Crosslinked carboxymethyl chitin hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical biopolymer materials, in particular to cross-linked carboxymethyl chitin hydrogel and a preparation method and application thereof, and the preparation method comprises preparation of non-cross-linked carboxymethyl chitin and cross-linking reaction. The preparation method of the non-crosslinked carboxymethyl chitin comprises the following steps: 1) alkalization: suspending chitin in an alkaline aqueous solution, and stirring and alkalizing at 5-30 DEG C; 2) etherification: dissolving substituted acetic acid or salt thereof, dropwise adding the dissolved substituted acetic acid or salt thereof into the alkalized system, and carrying out etherification reaction at 5-45 DEG C; and 3) separating and refining a crude product. The non-crosslinking carboxymethyl chitin prepared by the method can be used for a PEGDE crosslinking reaction, so that the PEGDE crosslinking carboxymethyl chitin with high viscoelasticity is obtained, and other carboxymethyl chitin with high substitution degree does not have the non-crosslinking carboxymethyl chitin. The elastic modulus of the cross-linked carboxymethyl chitin hydrogel obtained through the reaction is wide and dynamically adjustable, and meanwhile, the product has excellent moist heat sterilization stability and enzymatic degradation stability.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biopolymer materials, and specifically relates to a cross-linked carboxymethyl chitin gel and a preparation method and application thereof. Background Art

[0002] Hydrogels are three-dimensional polymer networks dispersed in water. They are flexible, retain their shape, and can absorb large amounts of water. They have a wide range of applications in biomedicine, environmental protection, the food industry, and other fields. Depending on the synthetic material, hydrogels are categorized as synthetic polymer hydrogels and natural polymer hydrogels. Natural polymers offer advantages such as good biocompatibility, environmental sensitivity, abundant sources, and low cost. However, they suffer from drawbacks such as a short lifetime in the human body due to enzymatic degradation. Therefore, the synthesis of polymer hydrogels through chemical methods has become a growing research hotspot.

[0003] Chitin is widely distributed in nature and exists in the cell walls of some fungi, some algae, and the bones and shells of shrimps, crabs, insects, etc. Its annual production on Earth is second only to cellulose, making it the second largest natural polysaccharide resource. Chitin is a natural biomass material that is biocompatible, biodegradable, and sustainable, and has potential applications in many clinical fields. However, due to the strong hydrogen bonds within and between its molecules, chitin is almost insoluble in any solvent (including acidic and alkaline aqueous solutions and various organic solvents), which makes chitin unable to be widely used. Carboxymethyl Chitosan (CMC) refers to a product obtained by S N Chitin derivatives are obtained by introducing carboxymethyl groups onto chitin's hydroxyl and / or amino groups via a 2-substitution reaction. The introduction of carboxymethyl groups disrupts the inherent hydrogen bonding within chitin, while also enhancing the water solubility of the carboxyl group or carboxylate itself. Therefore, this strategy addresses the solubility problem of chitin and has gained widespread recognition in the industry. Furthermore, compared to sodium hyaluronate, carboxymethyl chitin lacks specific enzymes in the body and has a longer residual lifespan than sodium hyaluronate, effectively reducing the frequency of injections. Its antibacterial properties also offer a significant advantage over sodium hyaluronate. Consequently, carboxymethyl chitin has found widespread medical applications, such as intra-articular injections, surgical adhesion prevention, and cosmetic procedures.

[0004] The introduction of carboxymethyl groups solves the solubility problem of chitin, but there are also some technical problems that remain unresolved. For example: 1) Current synthetic strategies often make it difficult to control the degree of substitution of carboxymethyl groups, resulting in either over-substitution (full substitution of O and N) or insufficient substitution, leading to poor solubility; 2) Excessive substitution increases solubility, but excessive solubility leads to poor viscoelastic properties; 3) When used for intra-articular injection, although the half-life of carboxymethyl chitin in the body is longer than that of sodium hyaluronate and the injection frequency is lower, multiple injections are still required; 4) When used for postoperative adhesion prevention, its aqueous solution has good fluidity in the body but insufficient adhesion. At the same time, its half-life is short, and hydrolysis in the body fluid environment will cause it to degrade prematurely, which is insufficient to cover a wound healing cycle; 5) In terms of chemical stability, non-cross-linked carboxymethyl chitin products cannot withstand terminal wet heat sterilization, and there are significant biosafety risks during clinical use, which may be related to excessive substitution.

[0005] Improving the biological and chemical stability of carboxymethyl chitin hydrogels through cross-linking is a major strategy to address these drawbacks. Currently, the main cross-linking agents used in biomedical materials include 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS). However, due to their potential toxicity, these cross-linkers are increasingly being replaced by natural products such as anthocyanins and lysine. Lysine, for example, has excellent biosafety and is an essential amino acid in the body, offering significant clinical advantages. The cross-linking reaction between amine cross-linkers and carboxymethyl chitin essentially involves the formation of an amide bond between amino and carboxyl groups. To achieve this, condensation agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) are added to the reaction system. However, the use of these condensation agents carries the risk of introducing impurities into the product. At the same time, polyethylene glycol (PEG), a linear or branched polyether, is a polymer that has been attracting much attention in the industry and is often used to covalently modify biomacromolecules to meet the needs of many pharmaceutical and biotechnology applications. Because PEG is a non-toxic, non-immunogenic polymer, it is not recognized by biomolecules such as proteins and can escape the mononuclear phagocyte system in the blood, so it is called a "stealth molecule." Studies have shown that PEG has low toxicity through various routes of administration.

