Bifidobacterium longum and application thereof

Abstract

The present application relates to the field of microbial technology, and discloses a bifidobacterium longum isolated from a PD-1 treatment effective tumor patient and application thereof. The present application uses bifidobacterium longum SYSU10 in combination with an immune checkpoint inhibitor to perform anti-tumor treatment on tumor-bearing mice. The bifidobacterium longum SYSU10 can stimulate the production of an anti-tumor immune protection response, remodel the tumor microenvironment, and convert a "cold" tumor into a "hot" tumor. Moreover, the bifidobacterium longum SYSU10 significantly enhances the treatment effect of the immune checkpoint inhibitor on colorectal cancer, can not only inhibit the in situ growth of a tumor but also inhibit the metastasis of tumor cells, and has good safety. The present application also provides a nano drug delivery system formed by the combination of the bifidobacterium longum SYSU10 and an anti-tumor active small molecule L-fucose. The bifidobacterium longum SYSU10 is combined with a functional nanoparticle by using the anaerobic tendency of the bifidobacterium longum SYSU10, and a high-specificity tumor targeted treatment scheme is provided.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bifidobacterium longum and its applications, specifically to a Bifidobacterium longum and its application in the preparation of nano-drug delivery systems. Background Technology

[0002] Over the past half-century, cancer patient survival has significantly improved due to a deeper understanding of cancer biology, better surgical outcomes, and increasingly effective multimodal chemotherapy and radiotherapy regimens. Cytotoxic drugs remain the primary treatment for most patients with advanced disease, but their treatment response is unpredictable, and treatment-related morbidity and mortality are quite high.

[0003] Next-generation personalized cancer treatments with better safety and fewer side effects are emerging, with immune checkpoint inhibitor (ICI) therapy being a prime example. ICI therapy has revolutionized traditional cancer treatment strategies, disrupted the landscape of radiotherapy and chemotherapy for tumors, and achieved breakthroughs in the treatment of metastatic solid malignancies. Currently identified immune checkpoints include programmed cell death 1 (PD-1) and its ligand (PD-L1), lymphocyte activation gene-3 (LAG3), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Blocking these immune checkpoints can release immune braking responses and effectively inhibit tumor immune escape.

[0004] However, responses to ICI treatment are heterogeneous, with reports showing an objective response rate of only 10%-30%. This indicates that a significant portion of patients do not respond to ICI treatment, severely reducing its clinical value. The factors determining patient response to ICI treatment have long puzzled researchers. With the development of gut microbiome and bioinformatics analysis technologies, the relationship between gut microbiota and ICI treatment heterogeneity has begun to emerge, providing new insights into improving anti-tumor immune responses and expanding the efficacy of ICI. However, the specific functions and molecular mechanisms of each component of the complex gut microbiome remain largely unknown. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a *Bifidobacterium longum* strain and its application in the preparation of nanomedicine delivery systems. This invention screens a novel *Bifidobacterium longum* strain, SYSU10, which can exert a synergistic anti-tumor effect when used in combination with immune checkpoint inhibitors. Its anti-tumor efficacy is verified through in vitro cell experiments and mouse experiments, and the molecular mechanism is revealed through various experimental methods, providing a new anti-tumor therapy and maximizing the clinical value of ICI treatment.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The primary objective of this invention is to provide a Bifidobacterium longum SYSU10, which was deposited on September 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25777, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The nucleotide sequence of the 16S rDNA of *Bifidobacterium longum* SYSU10 is shown in SEQ ID NO:1. The 16S rDNA of *Bifidobacterium longum* SYSU10 of this invention, compared with known *Bifidobacterium longum* strains (National Center for Biotechnology database), has at least three base mutation sites, and its closest phylogenetic relationship is *Bifidobacterium longum* subsp. *suillum* strain Su 851.

