumors. This further suggests that BPA may be cleared in the form of urine. Kulvik et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) also found that blood boron concentration decreased with time after boron drug infusion, and boron concentration in the brain changed little, which is consistent with our study. Therefore, during BNCT treatment, long- term stable drug administration may further increase tumor boron concentration.\u003c/p\u003e \u003cp\u003eWe studied the cytotoxicity and drug uptake of different concentrations and different times of BPA co-cultured with Hepa1-6 and HepG2. After co-culturing BPA with Hepa1-6 and HepG2 cells for 24 hours, it exerted obvious toxic effects on both types of cells, and these effects showed a concentration- and time-dependent relationship. The intracellular boron concentration is positively correlated with drug concentration. This provides the best conditions for cell irradiation experiments. This is the first report on the absorption and toxicity of boron in liver cancer cells. Hermawan et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) also showed preferential enrichment of boron concentration in breast cancer cells. Yoshida et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) found that in glioma cells, the uptake of BPA by cancer cells was significantly higher than that of normal cells without obvious toxicity. On this basis, Couto et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) has developed a bimodal therapeutic agent for glioblastoma, whose uptake is 1.7 times that of BPA and which significantly improves the survival rate. When the drug concentration is lower than 1 mg/mL, as the drug concentration increases, the degree of hemolysis gradually increases. The maximum hemolysis rate is 1.4%, but no obvious hemolysis is observed in vitro. This indicates that the drug has little effect on the stability of red blood cells at low concentrations and has a certain degree of safety.\u003c/p\u003e \u003cp\u003eFor BNCT treatment, a high boron content in tumors is the key to treatment. The boron concentration ratio of tumor/blood or tumor/normal tissue should be at least greater than 2 times. In the CDX model, we compared single injection for 30 minutes, 1 hour and 2 hours. The results showed that the boron concentration in tumors with single injection for 2 hours was significantly higher than that in blood and other tissues (heart, liver, spleen, lung, brain). The ratio of the tumor to the tissue is greater than 2. Wang et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) reported that the uptake of BPA in F98 glioma reached the maximum value 1 hour after administration. Huang et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) indicated that BNCT mainly eliminates radiation-resistant liver cancer cells by targeting DNA damage and repair responses. Alam\u0026oacute;n et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) synthesized the boron-rich selective epidermal growth factor receptor (EGFR)- inhibitor hybrid 1. In U87 cells and tumor-bearing rats, it exhibited an enrichment ability superior to that of BPA, and significantly prolonged the survival period of the rats after BNCT treatment. The possibility of boron distribution in tumor tissues is higher than that in blood and normal tissues. BNCT treatment of HCC is feasible. At the same time, a large number of clinical and experimental studies have proved that BPA is safe (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). At the same time, we did not observe any abnormal structures such as brain, heart, lung, liver, kidney, and spleen on the paraffin sections of normal tissues of mice after administration, which once again proves its safety at the histological level.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the results indicate that BPA is preferentially enriched in HCC cells without significant toxicity, providing a protocol and rationale for BNCT treatment of HCC. In the future, detailed molecular experiments are needed to deeply explore the molecular mechanism by which Boron Neutron Capture Therapy (BNCT) kills liver cancer cells.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eHepa1-6 and HepG-2 cells were purchased from Shanghai Beyotime Biotechnology Co., Ltd, which were cultured in DMEM (Dalian Meilun Biotechnology Co., Ltd) medium containing 10% fetal bovine serum (Wuhan Servicebio Technology Co., Ltd) at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. The Ethics committee of the First Hospital of Lanzhou University approved the animal experiment plan in this paper. Eight-week-old female SD rats and 6-week-old female nude rats were purchased from Lanzhou Institute of Animal Research in Gansu Province, and the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were conducted in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003cp\u003eMeasurements of the Boron Concentration.