[0006] Therefore, how to choose a more efficient, environmentally friendly and safe cross-linking method is also one of the important issues that need to be studied and explored in the preparation of cross-linked carboxymethyl chitin. Summary of the Invention

[0007] To address the aforementioned issues in the prior art, the present invention prepares a carboxymethyl chitin that can undergo a cross-linking reaction with polyethylene glycol diglycidyl ether (PEGDE) under mild conditions. Based on this, a new PEGDE-cross-linked carboxymethyl chitin is prepared, and its properties and uses are studied. The cross-linked carboxymethyl chitin hydrogel prepared by the present invention retains the original biological properties of carboxymethyl chitin while exhibiting a certain degree of elasticity and dynamic viscosity. Furthermore, the present invention addresses the difficulty of terminal sterilization of existing carboxymethyl chitin aqueous solutions.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention first provides a method for preparing non-cross-linked carboxymethyl chitin, comprising the following steps:

[0010] (1) Alkalization: suspend chitin in an alkaline aqueous solution and stir at 5-30°C for alkalization; the alkaline aqueous solution is a sodium hydroxide aqueous solution and / or a potassium hydroxide aqueous solution; the mass percentage of alkali in the alkaline aqueous solution is 30%-55%, preferably 30%-50%; the mass ratio of chitin to alkaline aqueous solution is 1.0:5.0-1.0:25.0, preferably 1.0:7.0-1.0:15.0; the alkalization temperature is preferably 10-30°C.

[0011] (2) Etherification: dissolving a substituted acetic acid or a salt thereof and adding it dropwise to the alkalized system at 5 to 45° C. to carry out an etherification reaction; the substituted acetic acid is at least one of iodoacetic acid, bromoacetic acid, and chloroacetic acid; and the molar amount (relative to the chitin repeating unit, i.e., N-acetyl-D-glucosamine) is 1.5 to 7.0 equivalents, preferably 3.0 to 5.0 equivalents;

[0012] Preferably, the etherification temperature is 15-35°C.

[0013] Preferably, substituted chloroacetic acid or its salt is dissolved in an alcohol solution for use; the alcohol solution is isopropanol and / or 2-butanol.

[0014] Based on the principle of halogen exchange, potassium iodide, sodium iodide, etc. can be added to promote the substitution reaction; since the reaction system is heterogeneous, phase transfer catalysts such as sodium dodecyl sulfonate and sodium dodecyl sulfate can be added.

[0015] (3) Crude product separation and purification: A crude product is obtained by adding ethanol to the reaction solution, and the crude product is redissolved and filtered to remove insoluble matter to obtain a crude product filtrate; aqueous ethanol is added to the crude product filtrate to obtain a refined product. The aqueous ethanol is 75% to 99% ethanol, preferably 80% to 95% ethanol. Aqueous ethanol can precipitate the target product while removing inorganic salts, thereby improving product quality. Similarly, the use of organic solvents such as isopropanol and acetone can achieve the same effect. In addition, the crude product filtrate can be ultrafiltered to remove small molecular impurities, and then freeze-dried to obtain a refined product.

[0016] The non-crosslinked carboxymethyl chitin obtained using the above preparation method has good solubility and unique chemical reactivity, reacting with compounds containing epoxy groups. It also exhibits unique rheological properties, with low elastic modulus, viscous modulus, and shear viscosity. For example, a hydrogel containing 2.0% carboxymethyl chitin exhibits an elastic modulus (2.5 Hz) of 4 to 53 Pa, a viscous modulus (2.5 Hz) of 6 to 42 Pa, and a shear viscosity (10 1 / s) of 2 to 12 Pa.s.

[0017] The present invention also provides a method for preparing a cross-linked carboxymethyl chitin hydrogel. The method uses a specific, low-viscoelastic, non-cross-linked carboxymethyl chitin prepared in the present invention as a raw material and cross-links it with PEG-DE to produce a highly viscoelastic cross-linked carboxymethyl chitin. Prior art literature reports on non-cross-linked carboxymethyl chitin / carboxymethyl chitosan, which are often highly carboxymethyl substituted, i.e., the chitin / chitosan's hydroxyl and / or amino groups are highly substituted. These chitins are typically highly carboxymethylated using a vastly excessive amount of chloroacetic acid and varying reaction temperatures to improve the product's water solubility. The present invention, however, achieves a non-cross-linked carboxymethyl chitin / carboxymethyl chitosan with a low degree of carboxymethyl substitution by adjusting parameters (including the amount of chloroacetic acid, reaction temperature, and reaction time), leaving some hydroxyl groups exposed (although some amino groups are also present). This type of carboxymethyl chitin exhibits good water solubility and low viscoelasticity. Furthermore, the hydrogel exhibits good water solubility and low viscoelasticity, and cross-linking with PEG-DE yields a highly viscoelastic carboxymethyl chitin that is stable to moist heat sterilization.

