A method for preparing tumor antigens based on BNCT and its application

Abstract

The present invention relates to the technical fields of chemical and biomedical tumor diagnosis and treatment, and provides a method for preparing tumor antigens based on BNCT and its application. The method of the present invention constructs boron nitride nanoparticles based on chemical vapor deposition and ultrasonic treatment in water, and then modifies the boron nitride nanoparticles with polyvinylpyrrolidone PVP to obtain BN@PVP. Finally, the Toll-like receptor agonist R837 is loaded into BN@PVP to obtain BN-R837@PVP, and the prepared BN-R837@PVP is applied to tumor BNCT treatment. When the method of the present invention is applied to BNCT treatment, it can improve the cross-presentation efficiency of dendritic cells, effectively activate the anti-tumor immunity of cells, cause the amplification of CD4+ cells and CD8+ T cells in tumors, increase the infiltration of CD4+ and CD8+ T cells in tumor tissues, effectively inhibit the growth and metastasis of distant tumors, and can also stimulate individuals to exhibit a strong memory T cell effect, and can induce highly efficient and long-term anti-tumor immunity in vivo, providing a new idea for the development of novel BNCT tumor vaccines.

Description

Technical Field

[0001] The present invention relates to the field of chemical and biomedical tumor diagnosis and treatment technology, and in particular to a method for preparing tumor antigens based on BNCT and an application thereof. Background Art

[0002] Boron neutron capture therapy (BNCT) is a binary precision treatment system at the cellular level that utilizes stable isotopes to 10 The high thermal neutron capture cross section of the B atom absorbs low-energy thermal neutrons to undergo nuclear fission, producing alpha particles and recoil lithium nuclei. 7 Since the penetration distance of α particles is only 4 to 9 μm, the energy of α particles can be deposited in a single cell, achieving specific killing of a single cell, which makes the use of high linear energy transfer (high-LET) an important treatment method for malignant tumors.

[0003] Currently, BNCT has been applied to malignant brain gliomas and some solid tumors with special anatomical structures that are difficult to treat, and has achieved good therapeutic effects. BNCT has many advantages as a tumor-specific treatment method, but its in vivo application has extremely strict requirements on the tumor targeting of boron drugs. 10 B atoms accumulate in tumor tissues to more than 20 ppm, and in order to reduce 10 The damage of B atoms to normal tissues needs to be controlled 10 The distribution ratio of boron atoms in tumor tissue and normal tissue is not less than 3, which seriously hinders the development of BNCT in vivo. However, the boron content required for BNCT in vitro is easy to achieve, and the accumulation of boron drugs in tumor cells has exceeded 20ppm at this stage, which has good in vitro BNCT conditions. Therefore, it is extremely important to develop in vitro BNCT based on low dependence on boron drugs and overcome the limitations of boron drugs on BNCT to promote the development and application of BNCT.

[0004] As a new type of tumor treatment, tumor vaccines are constructed based on in vitro bioengineering methods. Their therapeutic effects do not rely on targeted enrichment of drugs in vivo. The application of tumor vaccines provides a new research and application path for BNCT. Its principle is to use tumor antigens of tumor cells themselves with the assistance of adjuvants such as cytokines and chemokines to activate or enhance the body's own immune system to kill tumor cells, thereby achieving the purpose of controlling and eliminating tumors. In April 2010, the U.S. Food and Drug Administration (FDA) approved the use of Provenge / Sipuleucel-T in the treatment of advanced prostate cancer, marking the birth of the first therapeutic cancer vaccine. However, the immunogenicity of tumor cells themselves is low, and how to improve the immunogenicity of tumor cells has become the key to the preparation of tumor vaccines. Inducing immunogenic cell death (ICD) of tumor cells can promote the exposure of tumor antigens and enhance antigen presentation. The latest research has confirmed that radiotherapy, chemotherapy, and photothermal therapy can induce immunogenic cell death in tumor cells, promote antigen presentation, and activate T cell responses. Therefore, radiotherapy, chemotherapy, and photothermal therapy can all be used to prepare tumor vaccines. BNCT is a radiotherapy treatment method that efficiently kills tumor cells and induces immunogenic cell death in vitro, and has great application prospects in tumor vaccine development. Therefore, it is urgent to propose a tumor antigen preparation method based on BNCT and its application, and to develop tumor vaccines using in vitro BNCT, so as to provide an effective solution to the problem of over-reliance on boron drugs when BNCT is used in tumor treatment, and promote the development of anti-tumor immunity. Summary of the invention

[0005] In view of the problem that cells are difficult to activate to produce anti-tumor immune response during BNCT, the present invention adopts polyvinyl pyrrolidone (PVP)-modified boron nitride nanoparticles loaded with Toll-like receptor agonist R837 to prepare BN-R837@PVP, which effectively improves the cross-presentation efficiency of dendritic cells (DCs). By applying the obtained BN-R837@PVP to tumor BNCT treatment, it can activate a long-term and effective adaptive immune response, induce CD4 + Cells and CD8 + T cell expansion and increase in CD4 + and CD8 + T cell infiltration effectively inhibits the growth and metastasis of distant tumors and prolongs the individual's survival.

