Bifidobacterium-nano-selenium radiotherapy sensitizer as well as preparation method and application thereof

Abstract

The invention provides a bifidobacterium-nano-selenium radiotherapy sensitizer as well as a preparation method and application thereof. The bifidobacterium-nano-selenium radiotherapy sensitizer is prepared from a Na2SeO3 solution, a Vc solution and bifidobacterium longum. The radiotherapy sensitizer can accurately target tumors, remodel an immunosuppression microenvironment and enhance the radiotherapy effect, and is safe and effective. The synthesis mode of the material is simple and effective, and the thought of using a biological material as a radiotherapy sensitizer is widened; the nano-material has biological activity and is selectively accumulated in a tumor, so that the defects of poor targeting property and poor metabolism effect of a general nano-material serving as a radiotherapy sensitizer are overcome; bifidobacterium longum in human intestinal tracts is selected as a substrate of the system, so that the defects that conventional attenuated or engineering bacteria are large in side effect and poor in biocompatibility when serving as a delivery substrate are overcome.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic pharmaceutical chemistry, and particularly relates to a bifidobacterium-nano-selenium radiotherapy sensitizer, a preparation method of the bifidobacterium-nano-selenium radiotherapy sensitizer, and application of the bifidobacterium-nano-selenium radiotherapy sensitizer in anti-tumor and radiotherapy sensitization drugs. Background Art

[0002] Since X-rays were discovered and applied in the medical field, radiotherapy has become one of the main means of cancer treatment. Studies have shown that radiotherapy can induce immunogenic death of tumor cells and stimulate specific anti-tumor immune responses. However, high-energy radiation may also damage surrounding normal tissues and increase the risk of tumor recurrence after high-dose radiotherapy. The tumor microenvironment (TME) is crucial to tumor development. Immune cells, as key interstitial components, affect treatment response, and the flora in tumor tissue is also an important factor in the formation of TME and has a local effect on tumor progression. However, radiotherapy also promotes the infiltration of immunosuppressive cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) in tumors. Therefore, the development of safe and effective multifunctional radiosensitizers that can accurately target tumors, reshape the immunosuppressive microenvironment, enhance the effect of radiotherapy, is an urgent need in current clinical practice. Summary of the invention

[0003] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a safe and effective multifunctional radiotherapy sensitizer that can accurately target tumors, reshape the immunosuppressive microenvironment, enhance the effect of radiotherapy.

[0004] The present application provides a method for preparing a bifidobacterium-nanoselenium radiotherapy sensitizer, comprising the following steps:

[0005] (1) Preparing a bifidobacterium resuspension: taking Bifidobacterium longum and culturing it in a culture medium for 14-18 hours, removing the culture medium after centrifugation, and resuspending it in PBS to obtain a bifidobacterium resuspension;

[0006] (2) Preparation of BL@SeNPs radiosensitizer: Na2SeO3 solution and Vc solution were prepared with ultrapure water; in a clean bench, a culture bottle containing the Bifidobacterium resuspension was placed in a magnetic stirrer for continuous stirring, Na2SeO3 was slowly added and incubated, and then Vc solution was added and the reaction was continued; the system was centrifuged to obtain the precipitated BL@SeNPs radiosensitizer.

[0007] Furthermore, the system was centrifuged to obtain the precipitated BL@SeNPs radiosensitizer, which was then washed with PBS and finally resuspended with PBS to the volume before the reaction to obtain the BL@SeNPs radiosensitizer dispersed in PBS.

[0008] Specifically, in the final mixed solution of step (2), the final concentration of Na2SeO3 is 1-20 mM; in the final mixed solution of step (2), the final concentration of Vc is 4-80 mM.

[0009] Further, step (1) comprises: taking out the preserved strain, thawing it in a water bath, removing the supernatant, resuspending it with a freshly prepared bifidobacterium liquid culture medium, and blowing it evenly; adding the resuspended liquid to a culture bottle containing a bifidobacterium liquid culture medium, culturing and amplifying the bacteria overnight at a suitable temperature; centrifuging the bacteria in the culture bottle, washing it with PBS to remove the culture medium residue, and finally resuspending it with PBS, and blowing it evenly. The preparation method according to claim 4 is characterized in that the bifidobacterium liquid described in step (1) should be taken in a volume of 200 μL with an OD600 of about 0.6, resuspended in 10 mL of bifidobacterium liquid culture medium, and cultured in a 37°C constant temperature incubator for 12-16 hours.