[0018] Using the aforementioned carboxymethyl chitin as a raw material, a cross-linking reaction with PEGDE under mild conditions yielded a highly viscoelastic cross-linked carboxymethyl chitin. This highly viscoelastic cross-linked carboxymethyl chitin exhibits high thermal stability and is resistant to moist heat sterilization. Comparative experiments revealed that non-cross-linked carboxymethyl chitin / carboxymethyl chitosan with a high degree of carboxymethyl substitution did not cross-link with PEGDE, regardless of whether the amino groups were exposed. This indicates that the reaction site with PEGDE is the hydroxyl groups on the chitin / chitosan.

[0019] Specifically, the cross-linking reaction is carried out by using the prepared non-cross-linked carboxymethyl chitin as a raw material, PEGDE as a cross-linking agent, and PBS solution or purified water as a solvent without adding other substances.

[0020] The specific steps include:

[0021] (1) Dissolving: dissolving the non-cross-linked carboxymethyl chitin raw material prepared by the present invention in purified water or PBS to obtain a carboxymethyl chitin solution; wherein the final concentration of the carboxymethyl chitin is 1% to 10%, preferably 2% to 5%;

[0022] (2) Cross-linking: adding a cross-linking agent PEGDE to the carboxymethyl chitin solution prepared in step (1), stirring evenly and then performing a cross-linking reaction to prepare a PEGDE cross-linked carboxymethyl chitin hydrogel; the molar ratio of carboxymethyl chitin to PEGDE epoxy group is 1:0.05 to 1:2, preferably 1:0.3 to 1:0.8; the cross-linking reaction temperature is 20 to 50°C, and the cross-linking reaction time is 24 to 192 hours.

[0023] (3) Dialysis: The PEGDE cross-linked carboxymethyl chitin hydrogel prepared in step (2) is dialyzed using purified water or PBS; wherein the dialysis temperature is 10-40° C., the dialysate is replaced every 24 hours, and the dialysis time is 72-120 hours;

[0024] (4) Canning and Sterilization: The gel obtained after dialysis in step (3) is crushed, canned, and sterilized by moist heat to obtain a finished PEGDE cross-linked carboxymethyl chitin hydrogel preparation. The semi-finished carboxymethyl chitin hydrogel is filled into pre-filled syringes with a size of 1 to 10 mL using a filling machine and sterilized in a moist heat sterilizer (sterilization temperature 121° C., sterilization time 8 to 30 min).

[0025] The cross-linked carboxymethyl chitin hydrogel prepared by the preparation method of the present invention has unique rheological properties. By adjusting the concentration of carboxymethyl chitin and the amount of cross-linking agent in the reaction solution, the elastic modulus of the cross-linked carboxymethyl chitin hydrogel obtained by the reaction is wide and dynamically adjustable. At the same time, the product has excellent safety, wet heat sterilization stability and enzyme degradation stability.

[0026] The cross-linked carboxymethyl chitin hydrogel prepared by the invention can be used in medical treatment, cosmetics, filling and shaping, as well as post-surgical adhesion prevention, drug delivery and drug controlled release.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention has prepared a non-cross-linked carboxymethyl chitin through a large number of experimental explorations and optimizations. This carboxymethyl chitin not only ensures its excellent water solubility, but also avoids a series of problems caused by excessive carboxymethylation, such as the large amount of alkali and chloroacetic acid used in the production process, and the further reaction of carboxyl groups with chloroacetic acid to form esters.

[0029] (2) The non-crosslinked carboxymethyl chitin prepared by the present invention has unique rheological properties, and its elastic modulus, viscous modulus and shear viscosity are all low. Taking the hydrogel with a carboxymethyl chitin content of 2.0% as an example, its elastic modulus (2.5Hz) is 4-53Pa, the viscous modulus (2.5Hz) is 6-42Pa, and the shear viscosity (10S -1 )2~12Pa.S, there is room for further cross-linking to change the rheological properties.

[0030] (3) The non-cross-linked carboxymethyl chitin prepared by the present invention has unique chemical reactivity and can be used in PEG-DE cross-linking reactions, thereby obtaining highly viscoelastic PEG-DE cross-linked carboxymethyl chitin, which is not possessed by other highly substituted carboxymethyl chitins. The cross-linking reaction occurs spontaneously under mild conditions, without the need for acids, bases, condensing agents, or other catalysts. Post-processing is simple and easy to operate, and the process is more tolerant to production equipment, making it more conducive to commercial production.

[0031] (4) The PEGDE cross-linked carboxymethyl chitin hydrogel prepared by the present invention has a wide range of elastic moduli and is dynamically adjustable by adjusting the concentration of carboxymethyl chitin and the amount of cross-linking agent in the reaction solution. At the same time, the product has excellent wet heat sterilization stability and enzyme degradation stability.

