Preparation method and application of polypeptide-grafted sodium alginate-chitosan gel

Abstract

The present invention relates to the technical field of polypeptides, and discloses a preparation method and application of a polypeptide-grafted sodium alginate-chitosan gel, wherein abundant antibacterial quaternary ammonium salt groups are introduced into the molecular chain of sodium alginate, and then deamination reaction is carried out with the amide bond in the glutamine Gln unit in the collagen polypeptide, so that the collagen polypeptide is grafted into the sodium alginate to obtain a polypeptide-grafted sodium alginate containing a diquaternary ammonium salt. Glutaraldehyde is used as a cross-linking agent, and the polypeptide-grafted sodium alginate and chitosan are chemically cross-linked to form a gel, and the obtained polypeptide-grafted sodium alginate-chitosan gel contains an antibacterial diquaternary ammonium salt structure and an antibacterial polypeptide structure, which gives the gel material a long-lasting antibacterial property and has excellent hydrophilicity and water absorption.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptides, in particular to a preparation method of polypeptide-grafted sodium alginate-chitosan gel and application thereof. Background Art

[0002] Polypeptides are compounds connected by peptide bonds and are widely used in clinical drugs, health care products, etc. Combining polypeptide molecules with natural polymers such as chitosan, sodium alginate, hyaluronic acid, etc. has broad application prospects in drug carriers, antibacterial materials, medical and sanitary products, etc. For example, the Chinese patent document "Antibacterial gel covalently grafted with antibacterial polypeptides and preparation method thereof" with application number 201610280846.4 discloses that sodium alginate is activated and reacted with polyvinyl alcohol capped with furfurylamine and maleimide to obtain sodium alginate gel, and then the antibacterial polypeptide is grafted into the gel under ultraviolet light using a photoinitiator to obtain an antibacterial gel covalently grafted with antibacterial polypeptides, thereby achieving covalent grafting of antibacterial polypeptides into the gel and achieving long-term antibacterial ability.

[0003] Sodium alginate has good biocompatibility, is cheap and easy to obtain, and is widely used in antibacterial materials, biomedicine, etc. For example, the Chinese patent document "A sodium alginate quaternary ammonium salt hemostatic antibacterial agent and its preparation method and application" with application number 201910591877.5 discloses that by grafting epoxy alkyl chain quaternary ammonium salt or halogenated alkyl quaternary ammonium salt into sodium alginate, a sodium alginate material with antibacterial effect and coagulation effect is obtained, which can be applied to medical and health fields such as modified gauze and blood collection needles. The present invention grafts quaternary ammonium salt and collagen polypeptide into sodium alginate, and cross-links with chitosan to obtain a polypeptide-grafted sodium alginate-chitosan gel material with antibacterial properties, which is applied to biomedicine, health and hygiene and other fields. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] The invention provides a preparation method and application of polypeptide-grafted sodium alginate-chitosan gel with antibacterial property.

[0006] (II) Technical solution

[0007] A method for preparing a polypeptide-grafted sodium alginate-chitosan gel, the method comprising the following steps:

[0008] S1: Add di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly and then add an aqueous solution of chloroethylamine hydrochloride. After the reaction, adjust the pH of the solution to neutral, add acetone for precipitation, filter the solvent, wash the precipitate with acetone and ethanol in turn and dry to obtain di-quaternary ammonium salt sodium alginate.

[0009] S2: Prepare PBS buffer, add diquaternary ammonium sodium alginate, stir evenly, then drop PBS buffer containing collagen polypeptide and PBS buffer containing transglutaminase in sequence, wherein the amount of transglutaminase is 8-13% of the weight of collagen polypeptide. After the reaction, heat to reflux to inactivate the transglutaminase. After cooling, purify the product by dialyzing in a dialysis bag and dry it to obtain polypeptide-grafted sodium alginate.

[0010] S3: Add polypeptide-grafted sodium alginate to distilled water, stir evenly, then drop acetic acid solution containing chitosan, then drop 20-35% glutaraldehyde aqueous solution, control the reaction temperature between 60-85 °C, the reaction time between 3-6 h, filter the solvent after the cross-linking reaction, wash with distilled water, and obtain polypeptide-grafted sodium alginate-chitosan gel.

