阅读说明：本技术 一种双组分蛋白粘合剂及其制备方法和应用 (Bi-component protein adhesive and preparation method and application thereof ) 是由 贺超良 王天然 程学良 张震 陈学思 于 2021-10-13 设计创作，主要内容包括：本发明提供了一种双组分蛋白粘合剂及其制备方法和应用,粘合剂包括组分一和组分二；所述组分一中包含具有式(I)结构的重复单元和具有式(Ⅱ)结构的端基；所述组分二选自牛血清白蛋白、人血清白蛋白或明胶。本发明提供的粘合剂采用组分一和组分二,使得粘合剂具有高强度、高黏附性、无生物毒性；其能够应用于伤口修复、组织工程及药物缓释载体领域。(The invention provides a bi-component protein adhesive and a preparation method and application thereof, wherein the adhesive comprises a component I and a component II; the component one comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II); the second component is selected from bovine serum albumin, human serum albumin or gelatin. The adhesive provided by the invention adopts the component I and the component II, so that the adhesive has high strength, high adhesiveness and no biotoxicity; it can be applied to the fields of wound repair, tissue engineering and drug sustained-release carriers.)

1. A two-component protein adhesive comprises a first component and a second component;

the component one comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II):

the component two is selected from one or more of bovine serum albumin, human serum albumin and gelatin.

2. The two-component protein adhesive of claim 1, further comprising a solvent;

the solvent is selected from water, physiological saline, buffer solution, tissue culture fluid or body fluid.

3. The two-component protein adhesive according to claim 1, wherein the mass ratio of the first component to the second component is 1: 0.95-1.05.

4. The two-component protein adhesive according to claim 1, wherein the component(s) is (are) one or more compounds selected from the group consisting of compounds of formulae (iii) to (v):

wherein n is more than or equal to 50 and less than or equal to 100; m is more than or equal to 25 and less than or equal to 50; p is more than or equal to 67 and less than or equal to 133.

5. A method of preparing a two-component protein adhesive as claimed in any one of claims 1 to 4, comprising the steps of:

and mixing the component one solution and the component two solution to obtain the bi-component protein adhesive.

6. The method according to claim 5, wherein the mass-volume concentration of the first component in the first component solution is (1-50) g/mL;

the mass volume concentration of the second component is (1-50) g/mL.

7. The method according to claim 5, wherein the mixing temperature is 25 to 45 ℃.

8. Use of the two-component protein adhesive according to any one of claims 1 to 4 or the two-component protein adhesive prepared by the preparation method according to any one of claims 5 to 7 in wound repair, tissue engineering and drug sustained release carriers.

Technical Field

The invention belongs to the technical field of high polymer materials, and particularly relates to a bi-component protein adhesive as well as a preparation method and application thereof.

Background

In the clinic, the issue of surgical wound closure has attracted increasing attention. The global market value of clinically common methods such as sutures, staples, etc. is expected to exceed $ 150 billion per year in 2024. However, these methods often suffer from leakage, wound infection, weeping scabbing, scarring, and post-healing suture and staple removal. Therefore, in recent decades, tissue adhesives as alternatives to surgical sutures have received extensive attention and research because of their advantages of easy application, short application time, and little tissue damage.

Protein is an important substance widely existing in organisms, and has good biocompatibility and degradability. In addition, proteins have diverse structures and abundant functional groups, so that the proteins have good functionality. As one of the earliest tissue adhesives, fibrin glue consisted of a bicomponent composition of human thrombin and virally inactivated human fibrinogen, the major component of which was protein. Fibrin glue has good biodegradability and weak immunogenicity, and does not cause rejection reaction of organisms, so that fibrin glue is widely applied to clinic. However, fibrin glue has poor mechanical strength and cannot be applied to high-load repair. Other protein adhesives, such as the BioGlue tissue adhesive, which is a two-component adhesive composed of purified bovine serum albumin and glutaraldehyde, have also been used clinically. Therefore, it is very necessary to develop a new tissue adhesive having high strength and high adhesiveness.

Disclosure of Invention

In view of the above, the present invention provides a high-strength, high-adhesiveness and non-biotoxicity protein adhesive, which is applied in the fields of wound repair, tissue engineering and drug sustained release carriers.

The invention provides a bi-component protein adhesive, which comprises a first component and a second component;

the component one comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II):

the second component is selected from bovine serum albumin, human serum albumin or gelatin.