[0032] (5) In view of the unique rheological properties, enzyme degradation stability and terminal wet heat sterilization stability of the PEGDE cross-linked carboxymethyl chitin hydrogel prepared by the present invention, it can be applied in the fields of medical treatment, cosmetics, filling and shaping, postoperative adhesion prevention, drug delivery and drug controlled release. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the safety test result of intradermal injection, 2 hours after injection (ER erythema = 0, ED edema = 0).

[0034] Figure 2 This is the safety test result of intradermal injection, 72 hours after injection (ER erythema = 0.2, ED edema = 0). DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. Technology and methods known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and methods should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

[0037] In the following examples, the experimental methods without specific conditions are generally measured according to national standards; if there is no corresponding national standard, the general standard requirements or general methods are used.

[0038] Example 1

[0039] A method for preparing non-cross-linked carboxymethyl chitin, comprising the following steps:

[0040] (1) Alkalization: Weigh 12.00 g of chitin, add it to 120 g of a 50% potassium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 16.5 h.

[0041] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 27.90 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 5-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 hours. After the addition is complete, stir and react at 20°C for 41 hours.

[0042] (3) Crude product separation and purification: 600 mL of 80% ethanol was added to the reactant, stirred for 15 min, allowed to stand, and the supernatant was decanted. Then, 95% ethanol was added, stirred for 15 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0043] The crude carboxymethyl chitin was reconstituted in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter. The filtrate was concentrated by ultrafiltration, and 95% ethanol was added dropwise to precipitate the product. The product was then filtered and the filter cake dried under reduced pressure to obtain 8.25 g of white powdery solid carboxymethyl chitin. Elemental analysis revealed a degree of carboxymethyl substitution of 0.509.

[0044] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0045] Example 2

[0046] A method for preparing non-cross-linked carboxymethyl chitin, comprising the following steps:

[0047] (1) Alkalization: Weigh 16.00 g of chitin and add it to 122.5 g of a 35% sodium hydroxide aqueous solution. Stir continuously at 20° C. and alkalize for 23.2 h.

[0048] (2) Etherification: Add 160 mL of isopropanol to the alkalized system and stir for approximately 30 min. Weigh 37.16 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature below 20°C and control the temperature at 5-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 0.5 h. After the addition is complete, stir and react at 25°C for 49.4 h.

[0049] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 15 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 15 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0050] The crude carboxymethyl chitin was reconstituted in 10 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration, and 95% ethanol was added dropwise to precipitate the product. The product was then filtered and the filter cake was dried under reduced pressure to obtain 17.00 g of white powdery solid carboxymethyl chitin. Elemental analysis revealed a degree of carboxymethyl substitution of 0.729.

[0051] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0052] Example 3

[0053] A method for preparing non-cross-linked carboxymethyl chitin, comprising the following steps:

[0054] (1) Alkalization: Weigh 12.00 g of chitin, add it to 102 g of a 35% sodium hydroxide aqueous solution, and stir continuously at 10° C. to alkalinize for 17 h.

[0055] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 27.89 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 8-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 hours. After the addition is complete, stir and react at 20°C for 41 hours.

[0056] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 20 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 20 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0057] The crude carboxymethyl chitin was reconstituted in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP membrane. The filtrate was concentrated by ultrafiltration and then dropwise added with 95% ethanol to precipitate the product. The product was then filtered and the filter cake dried under reduced pressure to obtain 12.77 g of white powdery solid carboxymethyl chitin. Elemental analysis revealed a carboxymethyl degree of substitution of 0.698.

[0058] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0059] Example 4

[0060] A method for preparing non-cross-linked carboxymethyl chitin, comprising the following steps:

[0061] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 25° C. to alkalize for 22.3 h.

[0062] (2) Etherification: Add 160 mL of isopropanol to the alkalized system and stir for approximately 30 min. Weigh 37.18 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature below 20°C and control the temperature at 5-20°C to add the chloroacetic acid solution in isopropanol dropwise for 0.9 h. After the addition is complete, stir and react at 35°C for 21.8 h.

[0063] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 15 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 15 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0064] The crude carboxymethyl chitin was reconstituted in 10 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration, and 95% ethanol was added dropwise to precipitate the product. The product was then filtered and the filter cake was dried under reduced pressure to obtain 15.11 g of carboxymethyl chitin as a white powder. Elemental analysis revealed a degree of carboxymethyl substitution of 0.486.

[0065] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0066] Example 5

[0067] A method for preparing non-cross-linked carboxymethyl chitin, comprising the following steps:

[0068] (1) Alkalization: Weigh 12.03 g of chitin, add it to 171.4 g of a 35% potassium hydroxide aqueous solution, and stir continuously at 10° C. to alkalinize for 18 h.

[0069] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for approximately 30 min. Weigh 16.73 g (3.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to 10°C and control the temperature at 10-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6.1 h. After the addition is complete, stir and react at 20°C for 42 h.

