BSH fluorescent probe PET-CT nine-square grid cancer cell reagent boron concentration determination method

Abstract

This invention discloses a BSH fluorescent probe PET-CT nine-square grid method for determining boron concentration in cancer cells. Using a BSH fluorescent probe PET-CT instrument, tumor cells from the endoderm, mesoderm, and ectoderm are selected according to the nine-square grid theory, and then their IC50 and PET-CT values ​​are measured separately. The significance of this invention is to assist in achieving precise treatment with BNCT capture therapy, maximizing the therapeutic effect of BNCT therapy, and avoiding the ineffective treatment caused by clinicians and dosimeters misselecting boron drugs, thus preventing patients from undergoing repeated radiotherapy, increasing suffering, and extending treatment time and costs.

Description

Technical Field

[0001] This invention relates to the field of boron concentration determination technology, specifically to a method for determining boron concentration using a BSH fluorescent probe PET-CT nine-square grid cancer cell reagent. Background Technology

[0002] BSH is a thiol-containing boron cage compound with good hydrolytic and metabolic stability in biological environments. It has a high boron content and, at the same concentration and dose level, can carry more B10 to tumor tissue compared to BPA, making it one of the most effective BNCT drugs currently available. The BSH molecule contains 12 boron atoms; its full Chinese name is mercaptododecanoic acid. It was brought to Japan from the United States by Japanese neurosurgeon Tan Hatanaka. It was proven to have good distribution and aggregation in brain tumors and was first used in BNCT therapy for brain tumors in 1968, achieving some efficacy when irradiated during craniotomy. The United States and Japan have approved BSH for clinical research, but it has not yet met the conditions for market approval. Early clinical applications of BSH were limited because it cannot cross the blood-brain barrier, resulting in low tumor uptake and retention, and it also exhibits certain toxicity. BSH leaks from the fragile blood vessels of cancerous tissue into the interstitial fluid and only accumulates around cells, with a relatively weak ability to cross cell membranes. The use of BSH for brain tumors remains controversial. To address the challenge of BSH infiltration into cells, a novel BSH complex developed by Hideki Matsui of Japan for boron neutron capture therapy was employed. This complex involves modifying BSH with a short peptide, which then acts as a carrier to deliver BSH into the cell membrane. The complex is obtained by mixing acetylated A6K containing hydrophobic and basic amino acid residues with BSH in an aqueous solution. This process preserves the intracellular infiltration properties of acetylated A6K. The acetylated A6K-BSH complex is formed by direct or covalent bonding at the carboxyl terminus of acetylated A6K, resulting in a spherical shape with a diameter of approximately 20-200 nm, which is the ideal shape for cell introduction. A6K is a complex composed of nanoparticles and BSH boron drug, and compared to second-generation BSH, it can achieve ten times higher boron drug uptake within cancer cells.

[0003] To further determine the applicability of BSH in BNCT therapy, we investigated the molecular and cellular mechanisms of BSH transmembrane transport and used a PET-CT positron emission tomography scanner to detect boron concentration in selected tissues. Using a self-developed fluorescent probe, we established a method for determining whether BSH can be used in BNCT therapy by measuring boron concentration. This method is applicable, but not limited to, determining treatment options for malignant tumors such as bladder cancer, spleen cancer, melanoma, thyroid cancer, and oral cancer. In this invention, we disclose a method for determining the feasibility of BSH in BNCT therapy by using PET-CT to detect boron concentration in various tissues. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a BSH fluorescent probe PET-CT nine-square grid method for determining boron concentration in cancer cells, using PET-CT to measure the concentration of the fluorescent probe Cl. 37 -BSH is a method for determining boron concentration to assess suitability for BNCT therapy. It is used to determine whether ectodermal, mesodermal, and endoderm cancers are suitable for BNCT therapy. This helps clinicians quickly and accurately determine whether BNCT can be used to treat these cancer patients, saving clinicians' assessment time and buying them more treatment time.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: A BSH fluorescent probe PET-CT nine-square grid method for determining boron concentration in cancer cells. Using a BSH fluorescent probe PET-CT instrument, tumor cells from three germ layers (endoderm, mesoderm, and ectoderm) are selected according to the nine-square grid theory, and then their IC50 and PET-CT values ​​are detected separately. The method includes the following steps:

[0008] Step 1: Cell resuscitation. Cells are resuscitated by injecting Cl... 37 -BSH, heat the water bath to 37℃, sterilize the instrument, and add 4mL of McCoy s5A culture medium containing 10% fetal bovine serum and 1% penicillin antibody to the centrifuge tube; place the U-251 cell cryopreservation tube in the preheated water bath and shake it in the same direction from time to time until all the ice crystals in the tube melt into liquid, then sterilize; pipette the cryopreservation solution, aspirate the cell suspension, and centrifuge at 1000rpm / min for 5min in the centrifuge tube; discard the supernatant, add 1mL of culture medium to the precipitate, pipette and transfer it to a culture flask, add an appropriate amount of culture medium, and observe the cells;

[0009] Step 2: Culture and passage to obtain more cells for Cl... 37 To determine the -BSH boron concentration, the cells must first be cultured and passaged. After the adherent cells in the culture flask cover 80%–90% of the bottom, wash the cells twice with 3 mL of PBS, add 1.5 mL of 0.25% trypsin, shake and observe after 3–4 min. When the cells no longer clump together, add twice the amount of culture medium to stop digestion; centrifuge in a 15 mL centrifuge tube at 1000 rpm / min for 5 min; discard the supernatant, add 1 mL of LMcCoys s5A culture medium to the bottom, pipette to resuspend, take 1 / 3 and add 6 mL of culture medium;

[0010] Step 3: CCK-8 method for determining Cl 37Add 1.5 mL of 0.25% trypsin to U-251 cells (BSH ic50), centrifuge at 1000 rpm for 5 min, and observe. Seed 100 μL of 3k-7k / well cells in a 96-well plate and incubate at 37°C, 5% CO2, and 90% humidity for 24 hours. Prepare sample solutions of different concentration gradients, add 10 μL of each solution to three replicates in a 96-well plate, and incubate for 6, 12, 24, or 48 hours. Thaw and centrifuge CCK-8, and add 10 μL of CCK-8 solution to each well.