[0009] This invention collected fecal samples from cancer patients who responded to PD-1 inhibitor therapy and successfully isolated and identified a strain of Bifidobacterium longum SYSU10 by culturing under different conditions.

[0010] Hemolysis and drug resistance tests have demonstrated that the isolated Bifidobacterium longum SYSU10 has good safety and probiotic characteristics.

[0011] Intestinal colonization ability test, acid and bile salt resistance test and intestinal adhesion experiment demonstrated that Bifidobacterium longum SYSU10 has good industrialization potential.

[0012] Secondly, the present invention provides a formulation comprising a bacterial suspension containing the aforementioned Bifidobacterium longum SYSU10.

[0013] Preferably, the formulation is administered orally, via tail vein injection, intratumoral injection, or adjacent normal injection. The formulation containing *Bifidobacterium longum* SYSU10 of this invention can accumulate at the tumor site, exerting anti-tumor effects (inhibiting tumor growth and metastasis) and improving the patient's quality of life.

[0014] Preferably, the formulation further includes a pharmaceutically acceptable carrier and excipients, and the dosage form of the formulation includes at least one of lyophilized powder, tablets, capsules, granules, or injections. The formulation can be prepared as a food, beverage, food supplement, probiotic, or health food.

[0015] Preferably, the formulation comprises a bacterial suspension containing at least one combination of the above-mentioned live Bifidobacterium longum SYSU10, bacterial derivatives, or bacterial metabolites.

[0016] Thirdly, the present invention provides the application of the above-mentioned Bifidobacterium longum SYSU10 in the preparation of antitumor agents or drugs.

[0017] Fourthly, this invention provides the application of the above-mentioned Bifidobacterium longum SYSU10, or its derivatives, or its metabolites, in combination with immune checkpoint inhibitors in the preparation of antitumor drugs.

[0018] This invention combines Bifidobacterium longum SYSU10 with an immune checkpoint inhibitor to better suppress tumor growth. The enhanced therapeutic effect is measured by inhibiting tumor growth or reducing tumor metastases.

[0019] A composition is formed by combining Bifidobacterium longum SYSU10, its derivatives, or its metabolites with an immune checkpoint inhibitor, wherein Bifidobacterium longum SYSU10, its derivatives, or its metabolites can enhance the anti-tumor efficacy of the immune checkpoint inhibitor and inhibit tumor growth and metastasis.

[0020] Preferably, the tumor is one or more of the following: adrenocortical carcinoma, urothelial carcinoma of the bladder, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiple morphological glioma, glioma, head and neck cancer, chromophobe renal carcinoma, mixed renal carcinoma, renal cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, melanoma of the skin, gastric cancer, esophageal cancer, testicular cancer, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, and soft tissue sarcoma. The types of tumors mentioned above are not limited to those listed above, but also include malignant tumors, metastatic tumors, or non-metastatic tumors, as well as other conventional tumor types in the art.

[0021] In one specific embodiment of the present invention, the tumor is colorectal cancer.

[0022] In a preferred embodiment of the application described in this invention, the immune checkpoint inhibitor is one or more combinations of blocking agents that act on negative co-stimulatory / co-inhibitory molecules and / or their ligands of T cells; the ligand is selected from one of CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and DcR3. Preferably, the immune checkpoint inhibitor comprises a PD-1 monoclonal antibody.

[0023] Fifthly, this invention provides the application of the above-mentioned Bifidobacterium longum SYSU10 in the preparation of nano-drug delivery systems.

[0024] The sixth objective of this invention is to provide a nano-drug delivery system, which is formed by loading the above-mentioned Bifidobacterium longum SYSU10 nanoparticles to form a nano-drug delivery system.

[0025] This invention utilizes the anaerobic tendency of Bifidobacterium longum SYSU10 and combines it with functional nanoparticles to form a nano-drug delivery system, providing a highly specific tumor treatment option.

[0026] In a preferred embodiment of the nanomedicine delivery system of the present invention, the nanoparticles include L-fucoalbumin.