[0070] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 15 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 15 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0071] The crude carboxymethyl chitin was reconstituted in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter. The filtrate was concentrated by ultrafiltration, and 95% ethanol was added dropwise to precipitate the product. The product was then filtered and the filter cake dried under reduced pressure to obtain 10.05 g of white powdery solid carboxymethyl chitin. Elemental analysis revealed a carboxymethyl degree of substitution of 0.406.

[0072] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0073] Table 1 Preparation of carboxymethyl chitin and determination of modulus and shear viscosity

[0074]

[0075] It can be seen from Table 1 that according to the preparation scheme provided by the present invention, carboxymethyl chitin with low viscoelasticity and low shear viscosity can be prepared. The reaction characteristics of the carboxymethyl chitin will be investigated later.

[0076] Examples 6 to 11

[0077] The non-cross-linked carboxymethyl chitin prepared in Example 1 was dispersed in purified water and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin reached 2%. A cross-linking agent, polyethylene glycol diglycidyl ether (PEGDE), was then added to the system (molar ratio of carboxymethyl chitin:PEGDE epoxy = 1:0.05 to 1:1). After stirring uniformly, the mixture was allowed to react at 40°C. Samples were taken at different time points to measure the modulus (2.5 Hz) and shear viscosity (10 1 / s). The results are as follows.

[0078] Table 2 Changes in sample modulus and shear viscosity during the crosslinking reaction at a carboxymethyl chitin concentration of 2%

[0079]

[0080] The results showed that compared with the blank group, carboxymethyl chitin and PEGDE underwent cross-linking reaction after the addition of cross-linking agent. As the reaction time prolonged, the modulus and shear viscosity of the reaction solution increased significantly, and the reaction effect was most significant when the cross-linking agent dosage was 0.7 equivalent and 1.0 equivalent.

[0081] Examples 12 to 17

[0082] The non-crosslinked carboxymethyl chitin prepared in Example 1 was dispersed in purified water and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin was 3%. Then, a crosslinking agent, polyethylene glycol diglycidyl ether (PEGDE), was added to the system (the molar ratio of carboxymethyl chitin:PEGDE epoxy=1:0.05-1:1). After stirring uniformly, the mixture was allowed to stand at 30°C for reaction. The modulus (2.5 Hz) and shear viscosity (10s) of the samples were measured at different time points. -1 ), and the results are as follows.

[0083] Table 3 Changes in sample modulus and shear viscosity during the crosslinking reaction at a carboxymethyl chitin concentration of 3%

[0084]

[0085]

[0086] The results showed that the modulus and shear viscosity of the samples increased with increasing crosslinker addition. Compared to Examples 6-11, increasing the raw material concentration significantly improved the modulus and shear viscosity of the samples. When the reaction product was dialyzed against a PBS solution to a carboxymethyl chitin content of 2%, the modulus of the gel decreased, but its viscosity was well retained, which could further expand the application of the material.

[0087] Examples 18 to 23

[0088] The non-cross-linked carboxymethyl chitin prepared in Example 1 was dispersed in purified water and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin reached 4%. A cross-linking agent, polyethylene glycol diglycidyl ether (PEGDE), was then added to the system (molar ratio of carboxymethyl chitin: PEGDE epoxy value = 1:0.05 to 1:1). After stirring uniformly, the mixture was allowed to react at 25°C. Samples were taken at different time points to measure the modulus (2.5 Hz) and shear viscosity (10 1 / s). The results are shown below.

[0089] Table 4 Changes in sample modulus and shear viscosity during the crosslinking reaction at a carboxymethyl chitin concentration of 4%

[0090]

[0091] The results show that the modulus and shear viscosity of the reaction product increase with the increase in the amount of cross-linking agent added. Compared with Examples 12 to 17, the increase in the raw material concentration further increases the modulus and shear viscosity of the sample.

[0092] Examples 24 to 29

[0093] The non-cross-linked carboxymethyl chitin prepared in Example 1 was dispersed in purified water and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin reached 5%. A cross-linking agent, polyethylene glycol diglycidyl ether (PEGDE), was then added to the system (molar ratio of carboxymethyl chitin: PEGDE epoxy value = 1:0.05 to 1:1). After stirring uniformly, the system was allowed to react at 40°C. Samples were taken at different time points to measure the modulus (2.5 Hz) and shear viscosity (10 1 / s). The results are as follows.

[0094] Table 5 Changes in sample modulus and shear viscosity during the crosslinking reaction at a carboxymethyl chitin concentration of 5%

[0095]

[0096] The results show that the modulus and shear viscosity of the reaction product increase with the increase in the amount of cross-linking agent added. Compared with Examples 18 to 23, the increase in the raw material concentration significantly increases the modulus and shear viscosity of the sample.

[0097] It can be seen from Examples 6 to 29 that by adjusting the concentration of carboxymethyl chitin and the amount of cross-linking agent in the cross-linking reaction, gel materials with a wide range of moduli and shear viscosities can be prepared, which have application potential in the fields of medical treatment, cosmetics, filling and shaping, anti-adhesion after surgery, drug delivery, and sustained and controlled drug release.

