CN114470312A

CN114470312A - Non-leakage packaged yeast hydrogel and preparation method thereof

Info

Publication number: CN114470312A
Application number: CN202210103555.3A
Authority: CN
Inventors: 吴元子; 蔡静雯; 舒京晶; 李玲; 周凯强; 王旭伟; 吕艳玮
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-13
Anticipated expiration: 2042-01-28
Also published as: CN114470312B

Abstract

The invention provides a leak-free encapsulated yeast hydrogel and a preparation method thereof. The leakage-free encapsulated yeast hydrogel is prepared by constructing yeast engineering bacteria carrying human genes, carrying out in-situ oxidation on sugar chains on the surfaces of the obtained engineering yeast, initiating and carrying out specific crosslinking on macromolecules. The hydrogel can prevent leakage of yeast, reduce possible immune response when directly contacting human body, and accelerate wound healing. The invention has the advantages that: good biocompatibility, mild reaction and wide application prospect.

Description

Non-leakage packaged yeast hydrogel and preparation method thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a leak-free packaged yeast hydrogel and a preparation method thereof.

Background

In past studies, the use of conventional dressings such as medical absorbent cotton gauze has not been satisfactory. The liquid absorption amount is not large enough and needs to be replaced frequently. After imbibing, the dressing is easy to dry and easy to adhere to wounds to cause secondary wounds, so the dressing is gradually replaced by other dressings.

The biological dressing is improved greatly compared with the traditional dressing, wherein the hydrogel is a hydrophilic reticular polymer swelling body containing a large amount of water, has good water absorption, smooth surface, good biocompatibility, stable composition, low price and weak antigenicity. Hydrogels are widely used in a variety of fields, such as biosensors, drug delivery, and the like.

The living cell-macromolecule grafting method is a mode for constructing engineering bacteria, and is one of important technologies for widening the application advantages of living cells and macromolecules. The modified living cell is endowed with new physicochemical properties, and the complete encapsulation of the living cell is ensured, and the antibacterial performance is strong. The hydrogel constructed by living cells and macromolecules provides a cellular environment similar to a natural cytoplasmic matrix, and provides a very effective bacteriostatic environment for preventing wound infection. In recent years, many researchers report various biomedical applications such as drug release and wound repair by a living cell-polymer grafting method.

Yeast is a unicellular fungus that ferments sugars into alcohol and carbon dioxide, distributed throughout the natural world, and is a typical heterotrophic facultative anaerobic microorganism. Yeast is harmless, easy to grow, and can survive under aerobic and anaerobic conditions, far below human cytotoxic concentrations. The human growth factor as the main active component of the antibacterial material has multiple biological functions, and can be combined with receptors on cell membranes to promote a series of complex biochemical cascade reactions in cells to play a physiological role, promote cell division, and repair skin wounds, gastrointestinal ulcers, corneal injuries and the like.

The invention oxidizes the sugar chain on the surface of the engineering yeast, initiates and polymerizes monomers, and prepares hydrogel through chemical crosslinking of macromolecules. Zero leakage of the yeast is realized, possible immune reaction generated when the yeast is in direct contact with a human body is avoided, and the biocompatibility is good. The yeast cells have strong antagonistic and bacteriostatic abilities, and the possibility of wound infection is reduced.

The human growth factor is expressed in situ on the yeast cell to promote the autolysis of necrotic tissues and the growth of new tissues, and the in situ expression avoids the inactivation of the growth factor in a short-range release mode to keep lasting activity. The hydrogel formed by crosslinking has good stress and stretchability, can reduce the contact between the surface of a wound and the outside, is favorable for the permeation of nutrient substances and the discharge of metabolic waste, and accelerates the healing of the wound. The material with excellent antibacterial property and protein adsorption resistance can be further used in special environments such as diabetic wounds, ulcers and the like. The invention further promotes the development of biological material science and provides a new method for better application of microorganisms.

Disclosure of Invention

The invention aims to overcome the defects of the existing wound dressing, and provides a leak-free packaged yeast hydrogel and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a non-leakage encapsulated yeast hydrogel and a preparation method thereof are characterized by comprising the following steps:

(1) constructing engineering bacteria carrying growth factors yeast: connecting the human cell growth factor gene optimized by the yeast cell codon to a vector to construct a recombinant plasmid carrying the growth factor; introducing the recombinant plasmid carrying the growth factor into yeast competent cells, and screening out engineering bacteria of which the target genes are successfully integrated into a yeast genome; carrying out shake flask fermentation culture on the engineering bacteria to obtain yeast engineering bacteria for expressing growth factors;

(2) preparation of hydrogel: and (2) carrying out in-situ oxidation on the surface sugar chain of the engineering yeast obtained in the step (1), initiating, and carrying out specific crosslinking on the high molecules to prepare the leakage-free encapsulated yeast hydrogel.