[0027] In a preferred embodiment of the nano-drug delivery system of the present invention, the nano-drug delivery system is injected via the tail vein. Injecting the above-mentioned nano-drug delivery system via the tail vein can improve the therapeutic effect against tumors.

[0028] The nano-drug delivery system provided by this invention can significantly improve the shortcomings of conventional therapies, such as high toxicity and side effects, easy metastasis and recurrence, short duration, short survival, and poor quality of life; it also significantly improves the shortcomings of immune checkpoint monotherapy, such as limited range of tumor types and small number of drug-responsive individuals; and it significantly improves the shortcomings of immune checkpoint combined radiotherapy and chemotherapy, such as high toxicity and side effects and small number of drug-responsive individuals.

[0029] The treatment method provided by this invention has good therapeutic effects on the following patients: patients with tumors that are inoperable, have no available targeted drugs, and are unresponsive to radiotherapy, chemotherapy, etc.; patients with tumors that are unresponsive or resistant to single-agent immune checkpoint inhibitors (primary, adaptive, and acquired resistance); and patients with tumors that are unresponsive or resistant to immune checkpoint inhibitors combined with radiotherapy, chemotherapy, and targeted therapy (primary, adaptive, and acquired resistance).

[0030] Seventh objective: This invention provides a method for preparing the above-mentioned nano-drug delivery system, comprising the following steps:

[0031] S1. Inoculate the above-mentioned Bifidobacterium longum SYSU10 bacterial suspension into the culture medium. After 12 hours, it reaches the logarithmic growth phase. Use the Bifidobacterium longum SYSU10.

[0032] S2. Nanoparticles encapsulated with L-fucoalbumin were prepared by uniaxial electrostatic spraying process;

[0033] S3. Mix the Bifidobacterium longum SYSU10 collected in S1 with the nanoparticles prepared in step S2, then add EDC and NHS, and then incubate in an anaerobic environment at 37°C with shaking; wash and centrifuge, collect the lower precipitate, and obtain the nano-drug delivery system.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention utilizes *Bifidobacterium longum* SYSU10 in combination with an immune checkpoint inhibitor (PD-1 monoclonal antibody) to treat tumor-bearing mice. The *Bifidobacterium longum* SYSU10 screened in this invention can stimulate an anti-tumor immune protective response, remodeling the tumor microenvironment and transforming "cold" tumors into "hot" tumors. Furthermore, *Bifidobacterium longum* SYSU10 significantly enhances the therapeutic effect of immune checkpoint inhibitors on colorectal cancer, inhibiting not only in situ tumor growth but also tumor cell metastasis with good safety. This invention also provides a nanoparticle drug delivery system composed of *Bifidobacterium longum* SYSU10 and the anti-tumor active small molecule L-fucose. Utilizing the anaerobic tendency of *Bifidobacterium longum* SYSU10, it is combined with functional nanoparticles to provide a highly specific tumor-targeted therapy. Attached Figure Description

[0036] Figure 1 A basic morphological diagram of Bifidobacterium longum SYSU10;

[0037] Figure 2 Phylogenetic tree of Bifidobacterium longum SYSU10;

[0038] Figure 3 The graphs show the hemolysis test and drug resistance test results.

[0039] Figure 4 The result diagram is shown in Example 4;

[0040] Figure 5 Plate images of tissue homogenates from the tail vein Bifidobacterium longum group;

[0041] Figure 6 Flowchart of Example 5;

[0042] Figure 7 Tumor volume change curve in Example 5;

[0043] Figure 8 Image of mouse weight in Example 5;

[0044] Figure 9 Tumor weight statistics chart for Example 5;

[0045] Figure 10 Image of tumor metastases in Example 5;

[0046] Figure 11 Flow cytometry results of tumors in Example 5. Detailed Implementation

[0047] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0048] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0049] exist Figure 1-11 The terms “B. longum”, “long bifidobacterium”, “long bifidobacterium SYSU10”, and “Bifidobacterium longum” mentioned in the text all refer to Bifidobacterium longum SYSU10.

