CN113398323B

CN113398323B - Preparation method and application of sericin adhesive

Info

Publication number: CN113398323B
Application number: CN202110740024.0A
Authority: CN
Inventors: 王琳; 王征; 刘欢
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2022-05-03
Anticipated expiration: 2041-06-30
Also published as: WO2023273875A1; CN113398323A

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

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 a sericin adhesive.

Background

Clinical wound closure and wound soft tissue reconstruction, which involve reattachment of the soft tissue surfaces, are typically closed with surgical sutures. Surgical sutures tend to cause additional trauma, leakage. Therefore, it is urgent to find a biodegradable tissue adhesive which is easy to handle and does not need to be removed to replace the conventional surgical suture, and the hot tide for the research of medical adhesives is triggered. Since water accounts for about 70% of the human body, most of the biological materials implanted into the human body are in a wet environment, which requires the adhesive to have underwater adhesion capability. The failure of biomaterial adhesion in the human environment can be attributed to several factors. First, water molecules can form a thin hydration layer at the interface between the biomaterial and the tissue, thereby preventing the adherent groups on the biomaterial from contacting the tissue surface. Second, water molecules easily disrupt the non-covalent adhesive interactions between the biomaterial and the tissue. In addition, swelling or shrinkage of biomaterials (e.g., hydrogels) upon imbibition of water can also impair their adhesion to tissue. Therefore, it is of great interest to develop biomaterials that can achieve effective adhesion under humid conditions.

Sericin is a natural macromolecular protein wrapped on the surface layer of silk fibroin fiber, and due to the shortages of people on sericin understanding and the limitations of research for a long time, a large amount of sericin is treated as waste in the silk reeling industry every year, thereby wasting a large amount of precious natural resources and causing serious pollution to the environment. In recent years, as shown in CN 111188194 and the like, sericin has excellent biological properties such as low immunogenicity, biodegradability, oxidation resistance, cell adhesion, and thus has an excellent repairing effect on skin tissue, vascular tissue, and bone tissue damage.

In the ocean, mussels can be firmly adhered to various substrates such as rocks under the condition that the mussels are continuously attacked by sea waves, and the sense of inspiration is provided for the development of underwater adhesives. The DOPA group in mussel foot protein DOPA, which is believed to be the key to mussel adhesion, effects adhesion through non-covalent interactions (including hydrogen bonding, metal coordination, pi-pi interactions and cation-pi interactions). To overcome the destructive effect of water on adhesives, inspired by marine mussels, researchers introduced DOPA groups in the polymer chain to make adhesives, but the preparation of adhesives required either complex pre-modification processes or toxic oxidants/metal ions, limiting the potential biological applications of such adhesives. Therefore, how to prepare the underwater adhesive with better biocompatibility is a problem to be solved urgently.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method and application of an underwater sericin adhesive, which can promote the drainage of the tissue surface by adjusting the hydrophilic-hydrophobic water balance of the components of the adhesive and mainly solves the problems that the existing adhesive does not have underwater adhesive capacity, the adhesive strength is weak, the biocompatibility is poor and the like.

In order to solve the problems, the invention adopts the following technical scheme:

the preparation method of the sericin adhesive comprises the following steps

Preparing reduced sericin: dissolving sericin powder in a tris (2-carboxyethyl) phosphine 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution for reduction to obtain a reduced sericin solution;

preparation of sericin adhesive: mixing the reduced sericin solution with a tannic acid solution, and fully reacting to obtain a sericin adhesive;

in one mode, the device also comprises

Preparing sericin powder: placing silkworm cocoon in Na₂CO₃Stirring the solution to obtain a gelatin silk solution, removing impurities, dialyzing and freeze-drying.

In one embodiment, in the preparation of reduced sericin:

the pH value of the 4-ethoxyl piperazine ethanesulfonic acid buffer solution is 7.0-7.4,

dissolving tris (2-carboxyethyl) phosphine in 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution at a ratio of 1-30g/mL

In one embodiment, the reduced sericin solution is prepared by: dissolving sericin in tris (2-carboxyethyl) phosphine 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution to prepare 10-30% (W/V) reduced sericin solution.

