Carboxymethyl hydroxybutyl chitosan derivative as well as preparation method and application thereof

Abstract

The invention discloses a carboxymethyl hydroxybutyl chitosan derivative as well as a preparation method and application thereof, and belongs to the field of biomedical materials. Chitosan is subjected to carboxymethylation modification to obtain carboxymethyl chitosan, and then hydroxybutylation modification is carried out to obtain the carboxymethyl chitosan. Or the chitosan is firstly subjected to hydroxybutylation modification to obtain hydroxybutyl substituted chitosan, and then hydroxybutyl chitosan is subjected to carboxymethyl group modification to obtain the modified chitosan; or chitosan is subjected to hydroxybutylation and carboxymethylation modification at the same time to obtain the product. The derivative disclosed by the invention can be compatible with an osmotic pressure regulator of a strong-polarity ionic compound to obtain a thermo-sensitive solution with good performance, the solution is uniform and transparent at a low temperature, the gel is also uniform at a body temperature, the transparency is very high, the gel strength is relatively large, the problems of whitening and fragmentation do not exist, and the derivative can be well matched with the physiological environment of a human body. The material can be used as a temperature-sensitive cell carrier, a tissue engineering scaffold material, a drug controlled release material and a body surface superficial layer and deep layer tissue or in-vivo implant material.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a carboxymethyl hydroxybutyl chitosan derivative and a preparation method and application thereof. Background Art

[0002] Chitosan is a natural high molecular weight cationic polysaccharide with the advantages of good biocompatibility, no immunogenicity, no toxic side effects, and good biosafety. It also has multiple functions such as hemostasis, analgesia, antibacterial, and anti-infection, and can promote wound healing. It is easy to make into membranes and has good performance for preparing various dressings. After decades of continuous development, chitosan materials have gone through the first three generations of materials and have now developed to the fourth stage.

[0003] The first generation of chitosan materials is chitin extracted from the shells of crustaceans such as shrimps and crabs, and chitosan obtained by further deacetylation. Chitin is insoluble in general common solvents, and chitosan is soluble in some dilute acid solutions, but both are insoluble in neutral aqueous solutions. The second generation of chitosan materials is made into water-soluble materials by grafting soluble groups on the molecules of chitosan or chitosan. Representative materials are derivatives such as carboxymethyl chitosan, carboxymethyl chitosan, and chitosan quaternary ammonium salts. This type of material solves the problem that the first generation of chitosan materials are insoluble in water. In the medical field, it solves the problem that the acidic solution of the first generation of chitosan materials cannot enter the body for use, greatly expanding the application of chitosan materials in the human body.

[0004] The third generation of chitosan materials is made by grafting intelligent groups on the molecules of chitin or chitosan to make it an intelligent chitosan biomaterial, including temperature-sensitive intelligent materials, pH-sensitive biomaterials, pressure-sensitive biomaterials, magnetic-sensitive biomaterials, etc. Among them, the chitosan material obtained by grafting hydroxybutyl has been widely used for its excellent temperature sensitivity. Hydroxybutyl chitosan material not only solves the problem of water insolubility of the first generation of biomaterials, but also has temperature-sensitive properties. That is, its aqueous solution is in a solution state at low temperature (lower than its gel temperature), and after gradually heating up, it can be converted into a non-flowing gel state after being higher than its gel temperature, such as reaching body temperature of 37°C. This feature allows the material to be fixed and play a role for a long time after acting on the affected area. It can be used on wounds of almost any shape, including flat, wrinkled, gap-shaped, etc. After use, it is converted into a non-flowing gel attached to the wound surface, and will not be lost due to changes in body position, thereby achieving a long-term treatment effect, solving the problem that the second generation of carboxymethyl chitosan and other materials have high fluidity and are easy to leave the wound area that needs a barrier. Therefore, hydroxybutyl chitosan material has good wound healing and other biological activities.

[0005] The invention patent application with publication number CN 113908329 A discloses a hydroxybutyl chitosan hydrogel dressing and a preparation method thereof, wherein the auxiliary materials used in combination with hydroxybutyl chitosan are polyols containing three or more hydroxyl groups, such as glycerol, mannitol, sorbitol, etc., while highly polar ionic osmotic pressure regulators commonly used in pharmacopoeias, such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, etc., cannot be used in combination with hydroxybutyl chitosan preparations under normal conditions. This is because hydroxybutyl chitosan is compatible with the above-mentioned highly polar substances, and is a uniform and transparent flowing solution at low temperature, but becomes a white opaque gel when heated. The gel strength is low, and it is easy to break under the action of external force, and will not solidify again unless it is homogenized again at low temperature.

