Itaconylated carboxymethyl chitosan cross-linked gel as well as preparation method and application thereof

Abstract

The invention provides itaconylated carboxymethyl chitosan cross-linked gel as well as a preparation method and application of the itaconylated carboxymethyl chitosan cross-linked gel. The itaconylated carboxymethyl chitosan cross-linked gel is prepared by the following steps: obtaining an itaconylated carboxymethyl chitosan framework material with the cross-linking degree of 5-40% under an initiation system, and then carrying out ray irradiation to carry out free radical secondary cross-linking, thereby obtaining the itaconylated carboxymethyl chitosan cross-linked gel. 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, is compact and stable in structure, has higher crosslinking degree and elasticity modulus, is beneficial to material exchange and promotion of cell proliferation and adhesion, can be used for beautifying and moisturizing and intraoperative and postoperative vein hemostasis, and can be applied to the field of intraoperative and postoperative vein hemostasis. Wide application prospects are realized in the fields of biological materials and tissue engineering.

Description

Technical Field

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

[0002] Carboxymethyl chitosan (CMC) is obtained by etherifying chitosan with chloroacetic acid, overcoming the disadvantage of poor water solubility of chitosan. After the etherification reaction, -CH2COOH is linked to chitosan. According to different substitution sites, carboxymethyl chitosan can be divided into 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, which not only retains the advantages of chitosan itself, but also has excellent properties such as good water solubility, antibacterial property, permeability promoting property, adhesiveness, biocompatibility and degradability, and also has physiological functions such as hemostasis, wound healing and antibacterial.

[0003] Itaconic acid is an organic unsaturated dicarboxylic acid and an important metabolite produced by the mitochondrial tricarboxylic acid (TCA) cycle. It has anti-inflammatory, antibacterial, antioxidant and antiviral properties, and can also regulate the body metabolism and maintain the body homeostasis. Therefore, itaconylated carboxymethyl chitosan is obtained by grafting itaconic anhydride onto carboxymethyl chitosan, and then a crosslinked gel is obtained by free radical polymerization of double bonds. After partial degradation of the gel in the human body and cleavage of the amide bond, itaconic acid molecules will be released in their original form, thereby exerting their bioactive effects.

[0004] At present, the preparation methods of crosslinked gels that have been studied more are chemical crosslinking method, photo-crosslinking method and high-energy ray irradiation crosslinking method. Among them, the crosslinking agents used in the chemical crosslinking method, such as epoxides and divinyl sulfone, all have certain cytotoxicity, and adverse reactions will occur when the residual crosslinking agent is applied to the human body; the initiator 2959 used in the photo-crosslinking method has certain toxicity, and the active hydroxyl groups of the initiator 2959 will graft onto the polymer molecules after the polymerization is completed, resulting in poor biocompatibility of the gel prepared by it. For example, Patent CN115926359A discloses a double-bonded bletilla striata polysaccharide-carboxymethyl chitosan gel and its preparation method and application. By introducing double bonds into bletilla striata polysaccharide and carboxymethyl chitosan, double-bonded bletilla striata polysaccharide and double-bonded carboxymethyl chitosan are obtained, and then the two are mixed with an initiator and placed under ultraviolet light for in-situ curing to obtain a double-bonded bletilla striata polysaccharide-carboxymethyl chitosan gel. Although this method uses double-bond free radical polymerization to obtain a crosslinked gel, the introduction of the photoinitiator poses a safety risk and has poor biocompatibility. Patent CN106832129A discloses that itaconic acid and carboxymethyl chitosan are crosslinked in the presence of an initiator to obtain itaconic acid homopolymer grafted carboxymethyl chitosan gel nanoparticles, and Patent CN114316141A discloses a magnetic carboxymethyl chitosan / acrylic acid / itaconic acid copolymer hydrogel adsorbent and its preparation method and application, but none of them disclose directly using itacylated carboxymethyl chitosan as a raw material for double-bond free radical polymerization; in addition, although CN116731387A discloses reacting methacrylic anhydride with carboxymethyl chitosan to obtain methacrylated carboxymethyl chitosan, mixing it with lithium saponite, freeze-drying and then placing it under cobalt-60 γ-ray irradiation for a certain time for crosslinking, but its irradiation dose is only 1-10 kGy. The gel prepared in this way has a low crosslinking degree, uneven texture and poor stability. In addition, acrylic acid generated during the metabolism of methacrylic acid has strong irritation and sensitization, and it is on the list of group 3 carcinogens, and acrylic acid can only be obtained from petroleum raw materials, so its biocompatibility is poor.

[0005] It can be seen that there are few reports on directly using itacylated carboxymethyl chitosan as a raw material for double-bond free radical polymerization at present, and there are almost no reports on the crosslinking reaction conditions and properties of itacylated carboxymethyl chitosan crosslinked gels. In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The object of the present invention is to provide an itaconyl carboxymethyl chitosan crosslinked gel and its preparation method. The present invention uses a radical secondary crosslinking technology to prepare the itaconyl carboxymethyl chitosan crosslinked gel. Specifically, a double bond radical polymerization of itaconyl carboxymethyl chitosan is initiated by using an initiation system, and then the product is irradiated and secondarily crosslinked by rays. The preparation method involved in the present invention can be carried out under relatively mild conditions, and by controlling the degree of pre-crosslinking and adjusting the irradiation dose, a crosslinked gel with excellent properties can be prepared.

[0007] The technical solution provided by the present invention is as follows:

[0008] In the first aspect of the present invention, an itaconyl carboxymethyl chitosan crosslinked gel is provided. An itaconyl carboxymethyl chitosan backbone material with a crosslinking degree of 5-40% is obtained under an initiation system, and then irradiated by rays to perform radical secondary crosslinking, thereby preparing the itaconyl carboxymethyl chitosan crosslinked gel.

