A kind of temperature-sensitive modified chitosan, preparation method and application

Abstract

The present invention relates to the technical field of biomedical hydrogel materials, and particularly relates to a temperature-sensitive modified chitin, a preparation method and an application thereof. The temperature-sensitive modified chitin is characterized in that the chitin molecular chain contains substituent A and substituent B; the structural formula of the substituent A is formula (I-2), where m is any integer from 0 to 100; the substitution degree of the substituent A is 0 to 35%; the temperature-sensitive modified chitin uses any one of temperature-sensitive hydroxypropyl chitin, temperature-sensitive hydroxyethyl chitin, and temperature-sensitive hydroxybutyl chitin as the starting material, and uses compound 1-(oxirane-2-yl)-(PEG) m -carboxylic acid as the substitution group for synthesis; the injectable hydrogel prepared from the temperature-sensitive modified chitin has good mechanical properties, good stability, a low degradation rate, and excellent lubrication effects; it can be applied to adhesion in surgery, tissue filling, anti-adhesion after surgery, orthopedic treatment and other aspects.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a temperature-sensitive modified chitin, a preparation method and an application thereof. Background Art

[0002] Polymer gel is a system composed of a three-dimensional network or interpenetrating network formed by cross-linking polymerization of molecular chains and a solvent (usually water), which is similar to biological tissue; it has good hydrophilicity, excellent swelling properties and biocompatibility, etc., and has broad application prospects in the fields of biomedicine and tissue engineering materials. Chitin, as a polymer of acetylglucosamine, is the second largest renewable natural polymer after cellulose and has a linear structure; however, due to the effect of hydrogen bonding, chitin itself has a high degree of crystallinity and is difficult to dissolve in water or low-concentration acid-base solutions, and is not easily soluble in commonly used organic solvents, which limits its research, development and application. With the development of science and technology, it has been found that by reacting chitosan with a carboxylating agent in a sodium hydroxide-urea system, hydroxypropyl chitosan with pH sensitivity and temperature sensitivity, namely, thermosensitive and modified chitosan, can be obtained; and with the deepening of research, the preparation method of thermosensitive and modified chitosan has been further improved, and the types of thermosensitive and modified chitosan have become more and more, making chitosan derivatives have better biocompatibility, biodegradability, low toxicity and multiple biological activities, and gradually widely used in the fields of biomedical materials and tissue engineering.

[0003] The degradation products of chitin and its derivatives are mainly glucosamine, which is an important substance necessary for the synthesis of proteoglycans in the matrix of articular cartilage. It can selectively act on arthritis, block the pathological development of osteoarthritis to a certain extent, and play the role of anti-inflammatory, pain relief, improvement of joint function and prevention of the progression of osteoarthritis. Therefore, chitin and its derivatives have cartilage protection function, can promote the proliferation of chondrocytes, increase the expression level of cartilage matrix components, and inhibit cartilage degradation and synovial inflammation. At present, there are many medical chitosan products for intra-articular injection on the market in China, such as Qitjie (carboxymethyl chitosan, national medical device registration number 20173640026) and Libaoxi (national medical device registration number 20173130026) of Shanghai Qisheng Biological Products Co., Ltd.; these chitosan and its derivatives as intra-articular injections, their effects and working principles are similar to those of hyaluronic acid solutions; however, free hyaluronic acid is easily degraded by enzymes in body tissues and stays in the joint cavity for a very short time; chemically cross-linked hyaluronic acid has a large viscosity and is difficult to inject, and these chemical cross-linking agents are all toxic. In view of the problems of hyaluronic acid in the treatment of osteoarthritis and the application of chitosan and its derivatives as intra-articular injections, our company has developed a thermosensitive modified chitosan hydrogel intra-articular injection (patent number: CN202110609013.9) by combining the properties of thermosensitive modified chitosan hydrogel and hyaluronic acid. The intra-articular injection is prepared by simply mixing hyaluronic acid or its salt into hydroxypropyl chitin hydrogel. Although it overcomes the defects of easy loss and degradation of hyaluronic acid solution, prolongs the retention time of hyaluronic acid in the body, and reduces the number of injections; however, the intra-articular injection is cross-linked only by the hydrogen bonding force within and between the hydroxypropyl chitin molecules, and the cross-linking effect is not good, resulting in poor mechanical properties and insufficient stability, which in turn makes the intra-articular injection have poor elasticity, low tensile strength, insufficient viscosity, poor lubrication, and other problems. In addition, the intra-articular injection is only suitable for relieving or treating osteoarthritis, and has a small scope of application.

