CN113616805B - Chitosan hydrogel and preparation method and application thereof - Google Patents
Chitosan hydrogel and preparation method and application thereof Download PDFInfo
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- CN113616805B CN113616805B CN202110953536.5A CN202110953536A CN113616805B CN 113616805 B CN113616805 B CN 113616805B CN 202110953536 A CN202110953536 A CN 202110953536A CN 113616805 B CN113616805 B CN 113616805B
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 86
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 85
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000002253 acid Substances 0.000 claims abstract description 27
- 229940121363 anti-inflammatory agent Drugs 0.000 claims abstract description 23
- 239000002260 anti-inflammatory agent Substances 0.000 claims abstract description 23
- 230000008014 freezing Effects 0.000 claims abstract description 19
- 238000007710 freezing Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims abstract description 17
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims abstract description 10
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000011259 mixed solution Substances 0.000 claims abstract description 9
- 238000011534 incubation Methods 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000003513 alkali Substances 0.000 claims abstract description 3
- 239000008367 deionised water Substances 0.000 claims abstract description 3
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims abstract description 3
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 claims description 17
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 7
- 239000000178 monomer Substances 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
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- 230000008439 repair process Effects 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 8
- 208000037816 tissue injury Diseases 0.000 abstract description 8
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 206010052428 Wound Diseases 0.000 description 9
- 208000027418 Wounds and injury Diseases 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 7
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- 230000007547 defect Effects 0.000 description 4
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 4
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- 238000002386 leaching Methods 0.000 description 3
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- 235000009161 Espostoa lanata Nutrition 0.000 description 2
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- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
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, then mixing with an anti-inflammatory agent, and circularly freezing to obtain a primary hydrogel; (2) And (3) placing the initial hydrogel obtained in the step (1) in a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide for incubation, and then treating the initial hydrogel by an alkali solution and washing the initial hydrogel by 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.
Description
Technical Field
The invention belongs to the field of medical materials, and particularly relates to chitosan hydrogel, a preparation method and application thereof, in particular to chitosan hydrogel with good anti-inflammatory effect, and a preparation method and application thereof.
Background
Hydrogels are an aggregate of hydrophilic polymers and water that are intermediate between solid and liquid states and have three-dimensional space. Hydrogels have good mechanical properties, biocompatibility and biodegradability, wet and soft surfaces and fluid-filled internal environments facilitate cell adhesion, proliferation and differentiation, and surface modifications such as drug loading and growth factors are easily carried out using specific biomolecules, which make them ideal biomaterials 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 packaging, slow down microbial growth, and the like. At present, chitosan hydrogel is used for repairing skin tissues, but the problem of secondary inflammation after skin tissue injury cannot be solved.
CN105664239B discloses a method for preparing self-adhesive skin repair hydrogel. Firstly, the natural chitosan is subjected to quaternization modification, so that the natural chitosan has excellent sterilization performance. Secondly, BSA nano-particles embedded with growth factors for promoting skin regeneration are prepared by a desolvation method. Again, dopamine was grafted to the natural gelatin material. Finally, the quaternized modified chitosan, the BSA nano particles wrapping the growth factors and the dopamine grafted gelatin are crosslinked to form a hydrogel material which has multiple functions of adhesiveness, antibacterial property and promoting skin wound healing, so that the material is used for skin repair.
CN109381738A discloses a chitosan-based hydrogel, a preparation method and application thereof. The chitosan-based hydrogel is prepared by taking chitosan or derivatives thereof as raw materials, grafting a phthalic dihydroxy group compound on a main chain, and crosslinking by a crosslinking agent. The preparation method of the hydrogel comprises the following steps: firstly, grafting a dihydroxyl group on chitosan or a derivative thereof to enable a product to have wet adhesiveness; further, by the crosslinking reaction, a hydrogel material having sufficient mechanical strength and viscoelasticity is formed. The hydrogel material has the functions of wet adhesiveness, 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 existence of 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 chitosan hydrogel and a preparation method and application thereof, in particular to chitosan hydrogel with 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 aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing a chitosan hydrogel, the method comprising the steps of:
(1) Dissolving chitosan in an acid solution, then mixing with an anti-inflammatory agent, and circularly freezing to obtain a primary hydrogel;
(2) And (3) placing the initial hydrogel obtained in the step (1) in a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide for incubation, and then treating the initial hydrogel by an alkali solution and washing the initial hydrogel by deionized water to obtain the chitosan hydrogel.
