A modified bacterium, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a modified bacterium, its preparation method, and its application. The modified bacterium includes the bacterial matrix and a poorly soluble or insoluble, biologically acceptable metal compound modified on the surface of the bacterial matrix. Upon intratumoral injection, the modified bacteria of this invention can activate an anti-tumor immune response, achieving exceptionally superior anti-tumor therapeutic effects and inhibiting tumor metastasis and recurrence.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a modified bacterium, its preparation method, and its application. Background Technology

[0002] Bacterial therapy for tumors has a long history, but its development has been slow. It wasn't until the 1990s, with the use of BCG for bladder cancer, that bacterial therapy was brought back into the public eye. In recent years, attenuated Salmonella and Listeria, among other attenuated bacteria, have been developed for the treatment of various tumors. Bacterial therapy for tumors is also an extension of immunotherapy, using bacteria as a foreign substance to stimulate an immune response and further inhibit tumor growth. However, most of the bacteria currently used are attenuated live bacteria, which still carry high risks and a narrow safety window in clinical use. Inactivated bacteria, on the other hand, do not achieve the desired immune stimulation effect. Overall, current bacterial therapies for tumors struggle to achieve effective treatment at safe dosages. Summary of the Invention

[0003] The present invention aims to provide a modified bacterium, its preparation method and application, which has good safety and excellent immunostimulatory effect when preparing tumor therapeutic agents.

[0004] This invention involves mixing metal ions with live or inactivated bacteria (initially using attenuated Salmonella) and adjusting the pH of the solution to a weakly alkaline state. This causes insoluble or sparingly soluble metal ion compounds to adhere to the bacterial surface, forming modified bacteria. These modified bacteria are then injected intratumorally into a mouse tumor model, inhibiting tumor growth. Further mechanistic studies revealed that in situ injection of the modified bacteria elicits an anti-tumor immune response, thus achieving a therapeutic effect on tumors. When divalent metal ions are placed under alkaline conditions, they readily form hydroxides and precipitate. The bacterial surface, as a typical solid-liquid interface, provides nucleation sites for these precipitates, further promoting their precipitation. Once precipitated on the bacterial surface, the hydroxides are transformed into more stable oxides. With continuous precipitation and transformation of hydroxides, an oxide layer forms on the bacterial surface, resulting in inactivated bacteria modified with metal compounds. The deposition reaction described in this application involves mixing some metal ion salts with bacteria, introducing corresponding anions, and under suitable pH conditions, the metal ions and anions form sparingly soluble or insoluble metal compounds that attach to the surface of the bacteria. Under thorough stirring, the target product can continuously settle on the surface of the bacteria in the liquid, and finally, the bacteria with attached metal compounds form a uniform and stable suspension.

[0005] Further research revealed that, in addition to attenuated Salmonella, this technology can also be used for other types of bacteria, including Staphylococcus aureus, Escherichia coli, and Lactobacillus. Modified bacteria prepared using the same method from these bacteria can also activate the immune system and achieve good anti-tumor activity.

[0006] Further investigation was conducted to determine whether other sparingly soluble or insoluble metal compounds, besides metal hydroxides or oxides, could form an adhesion layer on bacterial surfaces and achieve similar immunostimulatory results when their solubility is reduced. Through experiments attempting to synthesize inactivated bacteria with various metal compounds attached, it was found that some metal compounds, after binding to the bacterial surface, could also activate immune cells, induce a strong anti-tumor immune response, and potentially produce an immune memory effect, reducing the probability of cancer metastasis and recurrence.

[0007] According to the technical solution of the present invention, the modified bacteria include the bacterial body and a poorly soluble or insoluble biologically acceptable metal compound modified on the surface of the bacterial body.

[0008] Furthermore, the bacterial organism is selected from one or more of Salmonella, Staphylococcus aureus, Escherichia coli, Lactobacillus, attenuated strains of Salmonella, attenuated strains of Staphylococcus aureus, attenuated strains of Escherichia coli, and attenuated strains of Lactobacillus.

