CN114657098A

CN114657098A - Modified microorganism, modification method and application thereof, and antitumor drug

Info

Publication number: CN114657098A
Application number: CN202210309278.1A
Authority: CN
Inventors: 张晗; 李田忠; 谢中建
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-06-24

Abstract

The invention discloses a modified microorganism, a modification preparation method and application thereof and an anti-tumor medicament, wherein the microorganism in the modified microorganism is specifically modified by a metal organic framework material, and the microorganism is an anaerobic or facultative anaerobic microorganism with anti-tumor activity; the modified microorganism can be used for treating tumor, can be enriched in tumor, can realize the combination of photodynamic therapy and immunotherapy, and has good tumor treatment effect, low toxicity and high safety.

Description

Modified microorganism, modification method and application thereof, and antitumor drug

Technical Field

The invention relates to the technical field of biomedicine, in particular to a modified microorganism, a modification method and application thereof and an anti-tumor drug.

Background

Cancer is a serious threat to human life and health, and effective treatment methods are still lacking at present. Colorectal cancer (CRC) ranks third in the world's high incidence cancer list with a mortality rate of about 10%. In developed countries, especially in europe, north america, australia and new zealand, the frequency of CRC causes is very high. The progression of the cancer is closely related to dietary habits, living conditions and environmental factors. Although early diagnosis and early treatment reduce the likelihood of tumor progression and recurrence, the efficacy of conventional anti-tumor approaches such as chemotherapy, radiation therapy and surgery remains less than satisfactory, particularly for malignant metastatic cancers. In order to effectively and chronically eliminate solid tumors and inhibit tumor metastasis, it is necessary and urgent to develop new cancer therapeutic drugs.

Nano materials have been widely used for drug delivery and treatment of chronic diseases due to their excellent drug-loading capabilities. Doxorubicin (DOX) -entrapped liposomes and poly (lactic-co-glycine) (PLGA) are typical formulations approved by the United states drug administration and have been successfully marketed and commercialized. However, drug delivery still has many defects, such as reduced tumor targeting, low accumulation of Tumor Microenvironment (TME), and poor drug release, which prevent the wide clinical application of traditional nanomaterials. A Metal Organic Framework (MOF) belongs to a porous nanostructure, the MOF is directly used for tumor treatment in the existing research, but the MOF cannot actively target the tumor hypoxia environment, and the problem of poor curative effect exists.

Although pathogenic bacteria are harmful to human health, some nonpathogenic strains have unique advantages in cancer treatment and are used for tumor treatment, for example, lactic acid bacteria and escherichia coli can be used for antitumor treatment in the prior art, but the treatment effect is not obvious, the tumor is eliminated slowly and the recurrence is easy.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a modified microorganism, a modification preparation method and application thereof, and an anti-tumor medicament.

In a first aspect of the invention, a modified microorganism is presented, the microorganism being modified with a metal organic framework material, the microorganism being an anaerobic or facultative anaerobic microorganism with anti-tumor activity.

The antitumor activity comprises inhibiting tumor cell proliferation, inducing tumor cell apoptosis, metabolizing to generate substances capable of inhibiting tumor cell proliferation or inducing tumor cell apoptosis, and stimulating tumor immune response of a host.

The microorganism modified according to the embodiment of the present invention has at least the following beneficial effects: the modified microorganism can be used for treating tumors, specifically, the microorganism with anti-tumor activity is usually anaerobic or facultative anaerobic microorganism, the interior of the tumor is naturally anoxic, the microorganism can be enriched in the anoxic part in the tumor, meanwhile, the modified metal organic framework material on the microorganism is carried into the tumor and released, tumor specific ROS can be generated based on the modified metal organic framework material, the modified microorganism has better infrared photodynamic therapy capability on the tumor, and the modified microorganism can activate cancer cell antigens generated by infrared photodynamic to trigger in-vivo innate immunity and specific immunity, so that the photodynamic therapy and the immunotherapy are combined, and the tumor treatment effect is good; and the modified microorganism has low toxicity and high safety.

