Medical biogel of microbial fructan and preparation method of medical biogel

Abstract

The invention belongs to the technical field of medical biogel preparation, and discloses medical biogel of microbial fructan and a preparation method of the medical biogel. The medical biogel of the microbial fructan comprises a hydroxyl functionalized non-woven fabric base layer, the gel layer is compounded with the base layer; the gel layer is of a three-dimensional network structure formed by crosslinking methylacryloylation modified microbial fermentation fructan and polylipoic acid through covalent bonds. The activity of sulfur free radicals at the tail end of the modified microbial fermentation fructan molecule is quenched through combination of a double bond on the modified microbial fermentation fructan molecule and a disulfide bond in lipoic acid, so that the lipoic acid-based material is more stable, and the adhesive force is improved. The medical biological glue based on the polylipoic acid can be effectively adhered to the surfaces of various human tissues, and can release lipoic acid molecules with antibacterial activity, so that the potential of the medical biological glue as an active medical biological glue is shown. And finally, by taking the cellulose non-woven fabric as a template, gelation is performed on the cellulose non-woven fabric, so that the overall material is endowed with 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, methyl acrylate modified 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 film. This kind of technology has proved the feasibility of giving materials reversible adhesion and self-healing ability through dynamic disulfide bond and double bond crosslinking strategy.

[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, which is difficult to withstand the mechanical stress of the skin wound environment, and is prone to damage, so the application range is mostly limited 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 exogenous drugs or functional molecules, rather than the inherent properties of 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 crosslinked 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 at least solve any 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: A medical biological glue of microbial fructan, comprising: 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 crosslinking of methyl acrylate modified microbial fermentation fructan and polysulfonic acid.

[0007] Preferably, the hydroxyl-functionalized nonwoven fabric includes any one of cellulose nonwoven fabric, viscose fiber nonwoven fabric, chitosan nanofiber nonwoven fabric, carboxymethyl cellulose nonwoven fabric, and hydroxyalkyl alkyl cellulose mixed ether ester nonwoven fabric.

[0008] Preferably, after the microbial fermented fructan is modified by methacrylamide, double bonds that can participate in polymerization are introduced into its molecular chain.

[0009] Preferably, the double bonds on the methacrylamide-modified microbial fermented fructan molecules are covalently cross-linked with the disulfide bonds of polythioctic acid.

[0010] Preferably, the mass ratio of methacrylamide-modified microbial fermented fructan to polythioctic acid is 1:(2-10).

[0011] Preferably, methacrylic anhydride is used to modify microbially fermented fructan by methacrylation.

[0012] Preferably, the mass ratio of methacrylic anhydride to microbial fermented fructan is 1:(2-5).

[0013] The second technical solution adopted in this invention is: A method for preparing medical bio-adhesives from microbial fructans, comprising: Modified microbially fermented fructan was obtained by reacting microbially fermented fructan with methacrylic anhydride under alkaline conditions. The lipoic acid, stabilizer, and modified microbial fermented fructan were dissolved in water, heated and stirred to initiate the ring-opening polymerization of lipoic acid and covalent cross-linking with the modified microbial fermented fructan to obtain a pregel solution. The pregel liquid is supported on a hydroxyl-functionalized nonwoven fabric and heated to form a three-dimensional network gel.

[0014] Preferably, the pH value of the alkaline conditions is 7.5 to 8.0.

[0015] Preferably, the stabilizer is tris(hydroxymethyl)aminomethane.