[0009] Furthermore, the bacterial organism is either a live bacterium or an inactivated bacterium, preferably an inactivated bacterium, and both the live bacterium and the inactivated bacterium contain attenuated bacteria.

[0010] Furthermore, the metal compound is modified onto the surface of the bacterial host through a deposition reaction to form an attachment layer.

[0011] Furthermore, the cation of the metal compound is selected from one or more of zinc, calcium, copper, iron, manganese, and magnesium; the anion is selected from one or more of carbonate, hydroxide, sulfide, and phosphate.

[0012] Furthermore, the metal compound is selected from one or more of zinc carbonate, calcium carbonate, copper carbonate, magnesium carbonate; zinc hydroxide, iron hydroxide, copper hydroxide, manganese hydroxide, magnesium hydroxide, zinc sulfide, copper sulfide, manganese sulfide; zinc phosphate, calcium phosphate, copper phosphate, iron phosphate, magnesium phosphate, and manganese phosphate.

[0013] A second aspect of the present invention provides a method for preparing the above-described modified bacteria, comprising the following steps:

[0014] S1: Preparation of bacterial suspension;

[0015] S2: Add a soluble metal salt solution to the bacterial suspension;

[0016] S3: Add a readily soluble aqueous solution of hydroxide, carbonate, or phosphate to a pH of 8-12, or add a readily soluble aqueous solution of sulfide, and react to obtain the modified bacteria.

[0017] Specifically, in step S2,

[0018] Add an easily soluble aqueous hydroxide solution to a pH of 8-12, and centrifuge after reaction to obtain modified bacteria with metal hydroxides or metal oxides attached to their surface; the easily soluble aqueous hydroxide solution is preferably a sodium hydroxide aqueous solution;

[0019] Add an aqueous solution of readily soluble carbonate to a pH of 8-12, and centrifuge after reaction to obtain modified bacteria with metal carbonates attached to their surface; the readily soluble carbonate aqueous solution is preferably a sodium carbonate aqueous solution;

[0020] Add a readily soluble phosphate aqueous solution to a pH of 8-12, and centrifuge after reaction to obtain modified bacteria with metal phosphates attached to their surface; the readily soluble phosphate aqueous solution is preferably a sodium phosphate aqueous solution;

[0021] Add an aqueous solution of readily soluble sulfide salt, react and centrifuge to obtain modified bacteria with metal sulfides attached to their surface; the readily soluble sulfide salt aqueous solution is preferably a sodium sulfide aqueous solution;

[0022] Furthermore, in step S2, a soluble metal salt solution is added to the bacterial suspension at a rate of 0.2-13.5 mmol of metal ions per billion bacteria.

[0023] A third aspect of the present invention provides a modified bacterial lyophilized powder, comprising a lyophilization protectant and the modified bacteria described above.

[0024] Furthermore, the freeze-drying protectant is selected from one or more of sucrose, trehalose, inositol, sucrose, inulin, dextran, maltodextrin, malt polysaccharide, and 2-hydroxypropyl-β-cyclodextrin.

[0025] Furthermore, the volume fraction of the freeze-drying protectant in the bacterial freeze-dried powder is 0.1-20%, preferably 1-5%.

[0026] A fourth aspect of the present invention provides the use of the above-described modified bacteria or the above-described modified bacterial lyophilized powder in the preparation of tumor therapeutic agents.

[0027] Compared with the prior art, the technical solution of the present invention has the following advantages: The present invention provides a modified bacterium, which can be used to inactivate bacteria, and the metal compounds on its surface can be metabolized by decomposition into ionic state, thus having better safety.

[0028] The modified bacteria provided by this invention have surface-modified insoluble metal compounds that neutralize the weakly acidic microenvironment of tumors, thereby enhancing the activity of immune cells at the tumor site, reducing tumor drug resistance, and improving therapeutic efficacy.