[0011] Preferably, in S1, the reaction temperature is controlled between 80-120° C., and the reaction time is controlled between 24-48 h.

[0012] Preferably, the amount of chloroethylamine hydrochloride in S1 is 40-90% of the mass of di-tertiary amine sodium alginate.

[0013] Preferably, the pH of the PBS buffer in S2 is 6-7.

[0014] Preferably, the amount of collagen polypeptide in S2 is 60-120% of the mass of diquaternary ammonium salt sodium alginate.

[0015] Preferably, in S2, the reaction temperature is controlled between 40-60° C., and the reaction time is controlled between 6-12 h.

[0016] Preferably, the amount of chitosan in S3 is 30-60% of the mass of the polypeptide-grafted sodium alginate.

[0017] Preferably, the preparation method of di-tertiary amine sodium alginate in S1 comprises the following steps:

[0018] S4: Add oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly and then add 3-dimethylamino-1-propylamine, wherein the mass of 3-dimethylamino-1-propylamine is 30-70% of the weight of oxidized sodium alginate, reflux at 60-80 °C for 24-48 h, then reduce the temperature to 30-40 °C and drop an aqueous solution of sodium borohydride, stir and reduce for 12-24 h, after the reaction, remove the solvent by distillation under reduced pressure, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0019] 3. Beneficial technical effects

[0020] 3-Dimethylamino-1-propylamine is used to react with oxidized sodium alginate having a dialdehyde structure, and then a quaternization reaction is carried out with chloroethylamine hydrochloride to obtain a diquaternary ammonium salt sodium alginate containing amino groups, thereby introducing rich antibacterial quaternary ammonium salt groups into the molecular chain of sodium alginate.

[0021] Transglutaminase is used to catalyze the deamination reaction of the amide bond in the glutamine Gln unit in the collagen polypeptide with the amino group in the polypeptide-grafted sodium alginate, thereby grafting the collagen polypeptide into the sodium alginate to obtain the polypeptide-grafted sodium alginate containing a diquaternary ammonium salt.

[0022] Using glutaraldehyde as a cross-linking agent, the polypeptide is grafted onto sodium alginate and chitosan to undergo chemical cross-linking to form a gel. The resulting polypeptide grafted sodium alginate-chitosan gel contains an antibacterial diquaternary ammonium salt structure and an antibacterial polypeptide structure, which gives the gel material long-lasting antibacterial properties. It also has excellent hydrophilicity and water absorption, expanding the application prospects of sodium alginate-chitosan gel materials in medical dressings, bandages, biological tissue engineering and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the preparation route of polypeptide grafted sodium alginate. DETAILED DESCRIPTION

[0024] The preparation method of oxidized sodium alginate with dialdehyde structure refers to: Journal "Chemical Reagents" 2009, 31(02), "Preparation and Performance Study of Novel Natural Cross-linking Agent Oxidized Sodium Alginate".

[0025] Add 5 g of sodium alginate to distilled water, add dropwise an aqueous solution containing 3.2 g of sodium periodate, react for 12 h in the dark, then add ethylene glycol to terminate the reaction, add sodium chloride and ethanol after the reaction to precipitate, and obtain oxidized sodium alginate containing a dialdehyde structure.

[0026] Example 1

[0027] (1) Add 5 g of oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 1.8 g of 3-dimethylamino-1-propylamine, reflux at 80 °C for 36 h, then reduce the temperature to 40 °C and dropwise add an aqueous solution containing 1 g of sodium borohydride, stir for 12 h, and after the reaction, distill under reduced pressure to remove the solvent, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0028] (2) Add 5 g of di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly, then add an aqueous solution containing 2.5 g of chloroethylamine hydrochloride, control the reaction temperature to 120 °C, and the reaction time to 24 h. After the reaction, adjust the pH of the solution to neutral, add acetone for precipitation, filter the solvent, wash the precipitate with acetone and ethanol in turn, and dry to obtain di-quaternary ammonium sodium alginate.

[0029] (3) Prepare a PBS buffer solution with a pH of 7, and add 3 g of diquaternary ammonium sodium alginate. After stirring evenly, add PBS buffer solution containing 2.5 g of collagen polypeptide and PBS buffer solution containing 0.22 g of transglutaminase dropwise in sequence. Control the reaction temperature to 60 °C and the reaction time to 6 h. After the reaction, heat to reflux to inactivate the transglutaminase. After cooling, purify the product by dialyzing in a dialysis bag and dry it to obtain polypeptide-grafted sodium alginate.