[0004] The invention provides a temperature-sensitive modified chitosan, a preparation method and application of the temperature-sensitive modified chitosan in an injectable hydrogel, so as to solve the problems of poor mechanical properties, insufficient stability and poor lubrication effect of the chitosan hydrogel in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a temperature-sensitive modified chitin, a preparation method and an application thereof in an injectable hydrogel, so as to solve the problems of poor mechanical properties, insufficient stability and poor lubrication effect of chitin hydrogel in the prior art.

[0006] The technical solution of the present invention is: a temperature-sensitive modified chitin, wherein the chitin molecular chain contains a substituent A and a substituent B; the structural formula of the substituent A is: Formula (I-2), wherein m is any integer from 0 to 100; the degree of substitution of the substituent A is 0 to 35%; the substituent B is any one of hydroxypropyl, hydroxyethyl, and hydroxybutyl; the degree of substitution of the substituent B is 5 to 50%.

[0007] Preferably, the degree of substitution of the substituent A is 5-30%; and m is any integer between 2-50.

[0008] Preferably, the substituent B is a hydroxypropyl group, and the degree of substitution of the hydroxypropyl group is 10-35%.

[0009] Preferably, the thermosensitive modified chitosan is prepared from any one of thermosensitive hydroxypropyl chitosan, thermosensitive hydroxyethyl chitosan and thermosensitive hydroxybutyl chitosan as a starting material, and the compound 1-(ethylene oxide-2-yl)-(PEG) m -Carboxylic acid is synthesized as a substituent group;

[0010] The substituent group is 1-(2-oxirane-1-yl)-(PEG) m -OH is synthesized as raw material.

[0011] The present invention also provides a method for preparing the above-mentioned temperature-sensitive modified chitosan, wherein the synthesis of the temperature-sensitive modified chitosan comprises the following steps:

[0012] 1) 1-(2-Oxiranyl)-(PEG) m - Preparation of carboxylic acids

[0013] a. Weigh 1-(2-oxirane-1-yl)-(PEG) m -OH is dissolved in the buffer solution, and succinic anhydride is slowly added under weak alkaline conditions while stirring to react and form activated 1-(ethylene oxide-2-yl)-(PEG) m - Crude product of carboxylic acid;

[0014] b. 1-(2-oxirane-1-yl)-(PEG) m The crude product of -carboxylic acid was dissolved in dichloromethane and separated by thin layer chromatography on silica gel plates to obtain 1-(oxiran-2-yl)-(PEG) m - carboxylic acid structure;

[0015] 2) Preparation of thermosensitive modified chitin

[0016] a. Weigh any one of the temperature-sensitive hydroxypropyl chitosan, the temperature-sensitive hydroxyethyl chitosan, and the temperature-sensitive hydroxybutyl chitosan, and dissolve it in pure water, and stir overnight at a temperature below 5° C. to obtain a clear and viscous polymer solution;

[0017] b. Under low temperature conditions, sodium hydroxide aqueous solution and 1-(2-oxirane-1-yl)-(PEG) m -carboxylic acid are slowly added to the polymer solution, respectively, and after stirring for 24 hours, a reaction solution containing a temperature-sensitive modified chitosan polymer is obtained;

[0018] c. Add hydrochloric acid solution to the temperature-sensitive modified chitosan solution, adjust the pH of the reaction system to 7-7.5, remove small molecules in the solution by dialysis, and obtain the temperature-sensitive modified chitosan after freeze-drying.

[0019] Preferably, the deacetylation degree of the temperature-sensitive hydroxypropyl chitosan, the temperature-sensitive hydroxyethyl chitosan, and the temperature-sensitive hydroxybutyl chitosan are all less than 15%.

[0020] Preferably, in the polymer solution, the mass ratio of the temperature-sensitive hydroxypropyl chitosan is 0.8 wt% to 5 wt%; the 1-(ethylene oxide-2-yl)-(PEG) m -The added amount of carboxylic acid is 0.5-5 times the molar number of chitin structural unit.

[0021] Preferably, the sodium hydroxide aqueous solution is a 0.5-2M sodium hydroxide aqueous solution; and the hydrochloric acid solution is a 0.5-1.5M hydrochloric acid aqueous solution.

[0022] Preferably, the degree of deacetylation of the temperature-sensitive modified chitosan is less than 20%.

