Temperature-sensitive modified chitin hydrogel injection as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a temperature-sensitive modified chitin hydrogel injection and a preparation method and application thereof, in the temperature-sensitive modified chitin hydrogel injection, the mass concentration of temperature-sensitive modified chitin is 0.1-10%; a chitin molecular chain of the temperature-sensitive modified chitin contains a hydrophilic substituent group and a hydrophobic substituent group at the same time; the degree of acetylation of the temperature-sensitive modified chitin is not lower than 75%. A molecular chain of the temperature-sensitive modified chitin contains a hydrophilic substituent group and a hydrophobic substituent group at the same time, so that the temperature-sensitive modified chitin hydrogel injection has good fluidity before low-temperature injection and can be uniformly distributed at an injection target part; after the gel is formed at the body temperature of an injection target part, the strength is higher, adjacent organs and tumor parts can be fully separated, and an excellent isolation effect is achieved; the radiation dose of a target region is improved, and the risk that surrounding healthy tissues and organs are damaged by radiation is reduced.

Description

Technical Field

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

[0002] Radiotherapy is one of the three conventional methods for treating malignant tumors; usually, a higher target dose can produce a higher tumor control effect; however, a higher target dose will cause a certain dose of radiation to the healthy tissues around the target area, causing certain radiation damage, and even some normal tissues will be severely damaged, such as abnormal symptoms in the gastrointestinal tract, urinary system, reproductive system and other tissues, which will affect the patient's health and quality of life for a long period of time. Therefore, it is usually necessary to use medical isolation glue to "space" the target area from the surrounding tissues. Hydrogel is a cross-linked hydrophilic polymer network with a high water content, which shows good biocompatibility and has been developed for use in a variety of clinical fields; in addition, radiation oncologists have tried to use injectable hydrogels as isolation and protection materials to achieve the effect of increasing the target dose while reducing the risk of radiation damage to surrounding tissues during radiotherapy, thereby improving the quality of life of patients. According to the content recorded in the literature "Research Progress on the Spacing and Protective Effect of Hydrogels in Tumor Radiotherapy" (DOI: 10.3760 / cma.j.cn112271-20230315-00076.), it can be known that two types of matrix hydrogels have been reported to be able to be used as "spacers" for tumor radiotherapy, namely polyethylene glycol (PEG) matrix hydrogels and hyaluronic acid (HA) matrix hydrogels; and, polyethylene glycol-based and hyaluronic acid-based related hydrogel products have been approved for marketing abroad as radiotherapy spacer hydrogels; relevant clinical trials have been conducted in China, but no related products are currently on the market. Among them, disposable, absorbable polyethylene glycol-based hydrogels, such as the product that received CE approval from the European Union in 2010 and FDA approval in April 2015 (DEN140030, SpaceOAR System, AUGMENIX, INC.), are used to reduce common side effects of radiotherapy for prostate cancer, cervical cancer, etc.; the hydrogel product is a two-component AB product, component A is a phosphate buffered aqueous solution of a polyethylene glycol derivative, and component B is an alkaline aqueous solution of a polyamino cross-linking agent; components A and B are sealed in different vials, and when used, they are injected between the patient's prostate (or cervix) and the adjacent rectal wall through a double mixing device syringe under B-ultrasound guidance, isolating the rectum from the irradiated area, reducing the radiotherapy dose of the rectum, and reducing the impact of radiotherapy on the rectum; however, the product is stored in a dry powder state, and the dry powder needs to be prepared into a solution before injection, and components A and B need to be mixed for use. FDA-approved hyaluronic acid-based gels, such as Teleflex (Cross-linked sodium hyaluronate gel), when using this gel, the gel needs to be crushed into particles and then filled into a 3ml syringe to make the product injectable, which is a relatively complicated operation; in addition, the gel product has high viscosity, and the injection force required during the injection process is large, which is not convenient to use.