[0029] This invention provides a modified bacterium that can activate immune cells through multi-pathway stimulation, induce a strong anti-tumor immune response, and produce an immune memory effect to reduce the probability of cancer metastasis and recurrence, making it a novel immune agonist. Attached Figure Description

[0030] Figure 1 Scanning electron microscope images of modified inactivated bacteria with different metal compounds attached;

[0031] Figure 2 Scanning electron microscope images of different bacteria before and after modification with manganese dioxide;

[0032] Figure 3 The relative activity statistics of CT26 cells after co-incubation with different samples for 12 hours are shown in the figure.

[0033] Figure 4 A statistical graph showing the proportion of mature cells after co-incubation of bone marrow-derived dendritic cells with different samples by flow cytometry.

[0034] Figure 5 The image shows the tumor growth curves in a mouse tumor model after treatment with bacteria modified with different metal compounds.

[0035] Figure 6 This is a statistical graph showing the bioluminescent signal intensity of reporter gene expression after STING pathway activation in different groups of reporter cells after 24 hours of incubation with different samples.

[0036] Figure 7 Survival curves of mice with subcutaneous colon cancer tumors who were cured by inactivated Salmonella modified with manganese dioxide and then re-inoculated with the same tumor on day 60. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] Example A: Preparation and basic morphological characterization of bacteria modified with different metal compounds

[0039] Example A1: Preparation of bacteria with zinc carbonate-modified surface

[0040] S1: Prepare an aqueous solution of zinc sulfate;

[0041] S2: After amplification culture, attenuated Salmonella (hereinafter referred to as FS) was centrifuged to obtain bacterial cells and washed to remove residual culture medium and other substances from the bacterial cells. Paraformaldehyde was then added to the bacterial cells for bacterial inactivation and morphological fixation. The fixed inactivated bacteria were collected by centrifugation and washed twice with sterile physiological saline. Finally, the inactivated bacterial cells were resuspended in physiological saline to a concentration of 24 MCF. The suspension was tested to confirm that there were no live bacteria in the suspension and it could be stored at low temperature for later use.

[0042] S3: Add 1 mL of 10 mM zinc sulfate solution and an appropriate amount of physiological saline to the inactivated bacterial suspension (original bacterial suspension turbidity is 24 MCF, approximately 7.2 billion bacteria / mL, volume is 1 mL), stir briefly at room temperature, then add sodium carbonate solution to make the pH 8-12, continue stirring for at least 1 hour, centrifuge to collect the precipitate, wash twice with sterile physiological saline, and finally resuspend in sterile physiological saline and store at low temperature. The product is ZnCO3@FS.

[0043] In the experiment, different initial concentrations and volume ratios of bacterial suspensions and metal ion salt solutions were added to explore the range of feed ratios that could form metal compound-modified bacteria. With the total number of bacteria in the reaction system fixed, the feed ratio for a relatively stable suspension was explored by adjusting the amount of metal ion salt. The results showed that when the bacterial count was 1 billion and the metal ion concentration exceeded 13.5 mmol, regardless of adjustments to the pH, concentration, and reaction time, the product remained an unsustainable precipitate, unsuitable for subsequent applications. Lower concentrations of the metal ion salt did not affect the resuspension of the product. The lowest feed ratio tested was 0.2 μmol of metal ions per 1 billion bacteria.

[0044] Example A2: Preparation of bacteria with surfaces modified with ferric hydroxide or copper hydroxide

[0045] The soluble iron ion salt solution and the inactivated bacterial suspension were prepared as described in S1-S2 of Example A1; the difference is:

[0046] S3: Add 10 mM ferric chloride or copper sulfate solution and an appropriate amount of physiological saline to the inactivated bacterial suspension (the original bacterial suspension has a turbidity of 24 MCF, approximately 7.2 billion bacteria / mL, and the sample volume here is 1 mL). Stir briefly at room temperature, then add sodium hydroxide solution or ammonia to adjust the pH to 8-12, continue stirring for at least 1 hour, centrifuge to collect the precipitate, wash twice with sterile physiological saline, and finally resuspend in physiological saline and store at low temperature. The products are Fe(OH)3@FS and Cu(OH)2@FS.

