Medical biological glue of microbial fructan and preparation method thereof

Abstract

The application belongs to the technical field of medical biological glue preparation, and discloses a medical biological glue of microbial fructan and a preparation method thereof. The medical biological glue of microbial fructan comprises a hydroxyl-functionalized non-woven fabric base layer and a gel layer compounded with the base layer; the gel layer is a three-dimensional network structure formed by covalent bond cross-linking of methyl acryloyl-modified microbial fermentation fructan and polysulfated fatty acid. The combination of the double bond on the modified microbial fermentation fructan molecule and the disulfide bond in the sulfated fatty acid quenches the terminal sulfur radical activity, so that the sulfated fatty acid-based material is more stable and the adhesion is improved. The medical biological glue based on polysulfated fatty acid can effectively adhere to various human tissue surfaces and release sulfated fatty acid molecules with antibacterial activity, showing the potential as an active medical biological glue. Finally, the cellulose non-woven fabric is used as a template to perform gelation thereon, so that the material as a whole has excellent mechanical strength and biocompatibility.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical biological glue, in particular to a medical biological glue of microbial fructan and a preparation method thereof. BACKGROUND

[0002] Medical biological glue is an indispensable part of modern medicine, and its development aims to provide the best healing environment for wounds. An ideal medical biological glue should have good biocompatibility, excellent mechanical properties, firm adhesion to tissue but painless peeling, and active anti-inflammatory and antibacterial ability.

[0003] In recent years, reversible adhesive hydrogels based on dynamic covalent bonds (such as disulfide bonds) have become a research frontier. For example, methacrylated chitosan and thioctic acid-modified chitosan are used for photocrosslinking, and dopamine-modified hyaluronic acid is introduced to construct a double-crosslinked network oral ulcer dressing film. This type of technology has proven the feasibility of giving materials reversible adhesion and self-healing ability through dynamic disulfide bond and double bond crosslinking strategies.

[0004] However, such systems based on traditional polysaccharides (such as chitosan and hyaluronic acid) still have significant limitations: first, their mechanical strength generally stays at the kilopascal (kPa) level, making it difficult to withstand the mechanical stress of the skin wound environment, and they are prone to breakage, limiting their application to low-stress environments such as the oral cavity. Second, the biological functional activity of the material (such as antioxidant and wound healing promotion) often depends on the additional addition of exogenous drugs or functional molecules, rather than being inherent to the material itself, which poses the risk of drug burst release, low loading rate, or potential toxicity. Third, the amino groups in the chitosan molecule may interfere with the formation efficiency and stability of the dynamic crosslinking network. Finally, existing technologies are difficult to achieve ultra-high mechanical strength, controllable reversible adhesion, inherent biological activity, and excellent stability in the same material. SUMMARY

[0005] The present application aims to solve at least one of the above technical problems, and provides a medical biological glue of microbial fructan and a preparation method thereof.

[0006] To achieve the above technical effects, the first technical solution adopted by the present application is:

[0007] A medical biological glue of microbial fructan, comprising:

[0008] a hydroxyl-functionalized non-woven fabric base layer; and a gel layer compounded with the base layer;

[0009] The gel layer is a three-dimensional network structure formed by covalent crosslinking of methacrylated microbial fermentation fructan and polysulfotocopherol.

[0010] Preferably, the hydroxyl-functionalized non-woven fabric includes any one of cellulose non-woven fabric, viscose non-woven fabric, chitin nanofiber non-woven fabric, carboxymethyl cellulose non-woven fabric, and hydroxyalkyl alkyl cellulose mixed ether ester non-woven fabric.

[0011] Preferably, the microbial fermentation levan is modified by methacrylation, and a double bond participating in polymerization is introduced on a molecular chain of the microbial fermentation levan.

[0012] Preferably, the double bond on the microbial fermentation levan modified by methacrylation is covalently crosslinked with a disulfide bond of the polysulfotaurine.

[0013] Preferably, a mass ratio of the microbial fermentation levan modified by methacrylation to the polysulfotaurine is 1: (2-10).

[0014] Preferably, methacrylic anhydride is used to modify the microbial fermentation levan by methacrylation.

[0015] Preferably, a mass ratio of the methacrylic anhydride to the microbial fermentation levan is 1: (2-5).

[0016] The second technical solution adopted by the present application is:

[0017] The method for preparing the medical biological glue of microbial levan includes:

[0018] The microbial fermentation levan is reacted with the methacrylic anhydride under alkaline conditions to obtain modified microbial fermentation levan.

