CN113046342A - Preparation method and application of polysaccharide-embedded bacterial spores - Google Patents
Preparation method and application of polysaccharide-embedded bacterial spores Download PDFInfo
- Publication number
- CN113046342A CN113046342A CN202110340786.1A CN202110340786A CN113046342A CN 113046342 A CN113046342 A CN 113046342A CN 202110340786 A CN202110340786 A CN 202110340786A CN 113046342 A CN113046342 A CN 113046342A
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- Prior art keywords
- polysaccharide
- spores
- cyclodextrin
- embedded
- bacterial
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Images
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Abstract
The invention provides a preparation method of polysaccharide-embedded bacterial spores, wherein the polysaccharide-embedded bacterial spores are prepared from first chemically modified polysaccharides and second chemically modified bacterial spores. The invention also provides application of the polysaccharide-embedded bacterial spores in preparation of anti-cancer drugs. The method is simple and convenient to operate, mild in reaction condition and easy to industrialize; the polysaccharide embedded bacterial spores obtained by the invention have long detention time in intestinal tracts and have obvious anticancer effect. The materials such as bacteria, polysaccharide and the like selected by the invention are approved to be used for food application, and meanwhile, the treatment system does not show any toxicity or side effect in research, so that the practicability is strong.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to a preparation method and application of chemically modified bacterial spores.
Background
The incidence of cancer has increased year by year due to irregular daily work and rest, environmental pollution, food safety, and other problems. Every year, a great deal of resources are used in biomedical research to develop new diagnostic techniques and effective cancer therapies. Surgical resection, radiation therapy, chemotherapy are common treatment strategies for cancer. However, these treatments, especially chemoradiotherapy, are always associated with various side effects, such as fatigue, loss of appetite. Heterogeneous cancer cells may develop resistance to chemotherapy, eventually leading to tumor recurrence and metastasis. In addition, the surgery process still suffers from relapse and metastasis, which brings great pain to patients. Therefore, there is an urgent need for an effective drug for treating cancer.
The study of bacterial microorganisms for the treatment of cancer is now very widespread. Bacteria can be genetically and chemically modified and accumulate and proliferate specifically in tumors. By virtue of these properties, bacteria can transport chemotherapeutic drugs to tumor tissues, thereby improving the safety and effectiveness of cancer therapy, but reducing the cytotoxic effects on normal cells. Bacteria, as a living organism, can also directly exert a significant tumor-inhibiting effect as metabolites thereof. However, genetic engineering can only transform a few bacterial species, and simultaneously, transgene expression is lost, which easily results in insufficient dosage of anticancer drugs, and the reasons limit the development of genetically engineered bacteria in cancer treatment. And the groups on the surface of the bacteria enable chemical modification to be more convenient, and the modification target is easy to realize. The bacterial surface chemical modification methods include electrostatic adsorption and click chemistry. Bacteria are negatively charged and, although positively charged chemical substances can be adsorbed, bacteria are easily aggregated. The click chemistry method is used for modifying bacteria, the bacteria are required to be supplemented with saccharides with azide groups, and the azide groups are introduced on the surfaces of the bacteria through bacterial metabolism, so that the azide-alkynyl addition reaction is started. These chemical modification means have high toxicity, which can seriously reduce the activity of bacteria and greatly influence the treatment effect of diseases.
Therefore, there is a need to develop a mild chemical method to modify bacteria and use for cancer treatment.
Disclosure of Invention
The present invention is directed to solving at least some of the problems of the prior art, and accordingly, in a first aspect of the invention, the invention provides a method for producing polysaccharide-embedded bacterial spores from a first chemically-modified polysaccharide and a second chemically-modified bacterial spore.
In one or more embodiments of the invention, the first chemical modification is a β -cyclodextrin graft modification; the second chemical modification is an adamantane modification.