[0098] Examples 30 to 33

[0099] The tolerance of cross-linked carboxymethyl chitin hydrogels to moist heat sterilization was investigated at 121°C for 8 minutes. The shear viscosities of the gels before and after sterilization are shown in Table 6. Example 30 utilizes the reaction solution from Example 6 (carboxymethyl chitin content 2%), while Examples 31-33 utilize the reaction solutions from Examples 16, 26, and 28, respectively, diluted to a carboxymethyl chitin content of 2%.

[0100] Table 6 Shear viscosity of cross-linked carboxymethyl chitin hydrogel before and after sterilization

[0101]

[0102] The results showed that the shear viscosity of the cross-linked carboxymethyl chitin hydrogel sample decreased by no more than 30% after wet heat sterilization, and the shear viscosity value remained at a high level. This indicates that the cross-linked carboxymethyl chitin obtained by the PEGDE cross-linking reaction using the non-cross-linked carboxymethyl chitin prepared by the present invention can withstand terminal wet heat sterilization, thereby improving the application safety and making the application range of this material wider.

[0103] In order to highlight the beneficial effects of the present invention, the following comparative examples are given.

[0104] Comparative Example 1

[0105] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0106] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and stir continuously at 10° C. to alkalinize for 17 h.

[0107] (2) Etherification: Add 160 mL of isopropanol to the alkalized system and stir for approximately 30 min. Weigh 37.18 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature below 10°C and control the temperature at 5-10°C to add the chloroacetic acid solution in isopropanol dropwise for 5.2 h. After the addition is complete, stir and react at 10°C for 41 h.

[0108] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 20 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 20 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0109] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 9.72 g of white powdery solid carboxymethyl chitin.

[0110] A 2.0% carboxymethyl chitin solution of the non-cross-linked carboxymethyl chitin of this comparative example was prepared in phosphate buffer. The elastic modulus (G', 2.5 Hz) was 139.4 Pa, the viscous modulus (G", 2.5 Hz) was 43.4 Pa, and the shear viscosity (η, 10 1 / s) was 26.2 Pa.s as measured by a rheometer.

[0111] Example 34

[0112] The enzymatic stability of the cross-linked carboxymethyl chitin hydrogel was investigated. A sodium acetate-glacial acetic acid buffer solution at pH 4.5 was prepared as a lysozyme buffer, and the lysozyme was prepared into an enzymatic solution. The lysozyme solution was added to the non-cross-linked hydrogel prepared in Comparative Example 1 with a 2% carboxymethyl chitin content, and to the cross-linked hydrogel prepared by diluting the reaction solution of Example 27 to a carboxymethyl chitin content of 2%, to a final lysozyme concentration of 200 IU / g. The hydrogel was then enzymatically hydrolyzed at 25°C. During this process, the changes in dynamic viscosity (10 1 / s) were measured. The results are shown in Table 7.

[0113] Table 7 Cross-linked carboxymethyl chitin hydrogel enzyme degradation stability data

[0114] Sample name Non-cross-linked carboxymethyl chitin solution Cross-linked carboxymethyl chitin hydrogel Shear viscosity (10 1 / s, Pa.S) 0h 25.16 13.64 Shear viscosity (10 1 / s, Pa.S) 24h 3.68 12.87 Decline 85.6％ 5.6％

[0115] The results showed that the shear viscosity of the non-cross-linked carboxymethyl chitin solution decreased by 85.6% after 24 hours, while that of the cross-linked carboxymethyl chitin solution decreased by only 5.6%, as shown in Table 7. This indicates that PEGDE cross-linking greatly improves the enzymatic degradation stability of carboxymethyl chitin, thereby increasing the retention time of cross-linked carboxymethyl chitin in the body and achieving a long-lasting effect.

[0116] Examples 35 to 38

[0117] The non-cross-linked carboxymethyl chitin prepared in Example 1 was dispersed in purified water or PBS and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin reached 2%. A cross-linking agent, polyethylene glycol diglycidyl ether (PEGDE), was then added to the system (molar ratio of carboxymethyl chitin: PEGDE epoxy value = 1:0.7). After stirring uniformly, the mixture was stirred or allowed to react at 40°C. Samples were taken at different time points to detect changes in modulus (2.5 Hz) and shear viscosity (10 1 / s). The results are shown in Table 8.

[0118] Table 8 Effects of purified water and PBS with stirring and standing on cross-linking reaction

[0119]

[0120]

[0121] The experimental results show that compared with the stirring reaction, the modulus and shear viscosity of the static reaction sample are slightly higher than those under the stirring reaction condition. The modulus of the sample obtained by using PBS as the solvent is higher than that obtained by using purified water as the solvent. Therefore, using PBS as the solvent and static reaction are more conducive to the occurrence of cross-linking reaction.

