Non-animal-derived carboxyethyl chitosan hydrogel for injection as well as preparation method and application of non-animal-derived carboxyethyl chitosan hydrogel

Abstract

The invention provides non-animal-derived carboxyethyl chitosan hydrogel for injection as well as a preparation method and application of the non-animal-derived carboxyethyl chitosan hydrogel, and belongs to the technical field of hydrogel. The preparation method comprises the following steps: modifying bacterial source chitosan with acrylic acid, and coupling the modified bacterial source chitosan with silk fibroin peptide to prepare silk fibroin peptide / non-animal source carboxyethyl chitosan; oligomeric hyaluronic acid is oxidized, cholesterol is modified and used for coating bisabolol, squalene and asiatic acid, and oxidized hyaluronic acid modified lipidosome is prepared; the non-animal-derived carboxyethyl chitosan hydrogel for injection is prepared by mixing the non-animal-derived carboxyethyl chitosan with carbohydrazide modified gelatin, silk fibroin peptide / non-animal-derived carboxyethyl chitosan and oxidized hyaluronic acid, has good moisturizing, antibacterial and hemostatic properties, provides a moist environment for wound surfaces, prevents microbial infection, promotes granulation tissue formation, and has a good application prospect. The drug release rate is changed by matching with the change of the pH value of the skin surface, the wound healing process is shortened, and the high water content of the hydrogel is combined with the moisturizing property of the chitosan to prevent secondary injury caused by wound adhesion.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, specifically to a non-animal-derived carboxyethyl chitosan injection hydrogel, its preparation method, and its application. Background Technology

[0002] Chitosan possesses excellent biocompatibility and is a commonly used material in skin dressings. To date, the vast majority of chitosan used in research has been derived from crustaceans. Animal-derived chitosan has a complex extraction and purification process, resulting in high acquisition costs; it is also limited by marine resources. Furthermore, it can cause allergic reactions, making it unsuitable for individuals with seafood allergies. Plant-derived chitosan can be extracted from various plants, such as Ganoderma lucidum, algae, and shiitake mushrooms, making it more widely available and sustainable. Its extraction and preparation do not require strong alkali treatment, making the process simpler and more environmentally friendly; and it has lower immunogenicity and better safety. Functionally, plant-derived chitosan not only has good biocompatibility, anti-inflammatory, and antioxidant stress-reducing properties, but also exhibits stronger antibacterial capabilities due to its lower molecular weight compared to animal-derived chitosan. Studies have found that chitosan derived from Ganoderma lucidum can effectively inhibit the activity and reproduction of Escherichia coli and Staphylococcus aureus. However, research on plant-derived chitosan is currently scarce. Summary of the Invention

[0003] The purpose of this invention is to provide a non-animal-derived carboxyethyl chitosan injection hydrogel, its preparation method, and its applications. This hydrogel possesses excellent moisturizing, antibacterial, and hemostatic properties, providing a moist environment for the wound, preventing microbial infection, promoting granulation tissue formation, and altering the drug release rate by responding to changes in the skin's pH level, thus shortening the wound healing process. Simultaneously, the high water content of the hydrogel, combined with the moisturizing properties of chitosan, prevents wound adhesion and secondary damage.

[0004] The technical solution of this invention is implemented as follows: This invention provides a method for preparing a non-animal-derived carboxyethyl chitosan injection hydrogel. The method involves modifying bacterial chitosan with acrylic acid and then coupling it with silk fibroin peptides to obtain silk fibroin peptide / non-animal-derived carboxyethyl chitosan. Oligomeric hyaluronic acid is oxidized, then modified with cholesterol and used to encapsulate bisabolol, squalene, and asiatic acid to obtain oxidized hyaluronic acid-modified liposomes. These liposomes are then mixed with carbazide-modified gelatin, silk fibroin peptide / non-animal-derived carboxyethyl chitosan, and oxidized hyaluronic acid to obtain a non-animal-derived carboxyethyl chitosan injection hydrogel.