[0006] And if only aqueous solution is used, or the solution prepared by the compatibility of polyols such as glycerol and mannitol, it can reach low temperature and be easy to flow, and after being warmed up to near body temperature, it is converted into a non-flowing gel, and the gel strength is relatively large, and the gel is not scattered. But after the hydroxybutyl chitosan hydrogel dressing using this type of formula is used for human body, especially in the position with more body fluids, such as the peritoneal cavity and the wound surface position with more exudate, it will gradually shrink and precipitate, and can only play a short-term gel effect. And when used as a cell culture medium, the phenomenon of precipitation will also occur, that is, the matching degree of the hydroxybutyl chitosan hydrogel dressing and the physiological environment of human body also has certain defects. Summary of the invention

[0007] In view of the above problems, an object of the present invention is to provide a hydroxybutyl chitosan derivative grafted with a carboxymethyl group, which can be combined with an osmotic pressure regulator of a strongly polar ionic compound (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, etc.) to obtain a temperature-sensitive solution with good performance, and when forming a gel at body temperature, it also presents a uniform and transparent state, has high gel strength, does not have the problem of whitening and fragmentation, and can be well matched with the physiological environment of the human body.

[0008] The carboxymethyl hydroxybutyl chitosan derivative of the present invention has the following structure:

[0009]

[0010] Wherein, R1, R2, R3, and R4 are one of the following groups:

[0011] H, acetyl, hydroxybutyl, carboxymethyl, 2-carboxymethoxybutyl.

[0012] Furthermore, R1 and R2 are H, hydroxybutyl or carboxymethyl; and R3 and R4 are H, acetyl, carboxymethyl or hydroxybutyl.

[0013] Furthermore, the R1 is hydroxybutyl, R2 is H; the R3 is H, and R4 is carboxymethyl.

[0014] The structural formula of the group is as follows:

[0015] Hydroxybutyl: CH3CH2CHOHCH2-; Carboxymethyl: -CH2COOH; 2-carboxymethoxybutyl: Acetyl:

[0016] The degree of substitution of the hydroxybutyl group is 1.0-4.0; the degree of substitution of the carboxymethyl group is 0.1-3.0.

[0017] The preferred degree of substitution of hydroxybutyl groups is 1.5-3.0; the preferred degree of substitution of carboxymethyl groups is 0.5-2.0.

[0018] The range of the carboxymethyl substitution degree / hydroxybutyl substitution degree in the carboxymethyl hydroxybutyl chitosan derivative is 0.1-1.5.

[0019] Above-mentioned carboxymethyl hydroxybutyl chitosan derivative forms solution with pure water, or with polyol compatibility such as glycerol, mannitol, even with the osmotic pressure regulating material compatibility of strong polar ionic compound (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, etc.), all can obtain the temperature-sensitive solution with good performance. That is, it is a solution that is easy to flow at low temperature, and it is a gel that does not flow at body temperature. And the solution at low temperature is homogeneous and transparent, and the gel at body temperature is also homogeneous, and transparency is very high, and gel strength is relatively large, does not exist the problem of bleaching and scattered, and can have good matching with human physiological environment. This material can also have good compatibility with cell culture fluid, has the effect as cell carrier, and is the cell carrier with temperature sensitivity that effect is more excellent.

[0020] Furthermore, the present invention also provides a hydrogel comprising the above carboxymethyl hydroxybutyl chitosan derivative, which comprises the following components by mass: carboxymethyl hydroxybutyl chitosan derivative: 0.1%-10%; osmotic pressure regulator: 0.1-10%; water: the balance.

[0021] The osmotic pressure regulator is a polyol such as glycerol and mannitol, or a strongly polar ionic compound such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, and phosphate buffer.

[0022] Another object of the present invention is to provide a method for preparing the above-mentioned carboxymethyl hydroxybutyl chitosan derivative. The chitosan derivative can be prepared by any of the following methods.