[0006] The present invention specifically adopts the following technical solutions:

[0007] A method for preparing tumor antigens based on BNCT, using polyvinylpyrrolidone (PVP)-modified boron nitride nanoparticles to load Toll-like receptor agonist R837, specifically comprising the following steps:

[0008] Step 1: Construction of boron nitride nanoparticles based on chemical vapor deposition and ultrasonic treatment in water

[0009] Boric acid and urea are dissolved in a methanol aqueous solution at a molar ratio of 1:3 to form a mixed solution, the mixed solution is stirred at 45°C for 12 hours, the mixed solution is recrystallized, the mixed solution is filtered to obtain a white crystalline powder, the white crystalline powder is placed in a quartz tube furnace for pyrolysis to obtain h-BNNs, and the h-BNNs are dissolved in water to obtain a dispersion system, the dispersion system is centrifuged and filtered, and then the dispersion system is ultrasonically treated in water using a probe ultrasonicator, and the dispersion system is freeze-dried to obtain boron nitride nanoparticles;

[0010] Step 2: Preparation of BN@PVP using boron nitride nanoparticles and polyvinylpyrrolidone (PVP)

[0011] The boron nitride nanoparticles are dissolved in water to obtain a BN solution, polyvinyl pyrrolidone (PVP) is added to the BN solution to form a mixture, and the mixture is stirred for 16 to 18 hours. The mixture is ultrasonically treated in water using a probe ultrasonicator, and then the mixture is centrifuged and washed at least 3 times to obtain polyvinyl pyrrolidone (PVP)-modified boron nitride nanoparticles (BN@PVP);

[0012] Step 3: Loading the Toll-like receptor agonist R837 into BN@PVP to prepare BN-R837@PVP

[0013] Boron nitride nanoparticles BN@PVP were dissolved in water to obtain a boron nitride nanoparticle solution, and then Toll-like receptor agonist R837 was dissolved in dimethyl sulfoxide (DMSO) and mixed with the boron nitride nanoparticle solution to obtain a mixed system, and the mixed system was stirred at 45°C for 12 hours, and then the mixed system was centrifuged at least 3 times to remove the Toll-like receptor agonist R837 not loaded on the boron nitride nanoparticles, thereby obtaining BN-R837@PVP loaded with Toll-like receptor agonist R837;

[0014] Step 4: Use a UV-visible spectrophotometer to measure BN-R837@PVP, obtain the characteristic absorption peak of R837 in the UV-visible absorption spectrum, and determine the loading rate of R837 in BN-R837@PVP.

[0015] Preferably, in step 1, the volume ratio of methanol to water in the methanol aqueous solution is 1:1.

[0016] Preferably, in step 1, when the white crystalline powder is pyrolyzed in a quartz tube furnace, the temperature of the quartz tube furnace does not exceed 1000°C.

[0017] Preferably, in step 1, when the boron nitride solution is subjected to ultrasonic treatment in water using a probe ultrasonicator, the temperature of the boron nitride solution is 4-37°C.

[0018] Preferably, in step 2, the concentration of the BN solution is 100 μg / mL, and the amount of polyvinylpyrrolidone PVP added is 1 mg / ml.

[0019] Preferably, in step 2, the average particle size of BN@PVP is 220 nm.

[0020] Preferably, in step 3, the concentration of the boron nitride nanoparticle solution is 0.1 mg / mL.

[0021] The above-mentioned BNCT-based tumor antigen preparation method is used in tumor BNCT treatment.

[0022] Preferably, it is used to activate anti-tumor immune response of cells.

[0023] Preferably, by increasing CD4 + Cells and CD8 + T cell infiltration inhibits tumor growth and metastasis.

[0024] The present invention has the following beneficial effects:

[0025] The present invention uses polyvinyl pyrrolidone (PVP) to modify hydrophilic boron nitride nanoparticles, and loads the Toll-like receptor agonist R837 into BN@PVP to prepare BN-R837@PVP, which effectively improves the cross-presentation efficiency of dendritic cells (DCs). BN-R837@PVP is applied to BNCT treatment, and the combined effect of BN-R837@PVP and neutron irradiation is used to treat tumor cells, activate the adaptive immune response of cells, and amplify the CD4 + Cells and CD8 + T cells, increasing CD4 + Cells and CD8 + At the same time, when the BN-R837@PVP prepared by the present invention is applied to BNCT, the tumor antigen based on BNCT can attack the individual to show a strong memory T cell effect, which can effectively inhibit the tumor in vivo for a long time, and achieve the induction of efficient and long-term anti-tumor immunity in BNCT, which provides a new idea for the development of new BNCT tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 ATP release and HMGB1 migration in 4T1 cells under different treatment conditions. Figure 1 In the figure, (a) shows the ATP release in 4T1 cells under different treatment conditions, and (b) shows the HMGB1 migration in 4T1 cells under different treatment conditions.

[0027] Figure 2 Antigen presentation in 4T1 cells under different treatment conditions. Figure 2 In the figure, (a) shows the co-stimulatory factor CD80 expressed on dendritic cells without BNCT treatment, and (b) shows the co-stimulatory factor CD80 expressed on dendritic cells after BNCT treatment.

[0028] Figure 3 The fine structure of BN nanoparticles. Figure 3 In the figure, Figure (a) is the XRD analysis result of BN nanoparticles, Figure (b) is the XPS analysis result of BN nanoparticles, Figure (c) is the UV-visible absorption spectrum of BN-R837@PVP, and Figure (d) is the horizontal hydrated particle size of BN-R837@PVP after loading R837.

