Ionic group-containing substituted hydroxybutyl chitosan derivative as well as preparation method and application thereof

Abstract

The invention discloses a substituted hydroxybutyl chitosan derivative containing an ionic group as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The chitosan derivative can be obtained by performing ion group modification on chitosan to obtain ion modified chitosan and then performing hydroxybutylation modification. Or chitosan is firstly subjected to hydroxybutylation modification to obtain hydroxybutyl substituted chitosan, and then the hydroxybutyl substituted chitosan is subjected to ionic group modification. The substituted hydroxybutyl derivative containing the ionic group can be compatible with an osmotic pressure regulator of an ionic compound with strong polarity to obtain a temperature-sensitive solution with good performance, the solution is uniform and transparent at low temperature, the gel at body temperature is also uniform, the transparency is very high, the gel strength is relatively large, the problems of whitening and fragmentation do not exist, and the preparation method is simple. And the method can be well matched with the human physiological environment. The material can be used as a temperature-sensitive cell carrier, a tissue engineering scaffold material and a superficial and deep body surface 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 class of ionic group-substituted hydroxybutyl chitosan derivatives 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 chitosan derivative grafted with hydroxybutyl and anionic and cationic groups at the same time, which can 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, phosphate buffer, 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 hydroxybutyl chitosan derivative containing ionic group substitution of the present invention has the following structure:

[0009]

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

[0011] H, hydroxybutyl, ionic group, acetyl.

[0012] The ionic groups are: carboxymethyl, carboxyethyl, methylcarboxymethyl, carboxypropyl, 2-carboxypropyl, 1,3-dicarboxypropyl, carboxybutyl, 3-carboxybutyl, 2-carboxymethylpropyl, sulfonic acid, sulfonic acid propyl, 2-hydroxysulfonic acid propyl, trimethylaminohydroxypropyl chloride, trimethylaminoacetyl chloride, and groups formed by replacing the hydrogen atoms on the hydroxybutyl group with the above ionic groups.

[0013] The structural formula of the group is as follows:

[0014] Hydroxybutyl: CH3CH2CHOHCH2-acetyl: Carboxymethyl: -CH2COOH Carboxyethyl: -CH2CH2COOH Methyl Carboxymethyl: Carboxypropyl: -CH2CH2CH2COOH; 2-Carboxypropyl: 1,3-Dicarboxypropyl: Carboxybutyl: -CH2CH2CH2CH2COOH; 3-carboxybutyl: 2-Carboxymethylpropyl: Sulfonic acid group: -SO3H Sulfonic acid propyl group: -CH2CH2CH2SO3H; 2-hydroxysulfonic acid propyl group: Trimethylamino Hydroxypropyl Chloride: Trimethylaminoacetyl chloride: 2-Carboxymethoxybutyl: 2-Carboxyethoxybutyl:

[0015] The degree of substitution of the hydroxybutyl group is 1.0-4.0; the degree of substitution of the ionic group is 0.1-3.0.

[0016] Preferably, the degree of substitution of the hydroxybutyl group is 1.5-3.0; the degree of substitution of the ionic group is 0.3-2.0.

[0017] Above-mentioned hydroxybutyl chitosan derivative forms solution with pure water, or with polyol compatibility such as glycerol, mannitol, even with the osmotic pressure regulating substance compatibility of strong polar ionic compound (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, phosphate buffer 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.

[0018] Furthermore, the present invention also provides a hydrogel comprising the above-mentioned hydroxybutyl chitosan derivative substituted with ionic groups, comprising the following components by mass: 0.1%-10% of hydroxybutyl chitosan derivative substituted with ionic groups; 0.1%-10% of osmotic pressure regulator; and the balance of water.

[0019] 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.

[0020] Another object of the present invention is to provide a method for preparing the above-mentioned hydroxybutyl chitosan derivative substituted with ionic groups. The chitosan derivative can be prepared by any of the following methods.

[0021] Method 1: First, chitosan is modified with ionic groups to obtain ion-modified chitosan, and then hydroxybutylated chitosan is modified to obtain hydroxybutyl chitosan derivatives containing ionic group substitution. The detailed steps are as follows:

[0022] (1) Dissolving or dispersing chitosan with a solvent, adding an ionic group modification reagent to react: a) connecting the modification reagent with the hydroxyl 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 ionic group-modified chitosan; or b) allowing an aldehyde-containing Schiff base modification reagent to react with the amino group to generate a Schiff base, which is then reduced 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, then adjusting the pH value and adding a reducing agent, reacting at a certain temperature for a certain time, and preliminarily purifying the product to obtain ionic group-modified chitosan; or c) allowing the modification reagent to react with the amino group to undergo a Michael reaction Addition and connection: adding an ion-containing 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 performing preliminary purification to obtain ionic group-modified chitosan; or d) connection by a ring-opening reaction reagent: adding an ion-containing ring-opening 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 performing preliminary purification to obtain ionic group-modified chitosan; (2) dissolving or dispersing the ion-modified 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 ionic group-substituted hydroxybutyl chitosan derivative.