[0122] Examples 39 to 43

[0123] The non-cross-linked carboxymethyl chitin prepared in Example 1 was dispersed in PBS and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin was 2%. Then, a cross-linking agent, polyethylene glycol diglycidyl ether (PEGDE), having different epoxy values was added to the system (molar ratio of carboxymethyl chitin:PEGDE epoxy value = 1:0.7). After stirring uniformly, the mixture was allowed to react at 40°C. Samples were taken to test changes in modulus (2.5 Hz) and shear viscosity (10 1 / s). The results are shown in Table 9.

[0124] Table 9 Effect of adding PEGDE crosslinkers with different epoxy values on the crosslinking reaction

[0125]

[0126] The results show that the higher the epoxy value of the added cross-linking agent (correspondingly the lower its molecular weight), the higher the modulus and shear viscosity of the cross-linked carboxymethyl chitin hydrogel sample obtained by the cross-linking reaction.

[0127] Example 44 Cross-linked hydrogel drug loading experiment

[0128] The non-crosslinked carboxymethyl chitin prepared in Example 1 was dispersed in PBS and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin was 3%. The crosslinking agent polyethylene glycol diglycidyl ether (PEGDE) was then added to the system (the molar ratio of carboxymethyl chitin: PEGDE epoxy value = 1:1.0), stirred uniformly, and then doxorubicin hydrochloride was added. The mixture was then allowed to react at 40°C for 96 hours. The crosslinked carboxymethyl chitin hydrogel obtained by the reaction was added to PBS for drug release rate testing. The results are shown in Table 10. The results show that the crosslinked hydrogel has an excellent sustained and controlled release effect on doxorubicin hydrochloride, indicating that the crosslinked hydrogel provided by the present invention has potential application value in the field of drug delivery.

[0129] Table 10 Drug release rate test of drug-loaded cross-linked hydrogel

[0130]

[0131] Example 45 Testing the safety of intradermal injection of products

[0132] Intradermal reactivity tests were conducted on New Zealand rabbits, using normal saline as a control, to investigate the nonspecific acute irritation of the product of the present invention (Example 42) to the skin and to evaluate the biocompatibility of the material. Prior to the experiment, the rabbits were weighed and hair was removed from both sides of the spine, covering an area approximately 15 cm x 20 cm. The skin was ensured to be clean and undamaged. Prior to injection, the skin at the depilated site was disinfected with 75% ethanol and allowed to dry.

[0133] Mark the injection site, with an interval of about 2cm between injection points; Dosage volume: about 0.2ml / injection point; Injection method: After disinfection, use the skin test needle to pierce the skin closely against the skin layer, then lift the needle upwards and pierce it slightly. When the needle cannot swing left and right, it means that the needle is in the skin. After withdrawing without reflux, you can slowly inject. After the injection, a small white skin bump can be seen on the surface of the skin. If the bump can be maintained for a certain period of time, it proves that the drug solution is indeed injected into the skin. Observe the skin around the injection point of all animals 2 and 72 hours after the injection, and judge the skin erythema and edema scores of the test and control sites according to the scoring system specified in the standard. Figure 1-2 The experimental results show that compared with normal saline, the product has no obvious irritation to New Zealand rabbits and has good biocompatibility.

[0134] In order to highlight the beneficial effects of the present invention, the following comparative examples are given.

[0135] Comparative Examples 2 to 5

[0136] Four portions of the non-cross-linked carboxymethyl chitin prepared in Example 1 were dispersed in purified water and stirred to dissolve uniformly until the final concentration of carboxymethyl chitin was 2%. Polyethylene glycol (PEG), polyethylene glycol diglycidyl ether (PEGDE), and 28-bromo-2,5,8,14,17,20,23,26-nonaoxaoctacosane (M-PEG9-Br, CAS 125562-30-3) were then added (carboxymethyl chitin monomer: additive monomer molar ratio = 1:0.7) and a blank control was used. After stirring uniformly, the mixture was allowed to react at 40°C. Samples were taken at different time points to test the changes in modulus (2.5 Hz) and shear viscosity (10 1 / s). The results are shown in Table 11. The data showed that after 64h and 88h of addition of PEG and M-PEG9-Br, the viscoelasticity of the reaction system did not change significantly, which was equivalent to that of the blank control group. This indicates that polymers without epoxy groups will not react with non-cross-linked carboxymethyl chitin, and the change in the viscoelastic modulus after mixing PEDGE and carboxymethyl chitin comes from the cross-linking reaction between PEGDE and non-cross-linked carboxymethyl chitin.

[0137] Table 11 Effects of different cross-linking agents on cross-linking reaction

[0138]

[0139]

[0140] Comparative Examples 6-7

[0141] Over-carboxymethylated carboxymethyl chitin (manufacturer: Hao Leyuan; batch number 200420; carboxymethyl substitution degree 1.3) was dispersed in PBS and stirred to dissolve uniformly to a final carboxymethyl chitin concentration of 3%. A crosslinking agent, polyethylene glycol diglycidyl ether (PEGDE, epoxy value: 0.5-0.55), was then added to the system (molar ratio of carboxymethyl chitin: PEGDE epoxy value = 1:0.7 and 1:1). After stirring uniformly, the mixture was allowed to react at 40°C. Samples were taken at different time points to detect changes in the modulus (2.5 Hz) and shear viscosity (10 1 / s) of the samples. The results are shown in Table 11.