[0019] The taurine, the stabilizer, and the modified microbial fermentation levan are dissolved in water, heated and stirred to initiate ring-opening polymerization of the taurine and covalent crosslinking of the modified microbial fermentation levan, and a pre-gel solution is obtained.

[0020] The pre-gel solution is carried on a hydroxyl-functionalized non-woven fabric, and the pre-gel solution is formed into a three-dimensional network gel by heating.

[0021] Preferably, the pH value of the alkaline condition is 7.5-8.0.

[0022] Preferably, the stabilizer is tris-hydroxymethyl aminomethane.

[0023] Preferably, the heating and stirring is stirring for 30-60 s in an environment of 60-70 DEG C.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The application quenches the terminal sulfur radical activity of lipoic acid by modifying the combination of double bond on microbial levan molecule and disulfide bond in lipoic acid, so that the lipoic acid-based material is more stable and the adhesion is improved. The prepared microbial levan medical biological glue can realize effective adhesion on the surface of various human tissues and release lipoic acid molecules with antibacterial activity, showing the potential as an active medical biological glue. Finally, a cellulose non-woven fabric is used as a template to perform gelation thereon, so as to endow the material with excellent mechanical strength and biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a physical diagram of the medical biological glue, and a diagram of adhesion on dry skin and wet skin;

[0027] Figure 2 It is the adhesion strength of the medical biological glue on different substrates;

[0028] Figure 3 It is the adhesion effect of the medical biological glue before and after glutathione treatment;

[0029] Figure 4 In a, the number of viable bacteria of E. coli and S. aureus under the same dilution factor before and after co-culturing with the medical biological glue sample; b is the SEM image of E. coli and S. aureus before and after co-culturing with the medical biological glue sample;

[0030] Figure 5 It is the cytotoxicity test result of the medical biological glue;

[0031] Figure 6 It is the tensile strength of the medical biological glue;

[0032] Figure 7 In a, it is a comparison diagram of the medical biological glue before and after self-repairing under the condition of 60°C; b is the stress-strain curve of the medical biological glue before and after self-repairing;

[0033] Figure 8 In a, it is the separation condition of ordinary gauze before and after use; b is the separation condition of the medical biological glue of Example 1 after use. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0035] The first embodiment of the application provides a medical biological glue of microbial levan, comprising:

[0036] a hydroxyl-functionalized nonwoven base layer; and a gel layer complexed with the base layer;

[0037] The gel layer is a three-dimensional network structure formed by covalent crosslinking of methylacrylated modified microbial fermentation levan and polysulfoacetic acid.

[0038] The embodiment of the present application forms a uniform and stable covalent crosslinking network by modifying microbial fermentation levan and polysulfoacetic acid, and is complexed with a hydroxyl-functionalized nonwoven fabric, thereby obtaining a mechanical strength of the medical biological glue far exceeding that of the existing hydrogel medical biological glue. The tensile strength can reach 20 MPa, which is nearly three orders of magnitude higher than that of the double-network hydrogel based on chitosan / hyaluronic acid, and can meet the stringent requirements of skin wounds on the toughness and tear resistance of the medical biological glue, thereby breaking through the bottleneck of the mechanical performance of flexible hydrogel materials.

[0039] It should be noted that since the present application utilizes the hydroxyl groups on the cellulose chain to crosslink with levan and other parts such as sulfoacetic acid, all hydroxyl-functionalized nonwoven fabrics can achieve the effect of the present application. For example, cellulose nonwoven fabric, viscose nonwoven fabric, chitin nanofiber nonwoven fabric, carboxymethyl cellulose nonwoven fabric, hydroxyalkyl alkyl cellulose mixed ether ester nonwoven fabric, etc.

[0040] The embodiment of the present application innovatively selects microbial fermentation levan as the core base material, rather than traditional chitosan or hyaluronic acid. The inherent, excellent antioxidant, anti-inflammatory and cell proliferation biological activities of microbial fermentation levan (Levan) are successfully integrated into the biological glue network, realizing the design of “material as function”. This enables the biological glue to provide sustained inflammation regulation and pro-healing microenvironment without additional loading of antibiotics or antioxidants, thereby avoiding the risks brought by the use of small molecule drugs. And by using the characteristic of only having hydroxyl groups on the Levan molecule, methylacrylation can precisely introduce polymerization sites, avoiding the potential interference of amino groups in the chitosan matrix on the ring-opening polymerization reaction of sulfoacetic acid, ensuring efficient and stable covalent crosslinking between double bonds and disulfide bonds, thereby forming a three-dimensional network with uniform structure and reliable performance.