The human cell growth factor gene in the step (1) of the preparation method comprises a human epidermal growth factor hEGF gene and a human vascular endothelial growth factor hVEGF gene; the codon-optimized hEGF gene sequence is shown in SEQ ID NO. 1; the codon optimized hVEGF gene sequence is shown in SEQ ID NO. 2.

The preparation of the hydrogel in the step (2) in the above preparation method comprises the following specific steps:

1) washing bacterial liquid: streaking the engineering yeast flat plate obtained in the step (1), putting the engineering yeast flat plate into an incubator for overnight culture at 30 ℃, picking a single colony on the flat plate by using a gun head, and performing shake amplification culture for 12 hours by using a liquid culture medium at 30 ℃ and 180 rpm; centrifuging the expanded and cultured bacterial liquid, adding PBS buffer solution for washing, centrifuging for 2 times, and adding 50 mL PBS buffer solution for resuspension to obtain yeast suspension;

2) surface sugar chain in-situ oxidation: adding an oxidant into the yeast suspension, carrying out ice bath for 30 minutes in a dark place, centrifuging after the reaction is finished, and adding a PBS (phosphate buffer solution) for heavy suspension to obtain an oxidized yeast suspension;

3) and (3) initiation: and adding an initiator into the oxidized yeast suspension, fully and uniformly mixing, carrying out a light-shielding reaction for 1 h at 37 ℃, centrifuging, and adding a PBS buffer solution for resuspension to obtain the initiated yeast suspension.

4) Specific crosslinking of macromolecules: adding yeast suspension, a cross-linking agent, CuBr, water, a hydrogel monomer and ascorbic acid which are initiated into a sealed round-bottomed bottle, mixing, denitrifying the bottle for 55min, exposing the mixed solution in the air to stop reaction, irradiating by 360 nm ultraviolet light, and specifically crosslinking for 30min to obtain transparent and elastic jelly, namely the non-leakage encapsulated yeast hydrogel.

Further, the concentration of the yeast suspension in step 1) in the above preparation method is: 1X 10⁸one/mL.

Further, in the above preparation method, the oxidizing agent in step 2) is sodium periodate (NaIO)₄) The final concentration was 2 mg/ml.

Further, in the above preparation method, the initiator in step 3) is 2-bromo-2-methylpropanoic acid-2-aminooxyethyl ester (ABM) at a final concentration of 1 mg/ml.

Further, in the above preparation method, the cross-linking agent in step 4) is ethylene glycol dimethacrylate and dimethacrylate, and the hydrogel monomer is any one of methacrylated hyaluronic acid, methacrylated gelatin and methacrylated chitosan.

Further, in step 4) of the above preparation method, 1mL of the initiated yeast suspension, 15. mu.L of ethylene glycol dimethacrylate as a crosslinking agent, 0.2mL of dimethacrylate, 6 mg of CuBr, 2mL of water, 50. mu.L of a hydrogel monomer, and 0.1 mL of ascorbic acid with a concentration of 0.074g/mL were added to the sealed round-bottomed flask and mixed.

A leak-free encapsulated yeast hydrogel prepared by the method described above.

Use of the above-described leak-free encapsulated yeast hydrogel in a wound dressing.

The invention has the following remarkable advantages:

(1) the method of the invention oxidizes and initiates sugar chains on the surface of the engineering yeast, and the leakage-free encapsulated yeast hydrogel is prepared by the specific mutual crosslinking of high molecular monomers. The non-leakage encapsulated yeast hydrogel can effectively ensure zero leakage of yeast, reduce possible immunoreaction generated when the yeast is directly contacted with a human body, and has good biocompatibility and mild reaction.

(2) The human growth factor on the yeast cell can effectively accelerate the growth of epithelial cells and the healing of wounds, and the human growth factor is used as an active ingredient of the antibacterial material and can keep the activity in a short-range release mode. The method can approximately obtain stable growth factor concentration by only controlling the yeast concentration, and accurately control the drug administration. Therefore, the preparation method has considerable application in the aspects of development of medical dressings, bioactive materials and the like.

