CN115737600B - Continuous hydrogen-producing biological microsphere and preparation method and application thereof - Google Patents

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

A kind of biological microspheres for continuous hydrogen production and its preparation method and application

Technical Field

The present invention relates to the technical field of biomicrosphere preparation, and in particular to a biomicrosphere that continuously produces hydrogen, and a preparation method and application thereof.

Background technique

Hydrogen therapy can inhibit tumor proliferation and metastasis, and at the same time reduce the adverse reactions secondary to tumor radiotherapy and chemotherapy, with almost no side effects. Controlling the effective accumulation of hydrogen in the lesion site is the key to ensuring the effectiveness of hydrogen therapy for tumors. However, high-pressure hydrogen is flammable and explosive, and it is difficult to be widely used in clinical practice at this stage. In addition, due to the low solubility of hydrogen, it can diffuse arbitrarily in the body, making it difficult for hydrogen molecules to effectively reach and accumulate in large quantities in deep lesion tissues, resulting in limited treatment effects.

At present, the hydrogen supply methods for hydrogen therapy include: inhalation of hydrogen-oxygen mixed gas, injection of hydrogen-rich saline, and oral administration of hydrogen-rich water. For example, patent CN201910163975.9 discloses a safe hydrogen inhaler that outputs a hydrogen-oxygen mixed gas with a hydrogen concentration of about 2% for patients to inhale, and the hydrogen diffuses into the human body through the respiratory system to treat diseases. However, ordinary hydrogen respirators generally have the disadvantages of low hydrogen-oxygen preparation efficiency, poor gas path smoothness, and low precision control of hydrogen-oxygen output. After a period of use, if the cleaning is not thorough, it is easy to bring infection risks to patients. Patent CN202121453005.1 discloses a hydrogen-rich water machine that pressurizes and mixes hydrogen with drinking water, outputs hydrogen-rich water with a hydrogen concentration of about 1.6ppm for patients to drink, and the hydrogen in the water diffuses into the human body through the digestive system to treat diseases. The hydrogen in hydrogen-rich water is easy to dissociate and dissipate at normal temperature and pressure, resulting in the patient's actual hydrogen intake often being lower than the expected value. In addition, hydrogen ingested by oral or inhalation will diffuse freely in the body, and it is difficult to reach a hydrogen concentration with therapeutic effects in deep tissues, and the effect is limited. The above hydrogen transfer method cannot target the tumor, resulting in a low hydrogen concentration at the tumor site, and the above hydrogen transfer method cannot achieve continuous hydrogen supply to the tumor site, resulting in unsatisfactory treatment effect.

The development of hydrogen supply nanocarriers (such as nano palladium hydride, magnesium boride nanosheets) provides an effective solution for tumor-targeted hydrogen supply. However, the loading capacity of nanoparticles is limited, and continuous hydrogen supply cannot be achieved. In addition, nanoparticles are difficult to degrade in the body, and some of them contain heavy metal ions with biological toxicity, and their safety cannot be guaranteed. Wan et al. constructed liposome nanoparticles (Wan WL, Lin YJ, Chen HL, et al. In situ nanoreactor for photosynthesizing _{H 2} gas to mitigate oxidative stress in tissue inflammation [J]. Journal of the American Chemical Society, 2017, 139 (37): 12923-12926), embedded chlorophyll a, ascorbic acid and gold nanoparticles, and catalytic hydrogen production can be achieved at the tumor site under infrared photocatalysis. However, the limited penetration depth of infrared light in the tissue body greatly limits its catalytic hydrogen production ability, and the liposome membrane is too permeable to hydrogen, resulting in rapid hydrogen escape and difficulty in effective accumulation. Therefore, how to achieve effective storage, targeted delivery and controlled release of hydrogen is of great significance to improving the therapeutic effect and clinical application of hydrogen.

Summary of the invention

The technical problem to be solved by the present invention is to provide a continuous hydrogen-producing biological microsphere and a preparation method and application thereof. Active hydrogen-producing microorganisms are embedded in calcium alginate, and carboxylate ions on the surface of calcium alginate react with amino groups on the surface of polylysine to form a polylysine semipermeable membrane on the surface of the calcium alginate gel microsphere encapsulating the hydrogen-producing microorganisms, thereby preparing hydrogen-producing biological microspheres that can achieve continuous hydrogen supply.

In order to solve the above technical problems, the present invention provides the following technical solutions:

The first aspect of the present invention provides a hydrogen-producing biological microsphere, which is composed of calcium alginate gel microspheres carrying hydrogen-producing microorganisms and polylysine semipermeable membranes coated on the surface thereof.