[0005] As a further improvement to the present invention, the following steps are included: S1. Preparation of non-animal-derived carboxyethyl chitosan: Mix bacterial chitosan and acrylic acid, add water, heat and stir to react, adjust the pH value of the reaction system, add organic solvent to precipitate, filter, wash, and dry to obtain non-animal-derived carboxyethyl chitosan; S2. Preparation of oxidized hyaluronic acid: Oligomeric hyaluronic acid was dissolved in water, an oxidizing agent was added, the mixture was stirred and reacted, ethylene glycol was added and stirring was continued, the product was dialyzed, and freeze-dried to obtain oxidized hyaluronic acid; S3. Preparation of oxidized hyaluronic acid modified cholesterol: Oxidized hyaluronic acid was dissolved in water, an emulsifier was added and dispersed evenly, a dimethylformamide solution containing cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine was added, and the reaction was carried out under inert gas protection by heating and stirring, dialyzing, and freeze drying to obtain oxidized hyaluronic acid modified cholesterol; S4. Preparation of oxidized hyaluronic acid modified liposomes: Bisabolol, squalene, asiatic acid, soybean lecithin and oxidized hyaluronic acid modified cholesterol were dissolved in tetrahydrofuran, heated and rotary evaporated to form a film, hydrated with ultrapure water, sonicated, filtered, and oxidized hyaluronic acid modified liposomes were obtained. S5. Preparation of silk fibroin peptide / non-animal carboxyethyl chitosan: Non-animal carboxyethyl chitosan was dissolved in water, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, the reaction was activated by stirring, then silk fibroin peptide was added, the coupling reaction was stirred, filtered, dialyzed, and freeze-dried to obtain silk fibroin peptide / non-animal carboxyethyl chitosan; S6. Preparation of carbazide-modified gelatin: Gelatin was added to water and heated to dissolve. Carbazide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, followed by the addition of an ethanol solution containing 1-hydroxybenzotriazole. The mixture was stirred and reacted, dialyzed, and freeze-dried to obtain carbazide-modified gelatin. S7. Preparation of hydrogel: Silk fibroin peptide / non-animal-derived carboxyethyl chitosan, carbazide-modified gelatin, and oxidized hyaluronic acid were dissolved in water to obtain solutions; oxidized hyaluronic acid-modified liposomes were added to the silk fibroin peptide / non-animal-derived carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining non-animal-derived carboxyethyl chitosan injection hydrogel.

[0006] As a further improvement of the present invention, the mass ratio of the bacterial chitosan and acrylic acid in step S1 is 1:1.5-2.5, the temperature of the heating and stirring reaction is 85-95℃, the time is 4-6h, the pH value of the reaction system is adjusted to 9-10, and the organic solvent is acetone.

[0007] As a further improvement of the present invention, the oxidant in step S2 is sodium periodate, the mass ratio of oligomeric hyaluronic acid to oxidant is 10:4-6, and the stirring reaction time is 1-3 hours.

[0008] As a further improvement of the present invention, the mass ratio of oxidized hyaluronic acid, emulsifier, cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in step S3 is 0.4-0.6:4-6:0.3-0.4:0.45-0.55:0.02-0.03, and the heating and stirring reaction temperature is 25-35℃, and the time is 40-48h.

[0009] As a further improvement of the present invention, the mass ratio of bisabolol, squalene, asiatic acid, soybean lecithin and oxidized hyaluronic acid modified cholesterol in step S4 is 0.5-1:0.2-0.4:0.3-0.6:20-30:8-10, the hydration time is 40-60 min, the ultrasonic power is 300-500 W, and the time is 10-20 min.

[0010] As a further improvement of the present invention, the mass ratio of non-animal-derived carboxyethyl chitosan, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and silk fibroin peptide in step S5 is 10:1-2:2-3:3-5.

[0011] As a further improvement of the present invention, in step S6, the mass ratio of gelatin, carbohydrazine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole is 2:1-1.8:0.2-0.4:0.2-0.4, and the stirring reaction time is 20-28h; in step S7, the mass ratio of silk fibroin peptide / non-animal-derived carboxyethyl chitosan, carbohydrazine-modified gelatin, oxidized hyaluronic acid, and oxidized hyaluronic acid-modified liposomes is 10:4-6:10-15:2-3.

[0012] This invention further protects a non-animal-derived carboxyethyl chitosan hydrogel for injection prepared by the above-described method.

[0013] This invention further protects the use of the above-mentioned non-animal-derived carboxyethyl chitosan hydrogel for injection in the preparation of a drug that promotes skin wound healing.

[0014] Among them, wounds include traumatic injuries and ulcerated wounds. Traumatic injuries include wounds to the skin's surface or deep layers caused by cosmetic procedures, surgery, or sharp objects.

[0015] The present invention has the following beneficial effects: Infection is a major cause of delayed wound healing. Chitosan's complex spatial structure contains active functional groups, exhibiting broad-spectrum antibacterial properties. It has varying degrees of inhibitory effects against many pathogenic microorganisms, such as bacteria and fungi, and promotes granulation tissue formation, thereby accelerating wound repair. The plant-derived Ganoderma lucidum chitosan of this invention not only possesses highly effective antibacterial capabilities but also exhibits low immunogenicity and safety, and is widely available and inexpensive.

[0016] This invention utilizes carboxyethylated Ganoderma lucidum chitosan, which retains its cationic structure while simultaneously introducing anionic groups into the molecule, thus exhibiting an amphoteric polymer overall. The cationic effect of this chitosan allows it to interact with negatively charged platelets and erythrocytes, activating platelets and causing erythrocyte aggregation, leading to blood clot formation and achieving a significant hemostatic effect.

