Sulfonic acid propyl hydroxybutyl chitosan derivative as well as preparation method and application thereof

Abstract

The invention discloses a sulfonic acid propyl hydroxybutyl chitosan derivative as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The chitosan derivative can be obtained by firstly carrying out sulfonic acid propyl group modification on chitosan to obtain sulfonic acid propyl chitosan and then carrying out hydroxybutylation modification. Or the chitosan is firstly subjected to hydroxybutylation modification to obtain hydroxybutyl substituted chitosan, and then the hydroxybutyl chitosan is subjected to sulfonic acid propyl group modification to obtain the modified chitosan. The sulfonic acid propyl hydroxybutyl derivative disclosed by the invention can be compatible with an osmotic pressure regulator of a strong-polarity ionic compound to obtain a temperature-sensitive solution with good performance, the solution is uniform and transparent at a low temperature, the gel is also uniform at a body temperature, the transparency is very high, the gel strength is relatively large, and the problems of whitening and fragmentation do not exist; 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 sulfonate propyl hydroxybutyl chitosan derivative and a preparation method and application thereof. Background Art

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

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

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

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

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

[0007] In view of the above problems, an object of the present invention is to provide a chitosan derivative grafted with a sulfonate propyl group and a hydroxybutyl group 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 sulfonic acid propyl hydroxybutyl chitosan derivative of the present invention has the following structure:

[0009]

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

[0011] H, acetyl, hydroxybutyl, sulfonate propyl.

[0012] Furthermore, R1 is hydroxybutyl or H, R2 is hydroxybutyl or H; R3 is H, acetyl, hydroxybutyl or sulfonate propyl, and R4 is H or sulfonate propyl.

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

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

[0015] Hydroxybutyl: CH3CH2CHOHCH2-; Sulfonic acid propyl: -CH2CH2CH2SO3H; Acetyl:

[0016] The substitution degree of the hydroxybutyl group is 1.0-4.0; the substitution degree of the sulfonic acid propyl group is 0.1-2.0.

[0017] The preferred substitution degree of hydroxybutyl is 1.5-3.0; the preferred substitution degree of sulfonate propyl is 0.3-1.8.

[0018] The above-mentioned sulfonic acid propyl hydroxybutyl chitosan derivative forms a solution with pure water, or is compatible with polyols such as glycerol and mannitol, or even with osmotic pressure regulating substances of strongly polar ionic compounds (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, phosphate buffer, etc.), and 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. Moreover, the solution at low temperatures 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. The material can also have good compatibility with cell culture fluid, and has the function of being a cell carrier, and is a cell carrier with better effect and temperature sensitivity.

[0019] Furthermore, the present invention also provides a hydrogel comprising the above-mentioned sulfonate propyl hydroxybutyl chitosan derivative, comprising the following components by mass: sulfonate propyl hydroxybutyl chitosan derivative: 0.1%-10%; osmotic pressure regulator: 0.1-10%; water: the balance.

[0020] 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.

[0021] Another object of the present invention is to provide a method for preparing the above-mentioned sulfonate propyl hydroxybutyl chitosan derivative. The chitosan derivative can be prepared by any of the following methods.

[0022] Method 1: First, chitosan is modified by sulfonate propylation to obtain sulfonate propyl chitosan, and then modified by hydroxybutylation to obtain sulfonate propyl hydroxybutyl chitosan derivatives. The detailed steps are as follows:

[0023] (1) dissolving or dispersing chitosan with a solvent, adding 1,3-propane sultone, reacting at a certain temperature for a certain time, and performing preliminary purification to obtain sulfonate propyl chitosan; (2) dissolving or dispersing sulfonate propyl 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 sulfonate propyl hydroxybutyl chitosan derivative.