Furthermore, the hydrogen-producing microorganism is one or more of domesticated Enterobacter aerogenes, Clostridium butyricum, and Chlamydomonas reinhardtii.

Furthermore, the domestication specifically includes: in the culture medium for culturing microorganisms, gradually increasing the proportion of the cell culture medium in the culture medium until the bacteria are fully adapted to the environment of the cell culture medium, thereby obtaining domesticated microorganisms.

The second aspect of the present invention provides a method for preparing the hydrogen-producing biomicrospheres described in the first aspect, comprising the following steps:

(1) mixing a sodium alginate solution with hydrogen-producing microorganisms to obtain a mixed solution, and dropping the mixed solution into a calcium chloride solution using an electrostatic drop method to form calcium alginate gel microspheres carrying hydrogen-producing microorganisms;

(2) The calcium alginate gel microspheres carrying hydrogen-producing microorganisms prepared in step (1) are immersed in a polylysine solution to react and obtain the hydrogen-producing biological microspheres.

Furthermore, in step (1), the density of hydrogen-producing microorganisms in the mixed solution is 5×10 ⁷ to 10 ⁹ cells/mL.

Furthermore, in step (1), the sodium alginate solution is obtained by dissolving sodium alginate in a sodium chloride solution; and the concentration of the sodium alginate solution is 1.3-2.0% (w/v).

Furthermore, in step (1), the concentration of the calcium chloride solution is 0.8-2.0% (w/v).

Furthermore, in step (1), the electrostatic drop method is specifically as follows: adding the mixed solution into a syringe, and using a syringe pump to uniformly drop the mixed solution into the calcium chloride solution under the action of an electrostatic field.

Furthermore, the voltage of the electrostatic field is 4.0 to 5.5 kV, and the flow rate of the syringe is 5 to 15 mL/h.

Furthermore, the specification of the syringe needle is 0.4-0.7 mm.

Furthermore, in step (1), the diameter of the calcium alginate gel microspheres is 100 to 300 μm.

Furthermore, in step (1), after the mixed solution is dropped into the calcium chloride solution to form gel microspheres for 10 to 30 minutes, the microspheres are collected by natural precipitation.

Furthermore, in step (2), the concentration of the polylysine solution is 0.05% to 0.2% (w/v).

Furthermore, the calcium alginate gel microspheres carrying hydrogen-producing microorganisms are immersed in a polylysine solution and placed on a shaker for reaction for 10 to 20 minutes; the rotation speed of the shaker is 20 to 100 rpm.

The present invention encapsulates live hydrogen-producing microorganisms in calcium alginate gel microspheres by an electrostatic droplet method, and utilizes carboxylate ions rich in the surface of calcium alginate to react with amino groups on the surface of polylysine to form a polylysine semipermeable membrane on the surface of the microspheres to prepare hydrogen-producing microbial microspheres; the live hydrogen-producing microorganisms inside the hydrogen-producing microbial microspheres ferment and produce hydrogen using glucose, pyruvate, etc. as substrates, and the semipermeable membrane on the surface of the microspheres has selective permeability to the fermentation substrate and hydrogen, so the microspheres can achieve continuous hydrogen supply.

The third aspect of the present invention provides a use of the hydrogen-producing biomicrospheres described in the first aspect in preparing hydrogen therapeutic drugs.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention provides a hydrogen-producing biological microsphere, which is formed by embedding hydrogen-producing microorganisms that have been domesticated and adapted to the physiological environment of the human body in calcium alginate gel, and coating the surface of the microsphere with a polylysine semipermeable membrane. The microorganisms in the hydrogen-producing biological microsphere ferment and produce hydrogen using glucose, pyruvate, etc. as substrates, and the fermentation substrate and hydrogen can selectively pass through the semipermeable membrane to achieve continuous hydrogen supply.

2. The hydrogen-producing biological microspheres prepared by the present invention have high hydrogen production in different culture media (including cell culture media) and are less affected by the external environment; the microorganisms are confined inside the microspheres under the action of the calcium alginate gel substrate and the semipermeable membrane, which effectively prevents the overflow of the microorganisms or their components from causing hypersensitivity reactions or biological toxicity, while protecting the microorganisms from being recognized and eliminated by the immune cells in the body, thereby achieving the best hydrogen production efficiency.

