CN116024113A - Modified bacteria and preparation method and application thereof - Google Patents
Modified bacteria and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicine, and in particular relates to a modified bacterium, a preparation method and application thereof. After the modified bacteria are injected in tumor, the anti-tumor immune response can be activated, the abnormally excellent anti-tumor treatment effect is realized, and the tumor metastasis and recurrence are inhibited.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a modified bacterium, a preparation method and application thereof.
Background
Bacteria have long been used for treating tumors, but until the 90 th century, bacillus calmette-guerin is used for treating bladder cancer of people, and bacteria therapy is not introduced into the public field, and in recent years, attenuated bacteria such as attenuated salmonella, attenuated listeria and the like are gradually developed for treating different tumors, and the bacteria therapy of tumors is also an extension therapy of immunotherapy, and immune response is initiated by the stimulation of bacteria, namely heterologous foreign substances, so that tumor growth is further inhibited. However, most of the currently used strains are live bacteria after attenuated treatment, and have higher risks in clinical use, a narrower safety window, and inactivated bacteria cannot achieve the due immunostimulation effect, so that the current tumor bacterial therapy is difficult to realize effective treatment under a safe dosage as a whole.
Disclosure of Invention
The invention aims to provide a modified bacterium, a preparation method and application thereof, and the modified bacterium has good safety and good immune stimulation effect when being used for preparing a tumor therapeutic preparation.
According to the invention, after metal ions are mixed with living bacteria or inactivated bacteria (attenuated salmonella is initially used), the pH value of the solution is regulated to be slightly alkaline, insoluble or indissoluble metal ion compounds can be attached to the surface of the bacteria to form modified bacteria, the modified bacteria are injected into a tumor model of a mouse in an intratumoral injection mode, the effect of inhibiting tumor growth can be achieved, and further mechanism researches show that the modified bacteria cause antitumor immune response of a body after in-situ injection, so that the effect of tumor treatment is achieved. When divalent metal ions are placed in alkaline conditions, hydroxides are extremely easy to form and precipitate, the bacterial surface serves as a typical solid-liquid interface, nucleation centers of precipitates can be provided, the precipitation of the hydroxides is further promoted, after the hydroxides are precipitated on the bacterial surface, the hydroxides are converted into more stable oxides again, and an oxide adhesion layer is formed on the bacterial surface along with continuous precipitation and conversion of the hydroxides, so that the inactivated bacteria modified by the metal compounds are obtained. The deposition reaction is that after some metal ion salts are mixed with bacteria and the like, corresponding anions are introduced, under the condition of proper pH, metal ions and anions form indissolvable or insoluble metal compounds and are attached to the surfaces of the bacteria, under the condition of sufficient stirring, target products can be continuously settled on the surfaces of the bacteria in the liquid, and finally, the bacteria attached with the metal compounds form uniform and stable suspension.
Further studies have found that this technique can be applied to other types of bacteria, including staphylococcus aureus, escherichia coli, lactobacillus, and the like, in addition to attenuated salmonella. Modified bacteria prepared on the basis of the same method using the above bacteria can also activate the immune system to obtain good antitumor activity.
It was further investigated whether other poorly soluble or insoluble metal compounds than metal hydroxides or metal oxides, when the solubility was reduced, could form an adhesion layer on the bacterial surface and achieve similar immunostimulatory results. By trying the synthesis of inactivated bacteria to which a plurality of metal compounds are attached, it is found that after a part of metal compounds are combined on the surface of the bacteria, immune cells can be activated, a strong anti-tumor immune response is induced, an immune memory effect can be possibly generated, and the probability of cancer metastasis and recurrence is reduced.
According to the technical scheme of the invention, the modified bacteria comprise a bacteria body and insoluble or insoluble biological acceptable metal compounds modified on the surface of the bacteria body.
Further, the bacterial body is selected from one or more of salmonella, staphylococcus aureus, escherichia coli, lactobacillus, salmonella attenuated strain, staphylococcus aureus attenuated strain, escherichia coli attenuated strain, and lactobacillus attenuated strain.