[0023] The present invention also provides application of the temperature-sensitive modified chitosan in injectable hydrogel.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] (1) The present invention provides a thermosensitive modified chitosan, a preparation method, and an application of the thermosensitive modified chitosan in an injectable hydrogel; the thermosensitive modified chitosan comprises a substituent A and a substituent B on its chitosan molecular chain, and the substituent A is a polyethylene glycol long-chain structure with a carboxylic acid group at the end; when the thermosensitive modified chitosan is prepared into an injectable hydrogel and injected into the body, the substituent A with the long-chain structure can increase the number of intermolecular and intramolecular hydrogen bonds and the force of hydrogen bonds of the thermosensitive modified chitosan molecule, thereby increasing the entanglement of the molecular chain, making the network structure of the injectable hydrogel more compact and the degree of cross-linking higher, thereby improving the mechanical properties of the injectable hydrogel, making the morphology of the injectable hydrogel closer to a solid state, and reducing its fluidity. It is beneficial for the injectable hydrogel to be better fixed at the injection site to prevent it from moving with the flow of human tissue fluid; at the same time, the improvement of mechanical properties can also make the hydrogel have a more excellent lubricating effect; secondly, the carboxylic acid group at the end of the substituent A can be cross-linked with calcium ions and magnesium ions in the human tissue fluid, which can further improve the mechanical properties of the injectable hydrogel and reduce its degradation rate, thereby reducing the injection frequency; the problems of poor mechanical properties, insufficient stability and poor lubrication effect of chitin hydrogel in the prior art are solved; and the injectable hydrogel has good biocompatibility and low cytotoxicity, and can be used for bonding and tissue filling during surgery, anti-adhesion after surgery, orthopedic treatment and other aspects.

[0026] (2) The present invention provides a thermosensitive modified chitosan and the use of the thermosensitive modified chitosan in the preparation of injectable hydrogels. The chitosan molecule contains both a substituent A and a substituent B on the molecular chain. The substituent A and the substituent B on the molecular chain can not only effectively increase the number of intermolecular hydrogen bonds of the thermosensitive modified chitosan molecule and increase the intermolecular entanglement, but also enable the thermosensitive modified chitosan molecule to form more intramolecular hydrogen bonds, so that the injectable hydrogel has good elasticity and improves the tensile ductility of the injectable hydrogel; thereby making it have an excellent lubricating effect.

[0027] (3) The present invention provides a thermosensitive modified chitosan and the application of the thermosensitive modified chitosan in the preparation of injectable hydrogels. The molecular chain of the chitosan molecule contains a long-chain substituent A. The number of hydrogen bonds within and between the molecules of the chitosan molecule and the force between the hydrogen bonds can be adjusted by adjusting the degree of substitution of the substituent A and the chain length of the substituent A, thereby adjusting the mechanical properties of the injectable hydrogel, so that the injectable hydrogel has a good lubricating effect and a low degradation rate, and has a good repair effect to meet the use requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0029] Figure 1 The temperature sensitivity test results of the injectable hydrogels P4 and P5 prepared in Examples 4 and 5 of the present invention and the injectable hydrogel Q2 prepared in Comparative Example 2;

[0030] Figure 2 The mechanical properties test results of the injectable hydrogels P1-P5 prepared in Examples 1-5 of the present invention and the injectable hydrogels Q1-Q2 prepared in Comparative Examples 1-2;

[0031] Figure 3 The degradation behavior test results of the injectable hydrogels P1-P5 prepared in Examples 1-5 of the present invention and the injectable hydrogels Q1-Q2 prepared in Comparative Examples 1-2;

[0032] Figure 4a Cell morphology and survival of NIH 3T3 cells when the injectable hydrogels P1, P2, P5 prepared in Examples 1, 2, and 5 of the present invention and the injectable hydrogel Q1 prepared in Comparative Example 1 were co-cultured with NIH 3T3 cells;

[0033] Figure 4b The proliferation of NIH 3T3 cells when the injectable hydrogels P1, P2, P5 prepared in Examples 1, 2, and 5 of the present invention and the injectable hydrogel Q1 prepared in Comparative Example 1 are co-cultured with NIH 3T3 cells. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments:

[0035] A temperature-sensitive modified chitin, wherein the chitin molecular chain comprises a substituent A and a substituent B; wherein the structure of the substituent A is In formula (I-2), m is any positive integer from 0 to 100; the substituent B is any one of hydroxypropyl, hydroxyethyl, hydroxybutyl and the like; the degree of substitution of the substituent A is 0 to 35%; and the degree of substitution of the substituent B is 5 to 50%.

[0036] The thermosensitive modified chitosan is prepared from any one of thermosensitive chitosans such as thermosensitive hydroxypropyl chitosan, thermosensitive hydroxyethyl chitosan, thermosensitive hydroxybutyl chitosan, etc. as a starting material, and a compound 1-(ethylene oxide-2-yl)-(PEG) is used. m -carboxylic acid as a substituent group; wherein the substituent group is a compound 1-(oxirane-2-yl)-(PEG) m -OH as raw material; compound 1-(2-oxirane-1)-(PEG)m -OH was purchased directly and / or in the form of epichlorohydrin, (PEG) m -OH and other compounds as raw materials.

