Double-layer heterogeneous biological adhesive as well as preparation method and application thereof

Abstract

The preparation method specifically comprises the following steps: sequentially adding arginine and lipoic acid into a gelatin solution to obtain a hydrogel pre-polymerized solution; heating the hydrogel prepolymerization liquid to initiate a lipoic acid ring-opening polymerization cross-linking reaction, and adding a poly (3, 4-ethylenedioxythiophene) dispersion liquid modified by polydopamine to obtain gelatin / polylipoic acid conductive hydrogel; the preparation method comprises the following steps: heating and melting a lipoic acid monomer, then adding a cross-linking agent for reaction, then adding metal salt for stirring, and pouring the obtained yellow transparent liquid on the surface of the gelatin / polylipoic acid conductive hydrogel to obtain the double-layer heterogeneous biological adhesive. The invention also discloses an application of the double-layer heterogeneous biological adhesive in a biomedical adhesive. According to the method, strong interface connection between the two layers is achieved through the similar compatibility principle and the hot melting pouring technology, and layering is avoided during use; and the prepared adhesive has good tissue adhesion, self-repairing performance and biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of biomedical materials, and specifically relates to a preparation method of a bilayer heterogeneous bioadhesive, and also relates to the bilayer heterogeneous bioadhesive and its application. Background Art

[0002] During the repair process of in-vivo tissue wounds, the injured tissue is prone to adhesion with normal tissue through an adhesive, which often leads to serious postoperative complications. Therefore, it is required that the bioadhesive can not only provide strong adhesion to the tissue surface, but also have anti-adhesion ability after bonding the wound to avoid postoperative adhesion problems.

[0003] To solve this problem, adhesives with a bilayer heterogeneous structure have been proposed in the prior art. This adhesive has improved this problem to a certain extent through the heterogeneous structure (tissue adhesion layer / anti-adhesion layer). However, the existing bilayer adhesives still face key technical bottlenecks: 1. Insufficient interfacial bonding strength between the two layers: There is often a problem of insufficient bonding strength between the layers in the bilayer structure, and separation is likely to occur during use; 2. Lack or insufficiency of self-healing ability: Many bilayer adhesives only have self-healing ability in a single layer, or neither layer has self-healing characteristics. This makes it difficult for the adhesive to meet the clinical requirements of long-term adhesion maintenance and cyclic load tolerance in wound closure scenarios. Summary of the Invention

[0004] The first object of the present invention is to provide a preparation method of a bilayer heterogeneous bioadhesive, and the prepared adhesive has good tissue adhesion, self-healing performance and biocompatibility.

[0005] The second object of the present invention is to provide the above-mentioned bilayer heterogeneous bioadhesive.

[0006] The third object of the present invention is to provide the application of the above-mentioned bilayer heterogeneous bioadhesive in biomedical adhesives.

[0007] The technical solution adopted by the present invention is that the preparation method of the bilayer heterogeneous bioadhesive is specifically implemented according to the following steps: Step 1, prepare a polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion; Step 2, sequentially add arginine and lipoic acid to the gelatin solution, and stir evenly to obtain a hydrogel prepolymer solution; Step 3, heat the hydrogel prepolymer solution obtained in Step 2 to initiate the ring-opening polymerization cross-linking reaction of lipoic acid, and after the reaction ends, add the polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythiolic acid conductive hydrogel; Step 4: Heat and melt the lipoic acid monomer to carry out ring-opening polymerization reaction, then add a crosslinking agent to carry out inverse vulcanization reaction, and then add a metal salt and stir to obtain a yellow transparent liquid. Step 5: Pour the yellow transparent liquid obtained in Step 4 onto the surface of the gelatin / polythioctic acid conductive hydrogel obtained in Step 3, and cool to obtain a bilayer heterogenous bioadhesive.

