Acylated anion group substituted hydroxybutyl chitosan derivative as well as preparation method and application thereof

Abstract

The invention discloses an acylated anion group substituted hydroxybutyl chitosan derivative as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The acylated anion group substituted hydroxybutyl chitosan derivative has the following structure: # imgabs0 #, wherein R1, R2, R3 and R4 are one of the following groups: H, an acyl group, hydroxybutyl, polyhydroxybutyl and an anion substituent group. The hydroxybutyl chitosan derivative disclosed by the invention can form a solution with pure water, or is compatible with polyhydric alcohols such as glycerol, mannitol and the like, and even is compatible with a strong-polarity ionic compound, so that a temperature-sensitive solution with good performance can be obtained; the temperature-sensitive cell carrier has good compatibility with a cell culture solution, can enable cells to survive and promote cell proliferation, and is a temperature-sensitive cell carrier and tissue engineering scaffold material with a better effect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to an acylated anionic group-substituted 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 common solvents, and chitosan is soluble in some dilute acid solutions, but both are insoluble in neutral aqueous solutions.

[0004] The second generation of chitosan materials is made into water-soluble materials by grafting soluble groups on chitin or chitosan molecules. Representative materials are carboxymethyl chitosan, carboxymethyl chitosan and chitosan quaternary ammonium salt derivatives. This type of material solves the problem of the first generation of chitosan materials being 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.

[0005] 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 materials such as the second generation of carboxymethyl chitosan have high fluidity and are easy to leave the wound area that requires a barrier. Therefore, hydroxybutyl chitosan material has good wound healing and other biological activities.

[0006] 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 compatible with hydroxybutyl chitosan are polyols containing three or more hydroxyl groups such as glycerol, mannitol, and sorbitol, while the strong polar ionic osmotic pressure regulators commonly used in pharmacopoeias such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, and sodium sulfate cannot be used for compatibility with hydroxybutyl chitosan preparations under normal conditions. This is because hydroxybutyl chitosan is compatible with the above-mentioned strong polar substances, and is a uniform and transparent flowing solution at low temperature, but becomes a white opaque gel when heated, and the gel strength is low. It is easy to break under the action of external forces, and will not solidify again unless it is homogenized again at low temperature. If only an aqueous solution is used, or a solution prepared by compatibility with polyols such as glycerol and mannitol, it can itself reach low temperature and easy flow, and after heating to near body temperature, it is converted into a non-flowing gel, and the gel strength is large, and the gel does not break. However, when the hydroxybutyl chitosan hydrogel dressing of this type is used on the human body, especially in areas with more body fluids, such as the abdominal cavity and wound areas with more exudate, it will gradually shrink and precipitate, and can only play a short-term gelling role. Moreover, when used as a cell culture medium, precipitation will also occur, that is, the matching degree of the above hydroxybutyl chitosan hydrogel dressing with the physiological environment of the human body still has certain defects.

[0007] In addition, there are amino groups in the unit structure of hydroxybutyl chitosan and its derivative materials. After the early reaction process, the deacetylation degree of this group is extremely high, almost reaching more than 90%, or even 100%. This amino group exists in various forms, which may be an unsubstituted primary amino form, or a secondary amino form monosubstituted by a hydroxybutyl group or the like, or even a tertiary amino form disubstituted by a hydroxybutyl group or the like. Since the above three forms of amino structures all carry positive charges when the pH is close to neutral conditions, and the amino groups carrying too much positive charge have an obstructive or even inhibitory effect on the growth of cells, and cannot significantly promote cell proliferation, they are not effective when used as cell carriers and tissue engineering scaffold materials. Summary of the invention

[0008] In view of the above problems, an object of the present invention is to provide an acylated anionic group-substituted hydroxybutyl chitosan derivative, 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, can be well matched with the physiological environment of the human body, and is compatible with a cell culture medium. When used as a cell culture medium, the cultured cells grow well.

[0009] The acylated anionic group-substituted hydroxybutyl chitosan derivative of the present invention has the following structure:

[0010]

[0011] Among them, R 1 , R 2 , R 3 , R 4 One of the following groups:

[0012] H, acyl group, hydroxybutyl, polyhydroxybutyl, anionic substituent group.

[0013] The anionic substituent group is selected from carboxymethyl, 2-carboxymethoxybutyl, 2-carboxymethylpolyoxybutyl, carboxyethyl, methylcarboxymethyl, carboxypropyl, 2-carboxypropyl, 1,3-dicarboxypropyl, carboxybutyl, 3-carboxybutyl, 2-carboxymethylpropyl, sulfonic acid, sulfonic acid propyl, and 2-hydroxysulfonic acid propyl.

[0014] The hydroxybutyl group is substituted in R 1 , R 2 , R 3 , R 4 The anionic group, such as carboxymethyl, is substituted at R 1 , R 2 , R 3 , R 4 The hydroxyl group substituted at the position or at the newly generated hydroxyl group on the hydroxybutyl group substituted at the above position, the latter forms an oxybutyl group connected to the anionic group, such as a 2-carboxymethoxybutyl group; the acyl group substituted at R 3 or R 4 Position, and in the same unit R 3 or R 4 A position is replaced at most once.

[0015] The acyl group is acetyl, propionyl or butyryl.

