Tissue adhesive based on mussel mucin as well as preparation method and application of tissue adhesive

Abstract

The invention discloses a tissue adhesive based on mussel mucin as well as a preparation method and application thereof. The tissue adhesive is prepared from mussel mucin and an anionic compound containing a hydrophobic group, the mussel worm electrostatic self-assembly bionic composite material is developed on the basis of triple bionic principles of dopa adhesion of mussel byssus, electrostatic self-assembly polymerization of sandburg worms and hydrophobic acting force of barnacles. The preparation method of the tissue adhesive is simple, the tissue adhesive can be obtained only by mixing and oscillating the two stock solutions, the gel forming condition is simple and easy to implement, the pH range and the stock solution concentration range required by gel forming are large, the gel forming range is wide, the raw materials required in the preparation process are few, and the cost is low. Only two key raw materials including the mussel mucin and the anionic compound containing the hydrophobic group are needed, and the prepared tissue adhesive is high in adhesion speed and high in adhesion strength, has good cell compatibility and biological safety and can promote wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of medical tissue adhesives, and in particular relates to a tissue adhesive based on mussel mucin and a preparation method and application thereof. Background Art

[0002] Tissue adhesives usually refer to certain polymers or proteins that usually use related groups to achieve covalent cross-linking with tissue functional groups under physiological conditions, mainly including cyanoacrylates, gelatins, polysaccharides, etc. In addition to covalent interactions, non-covalent interactions such as hydrogen bonds and hydrophobic interactions can also be used to produce tissue adhesion, which can work together with covalent interactions to achieve strong adhesion. Through covalent interactions and non-covalent interactions, the material and tissue form adhesion under physiological conditions, replacing surgical sutures for suturing on the body surface or inside the body. However, traditional tissue adhesives such as cyanoacrylates and fibrin glues have defects such as high toxicity of metabolites or poor mechanical strength and bonding strength.

[0003] Among marine organisms, three types of organisms, mussels, sandcastle worms and barnacles, can form extremely strong adhesion effects on the corresponding substrate surfaces. Existing studies have shown that the adhesion of mussels mainly depends on the dopa structure in the mussel mucin on its foot thread, which forms an adhesion effect on the substrate surface; the glue of sandcastle worms is composed of co-peptides with opposite charges, which can replicate the key chemical functions of natural glue, including electrostatic compounding and adhesion; barnacles rely on the lipids they secrete to form a strong hydrophobic layer on the substrate surface to form strong adhesion.

[0004] Based on the charged properties of polyelectrolytes, they have rich electrostatic interactions in aqueous solution, which makes polyelectrolyte aqueous solution show more unique phase behavior compared with neutral polymers. By regulating the strength of the interaction between polyelectrolyte chains, polyelectrolyte aqueous solution can undergo liquid-liquid phase separation. Polyelectrolytes with opposite charges aggregate due to the mutual attraction between polymer chains and cause liquid-liquid phase separation. Polyelectrolytes with opposite charges are enriched in the liquid phase with high polymer concentration (complex coacervate) and produce a liquid phase with low polymer concentration (supernatant).

[0005] Mussel mucin is a protein extracted from the byssus of the marine organism mussel. It has good biocompatibility. The protein molecular chain of mussel mucin carries a large number of dopa groups and positively charged lysine groups. It has multiple functions such as anti-inflammatory, antioxidant, pigmentation prevention, and wound healing. At the same time, due to the protein structure formed by the regular arrangement of its lysine groups and dopa groups, mussel mucin has extremely strong biological adhesion. In addition, the bonding strength of mussel mucin is not only related to its primary structure, but also depends on the assembly of higher-level structures, which is jointly determined by cohesion and adhesion. However, there is a basic blank in the research on the assembly of higher-level structures based on recombinant mussel mucin, which hinders the development and application of protein adhesives and medical coating materials. At the same time, the preparation method of existing tissue adhesives based on mussel mucin is complicated, requires a variety of raw materials, and has weak adhesion performance.

[0006] Based on the liquid-liquid phase separation principle of natural mussel mucin and the triple bionic inspiration of marine mussels, sandcastle worms and barnacle worms, a new triple bionic mussel mucin tissue adhesive was developed with mussel mucin as the main body, utilizing the electrostatic self-assembly of its lysine groups with anionic compounds containing hydrophobic groups and negative charges, as well as hydrophobic forces. This is of great significance for the multi-scale bionic construction of protein-based tissue adhesives. Summary of the invention

[0007] The technical problem to be solved by the present invention is that the preparation method of the tissue adhesive based on mussel mucin prepared in the prior art is complicated, the raw materials required are numerous, and the adhesion performance is not strong. A tissue adhesive based on mussel mucin and a preparation method thereof are provided. An anionic compound containing a hydrophobic group, such as an anionic surfactant, can electrostatically interact with lysine on the mussel mucin to form a liquid-liquid phase separation product, thereby obtaining a tissue adhesive with a higher molecular weight, extremely strong adhesion and complete dopa-active groups. Moreover, the preparation method is simple and requires less raw materials.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] A tissue adhesive based on mussel mucin, characterized in that the tissue adhesive consists of mussel mucin and an anionic compound containing a hydrophobic group.

