CN115737600B - Continuous hydrogen-producing biological microsphere and preparation method and application thereof - Google Patents
Continuous hydrogen-producing biological microsphere and preparation method and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/36—Hydrogen production from non-carbon containing sources, e.g. by water electrolysis
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- Medicinal Preparation (AREA)
Abstract
The invention discloses a continuous hydrogen-producing biological microsphere, a preparation method and application thereof, wherein the hydrogen-producing biological microsphere consists of calcium alginate gel microspheres carrying hydrogen-producing microorganisms and polylysine semipermeable membranes coated on the surfaces of the calcium alginate gel microspheres, and the preparation method comprises the following steps: (1) Mixing the sodium alginate solution with hydrogen-producing microorganisms to obtain a mixed solution, and dripping the mixed solution into a calcium chloride solution by using an electrostatic dripping method to form calcium alginate gel microspheres carrying hydrogen-producing microorganisms; (2) Reacting the microsphere with polylysine solution to obtain the hydrogen-producing biological microsphere. The preparation method is simple and has good controllability, the prepared hydrogen-producing biological microsphere can continuously release hydrogen by using living hydrogen-producing microorganisms in the microsphere and using tumor tissue metabolites such as glucose, pyruvic acid and the like as substrates, and can selectively pass through fermentation substrates and hydrogen by using the polylysine semipermeable membrane, and meanwhile, hypersensitivity or biotoxic effects caused by microbial overflow are prevented, so that a new thought is provided for treating tumors by clinical gases.
Description
Technical Field
The invention relates to the technical field of biological microsphere preparation, in particular to a continuous hydrogen-producing biological microsphere, and a preparation method and application thereof.
Background
The hydrogen therapy can inhibit proliferation and metastasis of tumor, and can relieve adverse reaction of tumor radiotherapy and chemotherapy, and has almost no side effect. The control of the effective accumulation of hydrogen at the focus part is the key for ensuring the hydrogen treatment effect of tumors. However, high-pressure hydrogen is inflammable and has explosion hazard, and is difficult to be widely applied to clinic at the present stage. Furthermore, because the solubility of hydrogen is low, hydrogen molecules can be diffused in the body at will, so that the hydrogen molecules are difficult to effectively reach and accumulate in a large amount in deep focus tissues, and the treatment effect is limited.
Currently, hydrogen supply routes for hydrogen therapy include: sucking hydrogen-oxygen mixture, injecting hydrogen-enriched physiological saline and orally taking hydrogen-enriched water. For example, patent CN201910163975.9 discloses a safe hydrogen absorber, which outputs a mixed gas of hydrogen and oxygen with a concentration of about 2% for inhalation by a patient, and hydrogen diffuses into the human body through the respiratory system to treat diseases. However, the common hydrogen breathing machine has the defects of low hydrogen and oxygen preparation efficiency, poor smoothness of a gas path, low accurate control degree of hydrogen and oxygen output and the like, and after a period of use, the patient is easy to be infected if the patient is not thoroughly cleaned. Patent CN202121453005.1 discloses a hydrogen-rich water machine, which is to mix hydrogen gas with drinking water under pressure, output hydrogen-rich water with hydrogen concentration of about 1.6ppm for patients to drink, and the hydrogen in the water diffuses into human body through digestive system to treat diseases. Hydrogen in hydrogen-rich water is prone to dissociation and escape at normal temperature and pressure, resulting in the actual hydrogen intake of patients often being lower than expected. In addition, the hydrogen taken in by oral or inhalation can be freely diffused in the body, so that the hydrogen concentration with therapeutic effect is difficult to reach in deep tissues, and the effect is limited. The above hydrogen transfer method cannot target a tumor, resulting in a lower hydrogen concentration at the tumor site, and the above hydrogen transfer method cannot realize continuous hydrogen supply to act on the tumor site, resulting in an unsatisfactory therapeutic effect.
