阅读说明：本技术 一种壳聚糖水凝胶及其制备方法和应用 (Chitosan hydrogel and preparation method and application thereof ) 是由 陈祥松 吴金勇 袁丽霞 葛远飞 王相勤 姚建铭 于 2021-08-19 设计创作，主要内容包括：本发明提供了一种壳聚糖水凝胶及其制备方法和应用,所述壳聚糖水凝胶的制备方法包括以下步骤：(1)将壳聚糖溶于酸溶液中,之后与抗炎剂混合,循环冷冻得到初试水凝胶；(2)将步骤(1)得到的初试水凝胶置于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液中孵育,之后经碱溶液处理、去离子水水洗得到所述壳聚糖水凝胶。本发明提供的壳聚糖水凝胶抗炎效果好,皮肤组织损伤修复效果好。(The invention provides a chitosan hydrogel and a preparation method and application thereof, wherein the preparation method of the chitosan hydrogel comprises the following steps: (1) dissolving chitosan in an acid solution, mixing with an anti-inflammatory agent, and circularly freezing to obtain an initial test hydrogel; (2) and (2) placing the initial test hydrogel obtained in the step (1) into a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide for incubation, and then treating with an alkali solution and washing with deionized water to obtain the chitosan hydrogel. The chitosan hydrogel provided by the invention has a good anti-inflammatory effect and a good skin tissue injury repair effect.)

1. The preparation method of the chitosan hydrogel is characterized by comprising the following steps of:

(1) dissolving chitosan in an acid solution, mixing with an anti-inflammatory agent, and circularly freezing to obtain an initial test hydrogel;

(2) and (2) placing the initial test hydrogel obtained in the step (1) into a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide for incubation, and then treating with an alkali solution and washing with deionized water to obtain the chitosan hydrogel.

2. The method of preparing a chitosan hydrogel according to claim 1, wherein said acid of step (1) comprises acetic acid;

preferably, the volume fraction of the acid in the step (1) is 0.8-1.2%.

3. The method for preparing the chitosan hydrogel according to claim 1 or 2, wherein the mass of the chitosan in the step (1) is 2.5-3.5% of the mass of the acid solution.

4. The method for preparing a chitosan hydrogel according to any one of claims 1 to 3, wherein said anti-inflammatory agent of step (1) comprises any one of N-acetylneuraminic acid, polysialic acid or protocatechuic acid or a combination of at least two thereof.

5. The method of preparing a chitosan hydrogel according to claim 4, wherein said anti-inflammatory agent of step (1) comprises a combination of at least two of N-acetylneuraminic acid, polysialic acid or protocatechuic acid;

preferably, the anti-inflammatory agent of step (1) is a combination of N-acetylneuraminic acid and polysialic acid.

6. The method for preparing the chitosan hydrogel according to any one of claims 1 to 5, wherein the molar ratio of the monomer of the chitosan to the anti-inflammatory agent in the step (1) is (9-11): 1;

preferably, the number of times of the cyclic freezing of step (1) is at least 3;

preferably, the freezing temperature of the circulation freezing in the step (1) is-22 to-18 ℃, and the single time is 11 to 13 hours;

preferably, the thawing temperature of the circulating freezing in the step (1) is 23-27 ℃, and the single time is 11-13 h.

7. The method for preparing a chitosan hydrogel according to any one of claims 1 to 6, wherein the pH of the mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step (2) in 2- (N-morpholino) ethanesulfonic acid is 5.5 to 6.5.

8. The method for preparing a chitosan hydrogel according to any one of claims 1 to 7, wherein the incubation time in step (2) is 11 to 13 hours.

9. A chitosan hydrogel prepared by the method of preparing a chitosan hydrogel according to any one of claims 1 to 8.

10. Use of the chitosan hydrogel of claim 9 in the preparation of a dermal tissue repair material.

Technical Field

The invention belongs to the field of medical materials, particularly relates to chitosan hydrogel and a preparation method and application thereof, and particularly relates to chitosan hydrogel with a good anti-inflammatory effect and a preparation method and application thereof.

Background

A hydrogel is an aggregate of a hydrophilic polymer and water in a solid and liquid state and having three-dimensional spaces. The hydrogel has good mechanical property, biocompatibility and biodegradability, the wet and soft surface and the internal environment filled with liquid are favorable for cell adhesion, proliferation and differentiation, and specific biomolecules are easy to use for surface modification such as drug loading and growth factor loading, so that the hydrogel becomes an ideal biomaterial for skin tissue repair. In addition, in the field of food industry, hydrogels can effectively control the release of flavor substances, reduce the water activity of food in the package, slow down microbial growth, and the like. At present, the chitosan hydrogel has been studied for repairing skin tissues, but the problem of secondary inflammation after the skin tissues are damaged cannot be solved.