[0029] Figure 4 The results of the cell uptake experiment of 4T1 cells. Figure 4 In the figure, Figure (a) shows the boron concentration in 4T1 cells at different incubation times with BN-R837@PVP, Figure (b) shows the boron concentration in 4T1 cells after 4T1 cells were incubated with different concentrations of BN-R837@PVP for 24 hours, Figure (c) shows the cell viability analysis results of 4T1 cells, and Figure (d) shows the CRT exposure in 4T1 cells after BNCT induction observed by CLSM.

[0030] Figure 5 Diagram for the design of a tumor antigen injection experiment in mice.

[0031] Figure 6 The Spaghetti curves of mice after injection of different cell vaccines.

[0032] Figure 7 The results of the metastasis inhibition experiment based on tumor antigens of BNCT. Figure 7 Figure (a) shows the number of lung nodules in mice of different groups, and Figure (b) shows the number of CD4 in the spleen of mice of different groups. + Statistical results of cell populations. Figure (c) shows CD8 + Statistical results of T cell populations.

[0033] Figure 8 These are the results of adoptive immunization experiments. Figure 8Figure (a) shows the CD3 in the spleen of each group of mice. + Cell population, Figure (b) shows the CD4 + Cell population, Figure (c) shows the CD8 + Cell population, Figure (d) shows the CD4 + T cells, Figure (e) shows the CD8 + Figure (f) shows the CD4 T cells in the tumor tissues of mice in each group. + Figure (g) shows the infiltration of TEM cells in the tumor tissues of mice in each group. + Infiltration of TEM cells. DETAILED DESCRIPTION

[0034] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments:

[0035] The present invention provides a method for preparing tumor antigens based on BNCT, using polyvinyl pyrrolidone (PVP)-modified boron nitride nanoparticles to load a Toll-like receptor agonist R837, which specifically comprises the following steps:

[0036] Step 1: Construction of boron nitride nanoparticles based on chemical vapor deposition and ultrasonic treatment in water

[0037] A methanol aqueous solution was prepared using 20 ml of methanol and 20 ml of water, and boric acid and urea were dissolved in the methanol aqueous solution at a molar ratio of 1:3 to form a mixed solution. The mixed solution was then stirred at 45°C for 12 hours and then recrystallized. The mixed solution was filtered to obtain a white crystalline powder, and the white crystalline powder was placed in a quartz tube furnace. The temperature in the quartz tube furnace was increased to 1000°C at a heating rate of 5°C / min. The white crystalline powder was placed in a quartz tube furnace and calcined and pyrolyzed in a nitrogen environment for 2 hours to obtain h-BNNs. The h-BNNs were then dissolved in water to obtain a dispersed system. The impurities in the h-BNNs were effectively removed by centrifuging and filtering the dispersed system. The dispersed system was then ultrasonically treated in water using a probe ultrasonicator to reduce the size of the h-BNNs, and the dispersed system was freeze-dried to obtain boron nitride nanoparticles.

[0038] Step 2: Preparation of BN@PVP using boron nitride nanoparticles and polyvinylpyrrolidone (PVP)

[0039] In order to improve the stability of boron nitride nanoparticles, boron nitride nanoparticles were dissolved in water to obtain a BN solution, and 5 mg of polyvinyl pyrrolidone (PVP) was added to 10 ml of a BN solution with a concentration of 10 μg / ml to form a mixture, which was stirred for 16 to 18 hours. The mixture was ultrasonically treated in water for 1 hour using a probe ultrasonicator, and the speed of the centrifuge was set to 10,000 rpm. The treated mixture was centrifuged in a centrifuge for 10 minutes and washed at least 3 times to fully remove excess polyvinyl pyrrolidone (PVP) in the mixture to obtain boron nitride nanoparticles (BN@PVP) modified with polyvinyl pyrrolidone (PVP).

[0040] Step 3: Loading the Toll-like receptor agonist R837 into BN@PVP to prepare BN-R837@PVP

[0041] Boron nitride nanoparticles BN@PVP were dissolved in water to obtain a boron nitride nanoparticle solution with a concentration of 0.1 mg / ml, and then 1 mg of Toll-like receptor agonist R837 was dissolved in dimethyl sulfoxide DMSO, and then mixed with the boron nitride nanoparticle solution to obtain a mixed system. The mixed system was stirred at 45°C for 12 hours, and the mixed system was centrifuged at least 3 times to remove the Toll-like receptor agonist R837 not loaded on the boron nitride nanoparticles, thereby obtaining BN-R837@PVP loaded with Toll-like receptor agonist R837.

[0042] Step 4: Use a UV-visible spectrophotometer to measure BN-R837@PVP, obtain the characteristic absorption peak of R837 in the UV-visible absorption spectrum, and determine the loading rate of R837 in BN-R837@PVP.