[0011] Preferably, the detection must be performed by separately detecting different Cl levels in tumor tissue, normal tissue, and blood of the aforementioned tumor cell suspension. 37 Concentration, and calculate the different B concentrations in tumor tissue, normal tissue, and blood. 10 Concentration, and finally the difference between tumor tissue and normal tissue B was calculated. 10 Concentration ratio (T / N), tumor tissue to blood B 10 Concentration ratio (T / B).

[0012] Preferably, regarding (T / N) and (T / B), when both T / N and T / B are >3, it can be considered that BPA boron drug is suitable for this type of cancer in BNCT treatment, that is, this type of cancer is an indication for BSH; when both T / N and T / B are <3, it can be considered that BSH boron drug is not suitable for this type of cancer in BNCT treatment, that is, this type of cancer is not an indication for BSH.

[0013] Preferably, in step one, the cells are cultured at 37°C, 5% CO2, and saturated humidity for 24 hours, and then observed again, with the medium changed every 2-3 days.

[0014] Preferably, in step two, the cells are cultured to 20 million to 30 million in an incubator at 37°C, 5% CO2, and saturated humidity.

[0015] Preferably, the method used in step three is to design Cl 37 The concentrations of -BSH were 300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, and 0.14 μg / g, with three replicates for each concentration. After incubation with CCK-8 for four hours, the absorbance was measured at 450 nm using a microplate reader. The results were processed and analyzed using Excel and Graphpad Pris.

[0016] Preferably, the fluorescent probe Cl 37 -BSH structure is a boron chloride cage compound.

[0017] Preferably, the boron chloride cage compound chlorine-37 is attached to the β-position of the thiol group, but a small amount is attached to the Y-position. Chlorine-37 attached to the Y-position has the same pharmacological effect as that attached to the β-position and yields the same results in concentration determination.

[0018] (III) Beneficial Effects

[0019] Compared with existing technologies, this invention provides a method for determining boron concentration in BSH fluorescent probe PET-CT nine-square grid cancer cell reagents, which has the following beneficial effects: This invention assists in achieving precise treatment with BNCT capture therapy, maximizing the therapeutic effect of BNCT therapy, and avoiding the ineffective treatment caused by clinicians and dosimeters misselecting boron drugs, resulting in repeated radiotherapy for patients, increasing suffering, treatment time, and costs. This invention uses a three-germ layer analysis method to determine a sensitive tumor type that can be used for BPA. First, different tumor cells are selected according to the three germ layers, and then IC50, PET-CT, etc. are performed on them respectively. The applicability of BSH to various tumors is evaluated by combining these detection methods with the three-germ layer analysis method of this invention, which facilitates drug selection. Detailed Implementation

[0020] To better understand the purpose, structure, and function of this invention, a more detailed description is provided of the boron concentration determination method for BSH fluorescent probe PET-CT nine-square grid cancer cell reagent.

[0021] This invention: A BSH fluorescent probe PET-CT nine-square grid method for determining boron concentration in cancer cells, using a BSH fluorescent probe PET-CT instrument and the fluorescent probe Cl. 37 -BSH has a boron chloride cage structure, with chloro-37 attached to the β-position of the thiol group, but a small amount attached to the Y-position. Chloro-37 attached to the Y-position and β-position have equivalent pharmacological effects and show the same results in concentration determination. Tumor cells from three different germ layers (endoderm, mesoderm, and ectoderm) were selected according to the nine-square grid theory, and their IC50 and PET-CT values ​​were analyzed separately. Analysis and recommendations were made regarding the appropriate use of BSH boron drugs.

[0022] Dosimeters and clinicians often encounter the problem of poor absorption of boron drugs by tumor cells during boron neutron therapy (BNCT), leading to suboptimal BNCT efficacy. This invention establishes a new analytical method to determine the appropriate use of BSH boron drugs, avoiding the situation where no alternative drugs are available when BPA is ineffective, resulting in treatment failure and requiring repeated radiotherapy, increasing patient suffering, treatment time, and costs.

[0023] This invention selects "nine-square grid" tumor cells as experimental subjects according to the three germ layers theory, clearly dividing cancer cells into nine equal parts, thereby making a systematic evaluation of boron drugs.

[0024] It includes the following steps:

[0025] Step 1: Cell resuscitation. Cells are resuscitated by injecting Cl... 37-BSH, heat the water bath to 37℃, sterilize the instrument, and add 4mL of McCoy s5A culture medium containing 10% fetal bovine serum and 1% penicillin antibody to the centrifuge tube; place the U-251 cell cryopreservation tube in the preheated water bath and shake it in the same direction from time to time until all the ice crystals in the tube melt into liquid, then sterilize; pipette the cryopreservation solution, aspirate the cell suspension, and centrifuge at 1000rpm / min for 5min; discard the supernatant, add 1mL of culture medium to the precipitate, pipette and transfer it to a culture flask, add an appropriate amount of culture medium, and observe the cells; culture at 37℃, 5% CO2, and saturated humidity for 24h, and observe the cells again, changing the medium every 2-3 days;

[0026] Step 2: Culture and passage to obtain more cells for Cl... 37 To determine the -BSH boron concentration, the cells must first be cultured and passaged. After the adherent cells in the culture flask cover 80%–90% of the bottom, wash the cells twice with 3 mL of PBS, add 1.5 mL of 0.25% trypsin, shake and observe after 3–4 min. When the cells no longer clump together, add twice the amount of culture medium to stop digestion; centrifuge in a 15 mL centrifuge tube at 1000 rpm / min for 5 min; discard the supernatant, add 1 mL of LMcCoys 5A culture medium to the bottom, pipette to resuspend, take 1 / 3 and add 6 mL of culture medium; culture in an incubator at 37℃, 5% CO2, and saturated humidity until 20 million to 30 million cells remain.

[0027] Step 3: CCK-8 method for determining Cl 37 Add 1.5 mL of 0.25% trypsin to U-251 cells (BSH ic50), centrifuge at 1000 rpm for 5 min, and observe. Seed 100 μL of 3kJ-7kJ / well in 96-well plates and incubate at 37°C, 5% CO2, and 90% humidity for 24 hours. Prepare sample solutions of different concentration gradients, add 10 μL of each solution to three replicates in 96-well plates, and incubate for 6, 12, 24, or 48 hours. Thaw and centrifuge CCK-8, and add 10 μL of CCK-8 solution to each well. Incubate at 37°C, 5% CO2, and 90% humidity for 0.5–4 hours. Measure the absorbance at 450 nm using a microplate reader, process and analyze the results.