[0008] The characteristics of the present invention also lie in that In Step 1, specifically: Dissolve dopamine in deionized water to obtain a dopamine solution; dissolve the initiator ammonium persulfate in deionized water to obtain an ammonium persulfate solution; dissolve 3,4-ethylenedioxythiophene in absolute ethanol to form a 3,4-ethylenedioxythiophene solution; mix the dopamine solution and the ammonium persulfate solution, and then mix with the 3,4-ethylenedioxythiophene solution, stir evenly, carry out a stirring reaction in an ice-water bath at 0°C - 5°C, after the reaction is completed, wash and centrifuge with deionized water until the supernatant becomes colorless and transparent, dry to obtain poly(3,4-ethylenedioxythiophene) nanoparticles modified with polydopamine, and disperse it in deionized water to obtain a dispersion of poly(3,4-ethylenedioxythiophene) modified with polydopamine.

[0009] The mass ratio of dopamine, 3,4-ethylenedioxythiophene, and ammonium persulfate is 0.04 - 10:1:0.1 - 6; the stirring reaction time is 3h - 24h, and the stirring speed is 200 - 600 rpm.

[0010] In Step 2, the mass percentage of gelatin in the hydrogel prepolymer solution is 10 - 30 wt%; the mass percentage of lipoic acid in the hydrogel prepolymer solution is 15 - 35 wt%; the mass percentage of arginine in the hydrogel prepolymer solution is 6 - 15 wt%.

[0011] In Step 3, the crosslinking reaction time is 30 - 60 min, and the crosslinking reaction temperature is 70 - 90°C.

[0012] In Step 4, the mass ratio of the lipoic acid monomer, the crosslinking agent, and the metal salt is 1:0.1 - 0.25:0.0003 - 0.001; the crosslinking agent is N,N'-methylenebisacrylamide; the metal salt is aluminum chloride; the polymerization reaction temperature is 80 - 120°C, and the polymerization reaction time is 10 - 60 min; the inverse vulcanization reaction temperature is 100 - 140°C, and the inverse vulcanization reaction time is 30 - 90 min; the stirring time is 5 - 20 min, and the stirring speed is 100 - 300 rpm.

[0013] The beneficial effects of the present invention are: (1) The preparation method of the present invention constructs a bioadhesive with different adhesions on both sides based on materials such as gelatin, polythioctic acid, arginine, and polydopamine-modified poly(3,4-ethylenedioxythiophene) nanoparticles and N,N'-methylenebisacrylamide. The prepared adhesive has good tissue adhesion, self-healing performance, and biocompatibility; no organic solvents are used in the preparation, improving the safety of the materials; in addition, the preparation process of this method is simple and easy to implement, the materials are inexpensive, and it is easy to manufacture on a large scale; (2) The double-layer heterogeneous bioadhesive prepared by the present invention realizes a strong interfacial connection between the two layers through the principle of similar compatibility and the hot-melt casting process, and will not delaminate during use; in addition, the shape of the double-layer heterogeneous bioadhesive can be realized by adjusting the form of the molding die and can be widely used. Description of the Drawings

[0014] Figure 1 It is a scanning electron micrograph of PDA-PEDOT nanoparticles prepared in Example 3.

[0015] Figure 2 It is a cross-sectional scanning electron micrograph of the double-layer heterogeneous bioadhesive prepared in Example 3.

[0016] Figure 3 It is a physical picture of the adhesion of the double-layer heterogeneous bioadhesive prepared in Example 3 to different substrates.

[0017] Figure 4 It is a graph of the adhesion strength results of the double-layer heterogeneous bioadhesive prepared in Example 3 to different substrates.

[0018] Figure 5 It is a physical picture of the self-healing of the double-layer heterogeneous bioadhesive prepared in Example 3.