[0047] Example A3: Preparation of modified bacteria with surface-coated manganese sulfide, zinc sulfide, and copper sulfide

[0048] S1: Prepare an aqueous solution of manganese chloride, copper sulfate, and zinc sulfate with a concentration of 10 mM;

[0049] S2: After amplifying and culturing the attenuated Salmonella, wash to obtain bacterial cells without culture medium. Do not use formaldehyde to treat the bacteria, and directly resuspend them in physiological saline to obtain a bacterial suspension of live bacteria.

[0050] S3: Add 10mM manganese chloride, copper sulfate, and zinc sulfate solutions, along with an appropriate amount of physiological saline, to the bacterial suspension (original bacterial suspension turbidity is 24 MCF, approximately 7.2 billion bacteria / mL, sample volume here is 1 mL). Stir at room temperature, then add sodium sulfide solution and continue stirring. Centrifuge to collect the precipitate, wash twice with sterile physiological saline, and finally resuspend in physiological saline to obtain MnS@FS, CuS@FS, and ZnS@FS, respectively, and store at low temperature.

[0051] Example A4: Basic Characterization of Modified Bacteria

[0052] The products from Examples A1-A3 were used to prepare scanning electron microscopy (SEM) samples. The surface morphology of bacteria modified with different metal compounds was characterized using SEM, and the results are as follows: Figure 1 As shown in the image, ZnCO3@FS, Fe(OH)3@FS, Cu(OH)2@FS, MnS@FS, ZnS@FS, and CuS@FS represent scanning electron microscope (SEM) images of Salmonella bacteria whose surfaces were modified with zinc carbonate, iron hydroxide, copper hydroxide, manganese sulfide, zinc sulfide, and copper sulfide, respectively. The results show that the bacterial surfaces modified with metal compounds all had a large amount of solid adhering to them, and there was not much solid residue in the environment. Furthermore, the morphology of the bacteria changed after different metal compounds were applied, depending on the crystal form of the metal compounds themselves, indicating that during the preparation process, the metal compounds adhered to the bacterial surface to form a coating layer.

[0053] Example A5: Preparation of other modified bacteria

[0054] Bacterial suspensions of *Escherichia coli* (FE), *Staphylococcus aureus* (F.SA), *Salmonella VPN20009* (FV), and *Lactobacillus* (FL) were prepared according to any one of steps S1-S2 in Examples A1-A3, and then modified with surface metal compounds, specifically:

[0055] S3: To prepare a manganese chloride solution, 1 mL of a 10 mM manganese chloride solution was added to a bacterial suspension (original bacterial suspension turbidity 24 MCF, approximately 7.2 billion bacteria / mL, volume 1 mL), along with an appropriate amount of physiological saline. The suspension was stirred at room temperature, then sodium hydroxide solution or ammonia was added to adjust the pH to 8-12. Stirring continued, and the precipitate was collected by centrifugation. The precipitate was washed twice with sterile physiological saline and finally resuspended in physiological saline. The morphology of different bacteria modified with manganese compounds was characterized using scanning electron microscopy. The results are as follows: Figure 2 As shown, FE represents *Escherichia coli*, F.SA represents *Staphylococcus aureus*, FS represents *Salmonella*, FL represents *Lactobacillus*, and FV represents *Salmonella* VNP20009. Bacteria modified with manganese dioxide are indicated by the prefix M@. Scanning electron microscopy images show a layer of material adhering to the bacterial surface, while the morphology and size of the bacteria themselves remain largely unchanged, indicating that various bacteria can form modified bacteria through the attachment of metal compounds.