[0016] The structural formula of the group is as follows:

[0017] Hydroxybutyl: CH 3 CH 2 CHOHCH 2 -; Polyhydroxybutyl: Carboxymethyl: -CH 2 COOH; 2-carboxymethoxybutyl: 2-Carboxymethyl polyoxybutyl: m=2-4; acetyl: Propionyl: Butyryl: Carboxyethyl: -CH 2 CH 2COOH; methyl carboxymethyl: Carboxypropyl: -CH 2 CH 2 CH 2 COOH; 2-Carboxypropyl: 1,3-Dicarboxypropyl: Carboxybutyl: -CH 2 CH 2 CH 2 CH 2 COOH; 3-carboxybutyl: 2-Carboxymethylpropyl: Sulfonic acid group: -SO 3 H; sulfonic acid propyl: -CH 2 CH 2 CH 2 SO 3 H; 2-Hydroxysulfonic acid propyl:

[0018] Among the above substituents, various anionic groups including carboxymethyl groups and acyl groups are terminating substituents. 1 , R 2 , R 3 , R 4 After the position of the hydroxybutyl group is replaced by this type of substituent, it can no longer continue to react with other substituents; while the hydroxybutyl group is a substituent that can continue to react, R 1 , R 2 , R 3 , R 4 The secondary hydroxyl group generated after the position is replaced by hydroxybutyl can continue to undergo hydroxybutylation reaction or react with anionic groups, such as carboxymethyl, to form polyhydroxybutyl or anionic group-connected oxybutyl groups, such as 2-carboxymethoxybutyl. In the present invention, "substitution degree" is defined as the number of various groups substituted on average per sugar unit, and the number of hydroxybutyl groups reacted in hydroxybutyl, polyhydroxybutyl and 2-carboxymethoxybutyl or other anionic group-modified oxybutyl groups is defined as "hydroxybutyl substitution degree"; the number of anionic groups reacted on anionic groups and anionic group-modified oxybutyl groups is defined as the corresponding "anionic group substitution degree", such as the number of carboxymethyl groups reacted on carboxymethyl and 2-carboxymethyl polyoxybutyl is defined as "carboxymethyl substitution degree". Therefore, although there are only four substitution positions on each chitosan derivative unit, the sum of the substitution degrees of each group can be greater than 4.

[0019] The degree of substitution of the hydroxybutyl group is 1.0-4.0; the degree of substitution of the anionic group is 0.1-3.0, and the degree of substitution of the acyl group is 0.05-0.95; further, the anionic group is a carboxymethyl group, and its degree of substitution is 0.1-3.0.

[0020] Preferably, the degree of substitution of hydroxybutyl is 1.2-3.0; the degree of substitution of anionic groups is 0.2-2.0, and the degree of substitution of acyl groups is 0.1-0.9; further, the anionic groups are carboxymethyl groups, and the degree of substitution thereof is 0.2-2.0.

[0021] More preferably, the degree of substitution of hydroxybutyl is 1.5-2.8; the degree of substitution of anionic groups is 0.3-1.8, and the degree of substitution of acyl groups is 0.2-0.8; further, the anionic groups are carboxymethyl groups, and the degree of substitution thereof is 0.3-1.8.

[0022] The above-mentioned acylated anionic group replaces the hydroxybutyl chitosan derivative and forms a solution with pure water, or is compatible with polyols such as glycerol and mannitol, or even with the osmotic pressure regulating material compatibility of strongly polar ionic compounds (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, etc.), and all can obtain a temperature-sensitive solution with good performance. That is, it is a solution that is easy to flow at low temperatures and a gel that does not flow at body temperature. And the solution at low temperatures is uniform and transparent, and the gel at body temperature is also uniform, and the transparency is very high, and the gel strength is relatively large, and there is no problem of bleaching and fragmentation, and good matching can be arranged with the human physiological environment. This material can also have good compatibility with cell culture fluid, has the effect as a cell carrier, and is a cell carrier with temperature sensitivity that is more excellent in effect. Acylated anion-substituted hydroxybutyl chitosan, based on the thermosensitivity of hydroxybutyl chitosan, the modification of anionic groups such as carboxymethyl improves its compatibility with physiological concentration salt solutions, and further acylated group modification replaces part of the amino group, reducing the number of charges of the amino group under neutral conditions, improving the cell compatibility of the gel, and being more conducive to the growth of cultured cells.

[0023] Furthermore, the present invention also provides a hydrogel comprising the above-mentioned acylated anionic group-substituted hydroxybutyl chitosan derivative, which comprises the following components by mass: acylated anionic group-substituted hydroxybutyl chitosan derivative: 0.1%-10%; osmotic pressure regulator: 0.1-10%; water: the balance.

[0024] The osmotic pressure regulator is one or more of polyols such as glycerol and mannitol, or strongly polar ionic compounds such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, and phosphate buffer.

[0025] Another object of the present invention is to provide a method for preparing the above-mentioned acylated anionic group-substituted hydroxybutyl chitosan, which can be prepared by any of the following methods.