[0009] Further, the initiation system is a redox system or a thermal decomposition system; the initiation system includes a radical initiator and an initiation assistant;

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

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

[0012] Further, the rays are γ rays, X rays or β rays, etc.

[0013] Further, the irradiation dose of the rays is 15-50 kGy.

[0014] The itaconyl carboxymethyl chitosan crosslinked gel can withstand high-dose ray irradiation, has high stability, and the final crosslinking degree reaches more than 60%, the elastic modulus is greater than 3000 Pa, and the in vitro coagulation index is below 10%.

[0015] The inventors found that by first subjecting itaconic acid acylated carboxymethyl chitosan to double bond free radical polymerization under an initiation system, a certain degree of pre-crosslinking reaction occurs, enabling the formation of an itaconic acid acylated carboxymethyl chitosan framework material with a crosslinking degree of 5-40% within a relatively short reaction time. Then, through gamma ray irradiation, the remaining double bonds in the chitosan framework material can cause the framework material not to degrade within a high irradiation dose range, but rather undergo secondary crosslinking, thereby obtaining an itaconic acid acylated carboxymethyl chitosan crosslinked gel with a stable structure and a high crosslinking degree. And during the secondary crosslinking by gamma rays, sterilization treatment is also carried out, saving subsequent processing steps and improving the overall reaction efficiency.

[0016] In a second aspect of the present invention, there is provided a method for preparing an itaconic acid acylated carboxymethyl chitosan crosslinked gel, comprising the following steps:

[0017] (1) Add an initiation system to the itaconic acid acylated carboxymethyl chitosan solution and carry out a pre-crosslinking reaction at 10-60°C for 20-40 minutes;

[0018] (2) Subject the pre-crosslinked product obtained in step (1) to secondary crosslinking by gamma ray irradiation.

[0019] Further, the reaction concentration of the itaconic acid acylated 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] Further, 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).

[0021] Further, when the initiation system is a redox system, the pH value range is 3.0-7.0, 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, 7.0.

[0022] Further, 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.

[0023] Further, step (1) includes taking an appropriate amount of itaconylated carboxymethyl chitosan, stirring and dissolving it, adding an initiator, adjusting the pH value, then adding an initiating aid, and stirring and reacting for 20 - 40 min to obtain a pre-crosslinked product of itaconylated carboxymethyl chitosan.

[0024] Further, in step (1), the amounts of the initiator and the initiating aid need to be added in a sufficient amount at one time, and cannot be added in small amounts multiple times, and the time interval between the addition of the initiator and the initiating aid is short.

[0025] In the pre-crosslinking stage of the present invention, by controlling the dosage of the free radical initiator, the reaction temperature and time, an itaconylated carboxymethyl chitosan framework material with a crosslinking degree of 5% - 40% is formed within a short reaction time, and then irradiated with rays, so that the gel can undergo secondary crosslinking, thereby obtaining a crosslinked gel with higher stability.

[0026] In the above pre-crosslinking process, due to the high reaction activity of the free radical initiator, while initiating the free radical polymerization of double bonds, it will also break the glycosidic bond in chitosan, resulting in cleavage. Thus, even if the polymerization reaction of the double bond is completed, due to the destruction of the glycosidic bond in chitosan, the target hydrogel cannot be formed. Therefore, if the dosage of the initiator is large or the duration of the initiation process is long, the glycosidic bond in itaconylated carboxymethyl chitosan will be broken; if the initiator is added in batches, due to the short half-life time of the existence of free radicals, the free radical concentration cannot reach the initiation dose, and the free radical polymerization of double bonds 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 once within a short time, so as to controllably achieve the pre-crosslinking degree within a suitable range. Further, the present invention controls the pre-crosslinking degree to be 5% - 40% to first obtain an itaconylated carboxymethyl chitosan framework material, so that the residual amount of double bonds is in the range of 60% - 95%; if the residual amount of double bonds is small, high-dose irradiation is likely to cause the glycosidic bond on itaconylated carboxymethyl chitosan to break, and if the residual amount of double bonds reaches more than 95%, the pre-crosslinking degree of itaconylated carboxymethyl chitosan is too low, it is difficult to form a network framework structure, the double bonds are in a disordered and scattered state, the distances of most double bonds are far, and only a small part are relatively close and can be crosslinked by irradiation, and the degree of secondary crosslinking is low; while the remaining double bond amount of 60% - 95% in the present invention can, on the one hand, enable the framework material to withstand a high-intensity irradiation dose and improve the crosslinking efficiency of double bonds, and on the other hand, can protect the glycosidic bond on itaconylated carboxymethyl chitosan from being damaged by rays during the irradiation process, and finally obtain a hydrogel material with a stable structure.

[0027] Furthermore, the preparation method further includes steps of crushing, dialyzing, and freeze-drying the pre-crosslinked product obtained in step (1).

[0028] Furthermore, for the crushing, a homogenizer is used to crush the itaconylated carboxymethyl chitosan pre-crosslinked product; for the dialysis, the gel is placed in a dialysis bag for dialysis; and for the freeze-drying, a vacuum freeze dryer is used for freeze-drying.

[0029] Furthermore, the crushing parameters of the homogenizer are 12000 rpm and the crushing time is 5 min. The dialysis solution is changed every 1 h. After the dialysis is completed, the product is placed in a vacuum freeze dryer for freeze-drying to obtain the freeze-dried powder of the itaconylated carboxymethyl chitosan pre-crosslinked gel.

