CN113398323B - A kind of preparation method and application of sericin adhesive - Google Patents

Abstract

The invention belongs to the field of medical biological composite materials, and particularly discloses a preparation method and application of a sericin adhesive, wherein the preparation method of the sericin adhesive comprises the following steps: preparing reduced sericin: dissolving sericin in a tris (2-carboxyethyl) phosphine solution for reduction to obtain a reduced sericin solution; preparation of sericin adhesive: the invention provides a novel preparation method of the sericin adhesive, which is simpler and more environment-friendly; the preparation of the adhesive with underwater adhesion capability by taking sericin as a raw material is realized; the silk gel jelly has good adhesive property and wound repair promoting property.

Description

A kind of preparation method and application of sericin adhesive

technical field

The invention belongs to the field of medical biological composite materials, and particularly relates to a preparation method and application of sericin adhesive.

Background technique

Surgical sutures are often used for clinical work involving reconnection of soft tissue surfaces, such as wound closure and trauma soft tissue reconstruction. But surgical suturing often results in additional trauma and leakage. Therefore, it is imperative to seek biodegradable tissue adhesives that are easy to handle and do not require removal to replace traditional surgical sutures, which has led to an upsurge in the study of medical adhesives. Since water accounts for about 70% of the human body, most of the biomaterials implanted in the human body are in a humid environment, which requires the adhesive to have underwater bonding ability. The reasons for the failure of biomaterial adhesion in the human environment can be attributed to several factors. First, water molecules can form a thin layer of hydration at the interface between the biomaterial and tissue, preventing adhesive groups on the biomaterial from contacting the tissue surface. Second, water molecules can easily disrupt non-covalent adhesive interactions between biomaterials and tissues. In addition, swelling or shrinking of biomaterials (eg, hydrogels) after water absorption can also weaken their adhesion to tissues. Therefore, it is of great significance to develop biomaterials that can achieve effective adhesion under humid conditions.

Sericin is a natural macromolecular protein wrapped on the surface of silk fibroin. For a long time, due to the lack of people's understanding of sericin and the limitation of research, a large amount of sericin is treated as waste in the silk reeling industry every year. It wastes a lot of precious natural resources and causes serious pollution to the environment. In recent years, as shown in CN 111188194, etc., sericin has good biological properties such as low immunogenicity, biodegradability, anti-oxidation, and cell adhesion, so sericin has good effects on skin tissue, vascular tissue, and bone tissue damage. Excellent repair effect.

In the ocean, mussels can adhere strongly to various substrates, such as rocks, under constant exposure to waves, inspiring the development of underwater adhesives. The dopa group DOPA in mussel podins is thought to be critical for mussel adhesion, which is achieved through non-covalent interactions including hydrogen bonding, metal coordination, π-π interactions and cation-π interactions Adhesion. In order to overcome the destructive effect of water on the adhesive, inspired by marine mussels, the researchers introduced DOPA groups into the polymer chain to prepare the adhesive, but the preparation of the adhesive requires a complex pre-modification process or toxic Oxidant/metal ions, limiting the potential biological applications of this type of adhesive. Therefore, how to prepare underwater adhesives with better biocompatibility is an urgent problem to be solved.

SUMMARY OF THE INVENTION

In view of the above-mentioned problems, the present invention provides a preparation method and application of an underwater sericin adhesive. By adjusting the hydrophilic and hydrophobic balance of the adhesive components, the drainage of the tissue surface is promoted, which mainly solves the problem that the existing adhesive does not have the ability of underwater adhesion. Weak adhesive strength, poor biocompatibility and other issues.

In order to solve the above problems, the present invention adopts the following technical solutions:

A preparation method of sericin adhesive, comprising the following steps

Preparation of reduced sericin: sericin powder is dry dissolved in tris(2-carboxyethyl)phosphine and reduced in 4-hydroxyethylpiperazineethanesulfonic acid buffer to obtain reduced sericin solution;

Preparation of sericin adhesive: The reduced sericin solution is mixed with the tannic acid solution, and the sericin adhesive is obtained after fully reacting;

In one way, it also includes

Dry preparation of sericin powder: silkworm cocoons are placed in Na ₂ CO ₃ solution, stirred to obtain glue silk solution, impurity removal, dialysis, and freeze-drying.

