Engineered probiotics loaded with Cu2-xSe nanoparticles in cells as well as preparation method and application of engineered probiotics

Abstract

The invention belongs to the technical field of probiotic preparations, and particularly relates to engineered probiotics loaded with Cu2-xSe nanoparticles in cells as well as a preparation method and application of the engineered probiotics. According to the application, the non-toxic probiotics are used as a bioreactor, and the engineered probiotics which are good in stability and have excellent photo-thermal performance and H2O2 catalysis performance and are loaded with the Cu2-xSe nanoparticles in cells are successfully constructed by regulating and controlling incubation conditions of the probiotics, the copper source and the selenium source. The compound can enrich tumor sites in a targeted manner, is efficiently taken by tumor cells, kills the tumor cells, induces M2 type TAMs to be reversely polarized into M1 type TAMs, improves inhibitory TIME, promotes TDLNs and DCs in TME to be mature, increases infiltration of CD8 + effector T cells and the number of CD8 + T cells and central memory T cells in the spleen, and achieves the anti-tumor effect. In addition, the long-term immune memory effect can be induced to be generated, and tumor recurrence is effectively inhibited.

Description

Technical Field

[0001] This application belongs to the technical field of probiotic preparations, and more specifically, relates to engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles and their preparation methods and applications. Background Art

[0002] The tumor immune microenvironment (TIME) is an essential component of the tumor microenvironment (TME) and plays an important role in the growth, invasion, and migration of tumor cells. The TIME infiltrates various immune cells, including tumor-associated macrophages (TAMs), cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, dendritic cells (DCs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and inhibitory / pro-inflammatory cytokines secreted by these cells. The interaction between various immune cells and the cytokines they secrete with tumor cells constitutes the cancer-immunity cycle (CI cycle) to specifically recognize and eliminate tumor cells. Immunotherapy is based on the CI cycle and inhibits tumor growth by activating or enhancing the body's own anti-tumor immune response. However, affected by the inhibitory TIME, the response rate of tumor immunotherapy is low, and the tumor treatment effect exerted is limited. Summary of the Invention

[0003] Aiming at the defects of the prior art, the purpose of this application is to provide engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles and their preparation methods and applications. In this application, non-toxic probiotics are used as bioreactors. By regulating the oxygen concentration, culture medium type, etc. when the probiotics are incubated with copper sources and selenium sources, a large amount of Cu 2-x Se nanoparticles are synthesized intracellularly, and engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles with good stability, excellent photothermal performance, and H2O2 catalytic performance are successfully constructed.

[0004] To achieve the above object, in a first aspect, the present application provides a method for preparing an engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles, comprising the following steps: Placing the probiotic, selenium source, and copper source in a culture medium containing glucose, incubating in an anaerobic environment, and synthesizing Cu 2-x Se nanoparticles intracellularly through redox reaction to obtain the above-mentioned engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles; wherein, x is 0 to 1.

[0005] Preferably, the above-mentioned probiotic is a probiotic with redox ability.

[0006] More preferably, the above-mentioned probiotic is one or more of Bifidobacterium, Lactobacillus, Saccharomyces cerevisiae, probiotic Escherichia coli, and probiotic Bacillus spores.

[0007] Preferably, the above-mentioned selenium source is one or more of selenite, sodium selenate, selenium dioxide, selenomethionine, selenocysteine, methylselenocysteine, selenium urea, sodium selenosulfate, selenoacetic acid, and selenourea.

[0008] Preferably, the above-mentioned copper source is a divalent copper salt, selected from one or more of EDTA-Cu(II), copper chloride, copper sulfate, and copper sulfate.

[0009] Preferably, in the incubation system, the concentration of the above-mentioned probiotic is 2×10 9 CFU / mL to 5×10 9 CFU / mL.

[0010] Preferably, in the incubation system, the final concentration of the above-mentioned copper source is 2 mmol / L to 4 mmol / L.

[0011] Preferably, in the incubation system, the molar ratio of the above-mentioned copper source to the above-mentioned selenium source is (1 to 2):1.

[0012] Preferably, the above-mentioned culture medium containing glucose is one or more of glucose buffer medium, MRS medium, MEM medium, and DMEM medium.

[0013] Preferably, the above-mentioned incubation time is 24 h to 48 h.

[0014] In a second aspect, the present application provides an engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles prepared by the above preparation method.

[0015] In a third aspect, the present application provides the use of the above-mentioned engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles in the preparation of an M2-type TAMs inverse polarization drug or as an M2-type TAMs inverse polarization drug.

[0016] Fourthly, the present application provides the use of the engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles in the use as or the preparation of anti-tumor drugs.

[0017] Fifthly, the present application provides an anti-tumor drug, which comprises the engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles.

[0018] Preferably, the above-mentioned tumors include one or more of liver cancer, breast cancer, colon cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, ovarian cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma, glioblastoma.

[0019] Generally speaking, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) The present application uses non-toxic probiotics as bioreactors. By regulating conditions such as the oxygen concentration and the type of culture medium when incubating probiotics with copper sources and selenium sources, a large amount of Cu 2-x Se nanoparticles are synthesized intracellularly, and an engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles with good stability, excellent photothermal performance and H2O2 catalytic performance is successfully constructed.

[0020] (2) The present application proves through experiments that the engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles can inhibit the polarization of TAMs into the M2 type, induce the reverse polarization of M2-type TAMs into the M1 type, promote the release of TNF-α and IL-6 pro-inflammatory factors by TAMs, and inhibit the release of TGF-β, and are suitable for use as or the preparation of drugs for the reverse polarization of M2-type TAMs.

[0021] (3) Compared with the single administration of Dead EcN and Cu 2-x Se NPs, the engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles provided by the present application (Cu 2-x Se@EcN) show a synergistic effect in the reverse polarization of M2-type TAMs, can promote the expression of M1-type related markers CD80 and CD86 and the release of pro-inflammatory factors TNF-α and IL-6, and inhibit the expression of M2-type related marker CD206 and the release of TGF-β. The synergistic effect between components makes the reverse polarization effect of M2-type TAMs significantly better than that of single Dead EcN and single Cu 2-x Se NPs.

[0022] (4) The engineered probiotics intracellularly loaded with Cu 2-xEngineered probiotics loaded with Se nanoparticles can be efficiently internalized by tumor cells, effectively kill tumor cells, trigger immunogenic cell death in tumor cells, and co-stimulate the maturation of DCs. The intracellular loading of Cu 2-x Engineered probiotics loaded with Se nanoparticles can rapidly target and accumulate in tumor sites after intravenous injection. Using thermo-assisted chemodynamic synergistic immunotherapy, it can induce the repolarization of M2-type TAMs into M1-type, enhance the body's immune response, improve the inhibitory TIME, and at the same time promote the maturation of DCs in tumor-draining lymph nodes (TDLNs) and TME, increase CD8 + effector T cell infiltration, and increase the number of CD8 + T cells and central memory T cells in the spleen, thus effectively inhibiting tumor growth and achieving anti-tumor effects. In addition, the intracellular loading of Cu 2-x Engineered probiotics loaded with Se nanoparticles can induce long-term immune memory effects, effectively inhibit the secondary growth of tumor cells, inhibit tumor recurrence, and at the same time have good biosafety, and are suitable for use as or in the preparation of anti-tumor drugs. Brief Description of the Drawings