[0043] Since BNCT treatment can trigger the immunogenic cell death of cancer cells to expose antigens, but due to the low absorption efficiency and low maturity, the cross-presentation between dendritic cells DCs and BNCT-treated cells is severely displayed, making it difficult for BNCT-treated cells to stimulate and produce effective anti-tumor immune responses. Therefore, in order to improve the efficacy of cross-presentation between cells, the present invention adopts a carrier-free strategy, using polyvinyl pyrrolidone PVP to modify hydrophilic boron nitride nanoparticles, combined with the installation of small molecule Toll-like receptor agonist R837 to prepare BN-R837@PVP, which significantly improves the cross-presentation efficiency of dendritic cells.

[0044] The BN-R837@PVP prepared by the method of the present invention, combined with neutron irradiation, can activate the adaptive immune response after being attacked by BNCT-treated cells in vivo, so that CD4 + Cells and CD8 + T cell expansion by increasing CD4 + Cells and CD8+ The infiltration of T cells inhibits tumor growth and metastasis and prolongs individual survival. In addition, this BNCT-based treatment also exhibits a powerful memory T cell effect, which can effectively and long-term suppress tumors in vivo, which is conducive to BNCT inducing long-term and effective anti-tumor immunity.

[0045] Example 1: Immune response of tumor cells after BNCT treatment

[0046] Calreticulin (CRT) exposure, high mobility group protein 1 (HMGB1) migration and ATP release were selected as three markers for detecting immunogenic cell death (ICD) in cells. The three markers of ICD were detected in BNCT-treated 4T1 cells to study whether BNCT treatment could induce immunogenic cell death.

[0047] 4T1 cells were incubated with BN@PVP at a concentration of 40 ppm for 24 h. The incubated 4T1 cells were divided into an experimental group and a control group. The 4T1 cells in the experimental group were treated with BNCT (using 2.57 x 10 8 cm -2 ·s -1 4T1 cells were irradiated with neutrons for 1 h), washed twice in cold PBS containing 0.5% FBS and 2% BSA, Alexa Fluor 647-coupled anti-CRT antibody and Hoechst were added to 4T1 cells at 4°C, CRT on the surface of 4T1 cells in the experimental and control groups was labeled with anti-CRT antibody, and cell nuclei were labeled with Hoechst 33342. After the 4T1 cells were left to stand for 1 h, the 4T1 cells were washed twice with cold PBS, and microscopic imaging and flow cytometry FACS analysis were performed on the experimental and control groups. CRT exposure of 4T1 cells was observed using confocal laser scanning microscopy (CLSM), and it was found that BNCT treatment significantly induced CRT exposure on the cell surface compared with the control group.

[0048] The 4T1 cells were then incubated with BN@PVP at B concentrations of 0 ppm, 10 ppm, 20 ppm, and 40 ppm for 24 h, and then neutron irradiated for 1 h to measure ATP release and HMGB1 migration in 4T1 cells under BN@PVP conditions with different B concentrations.

[0049] The ATP released from the supernatant of 4T1 cell culture medium was evaluated using the ATPlite 1step Luminescence Assay System to obtain the ATP released by 4T1 cells in DC2.4 cells, such as Figure 1(a) and then the migration level of HMGB1 in 4T1 cells under different conditions was evaluated by enzyme-linked immunosorbent assay (ELISA) using a commercial kit. Figure 1 (b) Based on the results of flow cytometry FACS, even if 10 CRT exposure on 4T1 cells was also observed at 10 ppm B concentration. 10 At the same concentration of B, a significant increase in ATP release and HMGB1 migration was also observed after BNCT treatment, while 4T1 cells treated with boron or neutron irradiation alone did not cause any significant increase in CRT exposure, ATP release and HMGB1 migration, all of which directly indicate that BNCT treatment can induce immunogenic cell death.

[0050] The presentation of exogenous antigens on the major histocompatibility complex (MHC) is called cross-presentation, which is crucial for the initiation of adaptive immune responses. In order to study the uptake ability of dendritic cells on BNCT-treated cells, a cell uptake experiment with a control group was set up. DC2.4 cells were cultured in 48-well plates and labeled with carboxyfluorescein succinimidyl amino ester CFSE. After 4T1 cells were co-incubated with BN@PVP with a boron concentration of 40 ppm for 24 hours, 4T1 cells were labeled with Cell TrackerTM Deep Red, and then the 4T1 cells were divided into an experimental group and a control group. The 4T1 cells in the experimental group were neutron irradiated for 1 hour, and the 4T1 cells in the control group were not neutron irradiated. After the 4T1 cells in the experimental group and the control group were co-cultured with DC2.4 cells for 24 hours, the DC2.4 cells were rinsed with pre-cooled PBS and re-plated for imaging. The phagocytosis of 4T1 cells in DC2.4 cells was observed using a confocal laser scanning microscope, and the uptake efficiency of DC2.4 cells in the control group and the experimental group was evaluated. The expression of co-stimulatory molecules CD80 and CD86 on DC2.4 cells was obtained, as shown in Figure 2. Figure 2 According to the results of the cell uptake experiment, compared with normal 4T1 cells, the red fluorescence signal in 4T1 cells after BNCT treatment increased slightly, indicating that DC2.4 cells had a low absorption rate of tumor antigens treated with BNCT. The expression of co-stimulatory factors CD80 and CD86 on dendritic cells was further determined to evaluate the DC 25,27 The maturity of dendritic cells was monitored and it was found that the co-stimulatory factors CD80 and CD86 did not change significantly, which proved that although BNCT treatment can induce ICD, the low efficiency of antigen presentation by dendritic cells is not conducive to the induction of immune response by BNCT treatment, which becomes the bottleneck of BNCT-induced immune response.