In one way, in the preparation of reduced sericin:

4-Hydroxyethylpiperazineethanesulfonic acid buffer pH 7.0-7.4,

Tris(2-carboxyethyl)phosphine was dissolved in 4-hydroxyethylpiperazine ethanesulfonic acid buffer at a ratio of 1-30 g/mL

In one way, the preparation of reduced sericin solution: sericin is dissolved in tris(2-carboxyethyl) phosphine in 4-hydroxyethylpiperazine ethanesulfonic acid buffer to make a concentration of 10-30% (W/V ) reduced sericin solution.

The dry sericin powder is dissolved in tris(2-carboxyethyl)phosphine solution at the ratio of 0.01-0.3g/mL,

The mass ratio of dry sericin powder to tris(2-carboxyethyl)phosphine is 1/20-1/30,

In one method, in the preparation of the sericin adhesive, the reduced sericin solution and the tannic acid solution are mixed in a volume ratio of (1-1.5):(1-2).

In one way, the sericin adhesive is being prepared:

The concentration of tannic acid solution is 0.05-0.5g/mL.

In one way, in the preparation of the sericin adhesive, the reduced sericin solution is mixed with the tannic acid solution and fully reacted to obtain the sericin adhesive.

The beneficial effects of the present invention are:

A relatively new preparation method of sericin adhesive is provided, which is simpler and environmentally friendly; the adhesive is prepared by using sericin as a raw material; and the sericin adhesive has good adhesion performance and wound surface repairing performance.

Description of drawings

Figure 1 is the performance display diagram of the sericin adhesive;

Fig. 2 is a graph showing the adhesion performance of sericin adhesive;

Figure 3 is a graph showing the adhesion performance of sericin adhesive to biological tissue;

Figure 4 is a graph showing the in vivo healing-promoting performance of sericin adhesive.

Figure 5 is a graph showing the degradation of sericin adhesive in vivo.

Figure 6 is a graph showing the biocompatibility of sericin adhesive.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings:

A preparation method of sericin adhesive, comprising the following steps

Preparation of reduced sericin: sericin powder is dry dissolved in tris(2-carboxyethyl)phosphine and reduced in 4-hydroxyethylpiperazineethanesulfonic acid buffer to obtain reduced sericin solution;

Preparation of sericin adhesive: The reduced sericin solution is mixed with the tannic acid solution, and the sericin adhesive is obtained after fully reacting;

In one way, it also includes

Dry preparation of sericin powder: silkworm cocoons are placed in Na ₂ CO ₃ solution, stirred to obtain glue silk solution, impurity removal, dialysis, and freeze-drying.

In one way, in the preparation of reduced sericin:

4-Hydroxyethylpiperazineethanesulfonic acid buffer pH 7.0-7.4,

Tris(2-carboxyethyl)phosphine was dissolved in 4-hydroxyethylpiperazine ethanesulfonic acid buffer at a ratio of 1-30 g/mL

The dry sericin powder is dissolved in tris(2-carboxyethyl)phosphine solution at the ratio of 0.01-0.3g/mL,

The mass ratio of dry sericin powder to tris(2-carboxyethyl)phosphine is 1/20-1/30,

In one way, the preparation of reduced sericin solution: sericin is dissolved in tris(2-carboxyethyl) phosphine in 4-hydroxyethylpiperazine ethanesulfonic acid buffer to make a concentration of 10-20% (W/V ) reduced sericin solution.

In one method, in the preparation of the sericin adhesive, the reduced sericin solution and the tannic acid solution are mixed in a volume ratio of (1-1.5):(1-2).

In one way, the sericin adhesive is being prepared:

The concentration of tannic acid solution is 0.05-0.5g/mL.