[0028] The method of application is as follows:

[0029] Design Cl 37 The concentrations of -BSH were 300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, and 0.14 μg / g, with three replicates for each concentration. After incubation with CCK-8 for four hours, the absorbance was measured at 450 nm using a microplate reader. The results were processed and analyzed using Excel and Graphpad Pris.

[0030] Analysis results:

[0031] Cell viability = [(experimental wells - blank wells) / (negative control wells - blank wells)] × 100%

[0032] Inhibition rate = [(Negative control wells - Experimental wells) / (Negative control wells - Blank wells)] × 100%

[0033] Data analysis revealed that the IC50 values ​​for the nine tumor cell types ranged from 1.519 to 1.594, indicating low drug toxicity suitable for BNCT treatment. Furthermore, calculations showed that the boron content in Cl37-BSH within all nine tumor cell types was greater than 20 μg / g, meeting the requirements for BNCT. Based on the IC50 calculations, a Cl37-BSH formulation was designed... 37 The concentration gradient of -BSH was 0, 16.5, 33, and 66 μg / g. Nine types of tumor cells and normal blood cells with logarithmic growth were seeded into culture dishes, and cytotoxicity experiments were performed to determine the optimal dosage concentration as 33 μg / g.

[0034] Furthermore, in the above practical process, the detection must be performed separately to detect different Cl levels in tumor tissue, normal tissue, and blood of the tumor cell suspension. 37 Concentration, and calculate the different B concentrations in tumor tissue, normal tissue, and blood. 10 Concentration, and finally the difference between tumor tissue and normal tissue B was calculated. 10 Concentration ratio (T / N), tumor tissue to blood B 10 The concentration ratio (T / B) is considered to be appropriate for BNCT treatment when both T / N and T / B are greater than 3, meaning that BPA boron is an indication for BSH treatment. When both T / N and T / B are less than 3, BSH boron is considered not appropriate for BNCT treatment, meaning that BSH treatment is not an indication for BSH treatment.

[0035] For a long time, there has been little research on the indications for BSH, and there has been much controversy, which has affected drug development and clinical use. This invention solves this problem by developing a three-germ-layer identification PET-CT detection method through a series of studies and experiments on BSH. This invention uses a three-germ-layer analysis method to determine a sensitive tumor type that can be used for BPA. First, different tumor cells are selected according to the three germ layers, and then they are tested separately by ic50, PET-CT, etc. The applicability of BSH to various tumors is evaluated by combining these detection methods with the three-germ-layer analysis method of this invention, which facilitates drug selection.

[0036] I. Selection of various cells from different germ layers:

[0037] Endoderm: 1. Liver cancer cells, 2. Bladder cancer cells, 3. Colorectal cancer

[0038] Mesodermis: 4. Kidney cancer cells, 5. Spleen cancer cells, 6. Lymphoma cells

[0039] Ectodermal cells: 7. Breast cancer cells, 8. Melanoma cells, 9. Head and neck tumor cells

[0040] Normal cells: 10. Blood cells, 11. Heart tissue cells

[0041] This experiment selected three types of ectodermal, mesodermal, and endodermal tumors for testing.

[0042] (I) Post-administration anatomical and pathological examination

[0043] The target organs after drug administration are the kidneys, spleen, liver, and central neurons.

[0044] (II) Pharmacokinetic Analysis

[0045] After entering the body, BSH is eliminated from the blood relatively quickly, while its uptake in the kidneys, intestines, and scalp is relatively high. Higher uptake in the kidneys indicates that BSH is mainly excreted through the urinary system, higher uptake in the intestines indicates that some BSH is metabolized in the digestive system, and higher uptake in the scalp may be related to the presence of sulfhydryl groups in the BSH molecule.

[0046] (III) Analysis of Cancer Types in Clinical Treatment

[0047] Clinical treatment practice shows that BSH, as a boron drug in BNCT, can be used in combination to treat liver cancer, bladder cancer, and brain cancers (such as gliomas), as well as some head and neck cancers (such as oral cancer). This indicates that BSH can accumulate in some cells of the liver, bladder, brain, and head and neck, mainly in the liver and bladder, with the most reports on its use in treating liver cancer.

[0048] (iv) Germ layer localization demonstration

[0049] Based on comprehensive pathological examination and clinical practice, BSH can be identified as primarily acting on the endoderm (liver, bladder, etc.) and can be classified as an endoderm boron drug, which differs from the ectoderm boron drug BPA. Furthermore, BSH also shows some therapeutic effect on ectoderm tumors such as brain tumors, malignant melanomas, breast cancer, and mesodermal kidney and spleen cancers.

[0050] This experiment aims to analyze drug toxicity to ensure that the drug is effective in treating patients without causing harm. The method involves first inoculating mice with U-251 tumor or head and neck tumor cells, then using positron emission tomography (PET) to detect the boron content in the mice. The data obtained from this method are analyzed to determine whether they meet the requirements of BNCT. Finally, the drug toxicity is analyzed to ensure that the drug is effective in treating patients without causing harm.

[0051] II. Determination of Cl37-BSH IC50 by CCK-8 method

[0052] 1. Cell resuscitation

[0053] Resuscitate cells to inject Cl 37 -BSH, heat the water bath to 37℃, sterilize the instrument, and add 4mL of McCoy s5A culture medium containing 10% fetal bovine serum and 1% penicillin antibody to the centrifuge tube; place the U-251 cell cryopreservation tube in the preheated water bath and shake it in the same direction from time to time until all the ice crystals in the tube melt into liquid, then sterilize; pipette the cryopreservation solution, aspirate the cell suspension, and centrifuge at 1000rpm / min for 5min in the centrifuge tube; discard the supernatant, add 1mL of culture medium to the precipitate, pipette and transfer it to a culture flask, add an appropriate amount of culture medium, and observe the cells;

