Itaconylated carboxymethyl chitosan cross-linked gel, preparation method therefor, and use thereof

Abstract

An itaconylated carboxymethyl chitosan cross-linked gel, a preparation method therefor, and a use thereof. The itaconylated carboxymethyl chitosan cross-linked gel is prepared by obtaining an itaconylated carboxymethyl chitosan framework material having a crosslinking degree of 5-40% in the presence of an initiating system and then carrying out ray irradiation for free radical secondary crosslinking. The preparation process is mild and controllable. By regulating and controlling the pre-crosslinking degree and the irradiation dose, the prepared gel has a continuous interpenetrating network structure. The structure is compact and stable and has a higher crosslinking degree and elastic modulus, thereby facilitating substance exchange and promoting cell proliferation and adhesion. The gel can be used for beautifying, moisturizing, and intraoperative and postoperative venous hemostasis and has wide application prospects in the fields of biological materials and tissue engineering.

Description

Itaconylated carboxymethyl chitosan cross-linked gel and its preparation method and application Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to an itaconylated carboxymethyl chitosan cross-linked gel and a preparation method and application thereof. Background Art

[0002] Carboxymethyl chitosan (CMC) is obtained by etherifying chitosan with chloroacetic acid, overcoming chitosan's poor water solubility. Following the etherification reaction, -CH2COOH groups are attached to chitosan. Depending on the site of substitution, carboxymethyl chitosan can be classified as N-carboxymethyl chitosan (N-CMC), O-carboxymethyl chitosan (O-CMC), N,O-carboxymethyl chitosan (N,O-CMC), and N,N-carboxymethyl chitosan (N,N-CMC). Carboxymethyl chitosan is a natural polysaccharide derivative that retains the advantages of chitosan while possessing excellent water solubility, antimicrobial properties, enhanced permeability, adhesion, biocompatibility, and degradability. It also possesses physiological functions such as hemostasis, wound healing, and antibacterial properties.

[0003] Itaconic acid, an organic unsaturated dicarboxylic acid, is a key metabolite of the mitochondrial tricarboxylic acid (TCA) cycle. It exhibits anti-inflammatory, antibacterial, antioxidant, and antiviral properties, and can also regulate metabolism and maintain homeostasis. Therefore, itaconic anhydride is grafted onto carboxymethyl chitosan to produce itaconylated carboxymethyl chitosan, which is then polymerized via double-bond free radical polymerization to form a cross-linked gel. This gel undergoes partial degradation in the human body, cleaving its amide bonds and releasing the itaconic acid molecules in their original form, thereby exerting its biological activity.

[0004] Currently, the most studied methods for preparing cross-linked gels are chemical cross-linking, photocross-linking, and high-energy irradiation cross-linking. The cross-linking agents used in the chemical cross-linking method, such as epoxides and divinyl sulfone, all have certain cytotoxicity, and residual cross-linking agents can produce adverse reactions when applied to the human body. The initiator 2959 used in the photocross-linking method has certain toxicity, and after polymerization, the active hydroxyl groups of the initiator 2959 will be grafted onto the polymer molecules, resulting in poor biocompatibility of the gel prepared therefrom. For example, patent CN115926359A discloses a double-bonded Bletilla striata polysaccharide-carboxymethyl chitosan gel, a preparation method, and its application. Double bonds are introduced into Bletilla striata polysaccharide and carboxymethyl chitosan to obtain double-bonded Bletilla striata polysaccharide and double-bonded carboxymethyl chitosan. These two are then mixed with an initiator and placed under ultraviolet light for in-situ curing to obtain the double-bonded Bletilla striata polysaccharide-carboxymethyl chitosan gel. Although this method utilizes double-bond free radical polymerization to obtain the cross-linked gel, the introduction of the photoinitiator poses safety risks and poor biocompatibility. Patent CN106832129A discloses cross-linking itaconic acid and carboxymethyl chitosan in the presence of an initiator to obtain itaconic acid homopolymer-grafted carboxymethyl chitosan gel nanoparticles. Patent CN114316141A discloses a magnetic carboxymethyl chitosan / acrylic acid / itaconic acid copolymer hydrogel adsorbent, its preparation method, and application. However, neither discloses using itaconylated carboxymethyl chitosan directly as a raw material for double bond free radical polymerization. Furthermore, although CN116731387A discloses reacting methacrylic anhydride with carboxymethyl chitosan to obtain methacrylated carboxymethyl chitosan, mixing it with laponite, freeze-drying it, and then irradiating it with cobalt-60 gamma rays for a certain period of time for cross-linking, the irradiation dose is only 1-10 kGy. The gel prepared in this manner has a low degree of cross-linking, a non-uniform texture, and poor stability. Furthermore, acrylic acid, produced during the metabolism of methacrylic acid, is highly irritating and sensitizing and is included in the list of Class III carcinogens. Acrylic acid can only be obtained from petroleum raw materials and has poor biocompatibility.

[0005] Therefore, there are few reports on double bond free radical polymerization using itaconylated carboxymethyl chitosan directly as raw material, and there are almost no reports on the crosslinking reaction conditions and properties of itaconylated carboxymethyl chitosan crosslinked gel. In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention provides an itaconylated carboxymethyl chitosan cross-linked gel and a method for preparing the same. The present invention utilizes a radical secondary crosslinking technique to prepare the itaconylated carboxymethyl chitosan cross-linked gel. Specifically, an initiator system is used to initiate double-bond free radical polymerization of the itaconylated carboxymethyl chitosan, followed by irradiation sterilization and secondary crosslinking of the product. The preparation method can be performed under relatively mild conditions and, by controlling the degree of pre-crosslinking and adjusting the irradiation dose, produces a cross-linked gel with excellent properties.