[0142] Table 11 Cross-linking reaction results of excessive carboxymethylated carboxymethyl chitin and cross-linking agent PEGDE

[0143]

[0144] The data show that, compared with Examples 15 and 16, in the presence of different cross-linking agents, the highly substituted carboxymethyl chitin did not undergo obvious cross-linking reaction, and the viscoelasticity of the system always remained at the initial level. This indicates that after the hydroxyl groups on the chitin core are over-substituted, they can no longer undergo cross-linking reaction with PEGDE. It also proves that when the carboxymethyl chitin of the present invention is cross-linked with PEGDE, its partially exposed 6-OH reacts with the epoxy group on the cross-linking agent to form a new ether bond.

[0145] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing non-cross-linked carboxymethyl chitin, characterized in that: The following steps are involved: (1) Alkalization: suspend chitin in alkaline aqueous solution and stir at 5-30°C to alkalize; (2) Etherification: dissolve substituted acetic acid or its salt and add dropwise to the alkalized system at 5-45°C for etherification reaction; (3) Crude product separation and purification.

2. The method for preparing non-cross-linked carboxymethyl chitin according to claim 1, wherein: The alkaline aqueous solution is a sodium hydroxide aqueous solution and / or a potassium hydroxide aqueous solution; the mass percentage of alkali in the alkaline aqueous solution is 30% to 55%, preferably 30% to 50%; the mass ratio of chitin to alkaline aqueous solution is 1.0:5.0 to 1.0:25.0, preferably 1.0:7.0 to 1.0:15.0; and the alkalization temperature is preferably 10 to 30°C.

3. The method for preparing non-cross-linked carboxymethyl chitin according to claim 1, wherein: The substituted acetic acid is at least one of iodoacetic acid, bromoacetic acid and chloroacetic acid; the molar amount of the substituted acetic acid is 1.5 to 7.0 equivalents, preferably 3.0 to 5.0 equivalents, relative to the chitin repeating unit N-acetyl-D-glucosamine; the etherification temperature is preferably 15 to 35°C.

4. The method for preparing non-cross-linked carboxymethyl chitin according to claim 3, wherein: Substituted chloroacetic acid or its salt is dissolved in an alcohol solution for use; the alcohol solution is isopropanol and / or 2-butanol.

5. The method for preparing non-cross-linked carboxymethyl chitin according to claim 1, wherein: In the step (2), based on the halogen exchange principle, potassium iodide or sodium iodide can be added to promote the substitution reaction; since the reaction system is heterogeneous, a phase transfer catalyst can be added, and the phase transfer catalyst includes at least one of sodium dodecyl sulfonate and sodium dodecyl sulfate.

6. The method for preparing non-cross-linked carboxymethyl chitin according to claim 1, wherein: The step (3) specifically comprises: adding an organic solvent dropwise to the reaction solution to obtain a crude product, re-dissolving the crude product, and filtering to remove insoluble matter to obtain a crude product filtrate; the organic solvent comprises at least one of ethanol, isopropanol, and acetone; An aqueous solution of an organic solvent is added dropwise to the crude product filtrate to obtain a refined product; or the crude product filtrate is ultrafiltered to remove small molecular impurities, and then freeze-dried to obtain a refined product.

7. The non-cross-linked carboxymethyl chitin prepared by the preparation method according to any one of claims 1 to 6, characterized in that: It can be cross-linked with polyethylene glycol diglycidyl ether (PEGDE).

8. A method for preparing a cross-linked carboxymethyl chitin hydrogel, characterized in that: The non-cross-linked carboxymethyl chitin according to claim 7 is used for a cross-linking reaction, wherein the cross-linking reaction is specifically carried out using the prepared non-cross-linked carboxymethyl chitin as a raw material, polyethylene glycol diglycidyl ether as a cross-linking agent, and PBS solution or purified water as a solvent, without adding other substances. The final concentration of carboxymethyl chitin in the reaction system is 1% to 10%, preferably 2% to 5%; the molar ratio of carboxymethyl chitin to PEGDE epoxy group is 1:0.05 to 1:2, preferably 1:0.3 to 1:0.8; The cross-linking reaction temperature is 20 to 50° C., and the cross-linking reaction time is 24 to 192 hours.

9. The cross-linked carboxymethyl chitin hydrogel prepared by the preparation method according to any one of claims 1 to 8, characterized in that: By adjusting the concentration of carboxymethyl chitin and the amount of the cross-linking agent, the elastic modulus of the obtained cross-linked carboxymethyl chitin hydrogel can be dynamically adjusted.

10. Use of the cross-linked carboxymethyl chitin hydrogel according to claim 9 in medical treatment, cosmetics, filling and shaping, post-surgical adhesion prevention, drug delivery and drug controlled release.

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