[0023] Method 2: First, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then, the hydroxybutyl-chitosan is modified by ionic groups to obtain the final hydroxybutyl-chitosan derivative containing ionic group substitution. The detailed steps are as follows:

[0024] (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 ionic 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 alkaline solution, and the reaction is carried out at a certain temperature for a certain time; or b) an ionic 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: an ionic Schiff base reaction reagent 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. 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 by Michael addition of a modifying reagent with an amino group: add an ion-containing Michael addition reaction reagent, 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 by a ring-opening reaction reagent: add an ion-containing ring-opening reaction reagent, adjust the pH value of the system with an alkaline solution or hydrochloric acid, and react at a certain temperature for a certain time; (3) 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 hydroxybutyl chitosan derivative substituted with an ionic group.

[0025] 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.

[0026] 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, sodium 3-chloro-2-hydroxypropanesulfonate, and 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0027] The ion-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.

[0028] The ion-containing Michael addition reaction reagent in step c) includes acrylic acid and methacrylic acid.

[0029] The ion-containing ring-opening reaction reagent in step d) includes: sodium 2,3-epoxypropanesulfonate, propane sultone, and 2,3-epoxypropyltrimethylammonium chloride.

[0030] The alkali is sodium hydroxide or potassium hydroxide, and the molar concentration of the alkali solution is 0.01-15 mol / L.

[0031] 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.

[0032] The pH value of the Michael addition reaction in step c) is in the range of 3-7.

[0033] 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.

[0034] The molar concentration of the alkaline solution in the hydroxybutylation reaction in step (2) is 0.01-15 mol / L.

[0035] The reaction temperature in step (1) or (2) is 0°C-70°C; the reaction time is 0.5h-120h.

[0036] The advantages of the present invention are:

[0037] 1. The 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 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 can obtain a temperature-sensitive solution with good performance: 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.

[0038] 2. The 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.

[0039] 3. The 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.

[0040] 4. The 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.

[0041] 5. The chitosan derivatives of the present invention can be used for superficial body surface, deep body surface tissue or implanted in the body. They 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

[0042] Figure 1 This is the infrared spectrum of the sample of Example 1 of the present invention.

[0043] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the sample in Example 1 of the present invention.

[0044] Figure 3 This is the infrared spectrum of the sample of Example 2 of the present invention.

[0045] Figure 4 This is the H NMR spectrum of the sample of Example 3 of the present invention.

[0046] Figure 5 This is the H NMR spectrum of the sample of Example 4 of the present invention.

[0047] Figure 6 This is the H NMR spectrum of the sample of Example 6 of the present invention.

[0048] Figure 7 This is the acid-base titration curve of the sample in Example 3 of the present invention.

[0049] Figure 8 This is a photograph of the state of the sample in Example 1 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.

[0050] Fig. 9 This is a photograph of the state of the sample of 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.

[0051] Fig.10 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% concentration solution and then heated to 37°C.

[0052] Fig.11 This is a test graph of the gel temperature of the sample of Example 5 of the present invention, which is prepared into a 5% solution using 0.85% sodium chloride solution.

[0053] Fig.12 This is a test graph of the gel temperature of the sample of Example 6 of the present invention, which is prepared into a 4% solution using a 0.85% sodium chloride solution. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments.

[0055] Embodiment 1:

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

[0057] (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.

[0058] Take a sample and measure its infrared spectrum, such as Figure 1 The peak positions are as follows: 1594 cm -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; 1051cm -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, 2922cm -1 Around, 2875cm -1 The three peaks on the left and right and 1461cm -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; 3367cm -1 The strong and broad absorption peaks on the left and right are the stretching vibration absorption peaks of O—H and N—H.