[0024] Method 2: First, chitosan is modified by hydroxybutylation to obtain hydroxybutyl-substituted chitosan; then, the hydroxybutyl chitosan is modified by sulfonate propyl group to obtain the final sulfonate propyl hydroxybutyl chitosan derivative. The detailed steps are as follows:

[0025] (1) chitosan is modified by hydroxybutylation 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, 1,3-propane sultone is added, and the reaction is carried out at a certain temperature for a certain time; (3) after the reaction is completed, the reaction system is stabilized at room temperature, placed in a dialysis bag for dialysis for a certain time, and taken out for freeze drying to obtain the final sulfonate propyl hydroxybutyl chitosan derivative.

[0026] 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.

[0027] The alkali is sodium hydroxide or potassium hydroxide.

[0028] The molar concentration of the alkaline solution is 0.01-15 mol / L.

[0029] The reaction temperature is 20°C-80°C.

[0030] The reaction time is 1h-120h.

[0031] The advantages of the present invention are:

[0032] 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 regulating substances of strongly polar ionic compounds (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, phosphate buffer, 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. Moreover, 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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

[0037] Figure 1 This is the infrared spectrum of the sample of Example 2 of the present invention.

[0038] Figure 2 This is the liquid nuclear magnetic resonance hydrogen spectrum of the sample in Example 3 of the present invention.

[0039] Figure 3 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.

[0040] Figure 4 This is a photograph of hydroxybutyl chitosan after being dissolved in 0.85% sodium chloride solution at low temperature to prepare a 3% solution and then heated to 37°C.

[0041] Figure 5 This is a test graph of the gel temperature of the sample of Example 4 of the present invention, which is prepared into a 4% solution using 0.85% sodium chloride solution. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments.

[0043] Embodiment 1:

[0044] (1) Disperse 50 g of chitosan in 100 mL of 50% sodium hydroxide solution, add 100 mL of isopropanol, stir for 2 h to make the dispersion uniform, add 100 mL of an aqueous solution containing 50 g of 1,3-propane sultone and 100 mL of purified water, react at 25 ° C for 2 h, adjust the pH value of the reacted material to neutral with sodium hydroxide solution, dialyze and freeze-dry to obtain 55.8 g of sulfonate propyl chitosan.

[0045] (2) Disperse 50 g of the obtained sulfonate propyl chitosan in 100 mL of 50% sodium hydroxide solution, stir for 2 h to make the dispersion uniform, then add 100 mL of isopropanol and 200 mL of 1,2-butylene oxide, and react at 70° C. for 12 h.

[0046] (3) The pH value of the reacted material was adjusted to neutral with hydrochloric acid, and 56.2 g of sulfonate propyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0047] Embodiment 2:

[0048] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756 A.

[0049] (2) 100 g of hydroxybutyl chitosan was dispersed in 300 mL of 50% sodium hydroxide solution. After uniform dispersion, 300 mL of an aqueous solution containing 100 g of 1,3-propane sultone was added, and the mixture was stirred at 20° C. for 72 h. The pH value of the reacted material was adjusted to neutral with sodium hydroxide solution, and 122.1 g of sulfonate propyl hydroxybutyl chitosan was obtained by dialysis and freeze drying.

[0050] Embodiment 3:

[0051] (1) Hydroxybutyl chitosan was prepared according to the method disclosed in patent application CN 102276756.

[0052] (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.

[0053] (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.

[0054] Embodiment 4:

[0055] (1) Disperse 50 g of chitosan in 1000 mL of an aqueous solution containing 11% potassium hydroxide and 8% guanidine hydrochloride, stir to make the dispersion uniform, freeze overnight and then take out and melt at low temperature to obtain a uniform solution, add 500 mL of 1,2-butylene oxide, react at 25° C. for 120 h, adjust the pH value of the reacted material to neutral with hydrochloric acid, dialyze and freeze-dry to obtain 58.0 g of hydroxybutyl chitosan.

[0056] (2) 50 g of the above hydroxybutyl chitosan was dispersed in 300 mL of 50% sodium hydroxide solution. After the dispersion was uniform, 300 mL of N,N-dimethylacetamide solution containing 100 g of 1,3-propane sultone was added and stirred at 25° C. for 6 h.