According to the chitosan hydrogel, the chitosan and the anti-inflammatory agent are combined, and the chitosan and the anti-inflammatory agent are connected through amidation reaction, so that the formed hydrogel has a pore structure, the sustained release of the anti-inflammatory agent in the delivery process can be ensured, the secondary inflammatory reaction after skin tissue injury can be relieved by the chitosan hydrogel, and the excellent effect of repairing skin tissue injury is achieved.
Preferably, the acid of step (1) comprises acetic acid.
Preferably, the acid of step (1) is present in a volume fraction of 0.8 to 1.2%.
Preferably, the mass of the chitosan in the step (1) is 2.5-3.5% of the mass of the acid solution.
The volume fraction of the acid may be 0.8%, 0.9%, 1%, 1.1%, 1.2%, or the like, 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%, or 3.5%, or the like, based on the mass of the acid solution, but the present invention is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value ranges are equally 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, for example, 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, etc., but not limited to the above-listed combinations, and other non-listed combinations within the above-listed combinations are equally applicable.
The selection of the anti-inflammatory agent improves the anti-inflammatory effect of the chitosan hydrogel and promotes the repair of skin injury tissues.
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 skin damaged tissues.
Preferably, the mass ratio of N-acetylneuraminic acid to polysialic acid is (1-3): (1-3), wherein the part of N-acetylneuraminic acid can be 1, 2 or 3, etc., and the part of polysialic acid can be 1, 2 or 3, etc., but the invention is not limited to the values listed above, and other non-listed values within the above-mentioned value range are equally 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 cycles of freezing in step (1) is at least 3.
Preferably, the freezing temperature of the circulating freezing in the step (1) is-22 to-18 ℃ and the single time is 11-13h.
Preferably, the thawing temperature of the cyclic freezing in step (1) is 23-27 ℃ and the single time is 11-13h.
Preferably, the pH of the mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 2- (N-morpholino) ethanesulfonic acid (MES) of N-hydroxysuccinimide (NHS) in step (2) is from 5.5 to 6.5.
Preferably, the incubation time of step (2) is 11-13h.
The molar ratio of the chitosan monomer to the anti-inflammatory agent may be 9:1, 9.5:1, 10:1, 10.5:1, 11:1, etc., the number of times of cyclic freezing may be 3 times, 4 times, 5 times, etc., the freezing temperature may be-22 ℃, -21 ℃, -20 ℃, -19 ℃ or-18 ℃, etc., the single time may be 11h, 11.5h, 12h, 12.5h, 13h, etc., the thawing temperature may be 23 ℃, 24 ℃, 25 ℃, 26 ℃ or 27 ℃, etc., the single time may be 11h, 11.5h, 12h, 12.5h, 13h, etc., the pH of the 2- (N-morpholino) ethanesulfonic acid mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide may be 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5 h, etc., and the like, but the single time may be 11h, 11.5h, 12.5h, 6.5 h, etc., or 13h, etc., and the number may not be limited to the other values recited in the ranges.
In a second aspect, the present invention provides a 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 skin tissue repair material.
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, connecting the chitosan and the anti-inflammatory agent through amidation reaction, so that the formed hydrogel has a pore structure, and can ensure that the anti-inflammatory agent is continuously released in the delivery process, so that the chitosan hydrogel can relieve secondary inflammatory reaction after skin tissue injury, has excellent skin tissue injury repairing effect and anti-inflammatory effect, and has NO cell inflammatory factor content as low as 70.10 +/-1.22 mu M or less; meanwhile, by selecting a specific anti-inflammatory agent, the anti-inflammatory effect of the chitosan hydrogel is further improved, and the repair of skin injury tissues is promoted.