[0003] Chitosan, as the second most abundant natural polymer material in nature, has good biocompatibility and biodegradability. Some of its derivatives have better biocompatibility, biodegradability and low toxicity while also being pH-sensitive or temperature-sensitive. They are gradually being widely used in fields such as biomedical materials and tissue engineering. For example, patent number CN201210220246.0 discloses a thermosensitive chitosan derivative - hydroxypentyl chitosan; patent number CN201410170871.8 discloses a method for homogeneously preparing low-deacetylation hydroxypropyl-modified chitosan; patent application number CN201810108226.1 discloses an injectable high-strength thermosensitive modified chitosan-based hydrogel, its preparation method and application, etc. These thermosensitive and modified chitin hydrogels usually have soft and lubricating properties and appropriate mechanical properties that enable them to be fixed in the injection site, and are used in bonding, tissue filling, and osteoarthritis treatment during surgery. However, perhaps because these hydrogels are usually low-strength, liquid-like, and relatively soft, it is difficult to think of using them as isolation glues for radiotherapy. In addition, there are no relevant patents or documents that disclose the use of injectable hydrogels made of thermosensitive and modified chitin as isolation and protection materials for tumor radiotherapy. Therefore, chitin needs to be further improved to expand its scope of application.

[0004] The present invention provides a temperature-sensitive modified chitin hydrogel injection and a preparation method and application thereof, so as to solve the problems in the prior art of existing isolation glue for radiotherapy, such as the need to prepare a solution before injection, inconvenient clinical operation, and large injection force required during injection. Summary of the invention

[0005] The purpose of the present invention is to provide a temperature-sensitive modified chitin hydrogel injection and its preparation method and application, so as to solve the problems existing in the prior art of existing isolation glue for radiotherapy, such as the need to prepare a solution before injection, inconvenient clinical operation, and large injection force required during injection.

[0006] The technical solution of the present invention is: a temperature-sensitive modified chitosan hydrogel injection, in which the mass concentration of the temperature-sensitive modified chitosan is 0.1-10%;

[0007] The chitosan molecular chain of the temperature-sensitive modified chitosan contains both hydrophilic substituents and hydrophobic substituents; the acetylation degree of the temperature-sensitive modified chitosan is not less than 75%.

[0008] Preferably, on the chitosan molecular chain of the thermosensitive modified chitosan, the degree of substitution of the hydrophilic substituent is 0.3-0.5, and the degree of substitution of the hydrophobic substituent is 0.05-0.20.

[0009] Preferably, the viscosity average molecular weight of the temperature-sensitive modified chitosan is 300-500 kDa.

[0010] Preferably, the hydrophilic substituent is any one of carboxymethyl, carboxyethyl and carboxypropyl; the hydrophobic substituent is any one or more of ethyl, propyl and butyl.

[0011] Preferably, the temperature-sensitive modified chitosan is synthesized using carboxymethyl chitosan, carboxyethyl chitosan, or carboxypropyl chitosan as starting materials and any one or more of ethyl halide, propyl halide, and butyl halide as hydrophobic group substitution reagents.

[0012] The present invention also provides a method for preparing the above-mentioned temperature-sensitive modified chitosan hydrogel injection, comprising the following steps:

[0013] S1. Weigh any one of carboxymethyl chitosan, carboxyethyl chitosan, and carboxypropyl chitosan, dissolve it in a sodium hydroxide solution, and stir overnight at a temperature below 5° C. to obtain a clear and viscous polymer solution;

[0014] S2, weighing a hydrophobic group substitution reagent, slowly adding the weighed hydrophobic group substitution reagent to the polymer solution under low temperature conditions, and stirring to obtain a reaction solution containing a temperature-sensitive modified chitosan polymer;

[0015] S3, adding hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the solution by dialysis, and preparing temperature-sensitive modified chitin after freeze-drying;

[0016] S4. Take the thermosensitive modified chitosan prepared above and dissolve it in injectable water to prepare a solution injection. The prepared solution injection forms a thermosensitive modified chitosan hydrogel under the condition of 20-40°C.

[0017] Preferably, in the reaction solution, the amount of the hydrophobic group substitution reagent added is 0.5-5 times the molar number of the chitin structural unit.

[0018] Preferably, the deacetylation degree of the carboxymethyl chitosan, the carboxyethyl chitosan and the carboxypropyl chitosan are all less than 20%.