[0041] Based on the inherent characteristics of the polysulfoacetic acid dynamic disulfide bond network, the medical biological glue provided by the embodiment of the present application can form strong adhesion on the wound surface (adhesion strength > 150 kPa), and under certain stimuli (such as antioxidants), the adhesion can change by more than 15 times, realizing the precise control of firm adhesion and painless debonding on demand, effectively preventing secondary damage when replacing the biological glue. The dynamic disulfide bond also enables the material to have rapid self-repairing ability, thereby prolonging the service life. At the same time, the stable covalent crosslinking network significantly slows down the degradation rate of the material, has a longer in vivo action time compared with traditional single natural polymer hydrogel, and can provide persistent wound protection.

[0042] The amount of modified microbial fermentation levan and polysulfide acetic acid does not need to be specially limited, and those skilled in the art can adaptively select according to the adhesion effect of the medical biological glue required. As some examples, the mass ratio of the methyl acrylate modified microbial fermentation levan and polysulfide acetic acid is 1: (2-10).

[0043] For the amount of methyl acrylate and microbial fermentation levan, the mass ratio is preferably controlled to be 1: (2-5), and if the amount exceeds this range, the dissolution effect of the product will be reduced, and if the amount is lower than this range, the grafting effect of the product and polysulfide acetic acid is limited.

[0044] The second embodiment provided by the present application is a preparation method of a medical biological glue of microbial levan, comprising:

[0045] The microbial fermentation levan and methyl acrylate react under alkaline conditions to obtain modified microbial fermentation levan;

[0046] The thioctic acid, the stabilizer and the modified microbial fermentation levan are dissolved in water, heated and stirred to initiate ring-opening polymerization of thioctic acid and covalent crosslinking with modified microbial fermentation levan to obtain a pre-gel solution;

[0047] The pre-gel solution is carried on a hydroxyl-functionalized non-woven fabric, and the pre-gel solution is formed into a three-dimensional network gel by heating.

[0048] In some preferred embodiments, the pH value of the alkaline condition is 7.5-8.0.

[0049] The stabilizer used is a commonly used component for improving the stability of thioctic acid in aqueous solution, such as tris-hydroxymethyl aminomethane.

[0050] The parameters of the heating and stirring do not need to be limited, and those skilled in the art can adaptively select suitable parameters according to the reaction raw materials, for example, stirring can be carried out in an environment of 60-70°C for 30-60s.

[0051] In order to make the technical solutions of the present application clearer, the preparation and performance of the medical biological glue are illustrated by a plurality of specific examples.

[0052] Microbial fermentation levan (Levan) is an extracellular EPS produced by Bacillus sp. SCU-E115 disclosed in patent CN115895930A. The methyl acrylate, thioctic acid and tris-hydroxymethyl aminomethane used are all purchased from Macklin.

[0053] Example 1 Preparation of a medical biological glue of microbial levan

[0054] Preparation of modified Levan: 2g of Levan was dissolved in 100ml of distilled water, 5.5mg of methacrylic anhydride was added, and the reaction was continued at room temperature for 24h. Sodium hydroxide was used to maintain the pH value of the reaction system at 8.0 during the reaction. After the reaction, the product was dialyzed with sterile water for 48h.

[0055] Preparation of modified Levan / lipoic acid pre-gel solution: 25g of lipoic acid, 10g of tris-hydroxymethyl aminomethane and 5g of modified Levan were dissolved in 50ml of deionized water, and stirred at 65°C for 30s to make it pre-gel.

[0056] Gelation of the pre-gel solution: first, according to the size of the polytetrafluoroethylene mold, the cotton fiber non-woven fabric was cut into corresponding size and shape, and placed in the mold. Then the pre-gel solution was poured into the mold, and finally gelled at 40°C for 4h, and finally demolded to obtain the medical biological glue. The physical picture of the medical biological glue is shown in Figure 1 .

[0057] Example 2 Preparation of medical biological glue of microbial levan

[0058] Preparation of modified Levan: 2g of Levan was dissolved in 100ml of distilled water, 4mg of methacrylic anhydride was added, and the reaction was continued at room temperature for 24h. Sodium hydroxide was used to maintain the pH value of the reaction system at 7.5 during the reaction. After the reaction, the product was dialyzed with sterile water for 48h.