In the invention, the mass ratio of the first component to the second component in the bi-component protein adhesive is 1: 0.95-1.05, preferably 1: 1.

in the present invention, a solvent is also included;

the solvent is selected from water, physiological saline, buffer solution, tissue culture fluid or body fluid; preferably a buffer solution. In a specific embodiment of the invention, the solvent is selected from PBS buffer solution.

In the present invention, the component (a) is specifically selected from one or more compounds having the structures of formula (iii) to formula (V):

wherein m, n and p are polymerization degrees; n is more than or equal to 50 and less than or equal to 100; m is more than or equal to 25 and less than or equal to 50; p is more than or equal to 67 and less than or equal to 133.

The invention provides a preparation method of the bi-component protein adhesive in the technical scheme, which comprises the following steps:

and mixing the component one solution and the component two solution to obtain the bi-component protein adhesive.

In the invention, the mass volume concentration of the first component in the first component solution is (1-50) g/mL; preferably (10-30) g/mL;

the mass volume concentration of the second component in the second component solution is (1-50) g/mL, preferably (10-30) g/mL.

In the invention, the mixing temperature is 25-45 ℃.

The invention provides an application of the double-component protein adhesive in the technical scheme or the double-component protein adhesive prepared by the preparation method in the technical scheme in wound repair, tissue engineering and drug slow-release carriers.

The invention provides a bi-component protein adhesive, which comprises a first component and a second component; the component one comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II); the second component is selected from bovine serum albumin, human serum albumin or gelatin. The adhesive provided by the invention adopts the component I and the component II, so that the adhesive has high strength, high adhesiveness and no biotoxicity; it can be applied to the fields of wound repair, tissue engineering and drug sustained-release carriers.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of 3, 4-bis (dibromomethyl) benzoic acid prepared in example 1 of the present invention;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate prepared in example 2 of the present invention;

FIG. 3 is a NMR spectrum of a terminal group-protected four-arm PEG derivative prepared in example 3 of the present invention;

FIG. 4 is a NMR spectrum of 4aPEG-OPA prepared in example 4 of the present invention;

FIG. 5 shows the results of mechanical strength tests of a bovine serum albumin adhesive with a mass volume concentration of 4 g/mL;

FIG. 6 shows the results of mechanical strength tests of a bovine serum albumin adhesive with a mass volume concentration of 8 g/mL;

FIG. 7 shows the results of mechanical strength tests of a gelatin binder having a mass volume concentration of 1.5 g/mL;

FIG. 8 shows the results of mechanical strength tests of a gelatin binder having a mass-volume concentration of 2 g/mL;

FIG. 9 shows the results of mechanical strength tests of a gelatin binder having a mass volume concentration of 4 g/mL;

FIG. 10 shows the results of in vitro degradation experiments for bovine serum albumin adhesive prepared in example 10 of the present invention;

FIG. 11 shows the results of in vitro degradation experiments for the BSA adhesive prepared in example 11 of the present invention;

FIG. 12 shows the results of a test of the adhesion of a human serum albumin adhesive to PMMA at a mass volume concentration of 10 g/mL;

FIG. 13 shows the results of a test of adhesion of human serum albumin adhesive to PMMA at a mass volume concentration of 15 g/mL;

FIG. 14 shows the result of a test for the adhesion of human serum albumin adhesive to PMMA at a mass volume concentration of 20 g/mL;

FIG. 15 shows the results of a test of adhesion of a human serum albumin binder to glass at a mass volume concentration of 10 g/mL;

FIG. 16 shows the results of a test of adhesion of a human serum albumin binder to glass at a mass volume concentration of 15 g/mL;

FIG. 17 shows the results of a test of adhesion of a human serum albumin binder to glass at a mass volume concentration of 20 g/mL;

FIG. 18 shows the results of a human serum albumin adhesive adhesion test to porcine casing at a mass volume concentration of 10 g/mL;

FIG. 19 shows the results of a human serum albumin adhesive adhesion test to porcine casing at a mass volume concentration of 15 g/mL;

FIG. 20 shows the results of a human serum albumin adhesive adhesion test to porcine casing at a mass volume concentration of 20 g/mL;

FIG. 21 shows the results of cytotoxicity of L929 cells with 4aPEG-OPA prepared in example 4 of the present invention at various concentrations;

figure 22 is a human serum albumin adhesive at a mass volume concentration of 15g/mL for rat skin wound closure.

