Pharmacokinetic, biodistribution, safety and efficacy studies of borophenylalanine (BPA) in BNCT in hepatocellular carcinoma cells and tumor-bearing mouse model

Abstract

Boron neutron capture therapy (BNCT), using boronophenylalanine (BPA) as the main boron carrier, is a dual-targeted particle radiotherapy at the cellular level. Although BPA shows clinical promise in liver cancer, there is relatively little basic research on its effect on hepatocellular carcinoma. Therefore, we systematically evaluated the uptake, safety, pharmacokinetics, and therapeutic efficacy of BPA. Boron uptake in hepatocellular carcinoma cells (Hepa1-6, HepG2) was quantified by ICP-AES, revealing concentration- and time-dependent accumulation (plateau at 6 h), while CCK-8 assays indicated significant cytotoxicity at 24 h. Pharmacokinetic studies in Sprague-Dawley (SD) rats showed rapid boron distribution (peak at 25 ± 5.8 min) with a blood clearance half-life of 74.71 ± 52.22 min. In tumor-bearing mouse 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. Cell viability significantly decreased after BPA-BNCT irradiation, and the tumor growth inhibition rate in mice reached 77%. BPA did not produce tissue damage in vivo, and there were no abnormalities in blood counts or liver or kidney function in vivo after irradiation. These findings suggest that BPA can be selectively enriched in hepatocellular tumors with good pharmacokinetics and therapeutic efficacy, supporting its clinical application in BNCT of hepatocellular carcinoma.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-14885-1.

Introduction

Liver cancer is one of the malignant tumors that pose a serious threat to human health worldwide, with up to 900,000 new cases occurring globally each year. Among them, hepatocellular carcinoma (HCC) accounts for 75–90% of primary liver cancer cases, and viral hepatitis infection is the main etiology¹. In the early stage of the disease, there are often no obvious symptoms, and most patients are already in the middle or late stages at the time of diagnosis, losing the opportunity for radical surgery². The overall prognosis of patients is poor. The first clinical report of BNCT for advanced recurrent hepatocellular carcinoma has demonstrated that it is more effective and safer than conventional therapies³. Therefore, the development of boron neutron capture therapy (BNCT) is crucial for improving the clinical management and treatment outcomes of liver cancer patients.

BNCT is a biologically targeted binary radiation therapy. First, boron compounds are delivered to tumor cells, followed by neutron irradiation to initiate a nuclear reaction (¹⁰B + n → ⁷Li + ⁴He (α) + 2.792 MeV), generating high-linear energy transfer α particles and recoil lithium-7 nuclei to kill tumor cells⁴. Due to the extremely short penetration range of these α and ⁷Li (< 5–10 μm), BNCT is able to significantly protect 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, malignant brain tumors, recurrent head and neck cancers, and malignant mesothelioma^5–9. The efficacy of BNCT depends on the preferential accumulation of boron in the tumor tissue, and the boron delivery agent must reach a tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) concentration ratio of more than 2¹⁰.

Fig. 1.

Neutrons combine with boron-bearing tumor cells to undergo nuclear fission.

Currently, most of the third-generation boron delivery agents are modified and improved based on BPA. Therefore, in-depth research on BPA can significantly facilitate the development and clinical translation of next-generation agents. The aim of this study was to investigate the effectiveness of BPA-BNCT in hepatocellular carcinoma cells and animals, and provide a foundation for the application of BNCT in liver cancer treatment. Carpano and Terada Shinichi^11,12 showed that significant boron uptake occurs in tumor tissues in mice 2–2.5 h after BPA injection. Tang’s team¹³ found that the distribution of boron in different tissues was correlated with the concentration of boron in the blood, with the exception of the brain, kidney and bladder. Chen and Zhang^14,15 found that BPA itself had little effect on cell viability, and no adverse reactions were observed in patients receiving intravenous infusion of BPA. In our study, a non-negligible toxicity of BPA to hepatocellular carcinoma was observed, so it is crucial to choose the appropriate co-culture time for irradiation. Its uptake was concentration-dependent, and it demonstrated targeting ability and therapeutic efficacy in tumor-bearing mouse models, providing a basis and dosimetric modeling for the application of BNCT in hepatocellular carcinoma treatment. These findings underscore the potential of BPA as a safe and effective boron delivery agent for BNCT in hepatocellular carcinoma.