3. The hydrogen-producing bio-microspheres prepared by the present invention can reach the site that needs hydrogen treatment (such as the tumor site) through interventional means, thereby achieving continuous hydrogen supply at the targeted site and reaching a hydrogen concentration that is effective for hydrogen treatment; in addition, tumor tissue metabolites can be used as fermentation substrates for microorganisms, so that the microspheres can continuously supply hydrogen at the tumor tissue site without the need for additional fermentation substrates. The continuous hydrogen-producing bio-microspheres provided by the present invention provide a new idea for clinical gas treatment of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the preparation process of hydrogen-producing biomicrospheres in Example 1;

FIG2 is an optical microscopic image of the hydrogen-producing biomicrospheres prepared in Example 1 cultured in #1 medium at 0 and 48 h;

FIG3 shows the growth of bacteria after the hydrogen-producing bio-microspheres prepared in Example 1 were cultured in #1 medium for 48 hours;

FIG4 shows the hydrogen production of MgB ₂ , domesticated Enterobacter aerogenes and hydrogen-producing biomicrospheres after culturing in different culture media for 48 hours;

FIG5 is a curve showing the change in hydrogen production of the hydrogen-producing biomicrospheres prepared in Example 1 in a #1 culture medium under continuous anaerobic fermentation at 37° C.

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used herein includes any and all combinations of one or more related listed items.

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

Example 1

This embodiment relates to the preparation of a hydrogen-producing biomicrosphere. The preparation process is shown in FIG1 , and the specific operations are as follows:

(1) After centrifuging the domesticated Enterobacter aerogenes at 8000 rpm, the mixture was mixed with 2 mL of 1.5% (w/v) sodium alginate solution to obtain a mixed solution, in which the density of Enterobacter aerogenes in the mixed solution was 1×10 ⁸ ; the mixed solution was drawn up using a syringe (needle of 0.5 mm), and the mixed solution was uniformly dripped into a calcium chloride solution using a syringe pump (flow rate of 8.2 mL/h) under the action of an electrostatic field (voltage of 4.5 kV), and gelled for 10 minutes, and 100 μM calcium alginate gel microspheres carrying Enterobacter aerogenes were collected by natural precipitation;

(2) The calcium alginate gel microspheres containing Enterobacter aerogenes collected in step (1) were mixed with 10 mL of 0.05% w/v polylysine solution at a volume ratio of 1:10, and placed in a shaker for reaction at a speed of 50 rpm for 10 min to form gel microspheres (hydrogen-producing biomicrospheres) with Enterobacter aerogenes encapsulated inside and a semipermeable membrane coated on the surface.

Example 2

This embodiment relates to the preparation of a hydrogen-producing biomicrosphere. The preparation process is shown in FIG1 , and the specific operations are as follows:

(3) After centrifuging the domesticated Enterobacter aerogenes at 8000 rpm, the mixture was mixed with 2 mL of 1.5% (w/v) sodium alginate solution to obtain a mixed solution, in which the density of Enterobacter aerogenes in the mixed solution was 5×10 ⁷ ; the mixed solution was drawn up using a syringe (needle of 0.5 mm), and the mixed solution was uniformly dripped into a calcium chloride solution using a syringe pump (flow rate of 8.2 mL/h) under the action of an electrostatic field (voltage of 4.5 kV), and gelled for 10 minutes, and 100 μM calcium alginate gel microspheres carrying Enterobacter aerogenes were collected by natural precipitation;

(4) The calcium alginate gel microspheres containing Enterobacter aerogenes collected in step (1) were mixed with 10 mL of 0.05% w/v polylysine solution at a volume ratio of 1:10, and placed in a shaker for reaction at a speed of 50 rpm for 10 min to form gel microspheres (hydrogen-producing biomicrospheres) with Enterobacter aerogenes encapsulated inside and a semipermeable membrane coated on the surface.

Performance Testing

Prepare medium #1: add K ₂ HPO ₄ , NH ₄ SO ₄ , MgSO ₄ , yeast extract and glucose to water, mix well to obtain medium #1, in which the concentration of K ₂ HPO ₄ is 6 g/L, the concentration of NH ₄ SO ₄ is 2 g/L, the concentration of MgSO ₄ is 0.4 g/L, the concentration of yeast extract is 5 g/L, and the concentration of glucose is 3 g/L.

1. Stability of Enterobacter aerogenes in microspheres

The hydrogen-producing biomicrospheres prepared in Example 1 were suspended in #1 culture medium and cultured in an incubator at 37°C for 48 h. The morphology of the microspheres before and after the culture was observed using an optical microscope. As shown in FIG2 , the density of Enterobacter aerogenes inside the microspheres increased significantly after 48 h of culture, which also indicates that Enterobacter aerogenes inside the microspheres can multiply in large quantities in the #1 culture medium environment.