Detailed Description

In order to further illustrate the present invention, a two-component protein adhesive, a method of preparing the same and applications thereof, provided by the present invention, are described in detail below with reference to examples, which should not be construed as limiting the scope of the present invention.

Example 1

12g of 3, 4-dimethylbenzoic acid and 57g N-bromosuccinimide were dissolved in 200mL of carbon tetrachloride. 1.62g of benzoyl peroxide was added to the reaction mixture and heated at 81 ℃ under reflux for 15 h. The resulting white precipitate was filtered while hot and washed three times with benzene and ether, respectively. The filtrates were combined and concentrated, and the residue was dissolved in 300mL of 10% sodium carbonate solution. Washed with dichloromethane, adjusted to pH 1 and then extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over sodium sulfate, filtered and concentrated in vacuo. Crystallizing and purifying in acetonitrile to obtain the 3, 4-bis (dibromomethyl) benzoic acid.

The nuclear magnetic resonance analysis of the obtained 3, 4-bis (dibromomethyl) benzoic acid is carried out, and figure 1 is a nuclear magnetic resonance hydrogen spectrum of the 3, 4-bis (dibromomethyl) benzoic acid prepared in example 1 of the invention.

Example 2

The 3, 4-bis (dibromomethyl) benzoic acid prepared in example 1 was dissolved in 180mL of 10% sodium carbonate solution and reacted at 70 ℃ for 4 h. The reaction mixture was acidified to pH 1 with concentrated hydrochloric acid in an ice water bath and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting solid was dissolved in 100mL of anhydrous methanol and treated with 750mg of scandium trifluoromethanesulfonate overnight at room temperature. The methanol was evaporated, the mixture was dissolved in 100mL of anhydrous acetonitrile, and 4.8g of NHS and 8.1g of EDC. HCl were added. The mixture was stirred at room temperature overnight and then concentrated in vacuo. The residue was dissolved in dichloromethane, washed with saturated brine and dried over magnesium sulfate. The crude product was purified by silica gel chromatography in n-hexane/ethyl acetate to give succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate.

The obtained 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester is subjected to nuclear magnetic resonance analysis, and fig. 2 is a nuclear magnetic resonance hydrogen spectrum of the 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester prepared in example 2 of the present invention.

Example 3

2g of amino-terminated four-armed polyethylene glycol and 514mg of succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate, prepared as in example 2, are dissolved in 25mL of anhydrous dichloromethane and 0.5mL of pyridine is added. The mixture was stirred at room temperature for 2 days and then precipitated in ice ethyl ether to obtain a terminal-protected four-arm polyethylene glycol derivative.

The obtained end group protected four-arm polyethylene glycol derivative is subjected to nuclear magnetic resonance analysis, and fig. 3 is a nuclear magnetic resonance hydrogen spectrum of the end group protected four-arm polyethylene glycol derivative prepared in example 3 of the invention.

Example 4

The end-group-protected four-arm polyethylene glycol derivative prepared in example 3 was dissolved in 5mL of deionized water, and 5mL of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 1 hour in the dark and then diluted to 50 mL. The mixture was dialyzed for 2 days and lyophilized to give a four-arm polyethylene glycol derivative having a structure represented by formula III (4 aPEG-OPA).

The obtained 4aPEG-OPA was subjected to nuclear magnetic resonance analysis, and FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of the 4aPEG-OPA prepared in example 4 of the present invention.

Example 5

The first component prepared in example 4 and bovine serum albumin were prepared into a solution with a mass volume concentration of 4g/mL with a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, and after being uniformly mixed by a vortex apparatus, the mixture was rapidly transferred to a rotational rheometer to measure the mechanical strength, and as a result, as shown in fig. 5, the mechanical strength of a bovine serum albumin adhesive with a mass volume concentration of 4g/mL was 600 Pa.

Example 6

The first component prepared in example 4 and bovine serum albumin were prepared into a solution with a mass volume concentration of 8g/mL using a PBS buffer solution with a pH of 7.4, and the two solutions were mixed in equal volumes, and after being uniformly mixed by using a vortex apparatus, the mixture was rapidly transferred to a rotational rheometer to measure the mechanical strength of the mixture, and as a result, as shown in fig. 6, the mechanical strength of a bovine serum albumin adhesive with a mass volume concentration of 8g/mL was 13000 Pa.