[0059] Preparation of modified Levan / lipoic acid pre-gel solution: 10g of lipoic acid, 4g of tris-hydroxymethyl aminomethane and 5g of modified Levan were dissolved in 50ml of deionized water, and stirred at 60°C for 30s to make it pre-gel.

[0060] Gelation of the pre-gel solution: first, according to the size of the polytetrafluoroethylene mold, the cotton fiber non-woven fabric was cut into corresponding size and shape, and placed in the mold. Then the pre-gel solution was poured into the mold, and finally gelled at 40°C for 4h, and finally demolded to obtain the medical biological glue.

[0061] Example 3 Preparation of medical biological glue of microbial levan

[0062] Preparation of modified Levan: 2g of Levan was dissolved in 100ml of distilled water, 10mg of methacrylic anhydride was added, and the reaction was continued at room temperature for 24h. Sodium hydroxide was used to maintain the pH value of the reaction system at 8.0 during the reaction. After the reaction, the product was dialyzed with sterile water for 48h.

[0063] Preparation of modified Levan / lipoic acid pre-gel solution: 50 g lipoic acid, 20 g tris-hydroxymethyl aminomethane and 5 g modified Levan were dissolved in 50 ml deionized water, and stirred at 70 °C for 60 s to make it pre-gel.

[0064] Gelation of pre-gel solution: first, according to the size of the polytetrafluoroethylene mold, the cotton fiber non-woven fabric was cut into corresponding size and shape, and placed in the mold. Then the pre-gel solution was poured into the mold, and finally gelled at 40 °C for 4 h, and finally demolded to obtain the medical biological glue.

[0065] Experimental example

[0066] The medical biological glue prepared in Example 1 was tested as follows:

[0067] (1) Test of adhesion effect of medical biological glue:

[0068] The prepared medical biological glue was cut into a 20 mm x 20 mm size sample, and was adhered to two 20 mm x 80 mm size glass / paper / pigskin / iron / plastic pieces, respectively, and was stretched at a speed of 20 mm / min with a universal tensile testing machine, and the stress-strain curve and corresponding adhesion strength were recorded. The test results are shown in Figure 2 It can be seen that the adhesion strength on glass can reach 173.16 kPa, and the adhesion strength on paper can reach 193.44 kPa at the maximum.

[0069] (2) Test of reversible adhesion effect of medical biological glue:

[0070] The prepared medical biological glue was cut into a 20 mm x 20 mm size sample, and was adhered to two 20 mm x 80 mm size pigskin, and some of the samples were not treated, and some were treated with glutathione solution (glutathione can reduce the disulfide bond in lipoic acid, so that the poly-lipoic acid chain in the adhesive material is broken, and the adhesion strength is reduced). The adhesion strength of the samples was recorded by stretching them at a speed of 20 mm / min with a universal tensile testing machine. The test results are shown in Figure 3 The adhesion strength of the samples before and after glutathione treatment can change by more than 15 times.

[0071] (3) Test of antibacterial effect of medical biological glue:

[0072] 10 mm x 10 mm size medical biological glue samples were taken, and were co-cultured with E. coli and S. aureus bacterial solution for 24 hours, respectively, and then the bacterial colonies were counted after dilution and coating of the bacterial solution in the control group and the sample group without co-culture. At the same time, the bacterial morphology of the related samples was observed by SEM. The relevant test results are shown in Figure 4As shown in FIG. 6, it can be seen that the medical biological glue has a good inhibitory effect on both bacteria.

[0073] (4) Cytotoxicity test of the medical biological glue:

[0074] Take 1 / 2 / 4 mg of the medical biological glue sample respectively, and immerse them in 100 ml of PBS buffer for 24 h to obtain the corresponding sample leachate. Next, co-culture them with the 293T cells after cultivation for 12 / 24 h, and finally use the kit to detect the cytotoxicity. The relevant test results are shown in FIG. 7. Figure 5 As shown in FIG. 7, it can be seen that the residual cell activity of all samples is maintained at more than 85%, indicating that it has no obvious cytotoxicity.

[0075] (5) Mechanical property test of the medical biological glue:

[0076] Take several pieces of 20 mm x 60 mm medical biological glue and non-gel non-woven fabric respectively, and use the universal tensile machine to stretch at a speed of 50 mm / min, and record the stress-strain curve. The relevant results are shown in FIG. 8. Figure 6 As shown in FIG. 8, it can be seen that the tensile strength of the medical biological glue is increased by more than 4 times, up to 20 MPa.