\u003c/p\u003e \u003cp\u003eThe tissue samples containing BPA were mixed with the appropriate concentrated nitric acid at 55\u0026deg;C for 1 hour and subsequently placed in a 55\u0026deg;C water bath for 4 hours. Once the tissue was completely dissolved, the solution was calibrated with ultra-pure water. The boron concentration in each sample was determined by using ICP-AES. The culture dishes were collected at different time points during the co-culture of cells and BPA. The cells were gently rinsed with physiological saline three times. An appropriate amount of concentrated nitric acid was added, and then the dishes were placed in an oven at 55\u0026deg;C and shaken for 40 minutes. The cells were collected with ultra-pure water and transferred into a 15 mL centrifuge tube. The volume was uniformly calibrated to 10 mL. Finally, the boron content was detected by ICP-AES.\u003c/p\u003e \u003cp\u003ePreparation of BPA-fructose complex solution.\u003c/p\u003e \u003cp\u003eThe \u003csup\u003e10\u003c/sup\u003eB-labeled BPA (\u003csup\u003e10\u003c/sup\u003eB-BPA) was provided by the Key Laboratory of Heavy Ion Radia-tion Biomedicine, Institute of Modern Physics, Lanzhou Academy of Sciences, China (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). BPA and fructose (Shanghai Beyotime Biotechnology Co., Ltd) were first mixed in ultra-pure water at a molar ratio of 1:1.5, and then 1N NaOH was added to pH 10.5 under continuous stirring, followed by titration to pH 7.6 with 1N HCl. Finally, the solution was filtered and sterilized using a 0.22 \u0026micro;m filter. The concentration of boron prepared was 1 mg/mL. All the BPA appearing in the subsequent experiments was prepared by the fructose-complex method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUptake and toxicity of BPA on Hepa1-6 and HepG2.\u003c/p\u003e \u003cp\u003eHepa1-6 is a murine hepatoma cell line derived from BW7756 hepatoma tumor, HepG2 is derived from human hepatocellular carcinoma tissue, and boron intake was determined during exponential growth phase. The cells in logarithmic growth phase were digested, and spread on 6-well plate. Co-cultured with BPA (at concentrations of 1, 5, 10, 25, 50, 100, 200 \u0026micro;g/mL) for 1, 3, 6, and 12 hours respectively. At the end of co-culture, the cells were washed three times with precooled saline, 500 \u0026micro;L concentrated nitric acid was added and placed in an oven at 55\u0026deg;C for 40 min with shaking, and then the volume was fixed with ultra-pure water. Boron concentration in cells using ICP-AES and expressed as \u0026micro;g/10\u003csup\u003e7\u003c/sup\u003e cells. The number of cells was counted with Neubauer\u0026rsquo;s chamber.\u003c/p\u003e \u003cp\u003eThe cells in logarithmic growth phase were digested, and 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e/mL cell suspension was spread on a 96-well plate, and 200 \u0026micro;L cell suspension was added to each well. After the cells were attached to the well, the supernatant was discarded, and the prepared BPA (0.1, 1, 5, 10, 25, 50, 100, 200, 500 \u0026micro;g/mL) was added to the 96-well plate, and 150 \u0026micro;L was added to each well. After 3, 6, 12, and 24 hours of co-culture, 100 \u0026micro;L of 10% CCK8 (a reagent for cell proliferation and cytotoxicity detection) was added. The samples were incubated in a 37\u0026deg;C incubator in the dark for 1\u0026ndash;3 hours. Then, the absorbance was measured at 450 nm using an enzyme-labeled instrument. Graphpad Prism was used for plotting.\u003c/p\u003e \u003cp\u003eIn vitro hemolysis of BPA.\u003c/p\u003e \u003cp\u003eBlood was collected from the orbital venous plexus of four nude rats respectively. The blood was centrifuged at 3000 rpm for 10 min, rinsed, and centrifuged repeatedly with normal saline, and red blood cells were collected and diluted to 4% red blood cell suspension with normal saline. Saline solutions containing 0.1, 0.2, 0.5, and 1mg/mL BPA were prepared. An equal volume of red blood cell suspension was mixed with various concentrations of BPA solutions. Co-cultured for 4 hours at 37\u0026deg;C. Then the red blood cell suspension with distilled water was set as the positive control group, and the red blood cell suspension with normal saline was set as the negative control. Then, the mixture was centrifuged at 3000 rpm for 10 min, measure the absorbance of the supernatant at 540 nm. According to the measured absorbance value, the hemolysis rate of each concentration of drug was calculated by the formula: Hemolysis rate (%) = (A sample-A negative) / (A positive-A negative) \u0026times;100%.\u003c/p\u003e \u003cp\u003eThe pharmacokinetic characteristics of boron in SD rats following tail vein injection of BPA.