[0030] (4) Add 10 g of polypeptide-grafted sodium alginate to distilled water, stir evenly, then add dropwise acetic acid solution containing 3 g of chitosan, and then add dropwise 20% glutaraldehyde aqueous solution, control the reaction temperature to be between 85 °C and the reaction time to be between 3 h. After the cross-linking reaction, filter the solvent, wash with distilled water, and obtain polypeptide-grafted sodium alginate-chitosan gel.

[0031] Example 2

[0032] (1) Add 5 g of oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 3.5 g of 3-dimethylamino-1-propylamine, reflux at 60 °C for 48 h, then reduce the temperature to 30 °C and dropwise add an aqueous solution containing 2 g of sodium borohydride, stir for 18 h, and after the reaction, remove the solvent by distillation under reduced pressure, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0033] (2) Add 5 g of di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly, then add an aqueous solution containing 4.5 g of chloroethylamine hydrochloride, control the reaction temperature to 80 °C, and the reaction time to 36 h. After the reaction, adjust the pH of the solution to neutral, add acetone for precipitation, filter the solvent, wash the precipitate with acetone and ethanol in turn, and dry to obtain di-quaternary ammonium sodium alginate.

[0034] (3) Prepare a PBS buffer solution with a pH of 6, and add 3 g of diquaternary ammonium sodium alginate. After stirring evenly, add PBS buffer solution containing 1.8 g of collagen polypeptide and PBS buffer solution containing 0.15 g of transglutaminase dropwise in sequence. Control the reaction temperature to 60 °C and the reaction time to 6 h. After the reaction, heat to reflux to inactivate the transglutaminase. After cooling, dialyze the product in a dialysis bag for purification and dry it to obtain polypeptide-grafted sodium alginate.

[0035] (4) Add 10 g of polypeptide-grafted sodium alginate to distilled water, stir evenly, then add dropwise acetic acid solution containing 4 g of chitosan, and then add dropwise 25% glutaraldehyde aqueous solution, control the reaction temperature to be between 85 °C and the reaction time to be between 3 h. After the cross-linking reaction, filter the solvent, wash with distilled water, and obtain polypeptide-grafted sodium alginate-chitosan gel.

[0036] Example 3

[0037] (1) Add 5 g of oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 3.5 g of 3-dimethylamino-1-propylamine, reflux at 60 °C for 24 h, then reduce the temperature to 40 °C and dropwise add an aqueous solution containing 2 g of sodium borohydride, stir for 18 h, and after the reaction, remove the solvent by distillation under reduced pressure, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0038] (2) Add 5 g of di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly, then add an aqueous solution containing 4.5 g of chloroethylamine hydrochloride, control the reaction temperature to 100 °C, and the reaction time to 36 h. After the reaction, adjust the pH of the solution to neutral, add acetone for precipitation, filter the solvent, wash the precipitate with acetone and ethanol in turn, and dry to obtain di-quaternary ammonium sodium alginate.

[0039] (3) Prepare a PBS buffer solution with a pH of 6, and add 3 g of diquaternary ammonium sodium alginate. After stirring evenly, add PBS buffer solution containing 3.6 g of collagen polypeptide and PBS buffer solution containing 0.45 g of transglutaminase dropwise in sequence. Control the reaction temperature to 40 °C and the reaction time to 12 h. After the reaction, heat to reflux to inactivate the transglutaminase. After cooling, dialyze the product in a dialysis bag for purification and dry it to obtain polypeptide-grafted sodium alginate.

[0040] (4) Add 10 g of polypeptide-grafted sodium alginate to distilled water, stir evenly, then add dropwise acetic acid solution containing 55 g of chitosan, and then add dropwise 30% glutaraldehyde aqueous solution, control the reaction temperature to be between 85 °C and the reaction time to be between 6 h. After the cross-linking reaction, filter the solvent, wash with distilled water, and obtain polypeptide-grafted sodium alginate-chitosan gel.