[0050] Example 1: Screening and Identification of Fecal Bacteria

[0051] In this embodiment, stool samples were collected from patients who responded to PD-1 inhibitor treatment.

[0052] I. Experimental Methods:

[0053] (1) Fecal collection: Add 20mL of bacterial cryopreservation solution to the cell culture flask, use sterile cotton swabs to collect the freshly excreted feces into the cell culture flask, seal the culture flask in an anaerobic bag, and store and transport it in an ice box.

[0054] (2) Dilution and plating: After shaking, aspirate 100 μL of fecal suspension into 900 μL of PBS buffer, mix thoroughly with a pipette, and then aspirate another 100 μL of suspension into 900 μL of PBS buffer. Repeat this process for 10 serial dilutions. Take 20 μL of each dilution and plating it onto MRS, MRS+MUP (1:1000, v / v), BHI, BHI+PB (1:500, v / v), and CBA plates, respectively. Incubate at 37°C under aerobic or anaerobic conditions for 48-72 h.

[0055] (3) Enrichment culture of single colonies: Select plates with 5-100 colonies, randomly pick a single colony with a disposable inoculation loop and put it into 20 μL PBS. After mixing by pipetting, take 15 μL of bacterial solution and spread it on the same plate. Incubate at 37℃ under aerobic or anaerobic conditions for 48-72 h. The remaining 5 μL of bacterial solution will be reserved for Gram staining identification.

[0056] (4) Bacterial passage: Use a disposable inoculation loop to scrape as much of the bacteria growing on the plate as possible, resuspend it in 200 μL of PBS buffer, and then spread 200 μL of bacterial solution back onto the same plate and incubate under the same conditions for 48-72 h.

[0057] (5) Bacterial cryopreservation: After the bacteria have been enriched to a sufficient quantity, the bacteria on the plate are collected with an inoculation loop into a sterile cryopreservation solution made of fetal bovine serum, BHI medium and glycerol, mixed by pipetting, and stored at -80°C. At the same time, an appropriate amount of bacteria is collected for extraction of bacterial genomic DNA.

[0058] (6) Gram staining identification: Add 5 μL of bacterial suspension prepared when picking single colonies to the center of a glass slide, fix by intermittent baking with an alcohol lamp, cover with crystal violet staining solution for 1 min, wash with running water, then add Lugol's iodine solution for 1 min, wash with running water, then cover the surface of the glass slide with decolorizing alcohol for 20-30 s, gently shaking the slide during this period, wash off the alcohol with running water, counterstain with safranin staining solution for 1 min, wash with running water, and observe the bacterial morphology under an optical microscope after the glass slide dries. If the colonies are impure and more than one bacterial morphology is present, the sample is streaked and cultured, and single colonies are picked again.

[0059] (7) Bacterial genomic DNA extraction: Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit in accordance with the instructions of Tiangen Biotech Co., Ltd., and the DNA concentration was determined using NanoDrop2000.

[0060] (8) 16S fragment amplification:

[0061] 1) Use 27F / 1492R primer pair, 27F sequence: 3'-AGAGTTTGATCMTGGCTCAG-5' 1492R sequence: 3'-GGTTACCTTGTTACGACTT-5'.

[0062] The reaction system is as follows:

[0063] LA Taq enzyme 15μL PCR Forward Primer (10 μM) 1.2μL PCR Reverse Primer (10μM) 1.2μL Bacterial genomic DNA 3μL <![CDATA[ddH2O]]> up to 30μL

[0064] 2) The PCR amplification program is as follows: 95℃ pre-denaturation for 60s; 95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 90s, cycle number 31; 72℃ further extension for 7min, end temperature set to 4℃.

