A biomedical adhesive system based on barnacle Trx-Balcp19k protein and its application

Abstract

The present invention discloses a biomedical adhesive system based on barnacle Trx-Balcp19k protein, which includes a first Trx-Balcp19k protein layer, a tannic acid layer, and a second Trx-Balcp19k protein layer, and the tannic acid layer is sandwiched between the first Trx-Balcp19k protein layer and the second Trx-Balcp19k protein layer. The present invention also provides an application of the above-mentioned biomedical adhesive system based on barnacle Trx-Balcp19k protein. By adding tannic acid and adopting a layer-by-layer coating method, the present invention improves the underwater / wet environment adhesion performance of Trx-Balcp19k protein and the adhesion performance to biological substrates, and has simple components, which is beneficial to the popularization and application of barnacle Trx-Balcp19k protein in the field of biomedical adhesives.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a barnacle-based biomedical adhesive system and its application. Background Art

[0002] In biomedical surgical wound suturing, there is an urgent need for biomedical adhesive materials with good biocompatibility and still maintaining adhesiveness in a humid physiological environment. The rich biological resources in the marine environment bring a huge material treasure house and inspiration to people. As an adhesion and fouling organism in the marine intertidal zone, barnacles rely on the bioadhesive secreted by them (the synergistic action of multiple proteins) to adhere and solidify, achieving its firm attachment on the underwater substrate. This bioadhesive secreted by barnacles has the characteristics of good biocompatibility and strong wet adhesion ability, which brings great inspiration to the development of biomedical adhesives. However, the bioadhesive secreted by barnacles is non-continuously secreted, making it difficult to collect and use in large quantities artificially. Currently, mainly by collecting, dissolving and analyzing the cured barnacle bioadhesive, protein components such as cp19k, cp20k, cp52k, cp68k, cp100k, etc. have been obtained, and then attempts are made to obtain the above-mentioned glue proteins through biosynthesis and carry out research on its adhesion and solidification mechanism. Among them, cp19k is the key interfacial adhesion protein. Some researchers have biosynthesized recombinant proteins and quasi-natural proteins from it and carried out research on its structure, properties and functions, etc.

[0003] For example, Patent CN105031717A obtains recombinant barnacle glue protein cp19k through the method of heterologous expression in Escherichia coli, and through a specific dialysis purification process, a glue-like substance with very good viscosity can be obtained. The self-assembly performance and adhesion performance of it have been studied. The results show that its adhesion ability and biocompatibility are good, and it has good application prospects in biomedical adhesion. After improving the dialysis method by the means disclosed in Patent CN109627308A to increase the protein yield, it is found that although the protein freeze-dried and dissolved in water still has strong bonding performance, the bonding performance of the protein in an underwater / wet environment is poor, and the bonding performance of the protein when acting on biological tissues is poor.

[0004] Therefore, improving the bonding performance of the key protein cp19k in the barnacle bioadhesive in an underwater / wet environment and improving the bonding performance of the protein cp19k when used for biological tissues are of great significance for the application of this protein in the field of biomedical adhesives. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology, and provide a biomedical adhesive system based on barnacle Trx-Balcp19k protein with excellent adhesive performance underwater / wet environment and excellent adhesive performance when acting on biological tissues. To solve the above technical problem, the technical solution proposed by the present invention is:

[0006] A biomedical adhesive system based on barnacle Trx-Balcp19k protein, comprising a first Trx-Balcp19k protein layer, a tannic acid layer and a second Trx-Balcp19k protein layer, wherein the tannic acid layer is sandwiched between the first Trx-Balcp19k protein layer and the second Trx-Balcp19k protein layer.

[0007] As a general technical concept, the present invention also provides an application of the above-mentioned biomedical adhesive system based on barnacle Trx-Balcp19k protein, using the biomedical adhesive system for adhesion between adjacent substrates.

[0008] In the above application, preferably, first coat the Trx-Balcp19k protein solution on the adjacent surfaces of the adjacent substrates respectively, then coat the tannic acid on the surface of at least one Trx-Balcp19k protein solution, and then press the adjacent substrates tightly and cure, that is, realize the adhesion between the adjacent substrates. When curing, the bonded area of the pressed substrates can be fixed with clips and cured at 37 °C for 24 h.

[0009] In the above application, preferably, the Trx-Balcp19k protein solution is first self-assembled. After the Trx-Balcp19k protein solution is self-assembled, the bonding effect will be better.