[0059] Take the sample and dissolve it in D2O solution containing 20% ​​DCl for NMR hydrogen spectrum test. The spectrum is as follows: Figure 2 . Due to the acidic solvent used, the chemical shift of the peak will be offset, but the relative position and peak shape of the peak are basically the same. Spectrum description: -0.86ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group, and 0.23ppm 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 in the carboxymethyl group, and 1.42ppm is presumed to be the absorption peak of the hydrogen atom on the methyne connected to the original hydroxyl group after the hydroxyl group of the hydroxybutyl group is replaced by the carboxymethyl group. The multiple superimposed absorption peaks between 1.5ppm and 2.5ppm are the absorption peaks of the hydrogen atoms on the 2nd, 3rd, 4th, 5th, and 6th carbons on the chitosan sugar ring and the two carbon atoms on the hydroxybutyl group connected to the 6th hydroxyl group. 3.38ppm is the absorption peak of the hydrogen atom connected to the 1st carbon on the chitosan sugar ring.

[0060] Embodiment 2:

[0061] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756 A.

[0062] (2) Take 50 g of hydroxybutyl chitosan and dissolve it in 200 mL of purified water and 50 mL of acetic acid. After uniform dissolution, add 100 mL of isopropanol solution containing 75 g of glyoxylic acid and react at 50° C. for 1 h. After the reaction, adjust the pH value of the material to 9-10 with sodium hydroxide solution, then add 40 mL of aqueous solution containing 20 g of potassium borohydride and react for 1 h.

[0063] (3) The reacted material was dialyzed and freeze-dried to obtain 58.2 g of hydroxybutyl / N-carboxymethyl chitosan.

[0064] Take samples for infrared spectrum test, such as Figure 3 The peak position is as follows: 1589cm -1 Left and right 1397cm -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; 2963cm -1 Around, 2929cm -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 on the left and right are the stretching vibration absorption peaks of O—H and N—H.

[0065] Embodiment 3:

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

[0067] (2) Take 50 g of hydroxybutyl chitosan and disperse it in 200 mL of purified water. After the dispersion is uniform, add 60 g of acrylic acid and stir the mixture at 25° C. for 96 h.

[0068] (3) The pH value of the reacted material was adjusted to about neutral with sodium hydroxide solution, and then dialyzed and freeze-dried to obtain 55 g of carboxyethyl hydroxybutyl chitosan.

[0069] The sample was dissolved in a D2O solution containing 20% ​​DCl and tested by liquid NMR hydrogen spectrum. The results are as follows: Figure 4. Due to the acidic solvent used, the chemical shift of the peak will be offset. -0.47ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group, and 0.14ppm 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. The multiple superimposed absorption peaks between 1.5ppm and 2.8ppm 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. 2.76ppm is the absorption peak of the hydrogen atoms on the methylene connected to the amino or hydroxyl group on the carboxyethyl group, and 1.53ppm is the absorption peak of the hydrogen atoms on the methylene connected to the carboxyl group on the carboxylethyl group. 3.71ppm is the absorption peak of the hydrogen atom connected to the carbon at position 1 on the chitosan sugar ring.

[0070] Embodiment 4:

[0071] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756 A.

[0072] (2) 100 g of hydroxybutyl chitosan was dispersed in 300 mL of 50% sodium hydroxide solution. After the dispersion was uniform, 400 mL of an aqueous solution containing 200 g of 1,3-propane sultone was added and the mixture was stirred at 30° C. for 24 h.

[0073] (3) The pH value of the reacted material was adjusted to neutral with sodium hydroxide solution, and 135.6 g of sulfonate propyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0074] Take the sample and dissolve it in D2O solution containing 20% ​​DCl to conduct nuclear magnetic resonance hydrogen spectrum test. The spectrum is as follows Figure 5 . Due to the acidic solvent used, the chemical shift of the peak will be offset. The absorption peak at -1.25ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group, and the absorption peak at -0.63ppm 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. The three peaks at 0.98ppm, -1.0ppm and -0.20ppm are the absorption peaks of hydrogen on the three carbon atoms (arranged in order from the sulfonic acid group to the imino group) on the substituted sulfonic acid propyl group. The absorption peak at 0.08ppm is the absorption peak of the hydrogen atom on the carbon connected to the nitrogen atom on the sugar ring. The multiple superimposed absorption peaks between 1.0ppm and 2.5ppm are the absorption peaks of 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. The absorption peak at 2.97ppm is the absorption peak of the hydrogen atom connected to the carbon at position 1 on the chitosan sugar ring.

[0075] Embodiment 5:

[0076] (1) 60 g of chitosan was dispersed in 200 mL of purified water, and then 200 mL of isopropanol was added. The mixture was stirred for 2 h to make it uniformly dispersed. 200 mL of isopropanol solution containing 90 g of pyruvic acid and 200 mL of purified water were added, and the pH value was adjusted to 4-5. The mixture was reacted at 30° C. for 2 h. The pH value of the reacted material was adjusted to 8-9 with sodium hydroxide solution, and then 20 mL of an aqueous solution containing 12 g of potassium borohydride was added. The mixture was reacted for 2 h. After dialysis and freeze-drying, 70.8 g of methyl carboxymethyl chitosan was obtained.