Dissolving sericin powder in tris (2-carboxyethyl) phosphine solution at a ratio of 0.01-0.3g/mL,

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

in one mode, in the preparation of the sericin adhesive, the reduced sericin solution and the tannic acid solution are prepared according to the following formula (1-1.5): (1-2) mixing in a volume ratio.

In one approach, in the preparation of the sericin binder:

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

In one mode, in the preparation of the sericin binder, a reduced sericin solution is mixed with a tannic acid solution, and the sericin binder is obtained after sufficient reaction.

The invention has the beneficial effects that:

provides a novel preparation method of the sericin adhesive, which is simpler and more environment-friendly; the preparation of the adhesive by taking sericin as a raw material is realized; the sericin adhesive has better adhesive property and wound repair promoting property.

Drawings

FIG. 1 is a graph showing the performance of a sericin adhesive;

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

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

figure 4 is a graph showing the in vivo healing performance of sericin adhesives.

Fig. 5 is a graph showing degradation of the sericin binder in vivo.

Fig. 6 is a display of sericin adhesive biocompatibility.

Detailed Description

The invention is further described below with reference to the accompanying drawings:

the preparation method of the sericin adhesive comprises the following steps

in one mode, the method also comprises

In one embodiment, in the preparation of reduced sericin:

in one embodiment, the reduced sericin solution is prepared by: dissolving sericin in tris (2-carboxyethyl) phosphine 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution to prepare 10-20% (W/V) reduced sericin solution.

In one approach, in the preparation of the sericin binder:

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

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

Some of the preparation methods are further described below:

1) extraction of sericin

Weighing Bombyx bombycis (white jade, Haoyue), oar Bombyx bombycis (A. mylitta) or Ricinus communis Bombyx bombycis), cutting into pieces, cleaning, and removing water.

Adding 20mL of 0.02mol/L Na into each gram of silkworm cocoon₂CO₃And (3) stirring the aqueous solution for 1 hour at the temperature of 100 ℃ to dissolve sericin to obtain a sericin solution.

Centrifuging to remove impurity components in the sericin solution, dialyzing for 12-72 hours to obtain a clear sericin solution, and freeze-drying to obtain sericin powder.

2) Preparation of reduced sericin

Dissolving the sericin obtained in the step 1) or commercialized sericin in a ratio of 0.1g/mL in 1-10mL of tris (2-carboxyethyl) phosphine solution (4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, pH 7.0-7.4), and reacting for 30 minutes to obtain a reduced sericin solution.

3) Preparation of tannic acid solution

Weighing 1g of tannic acid and dissolving in 10mL of ddH₂In O, preparing a 10% (W/V) tannic acid solution

4) Preparation of sericin adhesive

According to the volume ratio of 1: 1 mixing the reduced sericin solution with the pertannic acid solution, fully stirring to obtain a sericin adhesive, taking out, and then using ddH₂And washing for five times to obtain the sericin adhesive.

Besides the DOPA group, mussel foot protein contains a part of hydrophobic amino acids, and the exposed hydrophobic groups expel surface water molecules through hydrophobic interaction, thereby achieving continuous underwater adhesion. Based on the above theory, we utilized a reducing agent to open disulfide bonds in sericin to expose hydrophobic amino acids, which further interact with tannic acid via hydrogen bonds, van der waals forces and hydrophobic interactions to form a sericin binder. Effective underwater adhesion is achieved by promoting surface drainage, adjusting the hydrophilic-hydrophobic balance.

In subsequent researches, the adhesive property and the repair promoting property of the sericin adhesive are tested in vitro and in vivo, and the material is found to have better tissue adhesive property, can effectively promote the repair of a wound surface and has good biocompatibility.

