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

Abstract

The present invention provides a graft-modified carboxymethyl chitosan cross-linked gel, which is prepared by mixing a double-bond-modified carboxymethyl chitosan, a mercapto-terminated polyethylene glycol, an active component and a photoinitiator, subjecting the resulting mixture to illumination to obtain a cross-linked gel that has been subjected to cross-linking and grafting in one step, and crystallizing the cross-linked gel at a low degree of supercooling to obtain a freeze-dried sponge. The cross-linked gel obtained in the present application has an excellent mechanical strength, the freeze-dried sponge has a good water-retention rate and a large porosity, and the active component has a high embedding rate.

Description

A grafted modified carboxymethyl chitosan cross-linked gel and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a grafted modified carboxymethyl chitosan cross-linked gel and a freeze-dried sponge prepared by using the cross-linked gel, as well as a preparation method and application thereof. Background Art

[0002] Accidental or postoperative wounds and tissue defects left on the skin can cause pain, physiological dysfunction, scars left after wound healing, and changes in physical appearance, which can affect the patient's freedom of movement and quality of life. Therefore, it is important to promote rapid wound healing and maintain a clean and sterile environment for the wound during the healing process. Sponge is a solid preparation with light weight, high porosity, low density and strong liquid absorption capacity. It has good advantages in wound absorption, air permeability, and water retention. Chitosan is a product of the polysaccharide chitin with some acetyl groups removed. Due to its biodegradability, low toxicity, good biocompatibility, and antibacterial properties, it is widely used in food agriculture, biomedicine, beauty and health care and other fields.

[0003] Patent CN116585522A proposes a physical cross-linking sponge based on chitosan, which can be used for wound hemostasis. However, chitosan is insoluble in water and needs to be added with acid to promote dissolution, and sodium hydroxide is subsequently added for neutralization, which may result in acid or alkali residues. By loading bioactive ingredients, the sponge can be given effects such as cell growth promotion, differentiation, and antibacterial. Patent CN116510064A discloses a chitosan sponge with sea cucumber collagen and anti-inflammatory drugs, which is used for postoperative hemostasis after wisdom tooth surgery, but the introduction of active ingredients involves a multi-step process and two freeze-dryings, and the process flow is more complicated. Therefore, the washing step of removing acid and alkali residues and the multi-step addition of active ingredients are bound to increase production process and cost. In addition, in the existing technology, glutaraldehyde is most commonly used as a cross-linking agent for chitosan sponges, but its toxicity directly limits its application in biomedical applications, and the mechanical strength of the sponge obtained using physical cross-linking may be insufficient.

[0004] Introducing double bonds into chitosan and utilizing light-induced crosslinking offers improved safety. However, prior art methods for double-bond chitosan modification, such as those described in CN115926359A, primarily involve grafting with methacrylic acid and its derivatives. Acrylic acid, formed by the metabolism of acrylic acid derivatives in the body, is highly irritating and allergenic, potentially causing health problems. Furthermore, prior art methods for grafting double-bonded chitosan onto active ingredients often employ a two-step reaction: first, grafting the active ingredient onto the double-bonded chitosan, followed by a crosslinking reaction between the double-bonded chitosan and a crosslinking agent. This avoids competition between the grafting reaction and the crosslinking reaction, which can affect the efficiency of each reaction. However, during the two-step reaction process, some chitosan will self-crosslink to form a gel. As the first grafting reaction proceeds, the viscosity of the system will inevitably increase, resulting in the inability to fully mix with the crosslinker during the second cross-linking reaction, reducing the efficiency of the cross-linking reaction. The cross-linking reaction mainly occurs on the surface, resulting in poor mechanical properties of the final gel. Summary of the Invention

[0005] Based on the above reasons, the present application provides a grafted modified carboxymethyl chitosan cross-linked gel and its preparation method and application, and obtains a freeze-dried sponge material with uniform and fine surface morphology and porous structure through a suitable freezing process.

[0006] The first aspect of the present invention provides a graft-modified carboxymethyl chitosan cross-linked gel, which is obtained by a one-step cross-linking-grafting reaction (Simultaneous Crosslinking Grafted Technology, SCGT) of double-bond-modified carboxymethyl chitosan, thiol-terminated polyethylene glycol, an active ingredient and a photoinitiator under light conditions.

[0007] Preferably, the double-bond modified carboxymethyl chitosan is itaconylated carboxymethyl chitosan. Preferably, itaconic acid or itaconic anhydride is grafted onto carboxymethyl chitosan to form an amide bond to obtain itaconylated carboxymethyl chitosan.

[0008] The one-step method of the present invention is particularly suitable for itaconylated carboxymethyl chitosan. This is because, on the one hand, itaconic acid is a product of cell metabolism, and the use of itaconylated chitosan containing double bonds has better biocompatibility; on the other hand, during the cross-linking reaction with the cross-linking agent thiol-terminated polyethylene glycol, the itaconylated carboxymethyl chitosan has large cross-linking steric hindrance and controllable cross-linking speed. Therefore, the one-step method can simultaneously achieve grafting-cross-linking reactions without affecting the progress and efficiency of the grafting modification reaction and the cross-linking reaction. The active ingredients that undergo the grafting modification reaction can be uniformly dispersed and loaded into the interior of the gel, thereby improving the loading rate of the active ingredients. At the same time, the phenomenon of self-crosslinking of the carboxymethyl chitosan into a gel during the grafting modification process is avoided, and the carboxymethyl chitosan can always be uniformly mixed with the cross-linking agent, so that the cross-linking reaction can occur simultaneously inside and on the surface of the carboxymethyl chitosan matrix material, thereby improving the mechanical properties and biocompatibility of the cross-linked gel product.

[0009] The carboxymethyl chitosan is obtained by carboxymethylation reaction of chitosan, which solves the problem of water solubility of chitosan.

[0010] The double bond substitution degree of the double bond modified carboxymethyl chitosan is preferably greater than 80%, for example 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0011] Preferably, the molecular weight of the double-bond modified carboxymethyl chitosan can be any, for example 3000-30000 Da, such as 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000 Da, etc.

[0012] The carboxymethyl chitosan is selected from N-carboxymethyl chitosan, O-carboxymethyl chitosan or N,O-carboxymethyl chitosan, preferably O-carboxymethyl chitosan, more preferably 6-O-carboxymethyl chitosan.

[0013] The thiol-terminated polyethylene glycol serves as a cross-linking agent and undergoes a thiol-ene click cross-linking reaction with the double-bond-modified carboxymethyl chitosan.

[0014] Preferably, the polyethylene glycol can be a two-arm or multi-arm polyethylene glycol, such as a four-arm or eight-arm polyethylene glycol, and a two-arm polyethylene glycol is further preferred.