Further, the bacterial body is a living bacterium or an inactivated bacterium, preferably an inactivated bacterium, both of which comprise attenuated bacteria.
Further, the metal compound is modified on the surface of the bacterial body through a deposition reaction to form an adhesion layer.
Further, 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.
Further, the metal compound is selected from zinc carbonate, calcium carbonate, copper carbonate, magnesium carbonate; zinc hydroxide, iron hydroxide, copper hydroxide, manganese hydroxide, magnesium hydroxide, zinc sulfide, copper sulfide, and manganese sulfide; one or more of zinc phosphate, calcium phosphate, copper phosphate, iron phosphate, magnesium phosphate and manganese phosphate.
In a second aspect, the present invention provides a method for preparing a modified bacterium as described above, comprising the steps of,
s1: preparing bacterial suspension;
s2: adding a soluble metal salt solution to the bacterial suspension;
s3: adding an aqueous solution of a readily soluble hydroxide, carbonate or phosphate to a pH of 8-12, or adding an aqueous solution of a readily soluble sulfide to produce the modified bacteria.
Specifically, in the step S2,
adding an easily-soluble hydroxide aqueous solution to a pH of 8-12, and centrifuging after the reaction to obtain modified bacteria with metal hydroxide or metal oxide attached to the surface; the aqueous solution of the easily soluble hydroxide is preferably an aqueous solution of sodium hydroxide;
adding an easily-soluble carbonate aqueous solution to a pH of 8-12, and centrifuging after the reaction to obtain modified bacteria with metal carbonate attached to the surface; the aqueous solution of readily soluble carbonate is preferably an aqueous solution of sodium carbonate;
adding an easily-soluble phosphate aqueous solution to a pH of 8-12, and centrifuging after the reaction to obtain modified bacteria with metal phosphate attached to the surface; the aqueous solution of phosphate which is easily soluble is preferably an aqueous solution of sodium phosphate;
adding an easily-soluble sulfide salt aqueous solution, reacting and centrifuging to obtain modified bacteria with metal sulfide attached to the surface; the aqueous solution of the easily soluble sodium sulfide is preferably an aqueous solution of sodium sulfide;
further, in the step S2, a soluble metal salt solution is added to the bacterial suspension in an amount of 0.2 to 13.5mmol of metal ions per 10 million bacteria.
In a third aspect the invention provides a modified bacterial lyophilized powder comprising a lyoprotectant and a modified bacterium as described above.
Further, the lyoprotectant is selected from one or more of sucrose, trehalose, myose, white cotton sugar, inulin, dextran, maltodextrin, maltose and 2-hydroxypropyl-beta cyclodextrin.
Further, the volume fraction of the lyoprotectant in the bacterial lyophilized powder is 0.1-20%, preferably 1-5%.
In a fourth aspect, the invention provides a modified bacterium as defined above or a lyophilized powder of a modified bacterium as defined above for use in the preparation of a tumor therapeutic formulation.
Compared with the prior art, the technical scheme of the invention has the following advantages: the invention provides a modified bacterium, which can be an inactivated bacterium, and the metal compound on the surface of the modified bacterium can be metabolized by decomposing into an ionic state, so that the modified bacterium has better safety.
The modified bacteria provided by the invention have the advantages that the insoluble metal compound with the surface modified has the effect of neutralizing the weak acidic microenvironment of tumors, the activity of immune cells at tumor positions can be improved, the drug resistance of the tumors can be reduced, and the curative effect can be improved.
The invention provides a modified bacterium, which can activate immune cells, induce strong anti-tumor immune response and generate immune memory effect to reduce cancer metastasis and recurrence probability through multi-channel stimulation, and is a novel immune agonist.