Drawings

FIG. 1 shows the WB pattern of hEGF and hEGF-GS 115; wherein Lane 1: hEGF; lane 2: hEGF-GS 115.

FIG. 2 is an image of yeast cells in hEGF-methylpropenylhyaluronic acid (HAMA) hydrogel.

FIG. 3 is a graph showing that yeast cells in hEGF-methyl allenated hyaluronic acid (HAMA) hydrogel are completely encapsulated without release. 1-6 are culture solutions taken at different times (24 h, 48h, 72h, 96h, 120h and 144 h).

FIG. 4 is a graph showing the relationship between yeast concentration and hEGF release in hEGF-methylpropenoic hyaluronic acid (HAMA) hydrogel.

FIG. 5 is a graph showing the proliferation of yeast cells in hEGF-methylpropenoic hyaluronic acid (HAMA) hydrogel.

FIG. 6 is an inverted view of hEGF-methylpropanoylated hyaluronic acid (HAMA) hydrogel.

FIG. 7 is a graph of the effect of hEGF-methylalkylenehyaluronic acid (HAMA) hydrogel as a wound dressing on wound healing.

Detailed Description

In order to make the present invention more comprehensible, the present invention is further described in conjunction with the embodiments, and the embodiments described herein are only for explaining the related invention and do not limit the invention.

Example 1: construction of hEGF-Methylpropylenehyaluronic acid (HAMA) hydrogel

Construction of yeast engineering bacteria: connecting a yeast cell codon preference optimized human epidermal growth factor (hEGF) gene sequence (SEQ ID NO.1) to a pGAPZ alpha A vector to construct a recombinant plasmid carrying the hEGF; introducing the recombinant plasmid carrying the hEGF into a yeast competent cell GS115, and screening out engineering bacteria of which the hEGF gene is successfully integrated into a yeast genome; the yeast engineering bacteria for expressing the recombinant protein are obtained by adopting shake flask fermentation culture under the conditions of 30 ℃ and 12h and identified by a Western immunoblotting method (Western Blot).

2. Preparing hydrogel:

1) washing bacterial liquid: and (2) streaking the engineering yeast plate obtained in the step (1), and putting the engineering yeast plate into an incubator for overnight culture at 30 ℃. Single colonies on the plate were picked up by a tip and subjected to shake-propagation culture at 30 ℃ and 180 rpm for 12 hours in YPD liquid medium. Centrifuging the expanded bacterial solution, adding PBS buffer solution for washing, centrifuging for 2 times, adding 50 mL PBS buffer solution for resuspending to obtain the concentration of 1 × 10⁸Yeast suspension per ml;

2) surface sugar chain in-situ oxidation: in the yeast suspension obtained in the step (1)Adding sodium periodate (NaIO) as oxidant₄) Solution of sodium periodate (NaIO)₄) The final concentration is 2 mg/ml, ice bath is carried out for 30min in a dark place, centrifugation is carried out after the reaction is finished, PBS buffer solution is added for heavy suspension, and the concentration is 1 multiplied by 10⁸An oxidized yeast suspension per ml;

3) and (3) initiation: adding initiator 2-bromo-2-methylpropanoic acid-2-aminooxyethyl ester (ABM) into the oxidized yeast suspension to make ABM final concentration be 1 mg/mL, fully mixing uniformly, reacting at 37 deg.C in dark place for 1 h, centrifuging at 4 deg.C and 5000 rpm for 7 min, adding 10 mL PBS buffer solution, and resuspending to obtain 1 × 10⁸Individual/ml of yeast suspension elicited.

4) Specific crosslinking of macromolecules: adding 1mL of yeast suspension, 15 mu L of EGDMA, 0.2mL of PEGDMA, 6 mg of CuBr, 2mL of water, 50 mu L of methyl allenylated hyaluronic acid (HAMA) and 0.1 mL of ascorbic acid (0.074 g/mL) into a sealed round-bottomed bottle, denitrifying the bottle for 55min, exposing the mixed solution in the air to stop reaction, irradiating by ultraviolet light of 360 nm, and specifically crosslinking for 30min to obtain transparent elastic jelly, namely the non-leakage packaged yeast hydrogel.

Identifying the expression condition of the target protein on the engineering bacteria by using Western Blot, wherein Lane 1: there was a band at 12 kDa (6 kDa for hEGF and 6kDa for 6 His), and a band at 26 kDa for Lane2 as a GST tag, indicating that the hEGF antibody reacted with the hEGF sample antigen-antibody reaction. The successful expression of hEGF in the engineered bacteria is shown in FIG. 1.