[0015] More preferably, the thiol-terminated polyethylene glycol is a dithiol-terminated polyethylene glycol.

[0016] The molecular weight of the bis-thiol terminated polyethylene glycol can be any molecular weight known in the prior art, preferably any value in the range of 400-5000 Da, for example, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 Da, and more preferably any value in the range of 1000-3000 Da.

[0017] Under light conditions, the double bonds in the double-bond modified carboxymethyl chitosan act as cross-linking reaction sites to undergo cross-linking reaction with the cross-linking agent thiol-terminated polyethylene glycol, which can ensure mechanical strength while avoiding the use of glutaraldehyde and improving the biocompatibility of the gel material.

[0018] The active ingredient contains a sulfhydryl group.

[0019] The active ingredient is selected from a compound containing a sulfhydryl group, preferably one or both of a cysteine-containing polypeptide and a sulfhydryl-containing drug (eg, a sulfhydryl-modified drug).

[0020] Further preferably, the polypeptide is one or both of RGD and IKVAV polypeptides.

[0021] Further preferably, the cysteine-containing IKVAV polypeptide sequence is CGSIKVAV (SEQ ID NO. 1); the cysteine-containing RGD polypeptide sequence is CGGRGDS (SEQ ID NO. 2).

[0022] The drug is preferably an antibiotic, such as doxorubicin, penicillin, daunorubicin, and the like.

[0023] The active ingredient is grafted onto the double-bond modified carboxymethyl chitosan through a thiol-ene click reaction between the thiol group and the double bond.

[0024] The photoinitiator includes but is not limited to one or more of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959), riboflavin, riboflavin sodium phosphate, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or eosin Y, and I2959 and / or LAP are further preferred.

[0025] Preferably, the light is ultraviolet light, for example, ultraviolet light with a wavelength of 300-400 nm (preferably 365 nm).

[0026] The second aspect of the present invention provides a method for preparing the above-mentioned graft-modified carboxymethyl chitosan cross-linked gel, the preparation method comprising:

[0027] (1) forming a mixed solution of double-bond modified carboxymethyl chitosan, thiol-terminated polyethylene glycol, active ingredient and photoinitiator;

[0028] (2) subjecting the mixed solution of step (1) to light reaction to obtain graft-modified carboxymethyl chitosan cross-linked gel.

[0029] Preferably, the photoinitiator in step (1) includes but is not limited to one or more of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959), riboflavin, riboflavin sodium phosphate, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or eosin Y.

[0030] The double-bond modified carboxymethyl chitosan is itaconylated carboxymethyl chitosan. Preferably, the double-bond modified carboxymethyl chitosan is obtained by grafting itaconic acid or itaconic anhydride with carboxymethyl chitosan to form an amide bond.

[0031] Preferably, the polyethylene glycol can be a two-arm or multi-arm polyethylene glycol, such as a four-arm or eight-arm polyethylene glycol. Further preferably, the two-arm polyethylene glycol can be a bis-thiol-terminated polyethylene glycol.

[0032] The active ingredient contains a sulfhydryl group. Preferably, the active ingredient is selected from a compound containing a sulfhydryl group, preferably one or both of a cysteine-containing polypeptide and a sulfhydryl-containing drug (eg, a sulfhydryl-modified drug).

[0033] Further preferably, the polypeptide is one or both of RGD and IKVAV polypeptides.

[0034] The drug is preferably an antibiotic, such as doxorubicin, penicillin, daunorubicin, and the like.

[0035] The present invention uses a one-step crosslinking-grafting reaction to produce a grafted, modified carboxymethyl chitosan crosslinked gel. Crosslinking and grafting reactions occur simultaneously within and on the surface of the carboxymethyl chitosan, making the crosslinking and grafting reactions more uniform and effective. This ensures the mechanical properties of the grafted-crosslinked gel product and the grafting efficiency of the active ingredients, achieving effective encapsulation of the active ingredients.

[0036] The active ingredient is grafted onto the double-bond modified carboxymethyl chitosan through a thiol-ene click reaction between the thiol groups and the double bonds. The thiol-terminated polyethylene glycol acts as a crosslinking agent and undergoes a thiol-ene click crosslinking reaction with the double-bond modified carboxymethyl chitosan.

[0037] The mixing temperature is any value between 30-40°C, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40°C.

[0038] The mixing time is any value between 2 and 5 hours, for example, 2, 2.5, 3, 3.5, 4, 4.5 or 5 hours.

[0039] Specifically, a constant temperature shaker is used for uniform mixing, the rotation speed of the constant temperature shaker is 50-200 rpm, and the incubation time in the constant temperature shaker is 2-5 hours.

[0040] Preferably, in step (1), the double-bond modified carboxymethyl chitosan and the photoinitiator are first mixed evenly, and then the thiol-terminated polyethylene glycol and the active ingredient are added, and the mixture is placed in a constant temperature shaker for mixing. The temperature of the constant temperature shaker is 30-40° C., the speed of the constant temperature shaker is 50-200 rpm, and the mixing time is 1-3 h.

[0041] Preferably, in the mixed solution of step (1), the mass concentration of double-bond modified carboxymethyl chitosan is 1-5%, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%, etc.

[0042] The mass concentration of the thiol-terminated polyethylene glycol is 0.1-1%, for example, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1%, etc., preferably 0.5-1%.

[0043] The mass concentration of the active ingredient is 0.01-0.1%, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1%, etc., preferably 0.03-0.05%.

[0044] The mass concentration of the photoinitiator is 0.05-0.2%, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.2%, etc., preferably 0.05-0.1%.

[0045] Preferably, the step (2) further comprises degassing the mixed solution before the light irradiation reaction, which can be vacuum degassing.

[0046] Further preferably, vacuum degassing is carried out at a speed of 5000-20000 rpm, a vacuum degree of 0-30 Pa (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 Pa), and centrifugation for 2-20 min (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0047] Optionally, the degassed mixed solution is poured into a mold for light irradiation reaction.

[0048] Preferably, the illumination in step (2) is ultraviolet illumination, for example, ultraviolet illumination with a wavelength of 300-400 nm (optionally 365 nm).

[0049] Preferably, the light power is 50-500 mW / cm 2 Any value in, for example, 50, 80, 90, 100, 110, 150, 200, 300, 350, 400, 450, 500 mW / cm 2 wait.

[0050] Preferably, the illumination time is 1-15 min, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, preferably any value in the range of 2-10 min.