[0065] (9) Agarose gel electrophoresis: Prepare 25 mL of 1×TAE buffer containing 1% (w / v) agarose, melt it using a microwave oven, add 2.5 μL of GoldView nucleic acid dye, mix well, pour into the gel casting tank, insert the comb, and allow it to cool. After the agarose gel cools, remove the comb and place it in a horizontal electrophoresis tank. Add 5 μL of amplified DNA sample and 3 μL of LDL2000 DNA marker to each well, turn on the power, and perform electrophoresis at a constant voltage of 110V for 20 min. Then observe under a UV lamp to see if the amplified bands are uniform and if their size is around 1500 bp.

[0066] (10) 16S rRNA sequencing and result comparison: The amplified DNA 16S fragment samples, which were confirmed by agarose gel electrophoresis, were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After obtaining the sequencing results, the sequence was assembled using ChromasPro software, and the 16S rRNA sequence was blasted on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to preliminarily identify the type of bacteria.

[0067] II. Experimental Results

[0068] The strain identified by cell division was named *Bifidobacterium longum* SYSU10. *Bifidobacterium longum* SYSU10 was deposited on September 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25777, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The nucleotide sequence of the 16S rDNA of *Bifidobacterium longum* SYSU10 is shown in SEQ ID NO:1.

[0069] The basic morphological diagram of Bifidobacterium longum SYSU10 of the present invention is shown below. Figure 1 As shown. The phylogenetic tree of *Bifidobacterium longum* SYSU10 is as follows. Figure 2 As shown.

[0070] Example 2: Security Verification

[0071] I. Experimental Methods:

[0072] (1) Hemolytic activity: Take 10 μL of Bifidobacterium longum SYSU10 in logarithmic growth phase, inoculate it onto Columbia blood agar plates using the dilution streak method, and incubate at 37°C for 36 h to observe whether there is a hemolytic zone.

[0073] (2) Drug susceptibility: ① Use an inoculation loop to scrape one loopful of the test bacteria in the logarithmic growth phase and resuspend it in 2 mL of sterile PBS buffer; ② Use a sterile cotton swab to repeatedly dip the above bacterial suspension and spread it evenly on an MRS plate; ③ After the bacterial suspension on the plate is absorbed, use tweezers to place the drug susceptibility paper on the plate, incubate at 37°C for 36 h, and then use a ruler to measure the size of the inhibition zone near the drug susceptibility paper and record the results in mm.

[0074] II. Experimental Results:

[0075] The selected Bifidobacterium longum SYSU10 strain exhibits good safety profile, being non-hemolytic and sensitive to common antibiotics, as shown in the results. Figure 3 As shown.

[0076] Example 3: Preparation of L-fucoidan loaded with Bifidobacterium longum SYSU10 Sugar White Egg White nanoparticle drug delivery system

[0077] (1) Preparation of albumin aqueous solution: Weigh gelatin, place it in pure water and heat and stir until completely dissolved. Then, after the temperature drops to room temperature, add bovine serum albumin to obtain albumin polymer aqueous solution.

[0078] (2) Preparation of L-fucose albumin nanoparticles: L-fucose was added to an aqueous solution of albumin polymer and stirred to dissolve into a homogeneous solution to obtain an aqueous solution of L-fucose albumin; L-fucose albumin nanoparticles were prepared by uniaxial electrostatic spraying technology.

[0079] (3) Bifidobacterium longum SYSU10 was placed in MRS medium at 37°C for anaerobic culture. When it reached the logarithmic growth phase, it was centrifuged and resuspended in PBS to obtain a bacterial suspension.

[0080] (4) The bacterial suspension and L-fucoal albumin nanoparticle solution were mixed at a volume ratio of 1:1, and then EDC and NHS were added. The mixture was then anaerobically cultured at 37°C for 8 hours. The mixture was then centrifuged at 2000 rpm for 5 minutes, and the supernatant was washed with PBS to obtain the Bifidobacterium longum SYSU10-loaded L-fucoal albumin nanomedicine delivery system.