Drawings
FIG. 1 is a scanning electron microscope image of modified inactivated bacteria with different metal compounds attached;
FIG. 2 is a scanning electron microscope image of different bacteria before and after modification with manganese dioxide;
FIG. 3 is a graph showing the relative activity statistics of CT26 cells after 12 hours of incubation with different samples;
FIG. 4 is a statistical plot of the proportion of flow-detected mature cells after co-incubation of bone marrow-derived dendritic cells with different samples;
FIG. 5 is a graph showing tumor growth in mice after treatment of a mouse tumor model with bacteria modified with different metal compounds.
FIG. 6 is a graph showing bioluminescence signal intensity statistics of reporter gene expression following 24h incubation of reporter cells with different samples following activation of STING pathway in different groups;
FIG. 7 is a graph showing survival of mice vaccinated with the same tumor again on day 60 after a subcutaneous tumor model of colon cancer was cured with manganese dioxide-modified inactivated Salmonella.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example a: preparation and basic morphological characterization of bacteria modified by different metal compounds
Example A1: preparation of bacteria with surface modified by zinc carbonate
S1: preparing zinc sulfate aqueous solution;
s2: after the attenuated salmonella (hereinafter abbreviated as F.S) is amplified and cultured, the bacteria are obtained by centrifugation and are cleaned, the residual culture medium and other substances in the bacteria are removed, paraformaldehyde is added into the bacteria to inactivate bacteria and fix the shape, the immobilized inactivated bacteria are centrifugally collected, the bacteria are cleaned twice by sterile normal saline, finally the inactivated bacteria are resuspended into an inactivated bacteria suspension with the concentration of 24MCF by normal saline, and no living bacteria in the bacteria liquid is detected and confirmed, so that the bacteria can be preserved at low temperature for standby;
s3: adding 1mL of 10mM zinc sulfate solution and proper amount of physiological saline into inactivated bacterial suspension (turbidity of 24MCF, about 72 hundred million bacteria/mL, volume of 1 mL), stirring at room temperature for a short time, adding sodium carbonate solution to pH 8-12, stirring for not less than 1 hr, centrifuging, collecting precipitate, washing with sterile physiological saline twice, re-suspending with sterile physiological saline, and preserving at low temperature to obtain ZnCO product 3 @F.S。
In experiments, bacterial suspension and metal ion salt solution with different original concentrations and volume ratios are tried to be added, the range of the feeding ratio of bacteria modified by metal compounds can be explored, the total amount of bacteria in a reaction system is fixed, the feeding ratio of products to be more stable suspension is explored by adjusting the feeding amount of metal ion salt, and as a result, when the bacterial amount is 10 hundred million and the concentration of metal ions exceeds 13.5mmol, the products are non-suspensible precipitates no matter how the pH, the concentration and the reaction time of the reaction system are adjusted, and the subsequent application cannot be realized. At lower levels of metal ion salts involved in the reaction, which did not affect resuspension of the product, the lowest feed ratio tried in the experiment was 0.2. Mu. Mol of metal ions per 10 million bacteria.
Example A2: preparation of bacteria with surface modified by ferric hydroxide or cupric hydroxide
The method of S1-S2 in example A1 provides a soluble ionic salt solution of iron and an inactivated bacterial suspension; the difference is that:
s3: adding 10mM ferric chloride or cupric sulfate solution and appropriate amount of physiological saline into inactivated bacterial suspension (turbidity of original bacterial solution is 24MCF, about 72 hundred million bacteria/mL, sample volume is 1 mL), stirring briefly at room temperature, adding sodium hydroxide solution or ammonia water to adjust pH to 8-12, stirring continuously for not less than 1 hr, centrifuging, collecting precipitate, washing twice with sterile physiological saline, re-suspending with physiological saline, and preserving at low temperature to obtain Fe (OH) 3 @F.S、Cu(OH) 2 @F.S。
Example A3: preparation of modified bacteria with surface attached with manganese sulfide, zinc sulfide and copper sulfide
S1: preparing manganese chloride, copper sulfate and zinc sulfate aqueous solution with the concentration of 10mM;
s2: after the attenuated salmonella is amplified and cultured, the bacteria without a culture medium are obtained by cleaning, formaldehyde is not used for treating bacteria, and physiological saline is directly used for resuspension to obtain bacterial suspension of living bacteria;
s3: 10mM manganese chloride, copper sulfate and zinc sulfate solution and proper amount of physiological saline are respectively added into bacterial suspension (the turbidity of the original bacterial solution is 24MCF, about 72 hundred million bacteria/mL, the sample adding volume is 1 mL), stirring is carried out at room temperature, then sodium sulfide solution is added, stirring is continued, the precipitate is centrifugally collected, washed twice with sterile physiological saline, and finally, the suspension is resuspended with physiological saline to obtain MnS@F.S, cuS@F.S and ZnS@F.S respectively and the suspension is preserved at low temperature.