[0010] Among them, the mussel mucin is any one or a combination of mussel byssin and recombinant mussel byssin; the mussel byssin is any one or a combination of mussel byssin MFP1, mussel byssin MFP2, mussel byssin MFP3, mussel byssin MFP4, mussel byssin MFP5 and mussel byssin MFP6; the amino acid sequences of mussel byssin MFP1, mussel byssin MFP2, mussel byssin MFP3, mussel byssin MFP4, mussel byssin MFP5 and mussel byssin MFP6 are shown as SEQ ID No.3 to 8 respectively.

[0011] Among them, the recombinant mussel byssus protein is obtained by heterologously expressing the coding genes of the mussel byssus protein after fusion by genetic engineering means; the mussel byssus protein is a combination of any two or more of mussel byssus protein MFP1, mussel byssus protein MFP2, mussel byssus protein MFP3, mussel byssus protein MFP4, mussel byssus protein MFP5 and mussel byssus protein MFP6; preferably, the recombinant mussel byssus protein is the recombinant mussel mucin MFP151 obtained by heterologously expressing the coding genes of the mussel byssus protein MFP1 and mussel byssus protein MFP5 after fusion by genetic engineering means.

[0012] Specifically, the construction method of the recombinant mussel mucin MFP151 is as follows: the mussel byssus protein MFP1 encoding gene and the mussel byssus protein MFP5 encoding gene are fused to obtain a fusion gene, and then the fusion gene is codon-optimized according to the codon preference of Escherichia coli (to improve the correct expression rate of the recombinant mussel mucin MFP151 protein) to obtain the recombinant mussel mucin MFP151 encoding gene, and then a recombinant plasmid carrying the recombinant mussel mucin MFP151 encoding gene is constructed, and the recombinant plasmid is transformed into E. coli BL21 (DE3) for heterologous expression, and finally the recombinant mussel mucin MFP151 is obtained by fermentation and purification. The detailed construction method of the recombinant mussel bioadhesive fp-151 has been disclosed in the article Hwang SD, Gim Y, Yoo JH, et al. Practical recombinant hybrid mussel bioadhesive fp-151 [J]. Biomaterials, 2007, 28 (24): 3560-3568.

[0013] Among them, the amino acid sequence of the recombinant mussel mucin MFP151 is shown as SEQ ID NO.1; the nucleotide sequence of the coding gene of the recombinant mussel mucin MFP151 is shown as SEQ ID NO.2.

[0014] Wherein, the anionic compound containing a hydrophobic group is any one of polyglutamic acid, hyaluronic acid, DNA and a surfactant; the surfactant is any one of alkyl sulfates, alkyl sulfonates, alkylbenzene sulfonates, lignin sulfonates and succinate sulfonates; preferably, the anionic compound containing a hydrophobic group is polyglutamic acid or alkylbenzene sulfonate; further preferably, the anionic compound containing a hydrophobic group is polyglutamic acid or sodium dodecylbenzene sulfonate.

[0015] The present invention also provides a method for preparing the tissue adhesive based on mussel mucin, which is characterized by comprising the following steps:

[0016] (1) dissolving the mussel mucin in a buffer solution to obtain a mussel mucin solution; dissolving the anionic compound containing a hydrophobic group in a buffer solution to obtain a solution of the anionic compound containing a hydrophobic group;

[0017] (2) mixing the mussel mucin solution obtained in step (1) with a solution of an anionic compound containing a hydrophobic group to obtain a mixed system, shaking, centrifuging, and collecting a precipitate to obtain the product.

[0018] In step (1), the buffer is any one of acetic acid-sodium acetate buffer, Tris-HCl buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, glycine-hydrochloric acid buffer or phthalic acid-hydrochloric acid buffer, preferably acetic acid-sodium acetate buffer; the pH of the buffer is 3-9, and the concentration is 0.1mM-0.1M; preferably, the pH of the buffer is 5, and the concentration is 0.1mM.

[0019] In step (1), the concentration of the mussel mucin solution is 1 to 100 mg / mL, preferably 100 mg / mL; the concentration of the solution of the anionic compound containing a hydrophobic group is 0.1 to 100 mg / mL.

[0020] In step (2), the mass ratio of mussel mucin to anionic compound containing hydrophobic group in the mixed system is 1:0.1-1, preferably 1:0.5.