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% CO₂. 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 BALB/c nu 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.

Preparation of BPA-fructose complex solution

The ¹⁰B-labeled BPA (¹⁰B-BPA) was provided by the Key Laboratory of Heavy Ion Radia-tion Biomedicine, Institute of Modern Physics, Lanzhou Academy of Sciences, China. 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 1 N NaOH was added to pH 10.5 under continuous stirring, followed by titration to pH 7.6 with 1 N HCl. Finally, the solution was filtered and sterilized using a 0.22 μm filter. The concentration of boron prepared was 1 mg/mL.

Measurements of the boron concentration

The tissue samples containing BPA were mixed with the appropriate concentrated nitric acid at 55 °C for 1 h and subsequently placed in a 55 °C water bath for 4 h. 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 radio frequency power of the ICP-AES is 1300 W, the nebulizer flow rate is 0.8 L/min, the auxiliary gas flow rate is 0.2 L/min, and the spectral lines of 249 nm and 213 nm are selected for the determination of boron and phosphorus elements. Before measurement, the cell standard curve was examined by ICP-AES to ensure quantitative accuracy. Meanwhile, different concentrations of boron were added to normal saline, and the linear correlation between ICP-AES values and boron concentrations was detected. 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 min. 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.

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 logarithmic growth phase. The cells in logarithmic growth phase were digested, and spread on 6-well plate. Co-cultured with BPA (10, 100, 200,500,1000 µg/mL) for 3, 6, 12, and 24 h 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/10⁷ cells. The number of cells was counted with Neubauer’s chamber. The cells in logarithmic growth phase were digested, and 2.5 × 10⁴/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 (10, 100, 200, 500, 1000 µg/mL) was added to the 96-well plate, and 150 µL was added to each well. After 3, 6, 12, and 24 h 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 h. 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 Balb/c nu respectively. The blood was centrifuged at 3000 rpm for 10 min, rinsed, and centrifuged repeatedly with 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 1 mg/mL BPA were prepared. An equal volume of red blood cell suspension was mixed with various concentrations of BPA solutions. Co-cultured for 4 h 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: ×100 (A = absorbance at 540 nm).

The pharmacokinetic characteristics of boron in SD rats following intravenous injection of BPA

BPA was administered intravenously to 24 8-week-old female SD rats at a dose of 500 mg/kg. The injection was administered by 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 min after administration. ICP-AES was used for detection. 1mL 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.

The biodistribution of BPA in normal tissues and tumors of tumor-bearing mice

Construction of tumor-bearing mice: 1 × 10⁶ Hepa1-6 cells (suspended in 100 µL PBS) were injected into the right dorsoventral abdomen of 6-week-old BALB/c nu mice.

When the tumor volume of mice was between 100 and 200 mm³, 12 mice were randomly divided into 3 groups of 4 mice each. To assess boron uptake, BPA (500 mg/kg) was injected by tail vein and the mice were executed at 0.5, 1, and 2 h post-injection. Boron concentrations in 100 µl blood and tumor or normal tissue were measured using ICP-AES and expressed as µg/g tissue. The entire liver is located in non-tumor areas, and all samples are taken from normal liver tissue.