Example A4: basic characterization of modified bacteria
Scanning electron microscope samples were prepared from the products of examples A1-A3, and the surface morphology of bacteria modified with different metal compounds was characterized by Scanning Electron Microscopy (SEM), and the results are shown in FIG. 1. Wherein, the pictures are marked with ZnCO 3 @F.S、Fe(OH) 3 @F.S、Cu(OH) 2 The terms @ F.S, mnS@F.S, znS@F.S, cuS@F.S respectively denote the passage of a surfaceScanning electron microscope pictures of salmonella modified by zinc carbonate, ferric hydroxide, copper hydroxide, manganese sulfide, zinc sulfide and copper sulfide. The results show that a large amount of solids are adhered to the surface of the bacteria modified by the metal compound, excessive solid substances are not remained in the environment, and the morphology of the bacteria adhered with different metal compounds is changed according to the different crystal forms of the metal compounds, so that the metal compounds adhere to the surface of the bacteria to form a coating in the preparation process.
Example A5: preparation of modified other bacteria
Bacterial suspensions of E.coli (F.E), staphylococcus aureus (F.SA), salmonella VPN20009 (F.V), lactobacillus (F.L) were prepared according to the steps S1-S2 of any of examples A1-A3, followed by modification of the surface metal compounds, in particular:
s3: a solution of manganese chloride was prepared, 1mL of a 10mM concentration manganese chloride solution was added to a bacterial suspension (original bacterial solution turbidity of 24MCF, about 72 hundred million bacteria/mL, and volume of 1 mL), and an appropriate amount of physiological saline was added thereto, stirring was performed at room temperature, then the pH was adjusted to 8-12 by adding sodium hydroxide solution or aqueous ammonia, stirring was continued, the precipitate was collected by centrifugation, washed twice with sterile physiological saline, and finally resuspended with physiological saline, and the morphology of the different bacteria modified with manganese compounds was characterized by scanning electron microscopy, as shown in FIG. 2, wherein F.E represents E.coli, F.SA represents Staphylococcus aureus, F.S represents Salmonella, F.L represents Lactobacillus, F.V represents Salmonella VNP20009, and manganese dioxide modified bacteria were represented by the M@ prefix. The scanning electron microscope image shows that a layer of substances is attached to the surface of the bacteria, but the shape and the size of the bacteria are not obviously changed, which indicates that various bacteria can form modified bacteria through the attachment of metal compounds.
Example B: cell experiment
Example B1: cell relative Activity of CT26 cells (mouse colon cancer cell line) after incubation with different samples
CT26 cells were cultured at 10 4 The density of each hole is inoculated in a 96-well plate, the cell culture box is used for culturing overnight at 37 ℃, and when the cell is attached and the state is good, different concentrations are addedThe different samples prepared in examples A1-A3, wherein the bacteria were inactivated attenuated salmonella, were co-incubated at 37 ℃. Bacteria modified with different metal compounds, corresponding to metal compound concentrations of 1 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, the total amount of bacteria in the non-modified bacterial group being the same as the total amount of modified bacteria. After 12 hours incubation, the medium containing the sample was removed, excess sample was washed off with PBS, and the relative activity of the cells was measured by MTT assay, as shown in FIG. 3 (data for each concentration, from left to right: F.S, mnS@F.S, cuS@F.S, cu (OH) 2@F.S,ZnS@F.S,Fe (OH) 3@F.S,ZnCO3@F.S). At low concentrations, the modified bacteria did not show higher cytotoxicity, cells incubated with the modified bacteria still had more than 80% activity, with increasing concentrations, the effect on the relative activity of the cells was gradually shown, and when the concentration reached 40 μg/mL, the cell activity was still greater than 50%. The modified bacteria have high safety in a certain concentration range, and the safety is related to the use concentration.