[0019] Figure 6 It is a test graph of the CCK-8 cytotoxicity test of the double-layer heterogeneous bioadhesive prepared in Example 3. Specific Embodiments

[0020] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0021] The preparation method of the double-layer heterogeneous bioadhesive of the present invention is specifically implemented according to the following steps: Step 1, prepare a polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion; specifically: Dissolve dopamine (DA) in deionized water to obtain a dopamine solution; Dissolve the initiator ammonium persulfate in deionized water to obtain an ammonium persulfate solution; Dissolve 3,4-ethylenedioxythiophene (EDOT) in absolute ethanol to form a 3,4-ethylenedioxythiophene solution; Mix the dopamine solution and the ammonium persulfate solution, and then mix with the 3,4-ethylenedioxythiophene solution. Stir evenly, and carry out a stirring reaction in an ice-water bath at 0 °C - 5 °C for 3 h - 24 h with a stirring speed of 200 - 600 rpm. After the reaction is completed, wash and centrifuge with deionized water to remove impurities until the supernatant becomes colorless and transparent, and then dry to obtain poly(3,4-ethylenedioxythiophene) nanoparticles modified with polydopamine. Disperse it in deionized water to obtain a dispersion of poly(3,4-ethylenedioxythiophene) modified with polydopamine; The mass ratio of dopamine, 3,4-ethylenedioxythiophene, and ammonium persulfate is 0.04 - 10:1:0.1 - 6; The concentration of the dispersion of poly(3,4-ethylenedioxythiophene) modified with polydopamine (PDA-PEDOT) is 10 mg / mL - 100 mg / mL; Step 2, add arginine and lipoic acid to the gelatin solution in sequence. Use arginine to promote the dissolution of lipoic acid in water, and stir evenly to obtain a hydrogel prepolymer solution; The mass percentage of gelatin in the hydrogel prepolymer solution is 10 - 30 wt%; the mass percentage of lipoic acid in the hydrogel prepolymer solution is 15 - 35 wt%; the mass percentage of arginine in the hydrogel prepolymer solution is 6 - 15 wt%; Step 3, heat the hydrogel prepolymer solution obtained in Step 2 to initiate the ring-opening polymerization cross-linking reaction of lipoic acid. After the reaction is completed, add the dispersion of poly(3,4-ethylenedioxythiophene) modified with polydopamine obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythioctic acid conductive hydrogel; The cross-linking reaction time is 30 - 60 min, and the cross-linking reaction temperature is 70 - 90 °C; during the cross-linking reaction, the stirring speed is 100 - 300 rpm; Step 4, heat and melt the lipoic acid monomer to carry out a ring-opening polymerization reaction. The polymerization reaction temperature is 80 - 120 °C, and the polymerization reaction time is 10 - 60 min; then add a cross-linking agent to carry out an inverse vulcanization reaction. The inverse vulcanization reaction temperature is 100 - 140 °C, and the inverse vulcanization reaction time is 30 - 90 min. Then add a metal salt and stir for 5 - 20 min with a stirring speed of 100 - 300 rpm; obtain a yellow transparent liquid; The mass ratio of the lipoic acid monomer, the cross-linking agent, and the metal salt is 1:0.1 - 0.25:0.0003 - 0.001; The cross-linking agent is N,N'-methylenebisacrylamide (MBA); The metal salt is aluminum chloride (AlCl3); Step 5, pour the yellow transparent liquid obtained in Step 4 onto the surface of the gelatin / polythioctic acid conductive hydrogel obtained in Step 3, and cool to obtain a bilayer heterojunction bioadhesive.

[0022] The present invention constructs a bio - adhesive with different adhesions on both sides based on materials such as gelatin, polythioctic acid, arginine, and polydopamine - modified poly(3,4 - ethylenedioxythiophene) nanoparticles and N,N'-methylenebisacrylamide. The prepared adhesive has good tissue adhesion, self - healing performance, and biocompatibility.