The detailed description is as follows

EXAMPLE 1 preparation of sericin binder

Step 1) extraction of sericin

1. Weighing Bombyx Bombycis (white jade, Haoyue), oar Bombyx Bombycis (A. mylitta) or Ricinus communis Bombyx Bombycis), cutting into pieces, and adding ddH₂Cleaning O, centrifuging at 3500rpm for 5 minutes to remove water;

2. adding 20mL of 0.02mol/L Na into each gram of silkworm cocoon₂CO₃Placing the aqueous solution in a constant-temperature water bath kettle, and stirring for 1 hour at 100 ℃ to dissolve sericin to obtain a sericin solution;

3. transferring the obtained sericin solution into a 50mL centrifuge tube, centrifuging at 3500rpm for 5 minutes to remove impurity components in the sericin solution, and obtaining a clear solution;

4. the clear solution was transferred into a dialysis bag with a molecular cut-off of 3500Da in ddH₂Dialyzing in O for 72 hours, stirring slowly, and changing water every 6 hours;

5. transferring the sericin solution into a 50mL centrifuge tube, placing the centrifuge tube into liquid nitrogen for quick freezing for 5 minutes, then placing the centrifuge tube into a freeze dryer for freeze drying to obtain sericin powder, and placing the sericin powder into a refrigerator at the temperature of-20 ℃ for storage for later use.

Step 2) preparation of reduced sericin

Dissolving the sericin obtained in the step 1) in 1-10mL of tris (2-carboxyethyl) phosphine solution (4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, pH 7.0-7.4) according to the proportion of 0.1g/mL, and reacting for 30 minutes to obtain a reduced sericin solution;

step 3) preparation of tannic acid solution

Weighing 1g of tannic acid and dissolving in 10mL of ddH₂Preparing a tannic acid solution with the concentration of 10% (W/V) in O;

step 4) preparation of sericin adhesive

According to the volume ratio of 1: 1 mixing the reduced sericin solution with the pertannic acid solution, fully stirring to obtain a sericin adhesive, taking out, and then using ddH₂And O washing for five times to obtain the sericin adhesive.

Example 2 reduced sericin and sericin binder characterization test

In FIG. 1, the A diagram shows the sericin solution before and after the treatment with the reducing agent tris (2-carboxyethyl) phosphine, and B, C, D shows the mercapto content, the ultraviolet absorption spectrum and the zeta potential of sericin and reduced sericin, respectively.

The observation is that the sericin solution becomes turbid after the treatment with the reducing agent as shown in A in FIG. 1, the B graph shows that the content of thiol groups in the reduced sericin molecules is significantly increased, which indicates that the treatment with tris (2-carboxyethyl) phosphine as the reducing agent causes disulfide bond cleavage in the sericin molecules, resulting in an increase in the content of thiol groups, and the C graph in FIG. 1 shows that the absorption peak of hydrophobic amino acid at 280nm in the reduced sericin solution is enhanced, further indicating that the hydrophobic amino acid in the protein is everted due to the disulfide bond cleavage. Panel D of FIG. 1 shows that treatment with reducing agents caused the sericin solution to show that the zeta potential changes from negative to positive, indicating that hydrophobic amino acids are everted resulting in changes in the surface potential of the protein.

In FIG. 1, E is a diagram showing the preparation process of the sericin binder, and F is a diagram showing a real object of the prepared sericin binder (rSer-TA) showing a stringiness phenomenon of the sericin binder upon stretching. FIG. 1, G is a Fourier Infrared Spectroscopy (FTIR) of sericin binder at 1658cm as compared with sericin^-1Amide I bond of (2) to 1655cm^-1This indicates that the reducing agent treatment does not cause a change in the secondary structure of sericin, and H and I in FIG. 1 are X-ray photoelectron spectroscopy (XPS) of sericin and a sericin binder, and the results show that the peak of disulfide bond (HSSH) in sericin is located at 164.2eV, and the peak of S-C ═ O structure in sericin binder rSer-TA is located at 168.3eV, which indicates that a partially oxidized phenolic structure of tannic acid reacts with a mercapto group in sericin to form a Michael type adduct, which indicates that the sericin binder is formed based on intermolecular interaction and partial covalent interaction.

Example 3 testing of Underwater adhesion Capacity of sericin adhesive

First, the experimental process

1. Preparing a 1 cm-by-3 cm ceramic wafer, respectively coating a reducing sericin adhesive and a non-reducing sericin adhesive on the ceramic wafer, and then putting the ceramic wafer into water;

2. placing a 100g weight on the ceramic sheet, waiting for 30s, and waiting for bonding;

3. and lifting the weight, and observing the interface bonding condition and the ceramic wafer falling condition.