[0037] 1. Preparation of thermosensitive modified chitin and injectable hydrogel Example 1

[0038] A thermosensitive modified chitin, the structural formula is Formula (I-1), the substituent A is Formula (I-3), m is 4; the substituent B is hydroxypropyl; wherein the thermosensitive modified chitosan is synthesized using thermosensitive hydroxypropyl chitosan as a starting material and the compound 1-(ethylene oxide-2-yl)-(PEG)4-carboxylic acid as a substituent group.

[0039] 1) Preparation of 1-(2-oxirane-1-yl)-(PEG)4-carboxylic acid

[0040] a. Weigh 5 g of 1-(oxirane-2-yl)-(PEG)4-OH, dissolve the weighed 1-(oxirane-2-yl)-(PEG)4-OH in a buffer solution, and slowly add 2.4 g of succinic anhydride to react while stirring under weak alkaline conditions to form a crude product of activated 1-(oxirane-2-yl)-(PEG)4-carboxylic acid;

[0041] b. The crude product of 1-(oxirane-2-yl)-(PEG)4-carboxylic acid was dissolved in dichloromethane and separated using a thin layer chromatography silica gel plate to obtain 4.2 g of 1-(oxirane-2-yl)-(PEG)4-carboxylic acid.

[0042] The buffer solution is one of toluene, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, dichloromethane and the like; the pH value of the weak alkaline condition is 8-9, and the alkaline solution is one of triethylamine, diethylamine, tetrahydropyrrole, pyridine, sodium bicarbonate aqueous solution and the like. 1-(Oxiran-2-yl)-(PEG)4-OH can be purchased directly or obtained by synthesis, that is, synthesized using epichlorohydrin and (PEG)4-OH as raw materials.

[0043]

[0044] 2) Preparation of thermosensitive modified chitin

[0045] a. Weigh 2 g of temperature-sensitive hydroxypropyl chitosan and dissolve it in pure water. Stir overnight at a temperature below 5°C to obtain a clear and viscous polymer solution.

[0046] b. Under low temperature conditions, 10 mL of 2M sodium hydroxide aqueous solution and 4.2 g of 1-(2-ethylene oxide)-(PEG)4-carboxylic acid were slowly added to the polymer solution, and stirred for 24 hours to obtain a reaction solution containing a temperature-sensitive modified chitin polymer.

[0047] c. Add 1 M hydrochloric acid solution to the reaction solution, adjust the pH of the reaction solution to 7-7.5, remove small molecules in the solution through dialysis, and obtain 1.3 g of temperature-sensitive modified chitin after freeze-drying.

[0048] 0.02 g of the prepared temperature-sensitive modified chitin was taken for mass spectrometry detection. 1 The HNMR spectrum shows that the acetylation degree of the thermosensitive modified chitin product is 0.87, the substitution degree of substituent A is 0.24, and the substitution degree of substituent B is 0.28. The viscosity average molecular weight of the thermosensitive modified chitin is measured by an Ubbelohde viscometer to be Mη=520 kDa.

[0049] Take 1g of the prepared temperature-sensitive modified chitin and dissolve it in an aqueous solution to prepare a sol with a mass concentration of 2%; place the sol at 37°C to form an injectable hydrogel, P1. The injectable hydrogel is temperature sensitive, and the rheological results show that the injectable hydrogel has a reversible sol-gel transition behavior, and the sol-gel transition temperature of the injectable hydrogel is 16.3°C. The injectable hydrogel can be used for bonding and tissue filling during surgery, anti-adhesion after surgery, and treatment of osteoarthritis.

[0050] Example 2

[0051] A thermosensitive modified chitin, the substituent A is Formula (I-4), m is 16; the substituent B is hydroxypropyl; wherein the thermosensitive modified chitosan is prepared from thermosensitive hydroxypropyl chitosan as a starting material and from a compound 1-(ethylene oxide-2-yl)-(PEG) 16 -Carboxylic acid is synthesized as a substituent group.

[0052] 1) Preparation of 1-(2-oxirane-1-yl)-(PEG)16-carboxylic acid

[0053] a. Weigh 5 g of 1-(2-oxirane-1-yl)-(PEG) 16 -OH, and the weighed 1-(2-oxirane-1-yl)-(PEG) 16 -OH was dissolved in the buffer solution, and 2.4 g of succinic anhydride was slowly added under weak alkaline conditions while stirring to react to form activated 1-(2-ethylene oxide)-(PEG) 16 - Crude product of carboxylic acid;

[0054] b. 1-(2-oxirane-1-yl)-(PEG) 16 The crude product of -carboxylic acid was dissolved in dichloromethane and separated by thin layer chromatography on silica gel plate to obtain 4.2 g of 1-(oxiran-2-yl)-(PEG) 16 -carboxylic acid. The buffer is acetonitrile, the pH value of the weak alkaline condition is 8-9, and the alkaline solution selected is triethylamine. 1-(2-oxirane-1)-(PEG) 16 -OH can be purchased directly or obtained by synthesis, that is, using epichlorohydrin, (PEG) 16 -OH is synthesized as raw material.