[0056] Example B: Cell Experiment

[0057] Example B1: Relative cell viability of CT26 cells (mouse colon cancer cell line) after incubation with different samples

[0058] CT26 cells were at 10 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight at 37°C. Once cells were adherent and in good condition, different concentrations of samples prepared in Examples A1-A3 (containing inactivated, attenuated Salmonella bacteria) were added and co-incubated at 37°C. Bacteria modified with different metal compounds were introduced at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL. The total bacterial count in the unmodified bacterial group was the same as that in the modified bacterial group. After 12 hours of incubation, the culture medium containing the samples was removed, excess samples were washed away with PBS, and relative cell viability was detected using the MTT assay. The results are shown below. Figure 3 As shown (data for each concentration from left to right: FS, MnS@FS, CuS@FS, Cu(OH)2@FS, ZnS@FS, Fe(OH)3@FS, ZnCO3@FS). At low concentrations, the modified bacteria did not exhibit high cytotoxicity, and cells co-incubated with the modified bacteria still retained more than 80% viability. With increasing concentration, the modified bacteria gradually showed an effect on relative cell viability, and when the concentration reached 40 μg / mL, cell viability was still greater than 50%. This indicates that the safety of the modified bacteria is concentration-dependent, exhibiting high safety within a certain concentration range.

[0059] Example B2: Experiments on the maturation of myeloid dendritic cells stimulated by different samples

[0060] Dendritic cells (DCs) are highly efficient antigen-presenting cells in the body. Upon stimulation by certain factors or after taking up antigens, they differentiate into mature DCs. Mature DCs can effectively activate naive T cells, induce the generation of cytotoxic T lymphocytes, and secrete tumor necrosis factor-α (TNF-α), playing a crucial role in anti-tumor immune responses. Therefore, samples that can effectively stimulate DC maturation can enhance the body's anti-tumor immune response.

[0061] Bone marrow-derived stem cells were extracted from the bone marrow of C57BL / 6 mice. Colony-stimulating factor (GM-CSF) was added to promote the differentiation of stem cells into bone marrow-derived dendritic cells (BMDCs). BMDCs were then compared with different modified bacterial samples (each sample contained 3.6 x 10⁻⁶ bacteria). 7 The corresponding bare metal compound content was 1.5-2.7 μg. In the other groups without bacteria, the amount of the same metal compound was the same as that in the bacterial group modified with the same metal compound. For example, in the ZnS and ZnS@FS groups, the same amount of ZnS was used for co-incubation. After 12 hours, the maturity rate of BMDCs in different groups was detected.

[0062] The statistical results of the maturity ratio of BMDCs are shown in Figure 4 As shown in the diagram. Each experimental group represents a different substance added to the cell culture medium; the specific substances are as follows:

[0063] blank: blank control group;

[0064] FS: Unmodified inactivated bacteria; ZnS: Zinc sulfide suspension; ZnCO3: Zinc carbonate suspension; Fe(OH)3: Ferric hydroxide colloid; CuS: Copper sulfide suspension; Cu(OH)2: Copper hydroxide suspension; MnS: Manganese sulfide suspension;

[0065] ZnS@FS: Salmonella suspension modified with zinc sulfide; ZnCO3@FS: Salmonella suspension modified with zinc carbonate; Fe(OH)3@FS: Salmonella suspension modified with iron hydroxide; CuS@FS: Salmonella suspension modified with copper sulfide; Cu(OH)2@FS: Salmonella suspension modified with copper hydroxide; MnS@FS: Salmonella suspension modified with manganese sulfide.

[0066] Based on the statistical analysis of the DC cell maturation rate, it can be seen that the modified bacteria can better stimulate the maturation of BMDCs. The results of most groups are significantly different from the blank control group, and some experimental groups even obtained results that are better than the positive control group (2 μg / mL lipopolysaccharide, LPS). This indicates that the modified bacteria of this invention have a very good ability to stimulate DC cell maturation.