[0010] Specifically, the molar ratio of Na2SeO3 to Vc in the entire reaction system is 1:2-20.

[0011] Furthermore, in step (2), 0.4-0.6M Na2SeO3 solution and 1.8-2.2MVc solution are prepared with ultrapure water; in a clean bench, the culture bottle containing the heavy suspension is placed in a magnetic stirrer and continuously stirred at a stirring speed of 550-650rpm, and 0.4-0.6M Na2SeO3 is slowly added dropwise, totaling 39-43μL, so that the selenium content of the system is 9-11mM before reacting with Vc, incubated for 28-32min, and then 39-43μLVc solution is slowly added dropwise, at which time the system gradually changes from a white turbid state to an orange-red turbid state, and the reaction is continued for 3.5-4.5h until the color of the system no longer deepens; the system is centrifuged at a speed of 3800-4200rpm for 4-6min to obtain the precipitated BL@SeNPs radiotherapy sensitizer.

[0012] Furthermore, in step (2), 0.5M Na2SeO3 solution and 2MVc solution are prepared with ultrapure water; in a clean bench, the culture bottle containing the resuspended solution is placed in a magnetic stirrer and continuously stirred at 600rpm. After that, all operations are performed on the stirrer, and 0.5M Na2SeO3 is slowly added dropwise, totaling 41μL, so that the selenium content of the system is 10mM before reacting with Vc, and incubated for 30min; then 41μLVc solution is slowly added dropwise, at which time the system gradually changes from a white turbid state to an orange-red turbid state, and the reaction is continued for 4h until the color of the system no longer deepens; the system is centrifuged at 4000rpm for 5min to obtain the precipitated BL@SeNPs radiotherapy sensitizer, which is washed 3 times with 10mLPBS to remove unreacted Na2SeO3 and Vc, and finally resuspended with 4mL PBS to the volume before the reaction to obtain the BL@SeNPs radiotherapy sensitizer dispersed in PBS.

[0013] A Bifidobacterium-nano-selenium radiotherapy sensitizer prepared according to any of the preparation methods described above.

[0014] Application of the above-mentioned Bifidobacterium-nanoselenium radiotherapy sensitizer in radiotherapy sensitization drugs.

[0015] The improvements of this application bring the following advantages:

[0016] (1) The material synthesis method is simple and effective, which broadens the idea of ​​using biomaterials as radiosensitizers;

[0017] (2) They are biologically active and can accumulate in tumors through the system, thus overcoming the shortcomings of general nanomaterials as radiosensitizers, such as poor targeting and poor metabolic effects;

[0018] (3) Bifidobacterium longum, which is present in the human intestine, is selected as the base of the system, which improves the disadvantages of conventional attenuated or engineered bacteria as delivery bases, such as large side effects and poor biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a SEM morphology characterization image of the BL@SeNPS radiosensitizer in the examples of this application.

[0020] Figure 2 This is an AFM morphology characterization image of the BL@SeNPS radiosensitizer in the examples of this application.

[0021] Figure 3 This is the activation of BMDC cells by LET-SeNPs and BL@SeNPs radiosensitizers in the examples of this application.

[0022] Figure 4 Cell survival rate of 4T1 cells under different irradiation doses after incubation with BL@SeNPs and BL.

[0023] Figure 5 The synergistic antagonism diagram between BL@SeNPs and radiotherapy at different doses.

[0024] Figure 6 BL@SeNPs combined with radiotherapy inhibited the changes in tumor volume during treatment in tumor-bearing mice.

[0025] Figure 7 It is the expression level of cytokine IFN-γ in tumor homogenate supernatant. DETAILED DESCRIPTION

[0026] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] Example 1, a method for preparing a bifidobacterium-nanoselenium radiotherapy sensitizer, comprising the following steps:

[0028] (1) Prepare bifidobacterium liquid culture medium and plate culture medium. Take 48.92g of bifidobacterium solid culture medium and 1mL Tween-80 and dissolve them in 1L ultrapure water, sterilize them at 120℃ and 103.4kPa for 30min, and obtain a sterile bifidobacterium liquid culture medium. Take 48.92g of bifidobacterium solid culture medium, 15g of agar powder and 1mL Tw-80 and dissolve them in 1L ultrapure water, sterilize them at 120℃ and 103.4kPa for 30min. After cooling slightly, pour 4mL of liquid culture medium into a 10cm sterile dish in a clean bench to obtain a long bifidobacterium plate culture medium.