[0010] In the above application, preferably, during the self-assembly, the concentration of the Trx-Balcp19k protein solution is controlled to be 250-300 mg / mL, the pH is 4.5-10.6, the ionic strength I is 140-200 mM, the environmental temperature is 2-5 °C, and the self-assembly duration is 65-90 h. Controlling under the above self-assembly conditions is beneficial to obtaining a protein solution with good bonding performance.

[0011] In the above application, preferably, the coating amount of the Trx-Balcp19k protein solution on the substrate is controlled to be 2.1-2.7 mg per 0.5 cm 2 coating area (for the pasting area) (2.1-2.7 mg in total for both sides), and the coating amount of the tannic acid on the Trx-Balcp19k protein solution is 1-12 mg per 0.5 cm 2 is 1-12 mg.

[0012] In the above application, preferably, the substrate is biological tissue, and the adjacent substrates are adhered underwater or in a wet environment.

[0013] In the present invention, more preferably, the self-assembly conditions of the Trx-Balcp19k protein solution are as follows: the protein solution concentration is 300 mg / mL, pH = 7, ionic strength I = 150 mM, the environmental temperature is 4 °C, and the self-assembly duration is 3 days. When the coating area is 0.5 cm 2 the concentration of the Trx-Balcp19k protein solution is 300 mg / mL, and the total volume is 8 μL. The concentration of tannic acid and the corresponding volume are 1500 g / L, 8 μL; 500 g / L, 2 μL. When the coating area is enlarged, the solution dosage is enlarged proportionally.

[0014] The principle of the present invention is as follows: According to the theory that the Trx-Balcp19k protein in barnacle glue protein mainly exists as an interfacial protein between barnacle glue and an external substrate, and at the same time, using the property that barnacle glue protein forms a fibrous structure through self-assembly to enhance adhesion, it is extended to the Trx-Balcp19k protein. The Trx-Balcp19k protein that forms a fibrous structure through self-assembly forms a strong interfacial force with the interface. The non-covalent interactions (including hydrogen bond interaction, cation-π interaction, electrostatic interaction) between the fibrous Trx-Balcp19k protein and tannic acid form cohesion. The protein-tannic acid-protein structure formed by the above two forces will form a powerful combined force structure. This structure can isolate water molecules on the one hand in an underwater / wet environment, creating a dry microenvironment inside the adhesive, enabling the internal Trx-Balcp19k to remain on the interface and play an interfacial adhesion role; on the other hand, the distribution of tannic acid in the middle of the adhesive layer not only enhances the cohesion, but also can be coupled through hydrogen bonds, cation-π, etc. with the Trx-Balcp19k groups to enhance their interaction; in addition, tannic acid can also promote the transformation of the secondary structure of the Trx-Balcp19k protein into β-sheet, improving the interfacial adhesion performance.

[0015] It should be emphasized that the biomedical adhesive system of the present invention is a sandwich structure obtained by layer-by-layer coating of protein-tannic acid-protein. Only by adopting this sandwich structure can the underwater / wet environment adhesion performance of this biomedical adhesive system be improved and the adhesion performance acting on biological substrates be improved. If the Trx-Balcp19k protein solution and tannic acid are directly mixed, the bonding effect is not good. This may be because the interaction between tannic acid and the Trx-Balcp19k protein solution will be fully completed in the solution when directly mixed, resulting in the force existing only inside and unable to interact with the interface anymore, and the interfacial adhesion effect will become worse.

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] The biomedical adhesive system and application based on barnacle Trx-Balcp19k protein of the present invention comprehensively utilize the protein distribution of barnacle glue colloid, the adhesion behavior of barnacle glue protein, and the interaction between barnacle glue protein and tannic acid, and develop a method to improve the underwater / wet environment adhesion performance of Trx-Balcp19k protein and the adhesion performance on biological substrates by adding tannic acid and adopting a layer-by-layer coating method. Moreover, the components are simple, which is conducive to the popularization and application of barnacle Trx-Balcp19k protein in the field of biomedical adhesives. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is a transmission electron microscope photograph of the Trx-Balcp19k protein solution before and after self-assembly in Example 1 (A in the figure represents the photograph of the protein without self-assembly, and B represents the photograph of the protein after self-assembly).