[0051] The third aspect of the present invention provides a freeze-dried sponge, which is obtained by subjecting the grafted modified carboxymethyl chitosan cross-linked gel described in the first aspect or the grafted modified carboxymethyl chitosan cross-linked gel obtained by the preparation method of the second aspect to low supercooling crystallization treatment.

[0052] For example, the cross-linked gel and the mold are crystallized at a low supercooling temperature to uniformly crystallize the water in the cross-linked gel, and then the water is freeze-dried to finally obtain a freeze-dried sponge containing active ingredients.

[0053] The fourth aspect of the present invention provides a method for preparing a freeze-dried sponge, which comprises subjecting the grafted modified carboxymethyl chitosan cross-linked gel described in the first aspect or the grafted modified carboxymethyl chitosan cross-linked gel obtained by the preparation method of the second aspect to low supercooling crystallization treatment.

[0054] Preferably, the low supercooling crystallization includes at least two sections;

[0055] Crystal growth section: endpoint temperature is 0°C to -10°C, cooling rate is 0.04-0.16°C / min, and holding time is 60-120 min;

[0056] Crystallization fixed section: the end temperature is -10℃ to -20℃, the cooling rate is 0.08-0.2℃ / min, and the holding time is 60-120min.

[0057] Further preferably, before the crystal growth section, the following steps are further included:

[0058] In the equilibrium section, the terminal temperature is 4°C to 20°C, the cooling rate is 0.5-1°C / min, and the holding time is 30-60min.

[0059] Further preferably, after the crystallization fixing section, the method further comprises:

[0060] In the crystallization stable section, the end temperature is -20°C to -50°C, the cooling rate is 0.5-1°C / min, and the holding time is 180-480min;

[0061] and a heating section with an end temperature of -20°C to -5°C and a heating rate of 0.5-1°C / min.

[0062] In one embodiment of the present invention, the low supercooling crystallization includes the following five sections:

[0063] In the equilibrium section, the end temperature is 4°C to 20°C, for example, 20°C, 15°C, 10°C, 6°C, 4°C, etc. The cooling rate is 0.5-1°C / min and the holding time is 30-60min.

[0064] In the crystal growth section, the end temperature is 0°C to -10°C, for example, 0°C, -5°C, -10°C, etc., the cooling rate is 0.04-0.16°C / min, and the holding time is 60-120 min;

[0065] The crystallization fixed section has an endpoint temperature of -10°C (not included) to -20°C, such as -12°C, -15°C, -20°C, etc. The cooling rate is 0.08-0.2°C / min and the holding time is 60-120min;

[0066] In the crystallization stable section, the end temperature is -20°C (not included) to -50°C, such as -25°C, -30°C, -40°C, -50°C, etc., the cooling rate is 0.5-1°C / min, and the holding time is 180-480min;

[0067] In the heating section, the end temperature is -20°C to -5°C, for example, -20°C, -15°C, -10°C, -5°C, etc., and the heating rate is 0.5-1°C / min.

[0068] Preferably, the cooling rate of the crystallization growth section is lower than the cooling rate of the crystallization fixing section. This is because the crystallization growth section determines the size of the ice crystals generated. Therefore, the cooling rate needs to be strictly controlled. The cooling rate is 0.04-0.16℃ / min, which can control the uniform growth of the ice crystal nuclei at the same time and keep the size small. The ice crystals are fully fixed by the crystallization fixing section, and uniform, fine, stable, and size-controlled ice crystals can be formed, thereby improving the uniformity of the pore size of the final freeze-dried sponge product.

[0069] Preferably, the low supercooling crystallization further includes vacuum treatment, specifically, performing sublimation and desorption steps under vacuum conditions to obtain a freeze-dried sponge.

[0070] Further preferably, the vacuum degree is 0-30 Pa (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 Pa).

[0071] Further preferably, the sublimation treatment is carried out at the terminal temperature condition of the heating section, and the sublimation treatment time is 10-30h (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30h).

[0072] Further optionally, the parsing temperature is 0°C to 30°C (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30°C), the parsing time is 3-20h (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20h), and the heating rate is 2-5°C / min.

[0073] The fifth aspect of the present invention provides a secondary cross-linked gel, comprising irradiating the graft modified carboxymethyl chitosan cross-linked gel of the first aspect or the graft modified carboxymethyl chitosan cross-linked gel obtained by the preparation method of the second aspect to obtain a secondary cross-linked gel.

[0074] The cross-linked gel can withstand irradiation sterilization, and after secondary cross-linking by irradiation, the cross-linked gel has a denser network structure, thereby enhancing mechanical properties.

[0075] The irradiation uses gamma rays, X rays or beta rays.

[0076] The irradiation dose is 10-30 kGy, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 kGy.

[0077] The sixth aspect of the present invention provides a secondary cross-linked freeze-dried sponge, comprising irradiating the freeze-dried sponge of the third aspect or the freeze-dried sponge obtained according to the preparation method of the fourth aspect to obtain a secondary cross-linked freeze-dried sponge.

[0078] The irradiation uses gamma rays, X rays or beta rays.

[0079] The irradiation dose is 10-30 kGy, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 kGy.

[0080] The present invention can control the addition amount of active ingredients, double-bond modified carboxymethyl chitosan, cross-linking agent thiol-terminated polyethylene glycol, appropriate amount of photoinitiator and other raw materials. During the grafting-cross-linking reaction process, cross-linking reaction and grafting reaction occur simultaneously inside and on the surface of the carboxymethyl chitosan, and the cross-linking and grafting reactions are more uniform and effective. While ensuring the performance of the grafted-cross-linked gel product, various properties of the cross-linked gel or the final freeze-dried sponge can be accurately regulated.

[0081] Furthermore, the reasonable dosage of each component of the raw material can also maintain the molar ratio of thiol to double bond in the active ingredient within a certain range (such as 4%-18%). Therefore, there are "residual" double bonds in the cross-linked gel or freeze-dried sponge material. The residual double bonds enable the cross-linked gel or freeze-dried sponge to not only undergo a secondary cross-linking reaction during the irradiation sterilization process, but also quench the free radicals in the system, maintain the stability of the cross-linked gel or freeze-dried sponge skeleton, and thus obtain a cross-linked gel product or freeze-dried sponge with better mechanical properties.

[0082] A seventh aspect of the present invention provides the use of the cross-linked gel, the cross-linked gel obtained by the above-mentioned preparation method, the above-mentioned secondary cross-linked gel, the above-mentioned freeze-dried sponge, the freeze-dried sponge obtained by the above-mentioned preparation method, or the above-mentioned secondary cross-linked freeze-dried sponge in preparing tissue filling and repair materials or drug carriers. For example, the cross-linked gel can be used to prepare products for pharmaceutical, medical beauty, and cosmetic applications, such as soft tissue filling and repair.