[0051] Example 2: Characterization of BN nanoparticle structure in BN-R837@PVP

[0052] Due to the inefficiency of antigen presentation, BNCT is not enough to activate a strong enough immune response to kill the already formed tumors. Toll-like receptor agonists can promote antigen phagocytosis and maturation of dendritic cells, stimulate T cells and other lymphocytes, and are beneficial to the treatment of already formed cancers. The present invention uses a loaded Toll-like receptor agonist R837 to stimulate dendritic cell activation, maturation and migration to produce a strong tumor-specific immune response, thereby activating the innate immune response of the cells. Therefore, the present invention loads the Toll-like receptor agonist R837 into BN@PVP to prepare BN-R837@PVP.

[0053] The invention constructs boron nitride nanoparticles based on a chemical vapor deposition method and ultrasonic treatment in water. Boric acid and urea are dissolved in a methanol aqueous solution at a molar ratio of 1:3 to form a mixed solution. The mixed solution is stirred at 45°C for 12 hours and then the mixed solution is recrystallized. The mixed solution is filtered to obtain a white crystalline powder. The white crystalline powder is placed in a quartz tube furnace for pyrolysis to obtain h-BNNs. The h-BNNs are then dissolved in water to obtain a dispersed system. The dispersed system is centrifuged and filtered, and then ultrasonic treatment is performed in water on the dispersed system using a probe ultrasonic instrument to reduce the size of the h-BNNs and freeze-dry to obtain BN nanoparticles.

[0054] The fine structure of BN nanoparticles was characterized. The hydrodynamic size and zeta potential of BN nanoparticles were recorded under dynamic light scattering conditions at 25 °C. The step size of the Bruker D8 Advance instrument was set to 0.02° and the 2θ range was from 10° to 70°. ° The X-ray diffraction pattern of BN nanoparticles was recorded by Bruker D8 Advance instrument, and the Fourier transform infrared spectrum of BN nanoparticles was obtained by ThermoScientific Nicolet iN10 MX spectrometer to determine the BN nanoparticles in the range of 4000-5000 cm -1 The UV-visible spectrum of the BN nanoparticles was obtained by UV-visible spectrophotometer, and the specific surface area of ​​the BN nanoparticles was calculated based on Brunauer-Emmett-Teller.

[0055] In this embodiment, the fine structure of BN nanoparticles was observed using high-resolution transmission electron microscopy (HRTEM). Figure 3As shown in Figure 2, lattice fringes with a spacing of 0.333 nm were observed in HRTEM. The lattice fringes are relatively complex, indicating that the crystallinity of BN nanoparticles is low. By obtaining the X-ray diffraction pattern of BN nanoparticles, it can be seen that the 2θ angle of BN nanoparticles has broad diffraction peaks at 20-26° and 40-45°, which belong to the (002) and (100) interplanar spacings of h-BNNs (JCPDS851068). The X-ray photoelectron spectroscopy shows that the binding energies of N1s and B1s are 397.8 eV and 190.1 eV, respectively, which are close to those with BN nanoparticles. 3 and NB 3 The low-layer boron nitride of the triangular unit has a peak of 191.9 eV in the B1s spectrum belonging to the BO bond structure, and a peak of 190.6 eV belonging to the BN bond structure. Based on the XPS results, it can be seen that the elemental structure of the BN nanoparticles is B 1.0 C 0.1 N 0.7 O 0.3 .

[0056] Using N 2 The specific surface area and pore size distribution of BN@PVP were determined by adsorption-desorption and Barrett-Joyner-Halenda pore size distribution. The specific surface area of ​​BN@PVP was found to be 419.2 cm 3 / g, and the pore size is about 2.6nm. Due to the high specific surface area and mesoporous structure of BN@PVP, it can effectively promote drug loading, which is beneficial to the combined treatment of BNCT with other drugs (including chemotherapeutic drugs and immune adjuvants).

[0057] BN-R837@PVP was prepared by loading the Toll-like receptor agonist R837 onto boron nitride nanoparticles BN@PVP. PVP was used to encapsulate BN-R837 to increase stability and monodispersity. The characteristic absorption peaks of the Toll-like receptor agonist R837 in the UV-visible absorption spectrum at 244nm, 305nm and 319nm in BN-R837@PVP were observed, confirming that the Toll-like receptor agonist R837 had been successfully loaded. At the same time, UV-visible absorption measurement of BN-R837@PVP revealed that the drug loading efficiency of R837 in BN-R837@PVP was 9.35%, the average hydrated particle size of BN@PVP before drug loading was 220nm, and the horizontal hydrated particle size of BN-R837@PVP increased to 255nm after loading R837.

[0058] Example 3: Antigen presentation efficiency of BN-R837@PVP

[0059] Multiple groups of 4T1 cells were cultured in well plates. The 4T1 cells were incubated with different concentrations of BN@PVP and BN-R837@PVP for 2 h, 12 h and 24 h, respectively. The cells were rinsed twice with PBS to remove excess BN nanoparticles in the culture medium. 1 ml of trypsin solution was added to each culture medium, and the lysate in the culture medium was collected and mixed with 2 ml of nitric acid. After nitration at 200 °C for 30 min, the B content in the digestive fluid was determined by inductively coupled plasma mass spectrometry (ICP-OES).