[0021] In step (2), the oscillation time is 1 to 5 minutes; the purpose of the oscillation is to allow the mussel mucin to fully react with the anionic compound containing a hydrophobic group, so that a non-covalent interaction occurs and the two are combined with each other.

[0022] In step (2), the centrifugal speed is 8000-12000 rpm / min, and the time is 5-12 min. Preferably, the centrifugal speed is 12000 rpm / min, and the time is 10 min.

[0023] The mussel mucin and the anionic compound containing a hydrophobic group form a mussel mucin tissue adhesive through electrostatic interaction and other non-covalent interactions. The abundant lysine residues on the mussel mucin make the protein molecules positively charged under a pH environment lower than the isoelectric point of the protein, thereby generating electrostatic interaction with the anionic compound to obtain a mussel mucin tissue adhesive based on the liquid-liquid phase separation principle.

[0024] The present invention also claims to protect the use of the mussel mucin-based tissue adhesive in the preparation of products with the effect of promoting tissue healing; the products are medicines, medical devices or cosmetics.

[0025] Wherein, the fresh mussel mucin-based tissue adhesive is applied to the tissue surface and then cured for 90s to 300s.

[0026] The mussel mucin-based tissue adhesive of the present invention has strong adhesion strength both in dry environment and wet environment.

[0027] Beneficial effects:

[0028] 1. The mussel mucin used in the present invention exposes more lysine residues on its protein peptide chain, so that it can be positively charged in an acidic environment below the isoelectric point, and better interact electrostatically with anionic compounds containing hydrophobic groups; at the same time, by compounding the mussel mucin and anionic compounds containing hydrophobic groups in proportion, a strong electrostatic interaction occurs between the two, so that the tissue adhesive finally prepared has a stronger adhesion strength.

[0029] 2. By compounding mussel mucin with anionic compounds containing hydrophobic groups in proportion and subjecting them to strong electrostatic action, a liquid-liquid phase-separated tissue adhesive is obtained, which greatly protects the antioxidant, anti-inflammatory and other activities of the mussel mucin itself. The anti-inflammatory, antioxidant and adhesion effects of mussel mucin, synergistically with the hydrophobic force of anionic compounds containing hydrophobic groups, make the tissue adhesive have both strong adhesion and antioxidant, anti-inflammatory and other properties.

[0030] 3. The components of the present invention, namely, mussel mucin and anionic compounds containing hydrophobic groups, are safe and non-toxic, and the cross-linking mode between the components is non-covalent cross-linking, which further ensures the safety of the tissue adhesive finally prepared.

[0031] 4. The rheological properties of the tissue adhesive prepared by the present invention are different from those of ordinary hydrogels, and it has better fluidity. Therefore, the tissue adhesive of the present invention has excellent injectability and spreadability, and has good anti-inflammatory, antibacterial and antioxidant properties. It has the effect of inhibiting skin inflammation, and can also promote cell growth and proliferation, can significantly promote wound healing, and can also be used for daily skin care.

[0032] 5. The preparation method of the tissue adhesive of the present invention is simple. The tissue adhesive can be obtained by mixing and shaking two stock solutions. The gelling conditions are simple and easy. The pH range and stock solution concentration range required for gelling are large, the gelling range is wide, and the preparation process requires fewer raw materials. Only two key raw materials, mussel mucin and anionic compounds containing hydrophobic groups, are needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0034] Figure 1 The actual picture and scanning electron microscope picture of the recombinant mussel mucin MFP151-polyglutamic acid tissue adhesive prepared in Example 2; wherein, Figure A and Figure B are actual pictures of MFP151-polyglutamic acid tissue adhesive, and Figure C is a scanning electron microscope picture of MFP151-polyglutamic acid tissue adhesive, wherein the left picture is a scanning electron microscope picture of the surface of MFP151-polyglutamic acid tissue adhesive, and the right picture is a scanning electron microscope picture of the inside of MFP151-polyglutamic acid tissue adhesive.

[0035] Figure 2 It is a schematic diagram of the composite coagulation phenomenon of the recombinant mussel mucin and the anionic compound in the solution in Example 3 and a schematic diagram of the morphological characteristics of the coagulation system formed by the composite of the two under an optical microscope; wherein, Figure A is an initial state of the MFP151 solution and the SDBS solution and a diagram of the liquid-liquid phase separation phenomenon that occurs after the composite coagulation of the two; Figure B is an optical microscope image of the SDBS solution and the MFP151-SDBS coagulation, wherein the left figure is an optical microscope image of the SDBS solution, and the right figure is an optical microscope image of the MFP151-SDBS coagulation; Figure C is an optical microscope image of the polyglutamic acid solution and the MFP151-polyglutamic acid coagulation, wherein the left figure is an optical microscope image of the polyglutamic acid solution, and the right figure is an optical microscope image of the MFP151-polyglutamic acid coagulation.