[0041] Example 4

[0042] (1) Add 5 g of oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 3 g of 3-dimethylamino-1-propylamine, reflux at 80 °C for 36 h, then reduce the temperature to 40 °C and dropwise add an aqueous solution containing 2 g of sodium borohydride, stir and reduce for 12 h, after which the solvent is removed by distillation under reduced pressure, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0043] (2) Add 5 g of di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly, then add an aqueous solution containing 3.2 g of chloroethylamine hydrochloride, control the reaction temperature to 120 °C, and the reaction time to 24 h. After the reaction, adjust the pH of the solution to neutral, add acetone for precipitation, filter the solvent, wash the precipitate with acetone and ethanol in turn, and dry to obtain di-quaternary ammonium sodium alginate.

[0044] (3) Prepare a PBS buffer solution with a pH of 7, and add 3 g of diquaternary ammonium sodium alginate. After stirring evenly, add PBS buffer solution containing 3 g of collagen polypeptide and PBS buffer solution containing 0.3 g of transglutaminase dropwise in sequence. Control the reaction temperature to 50 °C and the reaction time to 8 h. After the reaction, heat to reflux to inactivate the transglutaminase. After cooling, purify the product by dialyzing in a dialysis bag and dry it to obtain polypeptide-grafted sodium alginate.

[0045] (4) Add 10 g of polypeptide-grafted sodium alginate to distilled water, stir evenly, then add dropwise acetic acid solution containing 6 g of chitosan, and then add dropwise 35% glutaraldehyde aqueous solution, control the reaction temperature to be between 75 °C and the reaction time to be between 3 h. After the cross-linking reaction, filter the solvent, wash with distilled water, and obtain polypeptide-grafted sodium alginate-chitosan gel.

[0046] Comparative Example 1

[0047] (1) Add 5 g of oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 2.8 g of 3-dimethylamino-1-propylamine, reflux at 70 °C for 24 h, then reduce the temperature to 40 °C and dropwise add an aqueous solution containing 1.5 g of sodium borohydride, stir for 18 h, and after the reaction, remove the solvent by distillation under reduced pressure, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0048] (2) Add 10 g of di-tertiary amine sodium alginate to distilled water, stir evenly, then add dropwise acetic acid solution containing 3.5 g of chitosan, and then add dropwise 30% glutaraldehyde aqueous solution, control the reaction temperature to be between 80 °C and the reaction time to be between 4 h. After the cross-linking reaction, filter the solvent, wash with distilled water, and obtain sodium alginate-chitosan material.

[0049] Comparative Example 2

[0050] (1) Add 5 g of oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 2.5 g of 3-dimethylamino-1-propylamine, reflux at 80 °C for 48 h, then reduce the temperature to 40 °C and dropwise add an aqueous solution containing 1.3 g of sodium borohydride, stir and reduce for 24 h, after which the solvent is removed by distillation under reduced pressure, wash the precipitate with acetone and dry to obtain di-tertiary amine sodium alginate.

[0051] (2) Add 5 g of di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly, then add an aqueous solution containing 2.8 g of chloroethylamine hydrochloride, control the reaction temperature to 80 °C, and the reaction time to 36 h. After the reaction, adjust the pH of the solution to neutral, add acetone for precipitation, filter the solvent, wash the precipitate with acetone and ethanol in turn, and dry to obtain di-quaternary ammonium sodium alginate.

[0052] (3) Add 10 g of diquaternary ammonium salt sodium alginate to distilled water, stir evenly, then add dropwise an acetic acid solution containing 3 g of chitosan, and then add dropwise a 25% aqueous solution of glutaraldehyde. Control the reaction temperature to be between 85 °C and the reaction time to be between 3 h. After the cross-linking reaction, filter the solvent, wash with distilled water, and obtain a diquaternary ammonium salt sodium alginate-chitosan material.

[0053] Weigh the sodium alginate-chitosan material, add it to distilled water and soak it for 12 h, then take it out and drain the surface water, weigh it, and calculate the water absorption W of the sodium alginate-chitosan material.

[0054] Water absorption W = (m1-m) / m. m1 is the mass of sodium alginate-chitosan material before water absorption, and m is the mass of sodium alginate-chitosan material after water absorption.