[0029] (2) Resuscitation of bacterial strains. After obtaining the lyophilized powder of Bifidobacterium longum, wipe the ampoule tube with medical alcohol in a medical clean bench according to the activation method. After opening, use a pipette to draw 500 μL of ultrapure water (ultrapure water is sterilized at 120°C and 103.4 kPa for 30 minutes) into the ampoule tube containing the lyophilized powder, and blow it evenly. Pipette 10 μL of bacterial solution and add it to the Bifidobacterium plate culture medium, use a coating rod to evenly spread it on the plate culture medium, then place the plate in an anaerobic culture bag, add a gas-producing bag and seal it, and culture it overnight at an appropriate temperature.

[0030] (3) Select colonies of appropriate size from the plate and add them to 10 mL of liquid culture medium. Shake the culture medium at an appropriate temperature. Take out 100 μL of the culture medium every 12 hours to measure the OD value at 600 nm and determine its dynamic growth curve.

[0031] (4) Preservation of bacterial strains. Pick out colonies of appropriate size from the plate and add them to 10 mL of bifidobacterium liquid culture medium, and amplify them by shaking at an appropriate temperature overnight. Add 5 mL of ultrapure water to 5 mL of pure glycerol, and sterilize at 120°C and 103.4 kPa for 30 minutes. Then mix equal volumes of the bacterial liquid and the glycerol-water mixture and shake well. In a 1 mL EP tube (sterilized at high temperature and high pressure in advance), transfer 200 μL of the mixture to each tube to obtain bacterial strains preserved in 25% glycerol, and then store them in a -80°C refrigerator.

[0032] (5) Recovery of Bifidobacterium longum. After taking out the stored strain from -80℃, thaw it quickly in a 35℃ water bath. After it is completely thawed, centrifuge it at 3000rpm for 5min to remove the supernatant, resuspend it with 200μL of freshly prepared Bifidobacterium liquid culture medium, and blow it evenly. Add all the resuspended liquid to a culture bottle containing 10mL of Bifidobacterium liquid culture medium, and culture and amplify the bacteria overnight at a suitable temperature.

[0033] (6) Preparation of BL@SeNPs radiosensitizer. (The culture bottle, PBS, and ultrapure water were sterilized at high temperature and high pressure). The bacterial solution in the culture bottle was centrifuged at 4000 rpm for 5 min. Washed with 10 mL PBS three times to remove the culture medium residue, and finally resuspended with 4 mL PBS and blown evenly. Prepare 0.5 M Na2SeO3 solution and 2 M Vc solution with ultrapure water. In the clean bench, the culture bottle containing the resuspended solution was placed in a magnetic stirrer and stirred continuously at 600 rpm (all subsequent operations were performed on the stirrer). Slowly add 0.5 M Na2SeO3 in total of 41 μL, so that the selenium content of the system was 10 mM before reacting with Vc, and incubate for 30 min. Then slowly add 41 μL Vc solution, at this time the system gradually changes from a white turbid state to an orange-red turbid state, and the reaction continues for 4 hours until the color of the system no longer deepens. The system was centrifuged at 4000 rpm for 5 min to obtain the precipitated BL@SeNPs radiosensitizer, which was washed 3 times with 10 mL PBS to remove unreacted Na2SeO3 and Vc, and finally resuspended with 4 mL PBS to the volume before the reaction to obtain the BL@SeNPs radiosensitizer dispersed in PBS. The BL@SeNPs were gradiently diluted, and the corresponding OD600 values ​​at different dilutions were measured using an ELISA instrument, and the corresponding McFarland turbidity values ​​were measured using a bacterial turbidity meter to convert the bacterial concentration corresponding to OD600 for later administration. The same method was used to calculate the bacterial concentration for the original Bifidobacterium longum.

[0034] Example 2

[0035] Repeat steps (1) to (4) of Example 1.