[0057] (3) The pH value of the reacted material was adjusted to neutral with sodium hydroxide solution, and 57.9 g of sulfonate propyl hydroxybutyl chitosan was obtained through dialysis and freeze-drying.

[0058] Example 5: Product structure identification and functional testing

[0059] The samples in Example 2 were subjected to infrared spectroscopy test. Figure 1 The peak positions are as follows: 1032cm -1 The absorption peaks on the left and right are the symmetrical vibration absorption peaks of O=S=O, 1316cm -1 The absorption peaks on the left and right are the stretching vibration absorption peaks of S=O, indicating the presence of sulfonic acid groups; 2963cm -1 Around, 2927cm -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; 3373cm -1 The strong and broad absorption peaks on the left and right are the stretching vibration absorption peaks of O—H and N—H.

[0060] The sample prepared in Example 3 was dissolved in a D2O solution containing 20% ​​DCl and subjected to H NMR spectrum testing. Figure 2. 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.

[0061] Example 6: Solution property test

[0062] The carboxymethyl hydroxybutyl chitosan material in Example 1 was dissolved in purified water and 0.85% sodium chloride solution, respectively, with a concentration of 3%, and was recorded as sample ① and sample ②. Hydroxybutyl chitosan prepared according to the method disclosed in patent application CN 102276756A was dissolved in purified water and 0.85% sodium chloride solution as a control, with a concentration of 3%, and was recorded as sample ③ and sample ④.

[0063]

[0064] The above data show that the sample in the 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.

[0065] Example 8: Preparation and performance testing of hydrogel

[0066] The sample of Example 4 was prepared into a 4% solution with 0.85% sodium chloride solution. The osmotic pressure was 312 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. Figure 5 As shown in the figure, the hydrogel solidifies into a non-flowing gel at body temperature, and is suitable for use as a dressing, a filling or a scaffold material inside or on the body surface, and can be used as a drug or cell carrier to load relevant therapeutic drugs or therapeutic cells to further enhance the therapeutic effect.

[0067] 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 sulfonate propyl hydroxybutyl chitosan derivative, characterized in that: It has the following structure: Wherein, R1, R2, R3, and R4 are one of the following groups: H, acetyl, hydroxybutyl, sulfonate propyl.

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

3. The sulfonate propyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The substitution degree of the hydroxybutyl group is 1.0-4.0; the substitution degree of the carboxyethyl group is 0.1-2.

0.

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

5. The method for preparing the sulfonate propyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: (1) dissolving or dispersing chitosan with a solvent, adding 1,3-propane sultone, reacting at a certain temperature for a certain time, and performing preliminary purification to obtain sulfonate propyl chitosan; (2) dissolving or dispersing sulfonate propyl 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 sulfonate propyl hydroxybutyl chitosan derivative.

6. The method for preparing the sulfonate propyl hydroxybutyl chitosan derivative according to claim 1, characterized in that: The following steps are involved: (1) chitosan is modified by hydroxybutylation according to the method disclosed in patent application CN 102276756A to obtain hydroxybutyl chitosan; (2) hydroxybutyl chitosan is dissolved or dispersed in a solvent, 1,3-propane sultone is added, and the reaction is carried out at a certain temperature for a certain time; (3) after the reaction is completed, the reaction system is stabilized at room temperature, placed in a dialysis bag for dialysis for a certain time, and taken out for freeze drying to obtain the final sulfonate propyl hydroxybutyl chitosan derivative.

7. The method for preparing the sulfonate propyl hydroxybutyl chitosan derivative according to claim 5 or 6, 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.

8. The method for preparing the sulfonate propyl hydroxybutyl chitosan derivative according to claim 5 or 6, characterized in that The alkali is sodium hydroxide or potassium hydroxide; the molar concentration of the alkali solution is 0.01-15 mol / L; the reaction temperature is 20° C.-100° C.; and the reaction time is 1 h-120 h.

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