In one or more embodiments of the invention, a method of preparing polysaccharide-embedded bacterial spores comprises the steps of:
step 1): preparation of aminated β -cyclodextrin: dissolving beta-cyclodextrin in NaOH solution, adding p-methylbenzenesulfonyl chloride, carrying out vacuum drying to obtain a white intermediate product, dissolving the obtained white intermediate product in ethylenediamine, stirring for 20-50h, adding acetone for washing, drying, recrystallizing in water at 60-90 ℃ for 3-5 times, and carrying out vacuum drying to obtain aminated beta-cyclodextrin;
step 2): preparation of oxidized polysaccharide: dissolving polysaccharide in deionized water, adding potassium periodate, reacting for 6-24h, dialyzing, and lyophilizing to obtain oxidized polysaccharide;
step 3): preparing polysaccharide grafted and modified by beta-cyclodextrin: dissolving the oxidized polysaccharide obtained in the step 2) and the aminated beta-cyclodextrin obtained in the step 1) in water, stirring for reaction for 10-20h, dialyzing, and freeze-drying to obtain beta-cyclodextrin grafted polysaccharide;
step 4): and (3) purifying bacterial spores: inducing bacteria to form spores by using a spore production culture medium, centrifugally collecting the spores, purifying the separated spores by a sucrose gradient centrifugation method, adding the spores into 35% and 65% sucrose solutions, forming 2 layers of precipitates in a centrifugal tube after centrifugation, extracting the precipitates, and dispersing the precipitates in PBS to obtain a purified bacterial spore solution; preferably, the concentration of the purified bacterial spore solution obtained in the step 4) is 5 × 108CFU mL-1。
Step 5): preparation of adamantane-modified bacterial spores: adding adamantane carboxylic acid into Phosphate Buffer Solution (PBS), dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the PBS, and mixing the two solutions to obtain a mixed solution; adding the purified bacterial spore solution obtained in the step 4) into the mixed solution, oscillating for 1-10h, centrifugally collecting precipitate, and dispersing the precipitate into PBS to obtain adamantane-modified bacterial spores; preferably, the concentration of the adamantane-modified bacterial spores obtained in the step 5) is 108CFU mL-1。
Step 6): preparation of polysaccharide-embedded bacterial spores: mixing the beta-cyclodextrin graft modified polysaccharide solution with the mass volume concentration of 0.1-10% prepared by taking the beta-cyclodextrin graft modified polysaccharide obtained in the step 3) as a solute with the adamantane modified bacterial spore obtained in the step 5), oscillating for 10-40min at 37 ℃ in a shaking table, and centrifuging and collecting solids to obtain the bacterial spore embedded by polysaccharide.
In one or more embodiments of the invention, the bacterial spores are selected from spores of one or more of yeast, probiotic bacteria, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes.
In one or more embodiments of the invention, the polysaccharide is selected from one or more of polyfructose, polygalactose, dextran, isomaltose, hyaluronic acid.
In one or more embodiments of the present invention, in the step 1), the weight ratio of the β -cyclodextrin to the p-toluenesulfonyl chloride is 2: 3.
in one or more embodiments of the present invention, in the step 2), the weight ratio of the polysaccharide to the potassium periodate is 10: 1.
in one or more embodiments of the invention, the weight ratio of the oxidized polysaccharide to the aminated β -cyclodextrin in step 3) is 1: 1-2, preferably 1: 1.08.
in one or more embodiments of the invention, in the step 6), the volume ratio of the adamantane-modified bacterial spores to the beta-cyclodextrin grafted polysaccharide solution is 2: 1.
in a second aspect of the invention, the invention provides the use of polysaccharide-embedded bacterial spores in the preparation of an anti-cancer medicament;
preferably, the cancer is colon cancer;
preferably, the polysaccharide-embedded bacterial spores are prepared by the preparation method of the first aspect of the invention.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention provides a mild bacterial surface chemical modification means, prepares bacterial spores embedded by polysaccharide, has simple and convenient operation and mild reaction conditions, and is easy for industrialization;
2. the bacterial spores embedded by the polysaccharide are obtained by combining the bacterial spores and the polysaccharide through the chemical action of the host and the guest, and are applied to the preparation of anti-cancer drugs, in particular to the preparation of anti-colon cancer drugs.