BNCT irradiation in hepatocellular carcinoma models: in vitro and in vivo evaluation

Hepa1-6 cells were seeded in T25 flasks at 5 × 10⁴ cells/cm² and cultured for 24 h. The medium was then replaced with 500 µg/mL BPA-supplemented medium (control: standard medium) for 6 h. After digestion of the cells with trypsin, the cell suspension was transferred to a 1.5 mL centrifuge tube and placed on a neutron irradiation sample shelf. Neutron irradiation was performed for 10–20 min using an RFQ accelerator with a beam current of 6 mA. Post-irradiation, cells were seeded at graded densities. CCK-8 assay: Cells in 96-well plates were analyzed at 24, 48, 72, and 96 h post-irradiation using an enzyme marker (450 nm absorbance) to calculate relative viability. Colony formation: Cells in 60 mm dishes were cultured for 14 days, fixed, and stained with crystal violet for quantification. For the subcutaneous tumor model, 1 × 10⁶ Hepa1-6 cells were injected into the dorsum of Balb/c nu. After 7 days, 20 mice were randomly divided into five groups: Control, BPA + 10 min BNCT, BPA + 20 min BNCT, 10 min neutron irradiation alone, 20 min neutron irradiation alone. Neutron irradiation (6 mA beam current) was administered 2 h after intravenous BPA injection (500 mg/kg). Tumor volume was measured using calipers and calculated as (length × width²)/2. Mice were monitored for 17 days post-treatment.

Safety of BPA in tumor-bearing mouse models

To verify the safety of BPA at the dose used in this experiment, six-week-old female Balb/c nu (9 animals; N = 3) were selected. Histological structures at different time points after intravenous injection of BPA (500 mg/kg) were observed. Blood routine, liver function, and renal function were measured at 72 h after BNCT irradiation.

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.

Results

BPA and ICP-AES standard curves

The structural formula of BPA is shown in Fig. 2A. ICP-AES values were measured with different cell counts and the calibration curves were linearly correlated with a correlation coefficient of R² = 0.9996 and Y = 2E6X − 0.0075 (Fig. 2B). The ICP-AES values were obtained by dissolving BPA in saline, and the ICP-AES values were also linearly correlated with different concentrations of BPA, R² = 0.9993, Y = 25.778X + 23,170 (Fig. 2C).

Fig. 2.

Standard curves of ICP-AES. (A) Structure of BPA. (B) Linear correlation between ICP-AES values and cell count. (C) Linear correlation between ICP-AES values measured in normal saline containing different concentrations of boron and boron concentrations.

Drug uptake and toxicity 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. Rapid drug uptake was observed during the initial phase of co-culture of both cells with the drug. At 6 h, boron in Heap1-6 and HepG2 cells reached 1.5 µg/10⁷cell and 1.3 µg/10⁷cell, and the uptake rate reached a maximum at 24 h. This indicates that the uptake of boron by Hepa1–6 and HepG2 cells is dependent on both concentration and time. To explore the toxicity of BPA on Hepa1-6 and HepG2 cells, we selected 5 concentration gradients from 0 to 1000 µg/mL for different times. The results showed that the lowest value of cell viability of Heap1-6 and HepG2 was as high as 90% at 6 h of co-culture, but after 24 h, the cell viability decreased significantly and reached a minimum value of 60%, and there was a positive correlation with both concentration and time (Fig. 3).

Fig. 3.

(A,B) The comparison of cell viability after co-culturing BPA with Hepa1-6 and HepG2 cells; the figure displays the cell viability values measured after 3 h, 6 h, 12 h, and 24 h of co-culture. Data were expressed as the means ± SD. N = 6. (C,D) Drug uptake of BPA at different concentrations and different times in Hepa1-6 and HepG2 cells. Data were expressed as the means ± SD. N = 6.

Pharmacokinetic and distribution of BPA in SD rats

After intravenous injection of BPA, the boron concentration in the blood of rats reached a maximum value of 3.3 µg/mL at 30 min and then decreased with time. As shown in Table 1, analyses using non-compartmental models revealed the following boron PK parameters: the clearance half-life of the drug in the body was 74.713 ± 52.22 min; the time taken for the drug to reach its maximum concentration in the body was 25 ± 5.774 min; the maximum plasma concentration of the drug in the body was 3.546 ± 0.303 µg/mL; and the area under the curve AUC of the drug amounted to 460. 707 ± 170.724 min × µg/mL, suggesting that the drug does not remain in the body for a long period of time. 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 intravenous injection, then showed a decreasing trend, and the drug was almost eliminated from the body at 150 min. However, the boron concentration in the kidneys reached a maximum of 22 µg/g at 90 min after administration. From 10 min to 150 min, the maximum concentration of BPA in the brain was only 2.6 µg/g (Fig. 4). 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 < 0.04), kidney (r = 0.680, P > 0.05), and brain (r = 0.854, p < 0.03) (Table 2). These results suggest that BPA may be excreted through the kidneys, and boron does not accumulate in tissues.