[0023] Method 1: First, chitosan is modified by carboxymethylation to obtain carboxymethyl chitosan, and then modified by hydroxybutylation to obtain carboxymethyl hydroxybutyl chitosan derivatives. The detailed steps are as follows:

[0024] (1) dissolving or dispersing chitosan with a solvent, adding chloroacetic acid, adjusting the pH value of the system with an alkaline solution, reacting at a certain temperature for a certain time, and obtaining carboxymethyl chitosan; (2) dissolving or dispersing carboxymethyl chitosan with a solvent, adding 1,2-butylene oxide and an alkaline solution, and reacting at a certain temperature for a certain time; (3) after the reaction is completed, stabilizing the reaction system at room temperature, placing it in a dialysis bag for dialysis for a certain time, taking it out and freeze-drying it to obtain the final carboxymethyl hydroxybutyl chitosan derivative.

[0025] Method 2: First, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then the hydroxybutyl chitosan is modified by carboxymethyl groups to obtain the final carboxymethyl hydroxybutyl chitosan derivative. The detailed steps are as follows:

[0026] (1) chitosan is modified by hydroxybutylation according to the method disclosed in patent application CN 102276756 to obtain hydroxybutyl chitosan; (2) hydroxybutyl chitosan is dissolved or dispersed with a solvent, chloroacetic acid is added, the pH value of the system is adjusted with an alkaline solution, and the reaction is carried out at a certain temperature for a certain time; (3) after the reaction is completed, the reaction system is stabilized at room temperature, placed in a dialysis bag for dialysis for a certain time, and taken out for freeze drying to obtain the final carboxymethyl hydroxybutyl chitosan derivative.

[0027] Method 3: Chitosan is modified by hydroxybutylation and carboxymethylation simultaneously to obtain the final carboxymethyl hydroxybutyl chitosan derivative. The detailed steps are as follows:

[0028] (1) dissolving or dispersing chitosan with a solvent, adding 1,2-butylene oxide and chloroacetic acid in sequence, adjusting the pH value of the system with an alkaline solution, and reacting at a certain temperature for a certain time; (2) after the reaction is completed, stabilizing the reaction system at room temperature, placing it in a dialysis bag for dialysis for a certain period of time, taking it out and freeze-drying it to obtain the final carboxymethyl hydroxybutyl chitosan derivative.

[0029] The solvent for dissolving or dispersing chitosan or hydroxybutyl chitosan is a combination of one or more of water, sodium hydroxide solution, potassium hydroxide solution, urea solution, guanidine hydrochloride solution, hydrochloric acid, nitric acid, methanol, formic acid, ethanol, acetic acid, acetonitrile, n-propanol, isopropanol, n-butanol, acetone, dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, and dioxane.

[0030] The alkali is sodium hydroxide or potassium hydroxide.

[0031] The molar concentration of the alkaline solution is 0.01-15 mol / L.

[0032] The reaction temperature is 0°C-100°C.

[0033] The reaction time is 0.5h-120h.

[0034] The advantages of the present invention are:

[0035] 1. The carboxymethyl hydroxybutyl chitosan derivative of the present invention can form a solution with pure water, or be compatible with polyols such as glycerol and mannitol, or even be compatible with an osmotic pressure regulator of a strongly polar ionic compound (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, etc.), and a temperature-sensitive solution with good performance can be obtained: that is, it is an easy-flowing solution at low temperature and a non-flowing gel at body temperature; and the solution at low temperature is uniform and transparent, and the gel at body temperature is also uniform, with high transparency, high gel strength, no whitening and fragmentation problems, and can be well matched with the physiological environment of the human body.

[0036] 2. The carboxymethyl hydroxybutyl chitosan derivative of the present invention can also have good compatibility with cell culture fluid, and is a cell carrier and tissue engineering scaffold material with better effect and temperature sensitivity.

[0037] 3. The carboxymethyl hydroxybutyl chitosan derivative of the present invention can be mixed with drugs in various dosage forms, including liquid preparations, powder preparations, etc., and after mixing, it is still a gel under physiological conditions, there is no precipitation problem, and the effect of slow release can be achieved.

[0038] 4. The carboxymethyl hydroxybutyl chitosan derivative of the present invention can meet the requirements of further filtration sterilization or wet heat sterilization after preparing the solution, and will not cause the material to precipitate due to temperature fluctuations, resulting in the inability to filter or agglomeration after wet heat sterilization.