3. The polysaccharide embedded bacterial spores obtained by the invention have long detention time in intestinal tracts and have obvious anticancer effect.
4. The selected bacteria can effectively enrich the tumor part, in addition, because the polysaccharide is easy to modify, the chemotherapy drug can be flexibly loaded into the system, and the polysaccharide generates short chain fatty acid with anticancer effect through bacterial spore fermentation, thereby safely and effectively inhibiting colon cancer. In various tumor models, the bacterial spore-polysaccharide system is found to be enriched in tumor tissues through oral administration, and the bacterial spore-polysaccharide system treatment shows satisfactory tumor inhibition effect and anti-invasion capacity in a mouse subcutaneous colon cancer model.
5. The selected bacteria and polysaccharide are approved for food application, and meanwhile, the treatment system does not show any toxicity or side effect in research, so that the practicability is strong.
Drawings
FIG. 1 is a schematic diagram of the present invention;
FIG. 2 is the NMR chart of dextran (dextran) according to the present invention, aminated β -cyclodextrin (EDA- β -CD) obtained in example 1, oxidized dextran (PAD), β -cyclodextrin grafted dextran (Dex-g- β -CD);
FIG. 3 is a confocal picture of spore, glucan, polysaccharide embedded bacterial spores (spore-glucan), wherein FIG. 3a is a confocal picture of spores; FIG. 3b confocal pictures of dextran; FIG. 3c is a confocal picture of polysaccharide embedded bacterial spores (spore-glucan);
FIG. 4 is a graph showing the results of fluorescence intensity of bacteria and spores after gastric lavage of fluorescence-labeled polysaccharide-embedded bacteria (spore-glucan), bacteria and spores, respectively, retained in the intestinal tract for 12 hours in example 2 of the present invention after fasting for 48 hours;
FIG. 5 is a graph showing the results of production of short chain fatty acids (propionic acid, n-butyric acid, valeric acid salt, isocaproic acid, caproic acid) by spores (spore-glucan) of polysaccharide-embedded bacteria and spores in example 3 of the present invention;
FIG. 6 is a graph showing the survival rate of cancer cells in the polysaccharide-embedded bacterial spore (spore-glucan) group and the spore group under the simulated intestinal hypoxia environment in example 3 of the present invention;
FIG. 7 is a graphical representation of the tumor volume size results for diclofenac and sporular-dextran @ diclofenac in example 4 of the present invention;
Detailed Description
The scheme of the present invention will be explained below with reference to examples and comparative examples. It will be understood by those skilled in the art that the following examples and comparative examples are illustrative of the present invention only and should not be construed as limiting the scope of the present invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
The schematic diagram of the technical scheme of the invention is shown in figure 1, and the diagram shows that the polysaccharide grafted and modified by beta-cyclodextrin and the bacterial spore modified by adamantane are prepared, then the polysaccharide grafted and modified by beta-cyclodextrin and the bacterial spore modified by adamantane have a host-guest chemical action to obtain the bacterial spore embedded by polysaccharide, and the polysaccharide is fermented by the bacterial spore to generate short-chain fatty acid with an anti-cancer effect, so that the cancer is safely and effectively inhibited.
Example 1
The preparation method of the mild polysaccharide-embedded bacterial spore comprises the following specific steps:
step 1): preparation of aminated β -cyclodextrin: dissolving 10g of beta-cyclodextrin in 100ml of 0.3M sodium hydroxide solution, adding 15g of p-toluenesulfonyl chloride for reaction, stirring at 4 ℃ for 1h, and filtering to remove unreacted p-toluenesulfonyl chloride. The precipitate was filtered and washed three times with 300mL of acetone and then dried in vacuo. The white powder was recrystallized 3 times from water at 80 ℃ and dried in vacuo at 40 ℃ for 48h to give a white intermediate.