[0019] Preferably, the carboxymethyl chitosan, the carboxyethyl chitosan and the carboxypropyl chitosan are synthesized by using chitosan molecules as raw materials and reacting with sodium chloroacetate, sodium chloropropionate and sodium chlorobutyrate respectively.

[0020] The present invention also provides the above-mentioned temperature-sensitive modified chitin hydrogel injection for use as a medical isolation glue in radiotherapy.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] (1) The present invention provides a thermosensitive modified chitosan hydrogel injection and a preparation method and application thereof. The thermosensitive modified chitosan in the thermosensitive modified chitosan hydrogel injection contains both hydrophilic substituents and hydrophobic substituents on its chitosan molecular chain, which enables the thermosensitive modified chitosan hydrogel injection to have good temperature sensitivity and good fluidity before low-temperature injection, and can be evenly distributed at the target injection site; and under body temperature conditions, the thermosensitive modified chitosan hydrogel injection has high strength and excellent isolation effect after gelation at the target injection site, and can fully separate the adjacent organs from the tumor site; thereby improving the target area. The radiation dose is reduced, the risk of radiation damage to surrounding healthy tissues and organs during radiotherapy is reduced, and the quality of life of patients is improved; at the same time, because the temperature-sensitive modified chitosan hydrogel injection is temperature-sensitive, that is, it dissolves in water under low temperature conditions and turns into a gel state at body temperature, it can be pushed and injected conveniently during injection; and it can achieve the effect of staying in the injection site and not losing; in addition, the temperature-sensitive modified chitosan hydrogel injection is a single component and does not need to be mixed. Compared with PEG hydrogel materials and cross-linked sodium hyaluronate gel materials, the two products are easier to inject and use; it solves the problems of existing isolation glue for radiotherapy, which needs to be prepared into a solution before injection, is inconvenient in clinical operation, and requires a large injection force during injection.

[0023] (2) The present invention provides a thermosensitive modified chitosan hydrogel injection and a preparation method and application thereof. The chitosan molecular chain of the thermosensitive modified chitosan contains both hydrophilic substituents and hydrophobic substituents. The introduced hydrophilic substituents, such as carboxymethyl, carboxyethyl, carboxypropyl, etc., can improve the hydrophilicity of the chitosan molecule, so that it can be dissolved in water under low temperature conditions. However, if only hydrophilic substituents are introduced into the chitosan molecular chain, the obtained modified chitosan is in a solution state after dissolution, is easy to flow, and cannot be fixed in place after injection. By introducing a hydrophobic substituent into the molecular chain, that is, introducing a hydrophilic substituent and a hydrophobic substituent into the chitosan molecular chain at the same time, the solubility of the obtained temperature-sensitive modified chitosan molecule can be changed under the synergistic effect of the hydrophilic substituent and the hydrophobic substituent, and the temperature-sensitive modified chitosan molecule has a large solubility and can form a stable solution. As the temperature rises, the solubility of the temperature-sensitive modified chitosan molecule decreases, and the solution state becomes a gel state; thus, after injection, the chitosan molecule can form a gel in situ and no longer flow, thereby playing the role of in-situ fixation and isolation protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0025] Figure 1 The storage modulus test results of the temperature-sensitive modified chitosan hydrogel injections A1-A5 prepared in Examples 1-5 of the present invention and the modified chitosan hydrogel injections B1-B2 prepared in Comparative Examples 1-2;

[0026] Figure 2 The degradation behavior test results of the temperature-sensitive modified chitosan hydrogel injections A1-A5 prepared in Examples 1-5 of the present invention and the modified chitosan hydrogel injections B1-B2 prepared in Comparative Examples 1-2;

[0027] Figure 3 This is a comparison chart of cell morphology when the thermosensitive modified chitosan hydrogel injections A1, A2, A5 prepared in Examples 1, 2, and 5 of the present invention and the modified chitosan hydrogel injection B1 prepared in Comparative Example 1 are co-cultured with NIH 3T3 cells;