[0007] The technical solutions provided by the present invention are as follows:

[0008] The first aspect of the present invention provides an itaconylated carboxymethyl chitosan cross-linked gel, which is prepared by obtaining an itaconylated carboxymethyl chitosan skeleton material with a cross-linking degree of 5 to 40% under an initiation system, and then irradiating it with radiation to perform free radical secondary cross-linking to obtain the itaconylated carboxymethyl chitosan cross-linked gel.

[0009] Furthermore, the initiation system is a redox system or a thermal decomposition system; the initiation system comprises a free radical initiator and an initiation auxiliary;

[0010] Furthermore, when the initiation system is a redox system, the free radical initiator is hydrogen peroxide, and the initiation aid is selected from sodium sulfite, ferrous sulfate or ascorbic acid;

[0011] Furthermore, when the initiation system is a thermal decomposition system, the free radical initiator is a persulfate such as ammonium persulfate, potassium persulfate, etc.; the initiation auxiliary is tetramethylethylenediamine, triethylamine or ethylenediamine.

[0012] Furthermore, the rays are gamma rays, X rays or beta rays, etc.

[0013] Furthermore, the radiation dose of the rays is 15-50 kGy.

[0014] The itaconylated carboxymethyl chitosan cross-linked gel can withstand high-dose radiation, has high stability, and has a final cross-linking degree of more than 60%, an elastic modulus greater than 3000 Pa, and an in vitro coagulation index of less than 10%.

[0015] The inventors discovered that by first subjecting itaconylated carboxymethyl chitosan to double-bond free radical polymerization under an initiation system, a certain degree of pre-crosslinking reaction occurs, resulting in an itaconylated carboxymethyl chitosan skeleton material with a crosslinking degree of 5 to 40% formed within a relatively short reaction time. Subsequently, irradiation is performed, and the remaining double bonds in the chitosan skeleton material prevent the skeleton material from degrading even within a high irradiation dose range, instead allowing secondary crosslinking to occur. This results in a structurally stable itaconylated carboxymethyl chitosan crosslinked gel with a high degree of crosslinking. Furthermore, sterilization is performed during the secondary crosslinking process, saving subsequent processing steps and improving overall reaction efficiency.

[0016] The second aspect of the present invention provides a method for preparing an itaconylated carboxymethyl chitosan cross-linked gel, comprising the following steps:

[0017] (1) Adding an initiator system to the itaconylated carboxymethyl chitosan solution and subjecting it to a pre-crosslinking reaction at 10-60°C for 20-40 minutes;

[0018] (2) The pre-crosslinked product obtained in step (1) is subjected to secondary crosslinking by irradiation with radiation.

[0019] Furthermore, the reaction concentration of the itaconylated carboxymethyl chitosan solution is 1-60 mg / mL (such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 30, 30.5, 40, 40.5, 50, 50.5, 60 mg / mL); preferably 20-50 mg / mL.

[0020] Furthermore, the initiation system includes a free radical initiator and an initiation auxiliary.

[0021] Furthermore, the reaction concentration of the free radical initiator is 14-165 μmol / mL (such as 14, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 165 μmol / mL).

[0022] Furthermore, when the initiation system is a redox system, the pH value ranges from 3.0 to 7.0, specifically 3.0, 3.5, 4.2, 5.3, 5.8, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0.

[0023] Furthermore, when the initiation system is a thermal decomposition system, the pH value ranges from 7.0 to 10.0, specifically 7.0, 7.2, 7.5, 7.8, 8.0, 8.5, 9.0, 9.6 and 10.0.

[0024] Furthermore, the step (1) comprises taking an appropriate amount of itaconylated carboxymethyl chitosan, stirring and dissolving it, adding a free radical initiator, adjusting the pH value, and then adding an initiation aid, stirring and reacting for 20-40 minutes to obtain an itaconylated carboxymethyl chitosan pre-crosslinked product.

[0025] Furthermore, in step (1), the amount of the free radical initiator and the initiation aid must be added in sufficient amounts at one time, and cannot be added in small amounts multiple times, and the time interval between the addition of the free radical initiator and the initiation aid is short.

[0026] In the pre-crosslinking stage, the present invention controls the amount of free radical initiator, reaction temperature and time to form an itaconylated carboxymethyl chitosan skeleton material with a crosslinking degree of 5% to 40% within a relatively short reaction time. Then, radiation irradiation is performed to enable the gel to undergo secondary crosslinking, thereby obtaining a cross-linked gel with higher stability.