[0023] Example 1 The preparation method of the double - layer heterogeneous bio - adhesive of the present invention is specifically implemented according to the following steps: Step 1: Dissolve 0.15 g of DA in 20 mL of deionized water, and dissolve 2 g of APS in 25 mL of deionized water; add 1 g of EDOT to 45 mL of absolute ethanol; after stirring the DA solution for 5 minutes, mix the DA solution and the APS solution, and then mix with the EDOT solution. Stir in an ice - water bath for 3 h until the solution turns blue - black. Use deionized water to wash and centrifuge to remove impurities, dry, and disperse the obtained PDA - PEDOT nanoparticles in deionized water to obtain a polydopamine - modified poly(3,4 - ethylenedioxythiophene) dispersion; Step 2: Dissolve 2 g of gelatin in 4 mL of deionized water, and successively add 0.5 g of arginine and 2 g of thioctic acid, and stir evenly at 45 °C to obtain a hydrogel prepolymer solution; Step 3: Heat the hydrogel prepolymer solution obtained in Step 2 to 75 °C and react for 90 min, add 2 mL of the 10 mg / mL PDA - PEDOT dispersion obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythioctic acid hydrogel; Step 4: Weigh 5 g of thioctic acid, heat it to 100 °C to make it completely molten, add 0.5 g of cross - linker (MBA) and react for 30 min, then add 2.5 mg of metal salt (AlCl3) and continue to stir for 5 min; obtain a yellow transparent liquid; Step 5: Pour the mixed solution obtained in Step 4 onto the surface of the hydrogel obtained in Step 3 while it is hot, and cool to obtain a double - layer heterogeneous bio - adhesive.

[0024] Example 2 The preparation method of the double - layer heterogeneous bio - adhesive of the present invention is specifically implemented according to the following steps: Step 1: Dissolve 0.1 g of DA in 20 mL of deionized water, and dissolve 1 g of APS in 25 mL of deionized water; add 1 g of EDOT to 45 mL of absolute ethanol; after stirring the DA solution for 5 minutes, mix the DA solution and the APS solution, and then mix with the EDOT solution. Stir in an ice - water bath for 6 h until the solution turns blue - black. Use deionized water to wash and centrifuge to remove impurities. Disperse the obtained PDA - PEDOT nanoparticles in deionized water to obtain a polydopamine - modified poly(3,4 - ethylenedioxythiophene) dispersion; Step 2: Dissolve 1.5 g of gelatin in 4 mL of deionized water, and successively add 0.8 g of arginine and 2.5 g of lipoic acid. Stir evenly at 45 °C to obtain a hydrogel prepolymer solution. Step 3: Heat the hydrogel prepolymer solution obtained in Step 2 to 85 °C and react for 80 min. Add 2 mL of the 20 mg / mL PDA-PEDOT dispersion obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythioctic acid hydrogel. Step 4: Weigh 5 g of lipoic acid, heat it to 110 °C to completely melt it, add 0.75 g of crosslinking agent (MBA) and react for 30 min, and then add 2 mg of metal salt (AlCl3) and continue to stir for 5 min. Step 5: Pour the mixed solution obtained in Step 4 onto the surface of the hydrogel obtained in Step 3 while it is hot, and cool to obtain a double-layer heterogeneous biological adhesive.