[0039] 5. The carboxymethyl hydroxybutyl chitosan derivative of the present invention can be used for superficial layer of body surface, deep layer of body surface tissue or implanted in the body. It can be used for chronic wound care, packing and hemostasis of deep wounds, tissue adhesion prevention and hemostasis after implantation or placement in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the infrared spectrum of the sample of Example 2 of the present invention.

[0041] Figure 2 This is the infrared spectrum of the sample of Example 6 of the present invention.

[0042] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 6 of the present invention.

[0043] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 7 of the present invention.

[0044] Figure 5 This is the acid-base titration curve of the sample in Example 6 of the present invention.

[0045] Figure 6 This is a photograph of the state of the sample in Example 6 of the present invention after being dissolved in 0.85% sodium chloride solution at low temperature to prepare a 3% concentration solution and then heated to 37°C.

[0046] Figure 7 This is a photograph of the state of the sample in Example 7 of the present invention after being dissolved in 0.85% sodium chloride solution at low temperature to prepare a 3% concentration solution and then heated to 37°C.

[0047] Figure 8 This is a photograph of the state of the hydroxybutyl chitosan sample after it was dissolved in 0.85% sodium chloride solution at low temperature to prepare a 3% solution and then heated to 37°C.

[0048] Fig. 9 This is a test graph of the gel temperature of the sample in Example 4 prepared with 0.85% sodium chloride solution to make a 5% solution.

[0049] Fig.10 This is a gel temperature test graph of the sample of Example 7, which was prepared into a 3% solution using 0.7% sodium chloride solution and 0.2M phosphate buffer. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments.

[0051] Embodiment 1:

[0052] (1) Disperse 30 g of chitosan in 100 mL of 50% sodium hydroxide solution, add 100 mL of isopropanol, stir for 2 h to make the dispersion uniform, add 100 mL of isopropanol solution containing 30 g of chloroacetic acid and 100 mL of purified water, react at 65 ° C for 6 h, adjust the pH value of the reacted material to neutral with hydrochloric acid, dialyze and freeze-dry to obtain 33.5 g of carboxymethyl chitosan.

[0053] (2) Disperse 22 g of the obtained carboxymethyl chitosan in 100 mL of 50% sodium hydroxide solution, stir for 2 h to make the dispersion uniform, then add 100 mL of isopropanol and 200 mL of 1,2-butylene oxide, and react at 40° C. for 48 h.

[0054] (3) The pH value of the reacted material was adjusted to neutral with hydrochloric acid at room temperature, and 28.1 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0055] Embodiment 2:

[0056] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0057] (2) Take 50 g of hydroxybutyl chitosan and disperse it in 200 mL of 50% sodium hydroxide solution. After the dispersion is uniform, add 200 mL of isopropanol solution containing 50 g of chloroacetic acid and stir at 80° C. for 2 h.

[0058] (3) The pH value of the reacted material was adjusted to neutral with dilute hydrochloric acid or dilute sodium hydroxide solution at room temperature, and 58.2 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0059] Embodiment 3:

[0060] (1) Disperse 100 g of chitosan in 800 mL of 50% sodium hydroxide solution and stir for 2 h to make it uniformly dispersed. First, add 1000 mL of 1,2-butylene oxide and stir to mix evenly. Then, add 100 g of chloroacetic acid, 1000 mL of purified water and 1000 mL of isopropanol and react at 30 °C for 120 h.

[0061] (2) The pH value of the reacted material was adjusted to neutral with hydrochloric acid at room temperature, and 130 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0062] Embodiment 4:

[0063] (1) Disperse 50 g of chitosan in 1000 mL of an aqueous solution containing 11% potassium hydroxide and 8% urea, stir to make the dispersion uniform, freeze overnight and then melt at low temperature to obtain a uniform solution, add 50 g of chloroacetic acid and 500 mL of 1,2-butylene oxide, and react at 25°C for 48 h.

[0064] (2) The pH value of the reacted material was adjusted to neutral with hydrochloric acid at room temperature, and 74.4 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0065] Embodiment 5:

[0066] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0067] (2) Take 50 g of hydroxybutyl chitosan and disperse it in 2000 mL of dimethyl sulfoxide aqueous solution (volume ratio 1:1). After uniform dispersion, add 30 g of chloroacetic acid and 40 g of sodium hydroxide, stir and react at 50° C. for 2 h. The reacted material is dialyzed and freeze-dried to obtain 55.6 g of carboxymethyl hydroxybutyl chitosan.