[0027] During the pre-crosslinking process, since the free radical initiator has a high reactivity, while initiating the double bond free radical polymerization, it also destroys the glycosidic bonds in the chitosan, causing them to break. Thus, even if the double bond completes the polymerization reaction, the target hydrogel cannot be formed because the glycosidic bonds of the chitosan are destroyed. Therefore, if the amount of initiator is large or the initiation process lasts for a long time, it will cause the glycosidic bonds in the itaconylated carboxymethyl chitosan to break; if the initiator is added in batches, due to the short half-life of the free radicals, the free radical concentration cannot reach the initiating dose, and thus the free radical polymerization of the double bond cannot be initiated. On this basis, the present invention controls the initiator to be added in a sufficient amount at one time and ensures that the pre-crosslinking process is completed in a short time, thereby achieving a controllable pre-crosslinking degree within an appropriate range. Furthermore, the present invention controls the degree of pre-crosslinking to 5% to 40%, thereby first obtaining an itaconylated carboxymethyl chitosan skeleton material so that the residual amount of double bonds is in the range of 60% to 95%. If the residual amount of double bonds is small, high-dose irradiation easily leads to the breakage of glycosidic bonds on the itaconylated carboxymethyl chitosan. If the residual amount of double bonds reaches more than 95%, the pre-crosslinking degree of the itaconylated carboxymethyl chitosan is too low, making it difficult to form a network skeleton structure. The double bonds are in a disordered and scattered state, most of the double bonds are far apart, and only a small number of relatively close double bonds can be crosslinked by irradiation, resulting in a low degree of secondary crosslinking. The residual double bonds of 60% to 95% in the present invention can, on the one hand, enable the skeleton material to withstand high-intensity irradiation doses and improve the crosslinking efficiency of the double bonds. On the other hand, it can also protect the glycosidic bonds on the itaconylated carboxymethyl chitosan from being destroyed by radiation during the irradiation process, thereby ultimately obtaining a hydrogel material with a stable structure.

[0028] Furthermore, the preparation method further comprises the steps of crushing, dialyzing and freeze-drying the pre-crosslinked product prepared in step (1).

[0029] Furthermore, the pulverization is performed by using a homogenizer to pulverize the itaconylated carboxymethyl chitosan pre-crosslinked product, the dialysis is performed by placing the gel in a dialysis bag for dialysis, and the freeze-drying is performed by using a vacuum freeze dryer for freeze-drying.

[0030] Furthermore, the homogenizer grinding parameter is 12000 rpm, the grinding time is 5 min, the dialysis solution is changed every 1 hour, and after the dialysis is completed, the product is placed in a vacuum freeze dryer for freeze drying to obtain itaconylated carboxymethyl chitosan pre-crosslinked gel freeze-dried powder.

[0031] Furthermore, in step (2), the ray is a γ ray, an X ray or a β ray, etc.

[0032] Furthermore, in step (2), the irradiation dose is 10 to 50 kGy.

[0033] Furthermore, the irradiation not only promotes secondary cross-linking, but also has a sterilizing effect.

[0034] Furthermore, after irradiation, the mixture is compounded with a buffer solution to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel.

[0035] Furthermore, the buffer solution is PBS with a pH of 7.0, and the compound concentration is 80-120 mg / mL, preferably 100 mg / mL.

[0036] The third aspect of the present invention provides the use of the itaconylated carboxymethyl chitosan cross-linked gel described in the first aspect or the itaconylated carboxymethyl chitosan cross-linked gel prepared by the method described in the second aspect.

[0037] Furthermore, the application is the use of itaconylated carboxymethyl chitosan cross-linked gel in the preparation of hemostatic materials, soft tissue filling materials, medicines or tissue engineering materials.

[0038] Specifically, the soft tissue filler can be used to eliminate wrinkles (such as wrinkles around the eyes, forehead wrinkles, frown lines, perioral wrinkles, nasolabial folds, tear grooves, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.), anti-aging, eliminate scars, repair wounds, and stop venous bleeding during and after surgery.

[0039] Specifically, the drug uses itaconylated carboxymethyl chitosan cross-linked gel as a carrier to achieve the purposes of sustained release, controlled release, targeted drug delivery, etc.

[0040] Specifically, the tissue engineering material can be a bone tissue engineering material, a cartilage tissue engineering material, a corneal tissue engineering material, a cardiovascular tissue engineering material, a liver tissue engineering material, a rapid hemostatic material, etc.

[0041] The present invention has the following beneficial effects:

[0042] 1. The present invention adopts a free radical secondary cross-linking technology to prepare an itaconylated carboxymethyl chitosan cross-linked gel. Specifically, the itaconylated carboxymethyl chitosan is first subjected to double-bond free radical polymerization under an initiation system to generate a certain degree of pre-cross-linking reaction, so that an itaconylated carboxymethyl chitosan skeleton material is formed within a relatively short reaction time. The chitosan skeleton material is then irradiated with radiation. The remaining double bonds in the chitosan skeleton material prevent the skeleton material from being degraded within a high irradiation dose range, but instead undergo free radical secondary cross-linking, thereby obtaining an itaconylated carboxymethyl chitosan cross-linked gel with a stable structure and a high degree of cross-linking. In addition, a sterilization treatment is also performed during the radiation secondary cross-linking process, which saves subsequent processing processes and improves the overall reaction efficiency.

[0043] 2. In the pre-crosslinking stage, the present invention controls the one-time addition of a sufficient amount of free radical initiator and ensures that the pre-crosslinking process is completed in a short time, thereby achieving a controllable pre-crosslinking degree within an appropriate range; further, by controlling the pre-crosslinking degree within 5% to 40%, an itaconylated carboxymethyl chitosan skeleton material is first obtained so that the residual amount of double bonds is within the range of 60%-95%. The remaining amount of double bonds can, on the one hand, enable the skeleton material to withstand high-intensity irradiation doses and improve the crosslinking efficiency of the double bonds; on the other hand, it can also protect the glycosidic bonds on the itaconylated carboxymethyl chitosan from being destroyed by radiation during the irradiation process, thereby finally obtaining a structurally stable hydrogel material.