[0030] Furthermore, in step (2), the ray is γ ray, X ray, β ray, etc.

[0031] Furthermore, in step (2), the irradiation dose is 10 - 50 kGy.

[0032] Furthermore, the irradiation can not only promote secondary crosslinking but also has a sterilization effect.

[0033] Furthermore, after irradiation, it is compounded with a buffer solution to obtain the final product, the itaconylated carboxymethyl chitosan crosslinked gel.

[0034] Furthermore, the buffer solution is PBS with a pH of 7.0, and the compounding concentration is 80 - 120 mg / mL, preferably 100 mg / mL.

[0035] In the third aspect of the present invention, there is provided the application of the itaconylated carboxymethyl chitosan crosslinked gel described in the first aspect or the itaconylated carboxymethyl chitosan crosslinked gel prepared by the method described in the second aspect.

[0036] Furthermore, the application is the application of the itaconylated carboxymethyl chitosan crosslinked gel in the preparation of hemostatic materials, soft tissue filling materials, drugs, or tissue engineering materials.

[0037] Specifically, the soft tissue filler can be used for eliminating wrinkles (such as periorbital wrinkles, forehead lines, glabellar lines, perioral lines, nasolabial folds, tear troughs, nasolabial folds, neck lines, hand wrinkles, stretch marks, etc.), anti-aging, scar elimination, wound repair, intraoperative and postoperative venous hemostasis, etc.

[0038] Specifically, the drug uses the itaconylated carboxymethyl chitosan crosslinked gel as a carrier to achieve purposes such as sustained release, controlled release, and targeted drug delivery.

[0039] Specifically, the tissue engineering materials may be bone tissue engineering materials, cartilage tissue engineering materials, corneal tissue engineering materials, cardiovascular tissue engineering materials, liver tissue engineering materials, rapid hemostatic materials, etc.

[0040] The present invention has the following beneficial effects:

[0041] 1. The present invention uses a free radical secondary crosslinking technology to prepare itaconylated carboxymethyl chitosan crosslinked gel; specifically, itaconylated carboxymethyl chitosan is first subjected to double bond free radical polymerization under an initiation system to carry out a certain degree of pre-crosslinking reaction, so that an itaconylated carboxymethyl chitosan framework material is formed within a relatively short reaction time, and then irradiated with rays. The remaining double bonds in the chitosan framework material can enable the framework material not to degrade within a high irradiation dose range, but to carry out free radical secondary crosslinking, thereby obtaining an itaconylated carboxymethyl chitosan crosslinked gel with a stable structure and high crosslinking degree; and sterilization treatment is also carried out during the ray secondary crosslinking process, saving subsequent treatment processes and improving the overall reaction efficiency.

[0042] 2. In the pre-crosslinking stage, the present invention controls the initiator to be added in a one-time sufficient amount and ensures that the pre-crosslinking process is completed in one time within a relatively short time, so as to controllably achieve the pre-crosslinking degree within a suitable range; further, by controlling the pre-crosslinking degree to be 5% - 40%, an itaconylated carboxymethyl chitosan framework material is first obtained, so that the residual amount of double bonds is in the range of 60% - 95%. The remaining amount of double bonds, on the one hand, can enable the framework material to withstand a high irradiation dose and improve the crosslinking efficiency of double bonds, and on the other hand, can also protect the glycosidic bonds on itaconylated carboxymethyl chitosan from being damaged by rays during the irradiation process, and finally obtain a hydrogel material with a stable structure.

[0043] 3. The preparation process of the present invention is mild and controllable. Itaconylated carboxymethyl chitosan is respectively initiated by a variety of initiation systems to carry out double bond free radical polymerization, and then the product is irradiated and sterilized and secondarily crosslinked by rays. The post-treatment process is simple; the prepared itaconylated carboxymethyl chitosan crosslinked gel has a continuous interpenetrating network structure and a dense and stable structure, has a higher elastic modulus and blood coagulation performance, is conducive to blood penetration, can promote material exchange, cell proliferation and adhesion, and can be used for beauty moisturizing, intraoperative and postoperative venous hemostasis and as a wound dressing, and has potential research value and broad application prospects in the field of biomaterials and tissue engineering. Description of the Drawings

[0044] Figure 1 It is the SEM diagram of crosslinked gel A3 in Example 3, where (a) is the scanning electron microscope result of crosslinked gel sponge A3, and (b) is the partial enlarged view of (a).

[0045] Figure 2Appearance shape of crosslinked gel A3 in Example 3.

[0046] Figure 3 Liver hemostasis model diagram of crosslinked gel A3 in Example 3.

[0047] Figure 4 Liver hemostasis model diagram of commercially available biological protein sponge Chuangbifu. Detailed implementation manners

[0048] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the technical field to which the present invention pertains.

[0049] The public contents of various publications, patents and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0050] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained commercially.