[0026] Method 1: First, chitosan is modified with ionic groups to obtain ion-modified chitosan, then hydroxybutylated to obtain hydroxybutyl chitosan derivatives substituted with ionic groups, and then acylated to obtain acylated anionic group-substituted hydroxybutyl chitosan derivatives. The detailed steps are as follows:

[0027] (1) Dissolving or dispersing chitosan with a solvent, adding an anionic group modification reagent to react: a) connecting the modification reagent with the hydroxyl group or amino group on the chitosan through a nucleophilic substitution reaction: adding a nucleophilic substitution reaction reagent, adjusting the pH value of the system with an alkaline solution, reacting at a certain temperature for a certain time, and obtaining anionic group-modified chitosan; or b) obtaining a Schiff base reaction between an aldehyde-containing Schiff base modification reagent and the amino group to generate a Schiff base, and then reducing it with a reducing agent: adding an ionic Schiff base reaction reagent, adjusting the pH value of the system with an alkaline solution, reacting at a certain temperature for a certain time, adjusting the pH value again and adding a reducing agent, reacting at a certain temperature for a certain time, and preliminarily purifying the product to obtain anionic group-modified chitosan; or c) connecting the modification reagent with the amino group through Michael addition: adding an anionic Michael addition reaction reagent, adjusting the pH value of the system with an alkaline solution or hydrochloric acid, reacting at a certain temperature for a certain time, and preliminarily purifying the product. The chitosan is modified by an anionic group and purified by a step of purification to obtain anionic group-modified chitosan; or d) connecting by a ring-opening reaction reagent: adding an anionic ring-opening reaction reagent, adjusting the pH value of the system with an alkali solution or hydrochloric acid, reacting at a certain temperature for a certain time, and performing preliminary purification to obtain anionic group-modified chitosan; (2) dissolving or dispersing the anionic modified chitosan with a solvent, adding 1,2-butylene oxide and an alkali solution, and reacting at a certain temperature for a certain time; (3) after the reaction, stabilizing the reaction system at room temperature, placing it in a dialysis bag for dialyzing for a certain time, taking it out and freeze-drying it to obtain anionic group-substituted hydroxybutyl chitosan derivatives; (4) dissolving or dispersing the anionic group-substituted hydroxybutyl chitosan derivatives with a solvent, adding an acylation reaction reagent or its solution, reacting at a certain temperature for a certain time, adding alkali solution to terminate the reaction, placing it in a dialysis bag for dialyzing for a certain time, taking it out and freeze-drying it to obtain the final acylated anionic group-substituted hydroxybutyl chitosan derivatives.

[0028] Method 2: First, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then, hydroxybutyl chitosan is modified by anionic groups to obtain hydroxybutyl chitosan derivatives containing anionic groups, and then acylated to obtain acylated anionic group-substituted hydroxybutyl chitosan derivatives. The detailed steps are as follows:

[0029] (1) According to the method disclosed in patent application CN 102276756 A, chitosan is subjected to hydroxybutyl modification to obtain hydroxybutyl chitosan; (2) the hydroxybutyl chitosan is dissolved or dispersed in a solvent, and an anionic group modification reagent is added to react: a) the modification reagent is connected with the hydroxyl group or amino group on the hydroxybutyl chitosan through a nucleophilic substitution reaction: a nucleophilic substitution reaction reagent is added, the pH value of the system is adjusted with an alkali solution, and the reaction is carried out at a certain temperature for a certain time; or b) an anionic Schiff base modification reagent containing an aldehyde group reacts with the amino group to generate a Schiff base, which is then reduced with a reducing agent: a Schiff base reaction reagent containing an anion is added, the pH value of the system is adjusted with an alkali solution, and the reaction is carried out at a certain temperature for a certain time. The reaction is carried out for a certain time, then the pH value is adjusted and a reducing agent is added, and the reaction is carried out at a certain temperature for a certain time; or c) the modification reagent is connected by Michael addition with the amino group: an anion-containing Michael addition reaction reagent is added, the pH value of the system is adjusted with an alkali solution or hydrochloric acid, and the reaction is carried out at a certain temperature for a certain time; or d) the reaction is connected by a ring-opening reaction reagent: an ion-containing ring-opening reaction reagent is added, the pH value of the system is adjusted with an alkali solution or hydrochloric acid, 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 dialyzing for a certain time, taken out and freeze-dried to obtain a hydroxybutyl chitosan derivative substituted with an anionic group. (4) the hydroxybutyl chitosan derivative substituted with an anionic group is dissolved or dispersed in a solvent, an acylation reaction reagent or its solution is added, the reaction is carried out at a certain temperature for a certain time, an alkali solution is added to terminate the reaction, the hydroxybutyl chitosan derivative substituted with an anionic group is obtained by acylation reaction.

[0030] Method 3: First, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then, acylation is performed to obtain an acylated hydroxybutyl chitosan derivative, and then anionic group modification is performed to obtain an acylated anionic group-substituted hydroxybutyl chitosan derivative. The detailed steps are as follows:

[0031] (1) chitosan is subjected to hydroxybutyl modification according to the method disclosed in patent application CN 102276756 A to obtain hydroxybutyl chitosan; (2) hydroxybutyl chitosan is dissolved or dispersed in a solvent, an acylation reaction reagent or its solution is added, the reaction is carried out at a certain temperature for a certain time, an alkali solution is added to terminate the reaction, the chitosan is placed in a dialysis bag for a certain time, and the chitosan is taken out and freeze-dried to obtain an acylated hydroxybutyl chitosan derivative; (3) the acylated hydroxybutyl chitosan derivative is dissolved or dispersed in a solvent, an anionic group modification reagent is added to react: a) the modification reagent is connected with the hydroxyl group or amino group on the hydroxybutyl chitosan by a nucleophilic substitution reaction: a nucleophilic substitution reaction reagent is added, the pH value of the system is adjusted with an alkali solution, and the reaction is carried out at a certain temperature for a certain time; or b) an anionic Schiff base modification reagent containing an aldehyde group reacts with the amino group to generate a Schiff base, Then reduce with a reducing agent to obtain: add a Schiff base reaction reagent containing ions, adjust the pH value of the system with an alkaline solution, react at a certain temperature for a certain time, then adjust the pH value and add a reducing agent, and react at a certain temperature for a certain time; or c) connect through Michael addition of a modifying reagent and an amino group: add a Michael addition reaction reagent containing anions, adjust the pH value of the system with an alkaline solution or hydrochloric acid, and react at a certain temperature for a certain time; or d) connect through a ring-opening reaction reagent: add a ring-opening reaction reagent containing anions, adjust the pH value of the system with an alkaline solution or hydrochloric acid, and react at a certain temperature for a certain time; (4) after the reaction is completed, stabilize the reaction system at room temperature, put it into a dialysis bag for dialysis for a certain time, take it out and freeze-dry it to obtain the final acylated anion group-substituted hydroxybutyl chitosan derivative.

[0032] Method 4: First, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then, hydroxybutyl chitosan is modified by anionic groups to obtain hydroxybutyl chitosan derivatives containing anionic group substitution, i.e., acylated anionic group-substituted hydroxybutyl chitosan derivatives. The detailed steps are as follows:

[0033] (1) hydroxybutylation of chitosan to obtain hydroxybutyl chitosan; (2) dissolving or dispersing hydroxybutyl chitosan in a solvent, adding an anionic group modification reagent to react: a) connecting the modification reagent with the hydroxyl group or amino group on the hydroxybutyl chitosan through a nucleophilic substitution reaction: adding a nucleophilic substitution reaction reagent, adjusting the pH value of the system with an alkaline solution, and reacting at a certain temperature for a certain time; or b) allowing an anionic Schiff base modification reagent containing an aldehyde group to react with the amino group to generate a Schiff base, which is then reduced with a reducing agent: adding an anionic Schiff base reaction reagent, adjusting the pH value of the system with an alkaline solution, and reacting at a certain temperature for a certain time. c) connecting by Michael addition reaction of modifying reagent with amino group: adding Michael addition reaction reagent containing anion, adjusting pH value of the system with alkaline solution or hydrochloric acid, and reacting at a certain temperature for a certain time; or d) connecting by ring-opening reaction reagent: adding ring-opening reaction reagent containing anion, adjusting pH value of the system with alkaline solution or hydrochloric acid, 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 dialysis bag for dialyzing for a certain time, taking it out and freeze-drying it to obtain the final acylated anion group-substituted hydroxybutyl chitosan derivative.

[0034] Method 5: First, chitosan is modified with anionic groups to obtain chitosan substituted with anionic groups; then, the chitosan substituted with anionic groups is modified with hydroxybutyl to obtain a hydroxybutyl chitosan derivative substituted with anionic groups, i.e., an acylated anionic group substituted hydroxybutyl chitosan derivative. The detailed steps are as follows:

[0035] (1) Chitosan is modified with anionic groups to obtain chitosan modified with anionic groups; a) a modification agent is connected with a hydroxyl group or an amino group on hydroxybutyl chitosan by a nucleophilic substitution reaction: a nucleophilic substitution reaction agent is added, the pH value of the system is adjusted with an alkali solution, and the reaction is carried out at a certain temperature for a certain time; or b) an ionic Schiff base modification agent containing an aldehyde group is reacted with an amino group to produce a Schiff base, which is then reduced with a reducing agent: an ionic Schiff base reaction agent containing an aldehyde group is added, the pH value of the system is adjusted with an alkali solution, the reaction is carried out at a certain temperature for a certain time, the pH value is adjusted again and a reducing agent is added, and the reaction is carried out at a certain temperature for a certain time; or c) a modification agent is used to The method comprises the following steps: connecting the chitosan derivatives by Michael addition reaction: adding a Michael addition reaction reagent containing anions, adjusting the pH value of the system with an alkaline solution or hydrochloric acid, and reacting at a certain temperature for a certain time; or d) connecting by a ring-opening reaction reagent: adding a ring-opening reaction reagent containing anions, adjusting the pH value of the system with an alkaline solution or hydrochloric acid, and reacting at a certain temperature for a certain time; (2) dissolving or dispersing the chitosan modified with anionic groups in a solvent, adding a hydroxybutyl group modification reagent for reaction: (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 acylated anionic group-substituted hydroxybutyl chitosan derivative.

[0036] Among them, the solvent for dissolving or dispersing chitosan or chitin, hydroxybutyl chitosan or chitin, and anionic group-modified chitosan or chitin 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.

[0037] The reagents for the nucleophilic substitution reaction in step a) include: chloroacetic acid, 3-chloropropionic acid, 2-chloropropionic acid, 4-chlorobutyric acid, 2-methyl-2-chloropropionic acid, 5-chlorobutyric acid, 2-methyl-4-chloropropionic acid, chlorosulfonic acid, sodium 3-chloropropanesulfonate, and sodium 3-chloro-2-hydroxypropanesulfonate.

[0038] The anion-containing Schiff base reaction reagent in step b) includes: glyoxylic acid, pyruvic acid, α-ketoglutaric acid; the reducing agent includes: sodium borohydride, lithium aluminum hydride, lithium borohydride, potassium borohydride, sodium cyanoborohydride, borane ammonia, etc.