[0044] 3. The preparation process of the present invention is mild and controllable. Double-bond free radical polymerization of itaconylated carboxymethyl chitosan is initiated by multiple initiation systems, and the product is then irradiated and sterilized and secondary cross-linked by radiation, and the post-processing process is simple. The prepared itaconylated carboxymethyl chitosan cross-linked gel has a continuous interpenetrating grid structure and a dense and stable structure, has a higher elastic modulus and coagulation performance, is conducive to blood penetration, can promote material exchange and cell proliferation and adhesion, can be used for beauty and moisturizing, intraoperative and postoperative venous hemostasis, and as a wound dressing, and has potential research value and broad application prospects in the fields of biomaterials and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a SEM image of the cross-linked gel A3 of Example 3, wherein (a) is the SEM result of the cross-linked gel sponge A3, and (b) is a partial magnified image of (a).

[0046] FIG2 shows the appearance of the cross-linked gel A3 of Example 3.

[0047] FIG3 is a diagram of the liver hemostasis model of the cross-linked gel A3 of Example 3.

[0048] FIG4 is a diagram of the liver hemostasis model of the commercially available biological protein sponge Chuangbifu. DETAILED DESCRIPTION

[0049] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0050] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

[0051] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0052] Example 1

[0053] 1.5 g of itaconylated carboxymethyl chitosan was weighed, 30 mL of deoxygenated purified water was added, and the mixture was stirred to completely dissolve. Then, 441.18 μmol (0.015 g) of hydrogen peroxide was added and stirred evenly, and the pH of the solution was adjusted to 3.0. Then, 77.48 μmol (0.014 g) of ascorbic acid was added, and the mixture was rapidly stirred and allowed to react at 10° C. for 40 minutes to obtain the itaconylated carboxymethyl chitosan pre-crosslinked product. The itaconylated carboxymethyl chitosan pre-crosslinked product was then pulverized using a homogenizer at 12,000 rpm for 5 minutes. The gel was then placed in a dialysis bag for dialysis, with the solution changed every hour. After dialysis, the product was lyophilized in a freeze dryer to obtain the itaconylated carboxymethyl chitosan pre-crosslinked gel lyophilized powder. The lyophilized powder was irradiated with γ rays for secondary cross-linking and sterilization at an irradiation dose of 15 kGy, and then compounded with PBS with a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as A1.

[0054] Example 2

[0055] 1.5 g of itaconylated carboxymethyl chitosan was weighed, added to 30 mL of deoxygenated purified water, and stirred to completely dissolve. 882.35 μmol (0.030 g) of hydrogen peroxide was then added, stirred evenly, and the pH of the solution was adjusted to 4.5. 83.01 μmol (0.023 g) of ferrous sulfate was then added, stirred rapidly, and allowed to react at 20° C. for 35 minutes to obtain an itaconylated carboxymethyl chitosan pre-crosslinked product. The itaconylated carboxymethyl chitosan pre-crosslinked product was then pulverized using a homogenizer at 12,000 rpm for 5 minutes. The gel was then placed in a dialysis bag for dialysis, with the solution changed every hour. After dialysis, the product was lyophilized in a freeze dryer to obtain a lyophilized itaconylated carboxymethyl chitosan pre-crosslinked gel powder. The freeze-dried powder was irradiated with β rays for secondary cross-linking and sterilization at an irradiation dose of 25 kGy, and then compounded with PBS with a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was recorded as A2.

[0056] Example 3

[0057] 1.5 g of itaconylated carboxymethyl chitosan was weighed, 30 mL of deoxygenated purified water was added, and the mixture was stirred to completely dissolve. Then, 2.647 mmol (0.090 g) of hydrogen peroxide was added and stirred evenly. The pH of the solution was adjusted to 6.3. Then, 446.43 μmol (0.056 g) of sodium sulfite was added. After rapid stirring, the mixture was reacted at 60° C. for 20 min to obtain the itaconylated carboxymethyl chitosan pre-crosslinked product. The itaconylated carboxymethyl chitosan pre-crosslinked product was then pulverized using a homogenizer at 12,000 rpm for 5 min. The gel was then placed in a dialysis bag for dialysis, with the solution changed every 1 h. After dialysis, the product was lyophilized in a freeze dryer to obtain the itaconylated carboxymethyl chitosan pre-crosslinked gel lyophilized powder. The lyophilized powder was irradiated with gamma rays for secondary cross-linking and sterilization at a dose of 45 kGy, and then compounded with PBS at a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as A3.

[0058] Example 4

[0059] 1.5 g of itaconylated carboxymethyl chitosan was weighed, 30 mL of deoxygenated purified water was added, and the mixture was stirred to completely dissolve. 789.47 μmol (0.180 g) of ammonium persulfate was then added and the pH of the solution was adjusted to 8.2 after stirring. 862.07 μmol (0.100 g) of tetramethylethylenediamine was then added, and the mixture was rapidly stirred and allowed to react at 45° C. for 30 min to obtain the itaconylated carboxymethyl chitosan pre-crosslinked product. The itaconylated carboxymethyl chitosan pre-crosslinked product was then pulverized using a homogenizer at 12,000 rpm for 5 min. The gel was then placed in a dialysis bag for dialysis, with the solution changed every 1 h. After dialysis, the product was lyophilized in a freeze dryer to obtain the itaconylated carboxymethyl chitosan pre-crosslinked gel lyophilized powder. The lyophilized powder was irradiated with gamma rays for secondary cross-linking and sterilization at a dose of 40 kGy, and then compounded with PBS at a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as A4.