[0060] 4T1 cells were incubated with BN@PVP and BN-R837@PVP, respectively, and then the cell uptake was measured. DC2.4 cells were cultured in a well plate and labeled with carboxyfluorescein succinimidyl amino ester. 4T1 cells were incubated with BN@PVP with a B element concentration of 40 ppm and BN-R837@PVP with a B element concentration of 40 ppm for 24 hours, respectively. 4T1 cells were labeled with CellTrackerTM Deep Red and divided into an experimental group and a control group. After the cells in the experimental group were neutron irradiated for 1 hour, the 4T1 cells in both the experimental and control groups were co-cultured with DC2.4 cells for 24 hours, the DC2.4 cells were rinsed with pre-cooled PBS and re-plated, and the efficiency of DC2.4 cell uptake was evaluated using a laser scanning confocal microscope. The results are shown in Figure 2. Figure 4 As shown in Figure 2, the concentration of B element in 4T1 cells increased in a time- and concentration-dependent manner. The loading of R837 had little effect on cell uptake. After incubating 4T1 cells with BN@PVP and BN-R837@PVP with a B concentration of 40 ppm for 24 h, the concentration of B element in 4T1 cells reached 10 6 The boron content of each cell exceeded 20 μg, which is 1000 times the minimum boron content required. It can be seen that BN@PVP and BN-R837@PVP do not affect the cell viability of 4T1 cells. In contrast, under neutron irradiation, the cell viability after treatment with BN-R837@PVP with a B element concentration of 40 ppm was reduced to less than 50%.

[0061] Further, the CRT exposure of cells treated with BN-R837@PVP and neutron irradiated was performed. DC2.4 cells were cultured in RPMI-1640 containing 10% FBS, 4T1 cells were incubated with BN-R837@PVP with a B element concentration of 40ppm for 24h and then neutron irradiated for 1h. After the neutron-irradiated 4T1 cells were co-incubated with DC2.4 cells for 24h, the PE-anti-mouse CD80 antibody or FITC-anti-mouse CD86 antibody in DC2.4 cells was stained to evaluate the maturity of DC2.4 cells, and fluorescence was detected by flow cytometry or laser scanning confocal microscopy. According to the CLSM observation results, BNCT and BN-R837@PVP+Neutron obviously caused the accumulation of CRT on the cell surface, and the ICD induced by BNCT was not interfered by R837. Meanwhile, compared with BN@PVP+Neutron, BN-R837@PVP+Neutron significantly increased the red fluorescence signal in DC2.4 cells, indicating that the loading of R837 on BN nanoparticles promoted the uptake of 4T1 cells by dendritic cells.

[0062] DC2.4 cells were then incubated with different 4T1 cells for 24 hours. The supernatant was centrifuged and the expression levels of cytokines TNF-α, IL-1β and IL-12 were determined using ELISA kits. The maturity of DCs was evaluated based on the expression of co-stimulatory factors CD80 and CD86 on DCs. According to the experimental results, BN-R837@PVP+Neutron significantly increased the expression of co-stimulatory factors CD80 and CD86 on DCs, indicating that BN-R837@PVP loaded with R837 can effectively stimulate the cross-presentation of tumor antigens based on BNCT by increasing the uptake and maturation of dendritic cells.

[0063] Example 4: Inhibition of distal tumor growth using BN-R837@PVP

[0064] The distal effect is based on the activation of the immune system to suppress distal tumors that have not been irradiated by neutrons. In order to study the anti-tumor effect of BN-R837@PVP in BNCT treatment in vivo, 4T1 cells with different treatments were inoculated twice in the right hind limbs of mice as tumor antigens, such as Figure 5 shown.

[0065] After 4T1 cells were co-cultured with BN@PVP with a B element concentration of 40 ppm and BN-R837@PVP with a B element concentration of 40 ppm for 24 h, the 4T1 cells were divided into an experimental group and a control group. The 4T1 cells in the experimental group were neutron irradiated for 1 h, and the treated cells were suspended in a mixture of PBS and matrigel. Six 4T1 cell vaccines were injected into the right hind limbs of female BALB / c mice (6-8 weeks old) on days 0 and 7, respectively. The 4T1 cells in the G1 group were not treated, the 4T1 cells in the G2 group were neutron irradiated, the 4T1 cells in the G3 group were treated with BN@PVP, the 4T1 cells in the G4 group were treated with BN@PVP and neutron irradiation, the 4T1 cells in the G5 group were treated with BN-R837@PVP, and the 4T1 cells in the G6 group were treated with BN-R837@PVP and neutron irradiation. The tumor size and weight of the mice were recorded after injection. During the experiment, when the tumor size in the mouse reached 3000mm 3 Or if the mice lose weight seriously, the experiment needs to be terminated in time. When the survival time of the mice in the experimental group reaches twice that of the mice in the control group, the experiment is stopped. During the experiment, spleen cells and single cells are isolated from the tumor tissues in the mice on the 38th day. After lysing the red blood cells, the obtained cells are counted, such as Figure 6 As shown, cell samples were stained and analyzed using flow cytometry.