Example B2: experiments of different samples to stimulate the maturation of myeloid-derived dendritic cells
DC cells are highly efficient antigen presenting cells in the body, differentiate into mature DC cells after being stimulated or ingested by certain factors, and the mature DC cells can effectively activate initial T cells, induce the generation of cytotoxic T lymphocytes, secrete tumor necrosis factor alpha (TNF-alpha) and the like, and play a key role in anti-tumor immune response. Therefore, samples that can effectively stimulate DC cell maturation can enhance the anti-tumor immune response of the body.
Bone Marrow-derived stem cells were extracted from C57BL/6 mouse Bone Marrow, colony stimulating factor (GM-CSF) was added to promote differentiation of stem cells into myeloid-derived dendritic cells (Bone Marrow-Derived Dendritic Cells, BMDCs), and BMDCs were isolated from different modified bacterial samples (the amount of bacteria in each sample was 3.6X10) 7 A plurality of; the corresponding bare metal compound content is 1.5-2.7 μg, and the same amount of the same metal compound as that of the bacterial group modified by the metal compound is contained in the rest group without bacteria, such as ZnS and ZnS@F.S groupEqual amounts of ZnS) were used to detect the maturation rate of BMDCs in different groups after 12 hours.
The statistical results of the maturation rate of BMDCs are shown in FIG. 4. Wherein each experimental group represents the addition of different substances to the cell culture medium, the specific substances are as follows:
blanc: blank control group;
F.S: an unmodified inactivated bacterium; znS: zinc sulfide suspension; znCO 3 : zinc carbonate suspension; fe (OH) 3 : an iron hydroxide colloid; cuS: copper sulfide suspension; cu (OH) 2 : copper hydroxide suspension; mnS: a manganese sulfide suspension;
ZnS@F.S: a suspension of zinc sulfide modified salmonella; znCO 3 @ F.S: a suspension of zinc carbonate modified salmonella; fe (OH) 3 @ F.S: ferric hydroxide modified salmonella suspension; cuS@F.S: a suspension of copper sulfide modified salmonella; cu (OH) 2 @ F.S: suspension of copper hydroxide modified salmonella; mnS@F.S: a suspension of manganese sulfide modified salmonella.
Combining statistics of the maturation ratio of DC cells, it can be seen that the modified bacteria are better able to stimulate the maturation of BMDCs, most of the group results are significantly different from the blank control group (blank), and individual experimental groups even obtain results superior to those of the positive control group (2 mug/mL lipopolysaccharide, LPS), indicating that the modified bacteria of the invention have very excellent ability to stimulate the maturation of DC cells.
Example C: animal experiment
Example C1: treatment experiment of different metal compound modified bacteria on mouse colon cancer tumor model
Inoculating colon cancer tumor cells on the back of BALB/c mice to establish a tumor model, and after the tumor size is 120mm 3 On the left and right, groups of 6 mice each were randomized, with different groups of mice receiving treatment with bacteria modified with different metal compounds. Single treatment was performed by intratumoral injection. Wherein the bacterial dosage among the control groups is 1.8-10 10 Each bacterium/kg body weight, according to the difference of binding efficiency of each metal compound and bacterium, metallizationThe dosage of the compound is 0.75mg/kg-1.35mg/kg body weight, and the concentration of metal ions is 300 mug/mL-1.08 mg/mL. The tumor volume change was recorded and a tumor growth curve was prepared, the results are shown in fig. 5, and the survival rate of the mice, the tumor inhibition rate was calculated, the statistical conditions of the survival rate and the calculation results of the tumor inhibition rate are shown in table 1. When mice were subjected to treatment experiments with simple metal compounds (consistent with the metal compound dose in the bacterial group modified with such metal compounds, e.g. ZnS and ZnS@F.S, containing equal amounts of ZnS), some of the metal compounds exhibited some toxicity, which resulted in a slow increase in tumor volume at the initial stage, but failed to eradicate the tumor or inhibit the growth of the tumor at the later stage, no significant toxicity was exhibited, no significant tumor growth inhibition was produced, and no effective tumor treatment was seen on day 19 at a tumor inhibition rate of less than 20%.