[0020] Figure 2 It is a schematic diagram of the bonding performance test in the underwater / wet environment in Example 1 (blank is the 300mg / mL Trx-Balcp19k protein solution, 7 is the protein solution with 8μL of 1500g / L tannic acid added in the layer-by-layer coating of the protein, TA7 is the 8μL of 1500g / L tannic acid solution, 16 is the protein solution with 2μL of 500g / L tannic acid added in the layer-by-layer coating of the protein, and TA16 is the 2μL of 500g / L tannic acid solution).

[0021] Figure 3 It is a schematic diagram of the adhesion performance test of the pigskin substrate in Example 2 (blank is the 300mg / mL Trx-Balcp19k protein solution, 7 is the protein solution with 40μL of 1500g / L tannic acid added in the layer-by-layer coating of the protein, TA7 is the 40μL of 1500g / L tannic acid solution, 16 is the protein solution with 10μL of 500g / L tannic acid added in the layer-by-layer coating of the protein, and TA16 is the 10μL of 500g / L tannic acid solution).

[0022] Figure 4 It is a schematic diagram of the bonding performance test after directly mixing Trx-Balcp19k and TA in Comparative Example 1.

[0023] Figure 5 Effects of Trx-Balcp19k, TA, and Trx-Balcp19k-TA at different concentrations on the viability of NIH / 3T3 cells (blank is a group without the sample solution, i.e., the concentration is 0).

[0024] Figure 6 Photographs of live and dead cell staining after stimulating NIH / 3T3 with Trx-Balcp19k, TA, and Trx-Balcp19k-TA at different concentrations (green represents live cells and red represents dead cells). Detailed implementation manners

[0025] To facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0026] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0027] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0028] Example 1:

[0029] The biomedical adhesive system based on barnacle Trx-Balcp19k protein in this example includes a first Trx-Balcp19k protein layer, a tannic acid layer, and a second Trx-Balcp19k protein layer. The tannic acid layer is sandwiched between the first Trx-Balcp19k protein layer and the second Trx-Balcp19k protein layer to form a protein-tannic acid-protein sandwich structure.

[0030] The application of the above-mentioned biomedical adhesive system based on barnacle Trx-Balcp19k protein is to use this biomedical adhesive system for adhesion between adjacent substrates. In this example, a glass substrate is selected as the substrate. Specifically, the Trx-Balcp19k protein solution is self-assembled and incubated for 3 days at a concentration of 300 mg / mL, a pH of 7, an ionic strength I of 150 mM, and an environmental temperature of 4°C. Transmission electron microscopy tests are performed on the protein before and after self-assembly under the same concentration and solution environment, as Figure 1 shown, the morphology of fibrous structures in the solution after self-assembly is significantly increased.

[0031] The obtained self-assembled protein is respectively coated on the coating areas (0.5 cm2 ) Apply evenly inside (total amount used: 8 μL), and then apply tannic acid with a certain concentration and corresponding amount on any layer of the applied Trx-Balcp19k protein solution. Align the coated areas of the two test substrates and fix the two test substrates with clips to form a protein-tannic acid-protein structure. Place them separately in water at 37°C and a humid environment (the test substrates are placed in a closed constant temperature water bath, above the water, and the water temperature is 37°C) for 24 h. The average viscous shear strength of Trx-Balcp19k-TA on glass in a humid environment was measured by a dynamic mechanical tester to be much higher than that of pure Trx-Balcp19k protein and pure TA solution with the corresponding volume concentration. The average shear strength of Trx-Balcp19k protein after adding 8 μL of 1500 g / L tannic acid and 2 μL of 500 g / L tannic acid in a humid environment was as high as 1.89 ± 0.51 MPa and 1.62 ± 0.27 MPa respectively, both more than 3 times the data of the control group. The average viscous shear strength of Trx-Balcp19k-TA on glass in an underwater environment was 0.28 ± 0.05 MPa after adding 8 μL of 1500 g / L tannic acid and 1.12 ± 0.17 MPa after adding 2 μL of 500 g / L tannic acid, while the Trx-Balcp19k control group and the TA control group could not achieve bonding and no data could be obtained. The above data are as Figure 2 shown.

[0032] Example 2:

[0033] The biomedical adhesive system based on barnacle Trx-Balcp19k protein in this example includes a first Trx-Balcp19k protein layer, a tannic acid layer, and a second Trx-Balcp19k protein layer. The tannic acid layer is sandwiched between the first Trx-Balcp19k protein layer and the second Trx-Balcp19k protein layer to form a protein-tannic acid-protein sandwich structure.