[0060] Example 5

[0061] 1.5 g of itaconylated carboxymethyl chitosan was weighed, 30 mL of deoxygenated purified water was added, and the mixture was stirred to completely dissolve. 1.11 mmol (0.300 g) of potassium persulfate was then added, and the pH of the solution was adjusted to 10.0 after stirring. 1.485 mmol (0.150 g) of triethylamine was then added, and the mixture was rapidly stirred and allowed to react at 30° C. for 30 min to obtain an itaconylated carboxymethyl chitosan pre-crosslinked product. The itaconylated carboxymethyl chitosan pre-crosslinked product was then pulverized using a homogenizer at 12,000 rpm for 5 min. The gel was then placed in a dialysis bag for dialysis, with the solution changed every 1 h. After dialysis, the product was lyophilized in a freeze dryer to obtain a lyophilized itaconylated carboxymethyl chitosan pre-crosslinked gel powder. The lyophilized powder was irradiated with X-rays for secondary cross-linking and sterilization at a dose of 30 kGy, and then compounded with PBS at a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as A5.

[0062] Example 6

[0063] 1.5 g of itaconylated carboxymethyl chitosan was weighed, added to 75 mL of deoxygenated purified water, stirred to completely dissolve, then 4.934 mmol (1.125 g) of ammonium persulfate was added, stirred evenly, and the pH of the solution was adjusted to 9.5. Then 8.52 mmol (0.511 g) of ethylenediamine was added, stirred rapidly, and allowed to react at 15° C. for 40 minutes to obtain an itaconylated carboxymethyl chitosan pre-crosslinked product. The itaconylated carboxymethyl chitosan pre-crosslinked product was then pulverized using a homogenizer at 12,000 rpm for 5 minutes. The gel was then placed in a dialysis bag for dialysis, with the solution changed every 1 hour. After dialysis, the product was placed in a freeze dryer and freeze-dried to obtain the itaconylated carboxymethyl chitosan pre-crosslinked gel freeze-dried powder. The lyophilized powder was irradiated with γ rays for secondary cross-linking and sterilization at an irradiation dose of 15 kGy, and then compounded with PBS at a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as A6.

[0064] Comparative Example 1

[0065] The pre-crosslinking reaction temperature was 2° C., and the rest of the reaction process was consistent with Example 3, to obtain the final product, itaconylated carboxymethyl chitosan crosslinked gel, which was designated as B1.

[0066] Comparative Example 2

[0067] The pre-crosslinking reaction temperature was 70° C., and the rest of the reaction process was consistent with Example 4, to obtain the final product, itaconylated carboxymethyl chitosan crosslinked gel, which was designated as B2.

[0068] Comparative Example 3

[0069] Weigh 1.5 g of itaconylated carboxymethyl chitosan and add 30 mL of deoxygenated purified water. Stir to completely dissolve the mixture, then freeze-dry in a freeze dryer to obtain a lyophilized powder. The lyophilized powder was irradiated with gamma rays at a dose of 45 kGy for cross-linking and sterilization. The lyophilized powder was then reconstituted with PBS (pH 7.0) at a concentration of 100 mg / mL to obtain the final itaconylated carboxymethyl chitosan cross-linked gel, designated B3.

[0070] Comparative Example 4

[0071] Only the phrase "irradiating the lyophilized powder with γ rays for secondary crosslinking and sterilization at a dose of 40 kGy" in Example 3 was replaced with "sterilizing the lyophilized powder with moist heat". The remaining preparation processes were consistent with those in Example 3, and the final product, itaconylated carboxymethyl chitosan cross-linked gel, was obtained, which was designated as B4.

[0072] Comparative Example 5

[0073] The irradiation dose was 55 kGy, and the rest of the preparation process was consistent with that in Example 3, to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as B5.

[0074] Comparative Example 6

[0075] The pre-crosslinking reaction time was 50 min, and the rest of the preparation process was consistent with Example 3, to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was designated as B6.

[0076] Comparative Example 7

[0077] The amount of hydrogen peroxide added was 26.47 mmol (0.900 g), the amount of sodium sulfite added was 4.464 mmol (0.560 g), and the rest of the preparation process was consistent with Example 3 to obtain the final product, itaconylated carboxymethyl chitosan cross-linked gel, which was recorded as B7.

[0078] Comparative Example 8

[0079] The initiator and initiation aid were added in three portions, once every 2 minutes. The rest of the preparation process was consistent with Example 3, and the final product, itaconylated carboxymethyl chitosan cross-linked gel, was obtained, which was designated as B8.

[0080] Performance Test 1 Determination of Residual Double Bonds in Gel

[0081] 40 mg of the itaconylated carboxymethyl chitosan pre-crosslinked products and the final itaconylated carboxymethyl chitosan cross-linked gel of samples A1 to A6 and B1 to B8 prepared in Examples 1 to 6 and Comparative Examples 1 to 8 were respectively weighed and subjected to hydrogen nuclear magnetic resonance (HNMR) spectrum tests. The HNMR test solvent was deuterated water to ensure that the samples were completely dissolved before testing.

[0082] The residual double bonds in the gel were measured to determine the double bond consumption of the gel, i.e., the degree of cross-linking. The results of the cross-linking degree measurement of the gel are shown in Table 1.

[0083] Table 1 Gel cross-linking degree determination results

[0084] Performance Test 2 Determination of Rheological Properties of Gel

[0085] The rheological properties of the itaconylated carboxymethyl chitosan pre-crosslinked products and the final itaconylated carboxymethyl chitosan crosslinked gels (A1-A6 and B1-B8) prepared in Examples 1-6 and Comparative Examples 1-8 were tested using a rotational rheometer. The rotational rheometer test parameters were: 2 mL sample, operating gap: 1000 μm, loading gap: 45,000 μm, operating temperature: 37°C, deformation: 1%, frequency: 0.9 Hz, and run time: 60 s. The rheological data for each gel are shown in Table 2.