[0066] After 7 days, untreated 4T1 cells were injected into the mice through their left hind limbs. The weight and tumor volume of the mice were monitored at regular intervals. During the experiment, the weight of the mice gradually increased. By comparing the mice injected with different 4T1 cells, it was found that the BNCT-based tumor antigens (BN@PVP+Neutron and BN-R837@PVP+Neutron) could significantly inhibit the tumor growth on the contralateral side of the mice, and the 4T1 cells treated with BN-R837@PVP+Neutron had the strongest tumor inhibition effect.

[0067] Tumor-infiltrating T cells (CD4 + Cells and CD8 + T cells) are crucial for anti-tumor immune response. By evaluating the changes in T cell infiltration in tumor tissues in mice, it can be found that CD4 + The T cell population increased from 1.61% to 6.82%, and the CD4 + The T cell population increased from 1.61% to 8.33%, an increase of approximately 4 and 5 times, respectively. +The proportion of T cells increased by about 5 times and 11.8 times, from 0.44% to 2.31% and 5.18%, respectively, indicating that tumor antigens based on BNCT can significantly increase the CD4 + T cells and CD8 + T cell infiltration, including CD4 + Cells and CD8 + The maximum expansion of T cells was triggered by tumor antigens based on BN-R837@PVP and neutron irradiation. At the same time, IHC staining also showed that the CD4 + Cells and CD8 + T cells increased significantly.

[0068] In summary, BNCT enhanced the anti-tumor immune activity, and treatment of 4T1 cells with BN-R837@PVP and neutron irradiation could induce CD4 + Cells and CD8 + Infiltration of T cells enhances resistance to tumor attack.

[0069] Example 5: Inhibition of cancer metastasis using BN-R837@PVP

[0070] In order to evaluate the effect of BN-R837@PVP applied in BNCT on the prevention of 4T1 cell lung metastasis, on the basis of Example 4, 4T1 cells expressing luciferase were injected into female BABL / c mice after two rounds of 4T1 cell vaccination, the weight of the mice was monitored in real time, and IVIS imaging was used to detect the occurrence of lung metastasis. According to the results of the 4T1 cell lung metastasis experiment, the inoculation of 4T1 cells treated with BN-R837@PVP+Neutron can significantly improve the survival rate of mice. Except for the mice injected into the G6 group, the fluorescent signals of 4T1 cells labeled with luciferase can be clearly observed in the lungs of the other five groups of mice, indicating that lung metastasis has occurred in the mice. Similarly, except for the mice in the BN-R837@PVP+Neutron group, the ex vivo lungs and panoramic HE staining images of the other five groups of mice showed obvious metastatic foci, and the number of lung metastatic nodules in the BN-R837@PVP+Neutron group of mice was significantly reduced, as shown in Figure 4. Figure 7 Further FACS analysis of T cell populations in the spleen of mice revealed that the CD4 + T cells increased, and CD8 + The T cell population did not change significantly, but the CD4 + Cells and CD8 +T cells were significantly expanded. Therefore, it can be concluded that the application of BN-R837@PVP prepared in the present application in tumor antigen therapy based on BNCT can significantly inhibit the occurrence of 4T1 cell lung metastasis.

[0071] Example 6: Verification of the long-term anti-tumor immune response produced by BN-R837@PVP in BNCT treatment

[0072] In order to verify the long-term anti-tumor immune response generated by the application of BN-R837@PVP in BNCT treatment, multiple groups of mice were used for adoptive immunization experiments. The mice that survived the immunization study in Example 5 were injected with spleen cells on the 70th day, and 6 non-immunized mice (8-10 weeks) were injected with spleen cells from the BN-R837@PVP+Neutron group (Group G3 in Example 5). At the same time, 6 non-immunized mice (8-10 weeks) were injected with spleen cells from the BN@PVP+Neutron group (Group G2 in Example 5). After 2 days, 4T1 cells were injected into each group of mice. The weight and tumor volume of each mouse were recorded once a day during the experiment. On the 30th day of the experiment, spleen cells and single cells were isolated from the tumor tissue of each mouse, and the obtained cells were counted after lysing the red blood cells, and the CD4 + T cells (helper T cells), antigen-specific CD8 + T cells (cytotoxic T lymphocytes), CD4 + Central memory T cells (CD4 + Tcm) and CD8 + Central memory T cells (CD8 + Tcm) and CD4 + Effector memory T cells (CD4 + Tem) and CD8 + Effector memory T cells (CD8 + Tem) for analysis.

[0073] During the experiment, there was no significant difference in the body weight of mice in each group. Compared with groups G1 and G2, the tumor growth of mice in group G3 was significantly inhibited. This shows that tumor antigens based on BN-R837@PVP and neutron irradiation have strong and long-term anti-tumor effects.

