Carboxymethyl Chitosan, Self-Healing Hydrogel and Its Preparation and Application

Abstract

The present invention discloses a carboxymethyl chitosan, and the total degree of substitution DS of the carboxymethyl chitosan t = 48% - 71%, DS- NH2 = 3% - 22%, DS ‑OH = 38% - 67%, and there is an insoluble region between pH = 5.82 - 8.15; the carboxymethyl chitosan has the structural formula shown in formula (I): #imgabs0#(I), wherein, R1 is selected from COCH3 or CH2COOH or H, and R2 is selected from CH2COOH or H. The present invention also discloses a self-healing hydrogel prepared using the carboxymethyl chitosan. The self-healing rate and self-healing mechanical properties of the self-healing hydrogel provided by the present invention are significantly improved compared with the prior art.

Description

Technical Field

[0001] The present invention relates to carboxymethyl chitosan, self-healing hydrogel and their preparation and application. Background Art

[0002] Hydrogel is a solid with a three-dimensional spatial structure composed of hydrophilic polymers crosslinked, which has shown its unique charm in various fields of life. The preparation of multifunctional self-healing hydrogels has been deeply favored by researchers. The self-healing mechanism of hydrogels includes two types. One is that after mechanical damage, it needs to rely on external stimuli such as light, heat or pH change to repair the damaged part. The second is to achieve self-repair through the interaction of internal functional groups. The former cannot achieve autonomous self-healing, while the latter can achieve autonomous healing through a dynamic network formed by dynamic covalent bonds such as imine bonds and dynamic non-covalent bonds including ionic bonds, hydrogen bonds, etc. Polysaccharide-based self-healing hydrogels constructed based on dynamic forces have attracted extensive attention from researchers and are mainly applied in biomedical fields such as wound dressings, drug delivery and targeted drug delivery, electronic skin, tissue engineering and hemostasis, etc.

[0003] Chitosan (CS) is obtained by deacetylation of chitin. It has rich sources, is cheap and easy to obtain, and is easy to modify. It has good antibacterial properties, biodegradability and biocompatibility. Preparing multifunctional hydrogels with CS as the substrate is a hot research topic. After O-carboxymethylation modification of CS, water-soluble carboxymethyl chitosan (CMCS) can be obtained. While introducing -COOH, a large number of -NH2 are retained. Coupled with the fact that the whole molecule is rich in -OH, CMCS can coordinate with metal ions, which is beneficial to the preparation of self-healing hydrogels through dynamic forces such as imine bonds, metal coordination bonds and hydrogen bonds.

[0004] Polyethylene glycol (PEG) is one of the most widely used hydrophilic polymers. It has biocompatibility, non-toxicity, protein drug resistance, non-immunogenicity and good water solubility, and has been widely applied in the fields of tissue engineering, organ protection, medicine, etc. Due to the extreme hydrophilicity of PEG, it is usually used in the form of hydrogels to absorb and retain water. Preparing self-healing hydrogels with chitosan or chitosan derivatives as the substrate and polyethylene glycol derivatives as the crosslinking agent is an important research field. Related research has proved that the hydrogel network based on the four-arm structure is more fracture-resistant than the two-arm network with the same crosslinking density.

[0005] Currently, the coordination of carboxymethyl chitosan (CMCS) with metal ions has also been widely studied and significant results have been achieved, including with Fe 3+ 、Al 3+ 、Ag + 、Cu 2+ 、Zn 2+ 、Ca 2+A salt solution such as etc. is used as a cross-linking agent to prepare a hydrogel, obtaining a series of self-healing hydrogels with functions such as injectability, multi-responsiveness, excellent mechanical properties, or antibacterial properties. However, most of the prepared self-healing hydrogels need to be improved in terms of self-healing rate and self-healing mechanical properties. Summary of the Invention

[0006] The present invention aims to provide a self-healing hydrogel with significantly improved self-healing rate and self-healing mechanical properties compared to the prior art. By selectively substituting the C-6 -OH and C-2 -NH2 of chitosan, the water solubility of chitosan is effectively improved. The carboxymethyl chitosan-based physical-chemical double-crosslinked self-healing hydrogel provided by the present invention improves the problems of poor mechanical properties and self-healing rate of self-healing hydrogels in the prior art.

[0007] As an aspect of the present invention, it relates to a carboxymethyl chitosan, and the total degree of substitution DS of the carboxymethyl chitosan t = 48% - 71%, DS- NH2 = 3% - 22%, DS -OH = 38% - 67%, and there is an insoluble region between pH = 5.82 - 8.15. The carboxymethyl chitosan has the structural formula shown in formula (I):

[0008]

[0009] Wherein,

[0010] R1 is selected from COCH3 or CH2COOH or H,

[0011] R2 is selected from CH2COOH or H.

[0012] Preferably, the total degree of substitution DS of the carboxymethyl chitosan t = 64%, DS- NH2 = 58%, DS -OH = 6%, and there is an insoluble region between pH = 6.60 - 7.40.

[0013] As another aspect of the present invention, it relates to a method for preparing the above carboxymethyl chitosan, including:

[0014] (1) Dissolve sodium hydroxide in water, and sequentially add chitosan (CS) and isopropanol to carry out the alkalization reaction of chitosan to obtain alkalized chitosan;

[0015] (2) Slowly add chloroacetic acid (MCAA) dissolved in isopropanol to the alkalized chitosan obtained in the above step (1) to carry out a selective grafting reaction, and then add absolute ethanol to quench the reaction;

[0016] (3) Perform suction filtration. Wash the solid obtained by suction filtration with EtOH (such as 80%) until the washing liquid is neutral, and dry it (such as at 70 °C) to obtain carboxymethyl chitosan.