Drawings
FIG. 1 is an electron micrograph of the chitosan hydrogel provided in example 1;
fig. 2 is an infrared spectrum of the chitosan hydrogel provided in example 1.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
In the following examples, polysialic acid was purchased from wuhan midbody photovalley green biotechnology limited.
Example 1
The embodiment provides a chitosan hydrogel, which is prepared by the following steps:
(1) Dissolving chitosan in acetic acid solution with volume fraction of 1% (mass ratio of 3:100), mixing with N-acetylneuraminic acid and polysialic acid (mass ratio of 1:1) (molar ratio: chitosan monomer (N-acetylneuraminic acid and polysialic acid) =10:1), and circularly freezing (freezing temperature-20 ℃ for 12h, thawing temperature 25 ℃ for 12 h) for 3 times to obtain initial hydrogel;
(2) And (3) placing the initial hydrogel obtained in the step (1) in a mixed solution (molar ratio of 1:1:10, pH is adjusted to 6) of 2- (N-morpholino) ethanesulfonic acid of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, incubating for 12 hours, and leaching by using a 1M sodium hydroxide solution to obtain the chitosan hydrogel, wherein an electron microscopic picture is shown in figure 1, and an infrared spectrum is shown in figure 2. Appears in FIG. 2 at 1641cm -1 Extra-long distance 1567cm -1 Corresponds to the telescopic vibration absorption peak of-CO-NH-and is 1637cm -1 Bending vibration peaks also appear, 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 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=9:1), and circularly freezing (freezing temperature-20 ℃ C., time 12h, thawing temperature 25 ℃ C., time 12 h) for 4 times to obtain initial hydrogel;
(2) And (3) placing the initial hydrogel obtained in the step (1) in a mixed solution (molar ratio of 1:1:10) of 2- (N-morpholino) ethanesulfonic acid of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH value to 5.5, and 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 acetic acid solution with volume fraction of 1.2% (mass ratio of 3.5:100), mixing with protocatechuic acid (molar ratio: chitosan monomer: protocatechuic acid=11:1), and circularly freezing (freezing temperature-20deg.C for 12h, thawing temperature 25 deg.C for 12 h) for 4 times to obtain initial hydrogel;
(2) And (3) placing the initial hydrogel obtained in the step (1) in a mixed solution (molar ratio of 1:1:10) of 2- (N-morpholino) ethanesulfonic acid of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH value to 5.5, and 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 the polysialic acid in step (1) was replaced with an equivalent amount of N-acetylneuraminic acid.
Example 5
This example provides a chitosan hydrogel prepared in the same manner as in example 1 except that the N-acetylneuraminic acid in step (1) was replaced with an equivalent 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 in step (1) were replaced with the same amount of protocatechuic acid.