[0083] The "crosslinking-grafting" mentioned in the present invention means that a crosslinking reaction and a grafting reaction are carried out simultaneously, for example, grafting is carried out during the crosslinking process, or crosslinking is carried out during the grafting process.

[0084] The "one-step method" described in the present invention means a one-step process from liquid to gel, i.e., the reactants are mixed (the reactants can be added simultaneously or separately), and then reacted simultaneously, so that the originally liquid mixture undergoes a reaction (such as cross-linking-grafting) to obtain a gel.

[0085] The "subcooling degree" mentioned in the present invention refers to the difference between the actual temperature of the condensate under certain pressure conditions and the theoretical crystallization temperature under the pressure.

[0086] Beneficial effects of the present invention:

[0087] (1) In the present invention, the active ingredient is connected to the itaconylated carboxymethyl chitosan main body and the thiol-terminated polyethylene glycol by a one-step crosslinking-grafting reaction (Simultaneous Crosslinking Grafted Technology, SCGT). The active ingredient forms a chemical bond with the itaconylated carboxymethyl chitosan and can be uniformly dispersed and loaded into the gel as the one-step crosslinking-grafting reaction proceeds. This not only avoids the problem of low mechanical strength of the gel caused by uneven mixing of the system in the two-step method, but also simplifies the active ingredient modification steps and ensures a higher active ingredient loading rate.

[0088] (2) The present invention incorporates thiol-terminated polyethylene glycol as a crosslinking agent. This allows the itaconylated carboxymethyl chitosan to fully contact and mix with the crosslinking agent within the crosslinked itaconylated carboxymethyl chitosan system, thereby improving the efficiency of the crosslinking reaction, reducing the amount of photoinitiator used, shortening the crosslinking time, and improving the mechanical properties of the crosslinked gel, as well as the porosity and mechanical strength of the freeze-dried sponge. Furthermore, the thiol-terminated polyethylene glycol can be metabolized in vivo into non-toxic products, thus exhibiting good biocompatibility.

[0089] (3) The present invention can achieve precise control of the various properties of the final freeze-dried sponge by controlling the ratio of itaconylated carboxymethyl chitosan, thiol-terminated polyethylene glycol, and photoinitiator and the photocrosslinking time; through low supercooling crystallization treatment, by controlling the cooling rate of the crystallization growth section and the crystallization fixed section, the cooling rate of the crystallization growth section is made lower than the cooling rate of the crystallization fixed section, the size and uniformity of ice crystal formation are optimized, and finally a freeze-dried sponge material with uniform surface morphology and dense pores can be obtained; the freeze-dried sponge prepared by the present invention has good liquid absorption and liquid retention capabilities, and by adding different active ingredients such as bioactive polypeptides, the freeze-dried sponge also has antibacterial and epithelial cell differentiation and migration promoting capabilities.

[0090] (4) The grafted modified carboxymethyl chitosan cross-linked gel or freeze-dried sponge prepared by the present invention can withstand irradiation sterilization and has good stability. Due to the presence of "residual" double bonds in the cross-linked gel or freeze-dried sponge material, it can not only undergo secondary cross-linking during the irradiation sterilization process, but also quench the free radicals in the system, thereby maintaining the stability of the skeleton of the cross-linked gel or freeze-dried sponge material and having more excellent mechanical properties. Therefore, the cross-linked gel or freeze-dried sponge can be directly used as a medical device.

[0091] (5) The cross-linked gel or freeze-dried sponge provided by the present invention has excellent mechanical properties, stability and the effect of promoting cell growth, which improves its ease of use in the fields of soft tissue filling, soft tissue repair, medical beauty, etc. and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0093] FIG1 is a schematic diagram of the cross-linking-grafting reaction between itaconylated carboxymethyl chitosan and thiol-containing polypeptides and dithiol-terminated polyethylene glycol.

[0094] FIG2 is an appearance diagram of the samples of Comparative Example 3 (A) and Example 6 (B) before vacuum treatment.

[0095] FIG3 is a diagram showing the appearance of the samples of Comparative Example 3 (A) and Example 6 (B) after vacuum treatment.

[0096] FIG4 is a SEM scan of the surface morphology of the samples of Comparative Example 3 (A) and Example 6 (B) after vacuum treatment.

[0097] FIG5 is an appearance diagram of the product obtained in step 3) of Comparative Example 1. DETAILED DESCRIPTION

[0098] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. The embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0099] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0100] The peptides IKVAV and RGD were purchased from Nanjing Peptide Biotechnology Co., Ltd. and prepared using conventional solid-phase peptide synthesis methods.

[0101] Peptide information: Cysteine-containing IKVAV polypeptide sequence CGSIKVAV (SEQ ID NO. 1); Cysteine-containing RGD polypeptide sequence CGGRGDS (SEQ ID NO. 2).

[0102] Itaconic acid-modified carboxymethyl chitosan is obtained by grafting itaconic acid or itaconic anhydride with carboxymethyl chitosan. For example, the preparation method can be referred to patent CN202311845241.1.

[0103] Example 1:

[0104] (1) Weigh 5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959) and dissolve it in 5 mL of purified water to prepare a 0.1% solution.

[0105] 125 mg of itaconylated carboxymethyl chitosan (double bond substitution degree 88%) was weighed and added to the prepared I2959 solution, and then placed in a constant temperature shaker at 37°C and 150 rpm for incubation for 3 h to obtain a 2.5% itaconylated carboxymethyl chitosan solution.

[0106] 25 mg of bis-thiol-terminated polyethylene glycol (molecular weight 2 kDa) and 2 mg of cysteine-containing IKVAV peptide (SEQ ID NO. 1) were weighed and added to the above-mentioned itaconylated carboxymethyl chitosan solution. The mixture was placed in a constant temperature shaker and incubated for 2 h to obtain a mixed solution (the contents of itaconylated carboxymethyl chitosan, bis-thiol-terminated polyethylene glycol, I2959, and cysteine-containing IKVAV peptide were 2.5%, 0.5%, 0.1%, and 0.04%, respectively).

[0107] (2) Use a vacuum centrifuge to degas the mixed solution at a speed of 10,000 rpm, a vacuum degree of 10 Pa, and centrifuge for 10 minutes. Then pour the mixed solution into a polytetrafluoroethylene mold, shake it left and right to fill the mold, and place it under a UV lamp (365 nm) for 10 minutes at a power of 100 mW / cm 2 , and obtain graft-modified carboxymethyl chitosan cross-linked gel.

[0108] The schematic diagram of the cross-linking-grafting reaction between itaconylated carboxymethyl chitosan, a cross-linking agent, and a thiol-containing polypeptide is shown in FIG1 .