[0036] Figure 3It is a physical picture of the turbid coagulation system formed by the reaction of the recombinant mussel mucin MFP151 and the anionic compound under different mass ratios in Example 4 and its OD value statistical chart; wherein, Figure A is a statistical chart of the OD values ​​of the coacervates formed by the reaction of the recombinant mussel mucin MFP151 with polyglutamic acid and SDBS under different mass ratios, and Figure B is a physical picture of the coagulation system formed by the reaction of the recombinant mussel mucin MFP151 with polyglutamic acid under different mass ratios.

[0037] Figure 4 The relationship curves between strain and modulus G' and G" obtained from the strain scanning test of the recombinant mussel mucin MFP151-SDBS tissue adhesives Ⅰ, Ⅱ and Ⅲ in Example 5.

[0038] Figure 5 It is a schematic diagram of the lap-shear tensile experimental method of the recombinant mussel mucin-SDBS tissue adhesive in Example 6 and a statistical diagram of its adhesion strength on different substrate surfaces; wherein, Figure A is a schematic diagram of the lap-shear tensile experimental method of the recombinant mussel mucin-SDBS tissue adhesive, and Figure B is a statistical diagram of the adhesion strength of the recombinant mussel mucin-SDBS tissue adhesive on different substrate surfaces.

[0039] Figure 6 This is a diagram showing the effect of investigating the biocompatibility of the recombinant mussel mucin-polyglutamic acid aggregate tissue adhesive on human skin in Example 7.

[0040] Figure 7 This is a real picture of the plugging effect of the recombinant mussel mucin-SDBS tissue adhesive under fluid pressure in Example 8; wherein, Figure A is the spray fluid state of physiological saline when the recombinant mussel mucin-SDBS tissue adhesive is not applied, and Figure B is the pressure resistance effect after the recombinant mussel mucin-SDBS tissue adhesive is applied. DETAILED DESCRIPTION

[0041] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0042] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0043] The polyglutamic acid used in the following examples was purchased from Nanjing Xuankai Biotechnology Co., Ltd., and its purity was cosmetic grade (≥95%); the sodium dodecylbenzene sulfonate (SDBS) used was purchased from MacLean's reagent, and its purity was AR analytical grade (≥99%).

[0044] The vortex oscillator used in the following examples was purchased from SCILOGEX Instruments, model SCI-VS.

[0045] The composition of the TSB liquid culture medium used in the following examples is: 17.0 g / L trypticase peptone, 3.0 g / L soybean papain hydrolysate, 5.0 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, 2.5 g / L glucose, pH 7.3±0.2.

[0046] The buffer used in the following examples is acetic acid-sodium acetate buffer with a pH of 5 and a concentration of 0.1 mM.

[0047] Example 1 Preparation of recombinant mussel mucin MFP151

[0048] Based on the codon usage preference of Escherichia coli, a codon-optimized MFP151 gene fragment was obtained by total synthesis (the amino acid sequence of the optimized recombinant mussel mucin MFP151 is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2), which was then cloned into the pET28a(+) (purchased from Thermo Fisher Scientific) expression vector with a strong promoter to obtain a recombinant plasmid. After transformation, screening, and sequencing identification, the correctly sequenced recombinant plasmid was introduced into the expression host E. coli BL21 (DE3) (purchased from Thermo Fisher Scientific) for heterologous expression, and finally a genetically engineered bacterium E. coli (pET28a-fp151) that can efficiently heterologously express MFP151 was successfully constructed. E. coli (pET28a-fp151) was fermented and purified to obtain the MFP151 recombinant mussel mucin. The specific fermentation and purification process is as follows:

[0049] A single colony of E. coli (pET28a-fp151) was picked from the freshly transformed plate, transferred to 200 mL LB liquid culture medium containing ampicillin (100 mg / mL), and cultured in a shaking incubator at 37°C and 200 rpm as a seed solution; the seed solution was transferred to 3.5 L TSB liquid culture medium containing ampicillin (100 mg / mL) for fermentation under the following conditions: temperature: 37°C; rotation speed: 400 rpm; ventilation volume: 1.0 vvm; pressure: 0.04-0.05 MPa. When the OD value of the bacteria in the TSB liquid culture medium reached 1.5, IPTG (isopropyl-β-D-thiogalactoside) was added to the TSB liquid culture medium at a final concentration of 1 mM for induction.