[0036] (5) Recovery of Bifidobacterium longum. After taking out the stored strain from -80℃, thaw it quickly in a 35℃ water bath. After it is completely thawed, centrifuge it at 3000rpm for 5min to remove the supernatant, resuspend it with 200μL of freshly prepared Bifidobacterium liquid culture medium, and blow it evenly. Add all the resuspended liquid to a culture bottle containing 10mL of Bifidobacterium liquid culture medium, and culture and amplify the bacteria overnight at a suitable temperature.

[0037] (6) Preparation of BL@SeNPs radiosensitizer. (The culture bottle, PBS, and ultrapure water were sterilized at high temperature and high pressure). The bacterial solution in the culture bottle was centrifuged at 4000 rpm for 5 min. It was washed three times with 10 mL PBS to remove the culture medium residue, and finally resuspended with 4 mL PBS and blown evenly. 0.4 M Na2SeO3 solution and 1.8 M Vc solution were prepared with ultrapure water. In the clean bench, the culture bottle containing the resuspended solution was placed in a magnetic stirrer and stirred continuously at 550 rpm (all subsequent operations were performed on the stirrer). 0.4 M Na2SeO3 was slowly added dropwise to a total of 39 μL, so that the selenium content of the system was 9 mM before reacting with Vc, and incubated for 28 min. Then 39 μL Vc solution was slowly added dropwise. At this time, the system gradually changed from a white turbid state to an orange-red turbid state. The reaction continued for 3.5 h until the color of the system no longer deepened. The system was centrifuged at 3800 rpm for 4 min to obtain the precipitated BL@SeNPs radiosensitizer. Wash with 10mL PBS three times to remove unreacted Na2SeO3 and Vc, and finally resuspend with 4mL PBS to the volume before the reaction to obtain BL@SeNPs radiosensitizer dispersed in PBS. BL@SeNPs were gradient diluted, and the corresponding OD600 values ​​at different dilutions were measured using an ELISA instrument. The corresponding McFarland turbidity values ​​were measured using a bacterial turbidity meter to convert the bacterial concentration corresponding to OD600 for later administration. The same method was used to calculate the bacterial concentration for the original Bifidobacterium longum.

[0038] Example 3

[0039] Repeat steps (1) to (4) of Example 1.

[0040] (5) Recovery of Bifidobacterium longum. After taking out the stored strain from -80℃, thaw it quickly in a 35℃ water bath. After it is completely thawed, centrifuge it at 3000rpm for 5min to remove the supernatant, resuspend it with 200μL of freshly prepared Bifidobacterium liquid culture medium, and blow it evenly. Add all the resuspended liquid to a culture bottle containing 10mL of Bifidobacterium liquid culture medium, and culture and amplify the bacteria overnight at a suitable temperature.

[0041] (6) Preparation of BL@SeNPs radiosensitizer. (The culture bottle, PBS, and ultrapure water were sterilized at high temperature and high pressure). The bacterial solution in the culture bottle was centrifuged at 4000 rpm for 5 min. It was washed three times with 10 mL PBS to remove the culture medium residue, and finally resuspended with 4 mL PBS and blown evenly. 0.6 M Na2SeO3 solution and 2.2 M Vc solution were prepared with ultrapure water. In the clean bench, the culture bottle containing the resuspended solution was placed in a magnetic stirrer and stirred continuously at 650 rpm (all subsequent operations were performed on the stirrer). A total of 41 μL of 0.6 M Na2SeO3 was slowly added dropwise, so that the selenium content of the system was 11 mM before reacting with Vc, and incubated for 32 min. Then 41 μL Vc solution was slowly added dropwise, and the system gradually changed from a white turbid state to an orange-red turbid state. The reaction continued for 4.5 h until the color of the system no longer deepened. The system was centrifuged at 4200 rpm for 6 min to obtain the precipitated BL@SeNPs radiosensitizer. Wash with 10mL PBS three times to remove unreacted Na2SeO3 and Vc, and finally resuspend with 4mL PBS to the volume before the reaction to obtain BL@SeNPs radiosensitizer dispersed in PBS. BL@SeNPs were gradient diluted, and the corresponding OD600 values ​​at different dilutions were measured using an ELISA instrument. The corresponding McFarland turbidity values ​​were measured using a bacterial turbidity meter to convert the bacterial concentration corresponding to OD600 for later administration. The same method was used to calculate the bacterial concentration for the original Bifidobacterium longum.