[0109] Example 2:

[0110] The contents of itaconylated carboxymethyl chitosan, bis-thiol terminated polyethylene glycol, I2959, and cysteine-containing polypeptide IKVAV were adjusted to 2.5%, 0.5%, 0.05%, and 0.04%, respectively. The remaining formula and steps were consistent with those in Example 1.

[0111] Example 3:

[0112] The contents of itaconylated carboxymethyl chitosan, bis-thiol terminated polyethylene glycol, I2959, and cysteine-containing polypeptide IKVAV were adjusted to 1%, 0.5%, 0.05%, and 0.04%, respectively. The remaining formula and steps were consistent with those in Example 1.

[0113] Example 4:

[0114] The contents of itaconylated carboxymethyl chitosan, bis-thiol terminated polyethylene glycol, I2959, and cysteine-containing polypeptide IKVAV were adjusted to 5%, 0.5%, 0.05%, and 0.04%, respectively. The remaining formula and steps were consistent with those in Example 1.

[0115] Example 5:

[0116] The contents of itaconylated carboxymethyl chitosan, bis-thiol terminated polyethylene glycol, I2959, and cysteine-containing polypeptide IKVAV were adjusted to 2.5%, 1%, 0.05%, and 0.04%, respectively. The remaining formula and steps were consistent with those in Example 1.

[0117] Example 6:

[0118] The cross-linked gel obtained in Example 1 was subjected to low supercooling crystallization according to the following steps:

[0119] Place in freeze dryer and set program:

[0120] Equilibrium section: endpoint temperature 4°C, cooling rate 1°C / min, holding time 30 min;

[0121] Crystal growth section: end temperature -10 °C, cooling rate 0.08 °C / min, holding time 120 min;

[0122] Crystallization fixed section: end temperature -20 °C, cooling rate 0.16 °C / min, holding time 60 min;

[0123] Crystallization stable section: end point temperature -40 °C, cooling rate 1 °C / min, holding time 300 min;

[0124] Heating section: end temperature -20°C, heating rate 0.5°C / min;

[0125] After low supercooling crystallization, turn on the vacuum pump of the vacuum freeze dryer, maintain the vacuum degree of the sample chamber at 10Pa, sublimate at -20℃ for 18h, then continue to heat to 20℃ (heating rate of 2℃ / min), continue to decompose for 8h, and obtain freeze-dried sponge.

[0126] Example 7:

[0127] The cross-linked gel obtained in Example 2 was subjected to low supercooling crystallization according to the following steps:

[0128] Place in freeze dryer and set program:

[0129] Equilibrium section: end temperature 15°C, cooling rate 1°C / min, holding time 30 min;

[0130] Crystal growth section: end temperature -5°C, cooling rate 0.12°C / min, holding time 80 min;

[0131] Crystallization fixed section: end temperature -15 °C, cooling rate 0.2 °C / min, holding time 100 min;

[0132] Crystallization stable section: end temperature -30 °C, cooling rate 1 °C / min, holding time 300 min;

[0133] Heating section: end temperature -10°C, heating rate 0.5°C / min;

[0134] After low supercooling crystallization, turn on the vacuum pump of the vacuum freeze dryer, maintain the vacuum degree of the sample chamber at 10Pa, sublimate at -10℃ for 18h, then continue to heat to 20℃ (heating rate of 2℃ / min), and continue to decompose for 8h to obtain freeze-dried sponge.

[0135] Example 8:

[0136] The freeze-dried sponge obtained in Example 7 was irradiated with gamma rays at a dose of 25 kGy.

[0137] Example 9:

[0138] The cysteine-containing polypeptide IKVAV was replaced with the cysteine-containing polypeptide RGD (SEQ ID NO. 2). Specifically, the contents of itaconylated carboxymethyl chitosan, bisthiol-terminated polyethylene glycol, I2959, and the cysteine-containing polypeptide RGD were 2.5%, 0.5%, 0.05%, and 0.04%, respectively. A cross-linked gel was prepared according to the steps of Example 1, and low supercooling crystallization was performed according to the steps of Example 6 to obtain a freeze-dried sponge.

[0139] Example 10:

[0140] To investigate the drug loading capacity of the sponge, a thiol-modified doxorubicin (HS-DOX, Xi'an Qiyue Biotechnology Co., Ltd.) was used as an example. Doxorubicin exhibits a strong UV absorption peak at 480 nm, facilitating release measurement. The contents of itaconylated carboxymethyl chitosan, bis-thiol-terminated polyethylene glycol, I2959, and HS-DOX were adjusted to 2.5%, 0.5%, 0.05%, and 0.04%, respectively. A cross-linked gel was then prepared according to the procedures of Example 1, and low-supercooling crystallization was performed according to the procedures of Example 6 to obtain a freeze-dried sponge.

[0141] Comparative Example 1:

[0142] 1) Weigh 5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959) and dissolve it in 5 mL of purified water to prepare a 0.1% solution.

[0143] 2) 125 mg of itaconylated carboxymethyl chitosan (double bond substitution degree 88%) and 2 mg of cysteine-containing IKVAV peptide were weighed and added to the I2959 solution prepared in step 1), and then incubated in a thermostatic shaker at 37°C, 150 rpm for 3 h to obtain a reaction product, wherein the contents of itaconylated carboxymethyl chitosan, I2959, and cysteine-containing IKVAV peptide were 2.5%, 0.1%, and 0.04%, respectively.

[0144] 3) Place the product of step 2) under ultraviolet light for 5 minutes at a power of 100 mW / cm 2 After the reaction, the product will be cross-linked and in a semi-gel state as shown in FIG5 .

[0145] 4) Add 25 mg of bis-thiol-terminated polyethylene glycol (molecular weight 2 kDa) to the light-irradiated product of step 3), place in a constant temperature shaker and continue incubation for 2 h. Use a vacuum centrifuge for degassing at a speed of 10,000 rpm and a vacuum degree of 10 Pa for 10 min. After degassing, place under ultraviolet light for 5 min (power 100 mW / cm 2 ).

[0146] Comparative Example 2:

[0147] The cross-linked gel was prepared by the two-step method of Comparative Example 1, and low supercooling crystallization was performed according to the steps of Example 6 to finally obtain a freeze-dried sponge.

[0148] Comparative Example 3:

[0149] The cross-linked gel obtained in Example 1 was taken and a freeze-dried sponge was prepared using a conventional freeze-drying process.