Step 2): 2.5g of the white intermediate obtained in step 1) were weighed out, dissolved in 15mL of ethylenediamine at room temperature and stirred for 48 h. Precipitating with 300mL acetone, filtering, washing for three times to obtain white powder, recrystallizing with water at 80 deg.C for 5 times, and vacuum drying to obtain aminated beta-cyclodextrin (EDA-beta-CD).
Step 3): preparation of oxidized polysaccharide: 1g dextran (dextran) was dissolved in 40mL deionized water, 0.1g potassium periodate was added, the reaction was carried out for 20 hours, dialyzed in water for 3 days, and the product was freeze-dried to obtain oxidized dextran (PAD).
Step 4): preparation of beta-cyclodextrin grafted modified glucan: 0.5g of oxidized dextran obtained in step 3) was dissolved in 10mL of distilled water, 0.54g of aminated β -cyclodextrin obtained in step 2) was dissolved in 20mL of water, and the above solutions were mixed and stirred for 24 hours. The resulting reaction solution was dialyzed against water for 3 days. Beta-cyclodextrin graft-modified dextran (Dex-g-beta-CD) was obtained by freeze-drying the precipitate.
Step 5): and (3) purifying bacterial spores: and (3) exciting clostridium butyricum to form spores by using a spore production culture medium, and centrifugally collecting the precipitated spores. Slowly adding the separated spores into a 65% sucrose and 35% sucrose solution, centrifuging, forming 2 layers of precipitates in a centrifuge tube, extracting the precipitates, and dispersing the precipitates in PBS to obtain a purified bacterial spore solution.
Step 6): preparation of adamantane-modified bacterial spores: 1mg of adamantanecarboxylic acid was added to 100ml of a phosphate buffer solution, and then 3.2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.3mg of N-hydroxysuccinimide were dissolved in 1ml of the phosphate buffer solution, and the above solutions were mixed and shaken at 37 ℃ for 30 minutes. 10mL of the purified bacterial spore solution (5X 10)8CFUmL-1) Adding into the reaction mixture, shaking the solution at 37 deg.C for 1h, centrifuging, and collecting precipitate. The precipitate was dispersed in 3ml of phosphate buffer to obtain adamantane-modified bacterial spores.
Step 7): preparation of polysaccharide-embedded bacterial spores: 1mL of the adamantane-modified bacterial spores (5X 10) obtained in step 6)8CFUmL-1) Grafting the modified glucan with 500 mu L of the beta-cyclodextrin obtained in the step 4) (2 mgmL)-1And) mixing the solutions, shaking at 37 ℃ for 15min, and centrifuging to collect the precipitate to obtain polysaccharide-embedded bacterial spores (spore-glucan).
Ammonia obtained in the above stepThe NMR spectrum of the beta-cyclodextrin (EDA-beta-CD), oxidized dextran (PAD), beta-cyclodextrin grafted dextran (Dex-g-beta-CD) is shown in FIG. 2 by1H-NMR demonstrated successful preparation of EDA-beta-CD, Dex-g-beta-CD and PAD.
FIG. 3 is a confocal picture of spore, glucan, and polysaccharide embedded bacterial spores (spore-glucan). Wherein (a) is a spore; (b, glucan, (c) spore-glucan, as shown in the figure, glucan was successfully embedded on the surface of the spore.
Example 2
The spore-glucan prepared in example 1 was used for the study of intestinal retention capacity, and the specific study method was as follows:
step 1): mice were fasted for 48 hours and then randomized into three groups, each containing 100 μ L of each of the polysaccharide-embedded bacterial spores (spore-glucan) obtained in example 1, clostridium butyricum (bacteria) obtained in example 1, and spores obtained in example 1, which were gastric gavage and fluorescently labeled.
Step 2): at different time points (1 h, 7h, 6h, 12h post-dose), 3 mice per group were euthanized and their intestines were taken and imaged with the IVIS system.