[0017] In the step (1), the viscosity of the chitosan is <200 mPa·s, the degree of deacetylation (DD) ≥ 95%, the alkalization temperature is room temperature, the time is 1 h, the mass ratio of sodium hydroxide to chitosan is 1:0.58 - 2.33, and the volume ratio of water to isopropanol is 1:4.

[0018] In the step (2), the mass ratio of the added chloroacetic acid to the mass of sodium hydroxide in the step (1) is 1:1. The volume ratio of the added isopropanol in the step (2) to the water in the step (1) is 1:1. The reaction time is 2 - 5 h, and the reaction temperature is 20 - 60 °C.

[0019] As another aspect of the present invention, it relates to a self-healing hydrogel, and the self-healing hydrogel is a physical-chemical double-crosslinked self-healing hydrogel based on carboxymethyl chitosan, wherein the carboxymethyl chitosan is the above-mentioned carboxymethyl chitosan.

[0020] As still another aspect of the present invention, it relates to a method for preparing the above self-healing hydrogel, including:

[0021] Add a metal salt solution (ZnCl2, FeCl3, AgNO3) and a 4r-PEG-CHO solution to an aqueous carboxymethyl chitosan solution (with mass fractions of 1.5%, 3%, 5%), and stir rapidly to prepare a physical-chemical double-crosslinked self-healing hydrogel.

[0022] Adjust the aqueous carboxymethyl chitosan solution to pH = 5 - 6 with 1% HOAc. The metal salt solution (ZnCl2, FeCl3, AgNO3) is prepared with 1% HOAc, and the concentration is 0.30 mol·L -1 , and the 4r-PEG-CHO solution is directly prepared with water, and the concentration is 0.03 mol·L -1 .

[0023] In the method for preparing the above self-healing hydrogel, it is preferred to add different volumes of ZnCl2 solution and 4r-PEG-CHO solution to 10 mL of 3% wt CMCS aqueous solution, and add water to adjust the solid content to 3.04%.

[0024] As still another aspect of the present invention, it relates to the application of the above self-healing hydrogel in wound dressings, drug delivery and targeted drug delivery, electronic skin, tissue engineering, and hemostatic drugs.

[0025] The present invention realizes the selective substitution of the C-6 position -OH and C-2 position -NH2 of chitosan, effectively improving the water solubility of chitosan. The present invention uses aldehyde-terminated tetra-arm polyethylene glycol (4r-PEG-CHO) and ZnCl2 as crosslinking agents together to prepare a self-healing hydrogel by constructing a physical-chemical double crosslinked network, improving the problems of poor mechanical properties and slow self-healing rate of the self-healing hydrogel. Description of the Drawings

[0026] Figure 1 1H NMR spectrum of the carboxymethyl chitosan prepared in Example 1 of the present invention; 1

[0027] Figure 2 FT-IR spectrum of the carboxymethyl chitosan prepared in Example 1 of the present invention;

[0028] Figure 3 pH titration curve of the carboxymethyl chitosan prepared in Example 1 of the present invention;

[0029] Figure 4 First-order and second-order derivative curves of the pH titration curve of the carboxymethyl chitosan prepared in Example 1 of the present invention;

[0030] Figure 5 FT-IR spectra of the freeze-dried hydrogels obtained from the 3% wt aqueous solution (A) of the carboxymethyl chitosan prepared in Example 1 of the present invention and those obtained in Examples 19 (B), 18 (C), and 11 (D);

[0031] Figure 6 Test graphs of the storage modulus and loss modulus for Examples 1 and 6-9 of the present invention

[0032] Figure 7 Test graphs of the storage modulus and loss modulus for the hydrogels in Group A;

[0033] Figure 8 Test graphs of the storage modulus and loss modulus for the hydrogels in Group B;

[0034] Figure 9 Change trend of the storage modulus for the gels in Group A;

[0035] Figure 10 Change trend of the storage modulus for the gels in Group B;

[0036] Figure 11 Macroscopic self-repair graph of the hydrogel prepared in Example 2;

[0037] Figure 12 Dynamic strain sweep graph of the hydrogel prepared in Example 2;

[0038] ​Figure 13 Alternating strain scan of the hydrogel prepared in Example 2;

[0039] Figure 14 Injectability test of the hydrogel prepared in Example 2;

[0040] Figure 15 pH sensitivity test of the hydrogel prepared in Example 16. Detailed implementation

[0041] The inventors found that chitosan could not form a self-healing hydrogel. Therefore, the inventors carried out carboxymethylation modification on chitosan. After mixing the alkalized alkaline chitosan and chloroacetic acid in an organic solvent, a carboxymethylation reaction was carried out to obtain carboxymethyl chitosan, which was used to prepare a self-healing hydrogel.