[0016] Preferably, the heating and stirring is performed at 60-70°C for 30-60 seconds.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention modifies the binding of double bonds on fructan molecules from microbial fermentation with disulfide bonds in lipoic acid, quenching the activity of terminal sulfur free radicals and making lipoic acid-based materials more stable and improving adhesion. The prepared microbial fructan medical bioadhesive can effectively adhere to the surfaces of various human tissues and release lipoic acid molecules with antibacterial activity, showing its potential as an active medical bioadhesive. Finally, using cellulose nonwoven fabric as a template, gelation is performed on it, giving the material excellent overall mechanical strength and biocompatibility. Attached Figure Description

[0018] Figure 1 The images show actual medical bio-adhesive and its adhesion to dry and moist skin. Figure 2 The adhesion strength of medical bio-adhesive on different substrates; Figure 3 The adhesion effect of medical bio-adhesive before and after glutathione treatment; Figure 4 In the middle section, a represents the number of viable bacteria of Escherichia coli and Staphylococcus aureus at the same dilution before and after co-culturing with the medical bio-adhesive sample; b represents the SEM images of Escherichia coli and Staphylococcus aureus before and after co-culturing with the medical bio-adhesive sample. Figure 5 The results are from a cytotoxicity test of medical bio-adhesive. Figure 6 The tensile strength of medical bio-adhesive; Figure 7 Image a shows a comparison of the medical bio-adhesive before and after self-healing at 60℃; image b shows the stress-strain curves of the medical bio-adhesive before and after self-healing. Figure 8 In Figure a, ordinary gauze separates from the wound before and after use; in Figure b, the medical bio-adhesive of Example 1 separates from the wound after use. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] The first embodiment of the present invention provides a medical bio-adhesive of microbial fructan, comprising: Hydroxyl-functionalized nonwoven base layer; and a gel layer composited with the base layer; The gel layer is a three-dimensional network structure formed by covalent cross-linking of methacrylated microbial fermented fructan and polythioctic acid.

[0021] This invention achieves a mechanical strength far exceeding that of existing hydrogel medical bio-adhesives by modifying fructan fermentation with polythiooctanoic acid to form a uniform and stable covalent cross-linked network, which is then composited with hydroxyl-functionalized nonwoven fabric. Its tensile strength reaches 20 MPa, nearly three orders of magnitude higher than chitosan / hyaluronic acid-based dual-network hydrogels. This meets the stringent requirements of skin wounds for the toughness and tear resistance of medical bio-adhesives, overcoming the bottleneck of insufficient mechanical properties in flexible hydrogel materials.

[0022] It should be noted that, since this invention utilizes the hydroxyl groups on the cellulose chain to crosslink with other components such as fructan and lipoic acid, all hydroxyl-functionalized nonwoven fabrics can achieve the effects of this invention. Examples include cellulose nonwoven fabrics, viscose fiber nonwoven fabrics, chitosan nanofiber nonwoven fabrics, carboxymethyl cellulose nonwoven fabrics, and hydroxyalkyl alkyl cellulose mixed ether ester nonwoven fabrics.

[0023] This invention innovatively uses microbially fermented fructan as the core substrate, instead of traditional chitosan or hyaluronic acid. The inherent and excellent antioxidant, anti-inflammatory, and cell proliferation-promoting bioactivities of microbially fermented fructan (Levan) are successfully integrated into the bio-adhesive network, achieving a "material-as-function" design. This allows the bio-adhesive to provide a continuous inflammatory regulation and healing-promoting microenvironment without the need for additional antibiotics or antioxidants, avoiding the risks associated with small molecule drug use. Furthermore, utilizing the fact that Levan molecules only have hydroxyl groups, polymerization sites can be precisely introduced through methacrylation, avoiding potential interference from the ring-opening polymerization of lipoic acid by the amino groups in the chitosan matrix. This ensures efficient and stable covalent cross-linking between double bonds and disulfide bonds, thereby forming a structurally uniform and reliable three-dimensional network.

[0024] Based on the inherent properties of the dynamic disulfide bond network of polythiooctanoic acid (PoTA), the medical bioadhesive provided in this invention can form strong adhesion (adhesion strength >150 kPa) on the wound surface. Under specific stimuli (such as antioxidants), its adhesion force can change significantly by more than 15 times, achieving precise control of on-demand firm adhesion and painless detachment, effectively preventing secondary damage when changing the bioadhesive. The dynamic disulfide bonds also enable the material to have rapid self-healing capabilities, extending its service life. At the same time, the stable covalent cross-linked network significantly slows down the degradation rate of the material, providing a longer in vivo action time compared to traditional single natural polymer hydrogels, thus offering long-lasting wound protection.