Table 1: survival of mice treated with bacteria modified with different metal compounds and tumor inhibition at day 19 post-inoculation
The results in fig. 5 and table 1 show that bacteria modified with certain metal compounds can inhibit tumor growth, and after treatment with bacteria attached with different metal compounds, the tumor volume increase of mice is significantly slowed, the survival rate is significantly increased, up to 2/3 survival rate is achieved, and the tumor inhibition rate is substantially above 60%. Taken together, it has been demonstrated that the use of most of the metal compound-modified bacteria of the present invention can achieve tumor therapeutic effects, whereas Fe hydroxide-modified bacteria do not exhibit significant tumor inhibition, indicating which metal compound-modified bacteria have an inhibitory effect on tumor growth, and how to select metal compounds modified on the surface of the bacteria is unpredictable.
Example D: immune stimulation mechanism study of modified bacteria
Example D1: demonstration of activation of STING pathway by modified bacteria at the cellular level
The modified bacteria of the invention have a multi-channel stimulation effect, and the embodiment is designed by taking salmonella modified by manganese compounds (manganese dioxide) as an example, and other metal ions have corresponding immune stimulation mechanisms. In this example, cells (STING) containing a reporter gene regulated by the activation of STING pathway were used + ) After the STING channel of the cell is stimulated and activated, the expression of the reporter gene luciferase is activated, the luciferase substrate can be catalyzed to emit bioluminescence signals, and the bioluminescence signals are stronger when the STING activation degree is higher. The cells were respectively treated with manganese dioxide-modified bacteria (M@F.S) and manganese dioxide (MnO) 2 ) Salmonella (F.S), manganese chloride (Mn) 2+ ) After 24 hours of incubation, the Positive Control (PC) group was added with a luciferase substrate and the bioluminescence signal intensities were measured, and the bioluminescence signal intensities of all groups were respectively compared with the PBS group. Null cells are cells that do not express the STING pathway, and therefore cannot induce luciferase expression by activating the STING pathway, and incubation of the cells with different samples proves that the samples and reagents themselves in the experiment cannot interfere with bioluminescence signals or induce luciferase expression. Bioluminescence signal intensity ratio statistics as shown in fig. 6, STING pathway was significantly activated in cells incubated with modified bacteria compared to control group; the close level of STING activation compared to the Positive Control (PC) group suggests that the modified bacteria are able to activate STING pathways effectively.
Example D2: cell experiments prove that the modified bacteria can activate TLR4 channels
In this example, cells containing a reporter gene regulated by TLR4 pathway activation (TLR 4+ TLR4 positive cells) are used, and when TLR4 is stimulated to activate, the expression of the reporter gene luciferase is activated, which catalyzes the luciferase substrate to generate a bioluminescence signal. The cells were incubated with different samples (n=3). After incubation, the intensity of bioluminescence signals is detected after the luciferase substrate is added, and then ratios of the intensities of bioluminescence signals of all groups to the PBS group are respectively made, so that the activation degree of the TLR4 can be judged, and the higher the ratio is, the higher the activation degree of the TLR4 is. The TLR4 negative cells do not express TLR4, so that the TLR4 negative cells are not stimulated and express luciferase, bioluminescence does not occur after a luciferin substrate is added, and the group of cells proves that all reagents or samples used in the experiment cannot interfere with bioluminescence signals or induce luciferase expression.