[0051] Example 1: Itaconic acid acylated carboxymethyl chitosan undergoes double bond free radical polymerization at pH = 3.0 and reaction temperature of 10 °C to obtain itaconic acid acylated carboxymethyl chitosan crosslinked gel A1

[0052] Weigh 1.5 g of itaconic acid acylated carboxymethyl chitosan, add 30 mL of deoxygenated purified water, stir to completely dissolve it, then add 441.18 μmol (0.015 g) of hydrogen peroxide, stir evenly and adjust the pH value of the solution to 3.0, then add 77.48 μmol (0.014 g) of ascorbic acid, quickly stir and place it at 10 °C for reaction for 40 min to obtain the pre-crosslinked product of itaconic acid acylated carboxymethyl chitosan. Then use a homogenizer to crush the pre-crosslinked product of itaconic acid acylated carboxymethyl chitosan, with the parameter of 12,000 rpm for 5 min, then place the gel in a dialysis bag for dialysis, change the liquid every 1 h, and after the dialysis is completed, place the product in a freeze dryer for freeze drying to obtain the freeze-dried powder of the pre-crosslinked gel of itaconic acid acylated carboxymethyl chitosan. Irradiate the freeze-dried powder with γ-rays for secondary crosslinking and sterilization operations, with the irradiation dose of 15 kGy, and then compound it with PBS with a pH of 7.0, and the compounding concentration is 100 mg / mL to obtain the final product of itaconic acid acylated carboxymethyl chitosan crosslinked gel, denoted as A1.

[0053] Example 2: The itaconylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 4.5 and a reaction temperature of 20 °C to obtain the itaconylated carboxymethyl chitosan crosslinked gel A2

[0054] Weigh 1.5 g of itaconylated carboxymethyl chitosan, add 30 mL of deoxygenated purified water, stir to completely dissolve it, then add 882.35 μmol (0.030 g) of hydrogen peroxide, stir evenly, adjust the pH value of the solution to 4.5, then add 83.01 μmol (0.023 g) of ferrous sulfate, quickly stir and place it at 20 °C for reaction for 35 min to obtain the itaconylated carboxymethyl chitosan pre-crosslinked product. Then use a homogenizer to crush the itaconylated carboxymethyl chitosan pre-crosslinked product, with the parameters of 12000 rpm and crush for 5 min. Then place the gel in a dialysis bag for dialysis, change the solution every 1 h. After dialysis is completed, place the product in a freeze dryer for freeze-drying to obtain the itaconylated carboxymethyl chitosan pre-crosslinked gel lyophilized powder. Irradiate the lyophilized powder with β-rays for secondary crosslinking and sterilization operations, with an irradiation dose of 25 kGy, and then compound it with PBS with a pH of 7.0, and the compounding concentration is 100 mg / mL to obtain the final product, the itaconylated carboxymethyl chitosan crosslinked gel, denoted as A2.

[0055] Example 3: The itaconylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain the itaconylated carboxymethyl chitosan crosslinked gel A3

[0056] Weigh 1.5 g of itaconylated carboxymethyl chitosan, add 30 mL of deoxygenated purified water, stir to completely dissolve it, then add 2.647 mmol (0.090 g) of hydrogen peroxide, stir evenly, adjust the pH value of the solution to 6.3, then add 446.43 μmol (0.056 g) of sodium sulfite, quickly stir and place it at 60 °C for reaction for 20 min to obtain the itaconylated carboxymethyl chitosan pre-crosslinked product. Then use a homogenizer to crush the itaconylated carboxymethyl chitosan pre-crosslinked product, with the parameters of 12000 rpm and crush for 5 min. Then place the gel in a dialysis bag for dialysis, change the solution every 1 h. After dialysis is completed, place the product in a freeze dryer for freeze-drying to obtain the itaconylated carboxymethyl chitosan pre-crosslinked gel lyophilized powder. Irradiate the lyophilized powder with γ-rays for secondary crosslinking and sterilization operations, with an irradiation dose of 45 kGy, and then compound it with PBS with a pH of 7.0, and the compounding concentration is 100 mg / mL to obtain the final product, the itaconylated carboxymethyl chitosan crosslinked gel, denoted as A3.

[0057] Example 4: The itaconylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 8.2 and a reaction temperature of 45 °C to obtain the itaconylated carboxymethyl chitosan crosslinked gel A4

[0058] Weigh 1.5 g of itaconylated carboxymethyl chitosan, add 30 mL of deoxygenated purified water, stir to completely dissolve it, then add 789.47 μmol (0.180 g) of ammonium persulfate, stir evenly and adjust the pH value of the solution to 8.2, then add 862.07 μmol (0.100 g) of tetramethylethylenediamine, quickly stir and place it at 45 °C for reaction for 30 min to obtain a pre-crosslinked product of itaconylated carboxymethyl chitosan. Then use a homogenizer to crush the pre-crosslinked product of itaconylated carboxymethyl chitosan, with the parameters of 12000 rpm and crush for 5 min. Then place the gel in a dialysis bag for dialysis, change the liquid once every 1 h. After dialysis is completed, place the product in a freeze dryer for freeze-drying to obtain a freeze-dried powder of the pre-crosslinked gel of itaconylated carboxymethyl chitosan. Irradiate the freeze-dried powder with γ-rays for secondary crosslinking and sterilization operations, with an irradiation dose of 40 kGy, and then compound it with PBS with a pH of 7.0, and the compounding concentration is 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan crosslinked gel, denoted as A4.

[0059] Example 5: Itaconylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 10.0 and a reaction temperature of 30 °C to obtain itaconylated carboxymethyl chitosan crosslinked gel A5

[0060] Weigh 1.5 g of itaconylated carboxymethyl chitosan, add 30 mL of deoxygenated purified water, stir to completely dissolve it, then add 1.11 mmol (0.300 g) of potassium persulfate, stir evenly and adjust the pH value of the solution to 10.0, then add 1.485 mmol (0.150 g) of triethylamine, quickly stir and place it at 30 °C for reaction for 30 min to obtain a pre-crosslinked product of itaconylated carboxymethyl chitosan. Then use a homogenizer to crush the pre-crosslinked product of itaconylated carboxymethyl chitosan, with the parameters of 12000 rpm and crush for 5 min. Then place the gel in a dialysis bag for dialysis, change the liquid once every 1 h. After dialysis is completed, place the product in a freeze dryer for freeze-drying to obtain a freeze-dried powder of the pre-crosslinked gel of itaconylated carboxymethyl chitosan. Irradiate the freeze-dried powder with X-rays for secondary crosslinking and sterilization operations, with an irradiation dose of 30 kGy, and then compound it with PBS with a pH of 7.0, and the compounding concentration is 100 mg / mL to obtain the final product, itaconylated carboxymethyl chitosan crosslinked gel, denoted as A5.