In some embodiments of the invention, the microorganism is selected from at least one of lactococcus lactis, escherichia coli, attenuated salmonella. Preferably, the microorganism is selected from lactococcus lactis. If the microorganism is selected from the group consisting of lactococcus lactis, the lactococcus lactis modified by the metal organic framework material can spontaneously enrich in tumors through the circulatory system, and the lactococcus lactis modified by the metal organic framework material can be used for photodynamic therapy under hypoxic conditions in tumors based on the modification of the metal organic framework material, and the modified lactococcus lactis can trigger a series of immune responses including natural immunity and T cell immunity of tumors, form an inflammatory tumor environment, and generate long-term immunity to the tumors. In addition, the microorganism can also adopt Escherichia coli, and the Escherichia coli modified by the metal organic framework material can be used for photodynamic therapy under tumor hypoxia condition, can trigger macrophage innate immunity and T cell adaptive immunity similar to lactococcus lactis, can form inflammatory tumor environment, and can generate long-term immunity to tumors.

In the process of photodynamic therapy of microorganisms modified by the metal organic framework material under the anoxic condition in tumors, the metal organic framework material can be used as a photosensitizer. In some embodiments of the invention, the metal in the metal-organic framework material is a transition metal. The size of the metal organic framework material can be controlled to be 1.0-2.0 μm, and preferably 1.5 μm.

In some embodiments of the invention, the transition metal is selected from any one of Co, Zn, Mn.

In some embodiments of the invention, the metal organic framework material is selected from at least one of ZIF-67, ZIF-8, mn (ii) -MOF, preferably ZIF-67, ZIF-8.

In a second aspect of the present invention, there is provided a method for modifying any one or more modified microorganisms, comprising: dispersing microorganisms into an organic ligand solution, and then adding a metal source solution for mixing reaction; or dispersing the microorganisms in a metal organic framework material solution. The microorganism modified by the metal organic framework material is prepared by the method, specifically, the metal organic framework material is positively charged, the surface of the microorganism is negatively charged, and the metal organic framework material can be modified and loaded on the surface of the microorganism through electrostatic interaction.

In some embodiments of the invention, the metal source solution is a transition metal salt solution; typically an aqueous solution of a transition metal salt.

In a third aspect of the present invention, an application of any one of the modified microorganisms in preparation of an antitumor drug is provided. The antitumor drug is suitable for common tumors such as colorectal cancer, lung cancer, breast cancer, cervical cancer and the like.

In a fourth aspect of the present invention, an anti-tumor drug is provided, which comprises any one of the above modified microorganisms.

In some embodiments of the present invention, the mode of administration of the anti-tumor drug is intravenous.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a graph showing a particle size distribution of LAB @ ZIF-67 obtained in example 1;

FIG. 2 shows zeta potential test results for LAB @ ZIF-67 obtained in example 1, ZIF-67 obtained in comparative example 1 and unmodified lactococcus lactis LAB;

FIG. 3 is a TEM image of LAB @ ZIF-67 obtained in example 1;

FIG. 4 is a UV-Vis spectrum of LAB @ ZIF-67 obtained in example 1, ZIF-67 obtained in comparative example 1, and unmodified lactococcus lactis LAB;

FIG. 5 shows the results of cytotoxicity assays for unmodified lactococcus lactis LAB and LAB @ ZIF-67 obtained in example 1;

FIG. 6 shows the results of the experiment on the endocytosis of LAB @ ZIF-67 obtained in example 1;

FIG. 7 is a graph showing results of measuring intracellular reactive oxygen species production in unmodified lactococcus lactis LAB, ZIF-67 obtained in comparative example 1, and LAB @ ZIF-67 obtained in example 1;

FIG. 8 shows macrophage polarization phenotype assay results for unmodified lactococcus lactis LAB, ZIF-67 obtained in comparative example 1, and LAB @ ZIF-67 obtained in example 1;

FIG. 9 is a graph of tumor growth in mice following different drug injections;

FIG. 10 shows the body weight measurements of mice after different drug injections;

FIG. 11 shows the H & E staining results of tissue sections of different organs of mice after different drug injections;

FIG. 12 shows the detection results of apoptosis fluorescence signals in mouse tumors after different drugs are injected;

FIG. 13 shows the result of Ki-67 antibody staining detection of cell proliferation in mouse tumors after different drug injections;

FIG. 14 shows the result of staining with CD4 antibody on tumor tissue sections of mice after different drug injections;