The development of hydrogen supply nano-carriers (such as nano palladium hydride and magnesium boride nano-sheets) provides an effective solution for tumor targeted hydrogen supply. However, the nanoparticle has limited loading capacity, continuous hydrogen supply cannot be realized, the nanoparticle is difficult to degrade in vivo, part of the nanoparticle containing heavy metal ions has biotoxicity, and the safety cannot be ensured. Wan et al constructed liposome nanoparticle (Wan W L,Lin Y J,Chen H L,et al.In situ nanoreactor for photosynthesizing H2 gas to mitigate oxidative stress in tissue inflammation[J].Journal of the American Chemical Society,2017,139(37):12923-12926), embedding chlorophyll a, ascorbic acid and gold nanoparticles, and can realize catalytic hydrogen production at tumor sites under infrared light catalysis. However, the limited penetration depth of infrared light in the tissue greatly limits the catalytic hydrogen production capacity, and the liposome membrane has high permeability to hydrogen, so that the hydrogen can escape quickly and is difficult to effectively accumulate. Therefore, how to realize effective storage, targeted delivery and controllable release of hydrogen has important significance for improving the hydrogen treatment effect and clinical application.
Disclosure of Invention
The invention aims to solve the technical problem of providing a continuous hydrogen-producing biological microsphere, a preparation method and application thereof, wherein calcium alginate is used for embedding active hydrogen-producing microorganisms, carboxylate ions on the surface of the calcium alginate react with amino groups on the surface of polylysine, a polylysine semipermeable membrane is formed on the surface of the calcium alginate gel microsphere for embedding the hydrogen-producing microorganisms, and the hydrogen-producing biological microsphere is prepared, so that continuous hydrogen supply can be realized.
In order to solve the technical problems, the invention provides the following technical scheme:
The first aspect of the invention provides a hydrogen-producing microorganism microsphere, which consists of a calcium alginate gel microsphere carrying hydrogen-producing microorganisms and a polylysine semipermeable membrane coated on the surface of the calcium alginate gel microsphere.
Further, the hydrogen-producing microorganism is one or more of domesticated enterobacter aerogenes, clostridium butyricum and chlamydomonas reinhardtii.
Further, the acclimatization is specifically: in the culture medium for culturing the microorganisms, the proportion of the cell culture medium in the culture medium is gradually increased until the bacteria completely adapt to the environment of the cell culture medium, and the domesticated microorganisms are obtained.
In a second aspect, the present invention provides a method for preparing hydrogen-producing biological microspheres according to the first aspect, comprising the steps of:
(1) Mixing the sodium alginate solution with hydrogen-producing microorganisms to obtain a mixed solution, and dripping the mixed solution into a calcium chloride solution by using an electrostatic dripping method to form calcium alginate gel microspheres carrying hydrogen-producing microorganisms;
(2) Immersing the calcium alginate gel microsphere carrying hydrogen-producing microorganism prepared in the step (1) into polylysine solution, and reacting to obtain the hydrogen-producing microorganism microsphere.
Further, in the step (1), the density of the hydrogen-producing microorganism in the mixed solution is 5×10 7～109 cells/mL.
Further, in the step (1), the sodium alginate solution is obtained by dissolving sodium alginate in sodium chloride solution; the concentration of the sodium alginate solution is 1.3-2.0% (w/v).
Further, in the step (1), the concentration of the calcium chloride solution is 0.8-2.0% (w/v).
Further, in the step (1), the electrostatic droplet method specifically includes: adding the mixed solution into a syringe, and dripping the mixed solution into the calcium chloride solution at a constant speed by using a syringe pump under the action of an electrostatic field.
Further, the voltage of the electrostatic field is 4.0-5.5 kv, and the flow rate of the injector is 5-15 mL/h.
Further, the specification of the syringe needle is 0.4-0.7 mm.
Further, in the step (1), the diameter of the calcium alginate gel microsphere is 100-300 μm.
Further, in the step (1), after the mixed solution is dropped into the calcium chloride solution to form gel microspheres for 10-30 min, the microspheres are collected by natural precipitation.
Further, in the step (2), the concentration of the polylysine solution is 0.05% -0.2% (w/v).
Further, immersing the calcium alginate gel microspheres loaded with hydrogen-producing microorganisms in a polylysine solution, and placing the solution on a shaking table for reaction for 10-20 min; the rotating speed of the shaking table is 20-100 rpm.