Example 7

The first component obtained in example 4 and gelatin were mixed in equal volumes with a PBS buffer solution having a pH of 7.4 to prepare a solution having a mass volume concentration of 1.5g/mL, and the mixture was mixed uniformly by a vortex apparatus and then rapidly transferred to a rotational rheometer to measure the mechanical strength, and as a result, referring to FIG. 7, the mechanical strength of a gelatin binder having a mass volume concentration of 1.5g/mL was 60 Pa.

Example 8

The first component obtained in example 4 and gelatin were mixed in equal volumes with a PBS buffer solution having a pH of 7.4 to prepare a solution having a mass volume concentration of 2g/mL, and the mixture was mixed uniformly by a vortex apparatus and then rapidly transferred to a rotational rheometer to measure the mechanical strength thereof, and as a result, referring to FIG. 8, the mechanical strength of a gelatin binder having a mass volume concentration of 2g/mL was 400 Pa.

Example 9

The first component obtained in example 4 and gelatin were mixed in equal volumes with a PBS buffer solution having a pH of 7.4 to prepare a solution having a mass volume concentration of 4g/mL, and the mixture was rapidly transferred to a rotational rheometer to measure the mechanical strength after being uniformly mixed by using a vortex apparatus, and as a result, the mechanical strength of the gelatin binder having a mass volume concentration of 4g/mL was 4000Pa, as shown in fig. 9.

Example 10

The first component prepared in example 4 and bovine serum albumin were mixed in equal volumes with a PBS buffer solution having a pH of 7.4 to give solutions having a mass volume concentration of 4g/mL (second component concentration) and 6g/mL (bovine serum albumin concentration), and the mixture was mixed uniformly with a vortex apparatus and then solidified at 37 ℃. After stabilization, a PBS buffer solution having a pH of 7.4 and a PBS buffer solution containing proteinase K at a concentration of 5U/mL were added thereto, respectively, and the mixture was incubated at 37 ℃. At a specific time point the liquid was blotted dry and the mass was weighed. Then, PBS buffer solution with pH 7.4 and PBS buffer solution of proteinase K with concentration 5U/mL were added, and the incubation was continued at 37 ℃. The degradation curve is shown in figure 10, and the results of in vitro degradation experiments show that the obtained bi-component protein adhesive has degradability and is beneficial to biomedical application.

Example 11

The first component prepared in example 4 and bovine serum albumin were mixed in equal volumes with a PBS buffer solution having a pH of 7.4 to give solutions having a mass volume concentration of 4g/mL (second component concentration) and 8g/mL (bovine serum albumin concentration), and the mixture was mixed uniformly with a vortex apparatus and then solidified at 37 ℃. After stabilization, a PBS buffer solution having a pH of 7.4 and a PBS buffer solution containing proteinase K at a concentration of 5U/mL were added thereto, respectively, and the mixture was incubated at 37 ℃. At a specific time point the liquid was blotted dry and the mass was weighed. Then, PBS buffer solution with pH 7.4 and PBS buffer solution of proteinase K with concentration 5U/mL were added, and the incubation was continued at 37 ℃. The degradation curve is shown in figure 11, and the results of in vitro degradation experiments show that the obtained bi-component protein adhesive has degradability and is beneficial to biomedical application.

Example 12

The first component prepared in example 4 and human serum albumin were prepared into solutions with a mass volume concentration of 10g/mL with a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, mixed uniformly with a vortex apparatus, coated onto a PMMA plate, and the other PMMA plate was placed thereon with a contact area of 26mm × 10mm, and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 12.

Example 13

The first component prepared in example 4 and human serum albumin were prepared into solutions with a mass volume concentration of 15g/mL with a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, mixed uniformly with a vortex apparatus, coated onto a PMMA plate, and the other PMMA plate was placed thereon with a contact area of 26mm × 10mm, and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 13.

Example 14

The first component prepared in example 4 and human serum albumin were prepared into solutions with a mass volume concentration of 20g/mL with a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, mixed uniformly with a vortex apparatus, coated onto a PMMA plate, and the other PMMA plate was placed thereon with a contact area of 26mm × 10mm, and cured at 37 ℃ for 24 hours. The adhesion was tested with a universal tester and repeated 3 times, and the results are shown in FIG. 14.