In one way, in the preparation of the sericin adhesive, the reduced sericin solution is mixed with the tannic acid solution and fully reacted to obtain the sericin adhesive.

Some physical properties of the sericin adhesive are shown in Figure 1.

Some of the preparation methods are further described below:

1) Extraction of sericin

Weigh silkworm cocoons (Bombyx mori cocoons (Baiyu, Haoyue), A. mylitta or castor cocoons, etc.), cut them into pieces, wash them, and remove moisture.

Add 20 mL of a 0.02 mol/L Na ₂ CO ₃ aqueous solution per gram of silkworm cocoons, and stir at 100° C. for 1 hour to dissolve the sericin to obtain a sericin solution.

The impurity components in the sericin solution are removed by centrifugation, dialyzed for 12-72 hours to obtain a clear sericin solution, and the sericin powder is obtained after lyophilization.

2) Preparation of reduced sericin

The sericin obtained in step 1) or commercialized sericin was dissolved in 1-10 mL of tris(2-carboxyethyl)phosphine solution (4-hydroxyethylpiperazineethanesulfonic acid) at a ratio of 0.1 g/mL. buffer, pH 7.0-7.4), react for 30 minutes to obtain a reduced sericin solution.

3) Preparation of tannic acid solution

Weigh 1 g of tannic acid and dissolve it in 10 mL of ddH ₂ O to make a tannic acid solution with a concentration of 10% (W/V).

4) Preparation of sericin adhesive

The reduced sericin solution is mixed with the pertannic acid solution in a volume ratio of 1:1, fully stirred to obtain a sericin adhesive, and after being taken out, washed with ddH ₂ O for five times to obtain the sericin adhesive.

In addition to the DOPA group, the mussel foot protein also contains some hydrophobic amino acids, and the exposed hydrophobic groups expel surface water molecules through hydrophobic interaction, thereby achieving continuous underwater adhesion. There are a small amount of disulfide-rich hydrophobic amino acids (cysteine/cystine) in the sericin molecular backbone. Based on the above theory, we use a reducing agent to open the disulfide bonds in sericin to expose the hydrophobic amino acids, which further interact with the sericin. Tannins form sericin adhesives through hydrogen bonding, van der Waals forces, and hydrophobic interactions. Efficient underwater adhesion is achieved by promoting surface drainage and regulating the hydrophilic-hydrophobic balance.

In the follow-up research, we tested the adhesive properties and repair-promoting properties of the sericin adhesive in vitro and in vivo, and found that the material has good tissue adhesion properties, which can effectively promote the repair of wounds. Biocompatibility.

Specific examples are as follows

Example 1 Preparation of sericin adhesive

Step 1) Extraction of sericin

1. Weigh silkworm cocoons (Bombyx mori cocoons (Bombyx mori, Haoyue), A. mylitta or castor cocoons, etc.), cut them into pieces, clean them with ddH ₂ O, and centrifuge at 3500rpm for 5 minutes to remove moisture;

2. Add 20mL of 0.02mol/L Na ₂ CO ₃ aqueous solution per gram of silkworm cocoons, place in a constant temperature water bath and stir at 100°C for 1 hour to dissolve sericin to obtain sericin solution;

3. Transfer the obtained sericin solution into a 50 mL centrifuge tube, and centrifuge at 3500 rpm for 5 minutes to remove impurities in the sericin solution to obtain a clear solution;

4. Transfer the clear solution into a dialysis bag with a molecular cut-off of 3500Da, dialyze it in ddH ₂ O for 72 hours, stir at a slow speed, and change water every 6 hours;

5. Transfer the sericin solution to a 50mL centrifuge tube, put it in liquid nitrogen for quick freezing for 5 minutes, and then put it into a freeze dryer. After freeze-drying, the sericin powder is obtained, which is stored in a -20°C refrigerator for later use.