[0077] (6) Self-repairing test of the medical biological glue:

[0078] Draw a scratch on the surface of the medical biological glue with a size of 20 mm x 60 mm, and perform self-repairing at 60°C. The pictures before and after repair are taken by a microscope. Finally, stretch the repaired medical biological glue sample with the universal tensile machine at a speed of 50 mm / min, and record the stress-strain curve. The relevant results are shown in FIG. 9. Figure 7 As shown in FIG. 9, it can be seen that the sample has basically completed repair, and the mechanical property has basically no change.

[0079] (7) Release test of lipoic acid active substance of the medical biological glue:

[0080] In the simulation system of the wet environment on the wound surface, use the ultraviolet spectrophotometer to test the absorbance of the solution at 330 nm to represent the concentration of lipoic acid. At the 24th hour, the release rate of lipoic acid is (31.97 ± 0.92)%, and at the 72nd hour, the release rate reaches (47.52 ± 1.53)%, proving that it has good sustained-release effect and can play a long-term effect in actual application.

[0081] (8) Wound management effect test of the medical biological glue:

[0082] First, in a clean and sterile surgical environment, a 20mm incision was made on the back of several Bama pigs using a scalpel, the control group was treated with ordinary gauze, the experimental group was treated with the medical biological glue in Example 1, and finally the front was treated with glutathione solution. The relevant results are shown in Figure 8 After 2h of treatment with the medical biological glue, it can be seen that the exudate on the gauze caused adhesion between the gauze and the scab of the wound, while the medical biological glue in the example can be easily removed without affecting the healing of the wound.

[0083] The above-described examples only express part of the embodiments of the present application, not all. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only to represent selected embodiments of the present application. For those of ordinary skill in the art, without departing from the concept of the present application, a number of variations and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A medical bioadhesive of microbial fructan, characterized in that, The application relates to a hydroxyl-functionalized non-woven fabric gel composite, which comprises: a hydroxyl-functionalized non-woven fabric base layer; and a gel layer compounded with the base layer; the gel layer is a three-dimensional network structure formed by covalent cross-linking of methylacrylated microbial fermentation levan and polysulfated fatty acid.

2. The medical bioadhesive of microbial levan as set forth in claim 1, characterized in that, The hydroxyl-functionalized non-woven fabric comprises any one of cellulose non-woven fabric, chitin nanofiber non-woven fabric and hydroxyalkyl alkyl cellulose mixed ether ester non-woven fabric.

3. The medical bioadhesive of microbial levan as set forth in claim 1 or 2, characterized in that, Double bonds on the molecule of the methylacrylated microbial fermentation levan are covalently cross-linked with the disulfide bond of polysulfated fatty acid.

4. The medical bioadhesive of microbial fructan according to claim 1, characterized in that, The mass ratio of the methylacrylated microbial fermentation levan and the polysulfated fatty acid is 1: (2-10).

5. The medical bioadhesive of microbial fructan according to claim 1, characterized in that, Methylacrylation modification is performed on the microbial fermentation levan by using methacrylic anhydride.

6. The medical bio-gum of microbial fructan according to claim 5, characterized in that, The mass ratio of the methacrylic anhydride and the microbial fermentation levan is 1: (2-5).

7. The method for preparing a medical bioadhesive of microbial levan according to any one of claims 1 to 6, characterized in that, The application further relates to a preparation method of the hydroxyl-functionalized non-woven fabric gel composite. The microbial fermentation levan is reacted with methacrylic anhydride under alkaline conditions to obtain modified microbial fermentation levan; sulfated fatty acid, a stabilizer and the modified microbial fermentation levan are dissolved in water, heated and stirred to initiate ring-opening polymerization of the sulfated fatty acid and covalent cross-linking of the modified microbial fermentation levan, thereby obtaining a pre-gel solution; the pre-gel solution is carried on the hydroxyl-functionalized non-woven fabric, and heating is performed to make the pre-gel solution form a three-dimensional network gel.

8. The method for preparing a medical bio adhesive of microbial levan according to claim 7, characterized by, The pH value of the alkaline condition is 7.5-8.

0.

9. The method for preparing a medical bio adhesive of microbial levan according to claim 7, wherein, The stabilizer is tris-hydroxymethyl aminomethane.

10. The method for preparing a medical bioadhesive of microbial levan according to claim 7, characterized in that, The heating and stirring is stirring for 30-60 s in a 60-70 DEG C environment.

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