[0017] The carboxyethylated Ganoderma lucidum chitosan of this invention can also be coupled with silk fibroin peptides through the coupling of carboxyl and amino groups to obtain carboxyethylated Ganoderma lucidum chitosan modified with silk fibroin peptides. Silk fibroin peptides exhibit low immunogenicity and excellent biocompatibility, promoting cell adhesion, proliferation, and differentiation. Their strong moisturizing properties are also beneficial for maintaining cell viability and function. Silk fibroin peptides also possess significant antioxidant properties, preventing oxidative damage to cells and tissues and shortening the wound healing cycle. However, they suffer from problems such as loose molecular chain arrangement and poor mechanical strength. Simultaneously, the thiol and amino groups on the silk fibroin peptides can enhance free radical scavenging ability, improve antioxidant and antibacterial properties, participate in thiol-alcohol or thiol-aldehyde crosslinking, and form Schiff bases, improving network stability.

[0018] Gelatin retains the arginine-glycine-aspartic acid sequence, exhibiting good cell adhesion and biodegradability, but its mechanical strength is poor, making it unsuitable for long-term applications. After modification with carbazide, it can react with aldehyde groups to form hydrazone bonds, endowing the hydrogel with good pH responsiveness and dynamic reversibility.

[0019] Oxidized hyaluronic acid, due to the presence of multiple aldehyde groups on its molecular chain, can undergo Schiff base or hydrazone bond reactions with amino or hydrazine groups. This provides adjustable cross-linking density while ensuring biocompatibility, making it an ideal cross-linking agent. Because both Schiff base and hydrazone bonds are dynamically reversible, the hydrogel's chemical bond structure remains intact during the initial alkaline inflammatory phase of the wound, forming a physical barrier to prevent bacterial infection and mechanical damage, while also maintaining a moist environment and absorbing wound exudate. During the mid-healing phase, the wound pH gradually becomes acidic, promoting chemical bond breakage and reducing the degree of cross-linking, thereby accelerating the release rate and promoting the release and transdermal absorption of oxidized hyaluronic acid-modified liposomes. Degradation products (amino and aldehyde groups) can be utilized by cells, while simultaneously providing space for the migration of fibroblasts and keratinocytes. In the late healing phase, the skin pH further decreases, the hydrogel rapidly degrades, and all the drug is released without hindering new tissue formation, eliminating the need for dressing removal and reducing pain and further injury.

[0020] Furthermore, low-molecular-weight hyaluronic acid-modified liposomes can penetrate deep into the skin, promoting skin metabolism and resulting in smooth and supple healed skin. Simultaneously, these hyaluronic acid-modified liposomes can participate in cross-linking reactions, leading to more uniform distribution within the hydrogel system. The encapsulation of bisabolol, squalene, and asiatic acid in the liposomes enhances their solubility and bioavailability. Bisabolol possesses anti-inflammatory, soothing, and skin-regenerating effects; squalene provides moisturizing and cell metabolism-promoting effects; and asiatic acid promotes collagen synthesis, has anti-inflammatory and antioxidant properties. Through synergistic action, they can inhibit the expression of pro-inflammatory factors, achieving an anti-inflammatory effect. In addition to antibacterial activity against pathogens, they also promote the proliferation of probiotics. Probiotics can combat infection by eliminating pathogenic microorganisms, preventing inappropriate inflammatory responses, and promoting wound healing. The liposome structure also enhances transdermal absorption.