\u003c/p\u003e \u003cp\u003eThirty 8-week-old female SD rats were intravenously injected with BPA at a dose of 500 mg/kg using a tail vein injection method. The injection was administered via intravenous push. Under sodium pentobarbital anesthesia, blood was collected from the apex of the heart, and organs such as the heart, liver, spleen, lung, kidney, and brain were harvested at 10, 20, 30, 60, 90, and 150 minutes after administration. ICP-AES was used for detection. One milliliter of blood was taken to determine the boron concentration, which was expressed as \u0026micro;g/g of blood or tissue. Pharmacokinetic parameters were calculated using Winnonlin software with a non-compartment model, and a curve of boron concentration as a function of time was plotted. Pearson correlation calculations were performed using all tissues as variables.\u003c/p\u003e \u003cp\u003eBiodistribution of BPA in normal tissues and tumors of CDX.\u003c/p\u003e \u003cp\u003eTo establish a subcutaneous animal model, Hepa1-6 cells (2\u0026times;10\u003csup\u003e7\u003c/sup\u003e/mL) in the exponential growth phase were subcutaneously injected (push injection) into 6week-old nude rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). To evaluate boron uptake, BPA (500 mg/kg) was injected via the tail vein and sacrificed 0.5, 1 and 2 h after injection. Boron concentrations in 100 \u0026micro;l blood and tumor or normal tissue were measured using ICP-AES and expressed as \u0026micro;g/g tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSafety of BPA in CDX.\u003c/p\u003e \u003cp\u003eIn order to verify the safety of BPA at the dose used in this study, we selected 9 nude rats bearing tumors and conducted 3 replicate experiments. The nude rats were injected with BPA (500 mg/kg) via the tail vein at 2 h after injection, and different organs were extracted and paraffin sections were prepared to observe histological damage.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Two-way analysis of variance (ANOVA) was performed to evaluate the significance of the differences. Winnonlin software was used to analyze the pharmacokinetic parameters. Statistical analysis included Graphpad Prism 8, SPSS26.0 statistical software, and p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Tanglong Zhang, Huanyu Zhang and Juntao Ran; Formal analysis, Pengcheng Zhang; Investigation, Tanglong Zhang and Huanyu Zhang; Meth-odology, Tanglong Zhang and Zhuoya Zhang; Project administration, Juntao Ran and Xiaodong Jing; Resources, Pengcheng Zhang and Ting Zhao; Software, Pengcheng Zhang; Supervision, Juntao Ran; Writing – original draft, Tanglong Zhang and Zhuoya Zhang; Writing – review \u0026amp; editing, Juntao Ran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded in part by and the National Natural Science Foundation of China (NSFC), grant number 82360583, the Lanzhou University Medical Research Innovation Capacity Enhancement Program, grant number lzuyxcx-2022-159, Natural Science Foundation of Gansu Province of China (Grant No. 23JRRA0952) and The First Hospital of Lanzhou University Intramural Fund, Excellent Doctoral Start-up Fund (Grant No. ldyyyn2022-91）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eInstitutional Review Board Statement: The animal study protocol was approved by The Committee on the Ethics of Animal Experiments of Lanzhou University First Hospital (LDYYLL2024-684).\u0026nbsp;We used isoflurane inhalation anesthetic to euthanize the experimental mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eata\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003evailability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Boyi Yu for his expert technical assistance in the animal modeling experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYura, Y. \u0026amp; Fujita, Y. Boron neutron capture therapy as a novel modality of radiotherapy for oral cancer: Principle and antitumor effect. \u003cem\u003eOral Sci. Int.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 9\u0026ndash;14 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorita, T. et al. Dynamic changes in 18F-borono-L-phenylalanine uptake in unresectable, advanced, or recurrent squamous cell carcinoma of the head and neck and malignant melanoma during boron neutron capture therapy patient selection. \u003cem\u003eRadiat. Oncol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 4 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan, T. L. et al. Using salvage Boron Neutron Capture Therapy (BNCT) for recurrent malignant brain tumors in Taiwan. \u003cem\u003eAppl. Radiat. Isot.\u003c/em\u003e \u003cb\u003e160\u003c/b\u003e, 109105 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirose, K. et al. Boron neutron capture therapy using cyclotron-based epithermal neutron source and borofalan (10B) for recurrent or locally advanced head and neck cancer (JHN002): An open-label phase II trial. \u003cem\u003eRadiother Oncol.