Step 2) Preparation of reduced sericin

The sericin obtained in step 1) was dissolved in 1-10 mL of tris(2-carboxyethyl) phosphine solution (4-hydroxyethylpiperazine ethanesulfonic acid buffer, pH 7.0～ 7.4), react for 30 minutes to obtain a reduced sericin solution;

Step 3) Preparation of tannic acid solution

Weigh 1 g of tannic acid and dissolve it in 10 mL of ddH ₂ O to prepare a tannic acid solution with a concentration of 10% (W/V);

Step 4) Preparation of sericin adhesive

The reduced sericin solution is mixed with the pertannic acid solution in a volume ratio of 1:1, fully stirred to obtain a sericin adhesive, and after being taken out, washed with ddH ₂ O for five times to obtain the sericin adhesive.

Example 2 Characterization and detection of reduced sericin and sericin adhesive

In Figure 1, picture A is the solution of sericin before and after treatment with reducing agent tris(2-carboxyethyl) phosphine, and B, C, and D are the sulfhydryl content, UV absorption spectrum, and zeta potential of sericin and reduced sericin, respectively.

As shown in Fig. 1 A, the sericin solution became cloudy after the reducing agent treatment, and the sulfhydryl group content in the reduced sericin molecule was significantly increased in Fig. B, indicating that the reducing agent tris(2-carboxyethyl) phosphine treatment caused sericin The disulfide bond in the molecule is broken, resulting in an increase in the content of sulfhydryl groups. Figure C in Figure 1 shows that the absorption peak of the hydrophobic amino acid in the reduced sericin solution is enhanced at 280 nm, which further indicates that the disulfide bond breakage causes the hydrophobic amino acid in the protein to turn out. Panel D in Figure 1 shows that the sericin solution caused by reducing agent treatment shows that the zeta potential changes from negative to positive, indicating that the eversion of hydrophobic amino acids leads to changes in the surface potential of the protein.

In Figure 1, E is the preparation process of the sericin adhesive, and Figure F is the actual picture of the prepared sericin adhesive (rSer-TA), which shows that the sericin adhesive has a wire drawing phenomenon during stretching. G in Fig. 1 is the Fourier transform infrared spectroscopy (FTIR) of sericin adhesive. Compared with sericin, the amide I bond of sericin adhesive at 1658 cm ^-1 is shifted to 1655 cm ^-1 , which indicates that the reducing agent treatment It will not cause changes in the secondary structure of sericin. H and I in Figure 1 are X-ray photoelectron spectroscopy (XPS) of sericin and sericin adhesive. The results show that the disulfide bond (HSSH) in sericin The peak is located at 164.2 eV, while the peak of the S—C=O structure in the sericin adhesive rSer-TA is located at 168.3 eV, which indicates that the partially oxidized phenolic structure in the tannic acid reacts with the sulfhydryl group in sericin to form a Michael-type compound. The above results indicate that the sericin adhesive is formed based on intermolecular interactions and partial covalent interactions.

Example 3 Test of underwater adhesion ability of sericin adhesive

1. The experimental process

1. Prepare a 1cm*3cm ceramic sheet, apply the reduced sericin adhesive and the non-reduced sericin adhesive on the ceramic sheet respectively, and then put it in water;

2. Place the 100g weight on the ceramic piece, wait for 30s, and wait for it to bond;

3. Lift the weight and observe the bonding of the interface and the falling off of the ceramic sheet.

2. Analysis of the results

As shown in Figure 2A, when the weight was lifted, the reduced sericin adhesive bonded the weight and the ceramic sheet together, and the two were still bonded together after 30s of water impact (Figure 2B), indicating that the reduced sericin adhesive Has underwater bonding ability. The non-reduced sericin adhesive cannot bond the weight and the ceramic sheet, indicating that it does not have the ability to bond underwater. The above results indicate that the reduced sericin adhesive has a certain hydrophobic group to enhance its underwater adhesive performance.