The SEM image of the prepared non-leakage encapsulated yeast hydrogel is shown in FIG. 2, and the prepared hydrogel is observed by using an optical microscope to study the state of yeast cells in the hydrogel. Before the hydrogel was prepared, the yeast was stained with rhodamine B. The results in FIG. 2 show that the yeast cells in the hydrogel were clearly observed under a microscope, the single cells were round and intact in cell shape, and the size was about 1 μm, and the yeast cells were observed in a budding state (in a square frame), and it was preliminarily considered that the yeast cells in the hEGF-methylpropenoic hyaluronic acid (HAMA) hydrogel had activity and growth and propagation ability.

Yeast cells in the hEGF-methylpropylene hyaluronic acid (HAMA) hydrogel were observed, and the encapsulation effect of the hydrogel on the yeast cells was investigated. And (3) dialyzing the prepared hEGF-methyl propylene hyaluronic acid (HAMA) hydrogel in PBS (phosphate buffer solution) solution containing 2 g/100ml of glucose for 5 h, wiping water on the surface of the gel in a sterile environment after dialysis is finished, culturing in a DMEM (DMEM) medium containing 5 ml for 6 days under the condition of 30 ℃, taking out and replacing the culture solution every 24h, coating the culture solution taken out at different times (24 h, 48h, 72h, 96h, 120h and 144 h) on the surface of a YPD (YPD) solid medium, culturing in an incubator at 30 ℃ for 36 h, and observing whether obvious colonies appear on the surface, wherein the result is shown in figure 3. FIG. 3 shows that no yeast colonies appear on the surface of the solid medium, and the result shows that the constructed hEGF-methyl propylene hyaluronic acid (HAMA) hydrogel has a good cell encapsulation effect and can encapsulate yeast cells without leakage.

The yeast cell concentration of the hEGF-methyl propyl hyaluronic acid (HAMA) hydrogel is controlled, the release amount of the hEGF in the hydrogel is measured by an ELISA kit, the release curve of the hEGF release amount slowly rises along with the increase of the yeast concentration, and the stable growth factor concentration of the hEGF-methyl propyl hyaluronic acid (HAMA) hydrogel can be approximately obtained by controlling the yeast concentration (see figure 4).

Yeast cells in hEGF-methyl propylated hyaluronic acid (HAMA) hydrogel were observed, and proliferation of yeast cells was examined. Will be enveloped with 1 × 10⁸The hydrogel of each yeast is cultured at 30 deg.C and 240 rpm, and the OD value of the bacterial liquid is measured at every 24h, and is 1 × 10⁸Individual yeasts are controls; the results are shown in FIG. 5. The results in FIG. 5 show that the hydrogel has no significant effect on the proliferation of yeast cells.

Macroscopic observations were made on hEGF-methylpropenoic hyaluronic acid (HAMA) hydrogels. The prepared hydrogel is placed in a transparent glass bottle and inverted, and the hydrogel is in a uniform, colorless and transparent gel shape and has certain viscosity. See figure 6 for an illustration of the success of hydrogel preparation.

Example 2: construction of hEGF-Methacryloylated gelatin (GelMA) hydrogels

1. Construction of yeast engineering bacteria: the same as in example 1.

2. Preparing hydrogel:

1) washing bacterial liquid: and (2) streaking the engineering yeast flat plate obtained in the step (1), and putting the engineering yeast flat plate into an incubator for overnight culture at 30 ℃. Single colonies on the plate were picked up by a tip and subjected to shake-propagation culture at 30 ℃ and 180 rpm for 12 hours in YPD liquid medium. Centrifuging the expanded bacterial solution, adding PBS buffer solution for washing, centrifuging for 2 times, adding 50 mL PBS buffer solution for resuspending to obtain the concentration of 1 × 10⁸Yeast suspension per ml;

2) surface sugar chain in-situ oxidation: adding sodium periodate (NaIO) as oxidant into yeast suspension₄) Sodium periodate (NaIO)₄) The final concentration is 2 mg/ml, ice bath is carried out for 30 minutes in a dark place, centrifugation is carried out after the reaction is finished, PBS buffer solution is added for heavy suspension, and the concentration is 1 multiplied by 10⁸Yeast suspension after oxidation per ml;

3) and (3) initiation: adding initiator 2-bromo-2-methylpropanoic acid-2-aminooxyethyl ester (ABM) into the oxidized yeast suspension to make ABM final concentration be 1 mg/mL, fully mixing uniformly, making reaction at 30 deg.C in the dark for 1 h, centrifuging at 30 deg.C and 5000 rpm for 7 min, then adding 10 mL PBS buffer solution to make resuspension to obtain 1 × 10 concentration⁸Individual/ml of yeast suspension elicited.