[0050] After the fermentation is completed, the cells are collected by centrifugation at 12000r / min and 4°C for 20min, and the collected cells are washed with phosphate buffered saline (PBS) and resuspended in PBS solution, and ultrasonically broken in an ice water bath, with a power of 400W, ultrasonic 3s, and an interval of 5s, and the total ultrasonic time is 30min to 60min. After the crushing is completed, the inclusion body precipitate is collected and resuspended in a 25% v / v acetic acid aqueous solution at 4°C and 12000r / min. The MFP151 protein is extracted by shaking at 4°C overnight, and the extract is centrifuged at 4°C and 12000r / min for 20min to collect the supernatant. The supernatant is dialyzed in a dialysis bag with a molecular weight cutoff of 3500Da in a 5% v / v acetic acid aqueous solution and a 1mM ascorbic acid aqueous solution at 4°C overnight, and then the purified MFP151 recombinant mussel mucin is obtained by freeze drying.

[0051] Example 2 Preparation of recombinant mussel mucin MFP151-polyglutamic acid tissue adhesive and recombinant mussel mucin MFP151-sodium dodecylbenzene sulfonate tissue adhesive

[0052] (1) Preparation of recombinant mussel mucin MFP151-polyglutamic acid tissue adhesive

[0053] Use buffer to prepare 100 mg / mL recombinant mussel mucin MFP151 solution and 100 mg / mL polyglutamic acid solution respectively, mix 500 μL of recombinant mussel mucin MFP151 solution and 500 μL of polyglutamic acid solution, place the mixed system on a vortex oscillator for high-speed oscillation for 1 min, ensure sufficient reaction, centrifuge at 12000 rpm for 10 min in a high-speed centrifuge, remove the supernatant, collect the precipitate, and obtain the recombinant mussel mucin MFP151-polyglutamic acid tissue adhesive.

[0054] The prepared MFP151-polyglutamic acid tissue adhesive was freeze-dried and then observed under a scanning electron microscope.

[0055] Figure 1 The following is a physical picture and a scanning electron microscope picture of the recombinant mussel mucin MFP151-polyglutamic acid tissue adhesive prepared in this example. Figure 1 As can be seen from Figures A and B, the recombinant mussel mucin MFP151-polyglutamic acid tissue adhesive is light yellow and has a certain viscosity; Figure 1 The SEM in Figure C clearly shows that the surface of the recombinant mussel mucin MFP151-polyglutamic acid coacervate is a dense membrane structure (left picture), which is very different from the porous structure of the hydrogel. After removing the outer membrane, the inner side shows tiny pores and wrinkles (right picture), indicating that the recombinant mussel mucin-polyglutamic acid adhesive of the present invention is closer to a glue adhesive.

[0056] (2) Preparation of recombinant mussel mucin MFP151-sodium dodecylbenzene sulfonate tissue adhesive

[0057] 100 mg / mL recombinant mussel mucin MFP151 solution, 30 mg / mL, 50 mg / mL and 70 mg / mL SDBS solution were prepared with buffer. 500 μL recombinant mussel mucin MFP151 solution was mixed with 500 μL 30 mg / mL, 500 μL 50 mg / mL and 500 μL 70 mg / mL SDBS solution to obtain mixed systems I, II and III, respectively, and then they were placed on a vortex oscillator for high-speed oscillation for 1 min to ensure sufficient reaction, centrifuged at 12000 rpm for 10 min, the supernatant was removed, and the precipitate was collected to obtain recombinant mussel mucin MFP151-SDBS tissue adhesives I, II and III.

[0058] Example 3 Observation of the coagulation system formed by the combination of recombinant mussel mucin MFP151 and anionic compounds containing hydrophobic groups

[0059] In order to better observe the coacervates formed by the complex of MFP151 and anionic compounds containing hydrophobic groups, a low-concentration MFP151 solution was prepared with a buffer solution, which was mixed with a low-concentration polyglutamic acid solution and a SDBS solution, respectively. After sufficient reaction, a coacervate system was formed, and the coacervates in the coacervate system were observed with an optical microscope. The specific experimental steps are as follows:

[0060] The recombinant mussel mucin MFP151 obtained in step (1) was prepared into a 1 mg / mL recombinant mussel mucin MFP151 solution with a buffer. A 1 mg / mL polyglutamic acid solution and a 1 mg / mL SDBS solution were prepared with a buffer. 2 mL of the above recombinant mussel mucin MFP151 solution was mixed with 2 mL of the polyglutamic acid solution and 2 mL of the SDBS solution, respectively, and placed on a vortex oscillator for high-speed oscillation for 1 to 5 minutes to fully react. It was found that the two mixed systems were turbid, as shown in FIG. Figure 2 As shown in Figure A, this is because the anionic compound and MFP151 have an electrostatic interaction, the two are combined to form a coacervate, and both carry hydrophobic groups, so liquid-liquid phase separation occurs. A small amount of the coacervate system obtained after sufficient reaction is taken out and observed under an optical microscope. The results are as follows Figure 2As shown in Figures B and C, the left picture in Figure B shows the phenomenon observed under an optical microscope of SDBS solution, which is only a single water phase and no coacervate appears. The right picture shows the morphological characteristics of MFP151-SDBS coacervate under an optical microscope. MFP151-SDBS coacervate appears due to liquid-liquid phase separation. The left picture in Figure C shows the phenomenon observed under an optical microscope of polyglutamic acid solution, which is only a single water phase and no coacervate appears. The right picture shows the morphological characteristics of MFP151-polyglutamic acid coacervate under an optical microscope. MFP151-polyglutamic acid coacervate appears due to liquid-liquid phase separation. Affected by the surface tension of the liquid and the micelles of anionic compounds, MFP151-SDBS coacervate and MFP151-polyglutamic acid coacervate are spherical, with a size between 0.1 and 5 μm, and the coacervate is not soluble in water and has a strong hydrophobic effect.

[0061] Example 4 Optimization of the mass ratio of mussel mucin to anionic compound in tissue adhesive

[0062] The amount of coacervate formed by the combination of mussel mucin and anionic compounds determines the yield of the final tissue adhesive. The yield of the coacervate is characterized by the OD value of the coacervate in the coacervate system. The mass ratio of recombinant mussel mucin MFP151 and anionic compounds used in the preparation of tissue adhesives is optimized by measuring the OD value of coacervate formed by the combination of recombinant mussel mucin MFP151 and anionic compounds at different mass ratios. The specific experimental steps are as follows:

[0063] 1 mg / mL MFP151 solution and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg / mL polyglutamic acid solutions were prepared with buffer. 2 mL of MFP151 solution was mixed with 2 mL of polyglutamic acid solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg / mL, respectively, and the mixture was sealed and placed on a vortex oscillator for 3 minutes to allow for sufficient reaction, thereby obtaining MFP151-polyglutamic acid coagulation systems 1 to 10, wherein the mass ratios of recombinant mussel mucin MFP151 to polyglutamic acid in each coagulation system were 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 and 1:1, respectively. The OD value of each mixed system was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer to characterize the amount of MFP151-polyglutamic acid aggregates formed.

[0064] At the same time, MFP151-SDBS coagulation systems 1 to 10 were prepared, and the OD value of each coagulation system was measured. The preparation steps and OD value determination method of the MFP151-SDBS coagulation system were the same as those of the MFP151-polyglutamic acid coagulation system.

[0065] Figure 3 The figures are actual pictures of the turbid coagulation system formed by the reaction of the recombinant mussel mucin MFP151 with anionic compounds under different mass ratios in this embodiment and its OD value statistical chart; wherein, Figure A is a statistical chart of the OD values ​​of the coacervates formed by the reaction of the recombinant mussel mucin MFP151 with polyglutamic acid and SDBS under different mass ratios, and Figure B is a actual picture of the coagulation system formed by the reaction of the recombinant mussel mucin MFP151 with polyglutamic acid under different mass ratios.

[0066] from Figure 3 The results show that for MFP151-SDBS coacervate, when the mass ratio of MFP151 to SDBS is 1:0.4-0.6, the OD value of the coacervate formed by the two is higher, indicating that at this ratio, the amount of MFP151-SDBS coacervate formed is more; for MFP151-polyglutamic acid coacervate, when the mass ratio of MFP151 to polyglutamic acid is 1:0.5-1, the OD value of the coacervate system formed by the two is higher, indicating that at this ratio, the amount of MFP151-polyglutamic acid coacervate formed is more. The maximum benefit can be obtained by producing and preparing tissue adhesives according to the above-mentioned mass ratio of mussel mucin to anionic compounds.

[0067] Example 5 Characterization of rheological properties of tissue adhesives formed by the reaction of recombinant mussel mucin and sodium dodecylbenzene sulfonate at different mass ratios

[0068] The TA HR10 rotational rheometer (purchased from TA Instruments, USA) was used to perform strain sweep tests on the recombinant mussel adhesive protein MFP151-SDBS tissue adhesives I, II, and III prepared in Example 2. In this example, a stainless steel disc with a diameter of 40 mm was selected to perform strain sweep tests on the recombinant mussel adhesive protein MFP151-SDBS tissue adhesives I, II, and III. The test frequency was 1 Hz, the test gap was set to 1 mm, and the test temperature was 25 °C. The storage modulus G' and loss modulus G" of the hydrodynamic data of the recombinant mussel adhesive protein MFP151-SDBS tissue adhesives I, II, and III (the mass ratios of MFP151 and SDBS were 1:0.3, 1:0.5, and 1:0.7, respectively) were determined through the strain sweep test. G' represents the elastic part, that is, the part stored in the deformation ability; G" represents the viscous part, that is, the part lost in the deformation ability. When G" < G', the elastic part is the main part, which is a gel; when G" > G', the viscous part is the main part, which is a sol; when G" = G', the viscosity and elasticity are equal, which is the sol-gel transition point. The linear viscoelastic region ranges of the MFP151-SDBS tissue adhesives I, II, and III at 25 °C can be obtained respectively through the strain sweep.