[0023] Figure 1 Shows the synthesis amount of copper selenide under different culture conditions of this application; where content a is the color of the bacterial solution, content b is the TEM image of the bacterial precipitate, and content c is the heating curve of the bacterial solution; Figure 2 Shows the synthesis amount of copper selenide under different substrate concentrations and culture times of this application; where content a, content b, and content c are the heating curves of the bacterial solution when the final Cu concentration is 2, 3, and 4 mmol / L, respectively; Figure 3 For Cu 2-x Characterization of Cu Figure 4 For Cu 2-x TEM-EDS map of Cu Figure 5 For Cu 2-x Crystal structure of Cu Figure 6 For Cu 2-x Survival ability of Cu 2-x Se@EcN, where content a is the single colony photo of EcN and Cu 6 diluted 10 2-xCLSM images of Se@EcN stained with DAPI and PI respectively; content c is Cu 2-x Hydrodynamic diameter of Se@EcN stored in PBS or medium at 4 °C for 7 days Figure 7 For Cu 2-x Temperature rise of Se@EcN; where content a is the temperature-time curve, content b is the photo of the end temperature, and content c is the photothermal heating-temperature cycle curve irradiated for 5 cycles Figure 8 For Cu 2-x Ability of Se@EcN to generate ROS in vitro; where content a is Cu at different concentrations 2-x UV-Vis absorption spectrum of the reaction solution at 500 - 800 nm after Se@EcN and MB react with 1.0 mM H2O2 for 10 min, content b is Cu 2-x Rate of ROS generation by Se@EcN with / without laser treatment Figure 9 For Cu 2-x Uptake and killing ability of Se@EcN by different cells; where content a is the proportion of DiO-labeled Cu 2-x Se@EcN co-incubated with 3T3 cells and H22 cells respectively, and then DiO + Proportion of cells, content b and content c are the survival rates of 3T3 cells and H22 cells co-incubated with different concentrations of Cu 2-x Se@EcN after co-incubation Figure 10 For Cu 2-x Antitumor effect of photothermal-assisted Se@EcN; where content a is the survival rate of H22 cells after incubation with Cu 2-x Se@EcN with / without laser treatment, content b is the intracellular ROS level of H22 cells after incubation with Cu 2-x Se@EcN under photothermal assistance Figure 11 For Cu 2-x Photothermal-assisted chemodynamic induction of ICD in tumor cells mediated by Se@EcN; where content a and content b are the ATP content and HMGB1 content of H22 cells after incubation with Cu 2-x Se@EcN with / without laser treatment Figure 12 For Cu under photothermal assistance 2-x Stimulation of BMDCs maturation by Se@EcN; where content a and content b are the cell supernatants collected after incubation of H22 cells with Cu 2-x Se@EcN with / without laser treatment and co-cultured with BMDCs, CD80 + CD86+ Cells, MHC-II + Proportion of cells; Figure 13 is Cu 2-x Ability of Cu Figure 14 is Cu 2-x Se@EcN to inhibit the polarization of M0 macrophages into M2 macrophages in vitro; where content a, content b, and content c are CD80 + , CD86 + and CD206 + Proportion of cells, and content d, content e, and content f are the levels of TNF-α, IL-6, and TGF-β respectively; Figure 15 is Cu 2-x Distribution of Cu 2-x Se@EcN in vivo after intravenous administration; where content a is the material distribution and semi-quantitative mean fluorescence intensity of tumor-bearing mice at different injection time points, and content b is the distribution of Cu Figure 16 is Cu 2-x Antitumor therapy mediated by intravenous administration of Cu Figure 17 is Cu 2-x Tumor photos of tumor-bearing mice on the 14th day after intravenous administration of Cu Figure 18 is Cu 2-x Stimulation of DCs in TDLNs by therapy mediated by Cu + Se@EcN; where content a and content b are the proportions of CD86 + and MHC-II + cells in DCs of tumor-bearing mice TDLNs; content c is the proportion of CD8 + in CD3 Figure 19 is Cu 2-x Stimulation of DCs and reverse polarization of macrophages in the TME by therapy mediated by Cu + Se@EcN; where content a and content b are the proportions of CD86 + and MHC-II + cells in DCs of tumor-bearing mice TME; content c is the proportion of CD8 +Ratio; Content d and content e are respectively the proportions of CD80 + CD86 + and CD206 + cells in macrophages of TEM in tumor-bearing mice; Figure 20 is the effect of Cu 2-x Se@EcN-mediated treatment on T cells in the spleen; where content a and content b are respectively CD3 + in CD8 + and CD44 + CD62L + proportions in T cells; Figure 21 is the inhibitory effect of Cu 2-x Se@EcN-mediated combined immunotherapy on the growth of secondary inoculated H22 tumors; where content a is the tumor growth curve of mice within 14 days after secondary inoculation, content b is the tumor size of mice on the 14th day after secondary inoculation, and content c is the level of effector memory T cells on the 14th day after secondary inoculation; Figure 22 is the biosafety of Cu 2-x Se@EcN; where content a, content b, content c, content d, and content e are respectively the changes in ALT, AST, AST / ALT, BUN, and CERA levels in the peripheral blood of mice after tumor treatment, and content f is the weight change curve of mice; In all the drawings, the same reference numerals are used to represent the same significance level, where: represents p < 0.05, represents p < 0.01, represents p < 0.001, represents p < 0.0001, represents no statistical difference. Detailed implementation manners

[0024] In order to make the objectives, technical solutions, and advantages of this application clearer, the following further elaborates on this application in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0025] In the description of this application, it should be understood that the term "and / or" is a relationship describing associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. The symbol " / " in this article represents an "or" relationship between associated objects, for example, A / B represents A or B.

[0026] In the description of the embodiments of the present application, unless otherwise specified, the meaning of "a plurality" refers to two or more.

[0027] The present application provides a method for preparing an engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles, comprising the following steps: Placing the probiotic, selenium source, and copper source in a culture medium containing glucose, incubating in an anaerobic environment, and synthesizing Cu 2-x Se nanoparticles intracellularly through redox reactions, thus obtaining the above-mentioned engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles; wherein, x is 0 to 1.

[0028] In some embodiments, the above-mentioned probiotic is a probiotic with redox ability, which can reduce the copper source and selenium source to low-valent copper and selenium, and then synthesize Cu 2-x Se nanoparticles.

[0029] The present application uses non-toxic probiotics as bioreactors to synthesize Cu 2-x Se nanoparticles intracellularly. By regulating conditions such as the oxygen concentration, culture medium type, substrate concentration, and incubation time when the probiotic is incubated with the copper source and selenium source, a large amount of Cu 2-x Se nanoparticles is synthesized intracellularly, and an engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles with good stability, excellent photothermal performance, and catalytic H2O2 performance is successfully constructed.

[0030] In some embodiments, the above-mentioned probiotic is one or more of Bifidobacterium, Lactobacillus, Saccharomyces cerevisiae, probiotic Escherichia coli (such as Escherichia coli Nissle 1917) and probiotic Bacillus (such as Bacillus licheniformis, Bacillus licheniformis ).

[0031] In some embodiments, the above-mentioned selenium source is one or more of selenite, sodium selenate, selenium dioxide, selenomethionine, selenocysteine, methylselenocysteine, selenium urea, sodium selenosulfate, selenoacetic acid, and selenourea.