[0055] 1-(Oxiran-2-yl)-(PEG) 16 The synthesis method of -OH comprises the following steps:

[0056] a. Weigh 10g of (PEG) 16 -OH, and weigh (PEG) 16 -OH was dissolved in anhydrous tetrahydrofuran buffer, 0.27 g of sodium hydride was slowly added while stirring in an ice bath, and stirred for 30 min in an ice bath to obtain a reaction solution;

[0057] b. Weigh 0.6 g of epichlorohydrin and add it to the above reaction solution under ice bath conditions, stir for 30 min, and perform substitution reaction to obtain 1-(2-oxirane-1-yl)-(PEG) 16 -OH crude product;

[0058] Among them, (PEG) 16 The molar ratio of -OH, sodium hydride and epichlorohydrin is 1:(0.4-0.8):(0.4-0.6);

[0059] c. 1-(2-oxirane-1-yl)-(PEG) 16 The crude product of -OH was dissolved in ethyl acetate or dichloromethane and separated by thin layer chromatography on silica gel plates to obtain 5.5 g of pure 1-(2-oxirane-1-yl)-(PEG) 16 -OH products. The main steps of separation and purification using thin layer chromatography silica gel plates are: i. Plate: Apply on the silica gel plate in strips with a width of less than 5 mm; ii. Develop: Use petroleum ether / ethyl acetate as a developing agent to remove unreacted (PEG) 16 -OH, epichlorohydrin and impurities produced by side reactions are separated. The impurities produced by side reactions are mainly (PEG) 16 The product in which the hydroxyl groups at both ends of -OH are combined with epichlorohydrin, that is, Impurity of formula (I-5).

[0060]

[0061] 2) Preparation of thermosensitive modified chitin

[0062] a. Weigh 2.5 g of thermosensitive hydroxypropyl carboxylate and dissolve it in pure water. Stir overnight at a temperature below 5°C to obtain a clear and viscous polymer solution.

[0063] b. Under low temperature conditions, 10 mL of 1.5 M sodium hydroxide aqueous solution and 5 g of 1-(2-oxirane-1-yl)-(PEG) 16 -acid were slowly added to the polymer solution, and after stirring for 24 hours, a reaction solution containing a temperature-sensitive modified chitosan polymer was obtained.

[0064] c. Add 1.5M hydrochloric acid solution to the reaction solution, adjust the pH of the reaction solution to 7-7.5, remove small molecules in the solution by dialysis, and obtain 1.5g of temperature-sensitive modified chitin after freeze-drying.

[0065] 0.02 g of the prepared temperature-sensitive modified chitin was taken for mass spectrometry detection. 1 The HNMR spectrum shows that the acetylation degree of the thermosensitive modified chitin product is 0.89, the substitution degree of substituent A is 0.23, and the substitution degree of substituent B is 0.28. The viscosity average molecular weight of the thermosensitive modified chitin is measured by an Ubbelohde viscometer to be Mη=510 kDa.

[0066] Take 1g of the prepared temperature-sensitive modified chitin and dissolve it in an aqueous solution to prepare a sol with a mass concentration of 2%; place the sol at 37°C to form an injectable hydrogel, P2. The injectable hydrogel is temperature sensitive, and the rheological results show that the injectable hydrogel has a reversible sol-gel transition behavior, and the sol-gel transition temperature of the injectable hydrogel is 18.9°C.

[0067] Example 3

[0068] A thermosensitive modified chitin, the substituent A is Formula (I-6), m is 6; substituent B is hydroxypropyl; the thermosensitive modified chitosan is synthesized using thermosensitive hydroxypropyl chitosan as a starting material and compound 1-(ethylene oxide-2-yl)-(PEG)6-carboxylic acid as a substituent group. This example uses the same synthesis method as in Example 2 above to prepare compound 1-(ethylene oxide-2-yl)-(PEG)6-carboxylic acid and the thermosensitive modified chitosan, and 1.2 g of thermosensitive modified chitosan is prepared. 0.02 g of the thermosensitive modified chitosan prepared above is taken for mass spectrometry detection. 1The HNMR spectrum shows that the acetylation degree of the thermosensitive modified chitin product is 0.88, the substitution degree of substituent A is 0.16, and the substitution degree of substituent B is 0.25. The viscosity average molecular weight of the thermosensitive modified chitin is measured by an Ubbelohde viscometer to be Mη=490 kDa.