[0042] Compared with the rich physical cross-linking methods, the chemical cross-linking methods of chitosan and its derivative self-healing hydrogels are mostly limited to Schiff base reactions. Preparing self-repairing hydrogels only using imine bonds will result in weak mechanical strength of the gel. Therefore, in the present invention, chitosan was carboxymethylated, and CMCS aqueous solution was cross-linked with different contents of tetra-armed polyethylene glycol with terminal aldehyde groups (4r-PEG-CHO) and ZnCl2 under weak acid conditions to prepare a series of pH-sensitive injectable and rapidly self-healing hydrogels. The introduction of metal ions improved the deficiency that preparing self-repairing hydrogels only using imine bonds would lead to weak mechanical strength of the gel.

[0043] The inventors first prepared carboxymethyl chitosans with different degrees of substitution, grafting sites, and solubility by changing the reaction time, reaction temperature, and feed ratio.

[0044] Experimental steps:

[0045] (1) Dissolve sodium hydroxide in water, and successively add chitosan (CS) and isopropanol to carry out the alkalization reaction of chitosan to obtain alkalized chitosan;

[0046] (2) Slowly add chloroacetic acid (MCAA) dissolved in isopropanol to the alkalized chitosan obtained in step (1) to carry out a selective grafting reaction. After reacting for a period of time, add absolute ethanol to quench the reaction;

[0047] (3) Carry out suction filtration, wash the solid obtained after suction filtration with 80% EtOH until the washing solution is neutral, and dry it at 70 °C to obtain carboxymethyl chitosan.

[0048] Some experimental parameters and experimental results are shown in Table 1.

[0049] The viscosity of the chitosan used is < 200 mPa·s, the degree of deacetylation (DD) is ≥ 95%, the alkalization temperature is room temperature, the time is 1 h, and the mass ratio of sodium hydroxide to chitosan is 1:0.58 - 2.33. In step (1), the volume ratio of water to isopropanol is 1:4. In step (2), the mass ratio of chloroacetic acid to sodium hydroxide is 1:1, the volume ratio of isopropanol to the water during alkalization is 1:1, the reaction time is 2 - 5 h, and the reaction temperature is 20 - 60 °C.

[0050] Table 1 Effects of reaction time, reaction temperature, and feed ratio on the degree of substitution and solubility of carboxymethyl chitosan

[0051]

[0052] 1-a-1-d. Effect of reaction time on CMCS;

[0053] 2-a-2-e. Effect of reaction temperature on CMCS;

[0054] 3-a-3-d.m cs :m NaOH :m MCAA Effect on CMCS.

[0055] As the reaction time increases, the DS of CMCS t shows a trend of first increasing and then decreasing, the DS -OH gradually decreases, the pH range of the insoluble region is relatively narrow, and when the reaction time is 4 h, CMCS with the highest DS can be obtained. As the reaction temperature increases, the DS t gradually decreases, the DS t rapidly decreases, and when the DS -OH is relatively high, the solubility of CMCS becomes poor. Controlling the temperature at 20 - 25 °C is beneficial to the substitution of -OH at the C-6 position. As the mass ratio of m -NH2 :m cs :m NaOH :m MCAA increases, the DS -OH gradually decreases, the DS t shows a trend of first remaining unchanged and then decreasing. Controlling the m cs :m NaOH :m MCAAIt is between 1:0.58:0.58 and 1:1.75:1.75, which is more conducive to the occurrence of the substitution reaction. In the present invention, the mass ratio of sodium hydroxide to chitosan in the alkalization reaction is 1:0.58 - 2.33, further preferably 1:0.58 and 1:1.75. The grafting reaction temperature is 20 - 60 °C, further preferably 20 - 25 °C, and the reaction time is 2 - 5 h, further preferably 3 - 5 h. Based on Table 1, the inventors used 1-a (Example 6), 1-b (Example 7), 1-c (Example 8), 3-a (Example 1), and 3-c (Example 9) for further experiments.

[0056] In the examples of the present invention, the reaction process for preparing carboxymethyl chitosan from chitosan can be referred to as follows:

[0057]

[0058] R = -COCH3 / -H,

[0059] R1 = -COCH3 / -CH2COOH / -H,

[0060] R2 = -CH2COOH / -H, and at least one of the structural units has R1 and R2 not both being H.

[0061] After obtaining carboxymethyl chitosan, a metal salt solution (ZnCl2, FeCl3, or AgNO3) and a 4r-PEG-CHO solution are added to aqueous carboxymethyl chitosan solutions with mass fractions of 1.5%, 3%, and 5% respectively, and stirred rapidly to prepare a physically-chemically double-crosslinked self-healing hydrogel.

[0062] After obtaining carboxymethyl chitosan, it is formulated into an aqueous solution with a pH range of 5 - 6 and a mass fraction of 1.5%, 3%, and 5%. A metal salt solution (ZnCl2, FeCl3, AgNO3) and a 4r-PEG-CHO solution are added, and all the raw materials are rapidly stirred at room temperature to form a physically-chemically double-crosslinked self-healing hydrogel within one minute.

[0063] In the present invention, the aqueous carboxymethyl chitosan solutions with mass fractions of 1.5%, 3%, and 5% are further preferably 3%, and the metal salt solution (ZnCl2, FeCl3, AgNO3) is further preferably ZnCl2. The inventors used 1-a (Example 1), 1-b (Example), 1-c (Example), 3-a (Example 2), and 3-c (Example 1) for further experiments.

[0064] The formation principle of the physically-chemically double-crosslinked self-healing hydrogel based on carboxymethyl chitosan in the present invention can be referred to as follows:

[0065]

[0066] The formation process of the hydrogel is that amino groups and aldehyde groups react to form Schiff base bonds, and Zn 2+ and -COO - undergo strong coordination and gradually crosslink to construct a physical-chemical double crosslinked network.