[0042] Example 4 Characterization of BL@SeNPs

[0043] Figure 1 This is the SEM morphology characterization of BL@SeNPS radiosensitizer. Figure 1 The results showed that the synthesized BL@SeNPs can have granular protrusions on the bacterial surface, which are evenly distributed and uniform in size, ranging from 50 to 70 nm. These results show that bifidobacteria interact with nano-selenium, and this example successfully prepared a new radiotherapy sensitizer of in situ biomineralized nano-selenium of Bifidobacterium longum (BL).

[0044] Figure 2 This is the AFM morphology characterization of BL@SeNPS radiotherapy sensitizer. Figure 2 The results of AFM characterization of BL@SeNPs showed that there were uniform convexities on the surface of bacteria, that is, the existence of particles. The surface height analysis of BL@SeNPs showed that the particle height was about 100nm, which was similar to the particle size obtained by electron microscopy.

[0045] Figure 3The results are to verify the activation of immune cell function by stimulating the maturation of bone marrow-derived dendritic cells (BMDCs) by BL@SeNPs. First, monodispersed cells from the femoral and tibial bone marrow of female C57BL / 6 mice (6-8 weeks old) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng / mL) and β-mercaptoethanol (5 ng / mL) to obtain bone marrow-derived dendritic cells (BMDCs) for 6-8 days.

[0046] Then, BMDC cells were treated with LET-SeNPs, BL@SeNPs, BL, or lipopolysaccharide (LPS, 1 μg / mL, as a positive control group) for 12 h. Afterwards, the cells were stained with fluorescently labeled antibodies (including CD11c-FITC, CD86-APC-Cy7, and CD80-PE) to assess the maturity of BMDCs and analyzed using flow cytometry. Figure 3 As shown, compared with the untreated group (ie, control), it was found that BL@SeNPs had a good stimulatory effect on BMDCs, reaching 46.1%, which was 1.5 times that of the Control group.

[0047] Figure 4 is the cell survival rate of 4T1 cells incubated with BL@SeNPs and BL at different irradiation doses,

[0048] Mouse breast cancer 4T1 cells were cultured in RPMI-1640 medium, and all medium were supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. The cells were cultured in an incubator at 37°C and 5% CO2. The MTT method was used to detect the killing effect of BL@SeNPs radiosensitizer combined with radiotherapy on 4T1 cells. The specific steps are as follows: 4T1 cells were inoculated on a 96-well plate at a cell density of 2000 cells / well and a culture medium volume of 100 μL / well. After the cells adhered to the wall, different concentrations of different drugs were added as a comparison. After incubation for 6 hours, the 96-well plate was treated with different doses of X-ray irradiation (0, 2, 4 Gy). After 48 hours of continuous culture, PBS was added at a volume of 20 μL / well for three times to remove the residual bacteria. 100 μL PBS was added to each well, and then MTT (5 mg / mL of thiazolyl blue, 25 μL per well) was added to it. After incubation for 4 hours, the upper layer of PBS in the 96-well plate was removed, and 150 μL of DMSO was added. After incubation and shaking for 10 minutes, the absorbance of each well at 570 nm was detected by an ELISA instrument, and the cell survival rate was finally calculated. The killing of 4T1 cells after 48 hours of treatment with BL@SeNPs and BL was detected. BL still had no toxic changes, while BL@SeNPs could effectively kill tumor cells and respond to changes in radiotherapy doses. The results of the cytotoxicity experiment showed that the designed and synthesized new radiotherapy sensitizer BL@SeNPs can play a role in radiotherapy sensitization as expected.

[0049] Figure 5 Yes (This step is the same as Figure 4 The IC is calculated based on the absorbance of each well at 570 nm detected by the microplate reader. 50 The values ​​are used to depict the synergistic and antagonistic effects of BL@SeNPs on different radiotherapy doses. ) Synergistic and antagonistic diagram of BL@SeNPs and radiotherapy at different doses. The isobologram was further used to analyze the interaction between BL@SeNPs and X-rays. When the action point is located on the isobologram, it means that X-rays and drugs have an additive effect; when the action point is located above the isobologram, the two are antagonistic; when it is located below the isobologram, the two are synergistic. The experimental results show that when BL@SeNPs is combined with radiotherapy (2, 4Gy), the anti-tumor effect on 4T1 cells is synergistic, that is, BL@SeNPs has a radiosensitizing effect.