[0021] The hydrogel prepared by this invention is hydrophilic and has a three-dimensional cross-linked network structure. It can swell in water to retain a large amount of moisture. This non-animal-derived carboxyethyl chitosan injection hydrogel has excellent moisturizing, antibacterial, and hemostatic properties, providing a moist environment for the wound, preventing microbial infection, promoting granulation tissue formation, and altering the drug release rate by responding to changes in the skin's pH value, thus shortening the wound healing process. Simultaneously, the high water content of the hydrogel, combined with the moisturizing properties of chitosan, prevents wound adhesion and secondary damage. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] This embodiment provides a non-animal-derived carboxyethyl chitosan injectable hydrogel, comprising the following steps: S1. Preparation of non-animal-derived carboxyethyl chitosan: 5g of Ganoderma lucidum chitosan and 7.5g of acrylic acid were mixed, added to 100mL of water, the pH was adjusted to 6, heated to 85℃, stirred for 4h, NaOH solution was added to adjust the pH of the reaction system to 9, 100mL of acetone was added to precipitate for 1h, filtered, washed, and dried to obtain non-animal-derived carboxyethyl chitosan. S2. Preparation of oxidized hyaluronic acid: Dissolve 10g oligomeric hyaluronic acid in 1L of water, add 4g sodium periodate, stir and react for 1h, add 10mL ethylene glycol and continue stirring for 1h, dialyze, freeze dry to obtain oxidized hyaluronic acid; S3. Preparation of oxidized hyaluronic acid modified cholesterol: 4g of oxidized hyaluronic acid was dissolved in 1L of water, 40g of Tween-80 was added and dispersed evenly, and 300mL of dimethylformamide solution containing 3g cholesterol, 4.5g dicyclohexylcarbodiimide and 0.2g 4-dimethylaminopyridine was added. Under nitrogen protection, the mixture was heated to 25℃ and stirred for 40h. After dialyzing and freeze-drying, oxidized hyaluronic acid modified cholesterol was obtained. S4. Preparation of oxidized hyaluronic acid modified liposomes: 0.5g bisabolol, 0.2g squalene, 0.3g asiatic acid, 20g soybean lecithin and 8g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 40min, and the mixture was sonicated at 300W for 10min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes. S5. Preparation of silk fibroin peptide / non-animal carboxyethyl chitosan: 10g of non-animal carboxyethyl chitosan was dissolved in 500mL of water, 1g of N-hydroxysuccinimide and 2g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and the reaction was stirred and activated for 30min. Then, 3g of silk fibroin peptide was added, and the coupling reaction was stirred and carried out for 8h. The mixture was filtered, dialyzed, and freeze-dried to obtain silk fibroin peptide / non-animal carboxyethyl chitosan. S6. Preparation of carbazide-modified gelatin: 2g of gelatin was added to 200mL of water, heated to 45℃, stirred and dissolved, 1g of carbazide and 0.2g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and then 20mL of ethanol solution containing 0.2g of 1-hydroxybenzotriazole was added. The mixture was stirred and reacted for 20h, dialyzed, and freeze-dried to obtain carbazide-modified gelatin; S7. Preparation of hydrogel: 10g of silk fibroin peptide / non-animal carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL silk fibroin peptide / non-animal carboxyethyl chitosan solution; 4g of carbazide-modified gelatin was dissolved in water to obtain a 30mg / mL carbazide-modified gelatin solution; 10g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; 2g of oxidized hyaluronic acid-modified liposomes were added to the silk fibroin peptide / non-animal carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining a non-animal carboxyethyl chitosan injection hydrogel.

[0025] Example 2

[0026] This embodiment provides a non-animal-derived carboxyethyl chitosan injectable hydrogel, comprising the following steps: S1. Preparation of non-animal-derived carboxyethyl chitosan: 5g of Ganoderma lucidum chitosan and 12.5g of acrylic acid were mixed, added to 100mL of water, the pH was adjusted to 6, heated to 95℃, and stirred for 6h. NaOH solution was added to adjust the pH of the reaction system to 10, 100mL of acetone was added to precipitate for 1h, filtered, washed, and dried to obtain non-animal-derived carboxyethyl chitosan. S2. Preparation of oxidized hyaluronic acid: Dissolve 10g oligomeric hyaluronic acid in 1L of water, add 6g sodium periodate, stir for 3h, add 10mL ethylene glycol and continue stirring for 1h, dialyze, freeze dry to obtain oxidized hyaluronic acid; S3. Preparation of oxidized hyaluronic acid modified cholesterol: 6g of oxidized hyaluronic acid was dissolved in 1L of water, 60g of Tween-80 was added and dispersed evenly, and 300mL of dimethylformamide solution containing 4g cholesterol, 5.5g dicyclohexylcarbodiimide and 0.3g 4-dimethylaminopyridine was added. Under nitrogen protection, the mixture was heated to 35℃ and stirred for 48h. After dialyzing and freeze-drying, oxidized hyaluronic acid modified cholesterol was obtained. S4. Preparation of oxidized hyaluronic acid modified liposomes: 1g bisabolol, 0.4g squalene, 0.6g asiatic acid, 30g soybean lecithin and 10g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 60min, and the mixture was sonicated at 500W for 20min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes. S5. Preparation of silk fibroin peptide / non-animal carboxyethyl chitosan: 10g of non-animal carboxyethyl chitosan was dissolved in 500mL of water, 2g of N-hydroxysuccinimide and 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and the reaction was stirred and activated for 30min. Then 5g of silk fibroin peptide was added, and the coupling reaction was stirred and carried out for 10h. The mixture was filtered, dialyzed, and freeze-dried to obtain silk fibroin peptide / non-animal carboxyethyl chitosan. S6. Preparation of carbazide-modified gelatin: 2g of gelatin was added to 200mL of water, heated to 45℃, stirred and dissolved, 1.8g of carbazide and 0.4g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and then 20mL of ethanol solution containing 0.4g of 1-hydroxybenzotriazole was added. The mixture was stirred and reacted for 28h, dialyzed, and freeze-dried to obtain carbazide-modified gelatin; S7. Preparation of hydrogel: 10g of silk fibroin peptide / non-animal carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL silk fibroin peptide / non-animal carboxyethyl chitosan solution; 6g of carbazide-modified gelatin was dissolved in water to obtain a 30mg / mL carbazide-modified gelatin solution; 15g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; 3g of oxidized hyaluronic acid-modified liposomes were added to the silk fibroin peptide / non-animal carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining a non-animal carboxyethyl chitosan injection hydrogel.

