A silk fibroin-based adhesive patch for sutureless repair of tissue wounds and a preparation method thereof

Abstract

The application discloses a silk fibroin-based adhesive patch for tissue wound sutureless repair and a preparation method thereof. The patch is composed of two layers which are tightly combined. The first layer is a high-toughness hydrogel matrix layer which is formed by cross-linking of methacrylated silk fibroin and acrylamide and adding a second network of sodium alginate-calcium ions. The second layer is a strong adhesion adhesive layer which is formed by self-assembly cross-linking of silk fibroin, polyethylene glycol and tannic acid. The silk fibroin-based adhesive patch prepared by the application has the advantages of excellent mechanical toughness, wet adhesion sealing property, biodegradability, biocompatibility and promotion of tissue sutureless repair. The adhesive patch is convenient to use, has high portability, can be self-adhered to a target tissue interface, avoids diffusion and penetration into non-damaged parts, and has a wide application prospect in soft tissue injury repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medical material preparation, and particularly relates to a silk fibroin-based adhesive patch for sutureless repair of tissue wounds and a preparation method thereof. BACKGROUND

[0002] Uncontrolled bleeding during surgery or trauma is one of the major causes of death in the world today. Trauma to internal soft tissues, particularly gastrointestinal perforation, peripheral nerve defect and internal organ bleeding, can cause serious life-threatening symptoms. For example, gastrointestinal perforation can cause leakage of liquid contents, acute peritonitis and sepsis, and even death; severe peripheral nerve injury can even cause paralysis and permanent disability. Traditionally, surgical suturing is often used as a standard technique to connect or close wound tissues. However, in clinical practice, surgical suturing still has inherent disadvantages, such as long time consumption and high requirement for surgical skills, especially when used for closure of gastrointestinal defects, such suturing has a high risk of leakage of body fluids / air from the treated gastrointestinal tissue, resulting in serious infection and even death. More seriously, surgical suturing causes secondary damage and local stress concentration to the surrounding tissue. Therefore, the development of innovative clinical alternative materials to replace suturing not only aims to prevent leakage of body fluids / air, but also to reduce local stress concentration and irreversible damage at the target wound site.

[0003] In order to solve the problems existing in suturing, tissue adhesive patches have been widely developed. The adhesive patch has the outstanding characteristics of convenient use and simple operation. The tissue adhesive patch can adaptively fit the biological tissue and form an interfacial adhesion with the tissue, promoting wound repair. In order to solve the above problems, the development of tissue adhesive patches with strong wet adhesion, excellent mechanical toughness and biodegradability has attracted more and more attention, which is expected to solve the problems existing in the current repair methods.

[0004] Silk fibroin is a biocompatible and biodegradable material with excellent physical properties, and is widely used in the fields of biomaterials, tissue engineering, regenerative medicine or medical devices. At present, the silk fibroin-based patches on the market are mostly microneedle patches. For example, Qizhenzhen et al. developed a silk fibroin microneedle transdermal patch for treating insomnia in the publication CN113679657A; and Lu Shenzhou et al. developed a silk fibroin insulin microneedle patch in the publication CN117838841A. However, the above-mentioned patches have slight deficiencies in mechanical toughness and wet adhesion performance. Therefore, it is urgent to develop a silk fibroin-based patch material with strong toughness and strong wet adhesion. SUMMARY

[0005] The present application aims at overcoming the defects of the prior art, and provides a tissue adhesive patch with excellent mechanical toughness, wet adhesion, biodegradability, biocompatibility and promotion of tissue seamless repair.

[0006] To achieve the above object, the present application adopts the following technical scheme:

[0007] A preparation method of a silk fibroin-based adhesive patch for tissue wound seamless repair, the patch is composed of two layers, the first layer is crosslinked by methacrylated silk fibroin and acrylamide, and sodium alginate and calcium ions are added to form a hydrogel matrix layer; the second layer is formed by self-assembly and crosslinking of silk fibroin, polyethylene glycol and tannic acid to form an adhesive layer.