[0054] 2. Cultivation and Transmission

[0055] To obtain more cells for Cl 37 To determine the -BSH boron concentration, the cells must first be cultured and passaged. After the adherent cells in the culture flask cover 80%–90% of the bottom, wash the cells twice with 3 mL of PBS, add 1.5 mL of 0.25% trypsin, shake and observe after 3–4 min. When the cells no longer clump together, add twice the amount of culture medium to stop digestion; centrifuge in a 15 mL centrifuge tube at 1000 rpm / min for 5 min; discard the supernatant, add 1 mL of LMcCoys s5A culture medium to the bottom, pipette to resuspend, take 1 / 3 and add 6 mL of culture medium;

[0056] 3. CCK-8 method for determining Cl 37 -BSH ic50

[0057] Add 1.5 mL of 0.25% trypsin to U-251 cells, centrifuge at 1000 rpm for 5 min, and observe; seed 100 μL of 3k-7k cells / well in a 96-well plate and incubate at 37℃, 5% CO2, and 90% humidity for 24 hours; prepare sample solutions of different concentration gradients, add 10 μL of each solution to three replicates in a 96-well plate, and incubate for 6, 12, 24, or 48 hours; thaw and centrifuge CCK-8, and add 10 μL of CCK-8 solution to each well.

[0058] III. PET images of Cl37-BSH in tumor-bearing mice

[0059] This experiment aims to analyze drug toxicity to ensure that the drug is effective in treating patients without causing harm. The method involves first inoculating mice with U-251 tumor or head and neck tumor cells, then using positron emission tomography (PET) to detect the boron content in the mice. The data obtained from this method are analyzed to determine whether they meet the requirements of BNCT. Finally, the drug toxicity is analyzed to ensure that the drug is effective in treating patients without causing harm.

[0060] 1. Material preparation

[0061] Required animal samples: multiple Kunming mice weighing (20±5g)

[0062] Reason for sample selection: high similarity to human genes

[0063] The purpose of this implementation plan is to better help us study the feasibility of boron drugs in the practical application of BNCT treatment in humans. Therefore, in order to obtain more accurate implementation results, the selection of animal samples must meet the characteristic of high genetic similarity to humans. If samples with low genetic similarity to humans (including but not limited to plants, fungi, bacteria, etc.) are selected, the feasibility of this implementation invention cannot be verified. Kunming mice have a genetic similarity of 85% with humans, and the base pairs on their chromosomes are also highly similar to those on human chromosomes.

[0064] 2. Dosing regimen

[0065] Tail vein administration solution: Dissolve in 10% DMSO-physiological saline to prepare a solution of 1 mg / kg. -1 Cl-BSH clarified solution, prepared immediately before use.

[0066] Since the required amount of DMSO to be injected is small, if it is injected directly without being prepared as a solution, the drug will adhere to the inner wall of the syringe, resulting in a large error in the injection. 10% physiological saline is used because its osmotic pressure is basically equal to that of the blood plasma tissue fluid of Kunming mice.

[0067] 3. Preparation of single-cell suspension

[0068] This implementation plan requires inoculating Kunming mice with U-251 tumor cells by inoculating them with U-251 single-cell suspension, and the number must be sufficient. Therefore, this implementation plan uses a U-251 single-cell suspension of 1000w cells / mL.

[0069] U-251 cells were passaged and cultured in culture dishes according to standard procedures. Cells in the logarithmic growth phase were transferred to sterile 15 mL centrifuge tubes using a pipette. After digestion with 3 mL of trypsin for 4 min, twice the volume of McCoy s5A culture medium was added to terminate the digestion. The cells were then centrifuged at 1000 rpm for 5 min, and the supernatant was discarded to remove the trypsin. 5 mL of PBS was added, and the cells were pipetted until homogeneous. The cells were then centrifuged at 1000 rpm for 5 min, and this process was repeated 2-3 times to remove dead cells or cell debris from the cell suspension. An appropriate amount of sterile PBS was added to the cell pellet at the bottom of the centrifuge tube, and 10 μL was transferred to a cell counting chamber (the cell counting chamber was pre-cleaned with 75% alcohol). The cell counting chamber was observed under an inverted microscope, and cell counts were performed using a counter. The cell count was calculated to determine if the required cell count had been reached. Finally, an appropriate amount of sterile PBS was added to prepare a U-251 single-cell suspension of 1000 cells / mL for tumor cell inoculation.

[0070] 4. Model Building

[0071] Nine mouse models were constructed by subcutaneous inoculation of U-251 cells into the right axilla of Kunming mice: The prepared cell suspension was placed in an ice-filled foam box (to maintain cell viability). After spraying the work surface with 75% alcohol, a 1×10⁻⁶ cell suspension was drawn up using a 1 mL disposable sterile syringe. 7 Inoculate each mouse with 0.1 mL of U-251 single-cell suspension (number of cells / mL); handle Kunming mice and insert a needle subcutaneously into the right abdomen, reaching the cavity of the right scapula. Slide the needle back and forth to ensure sufficient space for tumor cell concentration and growth, injecting until a roundish bulge forms. Rotate the needle to withdraw it (to prevent leakage and linear tumor cell growth); repeat the above procedure, inoculating all Kunming mice within one hour (to avoid decreased cell viability leading to a lower tumorigenesis rate); observe tumor formation every two days. When a distinct tumor nodule appears subcutaneously in the lateral right axilla of the Kunming mouse, measure the tumor length and width with calipers. When the tumor volume exceeds 40 mm², [the tumor is considered tumor-free]. 3 When the time is reached, it indicates that the liver cancer mouse model has been successfully established; repeat the above steps to establish the remaining 8 models.

[0072] 5. Drug administration and detection

[0073] Under isoflurane anesthesia, 1 mg / kg of Cl was administered. 37 -BSH clarified solution was injected into hepatocellular carcinoma mice via the tail vein. Static PET images were acquired for 10 minutes 60 minutes after injection. Signal processing and image reconstruction yielded Cl... 37 -BSH drug boron concentration distribution image; after static acquisition of PET image data, mice were immediately sacrificed and different organs (brain, tumor, blood, etc.) were taken, microwave digested and boron concentration was measured by ICP-OES.