The ratio of bioluminescence signal intensities of three parallel samples of different groups is shown in Table 2, and the positive control group is 3 mug/mL of MPLA which is incubated with cells, so that the TLR4 pathway stimulation effect is obvious. Wherein the modified bacteria (MnO) 2 @ F.S) was able to significantly stimulate TLR4 compared to the Blank control (Blank), as demonstrated by a fluorescence intensity ratio greater than 1; the modified bacteria showed stronger fluorescence intensity compared to the pure bacteria (F.S), indicating that the modified bacteria were better able to stimulate TLR4 pathways. The modified bacteria of the invention are described as having TLR 4-like effects.
Table 2: bioluminescence intensity ratio statistics table after adding luciferase substrate to different cell culture media
The activation of STING and TLR4 shows that the modified inactivated bacteria of the invention effectively activate the natural immune system, and help to relieve immunosuppression of the tumor microenvironment, thereby enhancing the anti-tumor immune response of the organism. Combining example D1 and example D2, the modified bacteria of the invention have the effect of a multichannel agonist, while enhancing the nonspecific immune response and the anti-tumor immune response, contributing to the synergistic anti-tumor immunotherapy.
Example E: treatment experiment of colon cancer tumor model after different bacteria are modified by metal compounds
Grouping:
a first group: blank: blank control group;
second group: sa: inactivating staphylococcus aureus;
third group: F.E: inactivating the escherichia coli;
fourth group: F.L: inactivating lactobacillus;
fifth group: mnO (MnO) 2 @ f.sa: manganese dioxide modified inactivated staphylococcus aureus;
sixth group: mnO (MnO) 2 @ F.E: inactivated escherichia coli modified with manganese dioxide;
seventh group: mnO (MnO) 2 @ F.L inactivated lactobacillus modified with manganese dioxide;
eighth group: mnO (MnO) 2 @ F.S inactivated salmonella modified with manganese dioxide.
Different bacteria were prepared as modified bacteria according to the preparation method of example A5, and a mouse subcutaneous colon cancer model treatment experiment was performed. The tumor inhibition rate of each group on day 17 after inoculation was calculated, the tumor inhibition rate of the blank group was 0, and the results of the tumor inhibition rates of the other groups are shown in table 3. The results show that different modified inactivated bacteria can well inhibit tumors, and the tumor inhibition rate is more than 5 times of that of single bacteria treatment. The modified bacteria of different types are shown to realize the treatment of tumors.
Table 3: statistical table of tumor suppression rates for groups on day 17 post-inoculation in compound-modified bacterial treatment mice experiments with or without manganese
| Group of | F.SA | F.E | F.L | F.S |
| Tumor inhibition rate | 14.58％ | 12.55％ | 17.22％ | 10.23％ |
| Group of | MnO 2 @F.SA | MnO 2 @F.E | MnO 2 @F.L | MnO 2 @F.S |
| Tumor inhibition rate | 67.08％ | 62.55％ | >100％ | >100％ |
Example F: antitumor immune memory effect of organism generated after treatment of tumor by modified bacteria
In order to verify the vaccine effect of the modified bacteria, a colon cancer tumor model of the mice is established, the attenuated salmonella modified by manganese dioxide is used for treatment, and finally subcutaneous tumors of the mice completely disappear, the proportion of memory T cells in peripheral blood of the mice is analyzed by a flow cytometer after the treatment is carried out, the tumor-cured mice by the modified bacteria, the average proportion of memory T cells in the body of the mice is 83.86 percent, and compared with a blank control group without any treatment (the average content of memory T cells in the body of the mice is 59.5 percent), the modified bacteria can induce the production of memory T cells and generate an immune memory effect.
Meanwhile, the same kind of tumor cells are inoculated again to the cured mice, the tumor survival condition of the mice is observed, the survival curve of the mice is shown in figure 7, the mice cured by the modified bacteria have no obvious cancer recurrence phenomenon after being inoculated with the tumor cells again, and the mice still have longer survival time, that is, the modified bacteria can inhibit the recurrence of the tumor, and the vaccine-like effect is produced.