Second, result analysis

As shown in fig. 2A, when the weight was lifted, the weight and the ceramic plate were bonded together by the reduced sericin adhesive, and the reduced sericin adhesive was still bonded together after 30 seconds of water impact (fig. 2B), indicating that the reduced sericin adhesive had underwater bonding ability. And the non-reducing sericin adhesive can not adhere the weight and the ceramic plate, which indicates that the weight and the ceramic plate have no underwater adhesion capability. The above results indicate that the reduced sericin binder helps to enhance the underwater adhesion performance thereof due to having a certain hydrophobic group.

Example 4 testing of the in vitro adhesive Capacity of a sericin adhesive

First, the experimental process

Bonding two substrate pieces (polymethyl methacrylate PMMA plate, iron plate, silicon plate and ceramic plate) by using a reducing sericin adhesive, and detecting the bonding strength of the adhesive by using a mechanical extensometer after curing for 30 min.

Second, result analysis

1. As shown in C-D of fig. 2, the base sheets bonded by the sericin adhesive all reached 0.5MPa or more in adhesive strength, showing strong adhesive strength.

2. As shown in fig. 2E-F, the bond strength of the sericin binder exhibited an increasing form as the content of tannic acid was increased.

Example 5 sericin adhesive ability to tissue test

First, the experimental process

1. Preparing pigskin, cutting tissues of small intestine and large intestine into a certain size;

2. respectively coating a reducing sericin adhesive and a non-reducing sericin adhesive on the surfaces of the three tissues, observing the adhesive strength of the adhesives by twisting the tissues, and further attaching the tissues on a substrate to detect the mechanical adhesive strength;

3. soaking the bonded tissue in water for 30min, taking out, observing the bonding strength of the bonding agent by torsion, and further detecting the mechanical bonding strength;

second, result analysis

As shown in FIG. 3A, the tissue bonded by the reduced sericin adhesive is still adhered to the tissue surface when being twisted with a large force, and the adhesive is still remained on the tissue surface after being soaked in water for 30min and is not peeled off when being twisted with a large force. FIG. 3B shows that the tissue bonded with the nonreducing binder shows the presence of gaps when twisted with a large force, and the binder falls off after 30min immersion in water. The above results demonstrate that the eversion of the hydrophobic groups results in a higher adhesive strength of the reduced sericin adhesive to tissues, further demonstrating that adjusting the hydrophilic-hydrophobic water balance of the adhesive can adjust its underwater adhesive ability.

As shown in fig. 3C-D, the adhesion strength of reduced sericin adhesive to tissue reached 0.1MPa, whereas the adhesion strength of non-reduced sericin adhesive to tissue was only about 0.02 MPa; figures 3E-F show that the bond strength of reduced sericin binder to tissue still reaches above 0.05MPa after soaking in water, while the binder of non-reduced sericin binder to tissue is completely lost.

Example 6 testing of the ability of a sericin adhesive to block tissue leakage

First, the experimental process

1. Preparing a section of large intestine, filling water, fastening two ends of the large intestine, and pricking a small hole with a syringe, wherein the water can be filled from the small hole body;

2. and (5) plugging the small hole by using the adhesive, and observing the leakage condition.

Second, result analysis

As shown in fig. 3G, the large intestine tissue leaks due to perforation, the reduced sericin adhesive is coated on the leakage opening part, and the adhesive is attached to the tissue surface by pressing for 5s, and meanwhile, the leakage is blocked;

the above results demonstrate that the reduced sericin binder has a certain blocking ability against tissue leakage.

Example 7 sericin adhesive animal wound adhesion test

First, testing of mouse liver hemostasis with sericin binder

As shown in fig. 4 a, the abdominal skin of the mouse was cut open to expose the liver, the liver was wiped with physiological saline, a petri dish with filter paper was placed under the liver, a syringe was inserted into the liver tissue, blood was discharged, an adhesive was applied to the bleeding site, and the bleeding of the wound was observed, and a 3M adhesive group was used as a control.