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
T1/2z	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.

Fig. 4.

(A) Time curve of blood boron concentration—BPA in rats. The dose of BPA was 500 mg/kg. Female rats were 8 weeks old. Blood was collected at 10, 20, 30, 60, 90, and 150 min after BPA injection for boron determination. Data are expressed as mean ± SD. N = 4. (B) The boron concentration-time curves of BPA in the heart(a), liver(b), spleen(c), lung(d), kidney(e), brain(f), and blood(g) of rats. The dose of BPA was 500 mg/kg. Female rats were 8 weeks old. Blood and tissues were collected at 10, 20, 30, 60, 90, and 150 min after BPA injection for boron determination. Data are expressed as mean ± SD. N = 4.

Table 2.

All tissues were used as variables for pearson 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 subcutaneous tumor-bearing mouse models

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 0.5, 1and 2 h post-administration. The results showed that 2 h after tail vein injection, BPA mainly accumulated in tumor tissues. 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 T/B and T/N (heart, liver, spleen, lung, brain) boron concentration ratios were 4.02, 2.28, 2.39, 2.13, 2.04, and 2.46, respectively (Fig. 5). 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 idea that boron is cleared through the kidneys. These findings suggest that boron is more likely to distribute to tumor tissues than to blood or normal tissues.

Fig. 5.

(A) Tumor-bearing mouse model. We selected female 6-week-old BALB/c nu mice. N = 4. The injection method used was intravenous push injection. The mice were divided into 3 groups, namely the 0.5-hour post-BPA injection group, 1-hour post-BPA injection group, and 2-hour post-BPA injection group. Boron concentrations in tumors and tissues of subcutaneously tumor-bearing mouse models. (B) Boron concentrations in tumors, blood, and tissues of heart, liver, spleen, kidney, lung and brain (0.5, 1, 2 h). Female mice were 6 weeks old. The dose of BPA was 500 mg/kg. Data are expressed as mean ± SD. N = 4. (C) Ratios of boron concentrations in tumor/blood or tumor/tissue. Female mice were 6 weeks old. The dose of BPA was 500 mg/kg. Blood and tissues were collected at 0.5, 1 and 2 h after BPA injection for boron measurement. Data are expressed as mean ± SD. N = 4.

BNCT irradiation in hepatocellular carcinoma models: in vitro and in vivo evaluation

BNCT shows obvious killing effect on Hepa1-6 cells. Compared to controls, the BPA-BNCT group exhibited significant killing effect starting at 48 h post-irradiation (P < 0.01), with cell viability in the BPA + 20 min irradiation group decreasing to 40% by 96 h. The colony formation test showed a similar trend: the control group exhibited dense colonies, whereas the BPA-BNCT group significantly reduced the rate of colony formation, especially in the BPA + 20 min group, which exhibited sparse colonies. In tumor-bearing mice, BPA-BNCT irradiation significantly suppressed tumor progression. By day 17 post-treatment, tumor volume in the BPA + 20 min group (440 mm³) was reduced by 77% compared to controls (1173 mm³). Notably, all BNCT-treated mice survived throughout the observation period without radiation-induced skin ulceration or other adverse effects (Fig. 6).

Fig. 6.