[0025] Example 3 The preparation method of the double-layer heterogeneous biological adhesive of the present invention is specifically implemented according to the following steps: Step 1: Dissolve 0.05 g of DA in 20 mL of deionized water, dissolve 1.5 g of APS in 25 mL of deionized water, add 0.75 g of EDOT to 45 mL of absolute ethanol, stir the DA solution for 5 minutes, then mix the DA solution and the APS solution, and then mix with the EDOT solution. Stir in an ice-water bath for 12 h until the solution turns blue-black. Wash with deionized water and centrifuge to remove impurities. The obtained PDA-PEDOT nanoparticles are dispersed in deionized water to obtain a polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion. Step 2: Dissolve 1.2 g of gelatin in 4 mL of deionized water, and successively add 1 g of arginine and 3.2 g of lipoic acid. Stir evenly at 45 °C to obtain a hydrogel prepolymer solution. Step 3: Heat the hydrogel prepolymer solution obtained in Step 2 to 90 °C and react for 60 min. Add 2 mL of the 50 mg / mL PDA-PEDOT dispersion obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythioctic acid hydrogel. Step 4: Weigh 5 g of lipoic acid, heat it to 100 °C to completely melt it, add 1 g of crosslinking agent MBA and react for 30 min, and then add 3 mg of metal salt AlCl3 and continue to stir for 5 min. Step 5: Pour the mixed solution obtained in Step 4 onto the surface of the hydrogel obtained in Step 3 while it is hot, and cool to obtain a double-layer heterogeneous biological adhesive.

[0026] Example 4 Furthermore, Figure 1It is the SEM image of the PDA-PEDOT nanoparticles prepared in Example 3. It can be found that they are spherical and dispersed from each other, indicating the successful loading of PDA. Figure 2 It is the cross-sectional SEM image of the bilayer heterogenous bioadhesive prepared in Example 3. It can be seen that there is seamless integration between the outer and inner hydrogels, indicating good interfacial property between the two layers.

[0027] Example 5 Further Figure 3 It is the actual adhesion image of the bilayer heterogenous bioadhesive prepared in Example 3 to different substrates. It can be seen that the adhesive has good adhesion to various materials such as pig skin. Figure 4 It is the adhesion strength result image of the bilayer heterogenous bioadhesive prepared in Example 3 to different substrates. Its peeling strength to pig skin can reach 41.3 N / m, and it can firmly adhere to biological tissues. Figure 5 It is the self-healing actual image of the bilayer heterogenous bioadhesive prepared in Example 3. Whether it is the outer layer or the inner layer, it can achieve self-healing at room temperature without any external stimulation and restore its original performance. Figure 6 It is the CCK-8 cytotoxicity test image of the bilayer heterogenous bioadhesive prepared in Example 3. Mouse fibroblasts were cultured and sampled by the extraction method, stained with CCK-8 reagent, and observed using a laser confocal microscope. The results show that after 5 days of co-culture, almost no dead mouse fibroblasts were detected in either the outer layer or the inner layer, indicating that both layers have excellent biocompatibility.

[0028] Example 6 The preparation method of the bilayer heterogenous bioadhesive of the present invention is specifically implemented according to the following steps: Step 1: Dissolve 0.03 g of DA in 20 mL of deionized water, and dissolve 0.8 g of APS in 25 mL of deionized water. Add 0.6 g of EDOT to 45 mL of absolute ethanol. After stirring the DA solution for 5 minutes, mix the DA solution and the APS solution, and then mix with the EDOT solution. Stir in an ice-water bath for 24 h until the solution turns blue-black. Use deionized water to wash and centrifuge to remove impurities. The obtained PDA-PEDOT nanoparticles are dispersed in deionized water to obtain a polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion; Step 2: Dissolve 1.8 g of gelatin in 4 mL of deionized water, and sequentially add 0.6 g of arginine and 2.5 g of lipoic acid, and stir evenly at 45 °C to obtain a hydrogel prepolymer solution; Step 3: Heat the hydrogel prepolymer solution obtained in Step 2 to 85 °C and react for 60 min. Add 2 mL of the 60 mg / mL PDA-PEDOT dispersion obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythiol hydrogel; Step 4: Weigh 5 g of lipoic acid, heat it to 130 °C until it is completely melted, add 1.5 g of crosslinking agent (MBA) and react for 40 min, and then add 4 mg of metal salt (AlCl3) and continue stirring for 5 min; Step 5: Pour the mixed solution obtained in Step 4 onto the surface of the hydrogel obtained in Step 3 while it is still hot, and a double-layer heterogeneous bioadhesive is obtained after cooling.