[0067] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention.

[0068] Example 1

[0069] Dissolve 3.48 g of NaOH in 12 mL of water, and successively add 6 g of chitosan (CS) and 48 mL of isopropanol (m NaOH :m CS = 0.58:1, V H2O :V i-PrOH = 1:4), stir at room temperature for 1 h for alkalization.

[0070] Dissolve 3.48 g of chloroacetic acid (MCAA) in 12 mL of isopropanol (m NaOH :m MCAA = 1:1, V H2O :V i-PrOH = 1:1), and gradually add it to the above alkalized system. Stir and react at 25 °C for 4 h, and add 50 mL of absolute ethanol to terminate the reaction.

[0071] Wash the solid with 80% ethanol until the washing liquid is neutral to remove excess alkali and salt, and dry it at 70 °C to remove ethanol and water to obtain white powdery carboxymethyl chitosan. The total substitution degree DS t = 64%, DS- NH2 = 6%, DS- OH = 58%, and it is insoluble between pH = 6.60 - 7.40.

[0072] Prepare a 3% aqueous solution of CMCS with the obtained carboxymethyl chitosan, and adjust the pH value of the system from 1% HOAc to 6. Prepare a 0.30 mol·L -1 FeCl3 solution with 1% HOAc. Prepare a 0.03 mol·L -1 4r-PEG-CHO solution with pure water.

[0073] Add 0.2 mL of FeCl3 and 0.4 mL of 4r-PEG-CHO to 10 mL of CMCS solution, add water to adjust the solid content to 3.04%, and stir rapidly at room temperature. No gel is formed, and red-brown Fe(OH)3 precipitates due to the hydrolysis of FeCl3.

[0074] Example 2

[0075] The preparation of carboxymethyl chitosan was the same as that in Example 1

[0076] The obtained carboxymethyl chitosan was formulated into a 3% CMCS aqueous solution, and the pH value of the system was adjusted to 6 with 1% HOAc. A 0.30 mol·L -1 ZnCl2 solution was prepared with 1% HOAc. A 0.03 mol·L solution was prepared with pure water -1 4r-PEG-CHO solution.

[0077] 0.2 mL of ZnCl2 and 0.4 mL of 4r-PEG-CHO were added to 10 mL of the CMCS solution, and water was added to adjust the solid content to 3.04%. It was rapidly stirred at room temperature, and a gel was formed within 1 min (denoted as A1).

[0078] Example 3

[0079] The preparation of carboxymethyl chitosan was the same as that in Example 1

[0080] The obtained carboxymethyl chitosan was formulated into a 3% CMCS aqueous solution, and the pH value of the system was adjusted to 6 with 1% HOAc. A 0.30 mol·L -1 AgNO3 solution was prepared with 1% HOAc. A 0.03 mol·L solution was prepared with pure water -1 4r-PEG-CHO solution.

[0081] 0.2 mL of AgNO3 and 0.4 mL of 4r-PEG-CHO were added to 10 mL of the CMCS solution, and water was added to adjust the solid content to 3.04%. It was rapidly stirred at room temperature, and a gel was formed within 1 min. Since AgNO3 is unstable, it is easily decomposed by light and easily reacts with reducing organic substances. After standing overnight, the three-dimensional structure of the hydrogel collapsed and turned into a black solution.

[0082] Example 4

[0083] The preparation of carboxymethyl chitosan was the same as that in Example 1

[0084] The obtained carboxymethyl chitosan was formulated into a 1.5% CMCS aqueous solution, and the pH value of the system was adjusted to 6 with 1% HOAc. A 0.30 mol·L -1 ZnCl2 solution was prepared with 1% HOAc. A 0.03 mol·L solution was prepared with pure water -1 4r-PEG-CHO solution.

[0085] 0.2 mL of ZnCl2 and 0.4 mL of 4r-PEG-CHO were added to 10 mL of the CMCS solution, and it was rapidly stirred at room temperature. Since the concentration of the CMCS solution was too low, the gel formation rate became slower, and a weak gel was formed within 4 min.

[0086] Example 5

[0087] The preparation of carboxymethyl chitosan is the same as that in Example 1

[0088] The obtained carboxymethyl chitosan was formulated into a 5% CMCS aqueous solution, and the pH value of the system was adjusted to 6 with 1% HOAc. 0.30 mol·L -1 ZnCl2 solution was prepared with 1% HOAc. 0.03 mol·L was prepared with pure water -1 4r-PEG-CHO solution.

[0089] 0.2 mL of ZnCl2 and 0.4 mL of 4r-PEG-CHO were added to 10 mL of CMCS solution, and the mixture was rapidly stirred at room temperature to form a gel within 1 min. Due to the significant increase in the viscosity of the system, stirring was difficult and it was not easy to form a uniform gel.

[0090] Example 6

[0091] 6.96 g of NaOH was dissolved in 12 mL of water, and 6 g of chitosan (CS) and 48 mL of isopropanol (m NaOH :m CS =1.75:1, V H2O :V i-PrOH =1:4) were added in sequence, and the mixture was stirred at room temperature to form an alkalization system, and alkalized for 1 h.