[0025] There is no special limitation on the amount of modified microbial fermented fructan and polythioctic acid used; those skilled in the art can make an appropriate selection based on the desired adhesion effect of the medical bioadhesive. As some examples, the mass ratio of methacrylamide-modified microbial fermented fructan to polythioctic acid is 1:(2-10).

[0026] The preferred mass ratio of methacrylic anhydride to microbially fermented fructan is 1:(2-5). If the amount exceeds this range, the solubility of the product will be reduced. If the amount is below this range, the grafting effect of the product with polythioctic acid will be limited.

[0027] The second embodiment of the present invention provides a method for preparing a medical bio-adhesive of microbial fructan, comprising: Modified microbially fermented fructan was obtained by reacting microbially fermented fructan with methacrylic anhydride under alkaline conditions. The lipoic acid, stabilizer, and modified microbial fermented fructan were dissolved in water, heated and stirred to initiate the ring-opening polymerization of lipoic acid and covalent cross-linking with the modified microbial fermented fructan to obtain a pregel solution. The pregel liquid is supported on a hydroxyl-functionalized nonwoven fabric and heated to form a three-dimensional network gel.

[0028] In some preferred embodiments, the pH value of the alkaline conditions is 7.5 to 8.0.

[0029] The stabilizers used are commonly used components that improve the stability of lipoic acid in aqueous solutions, such as tris(hydroxymethyl)aminomethane.

[0030] The heating and stirring parameters are not limited. Those skilled in the art can select appropriate parameters based on the adaptability of the reaction raw materials. For example, stirring can be performed at 60-70°C for 30-60 seconds.

[0031] To make the technical solution of the present invention clearer, the preparation and performance of the medical bio-adhesive are illustrated below through several specific embodiments.

[0032] Levan, a microbial fermented fructan, is an extracellular EPS produced by Bacillus sp. SCU-E115, as disclosed in patent CN115895930A. The methacrylic anhydride, thioctic acid, and tris(hydroxymethyl)aminomethane used were all purchased from Maclean's.

[0033] Example 1: Preparation of medical bio-adhesive based on microbial fructans Preparation of modified Levan: 2g of Levan was dissolved in 100ml of distilled water, and 5.5mg of methacrylic anhydride was added. The reaction was continued at room temperature for 24h. Sodium hydroxide was used to maintain the pH of the reaction system at 8.0 during the reaction. After the reaction was completed, the system was dialyzed with sterile water for 48h.

[0034] Preparation of modified Levan / lipoic acid pregel solution: Dissolve 25g lipoic acid, 10g tris(hydroxymethyl)aminomethane and 5g modified Levan in 50ml deionized water and stir at 65℃ for 30s to pregel it.

[0035] Gelation of the pre-gelling solution: First, according to the size of the PTFE mold, cut the cotton non-woven fabric to the corresponding size and shape, and place it in the mold. Then, pour the pre-gelling solution into the mold, and finally gel at 40℃ for 4 hours. Afterward, demold to obtain the medical bio-adhesive. See the actual image of the medical bio-adhesive. Figure 1 .

[0036] Example 2: Preparation of medical bio-adhesive based on microbial fructans Preparation of modified Levan: 2g of Levan was dissolved in 100ml of distilled water, and 4mg of methacrylic anhydride was added. The reaction was continued at room temperature for 24h. Sodium hydroxide was used to maintain the pH of the reaction system at 7.5 during the reaction. After the reaction was completed, the mixture was dialyzed with sterile water for 48h.

[0037] Preparation of modified Levan / lipoic acid pregel solution: Dissolve 10g lipoic acid, 4g tris(hydroxymethyl)aminomethane and 5g modified Levan in 50ml deionized water and stir at 60℃ for 30s to pregel it.

[0038] Gelation of the pregel solution: First, according to the size of the polytetrafluoroethylene mold, cut the cotton fiber nonwoven fabric to the corresponding size and shape, and place it in the mold. Then, pour the pregel solution into the mold, and finally gel at 40°C for 4 hours. Finally, demold to obtain the medical bio-adhesive.