Example 4 In vitro adhesion ability test of sericin adhesive

1. The experimental process

Two base sheets (polymethyl methacrylate PMMA sheet, iron sheet, silicon sheet, ceramic sheet) were bonded with reduced sericin adhesive, and the adhesive strength of the adhesive was tested with a mechanical tensile tester after curing for 30 minutes.

2. Analysis of the results

1. As shown in C-D in Figure 2, the adhesive strength of the substrate sheets bonded with sericin adhesives all reached more than 0.5MPa, showing a strong adhesive strength.

2. As shown in Figure 2, E-F, the bond strength of the sericin adhesive increases with increasing tannin content.

Example 5 Test of the adhesive ability of sericin adhesive to tissue

1. The experimental process

1. Prepare pig skin, cut small intestine and large intestine tissue to a certain size;

2. Apply the reduced sericin adhesive and the non-reduced sericin adhesive on the surfaces of the three tissues respectively, observe the adhesive strength of the adhesive by twisting the tissues, and further attach the tissues to the base sheet to test the mechanical adhesive strength;

3. Soak the bonded tissue in water for 30min, take it out, observe the bond strength of the adhesive by twisting, and further test the mechanical bond strength;

2. Analysis of the results

As shown in Figure 3A, the tissue bonded by the reduced sericin adhesive still adhered to the tissue surface when twisted vigorously, and the adhesive remained on the surface of the tissue after immersion in water for 30 min, and did not fall off when twisted vigorously. Figure 3B shows that the tissue bonded by the non-reducing adhesive showed the existence of gaps when twisted vigorously, and the adhesive fell off after immersion in water for 30 min. The above results indicate that the eversion of the hydrophobic group leads to a higher adhesive strength of the reduced sericin adhesive to the tissue, further indicating that adjusting the hydrophilic and hydrophobic balance of the adhesive can adjust its underwater adhesive ability.

Figures 3C-D show that the adhesive strength of reduced sericin adhesive to tissue reaches 0.1MPa, while that of non-reduced sericin adhesive to tissue is only about 0.02Mpa; Figures 3E-F show reduced sericin after immersion in water The adhesive strength of the adhesive to the tissue can still reach more than 0.05MPa, while the adhesive of the non-reduced sericin adhesive to the tissue is completely lost.

Example 6 Test of sealing ability of sericin adhesive to tissue leakage

1. The experimental process

1. Prepare a section of large intestine, fill it with water and tie both ends, poke a small hole with a syringe, and the water can pass through the small hole;

2. Take the adhesive to block the small hole and observe the leakage.

2. Analysis of the results

As shown in Figure 3G, the large intestine tissue leaked due to perforation. Apply reduced sericin adhesive to the leakage port, press for 5s, the adhesive was attached to the surface of the tissue, and the leakage was blocked at the same time;

The above results indicate that the reduced sericin adhesive has a certain ability to block tissue leakage.

Example 7 Animal Wound Adhesion Test of Sericin Adhesive

1. Hemostasis test of mouse liver with sericin adhesive

As shown in A in Figure 4, cut the abdominal skin of the mouse to expose the liver, wipe it with normal saline, place a petri dish padded with filter paper under the liver, pierce the syringe into the liver tissue, the blood flows out, and the adhesive The agent was smeared on the bleeding site, and the bleeding of the wound was observed, and the 3M adhesive group was used as a control.

2. Healing test of rat wound healing model with sericin adhesive

As shown in D in Figure 4, a 2cm wound was cut on the back of a 250g SD rat with scissors, and the surrounding oozing blood was wiped off with sterile medical gauze. Wound adhesion. And continue to raise for 14 days, record the wound healing. Surgically sutured animals and 3M adhesive bonded animals served as controls.

3. Sericin adhesive for hemostasis test of small intestinal leakage in rats

As shown in K in Figure 4, cut the abdominal skin of the rat, find the small intestine, use sterile medical gauze to absorb the surrounding liquid, pierce the syringe into the small intestine tissue, the body fluid flows out, apply adhesive to the bleeding site, observe The leakage of body fluids in the wound, the untreated group was used as a negative control.