[0028] Figure 4 These are the cell compatibility test results of the temperature-sensitive modified chitosan hydrogel injections A1, A2, A5 prepared in Examples 1, 2, and 5 of the present invention and the modified chitosan hydrogel injection B1 prepared in Comparative Example 1. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments:

[0030] A thermosensitive modified chitosan hydrogel injection, comprising a thermosensitive modified chitosan hydrogel. In the thermosensitive modified chitosan hydrogel injection, the mass concentration of the thermosensitive modified chitosan is 0.1-10%; and the acetylation degree of the thermosensitive modified chitosan is not less than 75%; the chitosan molecular chain of the thermosensitive modified chitosan contains both hydrophilic substituents and hydrophobic substituents. The hydrophilic substituent is one of carboxymethyl, carboxyethyl, carboxypropyl, etc.; the hydrophobic substituent is one or more of alkyl chains such as ethyl, propyl, butyl, etc. In addition, in order to make the thermosensitive modified chitin hydrogel have higher strength, elastic modulus, etc., the degree of substitution of hydrophilic substituents on the molecular chain of the thermosensitive modified chitin is 0.3-0.5, and the degree of substitution of hydrophobic substituents is 0.05-0.30; and the degree of substitution of hydrophobic substituents is preferably 0.05-0.20; the viscosity-average molecular weight of the thermosensitive modified chitin is preferably in the range of 300-500kDa.

[0031] The above-mentioned temperature-sensitive modified chitin is synthesized with chitosan derivatives such as carboxymethyl chitosan, carboxyethyl chitosan, and carboxypropyl chitosan as starting materials, and one or more alkyl halides such as ethyl halides, propyl halides, and butyl halides as hydrophobic group substitution reagents. Among them, chitosan derivatives such as carboxymethyl chitosan, carboxyethyl chitosan, and carboxypropyl chitosan can be purchased from the market, or can be synthesized by reacting chitosan molecules with sodium chloroacetate, sodium chloropropionate, sodium chlorobutyrate, etc. as raw materials. Alkyl halides such as ethyl halides, propyl halides, and butyl halides are all purchased from the market. In other embodiments, the hydrophobic substituent can also be a cyclic or chain alkyl such as isobutyl, isopropyl, and cyclohexyl; the hydrophilic substituent can also be an alkyl chain containing one or more hydroxyl groups such as hydroxypropyl and hydroxyethyl, or other alkyl chains containing acyl groups and oxygen atoms, etc.

[0032] Example 1

[0033] S1. Weigh 2 g of water-soluble carboxymethyl chitosan, dissolve the weighed carboxymethyl chitosan in a 2 M sodium hydroxide solution, and stir overnight at 0° C. (ice bath) to obtain a clear and viscous polymer solution. In other embodiments, the concentration of the sodium hydroxide solution may be in the range of 0.5-2.5 M. Alternatively, an alkaline solution such as a potassium hydroxide solution or a potassium carbonate solution may be used instead of the sodium hydroxide solution in this embodiment.

[0034] S2, weighing 3.8 g of iodine (C2H5-I), slowly adding the weighed iodine to the polymer solution under low temperature conditions, and stirring for 48 hours to obtain a reaction solution containing a temperature-sensitive modified chitosan polymer;

[0035] S3, adding a 0.5M hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the reaction solution by dialysis, and then freeze-drying to prepare 1.1g of temperature-sensitive modified chitin; in other embodiments, the concentration of the hydrochloric acid solution may be in the range of 0.5-2M;

[0036] 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 0.83, the substitution degree of carboxyl is 0.44, and the substitution degree of ethyl is 0.18; the viscosity average molecular weight of the thermosensitive modified chitin is Mη=390 kDa as measured by Ubbelohde viscometer;

[0037] S4, take 0.2g of the prepared thermosensitive modified chitin, and dissolve it in injectable water at low temperature to prepare a solution with a mass concentration of 1.5% of thermosensitive modified chitin; then, divide the solution into portions and prepare a thermosensitive modified chitin hydrogel injection, A1; then, store the injection A1 at a temperature of 2°C-8°C for standby use. The injection A1 can form a thermosensitive modified chitin hydrogel at 20-40°C, that is, after the injection A1 is injected into the body, under the action of body temperature, the injection A1 will form a thermosensitive modified chitin hydrogel at the injection site.