[0034] The application of the above biomedical adhesive system based on barnacle Trx-Balcp19k protein is to use this biomedical adhesive system for adhesion between adjacent substrates. In this example, the substrate is selected as a pigskin substrate. Specifically, the obtained Trx-Balcp19k protein solution is self-assembled and incubated for 3 days at a concentration of 300 mg / mL, pH of 7, ionic strength I of 150 mM, and environmental temperature of 4°C.

[0035] The obtained self-assembled protein is respectively applied to the coated areas (2.5 cm 2)Apply evenly inside (total amount used: 40 μL), and then apply tannic acid with a certain concentration and corresponding amount on any layer of the applied Trx-Balcp19k protein solution. Align the coated areas of the two test substrates and fix the two test substrates with clips to form a protein-tannic acid-protein structure. Place them separately in a humid environment at 37 °C for 24 h. Using a dynamic mechanical tester, it is measured that the shear strength on pig skin can reach above 0.025 MPa after adding 40 μL of 1500 g / L tannic acid and 10 μL of 500 g / L tannic acid, both far exceeding the control group (0.040 MPa > 0.012 MPa), indicating that Trx-Balcp19k-TA shows good results in the pig skin adhesion experiment.

[0036] In addition, the shear strength after adding 40 μL of 1500 g / L tannic acid and 10 μL of 500 g / L tannic acid also exceeds the shear strength of their respective TA groups. This shows that the shear strength of Trx-Balcp19k-TA is produced by the combined action of Trx-Balcp19k protein and TA components, and both have a significant improvement on the shear effect.

[0037] Comparative Example 1:

[0038] Dissolve Trx-Balcp19k protein in deionized water to prepare a protein solution with a concentration of 100 g·L -1 . After centrifuging to remove air bubbles, continuously blow and suck the system to make it a homogeneous liquid phase. Dissolve TA in deionized water to prepare a TA solution with a concentration of 100 g·L -1 . Mix the two solutions of protein and TA in a volume ratio of 3:7, pre-freeze at -20 °C and then freeze-dry at -50 °C to obtain a sample, named 3-7.

[0039] Dissolve Trx-Balcp19k protein in deionized water to prepare a protein solution with a concentration of 20 g·L -1 . After centrifuging to remove air bubbles, continuously blow and suck the system to make it a homogeneous liquid phase. Dissolve TA in deionized water to prepare a TA aqueous solution with a concentration of 100 g·L -1 . Mix the two solutions of protein and TA in a volume ratio of 1:1, pre-freeze at -20 °C and then freeze-dry at -50 °C to obtain a sample, named 3c.

[0040] Weigh 10 mg of the freeze-dried powder of Trx-Balcp19k-TA stored in a -20 °C refrigerator and place it at room temperature within the test area of a substrate. Then, add 10 μL of deionized water to it to form a gel. Use another substrate to spread the gel evenly within the test area (1 cm × 1 cm), and quickly overlap and fix the test areas of the two substrates with clips. Place them in an oven at 37 °C for 24 h. The substrates are steel sheets, aluminum sheets, and glass slides respectively. The measured shear strength is shown in Figure 4 . It can be seen from Figure 4 that the highest shear strength is only 1.1 MPa, which is lower than that of pure Trx-Balcp19k, indicating that directly mixing Trx-Balcp19k and TA results in poor adhesive performance (no adhesive performance underwater or in a humid environment).

[0041] To prove the effects of Trx-Balcp19k, TA, and Trx-Balcp19k-TA on cell viability, the present invention also provides the following cell compatibility tests:

[0042] (1) CCK-8 cell viability detection:

[0043] ① Cell resuscitation: Place the centrifuge tube containing cryopreserved NIH / 3T3 cells in a 37 °C water bath and quickly thaw for 1 min. Then, carefully resuspend the cells with 3 mL of cell culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution. Centrifuge at room temperature (1000 rpm, 5 min), and discard the supernatant. Then, carefully resuspend the cells with 4 mL of cell culture medium and pour them into a 6 cm diameter cell culture dish to spread evenly. Finally, culture them in a cell incubator (37 °C, 5% CO2).