\u003c/em\u003e \u003cb\u003e155\u003c/b\u003e, 182\u0026ndash;187 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki, M. et al. Feasibility of boron neutron capture therapy (BNCT) for malignant pleural mesothelioma from a viewpoint of dose distribution analysis. \u003cem\u003e*Int J. Radiat. Oncol. Biol. Phys. * **66**\u003c/em\u003e, 1584\u0026ndash;1589 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeck-Sickinger, A. G. et al. New Boron Delivery Agents. \u003cem\u003eCancer Biother Radiopharm\u003c/em\u003e. \u003cb\u003e38\u003c/b\u003e, 160\u0026ndash;172 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerlay, J. et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. \u003cem\u003eInt. J. Cancer\u003c/em\u003e. \u003cb\u003e144\u003c/b\u003e, 1941\u0026ndash;1953 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, S., Cao, Q., Wen, W. \u0026amp; Wang, H. Targeted therapy for hepatocellular carcinoma: Challenges and opportunities. \u003cem\u003eCancer Lett.\u003c/em\u003e \u003cb\u003e460\u003c/b\u003e, 1\u0026ndash;9 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarth, R. F., Mi, P. \u0026amp; Yang, W. Boron delivery agents for neutron capture therapy of cancer. \u003cem\u003eCancer Commun.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 35 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCouto, M. \u0026amp; Cerecetto, H. Advancements in the Synthesis and Biological Properties of Carboranes and HighBoron Related Compounds: A Comprehensive Exploration with Emphasis on BNCT Applications. \u003cem\u003eJ. Braz Chem. Soc.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21577/0103-5053.20240109\u003c/span\u003e\u003cspan address=\"10.21577/0103-5053.20240109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eBoron-Based Compounds: Potential and Emerging Applications in Medicine\u003c/em\u003e. (Wiley, Hoboken, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarpano, M. et al. Experimental Studies of Boronophenylalanine ((10)BPA) Biodistribution for the Individual Application of Boron Neutron Capture Therapy (BNCT) for Malignant Melanoma Treatment. \u003cem\u003eInt. J. Radiat. Oncol. Biol. Phys.\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e, 344\u0026ndash;352 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerada, S. et al. Boron uptake of boronophenylalanine and the effect of boron neutron capture therapy in cervical cancer cells. \u003cem\u003eAppl. Radiat. Isot.\u003c/em\u003e \u003cb\u003e197\u003c/b\u003e, 110792 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulvik, M. et al. Biodistribution of boron after intravenous 4-dihydroxyborylphenylalanine-fructose (BPA-F) infusion in meningioma and schwannoma patients: A feasibility study for boron neutron capture therapy. \u003cem\u003eAppl. Radiat. Isot.\u003c/em\u003e \u003cb\u003e106\u003c/b\u003e, 207\u0026ndash;212 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, F. et al. Evaluation of Pharmacokinetics of Boronophenylalanine and Its Uptakes in Gastric Cancer. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 925671 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J. et al. Unlocking the potential: phenylboronic acid as a nuclear-targeting boron agent for neutron capture therapy. \u003cem\u003eMed. Oncol.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, 104 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Z. et al. Biodistribution studies of boronophenylalanine in different types of skin melanoma. \u003cem\u003eAppl. Radiat. Isot.\u003c/em\u003e \u003cb\u003e163\u003c/b\u003e, 109215 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Z. et al. Boramino acid as a marker for amino acid transporters. \u003cem\u003eSci. Adv.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, e1500694 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNomoto, T. et al. Poly(vinyl alcohol) boosting therapeutic potential of p-boronophenylalanine in neutron capture therapy by modulating metabolism. \u003cem\u003eSci. Adv.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, eaaz1722 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki, M. et al. First attempt of boron neutron capture therapy (BNCT) for hepatocellular carcinoma. \u003cem\u003eJpn J. Clin. Oncol.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 376\u0026ndash;381 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulvik, M. E. et al. Boron biodistribution in beagles after intravenous infusion of 4-dihydroxyborylphenylalanine-fructose complex. \u003cem\u003eAppl. Radiat. Isot.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e, 975\u0026ndash;979 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHermawan, A. et al. Cellular uptake evaluation of pentagamaboronon-0 (PGB-0) for boron neutron capture therapy (BNCT) against breast cancer cells. \u003cem\u003eInvest. New. Drugs\u003c/em\u003e. \u003cb\u003e37\u003c/b\u003e, 1292\u0026ndash;1299 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida, F. et al. Difference in BPA uptake between glioma stem-like cells and their cancerous cells. \u003cem\u003eAppl. Radiat. Isot.