[0061] Example 6: Itaconylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 9.5 and a reaction temperature of 30 °C to obtain itaconylated carboxymethyl chitosan crosslinked gel A6

[0062] Weigh 1.5 g of itaconic acylated carboxymethyl chitosan, add 75 mL of deoxygenated purified water, stir to dissolve it completely, then add 4.934 mmol (1.125 g) of ammonium persulfate, adjust the pH value of the solution to 9.5 after stirring evenly, then add 8.52 mmol (0.511 g) of ethylenediamine, place it in a reaction at 15 °C for 40 min after rapid stirring to obtain a pre-crosslinked product of itaconic acylated carboxymethyl chitosan. Then use a homogenizer to crush the pre-crosslinked product of itaconic acylated carboxymethyl chitosan, with the parameter of 12,000 rpm for 5 min, then place the gel in a dialysis bag for dialysis, change the solution once every 1 h, and freeze-dry the product in a freeze-dryer after dialysis to obtain a freeze-dried powder of the pre-crosslinked gel of itaconic acylated carboxymethyl chitosan. Irradiate the freeze-dried powder with γ-rays for secondary crosslinking and sterilization operations, with an irradiation dose of 15 kGy, and then compound it with PBS with a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, the crosslinked gel of itaconic acylated carboxymethyl chitosan, denoted as A6.

[0063] Comparative Example 1: Itaconic acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of -20 °C to obtain itaconic acylated carboxymethyl chitosan crosslinked gel B1

[0064] The pre-crosslinking reaction temperature is 2 °C, and the rest of the reaction process is the same as that in Example 3 to obtain the final product, the crosslinked gel of itaconic acylated carboxymethyl chitosan, denoted as B1.

[0065] Comparative Example 2: Itaconic acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 8.2 and a reaction temperature of -20 °C to obtain itaconic acylated carboxymethyl chitosan crosslinked gel B2

[0066] The pre-crosslinking reaction temperature is 70 °C, and the rest of the reaction process is the same as that in Example 4 to obtain the final product, the crosslinked gel of itaconic acylated carboxymethyl chitosan, denoted as B2.

[0067] Comparative Example 3: Itaconic acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain itaconic acylated carboxymethyl chitosan crosslinked gel B3

[0068] Weigh 1.5 g of itaconic acylated carboxymethyl chitosan, add 30 mL of deoxygenated purified water, stir to dissolve it completely, place it in a freeze-dryer for freeze-drying to obtain a freeze-dried powder. Irradiate the freeze-dried powder with γ-rays for secondary crosslinking and sterilization operations, with an irradiation dose of 45 kGy, and then compound it with PBS with a pH of 7.0 at a compounding concentration of 100 mg / mL to obtain the final product, the crosslinked gel of itaconic acylated carboxymethyl chitosan, denoted as B3.

[0069] Comparative Example 4: Itaconic acid-acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain itaconic acid-acylated carboxymethyl chitosan crosslinked gel B4

[0070] Only replace "irradiate the lyophilized powder with γ-rays for secondary crosslinking and sterilization, with an irradiation dose of 40 kGy" in Example 3 with "sterilize the lyophilized powder by moist heat sterilization", and the rest of the preparation process is the same as that in Example 3 to obtain the final product, itaconic acid-acylated carboxymethyl chitosan crosslinked gel, denoted as B4.

[0071] Comparative Example 5: Itaconic acid-acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain itaconic acid-acylated carboxymethyl chitosan crosslinked gel B5

[0072] The irradiation dose is 55 kGy, and the rest of the preparation process is the same as that in Example 3 to obtain the final product, itaconic acid-acylated carboxymethyl chitosan crosslinked gel, denoted as B5.

[0073] Comparative Example 6: Itaconic acid-acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain itaconic acid-acylated carboxymethyl chitosan crosslinked gel B7

[0074] The pre-crosslinking reaction time is 50 min, and the rest of the preparation process is the same as that in Example 3 to obtain the final product, itaconic acid-acylated carboxymethyl chitosan crosslinked gel, denoted as B6.

[0075] Comparative Example 7: Itaconic acid-acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain itaconic acid-acylated carboxymethyl chitosan crosslinked gel B8

[0076] The hydrogen peroxide added is 26.47 mmol (0.900 g), and the sodium sulfite added is 4.464 mmol (0.560 g). The rest of the preparation process is the same as that in Example 3 to obtain the final product, itaconic acid-acylated carboxymethyl chitosan crosslinked gel, denoted as B7.

[0077] Comparative Example 8: Itaconic acid-acylated carboxymethyl chitosan undergoes double-bond free radical polymerization at pH = 6.3 and a reaction temperature of 60 °C to obtain itaconic acid-acylated carboxymethyl chitosan crosslinked gel B9

[0078] The initiator and the initiating assistant are added in 3 times, with an interval of 2 min for each addition. The rest of the preparation process is the same as that in Example 3 to obtain the final product, itaconic acid-acylated carboxymethyl chitosan crosslinked gel, denoted as B8.