[0092] 6.96 g of chloroacetic acid (MCAA) was dissolved in 12 mL of isopropanol (m NaOH :m MCAA =1:1, V H2O :V i-PrOH =1:1), and it was added dropwise to the above alkalization system, stirred and reacted at 20 °C for 5 h, and 50 mL of absolute ethanol was added to terminate the reaction.

[0093] Filtered by suction, the solid obtained by suction filtration was washed with 80% ethanol until the washing liquid was neutral to remove excess alkali and salt, and dried at 70 °C to remove ethanol and water, and white powdery carboxymethyl chitosan could be obtained, with a total substitution degree DS t =53%, DS -NH2 =15%, DS -OH =38%, and it was insoluble between pH = 5.87 - 7.53.

[0094] The obtained carboxymethyl chitosan was formulated into a 3% CMCS aqueous solution, and the pH value of the system was adjusted to 5 with 1% HOAc. 0.30 mol·L was prepared with 1% HOAc -1 ZnCl2 solution. 0.03 mol·L was prepared with pure water -1 4r-PEG-CHO solution.

[0095] Add 0.2 mL of ZnCl₂ solution and 0.4 mL of 4r-PEG-CHO solution to 10 mL of aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir at room temperature to form a hydrogel.

[0096] Example 7

[0097] Dissolve 6.96 g of NaOH in 12 mL of water, and successively add 6 g of chitosan (CS) and 48 mL of isopropanol (m NaOH :m CS =1.75:1, V H2O :V i-PrOH =1:4), and stir at room temperature to form an alkalization system, and alkalize for 1 h.

[0098] Dissolve 6.96 g of chloroacetic acid (MCAA) in 12 mL of isopropanol (m NaOH :m MCAA =1:1, V H2O :V i-PrOH =1:1), and dropwise add it to the above alkalization system, stir and react at 20 °C for 4 h, and add 50 mL of absolute ethanol to terminate the reaction.

[0099] Perform suction filtration, wash the solid obtained by suction filtration with 80% ethanol until the washing liquid is neutral to remove excess alkali and salt, and dry at 70 °C to remove ethanol and water, and white powdery carboxymethyl chitosan can be obtained. The total degree of substitution DS t =71%, DS -NH2 =4%, DS -OH =67%, and it is insoluble between pH = 5.99 - 8.15.

[0100] Prepare an aqueous CMCS solution with a mass fraction of 3% from the obtained carboxymethyl chitosan, and adjust the pH value of the system from 1% HOAc to 5. Prepare a 0.30 mol·L -1 ZnCl₂ solution with 1% HOAc. Prepare a 0.03 mol·L -1 4r-PEG-CHO solution with pure water.

[0101] Add 0.2 mL of ZnCl₂ solution and 0.4 mL of 4r-PEG-CHO solution to 10 mL of aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir at room temperature to form a hydrogel.

[0102] Example 8

[0103] Dissolve 6.96 g of NaOH in 12 mL of water, and successively add 6 g of chitosan (CS) and 48 mL of isopropanol (m NaOH :m CS =1.75:1, VH2O :V i-PrOH = 1:4), stir at room temperature to form an alkalization system, and alkalize for 1 h.

[0104] Dissolve 6.96 g of chloroacetic acid (MCAA) in 12 mL of isopropanol (m NaOH :m MCAA = 1:1, V H2O :V i-PrOH = 1:1), dropwise add it to the above alkalization system, stir and react at 20 °C for 3 h, and add 50 mL of absolute ethanol to terminate the reaction.

[0105] Filter by suction, wash the solid obtained by suction filtration with 80% ethanol until the washing liquid is neutral to remove excess alkali and salt, and dry at 70 °C to remove ethanol and water to obtain white powdery carboxymethyl chitosan, with a total substitution degree DS t = 48%, DS -NH2 = 3%, DS -OH = 45%, and it is insoluble between pH = 6.80 - 7.51.

[0106] Prepare a 3% CMCS aqueous solution with the obtained carboxymethyl chitosan, and adjust the pH value of the system from 1% HOAc to 6. Prepare a 0.30 mol·L -1 ZnCl2 solution with 1% HOAc. Prepare a 0.03 mol·L -1 4r-PEG-CHO solution with pure water.

[0107] Add 0.2 mL of ZnCl2 solution and 0.4 mL of 4r-PEG-CHO solution to 10 mL of CMCS aqueous solution, add water to adjust the solid content to 3.04%, and quickly stir at room temperature to form a hydrogel.

[0108] Example 9

[0109] Dissolve 6.96 g of NaOH in 12 mL of water, and successively add 6 g of chitosan (CS) and 48 mL of isopropanol (m NaOH :m CS = 1.75:1, V H2O :V i-PrOH = 1:4), stir at room temperature to form an alkalization system, and alkalize for 1 h.

[0110] Dissolve 6.96 g of chloroacetic acid (MCAA) in 12 mL of isopropanol (m NaOH :m MCAA = 1:1, V H2O :V i-PrOH = 1:1), dropwise add it to the above alkalization system, stir and react at 25 °C for 4 h, and add 50 mL of absolute ethanol to terminate the reaction.

[0111] Perform suction filtration. Wash the solid obtained by suction filtration with 80% ethanol until the washing liquid is neutral to remove excess alkali and salts. Dry it at 70 °C to remove ethanol and water, and white powdery carboxymethyl chitosan can be obtained, with a total substitution degree DS t = 66%, DS -NH2 = 22%, DS -OH = 44%, and it is insoluble between pH = 5.82 - 7.28.