[0039] The anion-containing Michael addition reaction reagent in step c) includes acrylic acid and methacrylic acid.

[0040] The anion-containing ring-opening reaction reagent in step d) includes: sodium 2,3-epoxypropanesulfonate and propane sultone.

[0041] The alkali is sodium hydroxide or potassium hydroxide, and the molar concentration of the alkali solution is 0.01-15 mol / L.

[0042] The pH value range of the Schiff base reaction in step b) is 3-7, and the pH value range of the reduction reaction is 8-14.

[0043] The pH value of the Michael addition reaction in step c) is in the range of 3-7.

[0044] The pH value of the ring-opening reaction in step d) is in the range of 0-5 when using the reagent propane sultone; and in the range of 7-14 when using the reagents sodium 2,3-epoxypropanesulfonate and 2,3-epoxypropyltrimethylammonium chloride.

[0045] The molar concentration of the alkaline solution in the hydroxybutylation reaction in step (2) is 0.01-15 mol / L.

[0046] The reaction temperature in step (1) or (2) is 0°C-70°C; the reaction time is 0.5h-120h.

[0047] The acylation reaction reagent is acetic anhydride, propionic anhydride or butyric anhydride.

[0048] The present invention also provides the use of the hydrogel of the acylated anionic group-substituted hydroxybutyl chitosan derivative for preparing wound dressings, postoperative anti-adhesion materials, tissue filling materials, tissue engineering skin scaffold materials, tissue engineering cartilage scaffold materials, tissue engineering bone scaffold materials, cell scaffold materials, etc.

[0049] The advantages of the present invention are:

[0050] 1. The 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 osmotic pressure regulators of strongly polar ionic compounds (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.

[0051] 2. The hydroxybutyl chitosan derivative of the present invention can also have good compatibility with cell culture medium, not only can make cells survive, but also can promote cell proliferation, and is a cell carrier and tissue engineering scaffold material with better effect and temperature sensitivity.

[0052] 3. The 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.

[0053] 4. The 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.

[0054] 5. The hydroxybutyl chitosan derivative of the present invention can be used for superficial body surface, deep 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

[0055] Figure 1 It is the infrared spectra of the samples of Comparative Example 1, Example 4, Example 5 and Example 6 of the present invention.

[0056] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 2 of the present invention.

[0057] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 4 of the present invention.

[0058] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 5 of the present invention.

[0059] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 6 of the present invention.

[0060] Figure 6 This is the hydrogen nuclear magnetic resonance spectrum of the sample of comparative example 1 of the present invention.

[0061] Figure 7 It is the acid-base titration curve of the sample of comparative example 1 of the present invention.

[0062] Figure 8 It is the acid-base titration curve of the sample of Example 4 of the present invention.

[0063] Fig. 9 This is the acid-base titration curve of the sample in Example 6 of the present invention.

[0064] Fig.10 This is a test graph of the gel temperature of the sample of Example 4 of the present invention, which is prepared into a 5% solution using a 0.85% sodium chloride solution.

[0065] Fig.11 This is a test graph of the gel temperature of the sample of Example 6 of the present invention, which is prepared into a 5% solution using a 0.85% sodium chloride solution.

[0066] Fig.12 Inverted fluorescence microscope photographs of live and dead cell staining were taken after culturing cells of the culture medium control group and samples of comparative example 1, example 4, example 5 and example 6 with 0.85% sodium chloride solution made into 5% solution for 0d, 1d, 3d and 6d. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments.

[0068] Embodiment 1:

[0069] (1) Disperse 50 g of chitosan in 300 mL of 50% sodium hydroxide solution, add 400 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 60° C. for 3 h, adjust the pH value of the reacted material to neutral with hydrochloric acid, dialyze and freeze-dry to obtain 57.6 g of carboxymethyl chitosan.

[0070] (2) Disperse 50 g of the obtained carboxymethyl chitosan in 200 mL of 50% sodium hydroxide solution, stir for 2 h to make it evenly dispersed, then add 200 mL of isopropanol and 300 mL of 1,2-butylene oxide, and react at 50° C. for 48 h.

[0071] (3) The pH value of the reacted material was adjusted to neutral with hydrochloric acid at room temperature, and 57.8 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0072] (4) Dissolve 20 g of the obtained carboxymethyl hydroxybutyl chitosan in 800 mL of isopropanol aqueous solution and stir to dissolve evenly. Add acetic anhydride isopropanol solution, containing 15 mL of acetic anhydride and 75 mL of isopropanol, and stir to react at 20° C. for 2 h. After the reaction is completed, adjust the pH to 8 with sodium hydroxide solution, dialyze and freeze-dry to obtain partially acetylated carboxymethyl hydroxybutyl chitosan.

[0073] Embodiment 2:

[0074] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0075] (2) 40 g of hydroxybutyl chitosan was dispersed in 150 mL of 50% sodium hydroxide solution. After the dispersion was uniform, 160 mL of isopropanol solution containing 35 g of chloroacetic acid was added and the mixture was stirred at 60° C. for 4 h.

[0076] (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 42.7 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0077] (4) Dissolve 10 g of the obtained carboxymethyl hydroxybutyl chitosan in 500 mL of isopropanol aqueous solution and stir to dissolve evenly. Add acetic anhydride isopropanol solution containing 6 mL of propionic anhydride and 50 mL of isopropanol, and stir to react at 30° C. for 3 h. After the reaction is completed, adjust the pH to 11 with sodium hydroxide solution, dialyze and freeze-dry to obtain partially propionylated carboxymethyl hydroxybutyl chitosan.