The invention discloses a method for preparing hydrogen-producing microorganism microspheres, which comprises the steps of coating living hydrogen-producing microorganisms in calcium alginate gel microspheres by an electrostatic liquid drop method, and forming polylysine semipermeable membranes on the surfaces of the microspheres by utilizing the reaction of carboxylate ions rich in the surfaces of calcium alginate and amino groups on the surfaces of polylysine; the living hydrogen-producing microorganism inside the hydrogen-producing microorganism microsphere takes glucose, pyruvic acid and the like as substrates for fermentation and hydrogen production, and the semipermeable membrane on the surface of the microsphere has selective permeability to the fermentation substrates and hydrogen, so that the microsphere can realize continuous hydrogen supply.
In a third aspect, the present invention provides an application of the hydrogen-producing biological microsphere in preparing a hydrogen therapeutic drug.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention provides a hydrogen-producing biological microsphere, which is obtained by embedding hydrogen-producing microorganisms acclimatized to the physiological environment of a human body into calcium alginate gel to form a microsphere and coating polylysine semipermeable membranes on the surfaces of the microsphere, wherein the microorganisms in the hydrogen-producing biological microsphere take glucose, pyruvic acid and the like as substrates for fermentation hydrogen production, and the fermentation substrates and hydrogen can selectively pass through the semipermeable membranes to realize continuous hydrogen supply.
2. The hydrogen-producing biological microspheres prepared by the invention have high hydrogen production in different culture mediums (including cell culture mediums) and are little influenced by external environment; microorganisms are limited in the microspheres under the action of the calcium alginate gel substrate and the semipermeable membrane, so that hypersensitivity or biotoxicity caused by the overflow of the microorganisms or components thereof is effectively prevented, and the microorganisms are protected from being identified and cleared by immune cells in the body, thereby playing the best hydrogen production efficiency.
3. The hydrogen-producing biological microsphere prepared by the invention can reach a part (such as a tumor part) to be treated by hydrogen through an intervention means, so that continuous hydrogen supply is realized at a target part, and the effective hydrogen concentration of hydrogen treatment is achieved; in addition, the tumor tissue metabolite can be used as a fermentation substrate of the microorganism, so that the microsphere can continuously supply hydrogen at the tumor tissue part without providing additional fermentation substrate. The continuous hydrogen-producing biological microsphere provided by the invention provides a new idea for treating tumors by clinical gas.
Drawings
FIG. 1 is a schematic diagram of the preparation process of hydrogen-producing biological microspheres in example 1;
FIG. 2 is an optical micrograph of hydrogen-producing microspheres prepared in example 1 cultured in culture medium #1 for 0 and 48 hours;
FIG. 3 shows the growth of bacteria after 48 hours of culture of hydrogen-producing microspheres prepared in example 1 in culture medium # 1;
FIG. 4 shows the hydrogen production of MgB 2, domesticated Enterobacter aerogenes and hydrogen-producing biological microspheres after 48h of culture in different media;
FIG. 5 is a graph showing the variation of hydrogen production amount of the hydrogen-producing microbial pellets prepared in example 1 in a #1 medium at 37℃for continuous anaerobic fermentation.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
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 1
The embodiment relates to a preparation method of hydrogen-producing biological microspheres, the preparation process is shown in figure 1, and the specific operation is as follows:
(1) Centrifuging the domesticated enterobacter aerogenes at 8000rpm, and mixing with 2mL of 1.5% (w/v) sodium alginate solution to obtain a mixed solution, wherein the density of the enterobacter aerogenes in the mixed solution is 1 multiplied by 10 8; sucking the mixed solution by using a syringe (the needle is 0.5 mm), dripping the mixed solution into the calcium chloride solution at a constant speed by using a syringe pump (the flow rate is 8.2 mL/h) under the action of an electrostatic field (the voltage is 4.5 kV), gelatinizing for 10min, and naturally precipitating to collect 100 mu M calcium alginate gel microspheres of the enterobacter aerogenes;
(2) Mixing the calcium alginate gel microspheres loaded with the enterobacter aerogenes collected in the step (1) with 10ml of 0.05% w/v polylysine solution in a volume ratio of 1:10, and placing the mixture in a shaking table to react for 10min at a rotating speed of 50rpm to form gel microspheres (hydrogen-producing biological microspheres) internally encapsulating the enterobacter aerogenes and surface-encapsulating the semipermeable membrane.