The results of the experiment after 12 hours of administration are shown in FIG. 4, and it is understood from the results of the experiment that the fluorescence intensity of bacterial spores embedded with residual polysaccharide in the digestive tract is about 19 times that of the bacteria and spores. The experiment result proves that the polysaccharide coating can prolong the retention time of spores in intestinal tracts.
Example 3
The polysaccharide-embedded bacterial spores (spore-glucan) prepared in example 1 were tested for their ability to produce Short Chain Fatty Acids (SCFAs) in vitro and for their cytotoxicity, and the specific study method was as follows:
step 1): the formation of Short Chain Fatty Acids (SCFAs) was verified by high performance liquid chromatography-mass spectrometry (HPLC-MS).
Step 2): CT26 cells were hypoxic cultured in 4 96-well plates, and polysaccharide-embedded bacterial spores (spore-glucan) prepared in example 1 and spores were added in concentration gradients, respectively, all at 37 ℃ and 20% CO2The contents were incubated for 24 hours in the dark. mu.L of MTT (5mg/mL) was added to each well, and after 4 hours of incubation, the medium was aspirated,add 150. mu.L of dimethyl sulfoxide (DMSO) per well. The microplate reader measures the absorbance of all wells at 570nm and calculates the corresponding cell viability.
The results of polysaccharide-embedded bacterial spores (spore-glucan) and spore production of short chain fatty acids (propionic acid, n-butyric acid, valerate, isocaproic acid, caproic acid) are shown in FIG. 5, and it can be seen that both the polysaccharide-embedded bacterial spores (spore-glucan) and spores produce a large amount of short chain fatty acids, especially n-butyric acid. In addition, glucan encapsulation significantly increases the amount of propionic, valeric, isocaproic and caproic acids produced by the spores.
As shown in fig. 6, in the simulated intestinal hypoxia environment, the polysaccharide-embedded bacterial spore (spore-glucan) group was found to have a cancer cell survival rate of less than half. This experimental phenomenon demonstrates the anticancer effect of sporulated dextran.
Example 4
The polysaccharide-embedded bacterial spore (spore-glucan) prepared in example 1 is loaded with a chemotherapeutic drug diclofenac, and the anti-tumor effect of the polysaccharide-embedded bacterial spore (spore-glucan) drug-loaded platform is studied, wherein the specific study scheme is as follows:
step 1): spore-dextran @ diclofenac was prepared by mixing Dex-g-beta-CD (1.0g), diclofenac (1.1g), N-hydroxysuccinimide solution (0.6g) prepared in example 1, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.1g) and N, N-dimethylformamide (25mL), stirring at room temperature for 24h, dialyzing against deionized water for 3 days, and lyophilizing to give a powder. 1mL of the adamantane-modified spore prepared in example 1 was added to 0.5mL of the powder solution (2 mg/mL). Shaking for 15 minutes at 37 ℃, and centrifuging to obtain the spore-glucan @ diclofenac.
Step 2): when the size of the subcutaneous CT26 tumor is about 100cm3In time, tumor mice were randomly divided into 2 groups of 100 μ L each of diclofenac and sporular-dextran @ diclofenac. Subcutaneous tumor size was recorded every other day during treatment.
The schematic diagram of the tumor volume results of diclofenac and spore-glucan @ diclofenac is shown in fig. 7, and the tumors of mice related to the spore-glucan @ diclofenac are much smaller than those of the diclofenac group, which indicates that the spore-glucan can remarkably enhance the anti-cancer effect of the chemotherapeutic drug diclofenac. This example illustrates the establishment of a sporulation-dextran drug-loaded platform that opens up a unique and novel treatment modality for colon cancer treatment.
Although the embodiments and comparative examples of the present invention have been shown and described above, it is understood that the above embodiments and comparative examples are illustrative and not to be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention.
Claims (10)
1. A method for preparing polysaccharide-embedded bacterial spores, wherein the polysaccharide-embedded bacterial spores are prepared from a first chemically-modified polysaccharide and a second chemically-modified bacterial spore.