[0086] Table 2 Determination results of gel rheological properties

[0087] Comparison of the data in Tables 1 and 2 reveals that, by controlling the pre-crosslinking degree within the range of 5-40% and then irradiating with radiation for secondary crosslinking, the crosslinking degree of the final itaconylated carboxymethyl chitosan crosslinked gels A1-A6 can be further increased. For example, in Example 1, when the pre-crosslinking degree was 5%, the crosslinking degree reached 60% after irradiation. Furthermore, increasing the irradiation dose further promotes the crosslinking of double bonds. For example, in Example 3, at an irradiation dose of 45 kGy, the crosslinking degree of double bonds reached 100%, with essentially no residual double bonds. Furthermore, the elastic moduli of the samples in the examples after irradiation were all above 3600 Pa, an increase of 127% to 148% over the elastic modulus before irradiation. This is because, after pre-crosslinking, the resulting gels still had residual double bonds that had not fully reacted (as shown in Table 1). Therefore, secondary crosslinking occurred after irradiation sterilization, and the increased crosslinking degree resulted in a denser network structure and a higher elastic modulus.

[0088] In addition, when the reaction temperature is 10-60°C, it is conducive to the free radical polymerization of double bonds. Within this range, the higher the reaction temperature, the higher the activity of the free radicals, which is conducive to the occurrence of free radical polymerization. However, as the cross-linking temperature is further increased, although the free radical activity is high, the gel undergoes free radical polymerization in a short time, and the pre-crosslinking degree of the gel can reach 90% (see Comparative Example 2), the high energy of the free radicals will destroy the glycosidic bonds in the gel, resulting in the failure of the gel pre-crosslinking. Therefore, the gel does not form a cross-linked skeleton in the first stage and has poor stability. Even if the remaining double bonds continue to undergo free radical polymerization during the irradiation sterilization process, the main chain of the gel main body chitosan has been broken during the pre-crosslinking, and the resulting gel still cannot form a cross-linked structure. Moreover, due to the small amount of double bonds remaining, the double bonds are difficult to protect the glycosidic bonds remaining during the pre-crosslinking, and the glycosidic bonds are also destroyed by the radiation. Therefore, its elastic modulus after irradiation is low, only 1.7 Pa, even lower than the elastic modulus before irradiation. In Comparative Example 1, since the pre-crosslinking temperature is too low, the free radical activity is poor, the energy is low, and the double bonds cannot be triggered to undergo free radical polymerization, resulting in a pre-crosslinking degree of only 3% for the gel, making it difficult to form a network skeleton structure. The double bonds are in a disordered and scattered state, and most of the double bonds are far apart. Only a small number of relatively close double bonds can be irradiated and crosslinked, resulting in a low degree of secondary crosslinking. Therefore, its elastic modulus after irradiation (313.2 Pa) is much lower than the elastic modulus in Example 3 (5043 Pa).

[0089] The gel B3 obtained in Comparative Example 3 was prepared using the same process as the gel A3 in Example 3, except that no initiator was added. The elastic moduli before and after irradiation were 20 Pa and 103.1 Pa, respectively, indicating a much lower elastic modulus than the gel formed in Example 3. This indicates that, while the system can form a gel upon irradiation without pre-crosslinking with an initiator, the lack of pre-crosslinking to form a network skeleton structure means that the double bonds are still in a disordered, scattered state. Most double bonds are far apart, and only a small number of relatively close ones can be crosslinked by irradiation. This means that only a small number of double bonds participate in the crosslinking reaction (double bond consumption rate 23%), resulting in a low degree of secondary crosslinking. Most double bonds return to their previous double bond state after exposure to the radiation energy ends, which is the main reason for the gel's lower elasticity. Therefore, the main function of pre-crosslinking is to pre-form the gel skeleton. The preliminary network skeleton structure formed can shorten the distance between double bonds, so that the residual double bonds can further react during irradiation. The efficiency of irradiation cross-linking is much higher than that without pre-crosslinking or with a low degree of pre-crosslinking, which ultimately increases the elasticity of the gel and also increases the stability of the gel.

[0090] Gel B4 obtained in Comparative Example 4 and Gel A3 in Example 3 were prepared using the same process. The difference was that Gel B4 was sterilized using moist heat. Its elastic modulus before sterilization was 2031 Pa, while that of Gel A3 after irradiation sterilization was 5043 Pa. However, the elastic modulus of Gel B4 after moist heat sterilization was only 1.3 Pa. This indicates that the moist heat sterilization process, at temperatures as high as 121°C, makes the gel unstable under these conditions, destroying its network structure. Consequently, its elastic modulus significantly decreases after sterilization. Therefore, this type of gel is not suitable for moist heat sterilization.

[0091] Gel B5, obtained in Comparative Example 5, was prepared using the same process as Gel A3 in Example 3, except that the radiation sterilization dose was increased to 55 kGy. Its elastic modulus before sterilization was 2031 Pa, but after sterilization it was only 265 Pa. This is because when the radiation dose was increased from 45 kGy in Example 3 to 55 kGy, the double bond consumption remained at 100%, but the further increase in radiation dose destroyed the glycosidic bonds in the gel, causing the gel's backbone to disintegrate. Consequently, the elastic modulus of cross-linked gel B5 after sterilization was significantly lower than that of A3.