[0032] In some embodiments, the above-mentioned copper source is a divalent copper salt, selected from one or more of EDTA-Cu(II), copper chloride, copper sulfate, and copper sulfate.

[0033] In some embodiments, the concentration of the above-mentioned probiotic is 2×10 9 CFU / mL to 5×10 9 CFU / mL.

[0034] In some embodiments, in the incubation system, the final concentration of the above copper source is 2 mmol / L to 4 mmol / L.

[0035] In some embodiments, the molar ratio of the above copper source to the above selenium source is (1 to 2):1.

[0036] In some embodiments, the above medium containing glucose can be, but is not limited to, glucose buffer medium, MRS medium, MEM medium, DMEM medium, etc.

[0037] In some embodiments, the above incubation time is 24 h to 48 h.

[0038] On the other hand, the present application also provides an engineered probiotic prepared by the above preparation method and intracellularly loaded with Cu 2-x Se nanoparticles.

[0039] TAMs play an important role in tumor growth, invasion and metastasis. There are mainly two subtypes of TAMs: the pro-inflammatory and tumoricidal M¹-like phenotype and the anti-inflammatory and pro-repair M²-like phenotype, which are considered potential anti-cancer targeting indicators. The engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles provided by the present application can inhibit the polarization of TAMs to the M² phenotype, induce the reverse polarization of M²-type TAMs to M¹-type, promote the release of TNF-α and IL-6 pro-inflammatory factors by TAMs, and inhibit the release of TGF-β. Based on this, the present application provides the use of the above engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles in the preparation or use of drugs for the reverse polarization of M²-type TAMs.

[0040] The engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles provided by the present application can be efficiently taken up by tumor cells and kill tumor cells. At the same time, it triggers immunogenic cell death (ICD) in tumor cells and co-stimulates the maturation of DCs. The engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles provided by the present application can rapidly target and enrich in the tumor site after intravenous injection, promote the maturation of DCs in tumor-draining lymph nodes (TDLNs) and the tumor microenvironment (TME), increase the infiltration of CD8 + effector T cells (CTLs), induce the reverse polarization of TAMs into pro-inflammatory M¹-type, and increase the number of CD8 + T cells and central memory T cells in the spleen. At the same time, it has a long-term immune memory effect, can inhibit the secondary growth of tumor cells, effectively inhibit tumor recurrence, and has good biosafety. Based on this, the present application provides the use of the above engineered probiotic intracellularly loaded with Cu 2-x Se nanoparticles in the preparation or use of anti-tumor drugs.

[0041] The engineered probiotics with intracellularly loaded Cu 2-x Se nanoparticles have excellent photothermal performance and catalytic H2O2 performance. When used as or in the preparation of anti-tumor drugs, a thermo-assisted chemodynamic synergistic immunotherapy strategy can be adopted to enhance the anti-tumor effect.

[0042] The present application also provides an anti-tumor drug, which comprises the above-mentioned engineered probiotics with intracellularly loaded Cu 2-x Se nanoparticles.

[0043] In some embodiments, the above-mentioned tumors include, but are not limited to, one or more of liver cancer, breast cancer, colon cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, ovarian cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma, and glioblastoma.

[0044] In some embodiments, the above-mentioned engineered probiotics with intracellularly loaded Cu 2-x Se nanoparticles can be included in the anti-tumor drug together with a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0045] In some embodiments, the above-mentioned carrier includes all kinds of solvents, dispersion media, water-in-oil or oil-in-water emulsions, aqueous compositions, liposomes, microspheres, and microsomes.

[0046] The present application does not make special limitations on the administration route and method of the above-mentioned anti-tumor drug, as long as the effects of the present application are shown. In some embodiments, the administration mode of the above-mentioned anti-tumor drug is intravenous administration or intratumoral administration, and its dosage form includes, but is not limited to, injections. In some embodiments, laser irradiation of the tumor tissue can be used to enhance the anti-tumor effect through the thermo-assisted chemodynamic synergistic immunotherapy strategy.

[0047] It should be understood that materials with the same or similar types, models, qualities, properties, or functions as the reagents and instruments used in the following embodiments can be used to implement the present application. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0048] The main reagents and material sources used in the following embodiments are as follows: Na2SeO3 was purchased from Aladdin, CuSO4·5H2O, EDTA-2Na, and IR-780 were all purchased from Sinopharm Group, DAPI, PI, and CCK-8 kits were all purchased from Beyotime Institute of Biotechnology, DMEM medium and RPMI 1640 medium were purchased from Hyclone, and RAW264.7 macrophages were purchased from the China Center for Type Culture Collection (Shanghai, China). Antibodies against CD80, CD86, CD206, CD45, CD3ε, CD4, CD8a, CD11c, CD44, CD62L, and CD11b were all purchased from BioLegend.

[0049] The following are the examples: Example 1 Intracellular loading of Cu 2-x Preparation of engineered probiotics loaded with Se nanoparticles 1. Experimental materials CuSO4·5H2O, EDTA-2Na, Na2SeO3, probiotics ( Escherichia coli Nissle 1917, EcN) 2. Experimental procedures Prepare glucose buffer medium: Accurately weigh 5 g of tryptone, 2.5 g of yeast extract, 5 g of NaCl, 3.675 g of C6H5Na3O7, 0.238 g of MgCl2, and 2 g of C6H 12 O6, and add 500 mL of ultrapure water.

[0050] Prepare a 0.2 mol / L CuSO4 solution (light blue), a 0.2 mol / L EDTA-2Na solution, and a 0.5 mol / L Na2SeO3 solution. Then mix the CuSO4 solution and the EDTA-2Na solution in a volume ratio of 1:1 and stir for 2.5 h to obtain an EDTA-Cu(II) solution with a final concentration of 0.1 mol / L (dark blue). Then filter the EDTA-Cu(II) solution and the Na2SeO3 solution through a 0.22 μm filter membrane to sterilize.

[0051] Inoculate the probiotics ( Escherichia coli Nissle 1917, EcN) into LB medium and incubate for 8 h, then add it to 50 mL of LB medium in a volume ratio of 1:100 to obtain a bacterial suspension cultured for 24 h.

[0052] Respectively pipette 48.9 mL of the bacterial suspension cultured for 24 h (adjust the EcN cell concentration to 2×10 9CFU / mL) were placed in different sterile vials. In the control group (Control), 1.1 mL of sterile ultrapure water was added, sealed, and incubated at 37 °C for 7 days; in the experimental group (LB+Normoxia), 1 mL of sterilized EDTA-Cu(II) solution with a concentration of 0.1 mol / L and 0.1 mL of Na2SeO3 solution with a concentration of 0.5 mol / L were added, sealed, and incubated at 37 °C for 7 days; in the experimental group (LB+N2), 1 mL of sterilized EDTA-Cu(II) solution with a concentration of 0.1 mol / L and 0.1 mL of Na2SeO3 solution with a concentration of 0.5 mol / L were added, nitrogen was introduced to remove the original air in the vial, sealed, and incubated at 37 °C for 3 days; in the experimental group (Glu+N2), 50 mL of the bacterial suspension cultured for 24 h was aspirated, centrifuged at 8000 rpm for 3 min, the supernatant medium was discarded, and then it was resuspended with 48.9 mL of glucose buffer medium (adjust the EcN cell concentration to 2×10 9 CFU / mL) were placed in a sterile vial, 1 mL of sterilized EDTA-Cu(II) solution with a concentration of 0.1 mol / L and 0.1 mL of Na2SeO3 solution with a concentration of 0.5 mol / L were added, nitrogen was introduced to remove the original air in the vial, sealed, and incubated at 37 °C for 24 h. Finally, the bacterial solutions of each group were collected, centrifuged to discard the supernatant, and resuspended after centrifugal washing three times with physiological saline.