Claims

1. Preparation method of bilayer heterogeneous biological adhesive, characterized in that, The implementation is specifically carried out according to the following steps: Step 1: Prepare a polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion; Step 2: Sequentially add arginine and lipoic acid to the gelatin solution, and stir evenly to obtain a hydrogel prepolymer solution; Step 3: Heat the hydrogel prepolymer solution obtained in Step 2 to initiate the ring-opening polymerization cross-linking reaction of lipoic acid. After the reaction is completed, add the polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion obtained in Step 1, stir evenly, and cool to obtain a gelatin / polythiolactic acid conductive hydrogel; Step 4: Heat and melt the lipoic acid monomer to carry out ring-opening polymerization reaction, then add a cross-linking agent to carry out inverse vulcanization reaction, and then add a metal salt and stir to obtain a yellow transparent liquid; Step 5: Pour the yellow transparent liquid obtained in Step 4 onto the surface of the gelatin / polythiolactic acid conductive hydrogel obtained in Step 3, and cool to obtain a bilayer heterogeneous bioadhesive.

2. The preparation method of the double-layer heterogeneous biological adhesive according to claim 1, characterized in that, In the said Step 1, specifically: Dissolve dopamine in deionized water to obtain a dopamine solution; dissolve ammonium persulfate as an initiator in deionized water to obtain an ammonium persulfate solution; dissolve 3,4-ethylenedioxythiophene in absolute ethanol to form a 3,4-ethylenedioxythiophene solution; mix the dopamine solution and the ammonium persulfate solution, and then mix with the 3,4-ethylenedioxythiophene solution, stir evenly, and carry out a stirring reaction in an ice-water bath at 0°C - 5°C. After the reaction is completed, wash and centrifuge with deionized water until the supernatant becomes colorless and transparent, and dry to obtain polydopamine-modified poly(3,4-ethylenedioxythiophene) nanoparticles, and disperse them in deionized water to obtain a polydopamine-modified poly(3,4-ethylenedioxythiophene) dispersion.

3. The preparation method of the double-layer heterogeneous biological adhesive according to claim 2, wherein The mass ratio of dopamine, 3,4-ethylenedioxythiophene, and ammonium persulfate is 0.04 - 10:1:0.1 - 6; the stirring reaction time is 3h - 24h, and the stirring speed is 200 - 600rpm.

4. The preparation method of the double-layer heterogeneous biological adhesive according to claim 1, wherein, In the said Step 2, the mass percentage of gelatin in the hydrogel prepolymer solution is 10 - 30wt%; the mass percentage of lipoic acid in the hydrogel prepolymer solution is 15 - 35wt%; The mass percentage of arginine in the hydrogel prepolymer solution is 6 - 15wt%.

5. The preparation method of the double-layer heterogeneous biological adhesive according to claim 1, characterized in that, In the said Step 3, the cross-linking reaction time is 30 - 60min, and the cross-linking reaction temperature is 70 - 90°C.

6. The preparation method of the double-layer heterogeneous biological adhesive according to claim 1, characterized in that, In the said Step 4, the mass ratio of lipoic acid monomer, cross-linking agent, and metal salt is 1:0.1 - 0.25:0.0003 - 0.001; the cross-linking agent is N,N'-methylenebisacrylamide; the metal salt is aluminum chloride; the polymerization reaction temperature is 80 - 120°C, the polymerization reaction time is 10 - 60min; the inverse vulcanization reaction temperature is 100 - 140°C, the inverse vulcanization reaction time is 30 - 90min; the stirring time is 5 - 20min, and the stirring speed is 100 - 300rpm.

7. A bilayer heterogeneous bioadhesive prepared by the preparation method of the bilayer heterogeneous bioadhesive according to any one of claims 1 - 6.

8. The application of the bilayer heterogeneous bioadhesive according to any one of claims 1 - 6 in a biomedical adhesive.

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