CN105664239B discloses a preparation method of self-adhesive skin-repairing hydrogel. Firstly, the natural chitosan is subjected to quaternization modification, so that the chitosan has excellent bactericidal performance. Secondly, BSA nanoparticles embedded with growth factors for promoting skin regeneration were prepared by a desolvation method. Thirdly, dopamine was grafted to the natural gelatin material. Finally, the quaternary amination modified chitosan, the growth factor-coated BSA nanoparticles and the dopamine grafted gelatin are crosslinked to form a hydrogel material, and the hydrogel material has multiple functions of adhesion, antibacterial property and promotion of skin wound healing, so that the hydrogel material is used for skin repair.

CN109381738A discloses a chitosan-based hydrogel and a preparation method and application thereof. The chitosan-based hydrogel is prepared by taking chitosan or derivatives thereof as raw materials, grafting o-dihydroxy group compounds on a main chain, and crosslinking through a crosslinking agent. The preparation method of the hydrogel comprises the following steps: firstly, grafting o-dihydroxy groups on chitosan or derivatives thereof to ensure that the product has wet adhesion; further, by the crosslinking reaction, a hydrogel material having sufficient mechanical strength and viscoelasticity is formed. The hydrogel material has the functions of wet adhesion, hemostasis and wound healing promotion, and has wide application prospects in the aspects of tissue engineering and skin repair.

The problem of secondary inflammation after skin tissue injury cannot be solved due to the existing chitosan hydrogel. Therefore, how to provide a chitosan hydrogel with good anti-inflammatory effect becomes a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a chitosan hydrogel and a preparation method and application thereof, and particularly provides a chitosan hydrogel with a good anti-inflammatory effect and a preparation method and application thereof. The chitosan hydrogel provided by the invention has a good anti-inflammatory effect and a good skin tissue injury repair effect.

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

in a first aspect, the present invention provides a method for preparing a chitosan hydrogel, comprising the steps of:

(1) dissolving chitosan in an acid solution, mixing with an anti-inflammatory agent, and circularly freezing to obtain an initial test hydrogel;

(2) and (2) placing the initial test hydrogel obtained in the step (1) into a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide for incubation, and then treating with an alkali solution and washing with deionized water to obtain the chitosan hydrogel.

The chitosan hydrogel is combined with the anti-inflammatory agent and connected with the anti-inflammatory agent through amidation reaction, so that the formed hydrogel has a pore structure, the anti-inflammatory agent can be ensured to be continuously released in the delivery process, the secondary inflammatory reaction after the skin tissue is damaged can be relieved through the chitosan hydrogel, and the chitosan hydrogel has an excellent effect of repairing the damaged skin tissue.

Preferably, the acid of step (1) comprises acetic acid.

Preferably, the volume fraction of the acid in the step (1) is 0.8-1.2%.

Preferably, the mass of the chitosan in the step (1) is 2.5-3.5% of that of the acid solution.

The volume fraction of the acid may be 0.8%, 0.9%, 1%, 1.1%, 1.2%, etc., and the mass of the chitosan may be 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, etc., based on the mass of the acid solution, but the present invention is not limited to the above-mentioned values, and other values not listed in the above-mentioned numerical range are also applicable.

Preferably, the anti-inflammatory agent in step (1) includes any one or a combination of at least two of N-acetylneuraminic acid, polysialic acid or protocatechuic acid, such as a combination of N-acetylneuraminic acid and polysialic acid, a combination of polysialic acid and protocatechuic acid, or a combination of N-acetylneuraminic acid and protocatechuic acid, but is not limited to the above-listed combinations, and other combinations not listed within the above-mentioned combinations are also applicable.

The anti-inflammatory agent is selected to improve the anti-inflammatory effect of the chitosan hydrogel and promote the repair of damaged tissues of the skin.

Preferably, the anti-inflammatory agent of step (1) comprises a combination of at least two of N-acetylneuraminic acid, polysialic acid or protocatechuic acid.

Preferably, the anti-inflammatory agent of step (1) is a combination of N-acetylneuraminic acid and polysialic acid.

The specific anti-inflammatory agent further improves the anti-inflammatory effect of the chitosan hydrogel and promotes the repair of damaged tissues of the skin.

Preferably, the mass ratio of N-acetylneuraminic acid to polysialic acid is (1-3) to (1-3), wherein the number of N-acetylneuraminic acid parts can be 1, 2 or 3, etc., and the number of polysialic acid parts can be 1, 2 or 3, etc., but the above-mentioned values are not limited, and other values not listed in the above-mentioned value range are also applicable.