[0053] 3. Experimental results The copper selenide content in each group was initially judged according to the color of the bacterial solution and preliminary observation by transmission electron microscopy. Through the photothermal heating experiment, 1 mL of the bacterial suspension of each group was taken respectively, irradiated with a 1064 nm laser at a power of 1.0 W / cm 2 , and the temperature was monitored with an infrared thermal imager during the process (frequency: 1 time / min). After 10 min, the irradiation and monitoring were stopped, and the copper selenide content in each group was evaluated.

[0054] As Figure 1 Content a, Figure 1 Content b shows, the bacterial solution of the Glu+N2 group was dark green, and a large number of irregular round nanostructures were contained in the cells. As Figure 1 Content c shows, the bacterial solution of the Glu+N2 group could be heated to above 40 °C under laser irradiation, much higher than the other three groups. In summary, copper selenide nanoparticles with photothermal effect were successfully synthesized in EcN cells in this application.

[0055] Example 2 Optimization of the synthesis conditions of engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles 1. Experimental materials The bacterial suspension prepared in Example 1 2. Experimental steps [[ID=3}} Absorb 50 mL of the bacterial suspension cultured for 24 h, centrifuge at 8000 rpm for 3 min to discard the supernatant medium, and then resuspend with 50 mL of glucose buffer medium. Add an equal amount of the resuspended bacterial suspension, an appropriate amount of sterilized EDTA-Cu(II) solution and Na2SeO3 solution to 5 vials respectively. Divide them into five groups according to the added substrate ion concentration. The final concentrations of Cu are: 0, 2, 3, 4, 5 mmol / L. Pass nitrogen to remove the original air in the vials, seal and incubate at 37 °C for 48 h.

[0056] 3. Experimental results During the incubation process, collect an appropriate amount of bacterial liquid every 12 h, and evaluate the synthesis amount of copper selenide in each group according to the above photothermal heating experiment. After the incubation is completed, collect the bacterial liquid of each group, centrifuge to collect the supernatant and bacterial precipitate, and preliminarily judge the remaining amount of the substrate in each group according to the color change of the supernatant.

[0057] It was found by observing the color of the bacterial liquid that the supernatant with a final Cu concentration of 5 mmol / L was blue-green, indicating that the added amount of the substrate EDTA-Cu(II) exceeded the utilization degree of the bacteria, and this concentration was discarded in the subsequent experiments. As Figure 2 Content a~ Figure 2 As shown in Content c, the temperature of the bacterial liquid increased faster and higher when the substrate concentration increased. When the final concentration of the substrate Cu was 4 mmol / L, the photothermal heating curves at 24, 36, and 48 h of incubation were similar. That is, when the final concentration of the substrate Cu was 4 mmol / L and cultured for 24 h, the Cu 2-x Se nanoparticles synthesized intracellularly by EcN were basically saturated. Collect the black-green bacterial precipitate synthesized at this time and record it as Cu 2-x Se@EcN for subsequent experiments.

[0058] Example 3 Cu 2-x Characterization of Se@EcN 1. Experimental materials Cu synthesized in Example 2 2-x Se@EcN, DAPI / PI, PRMI 1640 medium 2. Experimental procedures (1) Use a biological transmission electron microscope (TEM) and a field emission TEM (FTEM) to observe the shape, elements and other characteristics of Cu 2-x Se@EcN. Use a dynamic light scattering instrument (DLS) to detect the particle size and potential of Cu 2-x Se@EcN. Use an X-ray diffractometer (XRD) and an X-ray photoelectron spectrometer (XPS) to analyze the valence state of the freeze-dried powder of Cu 2-x Se@EcN.

[0059] (2) Take EcN as a control, and use EcN and Cu2-x The Se@EcN bacterial suspension was diluted 10 6 times and then spread on the plate, which was placed in an incubator for 48 h, and the number of colonies on the plate surface was recorded. DAPI and PI staining solutions were added to the bacterial suspensions of Cu 2-x Se@EcN and EcN, and they were incubated in the dark at room temperature for 30 min, centrifuged and washed three times with normal saline and then resuspended. The bacterial suspension was dropped onto a glass slide, covered with a coverslip and sealed, and the survival of bacteria was observed. After centrifuging and washing the Cu 2-x Se@EcN bacterial suspension, it was resuspended with PBS and PRMI 1640 medium respectively. Samples were taken every day, and the particle size distribution of the samples was detected by DLS to evaluate its stability.

[0060] 3. Experimental results As Figure 3 shown in Content a, Figure 3 Content b, the hydrodynamic diameters of EcN and Cu 2-x Se@EcN were 1110 nm and 955 nm respectively, and the Zeta potentials were -27.1 ± 1.1 mV and -23.2 ± 0.7 mV respectively, indicating that the Cu 2-x Se nanoparticles synthesized intracellularly by EcN had no obvious effect on the size and surface charge of EcN itself.

[0061] As Figure 4 shown in Content a, Figure 4 Content b (FTEM images and EDS spectra), Cu 2-x Se@EcN contained Cu, Se, S, N and P elements. Among them, S, N, and P were from EcN itself, and Cu and Se were from the synthetic substrates added during the EcN culture process. The distribution of Cu and Se elements was highly consistent with the distribution of highly contrasted irregular circular nanostructures in EcN. It indicated that the nanostructures in EcN were mainly composed of Cu and Se elements. The EDS results showed that the atomic ratio of Cu to Se was about 1.7:1.

[0062] As Figure 5 shown in the HRTEM images in Content a, the nanostructures in the Cu 2-x Se@EcN bacteria had clear and ordered lattice structures, and the lattice spacings were about 0.33 nm and 0.20 nm respectively. As Figure 5 shown in the XRD spectra in Content b, the peak spectrum of Cu 2-x Se@EcN matched the standard diffraction spectrum of Cu 2-x Se (JCPDS No.06-0680). The main diffraction peaks (111, 220, 311) of its sample were highly consistent with the XRD characteristic peaks of pure cubic Cu 2-x Se. As Figure 5The XPS spectrum shown in Content c shows that the 2p orbit of Cu is divided into 2p 3 / 2 and 2p 1 / 2 , where a set of peaks located at 930.5 eV and 951.6 eV correspond to Cu(I), and the peaks at 933.5 eV and 953.5 eV belong to Cu(II). The above results indicate that Cu 2-x in CuSe@EcN is in a mixed valence state, including Cu(I) and Cu(II).