[0077] (2) 60 g of the obtained methyl carboxymethyl chitosan was dispersed in 200 mL of 45% sodium hydroxide solution and stirred for 2 h to make it uniformly dispersed. Then, 200 mL of isopropanol and 500 mL of 1,2-butylene oxide were added and reacted at 40° C. for 48 h.

[0078] (3) The pH value of the reacted material was adjusted to neutral with hydrochloric acid, and 76.4 g of methyl carboxymethyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0079] Embodiment 6:

[0080] (1) Disperse 20 g of chitosan in 200 mL of 20% sodium hydroxide solution, stir for 2 h to make it evenly dispersed, add 20 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride and 400 mL of purified water, react at 50 ° C for 24 h, adjust the pH value of the reacted material to neutral with hydrochloric acid, dialyze and freeze-dry to obtain 24.3 g of hydroxypropyltrimethylammonium chloride chitosan.

[0081] (2) Disperse 20 g of hydroxypropyltrimethylammonium chloride chitosan in 100 mL of 50% sodium hydroxide solution, stir for 2 h to make it evenly dispersed, then add 100 mL of isopropanol and 200 mL of 1,2-butylene oxide, and react at 30° C. for 48 h.

[0082] (3) The pH value of the reacted material was adjusted to neutral with hydrochloric acid, and 28.5 g of trimethylaminohydroxypropylhydroxybutyl chitosan chloride was obtained through dialysis and freeze-drying.

[0083] Take the sample and dissolve it in D2O solution containing 20% ​​DCl for NMR hydrogen spectrum test. The spectrum is as follows: Figure 6. Due to the acidic solvent used, the chemical shift of the peak will be offset. -1.00ppm is the absorption peak of the methyl hydrogen atom on the hydroxybutyl group, and -0.42ppm 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. 1.37ppm is the absorption peak of the hydrogen atom on the methyl group directly connected to the nitrogen atom on the trimethylaminohydroxypropyl chloride group, indicating the presence of trimethylaminohydroxypropyl chloride groups in the sample. The multiple superimposed absorption peaks between 1ppm-2.7ppm (except the peak at 1.37ppm) are the superposition of the absorption peaks of the hydrogen atoms on the 3rd, 4th, 5th, and 6th carbon atoms on the chitosan sugar ring and the methylene and methine carbon atoms on the hydroxybutyl group and the trimethylaminohydroxypropyl chloride group that are directly connected to the nitrogen atom in sequence. 3.21ppm is the absorption peak of the hydrogen atom connected to the 1st carbon on the chitosan sugar ring.

[0084] Example 7: Acid-base titration test

[0085] The hydroxybutyl / N-carboxymethyl chitosan material prepared in Example 2 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 7 As shown. The solid line is the curve of the relationship between pH value and the volume consumed by the standard sodium hydroxide solution, and the dotted line is the curve of the relationship 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 three obvious jump points in the curve, which are the carboxyl titration end point and the different amino titration end points. Combined with the hydrochloric acid titration end point at pH 2.2, the carboxymethyl substitution degree can be calculated to be 0.95.

[0086] Example 8: Solution property test of materials

[0087] The material in Example 1 was dissolved in purified water and 0.85% sodium chloride solution at a concentration of 3%, as samples ① and ②. The sample prepared in Example 6 was dissolved in purified water and 0.85% sodium chloride solution at a concentration of 3%, as samples ③ and ④. Hydroxybutyl chitosan prepared according to the method disclosed in patent application CN 102276756 A was dissolved in purified water and 0.85% sodium chloride solution at a concentration of 3%, as samples ⑤ and ⑥ as controls.

[0088] Table 1 The status of each sample under different conditions

[0089]

[0090] The above data show that the sample in the above embodiment can form a uniform solution with a nearly isotonic sodium chloride solution after liquid preparation, and transform into a non-flowing gel state after heating to 37°C. The gel is still uniform and transparent and not easy to break, which meets clinical needs.

[0091] Example 9: Preparation and performance testing of hydrogel

[0092] The sample of Example 5 was prepared into a 5% solution with 0.85% sodium chloride solution. The osmotic pressure was 303 mOsmol / kg when tested by an osmometer. The gel temperature was 19.2°C when tested by a rheometer. The intersection of the storage modulus (G') and the loss modulus (G") is the gel temperature. Fig.11 The sample of Example 6 was prepared into a 4% solution with 0.85% sodium chloride solution. The osmotic pressure was 305mOsmol / kg when tested by an osmometer. The gel temperature was 26.0°C when tested by a rheometer. The intersection of the storage modulus (G') and the loss modulus (G") is the gel temperature. Fig.12 As shown in the figure, the above hydrogel solidifies into a non-flowing gel at body temperature, which is suitable for dressings, fillings or scaffold materials for use in the body 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.