[0027] Example 3

[0028] This embodiment provides a non-animal-derived carboxyethyl chitosan injectable hydrogel, comprising the following steps: S1. Preparation of non-animal-derived carboxyethyl chitosan: 5g of Ganoderma lucidum chitosan and 8g of acrylic acid were mixed, added to 100mL of water, the pH was adjusted to 6, heated to 90℃, and stirred for 5h. NaOH solution was added to adjust the pH of the reaction system to 9.5, 100mL of acetone was added to precipitate for 1h, filtered, washed, and dried to obtain non-animal-derived carboxyethyl chitosan. S2. Preparation of oxidized hyaluronic acid: Dissolve 10g oligomeric hyaluronic acid in 1L of water, add 5g sodium periodate, stir for 2h, add 10mL ethylene glycol and continue stirring for 1h, dialyze, freeze dry to obtain oxidized hyaluronic acid; S3. Preparation of oxidized hyaluronic acid modified cholesterol: Dissolve 5g of oxidized hyaluronic acid in 1L of water, add 50g of Tween-80 powder evenly, add 300mL of dimethylformamide solution containing 3.5g cholesterol, 5g dicyclohexylcarbodiimide and 0.25g 4-dimethylaminopyridine, heat to 30℃ under nitrogen protection, stir and react for 44h, dialyze, freeze dry to obtain oxidized hyaluronic acid modified cholesterol; S4. Preparation of oxidized hyaluronic acid modified liposomes: 0.7g bisabolol, 0.3g squalene, 0.5g asiatic acid, 25g soybean lecithin and 9g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 50min, and the mixture was sonicated at 400W for 15min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes. S5. Preparation of silk fibroin peptide / non-animal carboxyethyl chitosan: 10g of non-animal carboxyethyl chitosan was dissolved in 500mL of water, 1.5g of N-hydroxysuccinimide and 2.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and the reaction was stirred and activated for 30min. Then, 4g of silk fibroin peptide was added, and the coupling reaction was stirred and carried out for 9h. The mixture was filtered, dialyzed, and freeze-dried to obtain silk fibroin peptide / non-animal carboxyethyl chitosan. S6. Preparation of carbazide-modified gelatin: 2g of gelatin was added to 200mL of water, heated to 45℃, stirred and dissolved, 1.4g of carbazide and 0.3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and then 20mL of ethanol solution containing 0.3g of 1-hydroxybenzotriazole was added. The mixture was stirred and reacted for 24h, dialyzed, and freeze-dried to obtain carbazide-modified gelatin. S7. Preparation of hydrogel: 10g of silk fibroin peptide / non-animal carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL silk fibroin peptide / non-animal carboxyethyl chitosan solution; 5g of carbazide-modified gelatin was dissolved in water to obtain a 30mg / mL carbazide-modified gelatin solution; 12g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; 2.5g of oxidized hyaluronic acid-modified liposomes were added to the silk fibroin peptide / non-animal carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining a non-animal carboxyethyl chitosan injection hydrogel.

[0029] Comparative Example 1 The difference from Example 3 is that step S3 was not performed.

[0030] Includes the following steps: S1. Preparation of non-animal-derived carboxyethyl chitosan: 5g of Ganoderma lucidum chitosan and 8g of acrylic acid were mixed, added to 100mL of water, the pH was adjusted to 6, heated to 90℃, and stirred for 5h. NaOH solution was added to adjust the pH of the reaction system to 9.5, 100mL of acetone was added to precipitate for 1h, filtered, washed, and dried to obtain non-animal-derived carboxyethyl chitosan. S2. Preparation of oxidized hyaluronic acid: Dissolve 10g oligomeric hyaluronic acid in 1L of water, add 5g sodium periodate, stir for 2h, add 10mL ethylene glycol and continue stirring for 1h, dialyze, freeze dry to obtain oxidized hyaluronic acid; S3. Preparation of liposomes: 0.7g bisabolol, 0.3g squalene, 0.5g asiatic acid, 25g soybean lecithin and 9g cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 50min, and the mixture was sonicated at 400W for 15min. The mixture was then filtered through a 0.45μm microporous membrane to obtain liposomes. S4. Preparation of silk fibroin peptide / non-animal carboxyethyl chitosan: 10g of non-animal carboxyethyl chitosan was dissolved in 500mL of water, 1.5g of N-hydroxysuccinimide and 2.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and the reaction was stirred and activated for 30min. Then, 4g of silk fibroin peptide was added, and the coupling reaction was stirred and carried out for 9h. The mixture was filtered, dialyzed, and freeze-dried to obtain silk fibroin peptide / non-animal carboxyethyl chitosan. S5. Preparation of carbazide-modified gelatin: 2g of gelatin was added to 200mL of water, heated to 45℃, stirred and dissolved, 1.4g of carbazide and 0.3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and then 20mL of ethanol solution containing 0.3g of 1-hydroxybenzotriazole was added. The mixture was stirred and reacted for 24h, dialyzed, and freeze-dried to obtain carbazide-modified gelatin; S6. Preparation of hydrogel: 10g of silk fibroin peptide / non-animal carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL silk fibroin peptide / non-animal carboxyethyl chitosan solution; 5g of carbazide-modified gelatin was dissolved in water to obtain a 30mg / mL carbazide-modified gelatin solution; 12g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; 2.5g of liposomes were added to the silk fibroin peptide / non-animal carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining a non-animal carboxyethyl chitosan injection hydrogel.