[0008] The preparation method of the above-mentioned silk fibroin-based adhesive patch for tissue wound seamless repair specifically comprises the following steps:

[0009] (1) Preparation of hydrogel matrix layer: add acrylamide aqueous solution to the methacrylated silk fibroin solution, stir uniformly, then add sodium alginate aqueous solution, calcium sulfate dihydrate and photoinitiator and mix uniformly to obtain a hydrogel matrix layer precursor solution, pour the precursor solution into a mold and place it in a dark box, and irradiate it with ultraviolet light for 10 minutes to form a hydrogel matrix layer;

[0010] (2) Preparation of adhesive layer powder: mix silk fibroin solution and polyethylene glycol aqueous solution, add an equal volume of tannic acid aqueous solution, stir to obtain a solid gel material, freeze-dry the solid gel material, grind and sieve to obtain adhesive layer powder;

[0011] (3) Preparation of silk fibroin-based adhesive patch: coat the adhesive layer powder obtained in step (2) on the hydrogel matrix layer obtained in step (1), and place it in a constant temperature and humidity incubator to stand still to obtain a silk fibroin-based adhesive patch; the temperature in the constant temperature and humidity incubator is 35-38℃, and the relative humidity is 50-60%.

[0012] Further, the preparation method of the methacrylated silk fibroin in step (1) is as follows: degummed silk is dissolved with lithium bromide to obtain a silk fibroin solution, glycidyl methacrylate is then added, and the mixture is stirred in the dark for 3 hours, followed by dialysis with deionized water for 5-7 days, centrifugation, and taking the supernatant to obtain a methacrylated silk fibroin solution, the mass fraction of the methacrylated silk fibroin solution is 0.1%-5%.

[0013] Further, the mass fraction of the acrylamide aqueous solution in step (1) is 10%-50%, the mass fraction of the sodium alginate aqueous solution is 1%-5%, and the molar concentration of the calcium sulfate dehydrate is 0.1M-1M; the molar concentration of the photoinitiator is 0.001M-0.1M.

[0014] Further, the photoinitiator in step (1) is lithium phenyl-2,4,6-trimethylbenzoylphosphinate or alpha-ketoglutaric acid.

[0015] Further, the preparation method of the silk fibroin solution in step (2) is that degummed silk is dissolved with lithium bromide, and then deionized water dialysis is performed for 1-3 days, and the silk fibroin solution is obtained by centrifugation; the mass fraction of the silk fibroin solution is 1%-10%.

[0016] Further, the polyethylene glycol in step (2) is one of polyethylene glycol 500, polyethylene glycol 1000 and polyethylene glycol 2000; the mass fraction of the polyethylene glycol aqueous solution is 1%-5%.

[0017] Further, the volume ratio of the silk fibroin solution to the polyethylene glycol aqueous solution in step (2) is 5:1-1:5; the mass fraction of the tannic acid solution is 10%-30%.

[0018] Further, the standing time in step (3) is 1-20 minutes.

[0019] A silk fibroin-based adhesive patch for tissue wound sutureless repair prepared by the above preparation method.

[0020] The beneficial effects of the present application are:

[0021] (1) The tissue adhesive patch prepared by the present application has a two-layer structure, and the hydrogel matrix layer has strong mechanical toughness. On the basis of the methacrylated modified silk fibroin, acrylamide, sodium alginate, calcium sulfate dehydrate and photoinitiator are added to synthesize the hydrogel matrix layer. The tough hydrogel matrix layer uses the covalent crosslinking between the methacrylated modified silk fibroin and the acrylamide to maintain the elasticity, and uses the sodium alginate / calcium ion crosslinking as a recoverable sacrificial bond to effectively dissipate energy, so as to endow the gel with excellent mechanical toughness to adapt to dynamic physiological movement. The tensile elongation of the patch material of the present application is more than 200%, the toughness strength is 400 KJ / m 3 The above.

[0022] (2) The adhesive patch has strong wet tissue adhesion. The adhesive layer is self-assembled and cross-linked by silk fibroin solution, polyethylene glycol solution and tannic acid solution. The polyphenol structure contained in tannic acid can bond with the amide bond, amino group, sulfhydryl group and other functional groups in the tissue surface protein, thereby giving the adhesive patch strong adhesion on the tissue surface; and the double-layer structure makes the patch material of the application have excellent mechanical toughness and strong wet adhesion, and proves that it has wide application prospect in the adhesion and sealing of soft tissue injury such as gastrointestinal tract. The in-situ fitting characteristics of the patch material of the application also provide more convenience for application.

[0023] (3) The raw material has good biocompatibility and degradability.

[0024] (4) The application has the advantages of simple operation, easy batch production and good industrialization prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 : Adhesion strength of the silk fibroin-based adhesive patch in a wet environment.

[0026] Figure 2 : Adhesion strength of the silk fibroin-based adhesive patch on different tissues.

[0027] Figure 3 : Comparison of the adhesion strength of the silk fibroin-based adhesive patch and commercial adhesive on fresh pig skin.