FIG. 15 shows the result of staining with CD8 antibody on tumor tissue sections of mice after different drug injections;

FIG. 16 shows the distribution of lactococcus lactis in various organs and tumor tissues of a mouse 24 hours after drug injection using LAB @ ZIF-67 obtained in example 1.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

The synthesis method of the metal organic framework material ZIF-67 modified lactococcus lactis comprises the following steps:

1X10⁶Lactococcus lactis was dispersed in 1ml of 0.05mM of 2-methylimidazole (2-MIM) solution and gradually added dropwise with 1ml of 0.01mM of cobalt nitrate Co (NO) under mild stirring₃)₂And (3) solution. After the reaction, the mixture was stirred for 1h and then centrifuged at 5000rpm for 5min to obtain a metal organic framework ZIF-67 modified lactococcus lactis, denoted as LAB @ ZIF-67, which was washed three times with deionized water before subsequent use.

Comparative example 1

The embodiment synthesizes a metal organic framework material ZIF-67, and the synthesis method comprises the following steps: 0.1mM Co (NO) was added under gentle stirring₃)₂The solution (100. mu.L) was slowly added to a 0.5mM 2-MIM solution (100. mu.L) and the mixture was stirred for 1 h. Then centrifuging at 10000rpm for 10min, and collecting the precipitate to obtain the metal organic framework ZIF-67. ZIF-67 was rinsed three times with deionized water prior to subsequent use.

Test examples

(I) characterization of materials

The particle size of ZIF-67 modified lactococcus lactis (LAB @ ZIF-67) obtained in example 1 and the zeta potential of ZIF-67, unmodified lactococcus Lactis (LAB) and LAB @ ZIF-67 obtained in comparative example 1 were measured by a Nano Sizer S (Markov) apparatus, and the results are shown in FIGS. 1 and 2, respectively. As can be seen from FIG. 1, the hydrated particle size of the LAB @ ZIF-67 obtained in example 1 was about 1.5 μm; as can be seen from FIG. 2, the surface potential of lactococcus lactis was changed from-40 mV to +10mV after modification with ZIF-67, demonstrating that lactococcus lactis was successfully modified.

The LAB @ ZIF-67 obtained in example 1 was observed and examined by means of a JEMF200 (Japanese JEOL) Transmission Electron Microscope (TEM), and the results are shown in FIG. 3. Further, UV-visible spectrum scanning was performed on ZIF-67 prepared in comparative example 1, unmodified lactococcus Lactis (LAB) and LAB @ ZIF-67 prepared in example 1, respectively, using a Rayleigh-2601 spectrophotometer (China), and the results are shown in FIG. 4. As can be seen from FIG. 4, the modified lactococcus lactis (LAB @ ZIF-67) exhibited more absorption peaks in the characteristic absorption wavelength range of ZIF-67 than the unmodified lactococcus Lactis (LAB), confirming that lactococcus lactis has been successfully modified with ZIF-67.

(II) cell culture

The mouse colorectal cancer cell line CT-26(ATCC CRL-2638) and the mouse macrophage cell line RAW264.7(ATCC TIB-71) were obtained from Procell Biotech (Wuhan, China). CT-26 and RAW264.7 cells were cultured in high-glucose RPMI-1640(Gibco, USA) and DMEM medium (Gibco, USA), respectively. Cell culture medium was supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin solution (Gibco). Cells were passaged every 2-3 days.

(III) cell viability assay

Cell viability was determined by the CCK-8 method. Will be 1 × 10⁴The individual CT-26 cells were seeded into each well of a 96-well plate and lactococcus lactis was added at different concentrations; after 24h, 10. mu.L of CCK-8 solution was supplemented and the cells were cultured for a further 2 h; the absorbance at 450nm was recorded by a microplate reader. Cell viability was calculated as follows:

cell viability ═ a_LAB-A_BLANK)/(A₀-A_BLANK)×100％

Wherein A is_LABAbsorbance of the culture medium containing lactococcus lactis, A_BLANKAbsorbance of blank well, A₀The absorbance of the culture medium without lactococcus lactis was obtained.