[0067] Example C: Animal Experiment

[0068] Example C1: Therapeutic effect of bacteria modified with different metal compounds on a mouse colon cancer tumor model

[0069] A tumor model was established by inoculating colon cancer tumor cells into the back of BALB / c mice, and the tumor size was increased to 120 mm. 3 Mice were randomly divided into groups of six, with different groups receiving treatment with bacteria modified by different metal compounds. Treatment was administered via intratumoral injection. The bacterial dosage was kept constant across all groups at 1.8 x 10⁻⁶. 10 The number of bacteria / kg body weight was determined by the varying binding efficiency of each metal compound to the bacteria. Metal compound doses ranged from 0.75 mg / kg to 1.35 mg / kg body weight, and metal ion concentrations ranged from 300 μg / mL to 1.08 mg / mL. Tumor volume changes were recorded and tumor growth curves were plotted during the study. The results are as follows: Figure 5 As shown in Table 1, the survival rate of mice and the tumor inhibition rate were recorded simultaneously. When mice were treated with a simple metal compound (with the same dosage as the metal compound in the bacterial group modified with that metal compound, for example, the ZnS and ZnS@FS groups contained equal amounts of ZnS), some metal compounds exhibited certain toxicity, initially slowing tumor growth but failing to eliminate the tumor or inhibit its growth in later stages. Groups that did not show significant toxicity also did not show significant tumor growth inhibition; the tumor inhibition rate on day 19 was less than 20%, and these were considered as not achieving effective tumor treatment.

[0070] Table 1: Survival rate and tumor inhibition rate on day 19 post-inoculation in mice treated with bacteria modified with different metal compounds.

[0071]

[0072] Figure 5The results in Table 1 show that bacteria modified with certain metal compounds can inhibit tumor growth. After treatment with bacteria coated with different metal compounds, the increase in tumor volume in mice slowed significantly, and the survival rate increased significantly, reaching up to 2 / 3 survival, with tumor inhibition rates generally exceeding 60%. In summary, this demonstrates that bacteria modified with most of the metal compounds of this invention can achieve tumor treatment effects, while bacteria modified with Fe hydroxide did not show a significant tumor-inhibiting effect. This indicates that it is difficult to predict which metal compounds will inhibit tumor growth and how to select the metal compounds to modify the bacterial surface.

[0073] Example D: Study on the immunostimulatory mechanism of modified bacteria

[0074] Example D1: Demonstrating the activation of the STING pathway by modified bacteria at the cellular level

[0075] The modified bacteria of this invention exhibit multi-pathway stimulation effects. This embodiment uses Salmonella modified with a manganese compound (manganese dioxide) as an example; other metal ions have their corresponding immunostimulatory mechanisms. This embodiment utilizes cells containing a reporter gene regulated by the STING pathway (STING...). + The STING pathway in this cell is activated upon stimulation, which in turn activates the expression of the reporter gene luciferase, catalyzing the bioluminescent signal emitted by the luciferase substrate. The higher the degree of STING activation, the stronger the bioluminescent signal. This cell was then compared with bacteria modified with manganese dioxide (M@FS), manganese dioxide (MnO2), Salmonella (FS), and manganese chloride (MnO2). 2+ The cells were co-incubated with the positive control (PC) group. After 24 hours, luciferase substrate was added, and the bioluminescence signal intensity was measured. The bioluminescence signal intensity of each group was then compared with that of the PBS group. Null cells do not express the STING pathway and therefore cannot induce luciferase expression by activating the STING pathway. The incubation of these cells with different samples demonstrated that the samples and reagents themselves could not interfere with the bioluminescence signal or induce luciferase expression. The bioluminescence signal intensity ratio statistical graph is shown below. Figure 6 As shown, the STING pathway was significantly activated in cells co-incubated with the modified bacteria compared to the control group; the STING activation level was similar to that of the positive control (PC), indicating that the modified bacteria can effectively activate the STING pathway.

[0076] Example D2: Cell experiments demonstrate that modified bacteria can activate the TLR4 pathway.

[0077] In this embodiment, cells containing reporter genes regulated by the TLR4 pathway (TLR4+, TLR4-positive cells) were used. When TLR4 is stimulated and activated, it activates the expression of the reporter gene luciferase, which catalyzes the luciferase substrate to generate a bioluminescent signal. These cells were co-incubated with different samples (n=3). After incubation, the bioluminescent signal intensity was measured after adding the luciferase substrate. The bioluminescent signal intensity of each group was then compared with that of the PBS group to determine the degree of TLR4 activation; a higher ratio indicates a higher degree of TLR4 activation. TLR4-negative cells do not express TLR4 and therefore are not stimulated to express luciferase. The addition of the luciferase substrate did not produce bioluminescence. This group of cells demonstrated that none of the reagents or samples used in the experiment interfered with the bioluminescent signal or induced luciferase expression.