[0038] Example 2

[0039] S1. Weigh 2 g of water-soluble carboxyethyl chitosan, dissolve the weighed carboxyethyl chitosan in a 1.5 M sodium hydroxide solution, and stir overnight at 0° C. (ice bath) to obtain a clear and viscous polymer solution;

[0040] S2, weighing 4.1 g of iodopropane (C3H7-I), slowly adding the weighed iodopropane to the polymer solution under low temperature conditions, and stirring for 56 hours to obtain a reaction solution containing a temperature-sensitive modified chitosan polymer;

[0041] S3, adding a 1M hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the reaction solution by dialysis, and then freeze-drying to prepare 1.3 g of temperature-sensitive modified chitin;

[0042] 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 0.78, the substitution degree of carboxyethyl is 0.48, and the substitution degree of propyl is 0.13; the viscosity average molecular weight of the thermosensitive modified chitin is Mη=375 kDa measured by Ubbelohde viscometer;

[0043] S4, taking 0.2g of the prepared temperature-sensitive modified chitosan, and dissolving it in injectable water at low temperature to prepare a solution with a mass concentration of 2% of the temperature-sensitive modified chitosan; then, the solution was packaged and prepared into a temperature-sensitive modified chitosan hydrogel injection, A2; thereafter, the injection A2 was stored at a temperature of 2°C-8°C for standby use.

[0044] Example 3

[0045] S1. Weigh 2 g of water-soluble carboxypropyl chitosan, dissolve the weighed carboxypropyl chitosan in a 1 M sodium hydroxide solution, and stir overnight at 0° C. (ice bath) to obtain a clear and viscous polymer solution;

[0046] S2, weighing 4.5 g of iodine propane (C3H7-I), slowly adding the weighed iodine propane to the polymer solution under low temperature conditions, and stirring for 48 hours to obtain a reaction solution containing a temperature-sensitive modified chitin polymer;

[0047] S3, adding a 1M hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the reaction solution by dialysis, and then freeze-drying to prepare 1.3 g of a temperature-sensitive modified chitin with a degree of substitution of ;

[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 0.82, the substitution degree of carboxypropyl is 0.39, and the substitution degree of propyl is 0.15; the viscosity average molecular weight of the thermosensitive modified chitin is Mη=410 kDa as measured by Ubbelohde viscometer;

[0049] S4, taking 0.2g of the prepared temperature-sensitive modified chitosan, and dissolving it in injectable water at low temperature to prepare a solution with a mass concentration of 4% of temperature-sensitive modified chitosan; then, packaging the solution and preparing a temperature-sensitive modified chitosan hydrogel injection, A3; then, storing the injection A3 at a temperature of 2°C-8°C for use.

[0050] Example 4

[0051] S1. Weigh 2 g of water-soluble carboxypropyl chitosan, dissolve the weighed carboxypropyl chitosan in a 1 M sodium hydroxide solution, and stir overnight at 0° C. (ice bath) to obtain a clear and viscous polymer solution;

[0052] S2, weighing 2 g of iodopropane (C3H7-I) and 3 g of chlorobutane (C4H9-Cl), slowly adding the weighed iodopropane and chlorobutane to the polymer solution under low temperature conditions, and stirring for 50 hours to obtain a reaction solution containing a temperature-sensitive modified chitin polymer;

[0053] S3, adding a 1M hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the reaction solution by dialysis, and then freeze-drying to prepare 1.6 g of a temperature-sensitive modified chitin with a degree of substitution of;

[0054] 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 0.81, the substitution degree of carboxypropyl is 0.39, the substitution degree of propyl is 0.06, and the substitution degree of butyl is 0.08; the viscosity average molecular weight of the thermosensitive modified chitin is Mη=415 kDa measured by Ubbelohde viscometer;

[0055] S4, taking 0.2g of the prepared temperature-sensitive modified chitosan, and dissolving it in injectable water at low temperature to prepare a solution with a mass concentration of 3% of temperature-sensitive modified chitosan; then, packaging the solution and preparing a temperature-sensitive modified chitosan hydrogel injection, A4; then, storing the injection A4 at a temperature of 2°C-8°C for use.