[0069] Take 1g of the prepared temperature-sensitive modified chitin and dissolve it in an aqueous solution to prepare a sol with a mass concentration of 2%; place the sol at 37°C to form an injectable hydrogel, P3. The injectable hydrogel is temperature sensitive, and the rheological results show that the injectable hydrogel has a reversible sol-gel transition behavior, and the sol-gel transition temperature of the injectable hydrogel is 20.0°C.

[0070] Example 4

[0071] A thermosensitive modified chitin, the substituent A is Formula (I-7), m is 12; the substituent B is hydroxybutyl; wherein the thermosensitive modified chitosan is prepared from thermosensitive hydroxybutyl chitosan as a starting material and a compound 1-(ethylene oxide-2-yl)-(PEG) 12 -Carboxylic acid is synthesized as a substituent group.

[0072] This example uses the same synthesis method as in Example 2 to prepare compound 1-(2-oxirane-1-yl)-(PEG) 12 -carboxylic acid and the temperature-sensitive modified chitin, and 1.4 g of the temperature-sensitive modified chitin was prepared. 0.02 g of the temperature-sensitive modified chitin prepared above was taken for mass spectrometry detection. 1 The HNMR spectrum shows that the acetylation degree of the thermosensitive modified chitin product is 0.87, the substitution degree of substituent A is 0.18, and the substitution degree of substituent B is 0.23. The viscosity average molecular weight of the thermosensitive modified chitin measured by Ubbelohde viscometer is Mη=540 kDa.

[0073] Take 1g of the prepared temperature-sensitive modified chitin and dissolve it in an aqueous solution to prepare a sol with a mass concentration of 2%; place the sol at 37°C to form an injectable hydrogel, P4. The injectable hydrogel is temperature sensitive, and the rheological results show that the injectable hydrogel has a reversible sol-gel transition behavior, and the sol-gel transition temperature of the injectable hydrogel is 20.9°C.

[0074] Example 5

[0075] A thermosensitive modified chitin, the substituent A is Formula (I-8), m is 82; the substituent B is hydroxybutyl; wherein the thermosensitive modified chitosan is prepared from thermosensitive hydroxybutyl chitosan as a starting material and a compound 1-(ethylene oxide-2-yl)-(PEG)82 -Carboxylic acid is synthesized as a substituent group.

[0076] This example uses the same synthesis method as in Example 2 to prepare compound 1-(2-oxirane-1-yl)-(PEG) 82 -carboxylic acid and the temperature-sensitive modified chitin, and 1.1 g of the temperature-sensitive modified chitin was prepared. 0.02 g of the temperature-sensitive modified chitin prepared above was taken for mass spectrometry detection. 1 The HNMR spectrum shows that the acetylation degree of the thermosensitive modified chitin product is 0.89, the substitution degree of substituent A is 0.12, and the substitution degree of substituent B is 0.25. The viscosity average molecular weight of the thermosensitive modified chitin is measured by an Ubbelohde viscometer to be Mη=620 kDa.

[0077] Take 1g of the prepared temperature-sensitive modified chitin and dissolve it in an aqueous solution to prepare a sol with a mass concentration of 2%; place the sol at 37°C to form an injectable hydrogel, P5. The injectable hydrogel is temperature sensitive, and the rheological results show that the injectable hydrogel has a reversible sol-gel transition behavior, and the sol-gel transition temperature of the injectable hydrogel is 14.7°C.

[0078] Comparative Example 1

[0079] The purchased thermosensitive hydroxypropyl chitosan was dissolved in an aqueous solution to prepare a sol with a mass concentration of 2%; the sol was placed at 37°C to form an injectable hydrogel, Q1.

[0080] Comparative Example 2

[0081] The purchased thermosensitive hydroxybutyl chitosan was dissolved in an aqueous solution to prepare a sol with a mass concentration of 2%; the sol was placed at 37°C to form an injectable hydrogel, Q2.

[0082] 2. Testing of injectable hydrogel properties

[0083] 1. Temperature sensitivity test of injectable hydrogels

[0084] Take appropriate amounts of the injectable hydrogels P4 and P5 prepared in Examples 4 and 5 and the injectable hydrogel Q2 prepared in Comparative Example 2, and measure their gel rheology in a dynamic oscillation mode. Figure 1As shown, from top to bottom are the curves of the storage modulus (G') and loss modulus (G") of the injectable hydrogels P5, P4, and Q2 changing with temperature; when the storage modulus (G') change curve of the injectable hydrogels P5, P4, and Q2 intersects with the change curve of their loss modulus (G"), the injectable hydrogels P5, P4, and Q2 change from a sol state to a gel state, and the intersection of the two curves is the transition point of the injectable gel, and the temperature corresponding to the intersection is the sol-gel transition temperature of the injectable hydrogels P5, P4, and Q2; Figure 1 It can be seen that the transition temperatures of the injectable hydrogels P5, P4, and Q2 are 14.7°C, 20.9°C, and 24.6°C, respectively. It can be seen that when the substituent A is introduced into the temperature-sensitive chitin molecular chain and prepared into an injectable hydrogel, the injectable hydrogel still has a reversible sol-gel transition behavior and is temperature sensitive; and the sol-gel transition temperature of the injectable hydrogel decreases with the increase of the degree of substitution of the substituent A and the increase of the chain length of the substituent A.