[0031] Comparative Example 2 The difference from Example 3 is that bisabolol was not added in step S4.

[0032] Specifically as follows: S4. Preparation of oxidized hyaluronic acid modified liposomes: 1.4g squalene, 0.6g asiatic acid, 30g soybean lecithin and 10g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 60min, and the mixture was sonicated at 500W for 20min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes.

[0033] Comparative Example 3 The difference from Example 3 is that squalene was not added in step S4.

[0034] Specifically as follows: S4. Preparation of oxidized hyaluronic acid modified liposomes: 1.4g bisabolol, 0.6g asiatic acid, 30g soybean lecithin and 10g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 60min, and the mixture was sonicated at 500W for 20min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes.

[0035] Comparative Example 4 The difference from Example 3 is that asiatic acid was not added in step S4.

[0036] Specifically as follows: S4. Preparation of oxidized hyaluronic acid modified liposomes: 1g bisabolol, 1g squalene, 30g soybean lecithin and 10g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 60min, and the mixture was sonicated at 500W for 20min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes.

[0037] Comparative Example 5 The difference from Example 3 is that oxidized hyaluronic acid modified liposomes were not added in step S7.

[0038] Specifically as follows: S7. Preparation of hydrogel: 10g of silk fibroin peptide / non-animal carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL silk fibroin peptide / non-animal carboxyethyl chitosan solution; 5g of carbazide-modified gelatin was dissolved in water to obtain a 30mg / mL carbazide-modified gelatin solution; 12g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; carbazide-modified gelatin solution and oxidized hyaluronic acid solution were added to the silk fibroin peptide / non-animal carboxyethyl chitosan solution, and stirred to form a gel, thus obtaining a non-animal carboxyethyl chitosan injection hydrogel.

[0039] Comparative Example 6 The difference from Example 3 is that step S5 was not performed.

[0040] Includes the following steps: S1. Preparation of non-animal-derived carboxyethyl chitosan: 5g of Ganoderma lucidum chitosan and 8g of acrylic acid were mixed, added to 100mL of water, the pH was adjusted to 6, heated to 90℃, and stirred for 5h. NaOH solution was added to adjust the pH of the reaction system to 9.5, 100mL of acetone was added to precipitate for 1h, filtered, washed, and dried to obtain non-animal-derived carboxyethyl chitosan. S2. Preparation of oxidized hyaluronic acid: Dissolve 10g oligomeric hyaluronic acid in 1L of water, add 5g sodium periodate, stir for 2h, add 10mL ethylene glycol and continue stirring for 1h, dialyze, freeze dry to obtain oxidized hyaluronic acid; S3. Preparation of oxidized hyaluronic acid modified cholesterol: Dissolve 5g of oxidized hyaluronic acid in 1L of water, add 50g of Tween-80 powder evenly, add 300mL of dimethylformamide solution containing 3.5g cholesterol, 5g dicyclohexylcarbodiimide and 0.25g 4-dimethylaminopyridine, heat to 30℃ under nitrogen protection, stir and react for 44h, dialyze, freeze dry to obtain oxidized hyaluronic acid modified cholesterol; S4. Preparation of oxidized hyaluronic acid modified liposomes: 0.7g bisabolol, 0.3g squalene, 0.5g asiatic acid, 25g soybean lecithin and 9g oxidized hyaluronic acid modified cholesterol were dissolved in 200mL tetrahydrofuran, heated to 30℃, and rotary evaporated to form a thin film. 100mL ultrapure water was added for hydration for 50min, and the mixture was sonicated at 400W for 15min. The mixture was then filtered through a 0.45μm microporous membrane to obtain oxidized hyaluronic acid modified liposomes. S5. Preparation of carbazide-modified gelatin: 2g of gelatin was added to 200mL of water, heated to 45℃, stirred and dissolved, 1.4g of carbazide and 0.3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and then 20mL of ethanol solution containing 0.3g of 1-hydroxybenzotriazole was added. The mixture was stirred and reacted for 24h, dialyzed, and freeze-dried to obtain carbazide-modified gelatin; S6. Preparation of hydrogel: 10g of non-animal-derived carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL non-animal-derived carboxyethyl chitosan solution; 5g of carbazide-modified gelatin was dissolved in water to obtain a 30mg / mL carbazide-modified gelatin solution; 12g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; 2.5g of oxidized hyaluronic acid-modified liposomes were added to the non-animal-derived carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining a non-animal-derived carboxyethyl chitosan injection hydrogel.