4) Specific crosslinking of macromolecules: adding 1mL of yeast suspension, 15 mu L of EGDMA, 0.2mL of PEGDMA, 6 mg of CuBr, 2mL of water, 50 mu L of methacrylic gelatin (GelMA) and 0.1 mL of ascorbic acid (0.074 g/mL) into a sealed round-bottom bottle, denitrifying the bottle for 55min, exposing the mixed solution in the air to stop reaction, irradiating by 360 nm ultraviolet light, and specifically crosslinking for 30min to obtain a transparent elastic jelly, namely the non-leakage packaged yeast hydrogel.

Example 3: construction of hEGF-Methylpropylenechitosan (CSMA) hydrogel

1. Construction of yeast engineering bacteria: the same as in example 1.

2. Preparing hydrogel:

1) washing bacterial liquid: engineering obtained in the step (1)The yeast plates were streaked and placed in an incubator at 30 ℃ for overnight culture. Single colonies on the plate were picked up by a tip and subjected to shake-propagation culture at 30 ℃ and 180 rpm for 12 hours in YPD liquid medium. Centrifuging the expanded bacterial solution, adding PBS buffer solution for washing, centrifuging for 2 times, adding 50 mL PBS buffer solution for resuspending to obtain the concentration of 1 × 10⁸Yeast suspension per ml;

2) in-situ oxidation of surface sugar chains: adding sodium periodate (NaIO) as oxidant into yeast suspension₄) Sodium periodate (NaIO)₄) The final concentration is 2 mg/ml, ice bath is carried out for 30 minutes in a dark place, centrifugation is carried out after the reaction is finished, PBS buffer solution is added for heavy suspension, and the concentration is 1 multiplied by 10⁸Yeast suspension after oxidation per ml;

4) Specific crosslinking of macromolecules: adding 1mL of yeast suspension, 15 mu L of EGDMA, 0.2mL of PEGDMA, 6 mg of CuBr, 2mL of water, 50 mu L of methyl allenylated chitosan and 0.1 mL of ascorbic acid (0.074 g/mL) into a sealed round-bottomed bottle, denitrifying the bottle for 55min, exposing the mixed solution in the air to stop reaction, irradiating by ultraviolet light of 360 nm, and specifically crosslinking for 30min to obtain a transparent and elastic jelly, namely the non-leakage packaged yeast hydrogel.

EXAMPLE 4 construction of hVEGF-Methylpropylenehyaluronic acid (HAMA) hydrogel

1. Construction of yeast engineering bacteria: connecting a codon preference optimized human hVEGF gene sequence (SEQ ID NO.2) of a yeast cell to a pGAPZ alpha A vector to construct a recombinant plasmid carrying hVEGF; introducing the recombinant plasmid carrying the hVEGF into a yeast competent cell GS115, and screening out engineering bacteria of which the hVEGF gene is successfully integrated into a yeast genome; the yeast engineering bacteria for expressing the recombinant protein are obtained by adopting shake flask fermentation culture under the conditions of 30 ℃ and 12h and identified by a Western immunoblotting method (Western Blot).

2. Preparing hydrogel:

EXAMPLE 5 construction of hVEGF-methacrylated gelatin (GelMA) hydrogel

1. Construction of yeast engineering bacteria: the same as in example 4.

2. Preparing hydrogel:

1) washing bacterial liquid: and (2) streaking the engineering yeast flat plate obtained in the step (1), and putting the engineering yeast flat plate into an incubator for overnight culture at 30 ℃. Picking single colony on the plate with a gun head, and culturing with YPD liquidCulturing the culture medium at 30 deg.C and 180 rpm for 12 h. Centrifuging the expanded bacterial solution, adding PBS buffer solution for washing, centrifuging for 2 times, adding 50 mL PBS buffer solution for resuspending to obtain the concentration of 1 × 10⁸Yeast suspension per ml;

3) and (3) initiation: adding initiator 2-bromo-2-methylpropanoic acid-2-aminooxyethyl ester (ABM) into the oxidized yeast suspension to make the final concentration of ABM be 1 mg/mL, fully and uniformly mixing, carrying out dark reaction at 30 ℃ for 1 h, centrifuging at 30 ℃ for 7 min at 5000 rpm, then adding 10 mL PBS buffer solution for heavy suspension to obtain the yeast suspension with the concentration of 1 × 10⁸Individual/ml of yeast suspension elicited.