The growth of Enterobacter aerogenes inside the hydrogen-producing biomicrospheres after 48 hours of culture was observed using a fluorescence microscope. As shown in FIG3 , some Enterobacter aerogenes died, but a large number of surviving Enterobacter aerogenes were still present in the microspheres, and the surviving Enterobacter aerogenes were basically encapsulated inside the microspheres, and no obvious diffusion was found outside the microspheres. This also indicates that the preparation of hydrogen-producing biomicrospheres by the present invention can well confine Enterobacter aerogenes within the semipermeable membrane.

2. Hydrogen production efficiency of hydrogen-producing biomicrospheres

Using magnesium boride nanosheets (Leyan, item number 1268675) as a control, the hydrogen-producing biomicrospheres prepared in Example 1 and Enterobacter aerogenes were inoculated into two culture media #1 and 1640 (CORNING) for fermentation, and the amount of Enterobacter aerogenes in the microspheres was controlled to be the same as that of a single Enterobacter aerogenes. The hydrogen production was measured after 48 hours of culture. The test data are expressed as mean ± standard deviation, and compared with the control group using a two-tailed T test, ^*** P < 0.001, ^* P < 0.05. The test results are shown in Figure 4:

The 1640 culture medium is a cell culture medium. The domesticated Enterobacter aerogenes has fully adapted to the environment of the cell culture medium. The hydrogen production after 48 hours of culture in the 1640 culture medium is slightly higher than that of the microspheres and significantly higher than that of the magnesium boride nanosheets. This also shows that Enterobacter aerogenes and the hydrogen-producing biological microspheres prepared by the present invention can have high hydrogen production efficiency in an in vivo-like environment.

In #1 medium, the hydrogen production of the domesticated Enterobacter aerogenes decreased significantly compared to that in 1640 medium, and was much lower than that of the hydrogen-producing biomicrospheres. The hydrogen production of the hydrogen-producing biomicrospheres cultured in two different mediums for 48 hours was similar, both higher than that of the magnesium boride nanosheets. This also shows that the hydrogen-producing biomicrospheres prepared by the present invention isolate the Enterobacter aerogenes inside the microspheres from the external environment through calcium alginate gel embedding and semipermeable membrane wrapping, and their hydrogen production capacity will not be greatly affected by changes in the external environment, and have stable and efficient hydrogen production capacity.

3. Continuous hydrogen production capacity of hydrogen-producing bio-microspheres

The hydrogen-producing bio-microspheres prepared in Example 2 were inoculated into the #1 culture medium and continued to ferment anaerobicly at 37°C. The hydrogen production was measured every 24 hours and the culture medium was updated. The test data was expressed as mean ± standard deviation. The test results are shown in Figure 5:

The hydrogen production of the hydrogen-producing bio-microspheres increased rapidly in the first three days, from 2 nmol/mL to 23 nmol/mL, and the hydrogen production from the third to the sixth day was relatively stable, with the daily hydrogen production fluctuating between 20 and 27 nmol/mL. It can be seen that the hydrogen-producing bio-microspheres prepared by the present invention have a continuous hydrogen production capacity and a high hydrogen production capacity.

The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims (4)

1. A hydrogen-producing biological microsphere, characterized in that it is composed of calcium alginate gel microspheres carrying hydrogen-producing microorganisms and polylysine semipermeable membranes coated on the surface thereof; the hydrogen-producing microorganisms are one or more of domesticated Enterobacter aerogenes, Clostridium butyricum, and Chlamydomonas reinhardtii; The method for preparing the hydrogen-producing biomicrospheres comprises the following steps: (1) mixing a sodium alginate solution with hydrogen-producing microorganisms to obtain a mixed solution, and dropping the mixed solution into a calcium chloride solution using an electrostatic droplet method to form calcium alginate gel microspheres carrying hydrogen-producing microorganisms; (2) immersing the calcium alginate gel microspheres carrying hydrogen-producing microorganisms prepared in step (1) in a polylysine solution to react and obtain the hydrogen-producing biological microspheres; In step (1), the density of hydrogen-producing microorganisms in the mixed solution is 5 × 10 ⁷ ~1 × 10 ⁹ cells/mL; the sodium alginate solution is obtained by dissolving sodium alginate in a 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 step (2), the concentration of the polylysine solution is 0.05%~0.2% (w/v). 2. The hydrogen-producing biomicrospheres according to claim 1 are characterized in that in step (1), the electrostatic droplet method is specifically: adding the mixed solution into a syringe, and using a syringe pump to uniformly drop the mixed solution into the calcium chloride solution 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 syringe is 5~15 mL/h. 3. The hydrogen-producing biological microspheres according to claim 1, characterized in that in step (1), the diameter of the calcium alginate gel microspheres is 100-300 μm. 4. Use of the hydrogen-producing biomicrospheres according to any one of claims 1 to 3 in the preparation of drugs for treating tumors with hydrogen.