[0078] Embodiment 3:

[0079] (1) Disperse 100 g of chitosan powder in 900 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 1000 mL of purified water and 2000 mL of isopropanol and react at 60 °C for 24 h.

[0080] (2) The pH value of the reacted material was adjusted to neutral with hydrochloric acid at room temperature, and 110 g of hydroxybutyl chitosan was obtained by dialysis and freeze-drying.

[0081] (3) 30 g of the obtained hydroxybutyl chitosan was dispersed in 200 mL of 50% sodium hydroxide solution, 600 mL of isopropanol aqueous solution was added, and then 100 mL of isopropanol solution containing 20 g of chloroacetic acid was added to react at 50° C. for 6 h. The pH was adjusted to neutral with hydrochloric acid, and the mixture was dialyzed and freeze-dried to obtain partially acetylated carboxymethyl hydroxybutyl chitosan.

[0082] Embodiment 4:

[0083] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0084] (2) Take 20 g of hydroxybutyl chitosan, disperse it in 200 mL of 50% sodium hydroxide solution, add 2000 mL of dimethyl sulfoxide aqueous solution, add 130 mL of aqueous solution containing 35 g of chloroacetic acid, and stir at 50° C. for 4 h.

[0085] (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 22.9 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0086] (4) Dissolve 10 g of the obtained carboxymethyl hydroxybutyl chitosan in 500 mL of isopropanol aqueous solution and stir to dissolve evenly. Add acetic anhydride ethanol solution, containing 10 mL of acetic anhydride and 50 mL of anhydrous ethanol, and stir to react at 40° C. for 5 h. After the reaction is completed, adjust the pH to 8 with sodium hydroxide solution, dialyze and freeze-dry to obtain partially acetylated carboxymethyl hydroxybutyl chitosan.

[0087] Embodiment 5:

[0088] Steps (1) to (3) are the same as those in Example 4.

[0089] (4) Dissolve 10 g of the obtained carboxymethyl hydroxybutyl chitosan in 500 mL of isopropanol aqueous solution and stir to dissolve evenly. Add an isopropanol solution of acetic anhydride, containing 2.5 mL of acetic anhydride and 50 mL of isopropanol, and stir to react at 30° C. for 5 h. After the reaction is completed, adjust the pH to 8 with sodium hydroxide solution, dialyze and freeze-dry to obtain partially acetylated carboxymethyl hydroxybutyl chitosan.

[0090] Embodiment 6:

[0091] Steps (1) to (3) are the same as those in Example 4.

[0092] (4) Dissolve 10 g of the obtained carboxymethyl hydroxybutyl chitosan in 500 mL of isopropanol aqueous solution and stir to dissolve evenly. Add an isopropanol solution of acetic anhydride, containing 1.2 mL of acetic anhydride and 50 mL of isopropanol, and stir to react at 20°C for 3 h. After the reaction is completed, adjust the pH to 8 with sodium hydroxide solution, dialyze and freeze-dry to obtain partially acetylated carboxymethyl hydroxybutyl chitosan. Comparative Example 1:

[0093] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0094] (2) Take 20 g of hydroxybutyl chitosan, disperse it in 200 mL of 50% sodium hydroxide solution, add 2000 mL of dimethyl sulfoxide aqueous solution, add 130 mL of aqueous solution containing 35 g of chloroacetic acid, and stir at 50° C. for 4 h.

[0095] (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 22.9 g of carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0096] Comparative Example 2:

[0097] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0098] (2) Dissolve 10 g of the obtained hydroxybutyl chitosan in 500 mL of isopropanol aqueous solution and stir to dissolve evenly. Add an isopropanol solution of acetic anhydride, containing 10 mL of acetic anhydride and 50 mL of isopropanol, and stir to react at 20° C. for 3 h. After the reaction is completed, adjust the pH to 8 with sodium hydroxide solution, dialyze and freeze-dry to obtain partially acetylated hydroxybutyl chitosan.

[0099] Comparative Example 3:

[0100] (1) Disperse 100 g of chitosan powder in 900 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 1000 mL of purified water and 2000 mL of isopropanol and react at 60 °C for 24 h.

[0101] (2) The pH value of the reacted material was adjusted to neutral with hydrochloric acid at room temperature, and 110 g of hydroxybutyl chitosan was obtained by dialysis and freeze-drying.

[0102] Example 7: Product infrared spectrum test

[0103] Take samples to measure their infrared spectra. The spectra of the four samples of Comparative Example 1 (number 1 in the figure) and Examples 4 to 6 (numbers 2-4 in the figure) are as follows: Figure 1 The peak positions are as follows: 1600cm -1 Left and right 1414cm -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; 1026cm -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; 2963 cm -1 Around, 2926cm -1 Around, 2875cm -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; 3377cm -1 The strong and broad absorption peaks around 1600 cm-1 are the stretching vibration absorption peaks of O—H and N—H. -1 The absorption peaks around 1600 cm are relatively narrow, while the three samples of Examples 4 to 6 have undergone acetylation reaction. -1 The absorption peaks on the left and right are relatively broad, which is caused by the superposition and broadening of the absorption peaks of the carbonyl group of acetyl and the carbonyl group of carboxymethyl.