[0068] Embodiment 6:

[0069] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0070] (2) Take 50 g of hydroxybutyl chitosan, disperse it in N,N-dimethylformamide, add 20 g of chloroacetic acid and 30 g of sodium hydroxide after uniform dispersion, stir and react at 60° C. for 2 h, and dialyze and freeze-dry the reacted material to obtain 55.6 g of carboxymethyl hydroxybutyl chitosan.

[0071] Embodiment 7:

[0072] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0073] (2) Take 50 g of hydroxybutyl chitosan, disperse it in N,N-dimethylformamide, and after it is evenly dispersed, add 40 g of chloroacetic acid and 55 g of sodium hydroxide, stir and react at 60° C. for 2 h. The reacted material is dialyzed and freeze-dried to obtain 60.1 g of carboxymethyl hydroxybutyl chitosan.

[0074] Example 8: Product structure identification and performance testing

[0075] The samples prepared in Example 2 and Example 6 were subjected to infrared spectroscopy tests respectively. Figure 1 and Figure 2 The peak positions of the spectra of the two samples are exactly the same, and the peak positions are as follows: 1594cm -1 Left and right 1413cm -1 The absorption peaks on the left and right are the asymmetric and symmetric stretching vibration absorption peaks of carboxyl, indicating the presence of carboxyl; 1061cm -1 The absorption peaks on the left and right are the stretching vibration absorption peaks of the ether bond (C—O) generated by the primary alcohol, indicating that the hydroxybutylation or carboxylmethylation reaction mainly occurs at the C6 position; 2962 cm -1 Around, 2924cm -1 Around, 2874cm -1 The three peaks on the left and right and 1460cm -1 The absorption peaks on the left and right are the stretching vibration and bending vibration absorption peaks of the C-H bond, indicating the presence of hydroxybutyl; 3408cm -1 The strong and broad absorption peaks on the left and right are the stretching vibration absorption peaks of O—H and N—H.

[0076] The samples in Example 6 and Example 7 were dissolved in a D2O solution containing 20% ​​DCl and subjected to H NMR spectrum testing. The spectra were as shown in Figure 3 and Figure 4. Due to the acidic solvent used, the chemical shift of the peak will be offset, but the relative position of the peak and the peak type are basically the same. Take the liquid nuclear magnetic hydrogen spectrum of the sample of Example 7 as an example to illustrate, -0.31ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl, and 0.30ppm is the absorption peak of the methylene hydrogen atom on the hydroxybutyl, and the presence of the two indicates that the sample has the presence of a hydroxybutyl group. 3.07ppm is the absorption peak of the hydrogen atom on the methylene in the carboxymethyl group, and 1.86ppm is the absorption peak of the hydrogen atom on the methine connected to the original hydroxyl group after the hydroxyl group of the hydroxybutyl group is replaced with the carboxymethyl group. The multiple superimposed absorption peaks between 2ppm-3ppm are the absorption peaks of the hydrogen atoms on the two carbon atoms connected to the 6 hydroxyls on the 2, 3, 4, 5, 6 carbons on the chitosan sugar ring and the hydroxybutyl group. 3.83ppm is the absorption peak of the hydrogen atom connected to the 1 carbon on the chitosan sugar ring. Figure 4 and Figure 3 In comparison, the carboxymethyl substitution degree of the sample increased, and the two peaks at 1.86ppm and 3.07ppm increased accordingly.

[0077] Example 9: Acid-base titration test

[0078] The carboxymethyl hydroxybutyl chitosan prepared in Example 6 was dissolved in excess hydrochloric acid and titrated with a standard sodium hydroxide solution using a fully automatic potentiometric titrator. The titration curve was as follows: Figure 5 As shown. The solid line is the relationship curve between pH value and the volume consumed by the standard sodium hydroxide solution, and the dotted line is the relationship curve between pH value and volume derivative and the volume consumed by the standard sodium hydroxide solution. It can be seen from the figure that there are two obvious jump points in the curve. They are the carboxyl titration end point and the amino titration end point. Combined with the hydrochloric acid titration end point at pH 2.2, the carboxymethyl substitution degree can be calculated to be 1.33.