Preferably, the molar ratio of the monomer of the chitosan to the anti-inflammatory agent in the step (1) is (9-11): 1.

Preferably, the number of times of cyclic freezing in step (1) is at least 3.

Preferably, the freezing temperature of the circulation freezing in the step (1) is-22 to-18 ℃, and the single time is 11 to 13 hours.

Preferably, the thawing temperature of the circulating freezing in the step (1) is 23-27 ℃, and the single time is 11-13 h.

Preferably, the pH of the mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in 2- (N-morpholino) ethanesulfonic acid (MES) in step (2) is 5.5-6.5.

Preferably, the incubation time in step (2) is 11-13 h.

Wherein, the molar ratio of the monomer of the chitosan to the anti-inflammatory agent can be 9:1, 9.5:1, 10:1, 10.5:1 or 11:1, the times of circulating freezing can be 3 times, 4 times or 5 times, etc., the freezing temperature can be-22 ℃, 21 ℃, 20 ℃, 19 ℃ or-18 ℃, etc., the single time can be 11h, 11.5h, 12h, 12.5h or 13h, etc., the thawing temperature can be 23 ℃, 24 ℃, 25 ℃, 26 ℃ or 27 ℃, etc., the single time can be 11h, 11.5h, 12h, 12.5h or 13h, etc., the pH of the mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide can be 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4 or 6.5, etc., the incubation time may be 11h, 11.5h, 12h, 12.5h or 13h, but is not limited to the above-mentioned values, and other values not listed in the above-mentioned range of values are also applicable.

In a second aspect, the present invention provides the chitosan hydrogel prepared by the method for preparing chitosan hydrogel as described above.

In a third aspect, the invention also provides the use of the chitosan hydrogel as described above in the preparation of a material for skin tissue repair.

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

the invention provides a chitosan hydrogel, which is prepared by combining chitosan and an anti-inflammatory agent and connecting the chitosan and the anti-inflammatory agent through amidation reaction, so that the formed hydrogel has a pore structure, can ensure the sustained release of the anti-inflammatory agent in the delivery process, can relieve secondary inflammatory reaction after skin tissue injury, has excellent skin tissue injury repair effect and anti-inflammatory effect, and has the content of NO cell inflammatory factors below 70.10 +/-1.22 mu M; meanwhile, by selecting a specific anti-inflammatory agent, the anti-inflammatory effect of the chitosan hydrogel is further improved, and the repair of skin damaged tissues is promoted.

Drawings

FIG. 1 is an electron micrograph of a chitosan hydrogel provided in example 1;

FIG. 2 is an infrared spectrum of the chitosan hydrogel provided in example 1.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the following examples, polysialic acid was purchased from Wuhan's Keogogu Green Biotechnology, Inc.

Example 1

The embodiment provides a chitosan hydrogel, which is prepared by the following steps:

(1) dissolving chitosan in an acetic acid solution with the volume fraction of 1% (mass ratio of 3:100), mixing with N-acetylneuraminic acid and polysialic acid (mass ratio of 1:1) (the molar ratio is that chitosan monomers (N-acetylneuraminic acid and polysialic acid) is 10:1), and circularly freezing (freezing temperature is-20 ℃, time is 12h, unfreezing temperature is 25 ℃, time is 12h) for 3 times to obtain an initial test hydrogel;

(2) placing the initial test hydrogel obtained in the step (1) in a mixed solution (the molar ratio is 1:1:10, and the pH is adjusted to 6) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide 2- (N-morpholino) ethanesulfonic acid for incubation for 12h, and then leaching by using 1M sodium hydroxide solution to obtain the chitosanThe hydrogel is shown in FIG. 1 by electron micrograph and in FIG. 2 by infrared spectrum. Appearing at 1641cm in FIG. 2^-1Is positioned at 1567cm^-1Corresponding to the absorption peak of stretching vibration of-CO-NH-and is 1637cm^-1A flexural oscillation peak was also shown confirming the interaction of N-acetylneuraminic acid, polysialic acid and chitosan.

Example 2

The embodiment provides a chitosan hydrogel, which is prepared by the following steps:

(1) dissolving chitosan in an acetic acid solution with the volume fraction of 0.8% (mass ratio of 2.5:100), mixing with N-acetylneuraminic acid (molar ratio: chitosan monomer: N-acetylneuraminic acid is 9:1), and circularly freezing (freezing temperature is-20 ℃, time is 12h, thawing temperature is 25 ℃, time is 12h) for 4 times to obtain an initial test hydrogel;

(2) and (2) placing the initial test hydrogel obtained in the step (1) into a mixed solution (the molar ratio is 1:1:10, and the pH is adjusted to 5.5) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide, incubating for 11 hours, and then leaching by using a 1M sodium hydroxide solution to obtain the chitosan hydrogel.