[0050] Example 5, inhibition of breast cancer tumors in mice by the novel radiosensitizer of Bifidobacterium-nanoselenium provided by the present invention

[0051] (1) Experimental groups

[0052] To evaluate the inhibitory activity of the developed nanovaccine against mouse breast cancer tumors, 4T1 tumor-bearing mice were randomly divided into 6 groups (n=5): (G1) Saline group; (G2) RT group; (G3) LET-SeNPs group; (G4) BL+RT group; (G5) BL@SeNPs group; and (G6) BL@SeNPs+RT group.

[0053] (2) Dosage method

[0054] Tail vein injection, 3 days as a treatment cycle: drug administration on day 0, X-ray irradiation (irradiation dose of 2Gy, total 16Gy) on day 2, no operation on day 3, and treatment started with this cycle. During the treatment, tumor volume, mouse weight and mouse survival rate were recorded every other day. When the maximum average tumor volume in the group reached about 1600mm3, the treatment was stopped for a total of 25 days, and the mice were killed on day 26.

[0055] (3) Experimental content

[0056] (i) Evaluation of the proportion of immune cells in the tumor. The tumor tissue was removed and weighed, then cut into two halves, and one half was fixed with 4% paraformaldehyde for tissue sectioning. One half was ground through a membrane, centrifuged at 1200 rpm for 5 min, the supernatant was collected, and washed three times with PBS. For the ground supernatant, after appropriate dilution, ELISA kits were used to detect TNF-α and IFN-γ levels. For different immune cells (including CTL, MDSC, Neutrophil, Macrophages, Treg and other immune-related cells), different antibody staining schemes were used to stain tumor tissue cells, and then detected using a Beckman flow cytometer, and at least 10,000 cells were detected for each sample.

[0057] (ii) Immunofluorescence staining of tumor sections was performed using TUNEL-DAPI and Hoechst-HMGB1 immunofluorescence staining, and then photographed and analyzed using a fluorescence microscope.

[0058] (4) Experimental results

[0059] Figure 6The figure shows the changes in tumor volume during the treatment of tumor-bearing mice inhibited by BL@SeNPs combined with radiotherapy. First, the tumor volume of each group of tumor-bearing mice during the treatment was measured. It can be observed that BL@SeNPs combined with radiotherapy injected through the tail vein can effectively inhibit tumor growth. By the time of sacrifice, the average tumor volume of mice was controlled at 518mm3, while the average tumor volume of the Saline group grew as high as 1727mm3, which is 3.3 times the size of the tumor volume of mice in the BL@SeNPs group. The RT group also showed certain experimental results of tumor growth inhibition. The LET-SeNPs combined with radiotherapy group showed similar inhibitory effects to the BL combined with radiotherapy group.

[0060] Figure 7 ELISA method for detecting the expression level of cytokine IFN-γ in tumor homogenate supernatant = Take the ELISA kit out of the refrigerator, equilibrate at room temperature for 30 minutes, and gently mix all components. Add the tumor tissue homogenate supernatant and standard to the microwell ELISA plate pre-coated with anti-mouse IFN-γ antibody. Incubate the reaction plate at room temperature for 120 minutes to allow IFN-γ to bind to the solid phase antibody. Use washing solution to wash the reaction plate 4-6 times to remove unbound components and blot dry. Then add HRP-labeled anti-mouse IFN-γ antibody (enzyme-labeled antibody) to each well, incubate and wash again to form a sandwich complex of solid phase antibody-antigen-enzyme-labeled antibody. Then add color substrates A and B, the substrate produces a blue product under HRP catalysis, and then add stop solution, and the color changes to yellow. Finally, the absorbance (OD value) was measured at a wavelength of 450nm on an ELISA reader. The absorbance was positively correlated with the concentration of mouse IFN-γ in the sample to be tested. The standard curve was drawn using the OD value of the standard to calculate the concentration of mouse IFN-γ in the sample. The results showed that in the detection of IFN-γ in the tumor, the BL@SeNPs+RT group had the highest IFN-γ expression level of 533.6pg / mL, and the BL+RT group had a content of 420.8pg / mL, and the expression level was also high, with the RT group having a content of 282.4pg / mL, the LET-SeNPs+RT group having a content of 202.1pg / mL, and the BL@SeNPs group having a content of 260.2pg / mL.