[0074] 6. Experimental Data and Conclusions

[0075] It can be concluded that Cl 37 -Bron atoms in BSH are mainly concentrated in liver cancer cells, with no significant uptake in other normal tissues, and significant uptake at the tumor site; calculations show that injecting 1 mg / kg of Cl via the tail vein... 37 -BSH, Cl in liver cancer cells 37 The drug content of -BSH is 33.3 μg / g, so the boron concentration in liver cancer cells is about 21 μg / g. The number of boron atoms in tumor cells fully meets the treatment requirements of BNCT.

[0076] IV. Detection of Boron Content

[0077] This implementation plan aims to use mice to simulate the human body and help us study the feasibility of using boron drugs in the practical application of BNCT treatment in humans.

[0078] Feasibility analysis of BNCT technology:

[0079] To investigate the actual efficacy of boron drugs in human BNCT treatment, the first step is to determine how much boron drug is absorbed by the patient's tumor cells and in which parts of the body the boron drug is present. Since sufficient absorption of boron drug is necessary for BNCT technology to kill tumor cells via neutron beam irradiation, it is essential to first detect the boron drug content in the patient's body. This implementation method uses boron content measurement. Kunming mice are used as the human simulant in this study; therefore, detecting the boron content in the mice allows for the determination of the concentration and distribution of boron drug within them, thus simulating the concentration and distribution of boron drug in the patient's body and demonstrating the feasibility of BNCT technology in killing tumor cells through neutron beam irradiation.

[0080] Note: Kunming mice with nine types of cancer have been cultured in Technical Solution 2. The cancer-causing Kunming mice cultured in the same batch as those in Technical Solution 2 are used in this implementation process.

[0081] 1. Selection of methods for detecting boron content in mice

[0082] Since this implementation scheme simulates the human body environment, the selected detection method must possess the following characteristics:

[0083] ① It is important to consider the potential harm to patients from the reagents and methods used in the testing.

[0084] ② The testing time should not be too long (if the testing time is too long, it will delay the patient's treatment and worsen the patient's condition).

[0085] ③ Ease of use and cost of use

[0086] According to characteristic requirement 1, identification should not be performed using traditional chemical reactions to avoid harming patients.

[0087] According to characteristic requirement 2, if the fluorescence labeling method is used for determination, the half-life of the fluorescent reagent should not be too long.

[0088] Based on characteristic requirements 1-3, this implementation plan uses positron emission tomography (PET) to detect boron content in mice.

[0089] ④ Inject Cl 37 After BSH, you need to wait 8 hours. (After 8 hours, A6K will be almost completely decomposed.)

[0090] 2. Concentration detection using positron emission tomography (PET)

[0091] ① Principle of Positron Emission Tomography (PET):

[0092] By labeling a substance with a short-lived radioactive nuclide (such as F18), the accumulation of this substance in metabolism can reflect the state of life's metabolic activities, thereby achieving the purpose of diagnosis.

[0093] ② Advantages of positron emission tomography (PET):

[0094] (1) High sensitivity: PET is an imaging technique that reflects molecular metabolism. When the disease is in the early stage of molecular changes and the morphology and structure of the lesion area have not yet shown abnormalities, and MRI and CT examinations cannot make a clear diagnosis, PET examination can find the lesion and obtain three-dimensional images. It can also perform quantitative analysis to achieve early diagnosis, which is unmatched by other imaging examinations.

[0095] (2) High specificity: When MRI and CT scans find tumors in organs, it is difficult to determine whether they are benign or malignant. However, PET scans can make a diagnosis based on the high metabolic characteristics of malignant tumors.

[0096] (3) Whole body imaging: A single PET whole body imaging examination can obtain images of all areas of the whole body.

[0097] (4) Good safety: The radionuclides required for PET scans have a certain degree of radioactivity, but the amount of radionuclides used is very small, and the half-life is very short (about 12 minutes for some and about 120 minutes for others). After physical attenuation and biological metabolism, the radioactive dose is very short in the body of the examinee. The radiation dose of a whole-body PET scan is far less than that of a routine CT scan of a single area, so it is safe and reliable.

[0098] 3. Procedure for detecting boron content in mice using positron emission tomography (PET-CT)

[0099] This implementation method uses Cl 37 -BSH is a boron drug (synthetic route is in the appendix). Therefore, the protocol implemented in this study was to detect the concentration of B in Kunming mice and determine whether the drug could be used in BNCT therapy based on the calculated data.

[0100] ① Preparation of anesthetic drugs

[0101] Commonly used anesthetics include injectable anesthetics (such as trimethoprim, chloral hydrate, etc.) and inhaled anesthetics (such as isoflurane, sevoflurane, etc.). Gas anesthesia is more effective than intraperitoneal injection anesthesia. When the collection time is long, respiratory anesthesia should be used.

[0102] ② Gas anesthesia for small animals

[0103] The experimental animals were placed in anesthesia boxes, and the flow rate of medical oxygen in the anesthesia machine was adjusted to IL / min, and the concentration of isoflurane gas was 1% to 2% to induce the animals into a state of deep anesthesia.

[0104] ③ Small animal placement

[0105] The small animal is placed in the transmission system and sent to the scanning position. To prevent motion artifacts caused by the animal moving due to twitching for various reasons, the small animal is usually fixed on the animal bed. It is fixed by a three-dimensional frame structure or wrapped around it. The small animal is placed in a prone position so that the midsagittal plane of the head is parallel to the long axis of the body and the midline of the body coincides with the midline of the bed.

[0106] When high-resolution imaging of a specific part of a live animal is required, the area to be scanned should be placed in the center of the scanning bed, and a laser positioning device should be used for precise axial and horizontal positioning. The protective cover should be tightly closed, and preparation for PET-CT scanning should be made.

[0107] ④ Setting PET-CT scan parameters

[0108] Different parameters such as X-ray tube voltage, X-ray tube current, and exposure time are set according to the acquisition location and density; for example, a respiratory gating device is placed below the animal's chest cavity, and invasive ECG electrodes are fixed under the skin of the animal's limbs, and the position is adjusted to obtain a stable ECG signal.

[0109] ⑤ PET-CT image acquisition

[0110] (1) Radionuclide labeling in boron-containing drugs (Cl) used for BNCT 37 -BSH) molecules.

[0111] (2) After being injected into mice, the positrons generated during the decay of the radionuclide annihilate with the surrounding free electrons, emitting a 511 keV γ photon pair; the PET-CT system detects this γ photon pair, and after signal processing and image reconstruction, the boron concentration distribution image containing BSH drug can be obtained.