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 injury repair test:
the chitosan hydrogels provided in examples 1-8 were subjected to skin damage repair tests as follows: (1) establishing a wound model of the skin defect of the mice: BALB/c male mice (supplied by the university of Anhui medical science animal experiment center (license number: SYXK (Anhui) 2017-006)) were anesthetized with a solution of 1% pentobarbital by intraperitoneal injection, the back skin was washed with a cotton ball infiltrated with warm water after carefully removing the back hair of the mice with an electric hair cutter, and then the back skin of the mice was sterilized with a cotton ball of 75% ethanol by volume fraction. After the ethanol is completely volatilized, a puncher is used for manufacturing a full-layer skin defect wound surface with the diameter of 0.6cm on the left side and the right side of the back, a standard disc with the diameter of 0.6cm is attached beside the wound surface, and a digital camera is used for photographing and recording to calculate the initial wound surface area; (2) administration treatment: the chitosan hydrogel (1X 1 cm) provided in examples 1-8 was sterilized in 75% ethanol for 30 minutes, washed with PBS for 5 times, dried with sterile filter paper, and then attached to the wound surface after the bacterial liquid was added dropwise, and fixed by covering an adhesive film (5 mice per group); (3) determination of complete healing time of skin defect wound surface of mice: after wound making, the wound healing condition of each group of mice is observed every day, the complete epithelization of the wound is used as a healing standard, the number of days required by the complete healing of each wound is recorded, and the test result is 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 repairing effect; as can be seen from comparative examples 1, 4 to 8, 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 efficacy test:
taking NO cell inflammation as model, culturing RAW264.7 cells to logarithmic phase, digesting, resuspending and regulating cell concentration to 5×10 5 cells/mL, 100. Mu.L/well of cell suspension inoculated in 96-well plates at 5%CO 2 After overnight incubation at 37℃and removal of the medium, 200. Mu.L of lipopolysaccharide-containing medium was added to each well to stimulate the production of inflammatory factors, 20. Mu.g of the chitosan hydrogel provided in examples 1-8 were added to each test group, incubation was continued in the incubator for 48 hours, the supernatant was collected, absorbance was measured at 492nm, and 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 increases the anti-inflammatory effect of the chitosan hydrogel by using a combination of anti-inflammatory agents, while further increasing 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 as well as methods of making and using the same, but the present invention is not limited to, i.e., does not mean that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. The preparation method of the chitosan hydrogel is characterized by comprising the following steps of:
(1) Dissolving chitosan in an acid solution, then mixing with an anti-inflammatory agent, and circularly freezing to obtain a primary hydrogel;
(2) The initial hydrogel obtained in the step (1) is placed in a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide for incubation, and then the chitosan hydrogel is obtained through alkali solution treatment and deionized water washing;
wherein the anti-inflammatory agent of step (1) is a combination of N-acetylneuraminic acid and polysialic acid;
the freezing temperature of the circulating freezing in the step (1) is-22 to-18 ℃ and the single time is 11-13h;
the thawing temperature of the circulating freezing in the step (1) is 23-27 ℃, and the single time is 11-13h.
2. The method of preparing a chitosan hydrogel according to claim 1, wherein the acid of step (1) comprises acetic acid.
3. The method for preparing chitosan hydrogel according to claim 1, wherein the acid in step (1) has a volume fraction of 0.8-1.2%.
4. The method for preparing chitosan hydrogel according to claim 1, wherein the mass of chitosan in the step (1) is 2.5-3.5% of the mass of the acid solution.
5. The method of claim 1, wherein the molar ratio of the chitosan monomer to the anti-inflammatory agent in the step (1) is (9-11): 1.
6. The method of claim 1, wherein the number of cycles of freezing in step (1) is at least 3.
7. The method for preparing a chitosan hydrogel according to claim 1, wherein the pH of the mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2- (N-morpholino) ethanesulfonic acid of N-hydroxysuccinimide in step (2) is 5.5-6.5.
8. The method for preparing chitosan hydrogel according to claim 1, wherein the incubation time in step (2) is 11-13h.
9. A chitosan hydrogel prepared by the method for preparing a chitosan hydrogel according to any one of claims 1 to 8.
10. Use of the chitosan hydrogel according to claim 9 for the preparation of a skin tissue repair material.
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| CN103834033A (en) * | 2013-12-04 | 2014-06-04 | 江南大学 | Preparation method of polysialic acid-chitosan derivative hydrogel |
| CN108341977A (en) * | 2018-03-13 | 2018-07-31 | 武汉理工大学 | A kind of citric acid cross-linked chitosan hydrogel and preparation method thereof |
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| CN103834033A (en) * | 2013-12-04 | 2014-06-04 | 江南大学 | Preparation method of polysialic acid-chitosan derivative hydrogel |
| CN108341977A (en) * | 2018-03-13 | 2018-07-31 | 武汉理工大学 | A kind of citric acid cross-linked chitosan hydrogel and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages;Anahita Shahraz等;《Scientific Reports》;第1-17页，尤其是第1页摘要 * |
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