[0079] Performance Test 1: Determination of the residual amount of double bonds in the gel

[0080] Weigh 40 mg of the pre-crosslinked products and final products of itaconylated carboxymethyl chitosan crosslinked gels of samples A1 - A6 and B1 - B8 prepared in Examples 1 - 6 and Comparative Examples 1 - 8 respectively, and conduct nuclear magnetic resonance hydrogen spectrum tests on them. The nuclear magnetic resonance test solvent is deuterated water. Ensure that the samples are completely dissolved before testing.

[0081] By measuring the residual amount of double bonds in the gel, the double bond consumption amount of the gel, that is, the crosslinking degree, can be obtained. The crosslinking degree measurement results of the gels are shown in Table 1.

[0082] Table 1 Crosslinking degree measurement results of gels

[0083]

[0084]

[0085] Performance Test 2 Determination of rheological properties of gels

[0086] Take the pre-crosslinked products and final products of itaconylated carboxymethyl chitosan crosslinked gels of samples A1 - A6 and B1 - B8 prepared in Examples 1 - 6 and Comparative Examples 1 - 8 respectively, and test their rheological properties through a rotational rheometer. The rotational rheometer test parameters are: take 2 mL of the sample, operating gap: 1000 mm, loading gap: 45000 mm, operating temperature: 37 °C, strain: 1%, frequency: 0.9 Hz, operating time: 60 s. The rheological data of each gel obtained are shown in Table 2.

[0087] Table 2 Determination results of rheological properties of gels

[0088]

[0089]

[0090] It can be seen from a comparison of the data in Table 1 and Table 2 that for the above crosslinked gels A1 to A6, by controlling the crosslinking degree of pre-crosslinking within the range of 5-40%, followed by radiation irradiation for secondary crosslinking, the crosslinking degree of the final itaconylated carboxymethyl chitosan crosslinked gel can be further increased. For example, in Example 1, when the pre-crosslinking degree is 5%, the crosslinking degree can reach 60% after irradiation; and with the increase of the irradiation dose, the crosslinking of double bonds can be further promoted. For example, in Example 3, at an irradiation dose of 45 kGy, the crosslinking degree of double bonds reaches 100%, and there is basically no residue of double bonds. In addition, the elastic modulus of the samples in the examples after irradiation is all above 3600 Pa, which is increased by 127% to 148% compared with the elastic modulus before irradiation. The reason is that there are still residual double bonds in the gel obtained after pre-crosslinking that are not completely reacted (as shown in Table 1), so secondary crosslinking occurs in the gel after irradiation sterilization. The increase in its crosslinking degree makes the network structure of the gel denser and has a higher elastic modulus.

[0091] In addition, when the reaction temperature is 10-60 °C, it is beneficial to the free radical polymerization of double bonds. Within this range, the higher the reaction temperature, the higher the activity of free radicals, which is beneficial to free radical polymerization. However, with the further increase of the crosslinking temperature, although the free radical activity is high, causing the gel to undergo 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 free radicals will break the glycosidic bonds in the gel, resulting in the failure of pre-crosslinking of the gel. Therefore, no crosslinked skeleton is formed in the first stage of the gel, and its stability is poor; even during the irradiation sterilization process, the remaining double bonds continue to undergo free radical polymerization reactions, but since the main chain of the main body chitosan of the gel has been broken during pre-crosslinking, the resulting gel still cannot form a crosslinked structure. And due to the small amount of residual double bonds, the double bonds are difficult to protect the residual glycosidic bonds during pre-crosslinking, and the glycosidic bonds are also damaged by the rays. Therefore, its elastic modulus after irradiation is low, only 1.7 Pa, even lower than the elastic modulus before irradiation. For Comparative Example 1, due to the too low pre-crosslinking temperature, the free radical activity is poor and the energy is low, unable to initiate the free radical polymerization of double bonds, resulting in the pre-crosslinking degree of the gel being only 3%, making it difficult to form a network skeleton structure. The double bonds are in a disordered and scattered state, and the distance between most double bonds is far, and only a small part of the relatively close ones can be crosslinked by irradiation, so the degree of secondary crosslinking is low. Therefore, its elastic modulus after irradiation (313.2 Pa) is much lower than the elastic modulus in Example 3 (5043 Pa).

[0092] The gel B3 obtained in Comparative Example 3 has the same preparation process as the gel A3 in Example 3, except that no initiator is added. The elastic moduli before and after irradiation are 20 Pa and 103.1 Pa respectively. The elastic modulus of the formed gel is much lower than that of the gel formed in Example 3. It shows that under the condition of no pre-crosslinking with an initiator, although a gel can be formed during irradiation, due to the lack of pre-crosslinking to form a network skeleton structure, the double bonds are in a disordered and scattered state. The distances between most double bonds are far, and only a small number of relatively close ones can be irradiated and crosslinked, that is, only a small amount of double bonds participate in the crosslinking reaction (the double bond consumption rate is 23%). The degree of secondary crosslinking is low. After the ray energy reception ends, most double bonds return to the previous state of double bonds. This is also the main reason for the low elasticity of the gel. Therefore, the main role of pre-crosslinking is to pre-form a gel skeleton. The formed preliminary network skeleton structure can shorten the distance between double bonds, enabling the remaining double bonds to react further during irradiation. The efficiency of irradiation crosslinking is much higher than that without pre-crosslinking or with a low degree of pre-crosslinking, ultimately increasing the elasticity of the gel and also increasing its stability.