[0079] Example 10: Solution property test

[0080] The carboxymethyl hydroxybutyl chitosan in Example 6 was dissolved in purified water and 0.85% sodium chloride solution, respectively, with a concentration of 3%, as samples ① and ②. The carboxymethyl hydroxybutyl chitosan material in Example 7 was dissolved in purified water and 0.85% sodium chloride solution, respectively, with a concentration of 3%, as samples ③ and ④. And the hydroxybutyl chitosan prepared by the method disclosed in patent application CN102276756 was dissolved in purified water and 0.85% sodium chloride solution as a control, with a concentration of 3%, as samples ⑤ and ⑥.

[0081] Table 1 The status of each sample under different conditions

[0082]

[0083] The above data show that, depending on the sample preparation process, a thermosensitive gel that can solidify at 37°C can be obtained, and a thermosensitive solution that is fluid at 37°C can also be obtained, and the gel temperature is higher than 37°C.

[0084] Example 11: Degree of Substitution and Gel Temperature Test

[0085] The samples in Examples 1-7 were subjected to acid-base titration test, elemental analysis test, etc., respectively, to calculate the hydroxybutyl substitution degree and carboxymethyl substitution degree of the samples. Each sample was dissolved in water and 0.85% sodium chloride solution to form 3% and 5% solutions, respectively, and the pH value of the solution was adjusted to 7.0, and the gel temperature test was performed. The results are shown in Table 2.

[0086] Table 2 Relationship between the degree of hydroxybutyl substitution, degree of carboxymethyl substitution and gel temperature of each sample

[0087]

[0088] From the above data, it can be seen that in order to obtain a hydrogel that solidifies at 37°C, it is necessary to control certain substitution ratio conditions. Under the condition of a solution pH of 7.0: when water is used as the solvent, a 3% aqueous solution meets the requirement of solidification at 37°C, and it is necessary to control the carboxymethyl substitution degree / hydroxybutyl substitution degree ≤ 0.68. When this value is exceeded, the gel will remain liquid at 37°C. A 5% aqueous solution meets the requirement of solidification at 37°C, and it is necessary to control the carboxymethyl substitution degree / hydroxybutyl substitution degree < 0.84. When this value is exceeded, the gel will remain liquid at 37°C. When sodium chloride solution is used as the solvent, a 3% solution meets the requirement of solidification at 37°C, and it is necessary to control the carboxymethyl substitution degree / hydroxybutyl substitution degree ≤ 0.74. When this value is exceeded, the gel will remain liquid at 37°C. A 5% solution meets the requirement of solidification at 37°C, and it requires the carboxymethyl substitution degree / hydroxybutyl substitution degree ≤ 0.84.

[0089] According to the above results, although different conditions will affect whether the material solution can solidify at 37°C, the general rule is that the higher the ratio of carboxymethyl to hydroxybutyl substitution, the higher the gel temperature of the product, even exceeding 37°C. By controlling the degree of substitution of the two, the gel temperature of the material solution can be controlled. However, the ratio of carboxymethyl to hydroxybutyl substitution, which determines whether the material solution can gel at 37°C, is not fixed, but is affected by multiple factors such as pH value, ionic strength, solution concentration, and type of excipients.

[0090] Example 12: Preparation and performance testing of implantable hydrogels

[0091] The sample of Example 4 was prepared into a 5% solution with 0.85% sodium chloride solution. The osmotic pressure was 315 mOsmol / kg when tested by an osmometer. The gel temperature was 20.7°C when tested by a rheometer. Fig. 9The sample of Example 7 was prepared into a 3% solution with 0.7% sodium chloride solution and 0.2M phosphate buffer. The osmotic pressure was tested by an osmometer and was 308mOsmol / kg. The gel temperature was tested by a rheometer, which showed that there was no intersection between G' and G" between 4°C and 50°C, and G" was always above G', that is, the material was in a flowing liquid state within this temperature range, as shown in FIG. Fig.10 As shown. As can be seen from the figure, the gel temperature of the hydrogel prepared in the sample of Example 4 above is 20.7°C, which is suitable for dressings, fillings or scaffold materials for use in vivo and on the surface of the body, and can be used as a drug or cell carrier to load relevant therapeutic drugs or therapeutic cells to further enhance the therapeutic effect. The hydrogel prepared in the sample of Example 7 is a liquid between 4°C and 50°C, and the gel temperature exceeds the body temperature and cannot even gel. It is a flowing liquid at body temperature and can be used as a wound site on the surface or in the body that requires flowing liquid, such as intra-articular injection, filling in tooth extraction sockets, etc.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Ordinary technicians in the relevant field can still modify or replace the specific implementation methods of the present invention with equivalents by referring to the above embodiments. Any modifications or equivalent replacements that do not depart from the spirit and scope of the present invention are within the scope of protection of the claims of the present invention.