Example 2
The embodiment relates to a preparation method of hydrogen-producing biological microspheres, the preparation process is shown in figure 1, and the specific operation is as follows:
(3) Centrifuging the domesticated enterobacter aerogenes at 8000rpm, and mixing with 2mL of 1.5% (w/v) sodium alginate solution to obtain a mixed solution, wherein the density of the enterobacter aerogenes in the mixed solution is 5 multiplied by 10 7; sucking the mixed solution by using a syringe (the needle is 0.5 mm), dripping the mixed solution into the calcium chloride solution at a constant speed by using a syringe pump (the flow rate is 8.2 mL/h) under the action of an electrostatic field (the voltage is 4.5 kV), gelatinizing for 10min, and naturally precipitating to collect 100 mu M calcium alginate gel microspheres of the enterobacter aerogenes;
(4) Mixing the calcium alginate gel microspheres loaded with the enterobacter aerogenes collected in the step (1) with 10ml of 0.05% w/v polylysine solution in a volume ratio of 1:10, and placing the mixture in a shaking table to react for 10min at a rotating speed of 50rpm to form gel microspheres (hydrogen-producing biological microspheres) internally encapsulating the enterobacter aerogenes and surface-encapsulating the semipermeable membrane.
Performance testing
Preparation of #1 medium: adding K 2HPO4、NH4SO4、MgSO4, yeast extract and glucose into water, and mixing to obtain culture medium #1, wherein the concentration of K 2HPO4 in the culture medium #1 is 6g/L, NH 4SO4, the concentration of K 2HPO4 in the culture medium #1 is 2g/L, mgSO 4, the concentration of yeast extract is 0.4g/L, the concentration of glucose is 5g/L, and the concentration of glucose is 3g/L.
1. Stability of enterobacter aerogenes in microspheres
The hydrogen-producing organism microsphere prepared in example 1 was suspended in a #1 medium, cultured in an incubator at 37 ℃ for 48 hours, and the morphology of the microsphere before and after the culture was observed by an optical microscope, as shown in fig. 2, the density of enterobacter aerogenes inside the microsphere after the culture for 48 hours was significantly increased, which also indicates that the enterobacter aerogenes inside the microsphere can be propagated in a large amount in the #1 medium environment.
The growth condition of the enterobacter aerogenes in the hydrogen-producing biological microspheres after 48h of culture is observed by using a fluorescence microscope, as shown in fig. 3, a part of enterobacter aerogenes die, but a large amount of surviving enterobacter aerogenes still exist in the microspheres, the surviving enterobacter aerogenes are basically wrapped in the microspheres, and no obvious diffusion is found outside the microspheres, which also shows that the enterobacter aerogenes can be well limited in the semipermeable membrane by the hydrogen-producing biological microspheres prepared by the invention.
2. Hydrogen production efficiency of hydrogen-producing biological microspheres
The hydrogen-producing biological microsphere and the enterobacter aerogenes prepared in example 1 are respectively inoculated into two culture mediums #1 and 1640 (CORNING) for fermentation by taking magnesium boride nano-sheets (Leyan, cat# 1268675) as reference substances, the quantity of the enterobacter aerogenes in the microsphere is controlled to be the same as that of the enterobacter aerogenes singly, and the hydrogen production quantity is measured after 48 hours of culture. Test data are expressed as mean ± standard deviation, and ***P＜0.001,* P < 0.05 were compared to control using a two-tailed T-test. The test results are shown in fig. 4:
The 1640 culture medium is a cell culture medium, the domesticated enterobacter aerogenes completely adapts to the environment of the cell culture medium, and the hydrogen production amount of the culture medium in the 1640 culture medium is slightly higher than that of the microspheres and is obviously higher than that of the magnesium boride nano-sheets, which also shows that the enterobacter aerogenes and the hydrogen-producing biological microspheres prepared by the invention can have high hydrogen production efficiency in the in-vivo environment.
In the culture medium #1, the hydrogen production of the domesticated enterobacter aerogenes is obviously reduced compared with that in the culture medium 1640, and is far lower than that of the hydrogen-producing biological microspheres, and the hydrogen production of the hydrogen-producing biological microspheres cultured in two different culture mediums for 48 hours is similar and is higher than that of the magnesium boride nano-sheets. The hydrogen-producing biological microsphere prepared by the method is characterized in that the calcium alginate gel is embedded and the semipermeable membrane is wrapped, so that the enterobacter aerogenes in the microsphere is isolated from the external environment, the hydrogen production capacity is not greatly influenced by the change of the external environment, and the stable and efficient hydrogen production capacity is realized.