[0056] Example 5

[0057] S1. Weigh 2 g of water-soluble carboxypropyl chitosan, dissolve the weighed carboxypropyl chitosan in a 1 M sodium hydroxide solution, and stir overnight at 0° C. (ice bath) to obtain a clear and viscous polymer solution;

[0058] S2, weighing 1.5 g of iodine (C3H7-I) and 2 g of chlorobutane (C4H9-Cl), slowly adding the weighed iodine and chlorobutane to the polymer solution under low temperature conditions, and stirring for 72 hours to obtain a reaction solution containing a temperature-sensitive modified chitin polymer;

[0059] S3, adding a 1M hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the reaction solution by dialysis, and then freeze-drying to prepare 1.2g of a temperature-sensitive modified chitin with a degree of substitution of hydrophobic substituents;

[0060] 0.02 g of the prepared temperature-sensitive modified chitin was taken for mass spectrometry detection. 1The HNMR spectrum shows that the acetylation degree of the thermosensitive modified chitin product is 0.80, the substitution degree of carboxypropyl is 0.40, the substitution degree of ethyl is 0.08, and the substitution degree of butyl is 0.07; the viscosity average molecular weight of the thermosensitive modified chitin is Mη=430kDa measured by Ubbelohde viscometer;

[0061] S4. Take 0.2 g of the prepared temperature-sensitive modified chitosan and dissolve it in injectable water at low temperature to prepare a solution with a mass concentration of 3% of the temperature-sensitive modified chitosan. Then, divide the solution into portions and prepare a temperature-sensitive modified chitosan hydrogel injection, A5. Afterwards, store the injection A5 at a temperature of 2°C-8°C for use.

[0062] Comparative Example 1

[0063] Weigh 0.2g of the purchased carboxymethyl chitosan and dissolve it in injection water to prepare a solution with a mass concentration of 1.5% of modified chitosan. Then, the solution is packaged and prepared into a modified chitosan hydrogel injection, B1. At the same time, weigh 0.2g of the purchased carboxymethyl chitosan for mass spectrometry detection. 1 The acetylation degree of the modified chitosan product was calculated from the HNMR spectrum to be 0.83, the substitution degree of the carboxyl groups on the chitosan molecular chain to be 0.45, and the viscosity-average molecular weight of the temperature-sensitive modified chitosan was measured by an Ubbelohde viscometer to be Mη=380 kDa.

[0064] Comparative Example 2

[0065] Weigh 0.2g of the purchased carboxypropyl chitosan and dissolve it in injection water to prepare a solution with a mass concentration of 3% of modified chitosan. Then, the solution is packaged and prepared into modified chitosan hydrogel injection B2. At the same time, weigh 0.2g of the purchased carboxypropyl chitosan for mass spectrometry detection. 1 The acetylation degree of the modified chitin product was calculated from the HNMR spectrum to be 0.82, the substitution degree of the carboxypropyl group on the chitin molecular chain to be 0.39, and the viscosity-average molecular weight of the temperature-sensitive modified chitin was measured by an Ubbelohde viscometer to be Mη=410 kDa.

[0066] The temperature sensitivity, mechanical properties, degradability and cell compatibility of the injections A1-A5 prepared in the above Examples 1-5 and the injections B1-B2 prepared in Comparative Examples 1-2 were tested, and the specific test results are as follows:

[0067] 1. Temperature sensitivity test of thermosensitive modified chitin hydrogel injection