2. The method of claim 1, wherein the first chemical modification is a β -cyclodextrin graft modification; the second chemical modification is an adamantane modification.
3. The method of claim 1, comprising the steps of:
step 1): preparation of aminated β -cyclodextrin: dissolving beta-cyclodextrin in NaOH solution, adding p-methylbenzenesulfonyl chloride, carrying out vacuum drying to obtain a white intermediate product, dissolving the obtained white intermediate product in ethylenediamine, stirring for 20-50h, adding acetone for washing, drying, recrystallizing in water at 60-90 ℃ for 3-5 times, and carrying out vacuum drying to obtain aminated beta-cyclodextrin;
step 2): preparation of oxidized polysaccharide: dissolving polysaccharide in deionized water, adding potassium periodate, reacting for 6-24h, dialyzing, and lyophilizing to obtain oxidized polysaccharide;
step 3): preparing polysaccharide grafted and modified by beta-cyclodextrin: dissolving the oxidized polysaccharide obtained in the step 2) and the aminated beta-cyclodextrin obtained in the step 1) in water, stirring for reaction for 10-20h, dialyzing, and freeze-drying to obtain beta-cyclodextrin grafted and modified polysaccharide;
step 4): and (3) purifying bacterial spores: inducing bacteria to form spores by using a spore production culture medium, centrifugally collecting the spores, purifying the separated spores by a sucrose gradient centrifugation method, adding the spores into 35% and 65% sucrose solutions, forming 2 layers of precipitates in a centrifugal tube after centrifugation, extracting the precipitates, and dispersing the precipitates in PBS to obtain a purified bacterial spore solution;
step 5): preparation of adamantane-modified bacterial spores: adding adamantane carboxylic acid into Phosphate Buffer Solution (PBS), dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the PBS, and mixing the two solutions to obtain a mixed solution; adding the purified bacterial spore solution obtained in the step 4) into the mixed solution, oscillating for 1-10h, centrifugally collecting precipitate, and dispersing the precipitate into PBS to obtain adamantane-modified bacterial spores;
step 6): preparation of polysaccharide-embedded bacterial spores: mixing the beta-cyclodextrin graft modified polysaccharide solution with the mass volume concentration of 0.1-10% prepared by taking the beta-cyclodextrin graft modified polysaccharide obtained in the step 3) as a solute with the adamantane modified bacterial spore obtained in the step 5), oscillating for 10-40min at 37 ℃ in a shaking table, and centrifuging and collecting solids to obtain the bacterial spore embedded by polysaccharide.
4. The method for producing polysaccharide-embedded bacterial spores as claimed in any one of claims 1 to 3, wherein the bacterial spores are spores of one or more bacteria selected from yeast, probiotic bacteria, Clostridium butyricum, Lactobacillus, Bifidobacterium, and Actinomycetes.
5. The method of producing polysaccharide embedded bacterial spores as claimed in any one of claims 1 to 3, wherein the polysaccharide is selected from one or more of polyfructose, polygalactose, dextran, isomaltose and hyaluronic acid.
6. The method of claim 3, wherein in step 1), the weight ratio of β -cyclodextrin to p-toluenesulfonyl chloride is 2: 3.
7. the method of claim 3, wherein the weight ratio of polysaccharide to potassium periodate in step 2) is 10: 1.
8. the method of claim 3, wherein the weight ratio of oxidized polysaccharide to aminated β -cyclodextrin in step 3) is 1: 1 to 2.
9. The method for preparing polysaccharide-embedded bacterial spores as claimed in claim 3, wherein the volume ratio of the adamantane-modified bacterial spores to the beta-cyclodextrin grafted polysaccharide solution in step 6) is 2: 1.
10. the application of bacterial spores embedded by polysaccharide in the preparation of anti-cancer drugs;
preferably, the cancer is colon cancer;
preferably, the polysaccharide-embedded bacterial spore is prepared by the preparation method of any one of claims 1 to 9.
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