[0112] (3) After completing the static acquisition of PET-CT image data, 10 mL of blood was drawn from Kunming mice, and tumor tissue and growth tissue (heart of Kunming mice was selected in this experiment) were microwave digested. Finally, the boron concentration was determined by ICP-OES. During this implementation, at different FBY doses, the boron content in blood and tumor tissue measured by ICP-OES showed excellent correlation with the radioactive signal. This phenomenon indicates that the fluorescent probe (Cl) 37 The FBY of a given dose is almost identical to the PBY of a given drug dose in vivo. The fluorescent probe, namely Cl... 37 -BSH-dose FBY can completely mimic the phase distribution of drug-dose FBY in the patient's body, thereby dynamically and quantitatively detecting boron concentration.

[0113] (4) Analysis of B in tumor tissue 10 Concentration range, tumor / normal tissue B 10 Concentration ratio, tumor / blood B 10 The concentration ratio.

[0114] 4. Implementation Results and Data Analysis

[0115] Implementation results:

[0116] M BSH =164, M Cl37-BSH =200, M B10 =10,12M B10 =120(Cl) 37 -BSH contains 12

[0117] B 10 (atoms), M Cl37 =37

[0118] B 10 In Cl 37 -BSH content = 12M B10 ÷M Cl37-BSH =60%

[0119] Cl 37 In Cl 37 -BSH content = M Cl37 ÷M Cl37-BSH =18.5%

[0120] That is, the following relation holds:

[0121] (1)Cl 37 -BSH concentration * 0.6 = B 10 concentration

[0122] (2)Cl 37 -BSH concentration * 0.185 = Cl 37 concentration

[0123] Cl was measured using the above-mentioned positron emission tomography method. 37 The content in tumor tissue, normal tissue (heart) and blood of Kunming mice.

[0124] B can be obtained from relations (1)-(2). 10 The content in tumor tissue, normal tissue (heart) and blood of Kunming mice.

[0125] The calculation results are shown in the table below:

[0126]

[0127]

[0128] 5. The impact of short peptide A6K on implementation results

[0129] Due to the presence of the short peptide A6K, B 10 The percentage of BSH-A6K (120÷718=16.7%) is much smaller than that of B. 10 The proportion of BSH (60%) is so high that it is necessary to determine B. 10 The concentration needs to be adjusted by first removing the short A6K peptide from BSH-A6K.

[0130] A6K is a short peptide, and studies have shown that short peptides are broken down after entering cells. Therefore, we determined the time required for the breakdown of BSH-A6K.

[0131] Measurement process:

[0132] ① Prepare 30 bottles of 29ml cell simulation solution, and add 1ml of BSH-A6K at a concentration of 6mg / L to each bottle.

[0133] ② The protein content was measured using biuret reagent every half hour.

[0134] ③ The protein content data obtained from process 2 show that starting from the 14th culture flask (i.e., 7 hours), the calculated protein content is already very low. Starting from the 16th culture flask (i.e., 8 hours), the biuret reagent no longer turns purple, proving that A6-K is completely decomposed at this point. From this phenomenon, we can conclude that if PET-CT detection is performed 8 hours after injecting mice with BSH-A6K, the calculated B... 10The concentration is not affected by the short peptide A6K.

[0135] ④ If the 8-hour waiting period is not followed or the time is insufficient, the calculated B 10 Concentration can introduce errors (leading to a lower concentration), but for BNCT therapy, we expect boron concentrations to accumulate more in malignant tumors. Therefore, the fact that the actual boron concentration is higher than the calculated concentration does not affect the inference of the conclusion.

[0136] 6. Conclusion

[0137] B in tumor tissue 10 The content of all of them is greater than 25ug B 10 / g, consistent with B in tumor tissue 10 The concentration range reached 20-35 μg B 10 / g, Tumor (T) / Normal Tissue (N)B 10 The concentration ratios are all greater than 3, indicating tumor (T) / blood (B)B. 10 The concentration ratios were all greater than 3.5, indicating that BSH had therapeutic effects on tumors of all three germ layers (exodermal, mesodermal, and endoderm). However, BSH is more toxic than BPA, while BPA has good therapeutic effects on ectodermal tumors. Therefore, BSH, which is more toxic, should not be used as a boron drug for ectodermal tumors such as breast cancer; and B in liver cancer cells... 10 The highest content was found in endodermal tumor cells B. 10 The content is higher than that of other germ layers, indicating that BSH has the most significant therapeutic effect on endoderm, especially liver cancer. This boron drug is suitable for use in BNCT treatment of the above-mentioned tumors. In BNCT treatment of mesodermal tumors (such as lymphoma), this boron drug can be considered depending on the specific condition of the patient.

[0138] The following will further illustrate this with reference to specific embodiments.

[0139] Example 1

[0140] After cell resuscitation, the cells were cultured and passaged. The drug toxicity of Cl-BSH was detected using the CCK-8 assay, and the drug IC50 was determined. 50 Cl-BSH was then injected into a mouse model of glioma via the tail vein. PET-CT scans were performed, and signal processing and image reconstruction were used to further demonstrate the aggregation or distribution of BSH in the cells.

[0141] Detection of Cl using PET-CT 37 - PET images of BSH in tumor-bearing mice. The implementation method involves first inoculating mice with U-251 tumor or head and neck tumor cells, then using positron emission tomography to detect the boron content in the mice. The data obtained from the implementation method are analyzed to see if they meet the requirements of BNCT. Finally, the drug toxicity is analyzed to ensure that the drug has a therapeutic effect without causing harm to the patient.

[0142] After preparing a fluorescent probe from boron drugs using a self-developed method, its Cl- was observed using PET-CT. 37 The content was detected to calculate the tumor (T) / normal tissue (N) B ratio. 10 Concentration and tumor (T) / blood (B)B 10 The concentration ratio was determined, and the impact of error was evaluated (the error in this implementation was caused by the presence of the A6K short peptide). Finally, the results of PET-CT detection of Cl were obtained through testing. 37 - The BSH boron content needs to be waited for 8 hours to eliminate the error caused by the A6K short peptide.