[0150] The freeze-drying process is as follows: the cross-linked gel is directly placed at -20 ° C and frozen for 300 minutes; then the vacuum pump of the vacuum freeze dryer is turned on, the vacuum degree of the sample chamber is maintained at 10Pa, sublimated at -20 ° C for 18 hours, and then heated to 20 ° C (heating rate is 2 ° C / min), and continued to decompose for 8 hours to obtain a freeze-dried sponge.

[0151] Comparative Example 4:

[0152] In step (1), no dithiol-terminated polyethylene glycol (HS-PEG-HS) was added, and the rest was the same as in Example 1.

[0153] Test Example 1: Gelation Effect

[0154] The products of Example 1 and Comparative Example 4 were taken and the gel formation was observed. The results are shown in Table 1.

[0155] Table 1 Gelation performance

[0156] As can be seen from Table 1, in Example 1, the addition of the crosslinker HS-PEG-HS allowed for a simultaneous crosslinking reaction during the grafting modification reaction, resulting in rapid gelation. In contrast, in Comparative Example 4, where no crosslinker HS-PEG-HS was added and only the active polypeptide was grafted onto itaconylated carboxymethyl chitosan, the double bond reactivity was relatively low. Under the same illumination conditions (10 minutes of UV illumination), only partial gelation was achieved, and a three-dimensional network structure could not be fully formed. Therefore, the mechanical strength of the product could not be guaranteed (see the elastic modulus value in Test 2).

[0157] Test Example 2: Mechanical Strength of Cross-linked Gel

[0158] The cross-linked gels obtained in Examples 1-5 and Comparative Examples 1 and 4 were thoroughly broken up by pushing two 5 mL syringes against each other 200 times. The storage modulus G' and loss modulus G" of the different cross-linked gels were then measured using a rheometer. A larger storage modulus G' indicated higher mechanical strength of the gel sample. The results are recorded in Table 2.

[0159] Table 2 Storage modulus G' and loss modulus G" of cross-linked gels with different formulations

[0160] The data in Table 2 show that the content of itaconylated carboxymethyl chitosan, bis-mercapto-terminated polyethylene glycol, and I2959 are all positively correlated with the mechanical properties of the cross-linked gel. Specifically, as can be seen from Example 1 and Comparative Example 4, the mechanical strength of the cross-linked gel increases with the addition of bis-mercapto-terminated polyethylene glycol. The data from Examples 2 and 5 show that the strength of the cross-linked gel can be rationally controlled by adjusting the ratio of bis-mercapto-terminated polyethylene glycol. The data from Examples 3 and 4 show that the mechanical properties of the cross-linked gel samples also improve with increasing the amount of itaconylated carboxymethyl chitosan added. Similarly, the data from Examples 1 and 2 show that with increasing the amount of photoinitiator used, the degree of photocrosslinking increases, thereby improving the strength of the cross-linked gel.

[0161] Comparing the data from Example 1 and Comparative Example 1, the cross-linked gel sample prepared using a two-step method in Comparative Example 1 exhibits significantly reduced mechanical strength. This is because the system partially solidifies during the first grafting modification reaction, preventing the dithiol-terminated polyethylene glycol from fully mixing within the system during the second cross-linking step. The cross-linking agent can only disperse on the surface, resulting in a relatively low cross-linking reaction efficiency and a significant impact on the final gel strength of the gel product. In contrast, the cross-linked gel prepared using a one-step method in Example 1 exhibits a higher modulus and better mechanical properties. This is because the grafting-cross-linking reaction can occur simultaneously on the surface and within the itaconylated carboxymethyl chitosan, resulting in a higher cross-linking reaction efficiency and better mechanical properties for the prepared gel product. Comparative Example 4, however, lacks a cross-linking agent and only partially forms a gel, resulting in significantly reduced mechanical properties.

[0162] Test Example 3

[0163] The appearance comparison of the freeze-dried sponges of Example 6 and Comparative Example 3 before vacuum treatment is shown in Figure 2, the appearance comparison of the freeze-dried sponges after vacuum treatment is shown in Figure 3, and the SEM comparison is shown in Figure 4.

[0164] As can be seen from Figure 2, Comparative Example 3, prepared using a traditional freezing process, failed to control the cooling rate during the freezing process, resulting in anisotropic growth of a large number of crystal nuclei, excessively rapid ice crystal formation, and irregular nuclei growth, forming uneven crystal blocks or sheets (such as the circular area in Figure 2A). In contrast, Example 6, which used low supercooling crystallization, had an overall smooth and flat sponge surface. This was primarily due to the ability to control the cooling rate in the crystallization growth section, thereby controlling the growth of the ice crystal nuclei, forming uniform and controllable ice crystals, and fully securing the ice crystals in the crystallization fixing section.

[0165] Furthermore, it can be seen from Figure 3 that the surface morphology of the freeze-dried sponge sample of Comparative Example 3 after vacuum treatment is uneven, there are a large number of irregular areas, the solids are unevenly distributed, and the transmittance is inconsistent; while the freeze-dried sponge of Example 6 of the present application has a good morphology, is milky white as a whole, and has uniform thickness; further, it can be seen from the SEM Figure 4 of the freeze-dried sponge that the sponge structure of Comparative Example 3 is almost collapsed, and there is no complete and regular pore structure, which is also the reason for its uneven morphology and uneven solid distribution, which will eventually lead to poor water absorption and mechanical properties of the sponge; while the freeze-dried sponge of Example 6 of the present application has a rich pore structure and uniform pore size. This is because the low supercooling crystallization treatment is adopted, and the cooling rate of the gradient freezing is precisely controlled, so that the ice crystals are more uniform, controllable, and smaller in size, and the pore size of the final freeze-dried sponge product is uniform.

[0166] Test Example 4: Sponge Porosity and Water Absorption Performance Testing

[0167] Swelling Rate Test: Cut the freeze-dried sponge samples prepared in Example 6, Comparative Examples 2, and 3 into 1×1 cm pieces, accurately weigh them, and record the initial mass of the dry sponge. Place each piece in a 50 mL centrifuge tube, add 10 mL of 0.9% sodium chloride injection, and place them in a 37°C constant temperature oscillator for 5 hours. Then, use tweezers to remove the absorbed sponge, let it drain for 30 seconds, and then accurately weigh and record the weight. After weighing, place the sponge back in the tube. The swelling rate is calculated as follows:

[0168] Among them, m1 is the mass of the absorbent sponge, and m0 is the initial mass of the dry sponge.

[0169] Water retention rate test: Take the sponge sample after the swelling rate test, place it in a dry centrifuge tube, centrifuge it at 500 rpm for 3 minutes, then take out the sponge and weigh it. The water retention rate is calculated according to the following formula:

[0170] Where m2 is the mass of the sponge after centrifugation, and m0 is the initial mass of the dry sponge.