[0044] ② Passage the cells every 2 - 3 days: Observe the cell density under a microscope. When the cell density is about 80%, passage the cells. Aspirate the old cell culture medium in the culture dish, first rinse it with 1 - 2 mL of PBS, and then digest the adherent cells with 0.6 mL of 0.25% trypsin solution for about 1 - 2 min. When most of the cells have detached (observed as round cells under a microscope), add 3 - 6 mL of fresh cell culture medium to stop digestion. Use a pipette to gently pipette to mix the cells evenly, and collect the cells into a centrifuge tube and centrifuge at low speed to remove the supernatant. Finally, resuspend the cells with an appropriate amount of cell culture medium to obtain the mother liquor. Dilute the mother liquor by a certain ratio and transfer it to a new cell culture dish (10 cm in diameter) for continued culture (the passage ratio is generally 1:3).

[0045] ③ After the cells are stable, perform cell seeding: Take a part of the mother liquor of the NIH / 3T3 cell culture medium to be passaged and dilute it to a concentration of 2.5×10 4 cell·mL -1, equipped with cell suspension; inoculate the cell suspension into a 96-well plate (the blank control group inoculates cell culture medium), 200 μL per well (i.e., 5000 cells), and then transfer it to a cell incubator to grow for 12 h.

[0046] ④ Drug administration and stimulation: Replace the cell suspension in the 96-well plate with cell culture medium, and at the same time add medium extracts of Trx-Balcp19k, TA, and Trx-Balcp19k-TA with different masses, and the concentrations are 0, 0.5, and 5 μg·mL -1 , with a total volume of 100 μL, and then place it in the cell incubator again for 24 h; set 10 replicate samples for each concentration.

[0047] ⑤ Add 20 μL of CCK-8 reagent to the cell culture medium after drug administration, incubate at 37 °C for 2 h, and then measure the absorbance at 420 nm with an enzyme-linked immunosorbent assay reader. The calculation method of cell viability is as follows: regard the absorbance measured in the control group without adding any protein or TA as 100% cell viability; the cell viability under other final concentration conditions = the absorbance measured under this final concentration condition / the absorbance of the corresponding control group × 100%.

[0048] (2) Live and dead cell imaging:

[0049] ① Spread 200 μL of NHI / 3T3 cell suspension (concentration of 5×10 4 cell·mL -1 ) on a 96-well plate and grow for 24 h, then replace with fresh cell culture medium, and at the same time add 100 μL of Trx-Balcp19k, TA, and Trx-Balcp19k-TA solutions with gradient final concentrations (0, 0.5, and 5 μg·mL -1 ) to stimulate growth for another 24 h; set 3 replicate samples for each concentration in each group.

[0050] ② Place the frozen live cell green fluorescent reagent A and dead cell red fluorescent reagent B in a 37 °C water bath to thaw quickly, and then add 1 mL of reagent A to a centrifuge tube containing 1 μL of reagent B and mix evenly to prepare the staining solution.

[0051] ③ Add 200 μL of the staining solution to the cell culture medium on the 96-well plate, incubate at room temperature for 15 min, and then immediately take pictures and observe with a fluorescence microscope. The results are as Figure 5 、 Figure 6 shown.

[0052] As Figure 5As shown, the percentage activity of NIH / 3T3 cells remained above 100% after being stimulated with different concentrations of Trx-Balcp19k, TA, and Trx-Balcp19k-TA for 24 h. This indicates that TA and Trx-Balcp19k-TA within the tested concentration range do not inhibit the proliferation of NIH / 3T3 cells. Referring to the classification criteria of the toxicity grading method in the United States Pharmacopeia, materials with a cell viability value greater than 100% are classified as toxicity grade 0 to cells, that is, they can be regarded as non-toxic to cells.

[0053] As Figure 6 shown, compared with the live-dead cell imaging of the blank group, a large amount of green fluorescence was exhibited by 0.5 and 5 μg·mL -1 of Trx-Balcp19k, TA, and Trx-Balcp19k-TA. This indicates that the toxicity of the hydrogel of the Trx-Balcp19k-TA system to NIH / 3T3 cells can be completely ignored.

Claims

1. A biomedical adhesive system based on barnacle Trx-Balcp19k protein, characterized in that, It includes a first Trx-Balcp19k protein layer, a tannic acid layer, and a second Trx-Balcp19k protein layer, and the tannic acid layer is sandwiched between the first Trx-Balcp19k protein layer and the second Trx-Balcp19k protein layer.

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