[0112] Prepare an aqueous solution of CMCS with a mass fraction of 3% from the obtained carboxymethyl chitosan. Adjust the pH value of the system from 1% HOAc to 5. Prepare a 0.30 mol·L -1 ZnCl2 solution with 1% HOAc. Prepare a 0.03 mol·L -1 4r-PEG-CHO solution with pure water.

[0113] Add 0.2 mL of ZnCl2 solution and 0.4 mL of 4r-PEG-CHO solution to 10 mL of the CMCS aqueous solution, add water to adjust the solid content to 3.04%, and rapidly stir at room temperature to form a hydrogel

[0114] Example 10

[0115] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0116] Add 0.3 mL of ZnCl2 and 0.4 mL of 4r-PEG-CHO to 10 mL of the CMCS aqueous solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature. A hydrogel can be obtained within one minute (denoted as A2).

[0117] Example 11

[0118] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0119] Add 0.4 mL of ZnCl2 and 0.4 mL of 4r-PEG-CHO to 10 mL of the CMCS aqueous solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature. A hydrogel can be obtained within one minute (denoted as A3 in Group A and B3 in Group B).

[0120] Example 12

[0121] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0122] Add 0.5 mL of ZnCl₂ and 0.4 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a hydrogel (denoted as A4) within one minute.

[0123] Example 13

[0124] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0125] Add 0.6 mL of ZnCl₂ and 0.4 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain (denoted as A5).

[0126] Example 14

[0127] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0128] Add 0.4 mL of ZnCl₂ and 0.2 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a hydrogel (denoted as B1).

[0129] Example 15

[0130] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0131] Add 0.4 mL of ZnCl₂ and 0.3 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a hydrogel (denoted as B2).

[0132] Example 16

[0133] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0134] Add 0.4 mL of ZnCl₂ and 0.5 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a hydrogel (denoted as B4).

[0135] Example 17

[0136] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0137] Add 0.4 mL of ZnCl₂ and 0.6 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a physically and chemically double-crosslinked self-healing hydrogel based on carboxymethyl chitosan (denoted as B5) within one minute.

[0138] Example 18

[0139] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0140] Add 0.4 mL of ZnCl₂ to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a hydrogel (denoted as A0) within one minute.

[0141] Example 19

[0142] The preparation of carboxymethyl chitosan and other solutions is the same as in Example 1.

[0143] Add 0.4 mL of 4r-PEG-CHO to 10 mL of an aqueous CMCS solution, add water to adjust the solid content to 3.04%, and rapidly stir all the raw materials at room temperature to obtain a hydrogel (denoted as B0) within one minute.

[0144] Structure Characterization

[0145] Use a digital superconducting nuclear magnetic resonance spectrometer (400M, AVIII400 HD) from Bruker GmbH, Germany, with D₂O as the solvent, to perform 1 ¹H NMR testing on the carboxymethyl chitosan prepared in Example 1, and the results are as Figure 1 shown. Figure 1 The results show that the peak at 2.55 ppm is the proton absorption peak of -CH₂- in -NCH₂COOH (H-9), and the peak at 2.06 ppm is the proton absorption peak of the methyl group in -COCH₃ of the non-deacetylated part of CS (H-8), indicating that carboxymethylation has successfully occurred on the 6-OH of chitosan and partially on -NH₂.

[0146] Use a Fourier transform infrared spectrometer (Bruker Tensor 27, Germany), in transmission mode, with the KBr pellet method, to perform FT-IR testing on the carboxymethyl chitosan prepared in Example 1, and scan the range from 4000 to 400 cm -1 , and the results are as Figure 2 shown. Figure 2 The results show that the characteristic absorption peaks of CMCS are mainly at 1604 cm -1 、1421 cm -1 (-COO -In the vibration absorption peaks of C=O and C-O, it was proved that -COO - existed. The C-N vibration absorption peak at 1321 cm -1 did not increase significantly, indicating that carboxymethylation basically did not occur on -NH2, and most grafted on -OH.

[0147] The degree of substitution of the carboxymethyl chitosan prepared in Example 1 and Examples 6-9 was measured by conductometric titration. 0.05 g of the sample was dissolved in 50 mL of 0.10 mol·L -1 HCl solution, and 0.25 mol·L -1 NaOH was used to continuously titrate the system. The titration was carried out in parallel three times, and the pH value and the corresponding volume of the system were recorded to plot the potentiometric titration curve. Then, according to the first-order ( Figure 3 ) and second-order derivative curves ( Figure 4 ), the inflection point was found and calculated by the following formula:

[0148]

[0149] Among them, V1, V2, and V3 are the end points of the titrations of HCl, -COOH, and -NH3 + respectively. DS t is the total degree of substitution, DS -NH2 is the degree of substitution occurring on -NH2, and DS -OH is the degree of substitution occurring on -OH.

[0150] The experimental results showed that for CMCS in Example 1, DS t = 64%, DS -NH2 = 6%, DS -OH = 58%; for CMCS in Example 6, DS t = 53%, DS -NH2 = 15%, DS -OH = 38%; for CMCS in Example 7, DS t = 71%, DS -NH2 = 4%, DS -OH = 67%; for CMCS in Example 8, DS t = 48%, DS -NH2 = 3%, DS -OH = 45%; for CMCS in Example 9, DS t = 66%, DS- NH2 = 22%, DS- OH = 44%.