[0063] As Figure 6 shown in Content a, at the same dilution ratio, there are no obvious single colonies on the plate of the Cu 2-x Se@EcN group, while a large number of obvious single colonies grow on the plate of the EcN group, indicating that after the intracellular synthesis of Cu 2-x Se nanoparticles in EcN, its proliferation ability is significantly affected. The DAPI / PI live-dead bacteria staining results further verify this conclusion. PI can stain dead bacteria. As Figure 6 shown in Content b, the PI fluorescence of the Cu 2-xS e@EcN group basically overlaps with the DAPI fluorescence, indicating that the Cu 2-xS e@EcN group is basically composed of dead bacteria. As Figure 6 shown in Content c, the hydrodynamic diameter of Cu 2-x Se@EcN does not change significantly within 7 days when stored in PBS or culture medium at 4°C, indicating that Cu 2-x Se@EcN has good storage stability.

[0064] Example 4 Photothermal Performance and Catalytic H2O2 Performance of Cu 2-x Se@EcN 1. Experimental Materials Cu 2-x Se@EcN synthesized in Example 2 2. Experimental Procedures (1) Irradiate the Cu 2 Se@EcN bacterial suspension (Cu concentration is 50 μg / mL) with a 1064 nm laser at different power densities (0.5, 0.8, 1.0 W / cm 2-x ) for 10 min. Use an infrared thermal imager to detect the temperature rise during the experiment (frequency is 1 time / min), and calculate the temperature change value ΔT of each group. Stop irradiating after the bacterial solution temperature stabilizes, wait for it to cool to room temperature and then irradiate again, and detect a total of 5 on-off cycles to evaluate its photothermal stability.

[0065] (2) Mix the MB solution and the H2O2 solution (MB concentration is 1 mg / mL, H2O2 concentration is 1.0 mM), and add different concentrations of Cu 2-xSe@EcN bacterial suspension (Cu concentrations are 0, 10, 25, 50 μg / mL), after reacting for 10 min at room temperature in the dark, the supernatant was collected by centrifugation and its UV-Vis absorption spectrum in the wavelength range of 500 - 800 nm was detected to evaluate the ability of Cu 2-x Se@EcN to produce ROS in vitro.

[0066] (3) The mixture (MB concentration is 1 mg / mL, H2O2 concentration is 1.0 mM, Cu concentration is 10 μg / mL) was reacted for 15 min at room temperature in the dark, with / without laser irradiation (1.0 W / cm 2 ) during the reaction, obtaining the +Laser group and -Laser group. An appropriate amount of the reaction solution was taken every 3 min, centrifuged to collect the supernatant, and its absorbance at 660 nm was detected to evaluate the effect of photothermal assistance on the ability of Cu 2-x Se@EcN to produce ROS in vitro.

[0067] 3. Experimental results As Figure 7 shown in content a~ Figure 7 content c, Cu 2-x Se@EcN has excellent in vitro photothermal heating ability, and during the photothermal cycling process, ΔT is basically the same, indicating that Cu 2-x Se@EcN has good photothermal stability. H

[0068] As Figure 8 shown in content a, the MB solution has an obvious UV-Vis absorption spectrum at 500 - 800 nm; the UV-Vis absorption spectrum of the MB + H2O2 reaction solution has no obvious change; while after adding Cu 2-x Se@EcN, the UV-Vis absorption spectrum of the reaction solution (MB + H2O2 + Cu 2-x Se@EcN) gradually disappears, indicating that Cu 2-x Se@EcN can produce ROS to reduce MB. As Figure 8 shown in content b, the A / A0 decline rate of the +Laser group is faster, indicating that photothermal can promote the ability of Cu 2-x Se@EcN to produce ROS in vitro.

[0069] Example 5 Cu 2-x Se@EcN is taken up and killed by tumor cells H22 1. Experimental materials Cu 2-x Se@EcN, Dead EcN, H22 cells, 3T3 cells, DiO fluorescent dye 2. Experimental procedures According to the synthesis of Cu in Example 2 2-xUnder the conditions of Se@EcN, EcN was cultured without adding substrates. The bacterial solution was collected, centrifuged and washed 3 times with normal saline and then resuspended. The bacterial suspension was placed in a constant temperature water bath at 70 °C for 30 min to obtain Dead EcN.

[0070] (1)Cu 2-x Se@EcN and 1.0×10 -5 mol / L DiO fluorescent dye were incubated at 37 °C in the dark for 30 min, washed 3 times with PBS and resuspended to obtain a DiO-labeled Cu 2-x Se@EcN bacterial suspension. H22 cells were seeded in 12-well plates, and 2 mL of RPMI 1640 complete medium containing DiO-labeled Cu 2-x Se@EcN (Cu concentration was 0.075 μg / mL) was added. After incubation at 37 °C for 0, 4, 8, 12, and 24 h respectively, the medium was discarded, and the cells were collected by centrifugation and washed 2 times with PBS. The experimental steps for 3T3 cells were the same as above. Flow cytometry was used to detect the mean fluorescence intensity of H22 cells and 3T3 cells in each well to evaluate the uptake of Cu 2-x Se@EcN by H22 and 3T3 cells.

[0071] (2)H22 cells were seeded in 96-well plates, and 1***00 μL of RPMI1640 complete medium containing Dead EcN, Cu 2-x Se@EcN (the Cu concentration in Cu 2-x Se@EcN was 0, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2 μg / mL, and the protein concentrations of DeadEcN and Cu 2-x Se@EcN were the same) were added. After co-incubation at 37 °C for 6, 12, and 24 h, the cells were collected by centrifugation and washed 3 times with PBS. 100 μL of RPMI 1640 complete medium was added to each well to resuspend the cells, and then 10 μL of CCK-8 reagent was added. The cells were incubated at 37 °C for 2 h. The experimental steps for 3T3 cells were the same as above. The absorbance (A 450 nm) was measured using a microplate reader to calculate the survival rate to evaluate the killing ability of Cu 2-x Se@EcN on H22 cells.

[0072] 3. Experimental results As Figure 9 shown in Content a, the fluorescence intensity of DiO in H22 cells was significantly higher than that in 3T3 cells, indicating that the uptake ability of H22 cells for Cu 2- x Se@EcN was stronger than that of 3T3 cells. As Figure 9 shown in Content b, Figure 9As shown in content c, there was no significant change in the survival rate of 3T3 cells among different treatment groups; as the concentration of Cu 2-x Se@EcN increased, the survival rate of H22 cells gradually decreased, indicating that Cu 2-x Se@EcN had strong killing effect on tumor cell H22 and weak toxicity on normal cell 3T3.

[0073] Example 6 Photothermal-assisted Cu 2-x Se@EcN killing tumor cell H22 1. Experimental materials Cu 2-x Se@EcN, Dead EcN, H22 cells 2. Experimental procedures (1) Inoculate H22 cells into 96-well plates, add 100 μL of RPMI 1640 complete medium containing PBS, Dead EcN, and Cu 2-x Se@EcN respectively (the Cu concentration in Cu 2-x Se@EcN is 0, 0.05, 0.1, 0.2 μg / mL, and the protein concentrations of Dead EcN and Cu 2-x Se@EcN are the same). After co-incubation at 37°C for 12 h, each well in the laser group is irradiated with a 1064 nm laser (1.0 W / cm 2 2) for 5 min, and then continue to incubate for 4 h. After centrifugation to remove the materials and the original culture medium, wash 3 times with PBS. Add 100 μL of RPMI1640 complete medium to each well to resuspend the cells, and then add 10 μL of CCK-8 reagent. Incubate at 37°C for 2 h, and detect the absorbance (A 450 nm) of each well with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell survival rate.