[0081] II. Experimental Results:

[0082] The prepared L-fucoal albumin nanoparticles had a uniform size distribution, with a particle size of approximately 200-600 nm. The Bifidobacterium longum (SYSU10)-loaded L-fucoal albumin nanoparticle drug delivery system still retained Bifidobacterium longum activity; the results are as follows. Figure 5 As shown.

[0083] Example 4: Injection of Bifidobacterium longum SYSU10 suspension or long-term intravenous injection via tail vein. Testing the therapeutic efficacy of Bifidobacterium-loaded L-fucoalbumin nanomedicine against melanoma.

[0084] I. Experimental Methods:

[0085] 1. Experimental grouping: A total of 20 C57 mice (5 weeks old), female.

[0086]

[0087] 2. Administer C57 mice with ABX solution (100 mg / kg each of metronidazole, vancomycin, ampicillin, and neomycin sulfate) for 7 consecutive days.

[0088] 3. Subcutaneous injection of 5*10 mg / L per mouse 5 B16-F10 cells / 100μL.

[0089] 4. When the tumor volume reaches 50mm 3 (Tumor volume = length * width) 2 *0.5) Tumor size was measured starting from day 0 of the experiment.

[0090] 5. Starting from tumor implantation, every 3 days, each mouse was injected intravenously with 100 μL of PBS / Bifidobacterium longum SYSU10 / Bifidobacterium longum SYSU10 loaded with L-fucoal albumin nanomedicine / L-fucoal albumin nanomedicine.

[0091] 6. When the tumor reaches a maximum length of 1200mm 3 Mice were euthanized and dissected in a biosafety cabinet. The tissues were homogenized, spread onto BHI plates, and incubated at 37°C to observe whether Bifidobacteria grew.

[0092] II. Experimental Results:

[0093] Tail vein injection of Bifidobacterium longum (SYSU10) exhibits good tumor-targeting properties, accumulating at the tumor site. When combined with L-fucoalbumin nanoparticles to form a nano-drug delivery system, its anti-tumor effect is significantly enhanced, as shown in the results. Figure 4 As shown.

[0094] Example 5: Synergistic anti-cancer effect of Bifidobacterium longum SYSU10 and PD-1 inhibitor. rectal cancer

[0095] I. Experimental Methods (Flowchart shown) Figure 6 As shown):

[0096] 1. Experimental grouping: A total of 32 C57 mice (5 weeks old), half male and half female.

[0097] 2. αPD-1 (4 females + 4 males) 3. Bifidobacterium longum (4 females + 4 males) 4. Bifidobacterium longum + αPD-1 (4 females + 4 males)

[0098] 2. C57 mice were given ABX solution (100 mg / kg each of metronidazole, vancomycin, ampicillin, and neomycin sulfate) orally for 7 consecutive days, followed by oral administration of 10 mg / kg of the solution to each mouse daily. 8 100 μL of a CFU / mL suspension of Bifidobacterium longum.

[0099] 3. Subcutaneous injection of 5*10 mg / L per mouse 5 MC38 cells / 100μL

[0100] 4. When the tumor volume reaches 50mm 3 (Tumor volume = length * width) 2 *0.5) Tumor size was measured starting from day 0 of the experiment.

[0101] 5. Starting from the 12th day after tumor transplantation, administer intraperitoneal injections of αPD-1 (BioXcell, RMP1-14) every 3 days at a dose of 2 mg / kg for a total of 4 injections.

[0102] 6. Record body weight and collect fresh stool samples on days 0, 4, 8, 12, 16, 20, and 24. Store the samples at -80°C for subsequent sequencing.

[0103] 7. Record tumor volume on days 0, 3, 6, 9, 12, 15, 18, 21, and 24.

[0104] On the day of the autopsy:

[0105] 1. Weighing and recording of the tumor, plus taking a photo.