[0028] Figure 4 : In-vitro sealing effect diagram of the silk fibroin-based adhesive patch on different tissues. DETAILED DESCRIPTION

[0029] The technical solutions described in the application will be further described below in combination with specific embodiments, but the application is not limited to this.

[0030] Example 1

[0031] This embodiment discloses a preparation method of silk fibroin solution, and the specific steps are as follows:

[0032] After 2 L of deionized water was heated to boiling, 8.48 g of anhydrous sodium carbonate was added, and after it was fully dissolved, 20 g of domestic silkworm silk was added for degumming treatment for 30 minutes to obtain degummed silk. The degummed silk was taken out and washed with deionized water, and was dried in an oven at 65°C. The dried degummed silk was added to 50 mL of a lithium bromide solution (9.3M) and dissolved at 65°C for 3 hours to obtain a mixed solution. The mixed solution was poured into a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed with deionized water for 3 days. After dialysis was completed, the solution was centrifuged at 9000 rpm for 20 minutes at 4°C, the precipitate was discarded, and the supernatant was collected to obtain a silk fibroin solution, which was stored in a 4°C refrigerator in the dark for later use.

[0033] Example 2

[0034] This example discloses a method for preparing a methacrylated modified silk fibroin solution, and the specific steps are as follows:

[0035] After 3 L of deionized water was heated to boiling, 15.9 g of anhydrous sodium carbonate was added, and after it was fully dissolved, 120 g of domestic silkworm silk was added for degumming treatment for 60 minutes to obtain degummed silk. The degummed silk was washed with deionized water and dried in an oven at 65°C. Then the degummed silk was added to 200 mL of a lithium bromide solution (9.3M) and dissolved at 65°C for 1 hour, then 12 mL of glycidyl methacrylate was added, and the reaction was carried out at 60°C for 3 hours, with the whole process being carried out in the dark. The reacted solution was poured into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, and dialyzed with deionized water for 6 days. After dialysis was completed, the solution was centrifuged at 9000 rpm for 20 minutes at 4°C, the precipitate was discarded, and the supernatant was collected to obtain a methacrylated modified silk fibroin solution, which was stored in a 4°C refrigerator in the dark for later use.

[0036] Example 3

[0037] This example discloses a method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds, and the specific steps are as follows:

[0038] (1) Preparation of the hydrogel matrix layer: A 1% methacrylated modified silk fibroin solution and a 30% acrylamide solution were mixed, stirred uniformly, then a 2% sodium alginate aqueous solution, 0.75 M calcium sulfate dihydrate, and 0.075 M α-ketoglutaric acid were added and mixed to obtain a precursor solution of the hydrogel matrix layer. The precursor solution was poured into a mold and placed in a dark box, and irradiated with 365 nm ultraviolet light for 10 minutes to form a gel, thereby obtaining a hydrogel matrix layer.

[0039] (2) Preparation of the adhesive layer powder: 5% silk fibroin solution and 2% polyethylene glycol solution were mixed in a volume ratio of 1:1, and then an equal volume of 20% tannic acid solution was added and mixed uniformly. The liquid part was discarded, and the solid glue-like substance was freeze-dried at -80°C, ground, and sieved through a 200-mesh screen to obtain the adhesive layer powder.

[0040] (3) Preparation of the silk fibroin-based adhesive patch: The adhesive layer powder prepared above was coated on the hydrogel matrix layer. A constant temperature and humidity incubator was set to a temperature of 37°C and a relative humidity of 60%. After 5 minutes, a silk fibroin-based adhesive patch was obtained.

[0041] The tensile elongation of the silk fibroin-based adhesive patch of this example was 210%, and the toughness strength was 460 KJ / m 3 . The adhesion strength in a blood environment reached 653.31 J / m 2 (see Figure 1 ). The silk fibroin-based adhesive patch had high adhesion strength to different tissues (muscle, skin, stomach, and intestine) (see Figure 2 ). The above results fully demonstrate that the silk fibroin-based patch has excellent wet-state tissue adhesion performance. In comparison with fresh pig skin, the adhesion strengths of commercially available adhesives (3M-Tegaderm Hydrocolloid (Tegaderm), cyanoacrylate (Histoacryl), and 3M-Steri-Strip (3M-SS)) were all lower than that of the adhesive patch of the present application (see Figure 3 ). Sealing experiments on different tissues (pig intestine, pig stomach, and pig lung) in vitro showed that the silk fibroin-based patch had excellent tissue sealing performance in a wet-state environment (see Figure 4 ).