Example 15

The first component prepared in example 4 and human serum albumin were prepared into 10g/mL solutions by PBS buffer solution with pH 7.4, the two solutions were mixed in equal volumes, and after being mixed uniformly by a vortex apparatus, the mixture was coated on a glass plate, and another glass plate was placed thereon with a contact area of 26mm × 10mm and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 15.

Example 16

The first component prepared in example 4 and human serum albumin were prepared into 15g/mL solutions by PBS buffer solution with pH 7.4, the two solutions were mixed in equal volumes, and after being mixed uniformly by a vortex apparatus, the mixture was coated on a glass plate, and another glass plate was placed thereon with a contact area of 26mm × 10mm and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 16.

Example 17

The first component prepared in example 4 and human serum albumin were prepared into solutions with a mass volume concentration of 20g/mL respectively using a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, mixed uniformly using a vortex apparatus, coated onto a glass plate, and the other glass plate was placed thereon with a contact area of 26mm × 10mm, and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 17.

Example 18

The first component prepared in example 4 and human serum albumin were prepared into 10g/mL solutions by PBS buffer solution with pH 7.4, the two solutions were mixed in equal volumes, and after being mixed uniformly by a vortex apparatus, the mixture was coated on a PMMA plate with a pig intestine, and another PMMA plate with a pig intestine was placed thereon with a contact area of 26mm × 10mm and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 18.

Example 19

The first component prepared in example 4 and human serum albumin were prepared into solutions with a mass volume concentration of 15g/mL respectively with a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, mixed uniformly with a vortex instrument, coated on a PMMA plate with a pig intestine, and the other PMMA plate with a pig intestine was placed thereon with a contact area of 26mm × 10mm and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 19.

Example 20

The first component prepared in example 4 and human serum albumin were prepared into solutions with a mass volume concentration of 20g/mL respectively with a PBS buffer solution with a pH of 7.4, the two solutions were mixed in equal volumes, mixed uniformly with a vortex instrument, coated on a PMMA plate with a pig intestine, and the other PMMA plate with a pig intestine was placed thereon with a contact area of 26mm × 10mm and cured at 37 ℃ for 24 hours. The adhesion was tested in a universal tester and repeated 3 times, and the results are shown in FIG. 20.

Example 21

Mouse fibroblasts L929 were seeded at 8000 cells/well in 96-well plates, 200 μ L of complete medium (90% DMEM medium + 10% newborn bovine serum) was added per well, and incubated in an incubator for 24 h. After 24h, the plates were removed and added with 20. mu. LpH of 7.4 PBS buffer and 10-0.625 mg/mL of the first component prepared in example 4 to a final concentration of 1-0.0625 mg/mL (specifically 1mg/mL, 0.5mg/mL, 0.25 mg/mL, 0.125mg/mL, and 0.0625mg/mL), respectively, and incubated in an incubator for 24 h. And taking out the culture plate after 24h, sucking the culture medium away, washing the culture plate for 2-3 times by using PBS (phosphate buffer solution), adding 10% CCK-8 solution in a dark place, placing the culture plate in an incubator for incubation for 1h, and testing the absorbance of the culture plate at 450nm and 630nm by using an enzyme-labeling instrument. The cytotoxicity of the adhesive material with different concentrations on L929 cells is shown in figure 21, and the experimental result shows that the adhesive material has no biological toxicity and can be applied to clinic.

Example 22

After the SD rat was anesthetized with sodium pentobarbital, a wound of about 2cm was formed on the back, the first component obtained in example 4 and human serum albumin were mixed with a PBS buffer solution having a pH of 7.4 to give solutions having a mass volume concentration of 15g/mL, and the two solutions were mixed in equal volumes, mixed uniformly with a vortex apparatus, and applied to the wound, and it was found that the wound was effectively closed. The observation was carried out for 14 days, and photographs were taken on days 1, 4, 7, 10, and 14, and the results are shown in FIG. 22. The results show that the two-component protein adhesive prepared by the invention has excellent wound healing promotion capability.

From the above examples, it can be seen that the present invention provides a two-component protein adhesive comprising a first component and a second component; the component one comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II); the second component is selected from bovine serum albumin, human serum albumin or gelatin. The adhesive provided by the invention adopts the component I and the component II, so that the adhesive has high strength, high adhesiveness and no biotoxicity; it can be applied to the fields of wound repair, tissue engineering and drug sustained-release carriers.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.