[0039] Example 3: Preparation of medical bio-adhesive based on microbial fructans Preparation of modified Levan: 2g of Levan was dissolved in 100ml of distilled water, and 10mg of methacrylic anhydride was added. The reaction was continued at room temperature for 24h. Sodium hydroxide was used to maintain the pH of the reaction system at 8.0 during the reaction. After the reaction was completed, the system was dialyzed with sterile water for 48h.

[0040] Preparation of modified Levan / lipoic acid pregel solution: Dissolve 50g lipoic acid, 20g tris(hydroxymethyl)aminomethane and 5g modified Levan in 50ml deionized water and stir at 70℃ for 60s to pregel it.

[0041] Gelation of the pregel solution: First, according to the size of the polytetrafluoroethylene mold, cut the cotton fiber nonwoven fabric to the corresponding size and shape, and place it in the mold. Then, pour the pregel solution into the mold, and finally gel at 40°C for 4 hours. Finally, demold to obtain the medical bio-adhesive.

[0042] Experimental Example The medical bio-adhesive prepared in Example 1 was subjected to the following tests: (1) Adhesion effect test of medical bio-adhesive: The prepared medical bio-adhesive was cut into 20mm × 20mm strips, and each strip was adhered to two 20mm × 80mm pieces of glass / paper / pigskin / iron / plastic sheet. The strips were then stretched using a universal tensile testing machine at a speed of 20mm / min, and the stress-strain curves and corresponding adhesion strengths were recorded. The test results are as follows: Figure 2 As shown, its adhesion strength on glass can reach 173.16 kPa, and its adhesion strength on paper can reach a maximum of 193.44 kPa.

[0043] (2) Test on the reversible adhesion effect of medical bio-adhesive: The prepared medical bioadhesive was cut into 20mm × 20mm strips and adhered to two 20mm × 80mm pieces of pigskin. Some samples were left untreated, while others were treated with a glutathione solution (glutathione can reduce the disulfide bonds in lipoic acid, breaking the polylipoic acid chains in the adhesive material and reducing its adhesive strength). The strips were then stretched using a universal tensile testing machine at a speed of 20mm / min, and their adhesive forces were recorded. The test results are as follows: Figure 3 As shown, the adhesion force can change by more than 15 times before and after reversible treatment with glutathione.

[0044] (3) Antibacterial effect test of medical bio-adhesive: A 10mm × 10mm sample of medical bio-adhesive was taken and co-cultured with *Escherichia coli* and *Staphylococcus aureus* bacterial suspensions for 24 hours. Then, the bacterial suspensions from the control group (which did not undergo co-culture) and the sample group were diluted, spread, and colonies were counted. Simultaneously, bacterial cell morphology was observed using SEM on the relevant samples. The relevant test results are as follows: Figure 4 As shown, the medical bio-adhesive exhibits good inhibitory effects on both types of bacteria.

[0045] (4) Cytotoxicity test of medical bio-adhesive: 1 / 2 / 4 mg of medical bio-glue samples were taken and soaked in 100 ml of PBS buffer for 24 h to obtain the corresponding sample extracts. These extracts were then co-cultured with cultured 293T cells for 12 / 24 h, and finally, a kit was used to detect cytotoxicity. The relevant test results are as follows: Figure 5 As shown, the residual cell viability of all samples remained above 85%, indicating that they did not have significant cytotoxicity.

[0046] (5) Mechanical property testing of medical bio-adhesive: Several 20mm × 60mm pieces of medical bio-adhesive and ungelled nonwoven fabric were taken and stretched using a universal tensile testing machine at a speed of 50mm / min. The stress-strain curves were recorded. The relevant results are as follows:Figure 6 As shown, the tensile strength of the medical bio-adhesive is increased by more than 4 times, reaching 20 MPa.