[0042] Example 4

[0043] The preparation method of a silk fibroin-based adhesive patch for sutureless repair of tissue wounds is disclosed in this example, and the specific steps are as follows

[0044] (1) Preparation of the hydrogel matrix layer: 2% methacrylated silk fibroin solution and 40% acrylamide solution were mixed and stirred uniformly, and then 3% sodium alginate solution, 0.5M calcium sulfate dihydrate, and 0.05M α-ketoglutaric acid were added and mixed to obtain a hydrogel matrix layer precursor solution. The precursor solution was poured into a mold and placed in a dark box, and irradiated with 365nm ultraviolet light for 10 minutes to form a gel, obtaining a hydrogel matrix layer.

[0045] (2) Preparation of the adhesive layer powder: mix 3% silk fibroin solution and 3% polyethylene glycol solution in a volume ratio of 3:1, then add an equal volume of 30% tannic acid solution, mix well, discard the liquid part, freeze-dry the solid glue-like substance at -80°C, grind, and sieve through a 200-mesh screen to obtain the adhesive layer powder.

[0046] (3) Preparation of the silk fibroin-based adhesive patch: coat the adhesive layer powder prepared above on the hydrogel matrix layer, set the temperature of the constant-temperature and constant-humidity incubator to 35°C and the relative humidity to 50%, and place for 2 minutes to obtain the silk fibroin-based adhesive patch.

[0047] The tensile elongation of the adhesive patch of this example is 280%, and the toughness strength is 510 KJ / m 3 The adhesive strength in a blood environment reaches 712.56 J / m 2 .

[0048] Example 5

[0049] The preparation method of the silk fibroin-based adhesive patch for sutureless repair of tissue wounds is disclosed in this example, and the specific steps are as follows

[0050] (1) Preparation of the hydrogel matrix layer: mix 3% methacrylated silk fibroin solution and 20% acrylamide solution, stir well, then add 4% sodium alginate solution, 0.8 M calcium sulfate dihydrate, and 0.065 M α-ketoglutaric acid, mix well to obtain the hydrogel matrix layer precursor solution, pour the precursor solution into a mold and place it in a dark box, irradiate with 365 nm ultraviolet light for 10 minutes to form a gel, and obtain the hydrogel matrix layer.

[0051] (2) Preparation of the adhesive layer powder: mix 7% silk fibroin solution and 4% polyethylene glycol solution in a volume ratio of 1:5, then add an equal volume of 10% tannic acid solution, mix well, discard the liquid part, freeze-dry the solid glue-like substance at -80°C, grind, and sieve through a 200-mesh screen to obtain the adhesive layer powder.

[0052] (3) Preparation of the silk fibroin-based adhesive patch: coat the adhesive layer powder prepared above on the hydrogel matrix layer, set the temperature of the constant-temperature and constant-humidity incubator to 38°C and the relative humidity to 60%, and place for 18 minutes to obtain the silk fibroin-based adhesive patch.

[0053] The tensile elongation of the adhesive patch of this example is 320%, and the toughness strength is 580 KJ / m 3 The adhesive strength in a blood environment reaches 771.89 J / m2 .

[0054] Comparative Example 1

[0055] The preparation method of the silk fibroin-based adhesive patch for sutureless repair of tissue trauma is disclosed in the present comparative example, and the specific steps are as follows

[0056] (1) Preparation of the hydrogel matrix layer: uniformly stir the 1% methacrylated silk fibroin solution and the 30% acrylamide aqueous solution together, then add the 2% sodium alginate aqueous solution, 0.75 M calcium sulfate dihydrate, and 0.075 M α-ketoglutaric acid, mix well to obtain the hydrogel matrix layer precursor solution, pour the precursor solution into a mold and place it in a dark box, irradiate with 365 nm ultraviolet light for 10 minutes to form a gel, and obtain the hydrogel matrix layer.

[0057] (2) Preparation of the adhesive layer gelatinous substance: uniformly mix the 5% silk fibroin solution and the 2% polyethylene glycol aqueous solution in a volume ratio of 1:1, then add an equal volume of 20% tannic acid aqueous solution and mix well, discard the liquid part, and obtain the solid gelatinous substance.

[0058] (3) Preparation of the silk fibroin-based adhesive patch: coat the solid gelatinous substance prepared above on the hydrogel matrix layer, set the temperature of the constant temperature and humidity incubator to 37°C and the relative humidity to 60%, and place for 5 minutes to obtain the silk fibroin-based adhesive patch.

[0059] The tensile elongation of the adhesive patch prepared in the present comparative example is 60%, and the toughness strength is 78 KJ / m 3 , and the adhesion strength in the blood environment is 60.79 J / m 2 , which is much lower than that of the adhesive patch prepared in Example 3.