[0085] 2. Mechanical properties testing of injectable hydrogels

[0086] Under the condition of 37°C, the storage modulus of the injectable hydrogels P1-P5 prepared in Examples 1-5 and the injectable hydrogels Q1-Q2 prepared in Comparative Examples 1-2 were respectively detected and compared; as shown in Figure 4, the storage modulus of the injectable hydrogels P1-P3 prepared in Examples 1-3 was higher than that of the injectable hydrogel Q1 prepared in Comparative Example 1 at 37°C, indicating that the injectable hydrogels P1-P3 prepared in Examples 1-3 had better mechanical properties, better elasticity, and higher tensile strength than the injectable hydrogel Q1 prepared in Comparative Example 1; further indicating that the introduction of substituent A on the temperature-sensitive chitin molecular chain can significantly improve the mechanical properties of the hydrogel. The higher the mechanical properties of the injectable hydrogel, the closer the injectable hydrogel morphology is to the solid state, and its fluidity is weakened accordingly. When the hydrogel is injected into the body, the hydrogel can be better fixed at the injection site. Similarly, the injectable hydrogels P4 and P5 prepared in Examples 4 and 5 also have better mechanical properties, better elasticity, and higher tensile strength than the injectable hydrogel Q2 prepared in Comparative Example 2. However, the injectable hydrogel P5 prepared in Example 5 has too high mechanical properties, so that the network structure of the injectable hydrogel P5 is too tight, the network loses elasticity, and the injectable hydrogel P5 loses the characteristics of a gel; thus, it can be seen that the longer the chain length of the substituent A introduced into the temperature-sensitive chitin molecular chain, the better.

[0087] 3. Degradation behavior detection of injectable hydrogels

[0088] The injectable hydrogels P1-P5 prepared in Examples 1-5 and the injectable hydrogels Q1-Q2 prepared in Comparative Examples 1-2 were respectively immersed in a lysozyme buffer solution, such as Figure 3 As shown, the mass loss of injectable hydrogels P1-P5 is slower than that of injectable hydrogels Q1-Q2, indicating that their degradation rate is slow and they have good stability. Compared with the injectable hydrogel P2 prepared in Example 2, the injectable hydrogels P1 and P3 prepared in Example 1 and Example 3 have a higher degradation rate than the injectable hydrogel P2 prepared in Example 2. It can be seen that when the degree of substitution of the substituent A is close, the longer the chain length of the substituent A, the more the number of intermolecular and intramolecular hydrogen bonds, the stronger the entanglement of the molecular chain, the tighter the network structure of the injectable hydrogel, and the lower the degradation rate of the injectable hydrogel; therefore, the degradation rate of the hydrogel can be adjusted by adjusting the chain length of the substituent A.

[0089] 4. Cytocompatibility testing of injectable hydrogels

[0090] The injectable hydrogels P1, P2, P5 prepared in the above-mentioned Examples 1, 2, and 5, and the injectable hydrogel Q1 prepared in Comparative Example 1 were respectively extracted and co-cultured with NIH 3T3 cells for 24 hours to construct four experimental groups; at the same time, a blank control group was constructed, i.e., NIH 3T3 cells were cultured alone; the cell morphology of NIH 3T3 cells in each experimental group was observed, and the cell survival rate and cell proliferation of NIH 3T3 cells were detected. First, the NIH 3T3 cells were stained for live and dead cells, with green representing live cells and red representing dead cells, and observed by laser confocal microscopy; Figure 4a As shown, compared with the blank control group without injectable hydrogel, in the above four experimental groups, the NIH 3T3 cells in each experimental group had good cell morphology, and the number of red dead cells in each experimental group was very small, which indicated that the NIH 3T3 cells had a higher survival rate; Figure 4bAs shown, compared with the blank control group without injectable hydrogel, the proliferation of NIH3T3 cells in the above four experimental groups is close to the growth rate of NIH 3T3 cells in the blank control group, and there is no significant difference. This shows that the injectable hydrogels P1, P2, P5 prepared in the above Examples 1, 2, and 5 and the injectable hydrogel Q1 prepared in Comparative Example 1 and NIH 3T3 cells co-cultured will hardly affect the growth and proliferation of NIH 3T3 cells, that is, the injectable hydrogels P1, P2, P5 and Q1 have good compatibility with NIH 3T3 cells. It can be seen that the substituent A introduced into the temperature-sensitive chitin molecular chain prepares a temperature-sensitive modified chitin molecule, and prepares it into an injectable hydrogel, which has good biocompatibility and low cytotoxicity, and can be injected into the body for bonding, tissue filling, postoperative adhesion prevention, and treatment of osteoarthritis.