[0143] Neutron emitter emits neutrons onto 10 tumor (T) / normal tissue (N) B cells. 10 Concentration ratio and tumor (T) / blood (B)B 10 Two groups of mice with different concentrations of boron-10 were irradiated. The effect of boron-10 aggregation in tumors on the therapeutic effect was determined by efficacy evaluation, which corroborates the correlation between tumor (T) / normal tissue (N) in BNCT treatment. 10 Concentration ratio and tumor (T) / blood (B)B 10 The concentration ratio should be greater than 3-5.

[0144] Example 2

[0145] Determining the effect of boron-10 aggregation degree on therapeutic efficacy

[0146] 1. Material preparation:

[0147] Five mice with breast cancer and five mice with brain tumors were ordered.

[0148] 1 mg / kg Cl37-BSH

[0149] neutron emitter

[0150] Anesthetic

[0151] 2. Administration:

[0152] Ten mice were anesthetized with isoflurane and injected via the tail vein with 1 mg / kg of Cl37-BSH clarified solution.

[0153] 3. Determination of Boron-10 Aggregation Degree:

[0154] The mice were subjected to PET-CT examination using the procedure described in Example 3.

[0155] 4. Calculation of average value:

[0156] Using the calculation method in Example 3, the average values ​​of the tumor (T) / normal tissue (N) B10 concentration ratio and the tumor (T) / blood (B) B10 concentration ratio were calculated for two groups of mice (one group of breast cancer mice and one group of brain tumor mice).

[0157] 5. Implement data processing and analysis

[0158] The average B10 concentration ratio of tumor (T) to normal tissue (N) in 5 breast cancer mice was 1.27;

[0159] The average ratio of tumor (T) to blood (B) B10 concentration in 5 breast cancer mice was 1.93;

[0160] The average B10 concentration ratio of tumor (T) to normal tissue (N) in 5 mice with brain tumors was 4.04.

[0161] The average ratio of tumor (T) to blood (B) B10 concentration in 5 breast cancer mice was 4.37;

[0162] 6. Neutron beam irradiation:

[0163] Ten mice were irradiated with neutron beams of less than 5 eV emitted by a neutron emitter.

[0164] 7. Mouse disease monitoring and implementation conclusions:

[0165] All 10 mice showed significant improvement after neutron beam irradiation, and the tumors of 7 mice were cured. However, 3 of the mice with breast cancer experienced tumor recurrence and died at 17, 19, and 39 days after neutron beam irradiation. This phenomenon suggests that a higher tumor (T) / normal tissue (N) B10 concentration ratio and a higher tumor (T) / normal tissue (N) B10 concentration ratio indicate better treatment efficacy.

[0166] Therefore, in BNCT treatment, the ratio of tumor (T) to normal tissue (N) B10 concentration and the ratio of tumor (T) to blood (B) B10 concentration should be greater than 3-5.

[0167] Example 3:

[0168] The assay procedure implemented in this embodiment takes into account the influence of the short peptide A6K on the results during the determination of boron concentration in Cl37BSH using PET-CT. Due to the presence of the short peptide A6K, the proportion of B10 in BSH-A6K (120÷718=16.7%) is much smaller than the proportion of B10 in BSH (60%). Therefore, to determine the B10 concentration, it is necessary to remove the short peptide A6K from BSH-A6K first. A6K is a short peptide, and studies have shown that short peptides are decomposed after entering cells. Therefore, we determined the time required for the decomposition of BSH-A6K.

[0169] This measurement procedure involves the following steps:

[0170] ① Prepare 30 bottles of 29ml cell simulation solution, and add 1ml of BSH-A6K at a concentration of 6mg / L to each bottle.

[0171] ② The protein content was measured using biuret reagent every half hour.

[0172] It can be concluded that if mice are injected with BSH-A6K and then subjected to PET-CT detection 8 hours later, the calculated B10 concentration will not be affected by the short peptide A6K.

[0173] If the 8-hour waiting period is not observed or is insufficient, the calculated B10 concentration will be inaccurate (to be too low). However, for BNCT therapy, we expect boron concentration to accumulate more in malignant tumors. Therefore, the fact that the actual data is higher than the calculated boron concentration does not affect the conclusion.

[0174] It should be further explained that BPA and BSH in this invention are both adjuvant boron drugs for BNCT cancer treatment. The purpose of the method implemented in this invention is to produce BSH containing fluorescent reagents so that the concentration and accumulation sites of BSH in the body can be detected by PET-CT.

[0175] Synthesis of fluorescent BSH

[0176] Based on the selection and synthesis methods of fluorescent BPA in the fluorescent labeling method, it can be reasonably inferred that to detect BSH, one of the following two methods is required.

[0177] Method 1: Injecting groups containing radioactive atoms

[0178] Method 2: Replace a certain atom of the compound with a radioactive atom.

[0179] BSH structure:

[0180] Twelve boron atoms are interconnected to form a cage-like structure, with 11 boron atoms bonded to a -H group and 1 boron atom bonded to a -SH group. Therefore, this cage-like compound is called BSH. The cage-like boron structure consists of four layers, containing 1, 5, 5, and 1 boron atom respectively. (Adjacent boron layers are interconnected.) In the first layer (fourth layer), one boron atom is bonded to a thiol group, and the remaining boron atoms are each bonded to a hydrogen atom.

[0181] Selection of synthesis method:

[0182] Since the synthesized BSH cannot change its medicinal value (chemical properties, i.e., the main functional groups cannot be greatly changed), due to the special cage-like structure of BSH, the -SH functional group and the cage-like B atom cannot be destroyed. Therefore, it can only replace the H atoms bonded to the boron atom.

[0183] About -SH (thiol group)

[0184] The sulfur atom contains a large number of lone pairs of electrons, making the thiol group a strong electron-donating group. Due to the presence of the thiol group, the electron cloud density near the -SH group in the boron cage is high. Therefore, the BH bond in the second layer of B in the boron cage (the B bonded to the thiol group is the first layer) readily reacts with electrophilic reagents. Because of this property, this implementation scheme will consider breaking the BH bond in the second layer of B in the boron cage, allowing the -H to leave and be replaced by a fluorescent -R group.

[0185] Selection of -R group and electrophilic reagent

[0186] As can be seen from the above, the selection of -R requires the following properties:

[0187] 1. It has strong electrophilic properties.