Second, sericin adhesive rat wound healing model healing test

As shown in D in FIG. 4, a 2cm wound was cut on the back of an SD rat of about 250g with scissors, the peripheral blood was removed with a medical sterile gauze, and a reduced sericin adhesive was applied to the side of the epidermis and adhered for 30 seconds to observe the adhesion of the wound surface. And continuously feeding for 14 days, and recording the wound healing condition. Animals that were surgically sutured and 3M adhesive-bonded served as controls.

Third, sericin adhesive is tested to stop bleeding of leakage blocking of small intestine in rat body

As shown in fig. 4, K, the abdominal skin of the rat was cut open, the small intestine was found, the surrounding fluid was completely absorbed with medical sterile gauze, the syringe was inserted into the small intestine tissue, the body fluid was discharged, the adhesive was applied to the bleeding site, the leakage of wound body fluid was observed, and the untreated group was used as a negative control.

Fourth, result analysis

1. As shown in B, C in fig. 4, the bleeding time and the bleeding amount of the sericin binder used for the rat liver injury bleeding model are obviously lower than those of a control, which proves that the sericin binder has a good hemostatic effect and shows that the material has a certain effect on hemostasis of bleeding of internal organ organs.

2. As shown in E, F in FIG. 4, the wound healing rate of the sericin adhesive group is significantly higher than that of the control group when the sericin adhesive is used in the wound healing experiment, and the images G-J show that the wound healing quality of the sericin adhesive group is better, and hair follicles, sebaceous glands and sweat glands are generated, which is obviously better than that of the control group. Shows the superiority of the sericin adhesive for wound healing.

3. As shown in K, L in fig. 4, the sericin adhesive has good hemostatic and blocking effects when used in a mouse small intestine and liver bleeding leakage model, which shows that the sericin adhesive also has certain potential in small intestine and liver bleeding leakage.

Example 8 degradation of sericin adhesive in vivo

First, the experimental process

1. The skin of the rat's back was cut open, adhesive was applied to the back tissue, and the tissue was sutured and observed for 7 days.

2. The wound was opened on day 3, day 5 and day 7, respectively, and the degradation of the adhesive was observed.

Second, result analysis

As shown in fig. 5A, the sericin adhesive was wrapped with tissues at the third day, a certain inflammatory reaction appeared, the adhesive became small at the 5 th day and completely degraded at the 7 th day, indicating that the sericin adhesive has degradability in vivo. The staining results of the section of FIG. 5B show that the adhesive has no significant inflammatory response and toxicity.

First, result analysis

As shown in fig. 6B, the sericin binder did not cause blood cell disruption, demonstrating that the sericin binder has good blood compatibility, further indicating that the sericin gel has good biocompatibility.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a sericin adhesive comprises the steps of reducing sericin preparation and sericin adhesive preparation, and is characterized in that: the steps of reduced sericin preparation and sericin binder preparation are as follows:

preparation of sericin adhesive: and mixing the reduced sericin solution with the tannic acid solution, and fully reacting to obtain the sericin adhesive.

2. The method of claim 1, wherein the sericin binder comprises: preparing sericin powder: placing silkworm cocoon in Na₂CO₃Stirring the solution to obtain a gelatin silk solution, removing impurities, dialyzing and freeze-drying.

3. The method of claim 1, wherein the sericin binder comprises: the preparation method of the reduced sericin comprises the following steps:

the pH value of the 4-ethoxyl piperazine ethanesulfonic acid buffer solution is 7.0-7.4;

dissolving tris (2-carboxyethyl) phosphine in a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution at a ratio of 1-30 g/mL;

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

4. The method of claim 1, wherein the method comprises the steps of: the preparation method of the sericin adhesive comprises the following steps of: the tannic acid is dissolved in water to obtain a tannic acid solution with a concentration of 5-50%.

5. The method of claim 1, wherein the sericin binder comprises: the preparation method of the sericin adhesive comprises the steps of reducing sericin solution and tannic acid solution according to the ratio of (1-1.5): (1-2) mixing in a volume ratio.

6. Use of a sericin binder, characterized in that: the application of sericin adhesive in preparing hemostatic and wound surface adhesive material.