[0060] Since the solid gelatinous substance cannot diffuse and penetrate on the surface and inside of the hydrogel matrix layer, the contact bonding surface between the two is not tight enough, the two-layer structure of the obtained patch is poorly fitted, which leads to easy separation between the solid gelatinous substance and the hydrogel matrix layer during the stretching process, and the patch has poor performance. In comparison with Example 3, the use of adhesive layer powder is more conducive to the tight combination of the hydrogel matrix layer and the adhesive layer. After absorbing water, the adhesive layer powder fuses, uniformly diffuses and penetrates on the surface and inside of the gelatinous substance layer, which is more conducive to the tight combination with the hydrogel matrix layer. Compared with simply using the solid gelatinous substance prepared by the preparation method to coat, the two-layer structure of the patch obtained by coating the adhesive layer powder on the hydrogel matrix layer is more tightly fitted, and the performance is more uniform. The in-situ fusion and penetration characteristics of the adhesive layer powder after absorbing water play an important role in the excellent performance of the patch material of the present application, and the in-situ fitting effect is better.

[0061] The above description is only some embodiments of the present application, and any equivalent changes and modifications made according to the scope of the present application should be included in the scope of the present application.

Claims

1. A method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds, characterized in that: Specifically, the following steps are included: (1) Preparation of hydrogel matrix layer: Add acrylamide aqueous solution to methacrylated silk fibroin solution, stir evenly, then add sodium alginate aqueous solution, calcium sulfate dihydrate and photoinitiator and mix well to obtain hydrogel matrix layer precursor solution. Pour the precursor solution into mold and place it in dark box. Irradiate with ultraviolet light for 10 minutes to form hydrogel matrix layer. (2) Preparation of adhesive layer powder: Mix silk fibroin solution and polyethylene glycol aqueous solution, add an equal volume of tannic acid aqueous solution, stir to obtain solid colloidal substance, freeze dry the solid colloidal substance, grind it, and sieve it to obtain adhesive layer powder. (3) Preparation of silk fibroin-based adhesive patch: The adhesive layer powder obtained in step (2) is coated on the hydrogel matrix layer obtained in step (1), and placed in a constant temperature and humidity incubator to stand to obtain the silk fibroin-based adhesive patch; the temperature in the constant temperature and humidity incubator is 35℃-38℃ and the relative humidity is 50%-60%.

2. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds according to claim 1, characterized in that: The preparation method of the methacrylated silk fibroin solution in step (1) is as follows: degummed silk is dissolved in lithium bromide to obtain a silk fibroin solution, then glycidyl methacrylate is added, stirred in the dark for 3 hours, followed by dialyzing with deionized water for 5-7 days, centrifugation, and taking the supernatant to obtain the methacrylated silk fibroin solution. The mass fraction of the methacrylated silk fibroin solution is 0.1%-5%.

3. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds according to claim 1, characterized in that: In step (1), the mass fraction of the acrylamide aqueous solution is 10%-50%, the mass fraction of the sodium alginate aqueous solution is 1%-5%, the molar concentration of the calcium sulfate dihydrate is 0.1M-1M, and the molar concentration of the photoinitiator is 0.001M-0.1M.

4. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds according to claim 1, characterized in that: The photoinitiator mentioned in step (1) is lithium phenyl-2,4,6-trimethylbenzoylphosphinate or α-ketoglutaric acid.

5. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds according to claim 1, characterized in that: The silk fibroin solution described in step (2) is prepared by dissolving degummed silk in lithium bromide, then dialyzing with deionized water for 1-3 days, and centrifuging to obtain the silk fibroin solution; the mass fraction of the silk fibroin solution is 1%-10%.

6. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue trauma according to claim 1, characterized in that: The polyethylene glycol mentioned in step (2) is one of polyethylene glycol 500, polyethylene glycol 1000, and polyethylene glycol 2000; the mass fraction of the polyethylene glycol aqueous solution is 1%-5%.

7. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue wounds according to claim 1, characterized in that: The volume ratio of the silk fibroin solution to the polyethylene glycol aqueous solution in step (2) is 5:1 to 1:5; the mass fraction of the tannic acid aqueous solution is 10% to 30%.

8. The method for preparing a silk fibroin-based adhesive patch for sutureless repair of tissue trauma according to claim 1, characterized in that: The settling time mentioned in step (3) is 1-20 minutes.

9. A silk fibroin-based adhesive patch for sutureless repair of tissue trauma, prepared by the method described in any one of claims 1 to 8.

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