[0069] The results of the strain sweep test are as Figure 4 shown. It can be seen from the figure that: firstly, regardless of the mass ratio of MFP151 and SDBS, the loss modulus G" of the recombinant mussel adhesive protein MFP151-SDBS tissue adhesives I, II, and III is much greater than the storage modulus G'. The MFP151-SDBS tissue adhesives mainly undergo viscous deformation rather than elastic deformation, indicating that this adhesive tends to be a liquid-like sol rather than a solid-like hydrogel, making its fluidity better than that of ordinary hydrogels and enabling it to have good injectability and spreadability; secondly, the loss modulus and storage modulus of the MFP151-SDBS tissue adhesive II (i.e., the SDBS concentration is 50 mg / mL, and the mass ratio of MFP151 and SDBS is 1:0.5) are both greater than those of the MFP151-SDBS tissue adhesives I and III, and its curve is relatively flat, indicating that when the mass ratio of MFP151 and SDBS is 1:0.5, the tissue adhesive obtained by the aggregation of the recombinant mussel adhesive protein and SDBS has higher cohesion, can better cope with viscous deformation, and maintain its own stability.

[0070] Example 6 Adhesion of the recombinant mussel adhesive protein MFP151-SDBS tissue adhesive to different substrates

[0071] To determine the adhesion of the recombinant mussel adhesive protein MFP151-SDBS tissue adhesive, a lap-shear tensile experiment was carried out on it. The specific experimental method is as follows:

[0072] Take an appropriate amount of the recombinant mussel mucin MFP151-SDBS tissue adhesive II prepared in Example 2 and evenly apply it to the designated areas on two substrates of the same material. After applying, quickly bond the areas on the two substrates coated with the tissue adhesive together and fix them with a clip. Dry group: Place it flat in a fume hood for 12 hours to dry, and use a tensile tester to test the adhesion performance of the tissue adhesive on the surfaces of different substrates; wet group: After the tissue adhesive is dry, remove the clip, soak the two substrates bonded together in water for 7 days, and then use a tensile tester to test the adhesion performance of the adhesive on the surfaces of different substrates. The substrates selected in this experiment are stainless steel, aluminum, iron, ceramic and PC plastic plates, and the adhesion area is 10mm×10mm.

[0073] Figure 5 The schematic diagram of the lap-shear tensile test method of the recombinant mussel mucin-SDBS tissue adhesive of this embodiment and the statistical diagram of the adhesion strength on different substrate surfaces. The results show that the recombinant mussel mucin-SDBS tissue adhesive has a strong adhesion strength on the surfaces of different substrates, with an adhesion strength of 0.3MPa to 3MPa in a dry environment and an adhesion strength of 0.1MPa to 1MPa in a wet environment. Regardless of whether it is in a dry environment or a wet environment, the adhesion strength of the recombinant mussel mucin-SDBS tissue adhesive on the surface of the iron substrate is the highest, which is greatly related to the rich dopa group in the recombinant mussel mucin. The catechol structure of the dopa group can chelate with iron, greatly enhancing the cohesion of the recombinant mussel mucin-SDBS condensate molecules, thereby improving the adhesion strength of the recombinant mussel mucin-SDBS tissue adhesive on the surface of the iron substrate.

[0074] Example 7 Investigation of human skin biocompatibility of recombinant mussel mucin-polyglutamic acid tissue adhesive

[0075] Use buffer to prepare 100 mg / mL recombinant mussel mucin MFP151 solution and 50 mg / mL polyglutamic acid solution respectively, mix 500 μL of the above recombinant mussel mucin MFP151 solution with 500 μL of the above polyglutamic acid solution, place it on a vortex shaker for high-speed oscillation for 1 min to ensure sufficient reaction, centrifuge at 12000 rpm for 10 min, remove the upper clear liquid, smear the lower precipitate (tissue adhesive) on the resin film, cut it into a double eyelid patch shape, recruit 3 single eyelid volunteers, stick the patch on the upper eyelid for an eyelid patch experiment, and record the adhesion of the double eyelid patch after 6 hours and 12 hours, and ask and record the volunteers' usage experience.