[0074] In order to further reveal the matrix with high anti-tumor efficacy, the T cell population in the spleen of mice was analyzed by flow cytometry. According to the analysis results, compared with the mice in the G1 group, the CD3 + cells, CD4 + Cells and CD8 + The proportion of T cells increased significantly. Figure 8IHC was used to evaluate the expression of CD4 + Cells and CD8 + Compared with the G1 and G2 mice, the CD4 + Cells and CD8 + T cells increased significantly, and Foxp3+ cells decreased. FACS detected that mice in the G1 group showed tumor-infiltrating CD4 + Cell populations and CD8 + T cell populations were approximately 5.53% and 6.75%, respectively, while in the G3 group mice, CD4 + Cells and CD8 + The infiltration of T cells was 13.50% and 14.13%, respectively, due to the inclusion of CD4 + TEM cells and CD8 + Effector memory T cells, including TEM cells, also participate in the growth and metastasis of tumors, and are ultimately associated with prolonged survival. Using FACS to study the subgroups of TEM in tumors, compared with mice in the G1 group, the CD4 + TEM cells and CD8 + This proves that tumor antigens treated with BN-R837@PVP+Neutron can lead to the proliferation of T cells in the spleen and increase the number of CD4 + Cells and CD8 + The infiltration of T cells and the growth of the TEM population facilitate effective and long-term tumor suppression in vivo.

[0075] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing tumor antigens based on BNCT, It is characterized in that Boron nitride nanoparticles modified with polyvinyl pyrrolidone (PVP) are loaded with Toll-like receptor agonist R837, which specifically includes the following steps: Step 1: Construction of boron nitride nanoparticles based on chemical vapor deposition and ultrasonic treatment in water Boric acid and urea are dissolved in a methanol aqueous solution at a molar ratio of 1:3 to form a mixed solution, the mixed solution is stirred at 45°C for 12 hours, the mixed solution is recrystallized, the mixed solution is filtered to obtain a white crystalline powder, the white crystalline powder is placed in a quartz tube furnace for pyrolysis to obtain h-BNNs, and the h-BNNs are dissolved in water to obtain a dispersion system, the dispersion system is centrifuged and filtered, and then the dispersion system is ultrasonically treated in water using a probe ultrasonicator, and the dispersion system is freeze-dried to obtain boron nitride nanoparticles; Step 2: Preparation of BN@PVP using boron nitride nanoparticles and polyvinylpyrrolidone (PVP) The boron nitride nanoparticles are dissolved in water to obtain a BN solution, polyvinyl pyrrolidone (PVP) is added to the BN solution to form a mixture, and the mixture is stirred for 16 to 18 hours. The mixture is ultrasonically treated in water using a probe ultrasonicator, and then the mixture is centrifuged and washed at least 3 times to obtain polyvinyl pyrrolidone (PVP)-modified boron nitride nanoparticles (BN@PVP); Step 3: Loading the Toll-like receptor agonist R837 into BN@PVP to prepare BN-R837@PVP Boron nitride nanoparticles BN@PVP were dissolved in water to obtain a boron nitride nanoparticle solution, and then Toll-like receptor agonist R837 was dissolved in dimethyl sulfoxide (DMSO) and mixed with the boron nitride nanoparticle solution to obtain a mixed system, and the mixed system was stirred at 45°C for 12 hours, and then the mixed system was centrifuged at least 3 times to remove the Toll-like receptor agonist R837 not loaded on the boron nitride nanoparticles, thereby obtaining BN-R837@PVP loaded with the Toll-like receptor agonist R837; Step 4: Use a UV-visible spectrophotometer to measure BN-R837@PVP, obtain the characteristic absorption peak of R837 in the UV-visible absorption spectrum, and determine the loading rate of R837 in BN-R837@PVP.

2. A method for preparing tumor antigens based on BNCT according to claim 1, It is characterized in that In the step 1, the volume ratio of methanol to water in the methanol aqueous solution is 1:

1.

3. The method for preparing tumor antigens based on BNCT according to claim 1, It is characterized in that In the step 1, when the white crystalline powder is pyrolyzed in a quartz tube furnace, the temperature of the quartz tube furnace does not exceed 1000°C.

4. The method for preparing tumor antigens based on BNCT according to claim 1, It is characterized in that In the step 1, when the boron nitride solution is subjected to ultrasonic treatment in water using a probe ultrasonic instrument, the temperature of the boron nitride solution is 4-37°C.

5. The method for preparing tumor antigens based on BNCT according to claim 1, It is characterized in that In step 2, the concentration of the BN solution is 100 µg / mL, and the amount of polyvinylpyrrolidone (PVP) added is 1 mg / ml.

6. The method for preparing tumor antigens based on BNCT according to claim 1, It is characterized in that In step 2, the average particle size of BN@PVP is 220 nm.

7. The method for preparing tumor antigens based on BNCT according to claim 1, It is characterized in that In step 3, the concentration of the boron nitride nanoparticle solution is 0.1 mg / mL.

8. Use of BN-R837@PVP loaded with Toll-like receptor agonist R837 prepared by the BNCT-based tumor antigen preparation method according to claim 1 in the preparation of tumor BNCT therapeutic drugs.

9. Use of BN-R837@PVP loaded with Toll-like receptor agonist R837 prepared by the method for preparing tumor antigens based on BNCT according to claim 8 in the preparation of tumor BNCT therapeutic drugs, It is characterized in that Used to activate cells' anti-tumor immune response.

10. Use of BN-R837@PVP loaded with Toll-like receptor agonist R837 prepared by the method for preparing tumor antigens based on BNCT according to claim 8 in preparing tumor BNCT therapeutic drugs, It is characterized in that By increasing CD4 + Cells and CD8 + T cell infiltration inhibits tumor growth and metastasis.

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