Antitumor effects of BNCT combined with BPA in hepatocellular carcinoma models in vitro and in vivo. CK: Control; 10: 10 min neutron irradiation alone (BNCT); 20: 20 min neutron irradiation alone (BNCT); N10: BPA + 10 min BNCT irradiation; N20: BPA + 20 min BNCT irradiation. (A) In vitro cytotoxicity assay: Hepa1-6 cells were divided into five groups. BPA-treated groups were incubated with 500 µg/mL BPA for 6 h prior to irradiation. Cell viability was assessed at 24, 48, 72, and 96 h post-irradiation using CCK-8. **p < 0.01, ****p < 0.0001 compared to CK group. Data were expressed as the means ± SD. N = 6. (B) Colony formation assay: Post-irradiation cells were seeded into 60 mm dishes and cultured for 14 days. Colonies were stained with crystal violet and quantified. (C) In vivo tumor growth kinetics: Subcutaneous Hepa1-6 xenografts were established in the right dorsum of Balb/c nude mice. Female mice were 6 weeks old. Data are expressed as mean ± SD. N = 4. Tumor volume was recorded starting 7 days post-inoculation. At the third day of recording, BPA (500 mg/kg) was intravenously administered, followed by neutron irradiation 2 h later. Tumor volume was monitored and calculated as (length × width²)/2. (D) Representative tumors from control and irradiated groups at day 17 post-treatment.

Safety assessment of BPA

Tissues were collected for safety assessment at 2 h and 1 week after intravenous injection of BPA. Compared with the non-injection control group, histological results showed no abnormal structures in the heart, liver, spleen, lung, kidney, and brain. Hemolysis experiments with BPA revealed its low hemolytic toxicity. 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. The changes in body weight of the administered group and the control group of tumor-bearing mice were observed for 1 week, and it was found that there was no significant difference in body weight between the two groups (Fig. 7). At 72 h after irradiation, serum and whole blood were collected from control and BPA-BNCT mice, and blood routine, liver and kidney functions were examined. The results were within the normal range and no obvious damage was observed (Table 3).

Fig. 7.

(A) Paraffin sections of normal tissues from mice in the non-injection group and the intravenous injection BPA group. N = 3 (B) degree of hemolysis at different concentrations of BPA; The BPA concentration (0.1−1 mg/mL); Data were expressed as the means ± SD. N = 4. (C) Changes in body weight of BPA-injected and control groups of tumor-bearing mice. On the third day of recording body weight, the experimental group was injected with BPA 500 mg/kg, and the change in body weight was recorded continuously for 1 week. Data were expressed as the means ± SD. N = 4.

Table 3.

Routine blood tests and biochemistry.

Items	Control	BPA + 20 min BNCT	Unit	Reference range
WBC	1.8 ± 0.3	1.4 ± 0.3	109/L	0.8–10.6
RBC	5.1 ± 0.4	4.4 ± 0.4	1012/L	6.5–11.5
PLT	1533 ± 21.6	1565 ± 44.8	109/L	400–1600
HGB	128.7 ± 6.1	116.7 ± 7.4	g/L	110–165
ALT	48.2 ± 15.3	64.2 ± 15.9	U/L	10.06–96.47
AST	133.9 ± 14	154.2 ± 29.4	U/L	36.3-235.48
TBIL	9.6 ± 3.9	7.3 ± 2.1	µmol/L	6.09–53.06
BUN	21.4 ± 1.9	26.1 ± 5.1	mmol/L	44.42-224.77
CREA	26.5 ± 3.8	26.5 ± 3.8	µmol/L	10.91–85.09

Note: Data are expressed as mean ± SD. N = 3. Irradiated two hours after BPA (500 mg/kg) injection. Blood routine, liver function and renal function were detected 72 h after irradiation.