[0076] Figure 6The figure is a result of the human skin biocompatibility investigation of the recombinant mussel mucin-polyglutamic acid tissue adhesive of this embodiment. As can be seen from the figure, after 12 hours of eye skin adhesion experiment, none of the three volunteers had adverse reactions such as allergy, discomfort and double eyelid sticker shedding. Therefore, the recombinant mussel mucin-polyglutamic acid tissue adhesive of the present invention will not cause allergies and cytotoxicity on the surface of human skin, and its own biocompatibility is guaranteed.

[0077] Example 8 Investigation of the compressive effect of recombinant mussel mucin-sodium dodecylbenzene sulfonate tissue adhesive

[0078] Take an empty 1.5L Wahaha pure water bottle, poke a hole with a diameter of 3mm at a height of about 5cm from the bottom of the bottle with an awl, fill the bottle with saline, and observe and record the fluid state of the saline spraying out of the hole. Pour out the water in the bottle, apply the recombinant mussel mucin MFP151-SDBS tissue adhesive II prepared in Example 2 to cover the above hole, press for 5min, and after the tissue adhesive is firmly adhered and dried to set, fill the bottle with saline again, and observe the fluid state of the saline.

[0079] Figure 7 This is a physical picture of the plugging effect of the recombinant mussel mucin-SDBS tissue adhesive of this embodiment under fluid pressure. The experimental results show that the hole is blocked by the recombinant mussel mucin-SDBS tissue adhesive, and the physiological saline is no longer sprayed out, indicating that the recombinant mussel mucin-SDBS tissue adhesive of the present invention has a certain ability to resist liquid pressure.

[0080] The present invention provides a tissue adhesive based on mussel mucin and a method for preparing the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A tissue adhesive based on mussel mucin, characterized in that: The tissue adhesive consists of mussel mucin and an anionic compound containing a hydrophobic group.

2. The tissue adhesive based on mussel mucin according to claim 1, characterized in that The mussel mucin is any one or a combination of mussel byssin and recombinant mussel byssin; the mussel byssin is any one or a combination of mussel byssin MFP1, mussel byssin MFP2, mussel byssin MFP3, mussel byssin MFP4, mussel byssin MFP5 and mussel byssin MFP6; the amino acid sequences of mussel byssin MFP1, mussel byssin MFP2, mussel byssin MFP3, mussel byssin MFP4, mussel byssin MFP5 and mussel byssin MFP6 are shown as SEQ ID No. 3 to 8 respectively.

3. The tissue adhesive based on mussel mucin according to claim 2, characterized in that: The recombinant mussel byssus protein is obtained by heterologously expressing the coding gene of the mussel byssus protein by fusing it through genetic engineering means; the mussel byssus protein is a combination of any two or more of mussel byssus protein MFP1, mussel byssus protein MFP2, mussel byssus protein MFP3, mussel byssus protein MFP4, mussel byssus protein MFP5 and mussel byssus protein MFP6.

4. The mussel mucin-based tissue adhesive according to claim 1, characterized in that: The anionic compound containing a hydrophobic group is any one of polyglutamic acid, hyaluronic acid, DNA and a surfactant; the surfactant is any one of alkyl sulfate, alkyl sulfonate, alkylbenzene sulfonate, lignin sulfonate and succinate sulfonate.

5. The method for preparing a tissue adhesive based on mussel mucin according to any one of claims 1 to 4, characterized in that: The steps include: (1) dissolving the mussel mucin in a buffer solution to obtain a mussel mucin solution; dissolving the anionic compound containing a hydrophobic group in a buffer solution to obtain a solution of the anionic compound containing a hydrophobic group; (2) mixing the mussel mucin solution obtained in step (1) with a solution of an anionic compound containing a hydrophobic group to obtain a mixed system, shaking, centrifuging, and collecting a precipitate to obtain the product.

6. The preparation method according to claim 5, characterized in that: In step (1), the buffer is any one of acetic acid-sodium acetate buffer, Tris-HCl buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, glycine-hydrochloric acid buffer or phthalic acid-hydrochloric acid buffer; the pH of the buffer is 3 to 9, and the concentration is 0.1 mM to 0.1 M.

7. The preparation method according to claim 5, characterized in that: In step (1), the concentration of the mussel mucin solution is 1 to 100 mg / mL; the concentration of the solution of the anionic compound containing a hydrophobic group is 0.1 to 100 mg / mL.

8. The preparation method according to claim 5, characterized in that: In step (2), the mass ratio of mussel mucin to anionic compound containing hydrophobic group in the mixed system is 1:0.1-1.

9. The preparation method according to claim 5, characterized in that: In step (2), the centrifugal speed is 8000-12000 rpm / min, and the time is 5-12 min.

10. Use of the mussel mucin-based tissue adhesive according to any one of claims 1 to 4 in the preparation of a product having the effect of promoting tissue healing; the product is a medicine, a medical device or a cosmetic.

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