[0047] (6) Self-healing test of medical bio-adhesive: A scratch was made on the surface of a 20mm × 60mm medical bioadhesive sample, which was then subjected to self-healing at 60℃. Images before and after healing were taken under a microscope. Finally, the healed medical bioadhesive sample was stretched using a universal tensile testing machine at a speed of 50mm / min, and the stress-strain curves were recorded. The relevant results are as follows: Figure 7 As shown, the sample has been basically repaired, and its mechanical properties remain essentially unchanged.

[0048] (7) Release test of thioctic acid active substances in medical bio-adhesive: The medical bio-adhesive from Example 1 was placed in a simulated wet environment on a wound surface. The absorbance of the solution at 330 nm was measured using a UV spectrophotometer to indicate the concentration of thioctic acid. At 24 h, the thioctic acid release rate was (31.97 ± 0.92)%, and at 72 h, it reached (47.52 ± 1.53)%, demonstrating its good sustained-release effect and its ability to maintain effectiveness over a long period in practical applications.

[0049] (8) Test on the wound management effect of medical biological adhesive: First, in a clean and sterile surgical environment, 20mm incisions were made on the backs of several Bama pigs using a scalpel. The control group was treated with ordinary gauze, while the experimental group was treated with the medical bio-adhesive described in Example 1. Finally, before removing the incision, the incisions were treated with a glutathione solution. The relevant results are as follows... Figure 8 As shown, after 2 hours of treatment with medical bio-adhesive, the exudate on the gauze caused adhesion between the gauze and the scab on the wound. However, the medical bio-adhesive in the example could be easily peeled off without affecting the healing of the wound.

[0050] The embodiments described above illustrate only a portion of the implementation methods of the present invention, and not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A medical bio-adhesive based on microbial fructans, characterized in that, include: Hydroxyl-functionalized nonwoven base layer; and a gel layer composited with the base layer; The gel layer is a three-dimensional network structure formed by covalent cross-linking of methacrylated microbial fermented fructan and polythioctic acid.

2. The medical bio-adhesive of microbial fructan as described in claim 1, characterized in that, The hydroxyl-functionalized nonwoven fabric includes any one of cellulose nonwoven fabric, viscose fiber nonwoven fabric, chitin nanofiber nonwoven fabric, carboxymethyl cellulose nonwoven fabric, and hydroxyalkyl alkyl cellulose mixed ether ester nonwoven fabric.

3. The medical bio-adhesive of microbial fructan as described in claim 1 or 2, characterized in that, The double bonds on the methacrylamide-modified microbial fermented fructan molecules are covalently cross-linked with the disulfide bonds of polythiooctanoic acid.

4. The medical bio-adhesive of microbial fructan according to claim 1, characterized in that, The mass ratio of methacrylamide-modified microbial fermented fructan to polythioctic acid is 1:(2-10).

5. The medical bio-adhesive of microbial fructan according to claim 1, characterized in that, Methacrylation modification of microbially fermented fructan was performed using methacrylic anhydride.

6. The medical bio-adhesive of microbial fructan according to claim 5, characterized in that, The mass ratio of methacrylic anhydride to microbial fermented fructan is 1:(2-5).

7. The method for preparing medical bio-adhesive of microbial fructan as described in any one of claims 1-6, characterized in that, Include: Modified microbially fermented fructan was obtained by reacting microbially fermented fructan with methacrylic anhydride under alkaline conditions. The lipoic acid, stabilizer, and modified microbial fermented fructan were dissolved in water, heated and stirred to initiate the ring-opening polymerization of lipoic acid and covalent cross-linking with the modified microbial fermented fructan to obtain a pregel solution. The pregel liquid is supported on a hydroxyl-functionalized nonwoven fabric and heated to form a three-dimensional network gel.

8. The method for preparing medical bio-adhesive of microbial fructan as described in claim 7, characterized in that, The pH value of the alkaline conditions is 7.5 to 8.

0.

9. The method for preparing medical bio-adhesive of microbial fructan as described in claim 7, characterized in that, The stabilizer is tris(hydroxymethyl)aminomethane.

10. The method for preparing medical bio-adhesive of microbial fructan as described in claim 7, characterized in that, The heating and stirring are carried out at 60-70°C for 30-60 seconds.

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