[0074] (2) Inoculate H22 cells into 48-well plates at a concentration of 6×10 4 cells / well, add 1 mL of 1640 complete medium containing PBS, DeadEcN, and Cu 2-x Se@EcN respectively (the Cu concentration in Cu 2-x Se@EcN is 0.075 μg / mL, and the protein concentrations of Dead EcN and Cu 2- x Se@EcN are 13.35 μg / mL). After incubation for 6 h, each well in the laser group is irradiated with a 1064 nm laser (1.0 W / cm 2 2) for 5 min, and then wash with PBS. Resuspend the washed cells and detect the level of ROS generated by Cu 2-x Se@EcN with / without laser treatment in H22 cells by flow cytometry (FCM).

[0075] 3. Experimental results As Figure 10 shown in Content a, compared with PBS, PBS+Laser, Dead EcN, and Dead EcN+Laser, the proportion of live cells in the Cu 2-x Se@EcN group decreased significantly. After laser irradiation, the survival rate of H22 cells further decreased, indicating that under photothermal assistance, Cu 2-x Se@EcN has stronger killing power against cells. As Figure 10 shown in Content b, the ROS fluorescence intensity of Cu 2-x Se@EcN+Laser was significantly higher than that of other groups, indicating that after laser irradiation, Cu 2-x Se@EcN produced more ROS in H22 cells, so it has stronger killing power against H22 cells.

[0076] Example 7 Cu 2-x Se@EcN induces ICD in tumor cells and stimulates DCs maturation 1. Experimental materials Cu 2-x Se@EcN, Dead EcN, H22 cells, BMDCs cells 2. Experimental procedures (1) According to the experimental procedure (2) of Example 6, incubate H22 cells with 1640 complete medium containing PBS, Dead EcN, and Cu 2-x Se@EcN for 12 h. In the laser group, irradiate each well with a 1064 nm laser (1.0 W / cm 2 ) for 5 min, continue to incubate for 4 h, discard the medium, block the cells with 1% BSA for 30 min, add anti-CRT primary antibody and incubate for 1 h, incubate with Cy3-labeled secondary antibody in the dark for 40 min, and perform fixation, DAPI staining, and mounting according to the experimental procedure (3) of Example 5, and observe the CRT externalization of H22 cells in each group by FCM.

[0077] (2) Repeat step (1), where the final concentration of Cu in Cu 2-x Se@EcN is 0.1 μg / mL, and the protein concentrations of Dead EcN and Cu 2-x Se@EcN are 17.8 μg / mL. Centrifuge the liquid in the wells at 1400 rpm for 5 min, discard the cell pellet, and collect the supernatant. Treat the supernatant with an enhanced ATP detection kit and detect the ATP content released by H22 cells into the extracellular space with a multifunctional microplate reader. Treat the supernatant with an HMGB1 ELISA kit and detect the HMGB1 content released by H22 with an enzyme-linked immunosorbent assay reader.

[0078] (3) After sacrificing BALB / c mice, soak them in 75% alcohol for 5 min, transfer them to a laminar flow hood, dissect the hindlimb tibia and pubis of the mice, remove the surrounding muscle tissue, cut both ends of the leg bones, aspirate a small amount of PBS with a 1 mL sterile syringe, insert the needle into the bone marrow cavity, and flush out the bone marrow cells. Collect the PBS containing bone marrow cells, repeatedly disperse the cells, and then pass through a 200-mesh filter. Centrifuge the cell suspension and discard the supernatant. After lysing the cell pellet with red blood cell lysate for 3 min, centrifuge again, wash once with PBS, and then resuspend with induction medium. Culture at 37°C and 5% CO2 for 5 days, and replace half of the induction medium every two days to obtain immature BMDCs cells. According to the experimental procedure (2) of Example 6, incubate H22 cells with 1640 medium containing PBS, Dead EcN, Cu 2- x Se NPs, Cu 2-x Se@EcN with or without laser irradiation. The final concentration of Cu in Cu 2-x Se@EcN and Cu 2-x Se NPs is 0.1 μg / mL, and the protein concentration of Dead EcN and Cu 2-x Se@EcN is 17.8 μg / mL. Collect the supernatant and add it to a 12-well plate (containing 2×10 5 BMDCs / well) and culture at 37°C for 24 h. After centrifuging to collect the cells, wash and resuspend them, add mouse flow antibodies (CD11c, CD80, CD86, IA-IE), incubate in the dark for 30 min, wash twice with PBS, and finally resuspend the cell pellet with 300 μL PBS and transfer it to a flow tube. Detect the expression levels of surface-related markers of BMDCs in each group by FCM.

[0079] 4. Experimental Results As Figure 11 shown in Content a, Figure 11 Content b, the contents of ATP and HMGB1 released by cells in the Cu 2-x Se@EcN group and the Cu 2-x Se@EcN+Laser group were significantly higher than those in other groups, indicating that Cu 2-x Se@EcN kills cells by generating ROS intracellularly, inducing ICD in tumor cells, and the induction effect of Cu 2-x Se@EcN is stronger under photothermal assistance.

[0080] As Figure 12 shown in Content a, Figure 12 Content b, in the Cu 2-x Se@EcN group and the Cu 2-x Se@EcN+Laser group, CD80 + CD86+ Cells and MHC-II + The proportion of cells was significantly higher than that of other groups, showing a significant induction effect on the maturation of BMDCs.

[0081] Example 8 Cu 2-x Cu 1. Experimental materials Cu 2-x Se@EcN, Dead EcN, Cu 2-x Se NPs, RAW264.7 cells 2. Experimental procedures Mix 250 μL of Tris-HCl and 10 μL of EDTA solution, and then make up the volume to 5 mL with ultrapure water to obtain buffer solution A. Then add 5 mg of lysozyme powder to prepare a working solution (concentration: 1 mg / mL). Resuspend the Cu 2-x Se@EcN precipitate with the working solution with a volume of 1 / 10 of the original bacterial solution volume, and let it stand in a 4°C refrigerator. After the bacterial solution is significantly stratified, centrifuge to discard the supernatant, wash the black-green precipitate, and ultrasonically treat the bacterial suspension with an ultrasonic cell disruptor (power: 300 W, working / rest time: 3 s). After the ultrasonically treated bacterial solution is centrifuged at 8000 rpm for 3 min, collect the black-green supernatant, and then centrifuge at 14000 rpm for 20 min to collect the black-green precipitate, which is Cu 2-x Se NPs.

[0082] RAW264.7 cells are M0-type macrophages; adding LPS with a final concentration of 100 ng / mL and IFN-γ with a final concentration of 20 ng / mL to the bacterial suspension of RAW264.7 cells and culturing for 24 h can obtain M1-type macrophages; adding IL-4 with a final concentration of 20 ng / mL to the bacterial suspension of RAW264.7 cells and culturing for 24 h can obtain M2-type macrophages.