[0093] 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 hydroxybutyl chitosan derivative containing ionic groups, characterized in that: It has the following structure: Wherein, R1, R2, R3, and R4 are one of the following groups: H, hydroxybutyl, acetyl, and ionic groups; The ionic group is carboxymethyl, carboxyethyl, methylcarboxymethyl, carboxypropyl, 2-carboxypropyl, 1,3-dicarboxypropyl, carboxybutyl, 3-carboxybutyl, 2-carboxymethylpropyl, sulfonic acid, sulfonic acid propyl, 2-hydroxysulfonic acid propyl, trimethylaminohydroxypropyl chloride or trimethylaminoacetyl chloride.

2. The hydroxybutyl chitosan derivative containing ionic group substitution 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 ionic group is 0.1-3.

0.

3. A hydrogel comprising the hydroxybutyl chitosan derivative substituted with ionic groups according to claim 1, characterized in that: The invention comprises the following components by mass: 0.1%-10% of hydroxybutyl chitosan derivative substituted with ionic groups; 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.

4. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution as claimed in claim 1, characterized in that: The following steps are involved: (1) Dissolving or dispersing chitosan with a solvent, adding an ionic group modification reagent to react: a) connecting the modification reagent with the hydroxyl 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 ionic group-modified chitosan; or b) allowing an aldehyde-containing Schiff base modification reagent to react with the amino group to generate a Schiff base, which is then reduced 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, then adjusting the pH value and adding a reducing agent, reacting at a certain temperature for a certain time, and preliminarily purifying the product to obtain ionic group-modified chitosan; or c) allowing the modification reagent to react with the amino group to undergo a Michael reaction Addition and connection: adding an ion-containing 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 performing preliminary purification to obtain ionic group-modified chitosan; or d) connection by a ring-opening reaction reagent: adding an ion-containing ring-opening 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 performing preliminary purification to obtain ionic group-modified chitosan; (2) dissolving or dispersing the ion-modified 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 ionic group-substituted hydroxybutyl chitosan derivative.

5. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution as claimed in claim 1, characterized in that: The following steps are involved: (1) According to the method disclosed in patent application CN 102276756A, chitosan is subjected to hydroxybutyl modification to obtain hydroxybutyl chitosan; (2) the hydroxybutyl chitosan is dissolved or dispersed in a solvent, and an ionic 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 alkaline solution, and the reaction is carried out at a certain temperature for a certain time; or b) an ionic 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: an ionic Schiff base reaction reagent containing an aldehyde group 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. The reaction is carried out at room temperature for a certain period of time, and 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 period of time; or c) the modification reagent is connected by Michael addition with the amino group: an ion-containing Michael addition reaction reagent is added, the pH value of the system is adjusted with an alkaline solution or hydrochloric acid, and the reaction is carried out at a certain temperature for a certain period of time; or d) the connection is carried out 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 alkaline solution or hydrochloric acid, and the reaction is carried out at a certain temperature for a certain period of 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 period of time, taken out and freeze-dried to obtain the final hydroxybutyl chitosan derivative substituted with an ionic group.

6. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution as claimed in claim 4 or 5, 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.

7. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution as claimed in claim 4 or 5, characterized in that: 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, sodium 3-chloro-2-hydroxypropanesulfonate, and 3-chloro-2-hydroxypropyltrimethylammonium chloride.

8. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution as claimed in claim 4 or 5, characterized in that: The ion-containing Schiff base reaction reagent in step b) includes glyoxylic acid, pyruvic acid or α-ketoglutaric acid; the reducing agent includes sodium borohydride, lithium aluminum hydride, lithium borohydride, potassium borohydride, sodium cyanoborohydride or borane ammonia.

9. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution as claimed in claim 4 or 5, characterized in that: The ion-containing Michael addition reaction reagent in step c) includes acrylic acid or methacrylic acid.

10. The method for preparing the hydroxybutyl chitosan derivative containing ionic group substitution according to claim 4 or 5, characterized in that: The ion-containing ring-opening reaction reagent in step d) includes: sodium 2,3-epoxypropanesulfonate, propane sultone or 2,3-epoxypropyltrimethylammonium chloride.

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