Claims

1. A carboxymethyl hydroxybutyl chitosan derivative, characterized in that: It has the following structure: Wherein, R1, R2, R3, and R4 are one of the following groups: H, acetyl, hydroxybutyl, carboxymethyl, 2-carboxymethoxybutyl.

2. The carboxymethyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The R1 and R2 are H, hydroxybutyl or carboxymethyl; the R3 and R4 are H, acetyl, carboxymethyl or hydroxybutyl.

3. The carboxymethyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The degree of substitution of the hydroxybutyl group is 1.0-4.0; the degree of substitution of the carboxymethyl group is 0.1-3.

0.

4. The carboxymethyl hydroxybutyl chitosan derivative according to claim 1 or 3, characterized in that: The range of the carboxymethyl substitution degree / hydroxybutyl substitution degree in the hydroxybutyl chitosan derivative is 0.1-1.

5.

5. A hydrogel comprising the carboxymethyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The invention comprises the following components by mass: 0.1%-10% of carboxymethyl hydroxybutyl chitosan derivative; 0.1%-10% of osmotic pressure regulator; and water as the balance. The osmotic pressure regulator is glycerol, mannitol or sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, and phosphate buffer.

6. The method for preparing the carboxymethyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: (1) dissolving or dispersing chitosan with a solvent, adding chloroacetic acid, adjusting the pH value of the system with an alkaline solution, reacting at a certain temperature for a certain time, and obtaining carboxymethyl chitosan; (2) dissolving or dispersing carboxymethyl chitosan with a solvent, adding 1,2-butylene oxide and an alkaline solution, and reacting at a certain temperature for a certain time; (3) after the reaction is completed, stabilizing the reaction system at room temperature, placing it in a dialysis bag for dialysis for a certain time, taking it out and freeze-drying it to obtain the final carboxymethyl hydroxybutyl chitosan derivative.

7. The method for preparing the carboxymethyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: (1) chitosan is modified by hydroxybutylation according to the method disclosed in patent application CN 102276756 to obtain hydroxybutyl chitosan; (2) hydroxybutyl chitosan is dissolved or dispersed with a solvent, chloroacetic acid is added, the pH value of the system is adjusted with an alkaline solution, and the reaction is carried out at a certain temperature for a certain time; (3) after the reaction is completed, the reaction system is stabilized at room temperature, placed in a dialysis bag for dialysis for a certain time, and taken out for freeze drying to obtain the final carboxymethyl hydroxybutyl chitosan derivative.

8. The method for preparing the carboxymethyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: (1) dissolving or dispersing chitosan with a solvent, adding 1,2-butylene oxide and chloroacetic acid in sequence, adjusting the pH value of the system with an alkaline solution, and reacting at a certain temperature for a certain time; (2) after the reaction is completed, stabilizing the reaction system at room temperature, placing it in a dialysis bag for dialysis for a certain period of time, taking it out and freeze-drying it to obtain the final carboxymethyl hydroxybutyl chitosan derivative.

9. The method for preparing the carboxymethyl hydroxybutyl chitosan derivative according to any one of claims 6 to 8, characterized in that The solvent for dissolving or dispersing chitosan or hydroxybutyl chitosan is a combination of one or more of water, sodium hydroxide solution, potassium hydroxide solution, urea solution, guanidine hydrochloride solution, hydrochloric acid, nitric acid, methanol, formic acid, ethanol, acetic acid, acetonitrile, n-propanol, isopropanol, n-butanol, acetone, dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, and dioxane.

10. The method for preparing the carboxymethyl hydroxybutyl chitosan derivative according to any one of claims 6 to 8, characterized in that The alkali is sodium hydroxide or potassium hydroxide; the molar concentration of the alkali solution is 0.01-15 mol / L; the reaction temperature is 0° C.-100° C.; and the reaction time is 0.5 h-120 h.

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