EXAMPLE 6 construction of hVEGF-Methylpropylenechitosan (CSMA) hydrogel

1. Construction of yeast engineering bacteria: the same as in example 4.

2. Preparing hydrogel:

2）surface sugar chain in-situ oxidation: adding sodium periodate (NaIO) as oxidant into yeast suspension₄) Sodium periodate (NaIO)₄) The final concentration is 2 mg/ml, ice bath is carried out for 30 minutes in a dark place, centrifugation is carried out after the reaction is finished, PBS buffer solution is added for heavy suspension, and the concentration is 1 multiplied by 10⁸Yeast suspension after oxidation per ml;

4) Specific crosslinking of macromolecules: adding 1mL of yeast suspension, 15 mu L of EGDMA, 0.2mL of PEGDMA, 6 mg of CuBr, 2mL of water, 50 mu L of methyl allenylated Chitosan (CSMA) and 0.1 mL of ascorbic acid (0.074 g/mL) into a sealed round-bottomed bottle, denitrifying the bottle for 55min, exposing the mixed solution in the air to stop reaction, irradiating by ultraviolet light of 360 nm, and specifically crosslinking for 30min to obtain a transparent elastic jelly, namely the non-leakage packaged yeast hydrogel.

Application example 1

The non-leakage packaged yeast hydrogel prepared in example 1 was applied as a wound dressing to the wound surface of mice, and the dressing change was performed on

days

3, 6, 9, 12 and 15, respectively, and the wound dressing was photographed and observed. The experimental group was drier on the wound surface and scab faster than the control group as shown in fig. 7.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims

1. A non-leakage encapsulated yeast hydrogel and a preparation method thereof are characterized by comprising the following steps:

2. The method of claim 1, wherein: the human cell growth factor gene in the step (1) comprises a human epidermal growth factor hEGF gene and a human vascular endothelial growth factor hVEGF gene; the codon-optimized hEGF gene sequence is shown in SEQ ID NO. 1; the codon optimized hVEGF gene sequence is shown in SEQ ID NO. 2.

3. The method of claim 1, wherein: the preparation of the hydrogel in the step (2) comprises the following specific steps:

2) surface sugar chain in-situ oxidation: adding an oxidant into the yeast suspension, carrying out ice bath for 30min in a dark place, centrifuging after the reaction is finished, and adding a PBS (phosphate buffer solution) for heavy suspension to obtain an oxidized yeast suspension;

3) and (3) initiation: adding an initiator into the oxidized yeast suspension, fully and uniformly mixing, carrying out a light-shielding reaction at 37 ℃ for 1 h, centrifuging, and adding a PBS (phosphate buffer solution) for heavy suspension to obtain an initiated yeast suspension;

4. The method according to claim 3, wherein: the concentration of the yeast suspension in the step 1) is as follows: 1X 10⁸one/mL.

5. The method according to claim 3, wherein: in the step 2), the oxidant is sodium periodate, and the final concentration of the oxidant is 2 mg/ml.

6. The method according to claim 3, wherein: the initiator in the step 3) is 2-bromo-2-methylpropanoic acid-2-aminooxyethyl ester, and the final concentration is 1 mg/ml.

7. The method according to claim 3, wherein: the cross-linking agent in the step 4) is ethylene glycol dimethacrylate and dimethacrylate, and the hydrogel monomer is any one of methacrylic hyaluronic acid, methacrylic acid gelatin and methacrylic acid chitosan.

8. The method according to claim 3, wherein: in step 4), 1mL of initiated yeast suspension, 15. mu.L of crosslinking agent ethylene glycol dimethacrylate and 0.2mL of dimethacrylate, 6 mg of CuBr, 2mL of water, 50. mu.L of hydrogel monomer, and 0.1 mL of ascorbic acid with a concentration of 0.074g/mL are added into a sealed round-bottomed bottle and mixed.

9. A leakless encapsulated yeast hydrogel prepared by the method of any one of claims 1 to 8.

10. Use of the leak-free encapsulated yeast hydrogel of claim 9 in a wound dressing.