[0093] The gel B4 obtained in Comparative Example 4 has the same preparation process as the gel A3 in Example 3, except that the sterilization method of gel B4 is moist heat sterilization. Its elastic modulus before sterilization is 2031 Pa, and the elastic modulus of gel A3 after irradiation sterilization is 5043 Pa. However, the elastic modulus of gel B4 after moist heat sterilization is only 1.3 Pa, indicating that during moist heat sterilization, the temperature is as high as 121 °C, and the gel is not stable in this state, and the network structure is damaged. Therefore, its elastic modulus decreases significantly after sterilization. So this type of gel is not suitable for moist heat sterilization.

[0094] The gel B5 obtained in Comparative Example 5 has the same preparation process as the gel A3 in Example 3, except that the irradiation sterilization dose is increased to 55 kGy. Its elastic modulus before sterilization is 2031 Pa, and the elastic modulus after sterilization is only 265 Pa. The reason is that when the irradiation dose is further increased from 45 kGy in Example 3 to 55 kGy, with higher irradiation energy, the double bond consumption is still 100%, but the further increased irradiation dose will break the glycosidic bonds of the gel, causing the gel skeleton to disintegrate. Therefore, the elastic modulus of the crosslinked gel B5 after sterilization decreases significantly compared with A3.

[0095] The gel B6 obtained in Comparative Example 6 and the gel A3 in Example 3 have the same preparation process, except that the pre-crosslinking time is extended to 45 min. The extension of the pre-crosslinking time can make the crosslinking degree of gel B6 before irradiation as high as 90%, and the elastic modulus is as high as 3055 Pa, but the elastic modulus after irradiation is only 0.5 Pa. This is mainly because under the condition of extended pre-crosslinking time, the pre-crosslinking degree of the gel is relatively high, the double bonds basically react completely, and the remaining amount is only 10%. Therefore, during irradiation, a small amount of double bonds are difficult to effectively protect the glycosidic bonds. The irradiation rays cause the glycosidic bonds of the gel to break, and the gel degrades, resulting in a significant decrease in the elastic modulus of gel B6 after irradiation. Therefore, reasonably controlling the pre-crosslinking time and making the pre-crosslinking degree within a suitable range is also crucial for preparing gel materials.

[0096] The gel B7 obtained in Comparative Example 7 and the gel A3 in Example 3 have the same preparation process, except that the amounts of the initiator and the initiating assistant are increased; by increasing the usage amounts of the initiator and the assistant, the consumption of double bonds in gel B7 before irradiation is 97%, and the consumption of double bonds after irradiation sterilization is 100%. Although a high dose of the initiator can complete the double bond free radical polymerization reaction to a relatively high degree, the initiator at this dose destroys and breaks the glycosidic bonds of the gel due to its extremely strong oxidizing property, and fails to form a gel network, resulting in the failure of gel crosslinking.

[0097] 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 initiating assistant is changed to adding in small amounts multiple times. After adding a small amount of the initiator each time, fewer free radicals are generated in the system, and their energy is not enough to initiate the free radical polymerization of double bonds. Therefore, the consumption of double bonds in gel B8 during the pre-crosslinking process is only 2%. Since the initiator (such as hydrogen peroxide, peroxide, etc.) has strong oxidizing property, the initiator added in batches will destroy and break the glycosidic bonds of chitosan, resulting in the failure of final crosslinking. After irradiation sterilization, due to the failure of pre-crosslinking to form a network skeleton structure, there are also double bonds in a disordered and scattered state. The distances between most double bonds are far, resulting in difficult crosslinking reaction. Therefore, the consumption of double bonds in the gel only increases slightly, and its elastic modulus is extremely low.

[0098] Performance Test 3 SEM Determination of Gel

[0099] The pore structure of the gel was observed by scanning electron microscope. After freeze-drying the crosslinked gel obtained in Example 3, it was cut into small squares with a blade, fixed on the electron microscope stage with conductive glue, sputtered with gold for 60 s, and analyzed by field emission scanning electron microscope. The scanning electron microscope results of the crosslinked gel sponge A3 are shown in Figure 1 , and Figure b is a partial enlarged view of Figure a.

[0100] From Figure 1It can be seen that the crosslinked gel A3 has an interpenetrating macroporous network structure. This unique structure is beneficial to blood penetration, thus promoting hemostasis, and it can be used for intraoperative and postoperative venous hemostasis.

[0101] Performance Test 4 Gel Water Absorption Performance Test

[0102] Take 1 g of the crosslinked gels A1 - A6 prepared in Examples 1 - 6 and place them in a petri dish. Vertically add 1 mL of purified water to the gel, and record the time when the purified water is completely absorbed by the gel. The test results of the gel water absorption performance are shown in Table 3.

[0103] Table 3 Test Results of Gel Water Absorption Performance

[0104] Gel Name Water Absorption Time (seconds) A1 6s A2 4s A3 1s A4 2s A5 3s A6 5s

[0105] As can be seen from Table 3, the crosslinked gels of Examples 1 - 6 have a very fast water absorption rate, and can completely absorb 1 mL of purified water within 6 s, indicating that the crosslinked gels prepared by the preparation process of the present invention have a rich porous network structure, so they can achieve rapid water absorption. And a relatively fast water absorption rate is the premise and basis for the gel to quickly stop bleeding. Among them, gel A3 has the fastest water absorption rate, and it only takes 1 s to absorb 1 mL of purified water, which is also related to the most stable gel structure and the richest porous structure in Example 3.