[0083] (1) Seed M0-type macrophages in a 12-well plate, and add 2 mL of DMEM complete medium containing PBS, Dead EcN, Cu 2-x Se NPs, Cu 2-x Se@EcN (the Cu concentration in Cu 2-x Se NPs and Cu 2-x Se@EcN is 0.075 μg / mL, Dead EcN and Cu 2-xThe protein concentration of Cu + Se@EcN was 13.35 μg / mL), and at the same time, IL-4 with a final concentration of 20 ng / mL was added to each well. After incubation at 37 °C for 24 h, the cells were collected, washed twice, and then resuspended. Fixation Buffer fixative was added to the resuspended cell suspension, and fixed at room temperature in the dark for 20 min. After centrifugation to remove the supernatant, it was washed once with Permeabilization Wash Buffer, resuspended with 100 μL Permeabilization Wash Buffer, and stained by adding flow cytometry antibody (CD206) and incubating for 30 min. CD206 was detected by FCM 2-x The proportion of cells in RAW264.7 cells to evaluate the effect of Cu

[0084] Se@EcN on the polarization of M2 macrophages. 2-x (2) M2 macrophages were seeded in 12-well plates, and 2 mL of DMEM complete medium containing PBS, Dead EcN, Cu 2-x Se NPs, Cu 2-x Se@EcN (the Cu concentration in Cu 2-x Se NPs and Cu 2-x Se@EcN was 0.075 μg / mL, and the protein concentration of Dead EcN and Cu 2-x Se@EcN was 13.35 μg / mL) was added. After incubation at 37 °C for 24 h, the cell pellets were collected, washed twice, and then resuspended. Flow cytometry antibodies (CD80 and CD86) were added to the resuspended cell suspension, and incubated at room temperature in the dark for 30 min. Then, flow cytometry antibody (CD206) was added according to step (1) for staining. The expression levels of CD80, CD86, and CD206 on the surface of macrophages were detected using FCM. To evaluate the effect of Cu

[0085] Se@EcN on the reverse polarization of M2 macrophages.

[0086] (3) The supernatants after incubation for 24 h in step (2) were collected, and the supernatants were processed according to the experimental procedures of the ELISA kits for TNF-α, IL-6, and TGF-β respectively. The levels of cytokines secreted by macrophages were detected using an enzyme-linked immunosorbent assay reader. As Figure 13 shown, the proportion of CD206 2-x cells in the Cu + Se@EcN group in RAW264.7 cells was the lowest, indicating that Cu 2- x Se@EcN could inhibit the polarization of M0 macrophages into M2 type in vitro. As Figure 14 Content a~ Figure 14As shown in content c, Cu 2-x The expression levels of M1-related markers CD80 and CD86 on the surface of macrophages in the Cu Figure 14 Se@EcN group were the highest, and the expression level of the M2-related marker CD206 was the lowest. As Figure 14 shown in content d~ 2-x content f, the pro-inflammatory factors TNF-α and IL-6 secreted by M1 macrophages in the Cu 2- x Se@EcN group were significantly higher than those in other groups, and the anti-inflammatory factor TGF-β secreted by M2 macrophages was significantly lower than that in other groups. In summary, the Cu

[0087] Se@EcN group could inhibit the polarization of macrophages into the M2 type in vitro, induce the reverse polarization of M2 macrophages into the M1 type, release pro-inflammatory factors TNF-α and IL-6, and inhibit the release of TGF-β. 2-x Se@EcN accumulates at the tumor site 1. Experimental materials Cu 2-x Se@EcN, male BALB / c mice, IR-780 2. Experimental procedures (1) Incubate Cu 2-x Se@EcN and 0.1 mg / mL IR-780 fluorescent dye in the dark at 37°C for 30 min, wash 3 times with PBS and resuspend to obtain IR-780-labeled Cu 2-x Se@EcN. Subcutaneously inoculate H22 cells (2 million cells / mouse) on the right side of the back near the right hind limb of male BALB / c mice to establish a subcutaneous tumor model of H22-bearing mice. When the tumor volume is about 100 mm 3 ³, inject 100 μL of IR-780-labeled Cu 2-x Se@EcN bacterial suspension (Cu concentration is 4 μg / mL) via the tail vein. Record it as 0 h before injection. Use a live imaging instrument to collect the whole-body fluorescence images of the mice at 0, 0.5, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h after injection and perform semi-quantitative analysis on the fluorescence intensity of the tumor area. After the experiment, sacrifice the mice and collect the heart, liver, spleen, lungs, kidneys, and tumors. Use a live imaging instrument to collect the fluorescence images of the main organs and tumors and perform semi-quantitative analysis.

[0088] 3. Experimental results As Figure 15 shown in content a, Figure 15 content b, at 2 h after injection, Cu began to appear at the tumor site 2-xSe@EcN fluorescence signal. The fluorescence signal increases with the extension of injection time. The fluorescence signal intensity at the tumor site is significantly higher than that of other tissues at 168 h after injection. It shows that Cu 2-x Se@EcN can rapidly target tumor tissues and gradually accumulate after administration.

[0089] Example 10 Inhibitory effect of Cu 2-x Se@EcN on tumor-bearing mice 1. Experimental materials Cu 2-x Se@EcN, male BALB / c mice, picric acid 2. Experimental procedures Establish a subcutaneous tumor model of H22 in tumor-bearing mice according to Example 9. Divide the tumor-bearing mice into 6 groups, namely A: PBS group, B: PBS + Laser group, C: Dead EcN group, D: Dead EcN + Laser group, E: Cu 2-x Se@EcN group, F: Cu 2-x Se@EcN + Laser group. Intravenously inject 100 μL of the corresponding materials into the tumor-bearing mice respectively, and label the mice with picric acid. After 24 h of injection, anesthetize the mice in groups B, D, and F, and irradiate the subcutaneous tumor site with a 1064 nm laser at a power density of 1.0 W / cm 2 for 10 min. Record the body weight, tumor length (L) and width (W) (unit: mm) of the tumor-bearing mice in each group every day, and calculate the tumor volume of the mice according to the tumor volume-time curve (V = LW 2 / 2).

[0090] 3. Experimental results As Figure 16 shown in Content a, Figure 16 Content b, Figure 17 In the early stage of the experiment, Cu 2-x Se@EcN has an inhibitory effect on H22 tumors, but is prone to recurrence; Cu 2-x Se@EcN + Laser can significantly inhibit the growth of H22 tumors, and the tumor inhibitory effect is significantly better than that of other groups.

[0091] Example 11 Immune response of Cu 2-x Se@EcN on tumor-draining lymph nodes (TDLNs), tumor microenvironment (TME), and spleen of tumor-bearing mice 1. Experimental materials Tumor-bearing mice, flow antibodies (CD45, CD11c, CD8, CD80, IA-IB, CD45, CD3ε, CD8a, CD44, CD62L, CD11b, F4 / 80, CD80, CD86) 2. Experimental procedures (1) Group, administer drugs, and perform light treatment on tumor-bearing mice according to Example 9. After sacrificing the mice on the 5th day, dissect the tumor-draining lymph nodes (TDLNs) of each group of mice. Use the piston of a syringe to squeeze the TDLNs up and down to break the tissue and release the cells. Centrifuge and resuspend to obtain a TDLNs cell suspension. Divide the cell suspension into two groups. One group is added with flow antibodies (CD45, CD11c, CD8, CD80, IA-IB), incubated at room temperature in the dark for 30 min, washed, resuspended, filtered, and then transferred to a flow tube. Analyze the maturation of DCs in TDLNs by FCM; the other group is added with flow antibodies (CD45, CD3ε, CD8a), incubated at room temperature in the dark for 30 min, washed, resuspended, filtered, and then transferred to a flow tube. Analyze the proportion of CD8 + T cells in CD3 + T cells.