Example G: lyoprotectant studies of modified bacteria
The modified bacterial suspensions obtained in examples A1 to A3 and example A5 were mixed with various lyoprotectants and lyophilized, and the state of the lyophilized bacteria and whether the bacteria suspension was redispersible after the solvent was added were observed, wherein the observation results of the lyophilization after the mixing of the different ratios of the lyoprotectants for the manganese sulfide modified salmonella bacteria are recorded in table 4. Wherein, the redispersible sample, the lyoprotectant and the proportion thereof are defined as usable ranges, and the sample with better freeze-drying appearance and redispersible into suspension is used, and the lyoprotectant and the corresponding proportion thereof are set as preferable ranges.
The sucrose and the beta-cyclodextrin can enable the modified bacteria to have a good freeze-drying effect, and besides the sucrose and the beta-cyclodextrin, trehalose, myose, raffinose, inulin, dextran, maltodextrin and maltose can enable the samples to be resuspended after freeze-drying, and the properties of the samples are not changed, but lactose and mannitol cannot realize effective freeze-drying protection.
Table 4: mnS@F.S freeze-drying protection condition and post-freeze-drying state record
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (12)
1. A modified bacterium comprising a bacterial body and a poorly soluble or insoluble biologically acceptable metal compound modified on the surface of said bacterial body.
2. The modified bacterium of claim 1, wherein said bacterial body is selected from one or more of salmonella, staphylococcus aureus, escherichia coli, lactobacillus, salmonella attenuated strain, staphylococcus aureus attenuated strain, escherichia coli attenuated strain, and lactobacillus attenuated strain.
3. The modified bacterium of claim 1 or 2, wherein said bacterial body is a living bacterium or an inactivated bacterium.
4. The modified bacterium of claim 1, wherein said metal compound is modified by a deposition reaction to form an adhesion layer on the surface of said bacterial body.
5. The modified bacterium of claim 1 or 4, wherein the cation of said 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.
6. The modified bacterium of claim 1 or 4, wherein said metal compound is selected from the group consisting of zinc carbonate, calcium carbonate, copper carbonate, magnesium carbonate; zinc hydroxide, iron hydroxide, copper hydroxide, manganese hydroxide, magnesium hydroxide, zinc sulfide, copper sulfide, and manganese sulfide; one or more of zinc phosphate, calcium phosphate, copper phosphate, iron phosphate, magnesium phosphate and manganese phosphate.
7. A method for producing a modified bacterium according to any one of claim 1 to 6, comprising the steps of,
s1: preparing bacterial suspension;
s2: adding a soluble metal salt solution to the bacterial suspension;
s3: adding an aqueous solution of a readily soluble hydroxide, carbonate or phosphate to a pH of 8-12, or adding an aqueous solution of a readily soluble sulfide to produce the modified bacteria.
8. The method according to claim 7, wherein in the step S2, a soluble metal salt solution is added to the bacterial suspension in an amount of 0.2 to 13.5mmol of metal ion per 10 hundred million bacteria.
9. A modified bacterial lyophilized powder comprising a lyoprotectant and the modified bacterium of any one of claims 1-6.
10. The modified bacterial lyophilized powder of claim 9, wherein the lyoprotectant is selected from one or more of sucrose, trehalose, myose, raffinose, inulin, dextran, maltodextrin, maltose and 2-hydroxypropyl-beta cyclodextrin.
11. The modified bacterial lyophilized powder of claim 9, wherein the volume fraction of lyoprotectant in the bacterial lyophilized powder is 0.1-20%.
12. Use of a modified bacterium according to any one of claims 1 to 6 or a modified bacterium lyophilized powder according to any one of claims 9 to 11 in the preparation of a tumor therapeutic formulation.
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| CN114317377A (en) * | 2022-01-19 | 2022-04-12 | 南京大学 | Modified bacterium and preparation method and application thereof |
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