[0188] Reason: Because the electron cloud density of the boron cage is large, its electrophilicity is stronger, its electron-withdrawing ability is stronger, and it is easier to replace the hydrogen on the second layer B in the boron cage. The more stable the BR bond is, the higher the stability of the product formed.

[0189] 2. Does not react with thiol groups.

[0190] Reason: If the selected electrophilic reagent reacts with -SH, it will destroy the effective functional groups in BSH, thereby changing the chemical properties of BSH and rendering it ineffective for treatment.

[0191] 3. The selected reagents must not produce gaseous products or byproducts after the reaction. Electrophilic disintegrants must not be used as reaction reagents.

[0192] Reason: If the selected reagents produce gas or an electrophilic disintegrant is used as the reaction reagent, the boron sphere structure will break, resulting in a significant decrease in yield.

[0193] 4. The introduced -R group needs to contain a fluorescent probe.

[0194] Reason: The introduction of radioactive atoms allows the concentration of the drug in the body to be measured by PET-CT.

[0195] Therefore, hypochlorous acid is a suitable electrophilic reagent for this implementation scheme. One hypochlorous acid (HCl37O) molecule reacts with the BH bond, causing the BH bond to break and forming a B-Cl37 bond, generating the byproduct H2O (water), which satisfies the above requirements.

[0196] The significance of this invention is to assist in achieving precise treatment with BNCT capture therapy, maximizing the therapeutic effect of BNCT therapy, and avoiding the ineffective treatment caused by clinicians and dosimeters mistakenly selecting boron drugs, which would result in patients undergoing repeated radiotherapy, increasing their suffering, treatment time, and costs.

[0197] It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the protection scope of the present invention.

Claims

1. A BSH fluorescent probe-based PET-CT nine-square grid method for determining boron concentration in cancer cells, characterized in that: Using a BSH fluorescent probe PET-CT scanner, tumor cells from three germ layers (endoderm, mesoderm, and ectoderm) were selected according to the nine-square grid theory, and then their ic50 and PET-CT values ​​were analyzed separately. The process included the following steps: Step 1: Cell resuscitation. Cells are resuscitated by injecting Cl... 37 -BSH, heat the water bath to 37℃, sterilize the instrument, and add 4mL of 10% fetal bovine serum and 1% McCoys5A culture medium containing double antibiotics to the centrifuge tube; place the U-251 cell cryopreservation tube in the preheated water bath and shake it in the same direction from time to time until all the ice crystals in the tube melt into liquid, then sterilize; pipette the cryopreservation solution, aspirate the cell suspension, and centrifuge at 1000rpm / min for 5min in the centrifuge tube; discard the supernatant, add 1mL of culture medium to the precipitate, pipette and transfer it to a culture flask, add an appropriate amount of culture medium, and observe the cells; Step 2: Culture and passage to obtain more cells for Cl... 37 To determine the boron concentration of -BSH, cells must first be cultured and passaged. After the adherent cells in the culture flask cover 80%–90% of the bottom, wash the cells twice with 3 mL of PBS, add 1.5 mL of 0.25% trypsin, shake and observe after 3–4 min. When the cells no longer clump together, add twice the amount of culture medium to stop digestion; centrifuge in a 15 mL centrifuge tube at 1000 rpm / min for 5 min; discard the supernatant, add 1 mL of LMcCoys5A culture medium to the bottom, pipette to resuspend, take 1 / 3 and add 6 mL of culture medium; Step 3: CCK-8 method for Cl determination 37 Add 1.5 mL of 0.25% trypsin to U-251 cells (BSHic50), centrifuge at 1000 rpm for 5 min, and observe. Seed 100 μL of 3k-7k cells / well in a 96-well plate and incubate at 37°C, 5% CO2, and 90% humidity for 24 hours. Prepare sample solutions of different concentration gradients, add 10 μL of each solution to three replicates in a 96-well plate, and incubate for 6, 12, 24, or 48 hours. Thaw and centrifuge CCK-8, and add 10 μL of CCK-8 solution to each well.

2. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 1, characterized in that: The detection process must involve separately testing the Cl- concentrations of the aforementioned tumor cell suspension in tumor tissue, normal tissue, and blood. 37 Concentration, and calculate the different B concentrations in tumor tissue, normal tissue, and blood. 10 Concentration, and finally the difference between tumor tissue and normal tissue B was calculated. 10 Concentration ratio (T / N), tumor tissue to blood B 10 Concentration ratio (T / B).

3. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 2, characterized in that: Regarding (T / N) and (T / B), when both T / N and T / B are >3, it can be considered that BPA boron drugs are suitable for BNCT treatment of this type of cancer, that is, this type of cancer is an indication for BSH; when both T / N and T / B are <3, it can be considered that BSH boron drugs are not suitable for BNCT treatment of this type of cancer, that is, this type of cancer is not an indication for BSH.

4. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 1, characterized in that: In step one, after culturing the cells at 37°C, 5% CO2, and saturated humidity for 24 hours, observe the cells again, and change the medium every 2-3 days.

5. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 1, characterized in that: In step two, the cells are cultured to 20 to 30 million in an incubator at 37°C, 5% CO2, and saturated humidity.

6. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 1, characterized in that: In step three, the food is incubated at 37°C, 5% CO2, and 90% humidity for 0.5-4 hours; the absorbance is measured at 450 nm using an ELISA reader, and the results are processed and analyzed.

7. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 6, characterized in that: The method used in step three is to design Cl. 37 The concentrations of -BSH were 300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, and 0.14 μg / g, with three replicates for each concentration. After incubation with CCK-8 for four hours, the absorbance was measured at 450 nm using a microplate reader. The results were processed and analyzed using Excel and GraphpadPris.

8. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 1, characterized in that: The fluorescent probe Cl 37 -BSH structure is a boron chloride cage compound.

9. The method for determining boron concentration in a BSH fluorescent probe-based PET-CT nine-square grid of cancer cells according to claim 8, characterized in that: The boron chloride cage compound chlorine-37 is attached to the β-position of the mercapto group, but a small amount is attached to the Y-position. Chlorine-37 attached to the Y-position and the β-position have the same pharmacological effects and produce the same results in concentration determination.

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