[0078] The bioluminescence signal intensity ratios of the three parallel samples in different groups are shown in Table 2. The positive control group, co-incubated with 3 μg / mL MPLA, showed a significant TLR4 pathway stimulation effect. The modified bacteria (MnO2@FS) significantly stimulated TLR4 compared to the blank control group (Blank), as evidenced by a fluorescence intensity ratio greater than 1. Compared to the simple bacteria (FS), the modified bacteria exhibited stronger fluorescence intensity, indicating that the modified bacteria could better stimulate the TLR4 pathway. This demonstrates that the modified bacteria of this invention have a TLR4 agonist-like effect.

[0079] Table 2: Statistical table of bioluminescence intensity ratios after adding luciferase substrate to cell culture media of different groups

[0080]

[0081] The activation of STING and TLR4 indicates that the modified inactivated bacteria of this invention effectively activate the innate immune system, helping to relieve immunosuppression in the tumor microenvironment and thus enhancing the body's anti-tumor immune response. Combining Examples D1 and D2, the modified bacteria of this invention have the effect of multi-pathway agonists, simultaneously enhancing both non-specific and anti-tumor immune responses, thus contributing to enhanced anti-tumor immunotherapy.

[0082] Example E: Therapeutic experiments on colon cancer tumor models using different bacteria modified with metal compounds.

[0083] Grouping:

[0084] Group 1: Blank: Blank control group;

[0085] Group 2: F.SA: Inactivated Staphylococcus aureus;

[0086] Group 3: FE: Inactivated Escherichia coli;

[0087] Group 4: FL: Inactivated Lactobacillus;

[0088] Group 5: MnO2@F.SA: Inactivated Staphylococcus aureus modified with manganese dioxide;

[0089] Group 6: MnO2@FE: Inactivated Escherichia coli modified with manganese dioxide;

[0090] Group 7: Inactivated lactobacilli modified with MnO2@FL manganese dioxide;

[0091] Group 8: Inactivated Salmonella modified with manganese dioxide using MnO2@FS.

[0092] Different types of bacteria were prepared into modified bacteria according to the preparation method in Example A5, and used in a mouse subcutaneous colon cancer model treatment experiment. The tumor inhibition rate of each group on day 17 after inoculation was calculated. The tumor inhibition rate of the blank control group was 0, and the tumor inhibition rate results of other groups are shown in Table 3. The results showed that different types of modified inactivated bacteria could effectively inhibit tumors, with a tumor inhibition rate more than 5 times that of bacterial treatment alone. This indicates that the modified bacteria of the present invention can all achieve tumor treatment.

[0093] Table 3: Tumor inhibition rate of each group on day 17 after inoculation in mouse experiments with manganese-modified bacteria.

[0094] Group F.SA FE FL FS Tumor inhibition rate 14.58％ 12.55％ 17.22％ 10.23％ Group <![CDATA[MnO2@F.SA]]> <![CDATA[MnO2@F.E]]> <![CDATA[MnO2@F.L]]> <![CDATA[MnO2@F.S]]> Tumor inhibition rate 67.08％ 62.55％ >100％ >100％

[0095] Example F: Anti-tumor immune memory effect produced by the body after modified bacteria treat tumors

[0096] To verify the vaccine efficacy of the modified bacteria of this invention, a mouse colon cancer tumor model was established, and the mice were treated with attenuated Salmonella modified with manganese dioxide. The subcutaneous tumors in the mice eventually disappeared completely. On day 60 after treatment, the proportion of memory T cells in the peripheral blood of the mice was analyzed by flow cytometry. In mice whose tumors were cured by the modified bacteria, the average proportion of memory T cells among all T cells in peripheral blood leukocytes was 83.86%, which was significantly higher than that of the blank control group (where the average proportion of memory T cells among all T cells in peripheral blood leukocytes was 59.5%). This indicates that the modified bacteria of this invention can induce the production of memory T cells and generate an immune memory effect.