The cytotoxicity of unmodified lactococcus Lactis (LAB) and LAB @ ZIF-67 obtained in example 1 was tested by the above methods, respectively, by cell viability test,specifically, red light irradiation (647nm, 0.5W/cm) is carried out 4h after non-red light irradiation and addition of lactococcus lactis are respectively set²5min), and the vitality of each group was tested according to the above method, and the results are shown in fig. 5. In FIG. 5, LAB indicates that LAB of the unmodified lactococcus lactis is added and not irradiated with red light, LAB + RL indicates that the unmodified lactococcus lactis is added and irradiated with red light, LAB @ ZIF-67 indicates that LAB of the modified lactococcus lactis is added and not irradiated with red light, and LAB @ ZIF-67+ RL indicates that LAB of the unmodified lactococcus lactis is added and not irradiated with red light.

The results shown in fig. 5 indicate that lactococcus lactis, when modified and given infrared light irradiation, has significantly increased toxicity to better kill tumor cells.

(IV) cell endocytosis assay

The endocytosis kinetics of the modified lactococcus lactis by cells is traced by a fluorescent labeling method. After the LAB @ ZIF-67 entraps Cy7 micromolecules, the cells are incubated with CT-26 cells of a mouse colorectal cancer cell line for different times (including 0h, 0.5h, 1h, 2h, 3h, 6h, 9h, 12h and 24 h); the cells were then fixed with 4% paraformaldehyde (Solarbio, china), resuspended in PBS after fixation, and subjected to flow cytometry analysis, specifically recording the Cy7 fluorescence intensity exhibited by the APC-Cy7 channel on a BD Fortessa (usa) flow cytometer.

The LAB @ ZIF-67 prepared in example 1 was subjected to the endocytosis assay using the above method, and the results are shown in fig. 6. The experimental results shown in FIG. 6, where the fluorescent seal had shifted completely to the right after 0.5h, indicate that LAB @ ZIF-67 is endocytosed by the cell faster and is essentially complete within 30 min.

(V) detection of intracellular reactive oxygen species production

The ROS detection kit (S0033S) used in the detection process was purchased from Biyuntian Biotech Co., Ltd (China). Six experimental groups and two control groups are set, three groups of the six experimental groups respectively treat CT-26 cells for 24 hours by unmodified lactococcus Lactis (LAB), ZIF-67 obtained in comparative example 1 and LAB @ ZIF-67 obtained in example 1; the other three groups were treated separately from CT-26 cells for 24h in the first three groups, and irradiated with red light (647nm, 0.5W/cm) 4h after the treatment of the cells²，5min); one control group did not have any treatment of CT-26 cells, and the other control group only had red light irradiation (647nm, 0.5W/cm) of CT-26 cells²，5min)。

Then adding the DCFH probe into the culture solution, and continuing to culture for 20 min; cells were fixed with 4% paraformaldehyde, and the fixed cells were analyzed by flow cytometry, and DCF fluorescence was monitored quantitatively in the FITC channel.

The results of the measurement of cellular active oxygen production by the above method are shown in FIG. 7. The results of the assays shown in fig. 7 show that modified lactococcus lactis, whether or not given light, produces additional significant ROS signal peaks, unlike the other groups, indicating that modified lactococcus lactis can produce tumor-specific ROS; in addition, the test results showed that the modified lactococcus lactis LAB @ ZIF-67 obtained in example 1 could maximize the production of intracellular reactive oxygen species in combination with red light irradiation.

(VI) macrophage polarization

RAW264.7 cells were inoculated into 24-well dishes and divided into 8 groups, six experimental groups and two control groups, three of the six experimental groups were co-cultured by adding unmodified lactococcus Lactis (LAB), ZIF-67 obtained in comparative example 1, and modified lactococcus lactis (LAB @ ZIF-67) obtained in example 1, respectively, and the other three experimental groups were co-cultured based on the first three experimental groups and irradiated with red light (647nm, 0.5W/cm) after co-culturing for 4 hours ²5 min); one control group (NTC) was co-cultured without addition of other materials, and the other control group (NTC + RL) was co-cultured without addition of any materials, and the inoculated cells were irradiated with red light (647nm, 0.5W/cm)²，5min)。

After 24h of culture in each of the above groups, cells were collected and stained with FITC-labeled anti-F4/80 antibody (157309, Biolegend), APC-labeled anti-CD 80 antibody (104713, Biolegend), and APC-labeled anti-CD 206 antibody (141707, Biolegend); performing phenotype analysis on the polarized cells by using a flow cytometer; the F4/80+ and CD80+ cell populations were considered M1 macrophages. The experiment was carried out in the above manner, and the results are shown in FIG. 8. The results shown in FIG. 8 indicate that macrophages have a propensity to polarize the phenotype of M1 in the presence of LAB @ ZIF-67.