[0171] Porosity test: Cut the freeze-dried sponge samples prepared in Example 6, Comparative Examples 2 and 3 into 1×1 cm small pieces. Note that they need to be cut into standard rectangles. Use a vernier caliper to measure the length, width and thickness of the sponge, where the thickness is measured at 6 different thicknesses on the sponge, take the average value, and calculate the volume of the sponge. Record the initial mass of the dry sponge after accurate weighing. Place them in 50 mL centrifuge tubes, add 10 mL of anhydrous ethanol, and let them stand in a constant temperature oscillator at 37°C. After 5 hours, take out the sponge and drain it naturally for 30 seconds, and weigh the mass of the sponge after absorbing ethanol. The porosity is calculated as follows:

[0172] Where m0 and m1 are the masses of the sponge before and after absorbing anhydrous ethanol, v is the volume of the sponge, and ρ is the density of anhydrous ethanol, which is 0.77641 g / cm at 37 °C. 3 The swelling rate, water retention rate and porosity of the freeze-dried sponge samples prepared in different embodiments and comparative examples are shown in Table 3.

[0173] Table 3 Sponge properties of different embodiments and comparative examples

[0174] The freeze-dried sponge obtained by the grafting-crosslinking one-step method in Example 6 is more uniform and has a high cross-linking efficiency because the cross-linking reaction occurs inside and on the surface of the carboxymethyl chitosan. Its swelling rate and water retention rate are high, and the porosity can reach 71.3%, which is significantly higher than the freeze-dried sponge obtained by the two-step method in Comparative Example 2. The swelling rate, water retention rate, and porosity of the freeze-dried sponge obtained in Comparative Example 3 are significantly reduced. This can also be seen intuitively from the SEM image of Figure 4. The sponge structure of Comparative Example 3 almost collapses, and there is no complete and regular pore structure, which ultimately leads to a significant reduction in the sponge swelling rate, water retention rate, and porosity. This also shows that the low supercooling crystallization process has an important influence on the pore structure of the freeze-dried sponge.

[0175] Test Example 5: Effect of irradiation sterilization on sponge performance

[0176] The changes in swelling rate, water retention rate and porosity of the freeze-dried sponges obtained in Examples 7-8 before and after irradiation were calculated, as shown in Table 4.

[0177] Table 4 Changes in sponge properties before and after irradiation

[0178] The experimental results show that after irradiation treatment, the swelling rate and water retention rate of the sponge are reduced to a certain extent. This is because the present invention controls the amount and ratio of the active polypeptide and the double-bond modified chitosan, so that there are certain double bonds remaining in the final product. The cross-linking reaction can continue to occur during the irradiation process, and the sponge structure is denser, so the swelling rate and water retention rate are reduced.

[0179] Test Example 6: Active ingredient release test of sponge

[0180] Preparation of blank freeze-dried sponge: The other steps were the same as those in Example 6, except that no active ingredient was added.

[0181] BCA method for detecting polypeptide release: blank freeze-dried sponge, freeze-dried sponge containing active ingredient (Examples 6, 9, 10), and polypeptide-containing sponge prepared by two-step method (Comparative Example 2). Weigh the initial weight of each group of sponges, cut off about two-thirds and accurately weigh and record, cut into small pieces of about 1×1 cm, place them in three 50mL centrifuge tubes, add 10mL of 10mM PBS (pH=7.35) respectively, shake at 20℃ and 80rad / min for 24h, take 20μL of sample solution from each well and place it in a 96-well plate for BCA detection, and detect 5 parallel values ​​for each sample. After the test is completed, continue the reaction for 48h, take a sample and repeat the detection operation. After measuring the absorbance value, use the blank freeze-dried sponge as a control to deduct the absorbance, and calculate the amount of active ingredient released according to the standard curve prepared previously.

[0182] Table 5 Active ingredient release test of sponge

[0183] As can be seen from Table 5, the active ingredient encapsulation strategy designed by the present invention can increase the effective load of the active ingredient. The sponge products obtained by the one-step graft modification-crosslinking reaction have a polypeptide release rate of less than 25% in Examples 6 and 9, whether in 24 hours or 48 hours, indicating that more than 75% of the polypeptide can be stably encapsulated in the sponge, and the polypeptide loading efficiency is high. For the antibiotic active ingredient in Example 10, the 48h release rate is only 25.3%, which also has good loading efficiency. This is because the one-step method can simultaneously achieve graft modification-crosslinking reaction, without affecting the progress of the graft modification reaction and the crosslinking reaction, and the active ingredient of the graft modification reaction can be evenly dispersed and loaded into the interior of the gel, thereby increasing the loading rate of the active ingredient. At the same time, the one-step method can be evenly mixed with the crosslinking agent, so that the crosslinking reaction can occur simultaneously inside and on the surface of the carboxymethyl chitosan matrix material. The crosslinking reaction strength is high, and the sponge is not easy to disintegrate or collapse when immersed in water. The active ingredient can be stably loaded in the carboxymethyl chitosan matrix, so the release rate is low.

[0184] However, for the sponge sample of Comparative Example 2 prepared by the two-step method, the polypeptide release rate was close to 50% after 48 hours, indicating that the sponge had a poor ability to load active polypeptides. This was because during the two-step reaction, the reaction activity of the itaconic acid double bond was low, the polypeptide grafting ability was limited, the grafting reaction efficiency was low, and the solution system had partially gelled in the first grafting reaction, resulting in the cross-linking agent being unable to mix evenly with the system, causing the cross-linking reaction to occur only on the surface of the carboxymethyl chitosan. The final cross-linking strength was low, and the sponge disintegrated faster when immersed in water, making it impossible to effectively load the active ingredient.

[0185] Test Example 7: Cell proliferation assay using sponges containing IKVAV or RGD polypeptides

[0186] Cell preparation: Take frozen L929 mouse fibroblasts within 5 generations, continue to pass for 2-3 generations after thawing, and conduct experiments after the cells grow well (use 1640 complete medium to culture cells: RPMI1640 medium + 10% FBS + 1% P / S).

[0187] Sample preparation and cell inoculation: The blank freeze-dried sponge prepared in Test Example 6, the sponge containing 0.04% IKVAV or RGD (Example 6, Example 9), and the freeze-dried sponge prepared by the two-step method (Comparative Example 2) were cut into 1×1 cm squares, sterilized by 25 kGy irradiation, and placed in the bottom of a 24-well plate. Five replicates were prepared for each group. L929 cells grown to 80% confluence were taken, digested, centrifuged, and diluted to 10 μg / mL using RPMI1640 complete medium. 7 After that, 150ul of cell suspension (1.5×10 6Carefully drip the suspension onto the dry sample surface and let it sit for 5 minutes to allow the dry sample to fully absorb the cell suspension. Add 850 μl of 1640 complete medium close to the well wall. Then, place the well plate in a 37°C incubator for incubation.