[0068] Take appropriate amounts of the injections A1 and A3 prepared in the above-mentioned Examples 1 and 3 and the injections B1 and B2 prepared in Comparative Examples 1 and 2 and test their temperature sensitivity. The specific test method is as follows: take out the injections A1, A3, B1 and B2 stored for more than 1 hour from their storage temperature, push them into 4 mL EP tubes, and place them in a water bath at 20°C, 21°C, 22°C... for 60 seconds, then immediately take out and invert the EP tube to observe the flow of the solution in the EP tube; the lowest water bath temperature corresponding to the gel in the EP tube not flowing within 30 seconds is the gel transition temperature; that is, start the test from 20°C and test in sequence with a temperature gradient of 1°C as an increasing gradient. When the gel in the EP tube does not flow within 30 seconds, stop the test, and the water bath temperature at this time is the gel transition temperature; and when the temperature rises to 40°C, if the gel in the EP tube still does not flow within 30 seconds, it is determined that the injection is not temperature sensitive. The test results show that the transition temperatures of injection A1 and injection A3 are 28°C and 30°C respectively, while injection B1 and injection B2 do not show gelation transition within the test range (20°C-40°C). It can be seen that the introduction of hydrophilic substituents and hydrophobic substituents on the thermosensitive chitin molecular chain at the same time, and the preparation of it into a thermosensitive modified chitin hydrogel injection, the injection has a reversible sol-gel transition behavior and is temperature sensitive, while the injection without the introduction of hydrophobic substituents does not show the characteristics of thermosensitive transition within 40°C.

[0069] 2. Mechanical properties test of thermosensitive modified chitin hydrogel injection

[0070] At 37°C, the storage moduli of the injections A1-A5 prepared in Examples 1-5 and the injections B1-B2 prepared in Comparative Examples 1-2 were respectively tested and compared; Figure 1 As shown, since injections B1 and B2 do not form gel under the condition of 20-40°C, their storage modulus is very low; while injections A1-A5 prepared in Examples 1-5 have relatively high storage moduli after being converted into gel state; and the storage modulus of injections A3-A5 after being converted into gel state is significantly higher than 1000Pa; thus, it can be seen that injections A3-A5 prepared in Examples 1-5 have excellent mechanical properties after being converted into gel state; and when the injection is converted into gel state, the higher its mechanical properties, the closer the gel form of the injection is to the solid state, its fluidity and softness will be weakened, and the performance and stability of being fixed in place will be further improved; therefore, when injections A1-A5 are respectively injected into the body, the injections A1-A5 will form gel in the body, and the formed gel can not only be better fixed at the injection site, but also have an isolation effect.

[0071] 3. Degradation behavior detection of thermosensitive modified chitin hydrogel injection

[0072] The injections A1-A5 prepared in the above Examples 1-5 and the injections B1-B2 prepared in Comparative Examples 1-2 were placed in dialysis bags respectively; then, they were immersed in a lysozyme buffer at 37°C, and samples were taken out after each degradation period, and the samples were weighed after absorbing the water to detect the mass loss. Figure 2 As shown, the mass loss of the injections A1-A5 prepared in Examples 1-5 is slower than that of the injections B1-B2 prepared in Comparative Examples 1-2; further, it is shown that the degradation rates of the injections A1-A5 prepared in Examples 1-5 are relatively slow after being converted into a gel state, and they have good stability; and by comparing the injections A1-A5 prepared in Examples 1-5 with each other, it can be seen that the higher the mass concentration of the temperature-sensitive modified chitosan in the injection, the slower the degradation rate of the corresponding gel, that is, as the solid content of the gel in the injections A1-A5 increases, the degradation rate slows down.

[0073] 4. Cytocompatibility testing of thermosensitive modified chitin hydrogel injection

[0074] The thermosensitive modified chitin hydrogel injections A1, A2, A5 prepared in the above-mentioned Examples 1, 2, and 5, and the modified chitin hydrogel injection B1 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, with green representing live cells and red representing dead cells, and observed by laser confocal microscopy; Figure 3 As shown, compared with the blank control group, 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 showed that the NIH 3T3 cells had a high survival rate; Figure 4As shown, compared with the blank control group, the proliferation of NIH 3T3 cells in the above four experimental groups is close to the proliferation rate of NIH 3T3 cells in the blank control group, and there is no significant difference, which shows that the co-culturing of the thermosensitive modified chitosan hydrogel injections A1, A2, A5 prepared in the above Examples 1, 2, and 5 and the modified chitosan hydrogel injection B1 prepared in Comparative Example 1 with NIH 3T3 cells has almost no effect on the growth and proliferation of NIH 3T3 cells, that is, the above-mentioned thermosensitive modified chitosan hydrogel injections A1, A2, A5 and modified chitosan hydrogel injection B1 have good compatibility with NIH 3T3 cells. It can be seen that by introducing hydrophilic substituents and hydrophobic substituents into the thermosensitive chitin molecular chain, a thermosensitive modified chitin molecule is prepared, and it is prepared into a thermosensitive modified chitosan hydrogel injection. The thermosensitive modified chitosan hydrogel injection has good biocompatibility and low cytotoxicity, and can be injected into the body as an isolation glue for use in radiotherapy.