[0061] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a bifidobacterium-nanoselenium radiotherapy sensitizer, characterized in that: The following steps are involved: (1) Preparing a bifidobacterium resuspension: taking Bifidobacterium longum and culturing it in a culture medium for 14-18 hours, removing the culture medium after centrifugation, and resuspending it in PBS to obtain a bifidobacterium resuspension; (2) Preparation of BL@SeNPs radiosensitizer: Na2SeO3 solution and Vc solution were prepared with ultrapure water; in a clean bench, a culture bottle containing the Bifidobacterium resuspension was placed in a magnetic stirrer for continuous stirring, Na2SeO3 was slowly added and incubated, and then Vc solution was added and the reaction was continued; the system was centrifuged to obtain the precipitated BL@SeNPs radiosensitizer.

2. The preparation method according to claim 1, characterized in that: The system was centrifuged to obtain the precipitated BL@SeNPs radiosensitizer, which was then washed with PBS and finally resuspended with PBS to the volume before the reaction to obtain the BL@SeNPs radiosensitizer dispersed in PBS.

3. The preparation method according to claim 2, characterized in that: In the final mixed solution of step (2), the final concentration of Na2SeO3 is 1-20 mM; in the final mixed solution of step (2), the final concentration of Vc is 4-80 mM.

4. The preparation method according to claim 1, characterized in that: Step (1) comprises: taking out the stored bacterial strain, thawing it in a water bath, removing the supernatant, resuspending it with a freshly prepared bifidobacterium liquid culture medium, and blowing it evenly; adding the resuspended liquid to a culture bottle containing a bifidobacterium liquid culture medium, culturing and amplifying the bacteria overnight at a suitable temperature; centrifuging the bacteria in the culture bottle, washing it with PBS to remove the culture medium residue, and finally resuspending it with PBS, and blowing it evenly.

5. The preparation method according to claim 4, characterized in that: The bifidobacterium liquid described in step (1) should be taken in a volume of 200 μL with an OD600 of about 0.6, resuspended in 10 mL of bifidobacterium liquid culture medium, and cultured in a constant temperature incubator at 37° C. for 12-16 hours.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The molar ratio of Na2SeO3 to Vc in the whole reaction system is 1:2-20.

7. The preparation method according to any one of claims 1 to 5, characterized in that: In step (2), 0.4-0.6M Na2SeO3 solution and 1.8-2.2M Vc solution are prepared with ultrapure water; in a clean bench, the culture bottle containing the heavy suspension is placed in a magnetic stirrer and stirred continuously at a stirring speed of 550-650rpm, and 0.4-0.6M Na2SeO3 is slowly added dropwise, totaling 39-43μL, so that the selenium content of the system before reacting with Vc is 9-11mM, incubated for 28-32min, and then 39-43μLVc solution is slowly added dropwise, at which time the system gradually changes from a white turbid state to an orange-red turbid state, and the reaction is continued for 3.5-4.5h until the color of the system no longer deepens; the system is centrifuged at a speed of 3800-4200rpm for 4-6min to obtain the precipitated BL@SeNPs radiotherapy sensitizer.

8. The preparation method according to claim 7, characterized in that: In step (2), 0.5M Na2SeO3 solution and 2M Vc solution are prepared with ultrapure water; in a clean bench, the culture bottle containing the resuspended solution is placed in a magnetic stirrer and stirred continuously at 600rpm. After that, all operations are performed on the stirrer, and 0.5M Na2SeO3 is slowly added dropwise, totaling 41μL, so that the selenium content of the system is 10mM before reacting with Vc, and incubated for 30min; then 41μLVc solution is slowly added dropwise, at which time the system gradually changes from a white turbid state to an orange-red turbid state, and the reaction is continued for 4h until the color of the system no longer deepens; the system is centrifuged at 4000rpm for 5min to obtain the precipitated BL@SeNPs radiotherapy sensitizer, which is washed 3 times with 10mLPBS to remove unreacted Na2SeO3 and Vc, and finally resuspended with 4mL PBS to the volume before the reaction to obtain the BL@SeNPs radiotherapy sensitizer dispersed in PBS.

9. The Bifidobacterium-nano-selenium radiotherapy sensitizer prepared according to the preparation method according to any one of claims 1 to 5.

10. Use of the bifidobacterium-nanoselenium radiosensitizer according to claim 9 in radiosensitization drugs.

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