[0041] Comparative Example 7 The difference from Example 3 is that no carbazide-modified gelatin solution was added in step S7.

[0042] Specifically as follows: S7. Preparation of hydrogel: 15g of silk fibroin peptide / non-animal carboxyethyl chitosan was dissolved in water to obtain a 50mg / mL silk fibroin peptide / non-animal carboxyethyl chitosan solution; 12g of oxidized hyaluronic acid was dissolved in water to obtain a 70mg / mL oxidized hyaluronic acid solution; 2.5g of oxidized hyaluronic acid modified liposomes were added to the silk fibroin peptide / non-animal carboxyethyl chitosan solution, and after stirring and mixing evenly, the oxidized hyaluronic acid solution was added, and the mixture was stirred to form a gel, thus obtaining a non-animal carboxyethyl chitosan injection hydrogel.

[0043] Test Example 1 The non-animal-derived carboxyethyl chitosan injection hydrogels prepared in Examples 1-3 and Comparative Examples 1, 5-7 were subjected to performance tests, and the results are shown in Table 1.

[0044] (1) Bond strength: Using a 1.0 mL glass syringe, the hydrogel was extruded into the middle of a 4 cm long and 3 cm wide pigskin strip, with an overlap area of ​​1 cm × 3 cm between the two strips, to obtain the test sample. The test was conducted according to YY / T0729.1 Test Method for Adhesive Bonding Properties Part 1: Overlap-Shear Tensile Bearing Strength.

[0045] (2) In vitro degradation performance: The hydrogel was freeze-dried to obtain a dry gel. A sample of dry gel, weighing m0, was placed in an aqueous solution at 37°C and agitated at 100 rpm. At regular intervals, the sample was removed, freeze-dried, and weighed as m1. The degradation solution was then replaced. Complete degradation was considered achieved when (m0-m1 / m0) was greater than 0.99. The time to complete degradation was recorded.

[0046] (3) In vitro cytotoxicity: The hydrogel was tested according to the ISO 10993-5 standard test method.

[0047] (4) Tensile strength: The tensile properties of the hydrogel were tested at room temperature using a CMT4102 universal testing machine. The specimen diameter was 5 mm, the length was 30 mm, and the tensile rate was 50 mm / min.

[0048] Table 1

[0049] As shown in the table above, the non-animal-derived carboxyethyl chitosan injection hydrogels prepared in Examples 1-3 of this invention have good bonding strength and mechanical strength, short degradation time, and low cytotoxicity.

[0050] Test Example 2 Dissolve streptozotocin in 50 nmol / L sodium citrate buffer to a final streptozotocin solution concentration of 4 mg / mL.

[0051] Establishment of a mouse model of diabetes: SPF-grade 8-10 week old male C57BL / 6J mice were acclimatized for 7 days, then fasted for 6 hours and injected intraperitoneally with streptozotocin solution (50 mg / kg). The mice were then returned to their original cages and provided with normal food and 5% sucrose water. This injection was repeated for 5 consecutive days. One week after the injection, the mice were fasted for 6 hours, and fasting blood glucose was measured via tail vein. A fasting blood glucose level ≥11.1 mmol / L was considered a successful establishment of a diabetic model.

[0052] Construction of a mouse skin injury model: Diabetic mice were anesthetized by intraperitoneal injection of 20% urethane (5 ml / kg). Hair was removed from the backs of the mice using a hair removal device, depilatory cream was applied, and the skin was wiped with alcohol swabs. Two full-thickness skin lesions were symmetrically created on both sides of the mouse's back using an 8 mm diameter punch, and the skin around the lesions was secured with silicone sheets. The skin and silicone sheets were sutured.

[0053] Mice with successfully established skin injury models were randomly divided into a model group, Examples 1-3, and Comparative Examples 1-7. The model group was treated with physiological saline. Examples 1-3 and Comparative Examples 1-7 were injected with the corresponding non-animal-derived carboxyethyl chitosan injection hydrogel. The dosage was 2 mL per mouse. After 5 days, photographs of the skin wounds were taken, and the wound healing area and healing rate were calculated using ImageJ software.

[0054] Wound healing rate = [Original wound area (day 0) - Wound area (day 5)] ÷ Original wound area (day 0) × 100%.

[0055] The results are shown in Table 2.