Example 3

The embodiment provides a chitosan hydrogel, which is prepared by the following steps:

(1) dissolving chitosan in an acetic acid solution with the volume fraction of 1.2% (mass ratio of 3.5:100), mixing with protocatechuic acid (molar ratio: chitosan monomer: protocatechuic acid is 11:1), and circularly freezing (freezing temperature is-20 ℃, time is 12h, thawing temperature is 25 ℃, time is 12h) for 4 times to obtain an initial test hydrogel;

(2) and (2) placing the initial test hydrogel obtained in the step (1) into a mixed solution (the molar ratio is 1:1:10, and the pH is adjusted to 5.5) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide, incubating for 11 hours, and then leaching by using a 1M sodium hydroxide solution to obtain the chitosan hydrogel.

Example 4

This example provides a chitosan hydrogel prepared in the same manner as in example 1 except that polysialic acid in step (1) was replaced with an equal amount of N-acetylneuraminic acid.

Example 5

This example provides a chitosan hydrogel prepared in the same manner as in example 1 except that N-acetylneuraminic acid in step (1) was replaced with an equal amount of polysialic acid.

Example 6

This example provides a chitosan hydrogel prepared in the same manner as in example 1 except that N-acetylneuraminic acid and polysialic acid are replaced with the same amount of protocatechuic acid in step (1).

Example 7

This example provides a chitosan hydrogel prepared in the same manner as in example 1 except that N-acetylneuraminic acid in step (1) was replaced with the same amount of protocatechuic acid.

Example 8

This example provides a chitosan hydrogel prepared in the same manner as in example 1 except that the polysialic acid in step (1) was replaced with the same amount of protocatechuic acid.

Skin damage repair test:

the chitosan hydrogels provided in examples 1-8 were subjected to a skin lesion repair test as follows: (1) establishing a mouse skin defect wound model: BALB/c male mice (provided by Anhui medical university animal experiment center (license number: SYXK 2017-. After the ethanol is completely volatilized, a puncher is used for manufacturing a whole-layer skin defect wound surface with the diameter of 0.6cm on the left side and the right side of the back respectively, a standard disc with the diameter of 0.6cm is attached beside the wound surface, and a digital camera is used for shooting and recording to calculate the initial wound surface area; (2) administration treatment: the chitosan hydrogel (1 × 1cm) provided in examples 1 to 8 was sterilized in 75% ethanol by volume for 30 minutes, washed 5 times with PBS, dried with sterile filter paper, attached to the wound surface to which the bacteria liquid was added, and fixed by covering with adhesive patches (5 mice per group); (3) measuring the complete healing time of the skin defect wound of the mouse: after the wound is created, the wound healing conditions of the mice in each group are observed every day, the days required for the complete healing of each wound are recorded by taking the complete epithelization of the wound as a healing standard, and the test results are as follows:

group of	Days required for wound healing	Group of	Days required for wound healing
				Example 1	10.5	Example 5	16.7
Example 2	16.0	Example 6	16.5
				Example 3	15.8	Example 7	13.2
Example 4	17.0	Example 8	14.3

The data show that the chitosan hydrogel provided by the invention has excellent skin injury repair effect; comparing examples 1, 4-8, it can be found that the present invention improves the skin damage repair effect of the chitosan hydrogel by using a combination of anti-inflammatory agents, while further improving the skin damage repair effect of the chitosan hydrogel by using a combination of specific anti-inflammatory agents.

Anti-inflammatory effect test:

taking NO cell inflammation as a model, taking RAW264.7 cells to culture to logarithmic growth phase, digesting, resuspending and adjusting the cell concentration to 5 x 10⁵cells/mL, seeded with 100. mu.L/well of cell suspension in 96-well plates in 5% CO₂The test group was added with 20. mu.g of the chitosan hydrogel provided in examples 1 to 8, respectively, and incubated for 48 hours in the incubator, and the supernatant was collected, and the absorbance was measured at 492nm, and the NO cell inflammatory factor content was calculated using the NO working curve, as follows:

the data show that the chitosan hydrogel provided by the invention has excellent anti-inflammatory effect; comparing examples 1, 4-8, it can be seen that the present invention improves the anti-inflammatory effect of the chitosan hydrogel by using a combination of anti-inflammatory agents, while further improving the anti-inflammatory effect of the chitosan hydrogel by using a combination of specific anti-inflammatory agents.

The applicant states that the present invention is illustrated by the above examples to the chitosan hydrogel of the present invention and the preparation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.