[0104] Example 8: Product H NMR Spectrum Test

[0105] Dissolve the sample in D containing 20% ​​DCl 2 O solution was tested by nuclear magnetic resonance hydrogen spectrum test, and the spectrum of the sample in Example 2 is as follows Figure 2As shown. Due to the acidic solvent used, the chemical shift of the peak will be offset. The peak positions are as follows: 0.65ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group; 1.27ppm is the absorption peak of the methylene hydrogen atom on the hydroxybutyl group. The presence of the two indicates that the sample has a hydroxybutyl group. 4.06ppm is the absorption peak of the hydrogen atom on the methylene in the carboxymethyl group; the multiple superimposed absorption peaks between 3.0ppm-3.8ppm are the absorption peaks of the hydrogen atoms on the carbons at positions 3, 4, 5, and 6 on the chitosan sugar ring and the two carbon atoms connected to the 6-hydroxyl group on the hydroxybutyl group. 4.32ppm is the absorption peak of the hydrogen atom connected to the carbon at position 1 on the chitosan sugar ring. The new absorption peaks appearing at 2.07ppm and 0.87ppm are the absorption peaks of the methylene and methyl hydrogen atoms on the propionyl group, respectively. It is confirmed that the samples in the above embodiments contain hydroxybutyl groups, carboxymethyl groups and propionyl groups on the chitosan units at the same time.

[0106] Dissolve the sample in D containing 20% ​​DCl 2 O solution was tested by nuclear magnetic hydrogen spectrum, and the spectra of the three samples of Example 4 to Example 6 are as follows Figure 3 , Figure 4 and Figure 5 As shown. Due to the acidic solvent used, the chemical shift of the peak will be offset. The peak positions are as follows: 0.59ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group; 1.19ppm is the absorption peak of the methylene hydrogen atom on the hydroxybutyl group. The presence of the two indicates that the sample has a hydroxybutyl group. 3.97ppm is the absorption peak of the hydrogen atom on the methylene in the carboxymethyl group; the multiple superimposed absorption peaks between 3.0ppm-3.8ppm are the absorption peaks of the hydrogen atoms on the carbons at positions 3, 4, 5, and 6 on the chitosan sugar ring and the two carbon atoms connected to the 6-hydroxyl group on the hydroxybutyl group. 4.23ppm is the absorption peak of the hydrogen atom connected to the carbon at position 1 on the chitosan sugar ring. The new absorption peaks that appear at 1.85ppm and 1.72ppm are the absorption peaks of the methyl hydrogen atoms on the acetyl group. It is confirmed that the samples in the above embodiments contain hydroxybutyl groups, carboxymethyl groups and acetyl groups on the chitosan units at the same time.

[0107] Take the sample of comparative example 1 and dissolve it in D containing 20% ​​DCl 2 O solution was tested with nuclear magnetic hydrogen spectrum, and the sample spectrum was as follows Figure 6As shown. Due to the acidic solvent used, the chemical shift of the peak will be offset. The peak positions are as follows: -0.86ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group; -0.24ppm is the absorption peak of the methylene hydrogen atom on the hydroxybutyl group. The presence of the two indicates the presence of hydroxybutyl groups in the sample. 2.57ppm is the absorption peak of the hydrogen atom on the methylene group in the carboxymethyl group. The multiple superimposed absorption peaks between 1.5ppm and 2.5ppm are the absorption peaks of the hydrogen atoms on the carbons at positions 2, 3, 4, 5, and 6 on the chitosan sugar ring and the two carbon atoms connected to the 6-hydroxyl group on the hydroxybutyl group. 3.38ppm is the absorption peak of the hydrogen atom connected to the carbon at position 1 on the chitosan sugar ring.

[0108] Example 9: Acid-base titration test

[0109] The samples prepared in Comparative Example 1, Example 4 and Example 6 were dissolved in excess hydrochloric acid and titrated with a standard sodium hydroxide solution using a fully automatic potentiometric titrator. The titration curves were as follows: Figure 7 , Figure 8 and Fig. 9 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 2-3 jump points in the curve. The jump points at pH about 4.4 and 9.5 are the amino titration starting point and amino titration end point, respectively. The amount of titratable amino substances contained in the unit mass of the sample can be calculated. The titratable amino substances contained in the sample of comparative example 1 are 2.49×10 -3 mol / g, the titratable amino group contained in the sample of Example 4 is 1.24×10 -3 mol / g, the number of titratable amino groups decreased to 49.8% of the original. The titratable amino groups contained in the sample of Example 6 were 1.78×10 -3 mol / g, the number of titrable amino groups was reduced to 71.5% of the original. Primary amino groups and alkylated amino groups such as secondary and tertiary amino groups are still titrable. However, acetylated amino groups are not titrable, which also shows that the amino groups of the sample of comparative example 1 are partially acetylated after further reaction, and the degree of acetylation increases with the increase of the amount of acylating agent within a certain range.

[0110] Example 10: Preparation and performance testing of hydrogel

[0111] The samples of Comparative Example 2, Example 4 and Example 6 were prepared into 5% solutions with 0.85% sodium chloride solution. The samples of Example 4 and Example 6 were dissolved into transparent solutions. The gel temperature was tested by rheometer, and the results were 28.5℃ and 26.6℃ respectively. Fig.10 and Fig.11The test results show that the above samples meet the requirements of gelation below body temperature and are suitable for clinical application. The sample of Comparative Example 2 contains hydroxybutyl and acetyl groups but no carboxymethyl groups, which results in the material being able to be formulated into a solution at low temperature, but turning white after being heated to body temperature, and having no salt solubility, which is similar to the sodium chloride solution prepared with ordinary hydroxybutyl chitosan.