3. Continuous hydrogen production capability of hydrogen-producing biological microsphere
The hydrogen-producing biological microspheres prepared in example 2 were inoculated into medium #1, anaerobic fermentation was continued at 37 ℃ and the hydrogen production was measured and the culture broth was updated at 24h intervals, and the test data were expressed as mean ± standard deviation. The test results are shown in fig. 5:
The hydrogen production of the hydrogen-producing biological microspheres increases rapidly from-2 nmol/mL to-23 nmol/mL in the first 3 days, the hydrogen production on the 3 rd to 6 th days is relatively stable, and the hydrogen production per day fluctuates within 20-27 nmol/mL. Therefore, the hydrogen-producing biological microsphere prepared by the invention has continuous hydrogen production capacity and high hydrogen production capacity.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (4)
1. The hydrogen-producing biological microsphere is characterized by comprising calcium alginate gel microspheres carrying hydrogen-producing microorganisms and a polylysine semipermeable membrane coated on the surfaces of the calcium alginate gel microspheres; the hydrogen-producing microorganism is one or more of domesticated enterobacter aerogenes, clostridium butyricum and chlamydomonas reinhardtii;
the preparation method of the hydrogen-producing biological microsphere comprises the following steps:
(1) Mixing the sodium alginate solution with hydrogen-producing microorganisms to obtain a mixed solution, and dripping the mixed solution into a calcium chloride solution by using an electrostatic dripping method to form calcium alginate gel microspheres carrying hydrogen-producing microorganisms;
(2) Immersing the calcium alginate gel microspheres carrying hydrogen-producing microorganisms prepared in the step (1) into polylysine solution, and reacting to obtain the hydrogen-producing microorganism microspheres;
In the step (1), the density of hydrogen-producing microorganisms in the mixed solution is 5 multiplied by 10 7~1 × 109 cells/mL; the sodium alginate solution is prepared by dissolving sodium alginate in sodium chloride solution; the concentration of sodium alginate in the sodium alginate solution is 1.3-2.0% (w/v); the concentration of the calcium chloride solution is 0.8-2.0% (w/v);
in the step (2), the concentration of the polylysine solution is 0.05% -0.2% (w/v).
2. The hydrogen-producing biological microsphere according to claim 1, wherein in the step (1), the electrostatic droplet method specifically comprises: adding the mixed solution into a syringe, and dripping the mixed solution into the calcium chloride solution at a constant speed by using a syringe pump under the action of an electrostatic field; the voltage of the electrostatic field is 4.0-5.5 kv, and the flow rate of the injector is 5-15 mL/h.
3. The hydrogen-producing biological microsphere according to claim 1, wherein in the step (1), the diameter of the calcium alginate gel microsphere is 100-300 μm.
4. Use of hydrogen-producing biological microspheres according to any one of claims 1-3 for the manufacture of a medicament for the treatment of tumors with hydrogen.
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| CA2243352A1 (en) * | 1996-01-23 | 1997-07-31 | Mcgill University | Microencapsulated genetically engineered microorganisms for clinical application |
| CA2437250A1 (en) * | 2003-08-14 | 2005-02-14 | Ims Recherche Inc. | Microencapsulation of living cells in covalently crosslinked alginate-poly-l-lysine-alginate membranes |
| CN102101036A (en) * | 2009-12-18 | 2011-06-22 | 中国科学院大连化学物理研究所 | Alginate-epsilon-polylysine microcapsules and preparation and application thereof |
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| CA2243352A1 (en) * | 1996-01-23 | 1997-07-31 | Mcgill University | Microencapsulated genetically engineered microorganisms for clinical application |
| CA2437250A1 (en) * | 2003-08-14 | 2005-02-14 | Ims Recherche Inc. | Microencapsulation of living cells in covalently crosslinked alginate-poly-l-lysine-alginate membranes |
| CN102101036A (en) * | 2009-12-18 | 2011-06-22 | 中国科学院大连化学物理研究所 | Alginate-epsilon-polylysine microcapsules and preparation and application thereof |
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