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, BPA can be highly enriched in tumor cells with abnormal metabolism^16,17. By detecting the LAT1 level in liver cancer patients by immunohistochemistry, patients with strong boron accumulation ability can be screened out, thereby enabling personalized treatment. Although existing literature has demonstrated that BPA is transported by LAT1, the lack of direct functional inhibition experiments for LAT1 remains a limitation of this study. Future studies will also need to demonstrate its transport mechanism through LAT1 inhibitors. Suzuki et al.³ 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 liver right lobe tumor treated by BNCT was unchanged, and the liver 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 cellular and animal models, as well as the therapeutic efficacy after irradiation, 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. Based on the elimination half-life, combined with patient weight and body surface area, calculation of drug metabolism parameters in patients according to the principle of allometric scaling, providing a theoretical basis for clinical equivalent doses and infusion duration¹⁸. Haapaniemi et al.¹⁹ used a continuous 2-hour infusion of BPA at 400 mg/kg to maintain boron concentration in the blood during laryngeal cancer treatment. The selection of 500 mg/kg BPA is feasible in terms of dosage safety, with no early or late toxic and side effects observed. 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. The rapid absorption of the drug at the initial stage; which makes a consistent change in all tissues, and the accelerated clearance at the later stage (increased renal excretion rate), leads to the concentration change in the kidney showing an inconsistent trend with other tissues and blood. This further suggests that BPA may be cleared in the form of urine. Kulvik et al.²⁰ 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. To overcome the rapid metabolism of boron drugs, clinicians continuously infuse boron agents by intravenous drip before and during treatment to prolong drug retention and maintain therapeutic boron concentrations in target tissues^19,21. This represents the most commonly used delivery method for boron agents in current clinical practice: continuous intravenous infusion ensures that boron levels remain at peak values to meet therapeutic requirements. This approach provides a new strategy for enhancing the efficacy of BNCT in treating liver cancer.

We studied the cytotoxicity and drug uptake of different concentrations and different times of BPA co-cultured with Hepa1-6 and HepG2. This provides the best conditions for cell irradiation experiments. This is the first report on the absorption and toxicity of boron in hepatocellular carcinoma 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. For BNCT treatment, a high boron content in tumors is the key to treatment. The boron concentration ratio of T/B or T/N tissue should be at least greater than 2. In the tumor-bearing mouse models, we compared single injection for 0.5h, 1 h and 2 h. The results showed that the boron concentration in tumors with single injection for 2 h 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.²⁴ reported that the uptake of BPA in F98 glioma reached the maximum value 1 h after administration. Huang et al.²⁵ indicated that BNCT mainly eliminates radiation-resistant liver cancer cells by targeting DNA damage and repair responses. Alamón et al.²⁶ demonstrated that glioma treatment with BNCT significantly prolonged the survival of mice after BNCT. The possibility of boron distribution in tumor tissues is higher than that in blood and normal tissues.

After irradiation, we observed a significant inhibition of tumor growth and remarkable cell killing. BNCT treatment of liver cancer is feasible. At the same time, a large number of clinical and experimental studies have proved that BPA is safe²⁷. No abnormal structures were observed in paraffin sections of normal tissues of mice after drug administration, and the hemolysis rate of the drug showed no obvious abnormality. After BNCT irradiation, the blood routine, liver function, and renal function of mice were all within normal ranges, which further confirms the safety of BPA-BNCT. We observed the tissue response only 1 week after treatment, and future observation of subacute and delayed tissue effects on tissues after BNCT irradiation is necessary.

In this study, boron accumulation was assessed by ICP-AES, which provides reliable quantitative data but does not reflect the uptake dynamics at the single-cell level. In the future, we need to combine flow cytometry and single-cell techniques to further analyze the space-time heterogeneity of boron uptake, which will provide a more accurate basis for the targeted delivery of boron drugs.

Conclusions

In summary, the results show that BPA can preferentially accumulate in hepatocellular carcinoma cells and tumors. BPA-BNCT is safe and effective for the treatment of liver cancer, providing a protocol and theoretical basis for BNCT in liver cancer. In the future, detailed molecular experiments are needed to deeply explore the molecular mechanism by which BNCT kills hepatocellular carcinoma cells.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Z.T.L Conceptualization, Writing – reviewZ.H.Yand R.J.T Formal analysis, Z.P.C Investigation, Software, SupervisionZ.T.L and Z.H.Y Methodology, Writing – review Z. Z.Y and J.X.D Project administration, Writing – reviewR.J.T and Z.T Resources, Writing – original draft.

Funding

This research was funded in part by and the National Natural Science Foundation of China (NSFC), grant number 82360583, Gansu Provincial Science and Technology Major Program (25JRRA1244), 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, ldyyyn2023-6).

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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.