[0075] 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 hydrogel injection, characterized in that: In the thermosensitive modified chitosan hydrogel injection, the mass concentration of the thermosensitive modified chitosan is 0.1-10%; The chitosan molecular chain of the temperature-sensitive modified chitosan contains both hydrophilic substituents and hydrophobic substituents; the acetylation degree of the temperature-sensitive modified chitosan is not less than 75%.

2. A temperature-sensitive modified chitosan hydrogel injection according to claim 1, characterized in that: On the chitosan molecular chain of the temperature-sensitive modified chitosan, the substitution degree of the hydrophilic substituent is 0.3-0.5, and the substitution degree of the hydrophobic substituent is 0.05-0.

20.

3. The temperature-sensitive modified chitosan hydrogel injection according to claim 1, characterized in that: The viscosity average molecular weight of the temperature-sensitive modified chitosan is 300-500 kDa.

4. A temperature-sensitive modified chitosan hydrogel injection according to any one of claims 1 to 3, characterized in that: The hydrophilic substituent is any one of carboxymethyl, carboxyethyl and carboxypropyl; the hydrophobic substituent is any one or more of ethyl, propyl and butyl.

5. The temperature-sensitive modified chitosan hydrogel injection according to claim 4, characterized in that: The temperature-sensitive modified chitin is synthesized by taking carboxymethyl chitosan, carboxyethyl chitosan and carboxypropyl chitosan as starting materials and any one or more of ethyl halide, propyl halide and butyl halide as hydrophobic group substitution reagents.

6. A method for preparing the temperature-sensitive modified chitosan hydrogel injection according to claim 5, characterized in that: The following steps are involved: S1. Weigh any one of carboxymethyl chitosan, carboxyethyl chitosan, and carboxypropyl chitosan, dissolve it in a sodium hydroxide solution, and stir overnight at a temperature below 5° C. to obtain a clear and viscous polymer solution; S2, weighing a hydrophobic group substitution reagent, slowly adding the weighed hydrophobic group substitution reagent to the polymer solution under low temperature conditions, and stirring to obtain a reaction solution containing a temperature-sensitive modified chitosan polymer; S3, adding hydrochloric acid solution to the reaction solution, adjusting the pH of the reaction solution to 7-7.5, removing small molecules in the solution by dialysis, and preparing temperature-sensitive modified chitin after freeze-drying; S4. Take the thermosensitive modified chitosan prepared above and dissolve it in injectable water to prepare a solution injection. The prepared solution injection forms a thermosensitive modified chitosan hydrogel under the condition of 20-40°C.

7. The method for preparing the temperature-sensitive modified chitosan hydrogel injection according to claim 6, characterized in that: In the reaction solution, the amount of the hydrophobic group substitution reagent added is 0.5-5 times the molar number of the chitin structural unit.

8. The method for preparing the temperature-sensitive modified chitosan hydrogel injection according to claim 6, characterized in that: The deacetylation degree of the carboxymethyl chitosan, the carboxyethyl chitosan and the carboxypropyl chitosan are all less than 20%.

9. The method for preparing the temperature-sensitive modified chitosan hydrogel injection according to claim 6, characterized in that: The carboxymethyl chitosan, the carboxyethyl chitosan and the carboxypropyl chitosan are synthesized by using chitosan molecules as raw materials and reacting with sodium chloroacetate, sodium chloropropionate and sodium chlorobutyrate respectively.

10. The thermosensitive modified chitosan hydrogel injection according to claim 5 is used as a medical isolation glue in radiotherapy.

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