[0056] Table 2

[0057] As shown in the table above, the non-animal-derived carboxyethyl chitosan injection hydrogels prepared in Examples 1-3 of this invention have a good effect on promoting wound healing.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a non-animal-derived carboxyethyl chitosan injection hydrogel, characterized in that, After being modified with acrylic acid, the bacterial chitosan was coupled with silk fibroin peptides to prepare silk fibroin peptide / non-animal carboxyethyl chitosan. Oligomeric hyaluronic acid was oxidized, cholesterol was modified, and used to encapsulate bisabolol, squalene, and asiatic acid to prepare oxidized hyaluronic acid modified liposomes. These liposomes were then mixed with carbazide-modified gelatin, silk fibroin peptide / non-animal carboxyethyl chitosan, and oxidized hyaluronic acid to prepare non-animal carboxyethyl chitosan injection hydrogel.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of non-animal-derived carboxyethyl chitosan: Mix bacterial chitosan and acrylic acid, add water, heat and stir to react, adjust the pH value of the reaction system, add organic solvent to precipitate, filter, wash, and dry to obtain non-animal-derived carboxyethyl chitosan; S2. Preparation of oxidized hyaluronic acid: Oligomeric hyaluronic acid was dissolved in water, an oxidizing agent was added, the mixture was stirred and reacted, ethylene glycol was added and stirring was continued, the product was dialyzed, and freeze-dried to obtain oxidized hyaluronic acid; S3. Preparation of oxidized hyaluronic acid modified cholesterol: Oxidized hyaluronic acid was dissolved in water, an emulsifier was added and dispersed evenly, a dimethylformamide solution containing cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine was added, and the reaction was carried out under inert gas protection by heating and stirring, dialyzing, and freeze drying to obtain oxidized hyaluronic acid modified cholesterol; S4. Preparation of oxidized hyaluronic acid modified liposomes: Bisabolol, squalene, asiatic acid, soybean lecithin and oxidized hyaluronic acid modified cholesterol were dissolved in tetrahydrofuran, heated and rotary evaporated to form a film, hydrated with ultrapure water, sonicated, filtered, and oxidized hyaluronic acid modified liposomes were obtained. S5. Preparation of silk fibroin peptide / non-animal carboxyethyl chitosan: Non-animal carboxyethyl chitosan was dissolved in water, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, the reaction was activated by stirring, then silk fibroin peptide was added, the coupling reaction was stirred, filtered, dialyzed, and freeze-dried to obtain silk fibroin peptide / non-animal carboxyethyl chitosan; S6. Preparation of carbazide-modified gelatin: Gelatin was added to water and heated to dissolve. Carbazide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, followed by the addition of an ethanol solution containing 1-hydroxybenzotriazole. The mixture was stirred and reacted, dialyzed, and freeze-dried to obtain carbazide-modified gelatin. S7. Preparation of hydrogel: Silk fibroin peptide / non-animal-derived carboxyethyl chitosan, carbazide-modified gelatin, and oxidized hyaluronic acid were dissolved in water to obtain solutions; oxidized hyaluronic acid-modified liposomes were added to the silk fibroin peptide / non-animal-derived carboxyethyl chitosan solution, and after stirring and mixing evenly, the carbazide-modified gelatin solution and the oxidized hyaluronic acid solution were added, and the mixture was stirred to form a gel, thus obtaining non-animal-derived carboxyethyl chitosan injection hydrogel.

3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of the bacterial chitosan to acrylic acid is 1:1.5-2.5, the heating and stirring reaction temperature is 85-95℃, the time is 4-6h, the pH of the reaction system is adjusted to 9-10, and the organic solvent is acetone.

4. The preparation method according to claim 2, characterized in that, The oxidant in step S2 is sodium periodate, the mass ratio of oligomeric hyaluronic acid to oxidant is 10:4-6, and the stirring reaction time is 1-3 hours.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of oxidized hyaluronic acid, emulsifier, cholesterol, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 0.4-0.6:4-6:0.3-0.4:0.45-0.55:0.02-0.03, and the heating and stirring reaction is carried out at a temperature of 25-35°C for 40-48 hours.

6. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of bisabolol, squalene, asiatic acid, soybean lecithin, and oxidized hyaluronic acid modified cholesterol is 0.5-1:0.2-0.4:0.3-0.6:20-30:8-10, the hydration time is 40-60 min, the ultrasonic power is 300-500 W, and the time is 10-20 min.

7. The preparation method according to claim 2, characterized in that, The mass ratio of non-animal-derived carboxyethyl chitosan, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and silk fibroin peptide in step S5 is 10:1-2:2-3:3-5.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of gelatin, carbazide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 2:1-1.8:0.2-0.4:0.2-0.4, and the stirring reaction time is 20-28 h; in step S7, the mass ratio of silk fibroin peptide / non-animal-derived carboxyethyl chitosan, carbazide-modified gelatin, oxidized hyaluronic acid, and oxidized hyaluronic acid-modified liposomes is 10:4-6:10-15:2-3.

9. A non-animal-derived carboxyethyl chitosan injection hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The use of a non-animal-derived carboxyethyl chitosan injectable hydrogel as described in claim 9 in the preparation of a medicament for promoting skin wound healing.

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