(VII) animal raising

The animal experiments were conducted under the supervision of the ethics and welfare committee of the animal of Shenzhen university (approval No.: AEWC-2021012); the 6-8 week old Balb/c mice (Witongliwa) used in this study were housed in an SPF-scale animal facility.

(VIII) study on drug injection and tumor growth

Mice were randomly divided into six groups (n-4) and each mouse was injected subcutaneously with 100uLPBS containing 5X 10⁵And CT-26 cells. When the tumor volume reaches 50mm³On the left and right, mice in different groups received 100uL volume of tail vein injection (Control group, PBS, 1X 10)⁶LAB of unmodified lactococcus lactis, 1X10⁶LAB @ ZIF-67 from example 1, 0.1mM ZIF-67 from comparative example 1). Wherein, two groups are injected with ZIF-67, and two groups are injected with LAB @ ZIF-67; and one of the group of ZIF-67 and one of the group of LAB @ ZIF-67 were given 647nm light irradiation (0.5W/cm) on each of day 1, day 3 and day 5²5 min). Tumor volume and mouse body weight were recorded every other day until day 20, and the results are shown in fig. 9 and fig. 10, respectively. In addition, after final measurement, major organs (heart, liver, spleen, lung, kidney) and tumors were collected for hematoxylin-eosin (H)&E) The results of the staining are shown in FIG. 11.

The results obtained in FIG. 9 show that the modified lactococcus lactis LAB @ ZIF-67 obtained in example 1 has a slight tumor-inhibiting effect, and that the tumor growth is significantly inhibited after irradiation with infrared light. The results obtained in FIG. 10 show that modified lactococcus lactis LAB @ ZIF-67 is less toxic in mice. The results obtained in FIG. 11 show that modified lactococcus lactis LAB @ ZIF-67 has no significant effect on the major organ structure of mice under different conditions.

Experimental data in tumor growth research experiments are expressed as mean +/-variance, data analysis is carried out by GraphPad Prism 8 software, significance difference of experimental results is compared by student-t test, and statistical significance is shown when p value is less than 0.05. The statistical analysis of the significant differences in the experimental results of the volume change of the tumor obtained from the drug injection and tumor growth studies by the above method, the results are shown in fig. 9, where p represents the value of p < 0.05: has statistical significance, wherein p is less than 0.01; specifically, the P values of the statistical analysis results shown in fig. 9, which are expressed from left to right along the abscissa, are 0.005, 0.004, and 0.008, respectively, indicating that the experimental results obtained in fig. 9 have significant differences.

(nine) immunohistochemical staining of tumors

Cutting to about 1mm³After the injection of the medicines and the experimental treatment of tumor growth research, the tumor tissues of the mice are immersed in 4% paraformaldehyde overnight, then taken out and put into 30% sucrose solution, and then are frozen and stored for 1h at 4 ℃ and then embedded into paraffin; placing the paraffin-embedded tissue into a sodium citrate buffer solution, and heating at 50 ℃ for 30min for antigen retrieval; sections of approximately 5 μm thickness were cut with a microtome, and the slides were blocked with 200 μ L of 5% goat serum (Biyuntian Biotech, C0265) and allowed to stand at room temperature for 1 h; then incubated overnight at 4 ℃ with different antibodies (primary antibodies, about 200 μ L) as required for the different test items described below; the next day, different slides were washed three times with PBST buffer; then incubating with DAB substrate (200 μ L) for about 30min at room temperature according to different requirements of the following different test items; then washed three times by PBST buffer solution, and after the mounting by a mounting agent, FITC fluorescence or DAB color distribution is observed by a confocal microscope.