[0091] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the scope of protection of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A temperature-sensitive modified chitin, characterized in that: The chitin molecular chain contains substituent A and substituent B; The structural formula of the substituent A is: Formula (I-2), wherein m is any integer from 2 to 50; the degree of substitution of the substituent A is 0 to 35%; The substituent B is any one of hydroxypropyl, hydroxyethyl and hydroxybutyl; the substitution degree of the substituent B is 5-50%; The thermosensitive modified chitosan is prepared from any one of thermosensitive hydroxypropyl chitosan, thermosensitive hydroxyethyl chitosan and thermosensitive hydroxybutyl chitosan as a starting material, and the compound 1-(ethylene oxide-2-yl)-(PEG) is used as a starting material. m -Carboxylic acid is synthesized as a substituent group; The substituent group is 1-(2-oxirane-1-yl)-(PEG) m -OH is synthesized as raw material.

2. A temperature-sensitive modified chitosan according to claim 1, characterized in that: The degree of substitution of the substituent A is 5-30%.

3. A temperature-sensitive modified chitosan according to claim 1, characterized in that: The substituent B is a hydroxypropyl group, and the degree of substitution of the hydroxypropyl group is 10-35%.

4. A method for preparing the temperature-sensitive modified chitosan according to claim 1, characterized in that: The synthesis of the temperature-sensitive modified chitin comprises the following steps: 1) 1-(2-Oxiranyl)-(PEG) m - Preparation of carboxylic acids a. Weigh 1-(2-oxirane-1-yl)-(PEG) m -OH is dissolved in the buffer solution, and succinic anhydride is slowly added under weak alkaline conditions while stirring to react to form activated 1-(ethylene oxide-2-yl)-(PEG) m - Crude product of carboxylic acid; b. 1-(2-oxirane-1-yl)-(PEG) m The crude product of -carboxylic acid was dissolved in dichloromethane and separated by thin layer chromatography on silica gel plates to obtain 1-(oxiran-2-yl)-(PEG) m - carboxylic acid structure; 2) Preparation of thermosensitive modified chitin a. Weigh any one of the temperature-sensitive hydroxypropyl chitosan, the temperature-sensitive hydroxyethyl chitosan, and the temperature-sensitive hydroxybutyl chitosan, and dissolve it in pure water, and stir overnight at a temperature below 5° C. to obtain a clear and viscous polymer solution; b. Under low temperature conditions, sodium hydroxide aqueous solution and 1-(2-oxirane-1-yl)-(PEG) m -carboxylic acid are slowly added to the polymer solution, respectively, and after stirring for 24 hours, a reaction solution containing a temperature-sensitive modified chitosan polymer is obtained; c. Add hydrochloric acid solution to the temperature-sensitive modified chitosan solution, adjust the pH of the reaction system to 7-7.5, remove small molecules in the solution by dialysis, and obtain the temperature-sensitive modified chitosan after freeze-drying.

5. The method for preparing a temperature-sensitive modified chitosan according to claim 4, characterized in that: The deacetylation degree of the temperature-sensitive hydroxypropyl chitosan, the temperature-sensitive hydroxyethyl chitosan, and the temperature-sensitive hydroxybutyl chitosan are all less than 15%.

6. The method for preparing a temperature-sensitive modified chitosan according to claim 5, characterized in that: In the polymer solution, the mass ratio of the temperature-sensitive hydroxypropyl chitosan is 0.8wt% to 5wt%; the 1-(ethylene oxide-2-yl)-(PEG) m -The added amount of carboxylic acid is 0.5-5 times the molar number of chitin structural unit.

7. The method for preparing a temperature-sensitive modified chitosan according to claim 6, characterized in that: The sodium hydroxide aqueous solution is a 0.5-2M sodium hydroxide aqueous solution; the hydrochloric acid solution is a 0.5-1.5M hydrochloric acid aqueous solution.

8. The method for preparing a temperature-sensitive modified chitosan according to claim 7, characterized in that: The deacetylation degree of the temperature-sensitive modified chitin is less than 20%.

9. Use of the temperature-sensitive modified chitosan according to claim 1 in preparing injectable hydrogel.

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