[0188] Cell proliferation detection: After the sample inoculated with cells has been cultured for 48 hours, take a new 24-well plate and transfer the samples one by one to the new wells. Prepare serum-free RPMI1640 culture medium containing 10% CCK-8 reagent (Biyuntian, product number C0038), add 1 ml of the above culture medium to each well, shake gently, return to the incubator and culture for 1.5 hours, take out the well plate and shake it for 3 minutes every 20 minutes. Take it out after 1.5 hours, shake it thoroughly, take 100 μl of the supernatant and transfer it to a 96-well plate, 3 parallels for each sample, and then measure the absorbance at 450 nm with a microplate reader. At the same time, measure the absorbance of 100 μl serum-free RPMI1640 + 10% CCK8 solution as background subtraction. The final relative cell viability of each group is the average of five parallels.

[0189] Table 6 Cell proliferation assay containing IKVAV or RGD polypeptide sponges

[0190] Using a blank freeze-dried sponge containing no active ingredient as a benchmark, the test found that after adding IKVAV or RGD peptide, the freeze-dried sponge samples of Example 6 (containing 0.04% IKVAV) and Example 9 (containing 0.04% RGD) showed a stronger cell proliferation rate, and the relative cell viability reached almost 150%. The cell proliferation rate of the freeze-dried sponge of Comparative Example 2 was only 112.8%, which was significantly lower than the freeze-dried sponge prepared by the one-step method in Example 6 (144.3%). See Table 6, indicating that the freeze-dried sponge designed by the present invention can effectively load the cell growth-promoting polypeptide and release it during cell growth, thereby promoting cell proliferation on the freeze-dried sponge.

[0191] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A graft-modified carboxymethyl chitosan cross-linked gel, characterized in that: The graft-modified carboxymethyl chitosan cross-linked gel is obtained by a one-step cross-linking-grafting reaction of double-bond-modified carboxymethyl chitosan, thiol-terminated polyethylene glycol, active ingredients and a photoinitiator under light conditions.

2. The graft-modified carboxymethyl chitosan cross-linked gel according to claim 1, characterized in that: The active ingredient is selected from one or both of a polypeptide containing cysteine and a drug containing a sulfhydryl group; The polypeptide is preferably one or both of RGD and IKVAV polypeptides; and the drug is preferably an antibiotic.

3. The graft-modified carboxymethyl chitosan cross-linked gel according to claim 1, characterized in that: The double-bond modified carboxymethyl chitosan is itaconylated carboxymethyl chitosan; The thiol-terminated polyethylene glycol is a dithiol-terminated polyethylene glycol; The photoinitiator is selected from one or more of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959), riboflavin, riboflavin sodium phosphate, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or eosin Y; preferably I2959 and / or LAP.

4. A method for preparing the graft-modified carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) forming a mixed solution of double-bond modified carboxymethyl chitosan, thiol-terminated polyethylene glycol, active ingredient and photoinitiator; (2) subjecting the mixed solution of step (1) to light reaction to obtain graft-modified carboxymethyl chitosan cross-linked gel.

5. The preparation method according to claim 4, characterized in that In the mixed solution of step (1), the mass concentration of double-bond modified carboxymethyl chitosan is 1-5%; the mass concentration of thiol-terminated polyethylene glycol is 0.1-1%, preferably 0.5-1%; the mass concentration of active ingredient is 0.01-0.1%; and the mass concentration of photoinitiator is 0.05-0.2%, preferably 0.05-0.1%.

6. The preparation method according to claim 4 or 5, characterized in that The mixing temperature in step (1) is 30-40° C. and the mixing time is 2-5 h; The illumination in step (2) is ultraviolet illumination, and the illumination power is 50-500mW / cm 2 ; The illumination time is 1-15min.

7. A freeze-dried sponge, characterized in that: The freeze-dried sponge is obtained by subjecting the graft-modified carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 3 or the graft-modified carboxymethyl chitosan cross-linked gel obtained by the preparation method according to any one of claims 4 to 6 to low supercooling crystallization treatment.

8. A method for preparing the freeze-dried sponge according to claim 7, characterized in that: The low supercooling crystallization includes at least two sections; In the crystal growth section, the end temperature is 0°C to -10°C, the cooling rate is 0.04-0.16°C / min, and the holding time is 60-120min; In the crystallization fixed section, the end temperature is -10°C to -20°C, the cooling rate is 0.08-0.2°C / min, and the holding time is 60-120min; Preferably, the cooling rate of the crystal growth zone is lower than the cooling rate of the crystal fixing zone.

9. The preparation method according to claim 8, characterized in that The low supercooling crystallization comprises: Before the crystal growth section, it also includes: In the equilibrium section, the terminal temperature is 4°C to 20°C, the cooling rate is 0.5-1°C / min, and the holding time is 30-60min; After the crystallization fixing section, it also includes: In the crystallization stable section, the end temperature is -20°C to -50°C, the cooling rate is 0.5-1°C / min, and the holding time is 180-480min; and a heating section with an end temperature of -20°C to -5°C and a heating rate of 0.5-1°C / min.

10. A secondary cross-linked gel, characterized in that: The secondary cross-linked gel comprises irradiating the grafted modified carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 3 or the grafted modified carboxymethyl chitosan cross-linked gel obtained by the preparation method according to any one of claims 4 to 6 to obtain a secondary cross-linked gel; preferably, the irradiation uses γ rays, X rays or β rays, and the irradiation dose is 10-30 kGy.

11. A secondary cross-linked freeze-dried sponge, characterized in that: The secondary cross-linked freeze-dried sponge comprises irradiating the freeze-dried sponge according to claim 7 or the freeze-dried sponge obtained by the preparation method according to any one of claims 8 to 9 to obtain a secondary cross-linked freeze-dried sponge; preferably, the irradiation uses γ rays, X rays or β rays, and the irradiation dose is 10-30 kGy.

12. Use of the graft-modified carboxymethyl chitosan cross-linked gel according to any one of claims 1 to 3, the graft-modified carboxymethyl chitosan cross-linked gel obtained by the preparation method according to any one of claims 4 to 6, the freeze-dried sponge according to claim 7, the freeze-dried sponge obtained by the preparation method according to any one of claims 8 to 9, the secondary cross-linked gel according to claim 10, or the secondary freeze-dried sponge according to claim 11 in the preparation of tissue filling and repair materials or drug carriers.

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