[0112] Example 11: Three-dimensional cell culture experiment in hydrogel

[0113] The samples in Comparative Example 1, Example 4 and Example 6 were prepared into 5% solutions with isotonic sodium chloride solution. They were mixed with HK-2 cell fluid and added to 96-well plates. After solidification at 37°C, 37°C DMEM / F12 basal medium containing 10% fetal bovine serum and 1% double antibody was added to the surface of the plates. The plates were incubated in a constant temperature incubator at 37°C and 5% CO. 2 The cells were cultured under saturated humidity and medium as control. The results showed that the cells survived well in all hydrogels, but proliferated slowly in the hydrogel prepared by the sample in Comparative Example 1, while in the hydrogels prepared by the samples in Examples 4 and 6, the cells not only survived well but also proliferated significantly, especially at 6 days, the cells grew into sheets, and the number was more than that of the control group. The samples of the cultured cells were stained for live and dead cells, and the inverted fluorescence microscope photos were as follows: Fig.12 As shown. This shows that the acylated carboxymethyl hydroxybutyl chitosan material is more suitable for cell growth and has better cell survival performance, and can be used as a new type of cell scaffold material. Compared with chitosan materials, the presence of hydroxybutyl in the acylated carboxymethyl hydroxybutyl chitosan improves water solubility and produces temperature sensitivity; the presence of carboxyl group further improves its compatibility with physiological concentration salt solution; and the presence of acylated group replaces part of the amino group, reduces the number of charges of the amino group under neutral conditions, and improves the cell compatibility of the gel.

[0114] 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. An acylated anionic group-substituted hydroxybutyl chitosan derivative, characterized in that: It has the following structure: Wherein, R1, R2, R3, and R4 are one of the following groups: H, acyl group, hydroxybutyl, polyhydroxybutyl, anionic substituent group; The anionic substituent group is selected from carboxymethyl, 2-carboxymethoxybutyl, 2-carboxymethylpolyoxybutyl, carboxyethyl, methylcarboxymethyl, carboxypropyl, 2-carboxypropyl, 1,3-dicarboxypropyl, carboxybutyl, 3-carboxybutyl, 2-carboxymethylpropyl, sulfonic acid, sulfonic acid propyl, 2-hydroxysulfonic acid propyl; The acyl group is acetyl, propionyl or butyryl; The degree of substitution of the hydroxybutyl group is 1.0-4.0, the degree of substitution of the anionic substituent group is 0.1-3.0, and the degree of substitution of the acyl group is 0.05-0.

95.

2. The acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 1, characterized in that: The anionic substituent group is a carboxymethyl group.

3. The acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 2, characterized in that: The degree of substitution of hydroxybutyl is 1.2-3.0, the degree of substitution of carboxymethyl is 0.2-2.0, and the degree of substitution of acyl is 0.1-0.

9.

4. The acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 2, characterized in that: The degree of substitution of hydroxybutyl is 1.5-2.8, the degree of substitution of carboxymethyl is 0.3-1.8, and the degree of substitution of acyl is 0.2-0.

8.

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

6. The method for preparing the acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: Chitosan is firstly modified with ionic groups to obtain ion-modified chitosan, then modified with hydroxybutyl to obtain hydroxybutyl chitosan derivatives containing ionic group substitution, and then modified with acylation to obtain acylated anionic group-substituted hydroxybutyl chitosan derivatives.

7. The method for preparing the acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: Firstly, chitosan is modified by hydroxybutylation to obtain hydroxybutyl substituted chitosan; then, hydroxybutyl chitosan is modified by anionic groups to obtain hydroxybutyl chitosan derivatives containing anionic group substituted, and then acylated chitosan derivatives are obtained by acylation.

8. The method for preparing the acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: Firstly, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then, it is modified by acylation to obtain acylated hydroxybutyl chitosan derivatives; and then, anionic group modification is performed to obtain acylated anionic group-substituted hydroxybutyl chitosan derivatives.

9. The method for preparing the acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: Firstly, chitosan is modified by hydroxybutylation to obtain hydroxybutyl substituted chitosan; then, hydroxybutyl chitosan is modified by anionic groups to obtain hydroxybutyl chitosan derivatives containing anionic group substitution, namely, acylated anionic group substituted hydroxybutyl chitosan derivatives.

10. The method for preparing the acylated anionic group-substituted hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: Firstly, chitosan is modified with anionic groups to obtain chitosan substituted with anionic groups; then, the chitosan substituted with anionic groups is modified with hydroxybutyl to obtain a hydroxybutyl chitosan derivative substituted with anionic groups, namely, an acylated anionic group substituted hydroxybutyl chitosan derivative.

11. The method for preparing the acylated anionic group hydroxybutyl chitosan derivative according to any one of claims 6 to 10, characterized in that: The acylation reaction reagent is acetic anhydride, propionic anhydride or butyric anhydride.

12. Use of the acylated anionic group-substituted hydroxybutyl chitosan derivative hydrogel according to claim 5 for preparing wound dressings, postoperative anti-adhesion materials, tissue filling materials, tissue engineering skin scaffold materials, tissue engineering cartilage scaffold materials, tissue engineering bone scaffold materials, and cell scaffold materials.

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