[0106] Performance Test 5 Determination of Gel In Vitro Coagulation Index (BCI)

[0107] Take 50 mg of the freeze-dried powder samples of gels A1 - A6 after irradiation, and reconstitute them with PBS at pH 7.0 to 100 mg / mL. After they are fully swollen, set aside; take a 5 mL centrifuge tube, add 20 μL of anticoagulated rabbit blood and 2 μL of CaCl2 (0.25 M) solution to it, then add the swollen gel to it, and then place the centrifuge tube in an incubator at 37 °C for 5 min; then add 2 mL of purified water to the centrifuge tube along the wall, and then place the centrifuge tube in an incubator at 37 °C for 10 min. Finally, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the supernatant at 540 nm. The blank control group sample is anticoagulated rabbit blood plus 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:

[0108] BCI = OD 样品 / OD 空白 *100%

[0109] Table 4 BCI Values of Itaconylated Carboxymethyl Chitosan Crosslinked Gels

[0110] Sample Name BCI A1 10％ A2 8％ A3 3％ A4 5％ A5 6％ A6 9％ Blank 100％

[0111] It can be seen from Table 4 that 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 of the gel, indicating that the coagulation performance of the gel obtained by the preparation process of the present invention is good, and the BCI value of gel A3 is the smallest, only 3%, indicating that its coagulation ability is the strongest. The reasons are: (1) itaconylated carboxymethyl chitosan itself has cationic properties, and through the action of positive charges, it adheres and aggregates with negatively charged red blood cell surface substances, thereby quickly forming blood clots for hemostasis; (2) the gel forms an interpenetrating macroporous structure during the preparation process, which has a strong water absorption capacity, so the gel can quickly absorb free water into its own network structure and bind and fix the free water, causing the blood to aggregate due to rapid water loss, resulting in increased platelet concentration, and finally forming blood coagulation.

[0112] Performance test 6 Gel A3 in New Zealand white rabbit liver hemostasis model

[0113] A New Zealand white rabbit was selected and the rabbit was given a small amount of 2.5 mL / kg intravenous injection of chloral hydrate injection for general anesthesia before the operation. After the rabbit was anesthetized, the abdomen was depilated and disinfected, and the rabbit was fixed on the laboratory table in a supine position. The upper abdominal skin was cut open layer by layer to expose the left lobe of the liver, and sterile gauze was placed under the liver lobe to protect the surrounding tissues.

[0114] A 1.0*0.8 cm square incision was made on the surface of the left outer lobe of the liver with a surgical blade, and the liver tissue at the wound was cut off with a depth of 0.2 cm, resulting in obvious bleeding on the wound surface. After free bleeding for 10 seconds, the cross-linked gel A3 of Example 3 prepared in advance was pushed to the wound with a syringe to completely cover the wound. The blank control group was directly covered with 16 layers of 3*3 cm sterile gauze on the wound, and gently pressed with fingers (the force was controlled to ensure that the material did not fall off at the minimum pressure), and the timing was started at the same time to record the bleeding time. After hemostasis was completed, the gauze was removed, the hemostatic gel was rinsed with physiological saline, and then the abdominal cavity of the animal was sutured layer by layer with surgical sutures, and the state of the animal was observed.

[0115] like Figure 2 The shape and appearance of the itaconylated carboxymethyl chitosan crosslinked gel A3 of Example 3 are shown in the figure. The gel can flow slightly when placed vertically. During the test, 1.0 mL of itaconylated carboxymethyl chitosan crosslinked gel A3 was injected and then pressed to stop bleeding. The bleeding 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 the biological protein sponge Chuangbifu was pressed to stop bleeding for 1 minute, the wound continued to ooze blood. Figure 4。The results show that the hemostatic performance of itaconic acylated carboxymethyl chitosan crosslinked gel is significantly better than that of the commonly used biological protein sponge hemostatic agent on the market, indicating that the crosslinked gel of the present invention can be used for intraoperative and postoperative venous hemostasis, and has good clinical use effect and broad application prospects.

[0116] 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An itaconyl carboxymethyl chitosan crosslinked gel, characterized in that, The itaconyl carboxymethyl chitosan crosslinked gel is prepared by obtaining an itaconyl carboxymethyl chitosan framework material with a crosslinking degree of 5-40% under an initiation system, and then performing free radical secondary crosslinking through radiation irradiation.

2. The itaconic acylated carboxymethyl chitosan crosslinked 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 assistant.

3. The itaconic acylated carboxymethyl chitosan crosslinked 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 assistant 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 assistant is tetramethylethylenediamine, triethylamine or ethylenediamine.

4. The itaconic acylated carboxymethyl chitosan crosslinked gel according to any one of claims 1-3, characterized in that, The radiation is γ-ray, X-ray or β-ray; and / or, the irradiation dose of the radiation is 15-50 kGy.

5. A method for preparing the itaconyl carboxymethyl chitosan crosslinked gel according to any one of claims 1-4, characterized in that, The method comprises the following steps: (1) Adding an initiation system to an itaconyl carboxymethyl chitosan solution, and performing a pre-crosslinking reaction at 10-60 °C for 20-40 min; (2) Irradiating the pre-crosslinked product obtained in step (1) with radiation for secondary crosslinking.

6. The preparation method according to claim 5, characterized in that, The concentration of the itaconyl 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 Step (1) includes taking an appropriate amount of itaconylated carboxymethyl chitosan, stirring and dissolving it, adding a free radical initiator, adjusting the pH value, then adding an initiation assistant, and stirring and reacting for 20-40 min 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 3.0-7.0; or when the initiation system is a thermal decomposition system, the pH value is 7.0-10.

0.

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

10. Use of the itaconyl carboxymethyl chitosan crosslinked gel according to any one of claims 1-4 or the itaconyl carboxymethyl chitosan crosslinked gel prepared by the preparation method according to any one of claims 5-9 in the preparation of a hemostatic material, a soft tissue filling material, a drug or a tissue engineering material.

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