[0092] Gel B6 obtained in Comparative Example 6 follows the same preparation process as Gel A3 in Example 3, except that the pre-crosslinking time was extended to 45 minutes. This extended pre-crosslinking time allowed Gel B6 to achieve a crosslinking degree of up to 90% before irradiation and an elastic modulus of 3055 Pa. However, the elastic modulus after irradiation was only 0.5 Pa. This is primarily due to the fact that with the extended pre-crosslinking time, the gel's pre-crosslinking degree is high, and the double bonds are essentially completely reacted, with only 10% remaining. Therefore, during irradiation, the small amount of double bonds fails to effectively protect the glycosidic bonds. The irradiation ruptures the glycosidic bonds in the gel, causing gel degradation and a significant decrease in the elastic modulus of Gel B6 after irradiation. Therefore, properly controlling the pre-crosslinking time to maintain a suitable pre-crosslinking degree is crucial for preparing gel materials.

[0093] The gel B7 obtained in Comparative Example 7 and the gel A3 in Example 3 were prepared using the same process, except that the amounts of initiator and initiation aid were increased. By increasing the amounts of initiator and initiation aid, the double bond consumption of gel B7 before irradiation was 97%, and after irradiation sterilization, the double bond consumption was 100%. Although a high dose of initiator can complete the double bond free radical polymerization reaction to a high degree, the initiator at this dose, due to its extremely strong oxidizing property, destroyed and broke the glycosidic bonds of the gel, preventing the formation of a gel network and resulting in gel cross-linking failure.

[0094] The gel B8 obtained in Comparative Example 8 and the gel A3 in Example 3 have the same preparation process, except that the feeding method of the initiator and the initiation auxiliary agent is changed to multiple additions in small amounts. After a single small amount of initiator is added, the system produces fewer free radicals, and its energy is insufficient to initiate free radical polymerization of double bonds. Therefore, the double bond consumption of gel B8 during the pre-crosslinking process is only 2%. Since the initiator (such as hydrogen peroxide, peroxide, etc.) has strong oxidizing properties, the initiator added in batches will destroy the glycosidic bonds of chitosan and cause them to break, resulting in the ultimate crosslinking failure. After irradiation sterilization, due to the failure of pre-crosslinking to form a network skeleton structure, the double bonds are in a disordered and scattered state. Most of the double bonds are far apart, which makes the crosslinking reaction more difficult. Therefore, the double bond consumption of the gel only increases slightly, so its elastic modulus is extremely low.

[0095] Performance Test 3 SEM determination of gel

[0096] The pore structure of the gel was observed using a scanning electron microscope. The cross-linked gel obtained in Example 3 was freeze-dried, cut into small cubes with a razor blade, fixed to an electron microscope stage with conductive adhesive, and sprayed with gold for 60 seconds. The resulting analysis was performed using a field-emission scanning electron microscope. The scanning electron microscope results for cross-linked gel sponge A3 are shown in Figure 1, with Figure b being a partial magnification of Figure a.

[0097] As shown in Figure 1, the cross-linked gel A3 has an interpenetrating macroporous network structure. This unique structure is conducive to blood penetration, thereby promoting hemostasis, and can be used for intraoperative and postoperative venous hemostasis.

[0098] Performance test 4 Gel water absorption performance test

[0099] 1 g of cross-linked gels A1-A6 prepared in Examples 1-6 were placed in a watch glass. 1 mL of purified water was added vertically to the gels, and the time it took for the purified water to be completely absorbed by the gels was recorded. Table 3 shows the test results of the gel water absorption performance.

[0100] Table 3 Test results of water absorption performance of gel

[0101] As shown in Table 3, the cross-linked gels of Examples 1-6 absorbed water very quickly, all completely absorbing 1 mL of purified water within 6 seconds. This indicates that the cross-linked gels prepared using the present invention possess a rich porous network structure, enabling rapid water absorption. Fast water absorption is a prerequisite and foundation for a gel's ability to rapidly stop bleeding. Gel A3 exhibited the fastest water absorption rate, absorbing 1 mL of purified water in just 1 second. This is also related to the fact that the gel of Example 3 has the most stable structure and the richest porous structure.

[0102] Performance Test 5 Determination of Gel In Vitro Coagulation Index (BCI)

[0103] Take 50 mg of the irradiated gel A1-A6 freeze-dried powder sample, reconstitute it with PBS with a pH of 7.0 to 100 mg / mL, and wait for it to fully swell before use; take a 5 mL centrifuge tube, add 20 ul of anticoagulated rabbit blood and 2 ul of CaCl2 (0.25M) solution to it, then add the swollen gel to it, and then incubate the centrifuge tube at 37°C for 5 minutes; then add 2 mL of purified water to the centrifuge tube, and then incubate the centrifuge tube at 37°C for 10 minutes, and finally use an enzyme marker to detect the absorbance of the supernatant at 540 nm. The blank control group sample is anticoagulated rabbit blood with CaCl2 solution. Calculate the BCI value of the sample, and the calculation results are shown in Table 4. The BCI calculation formula is as follows:

[0104] BCI=OD 样品 / OD 空白 *100%

[0105] Table 4 BCI values ​​of itaconylated carboxymethyl chitosan cross-linked gel

[0106] As shown in Table 4, the BCI values ​​of cross-linked gels A1 to A6 are all within 10%, and the smaller the BCI value, the stronger the hemostatic gel, indicating that the gel prepared by the present invention has good coagulation performance. Gel A3 has the smallest BCI value, only 3%, indicating that it has the strongest coagulation ability. The reasons are: (1) Itaconylated carboxymethyl chitosan itself has cationic properties. Through the action of positive charge, it adheres and aggregates with negatively charged red blood cell surface substances, thereby quickly forming blood clots to stop bleeding; (2) The gel forms an interpenetrating macroporous structure during the preparation process, which has a strong water absorption capacity. Therefore, the gel can quickly absorb free water into its own network structure and bind the free water, causing the blood to lose water rapidly, resulting in increased platelet concentration and aggregation, and finally forming blood coagulation.