[0151] Using a Fourier transform infrared spectrometer, in transmission mode, the structures of the hydrogels (Examples 4, 11, 12) after water removal by the freeze dryer and the CMCS solution (Example 2) were characterized. The KBr pellet method was used, and the scanning range was 4000 - 400 cm -1 , and the experimental results are as Figure 5 shown. By comparing the absorption peaks of A and B in the range of 1637 - 1564 cm -1 , it was found that the absorption peaks became narrower. This is because the formation of imine bonds (C=N) weakened the N-H stretching vibration. After adding ZnCl2 (C, D), the absorption peaks in this range became wider again, proving that Zn 2+ formed metal coordination bonds with -COOH, and the C-O vibration absorption peak of -OH on CMCS at 1075 cm -1 weakened, proving that Zn 2+ coordinated with -OH. It shows that both imine bonds and metal bonds exist in this hydrogel.

[0152] Performance testing

[0153] Rheological testing

[0154] The storage modulus (G') and loss modulus (G") of the gel were tested by a rotational rheometer (MARS III, Haake Karlsruhe, Germany). First, the hydrogel sample was placed on a flat plate and then cut into a hydrogel disk with a diameter of 60 mm and a thickness of 1.5 mm. Frequency scanning was carried out under the conditions of a strain of 1% and a temperature of 25 °C, and the scanning range was 10 - 100 Rad / s.

[0155] The rheological tests were carried out on the hydrogels prepared in Example 2 and Examples 6 - 9 ( Figure 6 ), and the magnitude of their storage modulus (G') was Example 2 > 7 > 9 > 6 > 8. It can be seen that at the same crosslinker content, the storage modulus of the hydrogel is related to the substitution degree of -OH and the pH value of the solution. The substitution of -NH2 is not conducive to the formation of imine bonds. Example 2 has better water solubility under the condition of a higher -OH substitution degree and can prepare hydrogels under higher pH conditions. Due to the influence of water solubility, Examples 6 - 9 have a lower pH when preparing hydrogels, and the formed imine bonds are more unstable and the metal bonds are weaker.

[0156] The dynamic frequency scanning test results of the hydrogels in Group A (Examples 2, 10 - 13, 18) and Group B (Examples 11, 14 - 17, 19) are respectively as Figure 7 , Figure 8 shown. The storage modulus (G') of each sample is greater than the loss modulus (G") within the tested frequency scanning range, proving that all samples exist in the form of solids and have a stable three-dimensional network structure of hydrogels.

[0157] The G' of two groups was analyzed respectively: The changing trend of G' in Group A is as Figure 9 shown. For the hydrogels of Examples 2, 10 - 12, with the increase of Zn 2+ content, the cross - linking density increases, and G' also increases. However, when the content of Zn 2+ is further increased, the G' of Example 13 decreases instead. This is because at low concentrations, Zn 2+ tends to coordinate with -COO - , and the interaction between Zn 2+ and -COO - is stronger than the bonding with -OH and -NH2. While at high concentrations, -OH and -NH2 participate in coordination more, thus G' decreases; The G' of all gels containing Zn 2+ is higher than that of Example 18, that is, the gel containing only 4r - PEG - CHO, indicating that the addition of Zn 2+ helps to increase G', and the gel has better elasticity.

[0158] The changing trend of G' in Group B is as Figure 10 shown. The influence of the content of 4r - PEG - CHO on the G' of the gel is relatively complex. Mainly because it is a flexible chain segment itself, which will reduce the hardness of the gel, while at the same time it can increase the number of cross - linking points and enhance the mechanical properties of the gel. Eventually, the size of G' is affected by both of them. The G' of the gels of Examples 14, 15, and 11 decreases in turn, mainly due to the influence of flexibility. The G' of the gels of Examples 11, 16, and 17 shows a trend of first increasing and then decreasing, which is caused by the uncertainty of the influence of flexibility and cross - linking density on the gel; The G' of the gels is either enhanced or weakened compared with Example 19, proving the two - sided nature of the influence of 4r - PEG - CHO on the gel properties.

[0159] Therefore, by regulating the content of the cross - linker and constructing a physical - chemical double cross - linked network, the mechanical properties of the gel can be effectively enhanced.

[0160] Self - healing performance test

[0161] Taking the sample of Example 2 as an example, the self - repair performance of the hydrogel was verified by macroscopic self - repair test and quantitative test. In the macroscopic self - repair test, three cylindrical plastic molds were prepared. Two of them were filled with samples stained with rhodamine 6G, and the other one was filled with an unstained sample. They were stacked cross -wise, as Figure 11As shown, the healing condition was examined after placing at 25 °C for 1 min. In the quantitative test, an alternating strain sweep test was carried out by a dynamic rheometer (Discovery HR-2, TA Instruments, USA) to examine the self-healing property of the gel. First, the temperature was controlled at 25 °C, the scanning frequency was 10 rad / s, and a dynamic strain sweep was performed in the strain range of 1% - 1500% to determine the sol-gel transition point of the gel. Then, it was tested at a large strain of 700% for 120 s, and then at a small strain of 1% for 120 s. The test was repeated 3 times, and the self-healing ability was judged by observing the changes in the storage modulus and loss modulus.