[0055] m (g) <![CDATA[m1(g)]]> W (g / g) Example 1 1 18.1 17.1 Example 2 1 29.9 28.9 Example 3 1 17.0 16.0 Example 4 1 21.2 20.2 Comparative Example 1 1 13.1 12.1 Comparative Example 2 1 19.6 18.6

[0056] Antibacterial performance test:

[0057] The sodium alginate-chitosan materials in the embodiments and comparative examples were added to beef nutrient broth, and then 0.5 mL of the Candida albicans suspension was added dropwise to the beef nutrient broth, and the mixture was shaken at 37°C for 10 min, 30 min, and 60 min, respectively, as the experimental group; no sodium alginate-chitosan material was added as the blank group.

[0058] Beef extract peptone medium was added to a sterile culture dish, and then 0.1 mL of the culture medium of Candida albicans was taken from each experimental group and the blank group, and cultured in a constant temperature incubator at 37 °C for 24 h. After culture, the live bacteria were counted and the inhibition rate Q was determined.

[0059] The inhibition rate Q = (C-C1) / C×100%. C is the number of viable bacteria in the blank group after cultivation, and C1 is the number of viable bacteria in the experimental group after cultivation.

[0060]

[0061] The above-mentioned specific implementation modes and examples only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a polypeptide-grafted sodium alginate-chitosan gel, characterized in that: The preparation method comprises the following steps: S1: Add di-tertiary amine sodium alginate to N,N-dimethylformamide, stir evenly, then add an aqueous solution of chloroethylamine hydrochloride, adjust the pH of the solution to neutral after the reaction, precipitate, filter, wash and dry to obtain di-quaternary ammonium salt sodium alginate; S2: preparing PBS buffer, adding diquaternary ammonium sodium alginate, stirring evenly, and then dropping PBS buffer containing collagen polypeptide and PBS buffer containing transglutaminase in sequence, wherein the amount of transglutaminase used is 8-13% of the weight of the collagen polypeptide, heating and refluxing after the reaction to inactivate the transglutaminase, dialysis purification and drying to obtain polypeptide-grafted sodium alginate; S3: adding polypeptide grafted sodium alginate to distilled water, stirring evenly, adding dropwise acetic acid solution containing chitosan, and then adding dropwise 20-35% glutaraldehyde aqueous solution, controlling the reaction temperature between 60-85°C, the reaction time between 3-6 h, filtering and washing after the cross-linking reaction, and obtaining polypeptide grafted sodium alginate-chitosan gel; The preparation method of di-tertiary amine sodium alginate in S1 comprises the following steps: Add oxidized sodium alginate containing a dialdehyde structure to ethanol, stir evenly, then add 3-dimethylamino-1-propylamine, wherein the mass of 3-dimethylamino-1-propylamine is 30-70% of the weight of oxidized sodium alginate, reflux at 60-80°C for 24-48h, then reduce the temperature to 30-40°C and dropwise add an aqueous solution of sodium borohydride, stir and reduce for 12-24h, and after the reaction, distill under reduced pressure, wash and dry to obtain di-tertiary amine sodium alginate.

2. The method for preparing the polypeptide grafted sodium alginate-chitosan gel according to claim 1, characterized in that: In S1, the reaction temperature is controlled between 80-120° C., and the reaction time is controlled between 24-48 h.

3. The method for preparing the polypeptide grafted sodium alginate-chitosan gel according to claim 1, characterized in that: The amount of chloroethylamine hydrochloride in S1 is 40-90% of the mass of di-tertiary amine sodium alginate.

4. The method for preparing the polypeptide grafted sodium alginate-chitosan gel according to claim 1, characterized in that: The pH of the PBS buffer in S2 is 6-7.

5. The method for preparing the polypeptide grafted sodium alginate-chitosan gel according to claim 1, characterized in that: The amount of collagen polypeptide in S2 is 60-120% of the mass of diquaternary ammonium salt sodium alginate.

6. The method for preparing the polypeptide grafted sodium alginate-chitosan gel according to claim 1, characterized in that: In S2, the reaction temperature is controlled between 40-60° C., and the reaction time is controlled between 6-12 h.

7. The method for preparing polypeptide-grafted sodium alginate-chitosan gel according to claim 1, characterized in that: The amount of chitosan in S3 is 30-60% of the mass of the polypeptide-grafted sodium alginate.

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