\u003c/em\u003e \u003cb\u003e164\u003c/b\u003e, 109234 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCouto, M. et al. Bimodal Therapeutic Agents Against Glioblastoma, One of the Most Lethal Forms of Cancer. \u003cem\u003eChemistry\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 14335\u0026ndash;14340 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. E. et al. Evaluation of 4-borono-2-18F-fluoro-L-phenylalanine-fructose as a probe for boron neutron capture therapy in a glioma-bearing rat model. \u003cem\u003eJ. Nucl. Med.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 302\u0026ndash;308 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, C. Y. et al. Boron Neutron Capture Therapy Eliminates Radioresistant Liver Cancer Cells by Targeting DNA Damage and Repair Responses. \u003cem\u003eJ. Hepatocell Carcinoma\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 1385\u0026ndash;1401 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlam\u0026oacute;n, C. et al. A Potential Boron Neutron Capture Therapy Agent Selectively Suppresses High-Grade Glioma: In Vitro and in Vivo Exploration. \u003cem\u003eMol. Pharm.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 2702\u0026ndash;2713 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalouff, T. D. et al. Boron Neutron Capture Therapy: A Review of Clinical Applications. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 601820 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Boron neutron capture therapy, Distribution of drugs, HCC, Hepa1-6 cells, HepG2, BPA","lastPublishedDoi":"10.21203/rs.3.rs-6184213/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6184213/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBoron neutron capture therapy (BNCT) enables dual-targeted heavy-ion radiotherapy at the cellular level, with boronophenylalanine (BPA) as a key boron carrier. Although BPA shows clinical promise in liver cancer, there is relatively little basic research on its effect on hepatocellular carcinoma. Here, we systematically evaluated BPA uptake, safety, and pharmacokinetics. Boron uptake in HCC cells (Hepa1-6, HepG2) was quantified via ICP-AES, revealing concentration- and time-dependent accumulation (plateau at 6 h), while CCK-8 assays indicated significant cytotoxicity at 24 h. Hemolysis experiments confirmed BPA safety (0.1\u0026ndash;1 mg/mL), and pharmacokinetic studies in Sprague-Dawley rats showed rapid boron distribution (peak at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 min) with a blood half-life of 74.71\u0026thinsp;\u0026plusmn;\u0026thinsp;52.22 min. In cell-derived xenograft models, BPA achieved tumor-specific targeting, with tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) ratios exceeding 2 and 4, respectively, at 2 h post-injection, followed by rapid systemic clearance. These findings demonstrate BPA selective enrichment in liver tumors, favorable pharmacokinetics, and low toxicity, supporting its clinical utility in BNCT for HCC.\u003c/p\u003e","manuscriptTitle":"Pharmacokinetic, Biodistribution, and toxicity Study of Boronophenylalanine in hepatocellular carcinoma cells and Tumor- Bearing Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 19:47:25","doi":"10.21203/rs.3.rs-6184213/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-15T05:40:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T11:45:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T02:34:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T17:50:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T11:23:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50746162116175197240420245638222635289","date":"2025-04-29T04:07:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171096890957280066746363453502781257681","date":"2025-04-28T23:31:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304892020018361930676698603780117696854","date":"2025-04-28T22:00:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281360314934501641794148146137606852337","date":"2025-04-28T21:31:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T21:17:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T10:29:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-20T15:17:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T04:37:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-08T12:49:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d00496fd-5308-4a2a-b250-0501ff533099","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47870020,"name":"Biological sciences/Cancer"},{"id":47870021,"name":"Biological sciences/Cell biology"},{"id":47870022,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-08-04T09:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 19:47:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6184213","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6184213","identity":"rs-6184213","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}