4. Analysis of results

1. As shown in B and C in Figure 4, the bleeding time and bleeding volume of the sericin adhesive used in the rat liver injury bleeding model are significantly lower than those of the control, which proves that the sericin adhesive has a good hemostatic effect, indicating that the material bleeds in internal organs Has a certain effect on hemostasis.

2. As shown in E and F in Figure 4, sericin adhesive was used in the wound healing experiment. The wound healing rate in the sericin adhesive group was significantly higher than that in the control group. Figures G-J show that the sericin adhesive group wound healing. The quality is better, with the generation of hair follicles and sebaceous glands and sweat glands, which is significantly better than the control group. The superiority of sericin adhesive for wound healing is shown.

3. As shown in K and L in Figure 4, sericin adhesive has good hemostatic and occlusion effects when used in mouse small intestine and liver hemorrhage and leakage models, indicating that sericin adhesive is also used in small intestine and liver bleeding leakage. have some potential.

Example 8 In vivo degradation of sericin adhesive

1. The experimental process

1. Cut the skin on the back of the rat, apply adhesive to the back tissue, suture the tissue and observe for 7 days.

2. The wounds were opened on the 3rd day, the 5th day and the 7th day respectively to observe the degradation of the adhesive.

2. Analysis of the results

As shown in Figure 5A, the sericin adhesive was wrapped by tissue on the third day, and there was a certain inflammatory response. The adhesive became smaller on the 5th day, and completely degraded on the 7th day, indicating that the sericin adhesive is degradable in vivo. . Figure 5B section staining results show that the adhesive has no obvious inflammatory response and toxicity.

1. Analysis of the results

As shown in Fig. 6B, the sericin adhesive did not cause blood cells to rupture, proving that the sericin adhesive has good blood compatibility, further indicating that the sericin jelly has good biocompatibility.

Finally, it should be noted that the above are only preferred examples of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still understand the foregoing. The technical solutions described in the embodiments are modified, or some technical features thereof are equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (6)

1. A preparation method of sericin adhesive, comprising preparation of reduced sericin and preparation of sericin adhesive, characterized in that: the steps of preparation of reduced sericin and preparation of sericin adhesive are as follows: Preparation of reduced sericin: sericin powder is dry dissolved in tris(2-carboxyethyl)phosphine and reduced in 4-hydroxyethylpiperazineethanesulfonic acid buffer to obtain reduced sericin solution; Preparation of sericin adhesive: The reduced sericin solution is mixed with the tannic acid solution, and the sericin adhesive is obtained after sufficient reaction. 2. The preparation method of a sericin adhesive according to claim ₁ , characterized in that: dry preparation of sericin powder: silkworm cocoons are placed in _Na2CO3 solution, stirred to obtain glue silk solution, impurity removal, dialysis, freezing Dry. 3. The preparation method of a sericin adhesive according to claim 1, wherein the preparation method of the reduced sericin is as follows: 4-Hydroxyethylpiperazine ethanesulfonic acid buffer pH is 7.0-7.4; Tris(2-carboxyethyl)phosphine was dissolved in 4-hydroxyethylpiperazine ethanesulfonic acid buffer at a ratio of 1-30g/mL; The dry sericin powder is dissolved in tris(2-carboxyethyl)phosphine solution at a ratio of 0.01-0.3 g/mL. 4. The preparation method of a sericin adhesive according to claim 1, characterized in that: the preparation method of the sericin adhesive is as follows, wherein the tannic acid solution is prepared: the tannic acid is dissolved in water to prepare a concentration of 5 -50% tannic acid solution. 5. The preparation method of a sericin adhesive according to claim 1, characterized in that: in the preparation method of the sericin adhesive, the reduced sericin solution and the tannic acid solution are according to (1-1.5): (1- 2) Volume ratio mixing. 6. An application of a sericin adhesive, characterized in that: the application of the sericin adhesive in the preparation of hemostasis and wound surface adhesive materials.

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