[0097] Simultaneously, the cured mice were re-inoculated with the same tumor cells, and the tumor survival status of the mice was observed. The mouse survival curve is shown in Figure 1. Figure 7As shown, mice cured by the modified bacteria did not show significant cancer recurrence after being inoculated with tumor cells again and still had a relatively long survival period. In other words, the modified bacteria can inhibit tumor recurrence and produce a vaccine-like effect.

[0098] Example G: Study on Lyophilization Protectants for Modified Bacteria

[0099] The modified bacterial suspensions obtained in Examples A1-A3 and Example A5 were mixed with various lyophilization protectants and then lyophilized. The state of the lyophilized suspensions and whether they could be redispersed into bacterial suspensions after the addition of solvents were observed. The observation results of lyophilization of manganese sulfide-modified Salmonella with different proportions of lyophilization protectants are recorded in Table 4. The lyophilization protectants and proportions of samples that can be redispersed are defined as the usable range. The lyophilization protectants and corresponding proportions used for samples with good lyophilized morphology and the ability to be redispersed into suspensions are defined as the preferred range.

[0100] Sucrose and β-cyclodextrin can enable modified bacteria to have good freeze-drying effects. In addition to sucrose and β-cyclodextrin, trehalose, inositol, sucrose, inulin, dextran, maltodextrin, and malt polysaccharide can all enable the samples to be resuspended after freeze-drying without changing their properties. However, lactose and mannitol cannot achieve effective freeze-drying protection.

[0101] Table 4: Freeze-drying protection conditions and state records after freeze-drying of MnS@FS

[0102]

[0103]

[0104] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A modified bacterium, characterized in that, It consists of the bacterial body and a poorly soluble or insoluble biologically acceptable metal compound modified on the surface of the bacterial body; The metal compound is selected from one of zinc carbonate, copper hydroxide, zinc sulfide, copper sulfide, manganese sulfide, and manganese dioxide; the bacterial culture is selected from an inactivated attenuated Salmonella strain. The metal compound is deposited onto the surface of the bacterial host to form an attachment layer.

2. A modified bacterium, characterized in that, It consists of the bacterial body and a poorly soluble or insoluble biologically acceptable metal compound modified on the surface of the bacterial body; The metal compound is manganese dioxide, and the bacterial organism is selected from one of inactivated Salmonella, inactivated Staphylococcus aureus, inactivated Escherichia coli, and inactivated Lactobacillus. The metal compound is deposited onto the surface of the bacterial host to form an attachment layer.

3. A method for preparing the modified bacteria according to any one of claims 1-2, characterized in that, Includes the following steps, S1: Preparation of bacterial suspension; S2: Add a soluble metal salt solution to the bacterial suspension; specifically, add a soluble metal salt solution to the bacterial suspension at a rate of 0.2-13.5 mmol of metal ions per billion bacteria. S3: Add an aqueous solution of readily soluble hydroxide or carbonate to a pH of 8-12, or add an aqueous solution of readily soluble sulfide, and react to obtain the modified bacteria.

4. A modified bacterial freeze-dried powder, characterized in that, Includes lyophilization protectants and modified bacteria as described in any one of claims 1-2.

5. The modified bacterial freeze-dried powder as described in claim 4, characterized in that, The freeze-drying protectant is selected from one or more of sucrose, trehalose, inositol, sucrose, inulin, dextran, maltodextrin, malt polysaccharide, and 2-hydroxypropyl-β-cyclodextrin.

6. The modified bacterial freeze-dried powder as described in claim 4, characterized in that, The volume fraction of the freeze-drying protectant in the bacterial freeze-dried powder is 0.1-20%.

7. The use of the modified bacteria as described in any one of claims 1-2 or the lyophilized powder of the modified bacteria as described in any one of claims 4-6 in the preparation of intratumoral injection formulations for the treatment of colon cancer.

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