[0106] 2. Collect blood and serum (incubate at 4℃ for 30 min, then centrifuge at 3000 rpm for 10 min), and store the supernatant at -80 for subsequent experiments.

[0107] 3. Arrange the tumors in each group on the board in descending order of size, then clean the tumors with filter paper and weigh them.

[0108] 4. Each group of tumors (partial), spleen (partial), liver, lungs, and lymph nodes were preserved in paraformaldehyde for subsequent IHC embedding.

[0109] 5. A portion of the tumor and spleen from each group were sampled for flow cytometry. The tumor was stained with CD3-FITC and CD8a-PE.

[0110] 6. After gently cutting the tumor into small pieces in a 6-well plate with ophthalmic scissors, place it on a 200-mesh cell sieve and gently crush the tumor with a 10mL syringe needle. After squeezing, rinse the cell sieve with a small amount of DMEM culture medium (or PBS) and rinse the cell suspension into a 50mL centrifuge tube, and then aliquot it into 1.5mL centrifuge tubes.

[0111] 7. Wash twice with PBS (the second wash with PBS solution containing 4% FBS) at 3200 rpm for 3 min at 4℃. After the second wash, discard the supernatant and resuspend the sample in PBS solution containing 4% FBS + antibody (100 μL per sample).

[0112] 8. Tumor stained with CD3+CD8a:

[0113] (1) Incubate the antibody in a black EP tube in a light-proof box. Incubate the cells with the antibody in a dark room. Refer to the instructions for the amount of antibody used. Add 100μL of 1uLCD3 and 1.5μL of LCD8a and incubate for 30-40 minutes. (2) After staining, add 1mL of 4%FBS in PBS, centrifuge to remove the supernatant, and then add 1mL of 4%FBS in PBS to resuspend the cells.

[0114] II. Experimental Results:

[0115] The anti-tumor immune protective response stimulated by *Bifidobacterium longum* SYSU10 increases CD8+ T cell infiltration into tumor sites, remodels the tumor microenvironment, and transforms "cold" tumors into "hot" tumors. Combining *Bifidobacterium longum* SYSU10 with immune checkpoint inhibitors significantly enhances the inhibitory effect of immune checkpoint inhibitors on colorectal tumors. The effects of each group on tumor volume changes are as follows: Figure 7 As shown, the mouse's weight is as follows Figure 8 As shown, tumor weight statistics are as follows: Figure 9 As shown. The impact of each group on tumor metastases is as follows. Figure 10 As shown. The flow cytometry results for each group of tumors are as follows. Figure 11 As shown.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nanomedicine delivery system, characterized in that, Using Bifidobacterium longum ( Bifidobacterium longum SYSU10 supports nanoparticles to form a nano-drug delivery system; The Bifidobacterium longum ( Bifidobacterium longum SYSU10 was deposited on September 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25777 and address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The nanoparticles include L-fucoalbumin.

2. The nanomedicine delivery system as described in claim 1, characterized in that, The nano-drug delivery system is administered via tail vein injection.

3. A method for preparing a nano-drug delivery system as described in claim 1 or 2, characterized in that, Includes the following steps: S1, the aforementioned Bifidobacterium longum ( Bifidobacterium longum SYSU10 bacterial culture was inoculated into the culture medium, and after 12 hours it reached the logarithmic growth phase. Bifidobacterium longum (…) was then collected. Bifidobacterium longum ) SYSU10 is used; S2. Nanoparticles encapsulated with L-fucoalbumin were prepared by uniaxial electrostatic spraying process; S3, the Bifidobacterium longum collected in S1 ( Bifidobacterium longum SYSU10 is mixed with the nanoparticles obtained in step S2, and then EDC and NHS are added. The mixture is then shaken and incubated in an anaerobic environment at 37°C. After washing and centrifugation, the lower precipitate is collected to obtain the nano-drug delivery system.

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