Fluorescence labeling detection of apoptotic cells within tumors: incubating by using an HRP-labeled TUNEL antibody, and directly carrying out microscopic imaging without incubating with a DAB substrate on the second day;

detection of proliferating cells in tumors: the antibody can be incubated by using an anti-Ki-67 antibody marked by HRP, and the DAB substrate is used for color development detection;

detection of intratumoral CD4+ and CD8+ cells: the cells were incubated with HRP-labeled anti-CD 4 and anti-CD 8 antibodies, respectively, and detected by color development with DAB substrate.

The results of fluorescence labeling detection of apoptotic cells in tumors as described above are shown in FIG. 12. The results shown in fig. 12 indicate that the modified lactococcus lactis LAB @ ZIF-67 significantly promotes apoptosis under infrared light irradiation.

The results of the detection of proliferating cells in tumor by the above method are shown in FIG. 13. The results shown in FIG. 13 indicate that modified lactococcus lactis LAB @ ZIF-6 significantly inhibited cell proliferation under infrared light conditions.

The results of the above assays for intratumoral CD4+ and CD8+ cells are shown in fig. 14 and 15, respectively. The results shown in fig. 14 demonstrate that lactococcus lactis LAB and modified lactococcus lactis LAB @ ZIF-67 can recruit CD4 positive T cells to the tumor microenvironment; the results shown in FIG. 15, in turn, indicate that lactococcus lactis LAB and modified lactococcus lactis LAB @ ZIF-67 can recruit CD8 positive T cells to the tumor microenvironment.

(Ten) enrichment Effect test experiment

Mice were injected subcutaneously with 100uL PBS containing 5X 10⁵CT-26 cells, when the tumor volume reaches 50mm³On the left and right, mice received a further tail vein injection (1X 10) of 100. mu.L volume⁶LAB @ ZIF-67 obtained in example 1) was injected for 24 hours, and then major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected, modified lactococcus lactis LAB @ ZIF-67 in each organ and tumor tissue were extracted and cultured on an agar plate containing M17 medium, and after 24 hours, the number of colonies growing on the plate was observed to determine the distribution of lactococcus lactis in different organs and tumor tissues of mice, and the results are shown in FIG. 16. Test results show that the modified lactococcus lactis LAB @ ZIF-67 can be spontaneously enriched in tumors.

According to the method, the prepared metal organic framework material modified microorganism (LAB @ ZIF-67) can be enriched in the tumor, can generate tumor specific ROS, has good infrared photodynamic therapy capability on the tumor, can activate and initiate natural immunity and specific immunity in vivo, realizes linkage of photodynamic therapy and immunotherapy, improves the tumor therapy effect, and has low toxicity and high safety. Therefore, the microorganism modified by the metal organic framework material can be used for preparing an anti-tumor medicament, and the application further provides the anti-tumor medicament which comprises the microorganism modified by the metal organic framework material and can be administrated by intravenous injection.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A modified microorganism, wherein the microorganism is modified with a metal organic framework material, and wherein the microorganism is an anaerobic or facultative anaerobic microorganism having anti-tumor activity.

2. The modified microorganism of claim 1, wherein the microorganism is selected from at least one of lactococcus lactis, escherichia coli, and salmonella attenuated.

3. The modified microorganism of claim 1 or 2, wherein the metal in the metal-organic framework material is a transition metal.

4. The modified microorganism according to claim 3, wherein the transition metal is selected from any one of Co, Zn, Mn.

5. The modified microorganism of claim 4, wherein the metal organic framework material is selected from at least one of ZIF-67, ZIF-8, mn (ii) -MOF.

6. A method for modifying a modified microorganism as claimed in any one of claims 1 to 5, which comprises: dispersing microorganisms into an organic ligand solution, and then adding a metal source solution for mixing reaction; or dispersing the microorganisms in a metal organic framework material solution.

7. The method for modifying a modified microorganism according to claim 6, wherein the metal source solution is a transition metal salt solution.

8. Use of a modified microorganism according to any one of claims 1 to 5 for the preparation of an anti-tumor medicament.

9. An antitumor agent comprising the modified microorganism according to any one of claims 1 to 5.

10. The antitumor agent as claimed in claim 9, wherein the administration mode of said antitumor agent is intravenous administration.