[0107] Performance Test 6 Gel A3 in New Zealand White Rabbit Liver Hemostasis Model

[0108] A New Zealand white rabbit was selected for general anesthesia. Preoperatively, the rabbit received multiple small intravenous injections of chloral hydrate at a dose of 2.5 mL / kg. After anesthesia, the rabbit's abdomen was depilated and disinfected, and the rabbit was secured in a supine position on a laboratory table. The upper abdominal skin was incised, and the laparotomy was performed layer by layer to expose the left lobe of the liver. Sterile gauze was placed beneath the liver to protect the surrounding tissue.

[0109] A 1.0 x 0.8 cm square incision was made on the surface of the left lateral lobe of the liver using a scalpel blade. Liver tissue was removed from the wound to a depth of 0.2 cm, resulting in significant bleeding from the wound. After 10 seconds of free bleeding, the cross-linked gel A3 (Example 3) prepared in advance was syringed into the wound to completely cover it. For the blank control group, 16 layers of 3 x 3 cm sterile gauze were directly covered on the wound and gently pressed with the fingers (the force was controlled to the minimum pressure to ensure that the material did not fall off). A timer was started simultaneously to record the bleeding time. After hemostasis was complete, the gauze was removed, the hemostatic gel was rinsed with physiological saline, and the animal's abdominal cavity was sutured layer by layer with surgical sutures, and the animal's condition was observed.

[0110] As shown in Figure 2, the shape and appearance photos of the itaconylated carboxymethyl chitosan cross-linked gel A3 of Example 3 are shown. The gel can flow slightly when placed vertically. During the test, 1.0 mL of itaconylated carboxymethyl chitosan cross-linked gel A3 was injected and then pressed to stop bleeding. The hemostasis time was about 30 seconds, and the wound stopped bleeding. The hemostatic effect was good. The specific hemostatic effect is shown in Figure 3. After pressing to stop bleeding with the biological protein sponge Chuangbifu for 1 minute, the wound continued to ooze blood. The effect is shown in Figure 4. The results show that the hemostatic performance of the itaconylated carboxymethyl chitosan cross-linked gel is significantly better than the commonly used biological protein sponge hemostatic agents on the market, indicating that the cross-linked gel of the present invention can be used for intraoperative and postoperative venous hemostasis, and has good clinical use effect and has broad application prospects.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An itaconylated carboxymethyl chitosan cross-linked gel, characterized in that: The itaconylated carboxymethyl chitosan cross-linked gel is prepared by obtaining an itaconylated carboxymethyl chitosan skeleton material with a cross-linking degree of 5 to 40% under an initiation system, and then irradiating it with rays to perform free radical secondary cross-linking.

2. The itaconylated carboxymethyl chitosan cross-linked gel according to claim 1, characterized in that: The initiation system is a redox system or a thermal decomposition system, and the initiation system includes a free radical initiator and an initiation auxiliary.

3. The itaconylated carboxymethyl chitosan cross-linked gel according to claim 2, characterized in that: When the initiation system is a redox system, the free radical initiator is hydrogen peroxide, and the initiation aid is sodium sulfite, ferrous sulfate or ascorbic acid; When the initiation system is a thermal decomposition system, the free radical initiator is a persulfate, preferably ammonium persulfate or potassium persulfate; the initiation aid is tetramethylethylenediamine, triethylamine or ethylenediamine.

4. The itaconylated carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 3, characterized in that: The rays are γ rays, X rays or β rays; and / or the irradiation dose of the rays is 15-50 kGy.

5. A method for preparing the itaconylated carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (1) adding an initiator system to the itaconylated carboxymethyl chitosan solution and subjecting the pre-crosslinking reaction to a temperature of 10 to 60° C. for 20 to 40 minutes; (2) The pre-crosslinked product obtained in step (1) is subjected to secondary crosslinking by irradiation with radiation.

6. The preparation method according to claim 5, characterized in that: The concentration of the itaconylated carboxymethyl chitosan solution is 1-60 mg / mL, preferably 20-50 mg / mL.

7. The preparation method according to claim 5 or 6, characterized in that: The step (1) comprises taking an appropriate amount of itaconylated carboxymethyl chitosan, stirring and dissolving it, adding a free radical initiator, adjusting the pH value, and then adding an initiator aid, stirring and reacting for 20-40 minutes to obtain a pre-crosslinked product; preferably, the reaction concentration of the free radical initiator is 14-165 μmol / mL.

8. The preparation method according to claim 7, characterized in that: When the initiation system is a redox system, the pH value is 3.0 to 7.0; or when the initiation system is a thermal decomposition system, the pH value is 7.0 to 10.

0.

9. The preparation method according to claim 8, characterized in that: The preparation method further comprises the step of compounding the irradiated product in step (2) with a buffer solution.

10. Use of the itaconylated carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 4 or the itaconylated carboxymethyl chitosan cross-linked gel prepared by the preparation method according to any one of claims 5 to 9 in the preparation of hemostatic materials, soft tissue filling materials, drugs or tissue engineering materials.

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