[0092] (2) Sacrifice the mice according to step (1). Cut the tumor samples into small pieces and digest with collagenase for 40 min. After filtration, centrifugation, and treatment with erythrocyte lysate, centrifuge and resuspend to obtain a tumor cell suspension. Analyze the maturation of DCs in the tumor, the proportion of CD8 + T cells in CD3 + T cells according to step (1). In addition, add flow antibodies (CD11b, F4 / 80, CD80, CD86) to the cell suspension, incubate at room temperature in the dark for 30 min, and then stain with flow antibody (CD206) according to the experimental step (1) of Example 8. Detect the proportion of M1 and M2 macrophages in the tumor by FCM.

[0093] (3) Sacrifice the mice according to step (1). Dissect the spleen samples of each group of mice. After extrusion, filtration, centrifugation, and treatment with erythrocyte lysate, centrifuge and resuspend to obtain a spleen cell suspension. Then add mouse flow antibodies (CD45, CD3ε, CD4, CD8a, CD44, CD62L). Prepare corresponding single-positive tubes and negative tubes separately, incubate at room temperature in the dark for 30 min, wash, resuspend, filter, and then transfer to a flow tube. Detect the proportion of CD8 + T cells in CD3 + T cells, and the proportion of CD44 + in CD8 + CD62L + T cells in CD8

[0094] 3. Experimental results As Figure 18 shown in content a~ Figure 18 content c, in TDLNs, in the Cu 2-x Se@EcN+Laser group, CD86 +Cells and MHC-II + The proportion of cells in DCs and CD8 + The proportion of T cells was significantly higher than that of other groups, indicating that Cu 2-x Se@EcN+Laser could efficiently stimulate the maturation of DCs in TDLNs and increase the proportion of CD8 + T cells.

[0095] As Figure 19 shown in content a~ Figure 19 content e, in TEM, the proportion of CD86 2-x cells and MHC-II + cells in DCs, and the proportion of CD8 + T cells, M1 macrophages (CD80 + CD86 + CD86 + ) were significantly higher than those of other groups; the proportion of M2 macrophages (CD206 + ) was lower than that of other groups, indicating that Cu 2-x Se@EcN could promote the activation of DCs in TME, increase the infiltration of CD8 + T cells, induce the repolarization of M2 macrophages into M1 type, thereby enhancing the anti-tumor immune response.

[0096] As Figure 20 shown in content a, Figure 20 content b, in the spleen, the proportion of CD8 2-x T cells, CD44 + CD62L + memory T cells in the Cu + Se@EcN+Laser group was significantly higher than that of other groups, which could significantly increase the number of CD8 + T cells in the spleens of tumor-bearing mice and increase the number of memory T cells.

[0097] Example 12 Long-term immune protection of Cu 2-x Se@EcN on tumor-bearing mice 1. Experimental materials Tumor-bearing mice, flow antibodies (CD45, CD3ε, CD8a, CD44, CD62L) 2. Experimental procedures Treat tumor-bearing mice according to Group F in the experimental procedure of Example 10. Record the day of administration as Day 0. Select healthy mice of the same age as those in Group F as the control group (Naive) on Day 60. On Day 61, inoculate the two groups of mice according to Group F in the experimental procedure of Example 10. Starting from Day 71, record the length (L) and width (W) (unit: mm) of the tumors of each group of mice every day until Day 84. Calculate the tumor volume of the mice according to the tumor volume-time curve (V = LW 2 / 2). Then sacrifice the mice, remove the spleens, and process them according to the experimental procedure (3) of Example 11. Add flow antibodies (CD45, CD3ε, CD8a, CD44, CD62L) to detect the proportion of CD44 + in CD8 + CD62L - T cells in the spleen.

[0098] 3. Experimental results As Figure 21 shown in Content a~ Figure 21 Content c, compared with untreated mice (Naive), the mice in the Cu 2-x Se@EcN+Laser group had no obvious tumor growth after secondary inoculation with H22 tumor cells, and the proportion of CD44 + CD62L - effector memory T cells in CD8 + T cells increased, indicating that Cu 2-x Se@EcN+Laser has a long-term immune protection effect on tumor-bearing mice.

[0099] Example 13 Biosafety of Cu 2-x Se@EcN 1. Experimental materials Collect the orbital blood, heart, liver, spleen, lung and kidney of the tumor-bearing mice after the experiment in Example 10.

[0100] 2. Experimental procedures Separate the orbital blood for blood biochemical index detection. Fix, embed and section the heart, liver, spleen, lung and kidney, and then perform H&E staining to observe the toxic and side effects of the materials on the organs.

[0101] 3. Experimental results As Figure 22 shown in Content a~ Figure 22 Content f, there was no significant difference in alanine aminotransferase (ALT), aspartate aminotransferase (AST), AST / ALT, blood urea nitrogen (BUN) and creatinine (CERA) in the serum of the mice in the Cu 2-x Se@EcN+Laser group and the body weight of the mice compared with the PBS group, indicating that Cu 2-xSe@EcN+Laser intravenous administration does not produce liver, myocardial, or renal toxicity, has no toxic or side effects on the body, and has good biosafety.

[0102] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application should be included within the protection scope of the present application.

Claims

1. A method for preparing engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles, characterized in that, It includes the following steps: Place probiotics, a selenium source, and a copper source in a medium containing glucose and incubate them in an anaerobic environment. Through redox reactions, Cu 2-x Se nanoparticles are synthesized intracellularly, and the engineered probiotics intracellularly loaded with Cu 2-x Se nanoparticles can be obtained; where x is from 0 to 1.

2. The preparation method according to claim 1, characterized in that, The probiotic is a probiotic with redox ability; Preferably, it is one or more of Bifidobacterium, Lactobacillus, yeast, probiotic Escherichia coli, and probiotic Bacillus spore.

3. The preparation method according to claim 1, characterized in that, The selenium source is one or more of selenite, sodium selenate, selenium dioxide, selenomethionine, selenocysteine, methylselenocysteine, selenium urea, sodium selenosulfate, selenoacetic acid, and selenourea.

4. The preparation method according to claim 1, characterized in that The copper source is a divalent copper salt, selected from one or more of EDTA-Cu(II), copper chloride, copper sulfate, and copper sulfate.

5. The preparation method according to any one of claims 1 to 4, characterized in that, In the incubation system, the concentration of the probiotic is 2×10 9 CFU / mL to 5×10 9 CFU / mL; and / or, The final concentration of the copper source is 2 mmol / L to 4 mmol / L; and / or, The molar ratio of the copper source to the selenium source is (1-2):

1.

6. The preparation method according to claim 1, characterized in that, The medium containing glucose is one or more of glucose buffer medium, MRS medium, MEM medium, and DMEM medium; and / or, The incubation time is 24 h to 48 h.

7. An engineered probiotic with intracellularly loaded Cu 2-x Se nanoparticles prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the engineered probiotics loaded with intracellular Cu 2-x Se nanoparticles in the use or preparation of drugs for reversing polarization of M2-type TAMs.

9. Use of the engineered probiotics loaded with intracellular Cu 2-x Se nanoparticles in the manufacture or preparation of anti-tumor drugs.

10. An anti-tumor drug, characterized in that, It includes the engineered probiotics with intracellularly loaded Cu 2-x Se nanoparticles as described in claim 7; The tumors include one or more of liver cancer, breast cancer, colon cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, ovarian cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma, and glioblastoma.

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