[0162] In the macroscopic self-healing experiment ( Figure 11 ), three molds were connected by the healed hydrogel and did not separate under the action of gravity, indicating that the hydrogel can achieve self-healing within a short time (<1 min). The results of the dynamic strain sweep are shown in Figure 12 . As the strain was gradually increased, the three-dimensional structure of the hydrogel was damaged, and G”>G’, indicating its shear-thinning property. Then, an alternating strain sweep test was carried out by controlling the large strain at 700% and the small strain at 1% ( Figure 13 ). In three cycles, the magnitudes of the storage modulus and loss modulus also changed periodically, indicating that the hydrogel can repair its damaged part and reconstruct its three-dimensional structure after the structure is damaged. The G’ in the second and third cycles was much smaller than that in the first cycle, mainly because the gel viscosity was too strong and overflowed during the test. By comparing the data of the second and third cycles, it can be proved that the gel can basically achieve self-healing after being damaged and recover to the G’ before damage.

[0163] Injectability test

[0164] The sample of Example 2 was selected for the injectability test. First, an appropriate amount of the sample was stained with rhodamine 6G for observation, and then the sample was sucked into a syringe and extruded through a needle to observe injectability. The syringe specification was 1 mL and the needle diameter was 8 mm.

[0165] The experimental results are shown in Figure 14 . The hydrogel could uniformly pass through a needle with a diameter of 8 mm and write the word "FPL", indicating that the hydrogel has injectability. This is because when the gel passes through a fine needle, the pressure causes partial dissociation of the imine bond and metal coordination bond, weakening the internal force of the gel, so it can be extruded and molded under a higher pressure.

[0166] pH sensitivity test

[0167] Take 2 mL of the hydrogel sample of Example 16 stained with rhodamine 6G, add 0.3 mL of 0.25 mol·L -1 HCl to destroy the three-dimensional spatial structure of the hydrogel, and then successively add 0.1 mL of 0.25 mol·L -1NaOH, 0.1 mL of 0.25 mol·L -1 HCl, observe the state of the sample and judge its pH sensitivity.

[0168] The experimental results are as Figure 15 shown. The hydrogel showed good pH sensitivity under the condition of pH < 6. When adjusted to strong acidity by adding HCl, the imine bond hydrolyzed, and the interaction force between Zn 2+ and -COOH weakened, the three-dimensional network structure of the hydrogel was damaged, and it was in a fluid state. After adding NaOH, chemical bonds and metal bonds were re-formed, and the three-dimensional structure was restored, showing a solid state. Repeating the addition of HCl and NaOH again, the hydrogel could still dissociate and re-form, proving its good pH sensitivity.

[0169] In summary, in this study, CMCS was used as the substrate, 4r-PEG-CHO and ZnCl2 were used as cross-linking agents to construct a physical-chemical double cross-linked network, and a new injectable self-healing CMCS / 4r-PEG-CHO / Zn 2+ hydrogel with pH sensitivity was successfully prepared. When the Schiff base bond and the metal bond are combined, the G' of the gel can be effectively improved by regulating the content of the cross-linking agent, enhancing its elastic mechanical properties. At the same time, due to the increase in the number of cross-linking points in the dynamic network, the gel not only maintains its own mechanical properties but also has excellent self-repair performance, proving the positive effect of the combination of metal bonds and imine bonds on the gel performance, providing a new idea for improving the disadvantages of weak mechanical properties and slow self-healing rate of self-healing hydrogels.

[0170] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Self-healing hydrogel, characterized in that, The self-healing hydrogel is a carboxymethyl chitosan-based physical-chemical double-crosslinked self-healing hydrogel, wherein the total degree of substitution DS of the carboxymethyl chitosan t = 48% - 71%, DS- NH2 = 3% - 22%, DS -OH = 38% - 67%, and there is an insoluble region between pH = 5.82 - 8.15; the carboxymethyl chitosan has the structural formula shown in formula (I): （I）, Among them, R1 is selected from COCH3 or CH2COOH or H, R2 is selected from CH2COOH or H; The formation process of the self-healing hydrogel is that the amino group of the 3 wt% carboxymethyl chitosan aqueous solution reacts with the aldehyde group of the 4r-PEG-CHO solution to form a Schiff base bond, and Zn 2+ and -COO - undergo strong coordination and gradually crosslink to construct a physical-chemical double crosslinked network.

2. A method for preparing the self-healing hydrogel according to claim 1, characterized in that, It includes: Adding a metal salt solution and a 4r-PEG-CHO solution to a 3wt% carboxymethyl chitosan aqueous solution, and rapidly stirring to prepare a physical-chemical double-crosslinked self-healing hydrogel; the metal salt solution is a ZnCl2 solution.

3. The method according to claim 2, wherein The carboxymethyl chitosan solution was adjusted to pH = 5 - 6 with 1% HOAc, and the metal salt solution was prepared with 1% HOAc at a concentration of 0.30 mol•L -1 , and the 4r-PEG-CHO solution was prepared with water at a concentration of 0.03 mol•L -1 .

4. The method according to claim 2, wherein Adding different volumes of ZnCl2 solution and 4r-PEG-CHO solution to 10 mL of 3wt% carboxymethyl chitosan aqueous solution, and adding water to adjust the solid content to 3.04%.

5. The application of the self-healing hydrogel according to claim 1 in the preparation of wound dressings, drug delivery and targeted drug delivery, electronic skin, tissue engineering and hemostatic drugs.

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