Preparation and application of proteoglycan colloid material capable of rapidly stopping bleeding and promoting wound healing

Abstract

The invention discloses preparation and application of a proteoglycan colloid material capable of rapidly stopping bleeding and promoting wound healing, and belongs to the field of biological materials. Basic amino acid rich in lysine or arginine is designed and expressed through molecular engineering, and then basic protein and hyaluronic acid with negative electricity are combined through a supramolecular assembly method to prepare a proteoglycan material with antibacterial ability. Then, recombinant mucoprotein rich in tyrosine is designed and expressed, and a DOPA adhesion group is formed through tyrosinase modification, so that the instant adhesion of the tissue adhesion material is enhanced. The amino acid of the basic protein remarkably enhances the interaction of various molecules of proteoglycan, meanwhile, the basic amino acid endows the tissue adhesive with excellent antibacterial ability, and the tissue adhesive provided by the invention has instant tissue adhesion performance, excellent biocompatibility and excellent antibacterial ability and hemostatic performance, and is suitable for being used in the field of medical instruments. The healing of different types of bleeding scenes and irregular wounds is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of protein glycosaminoglycan colloidal material preparation and application of fast hemostasis and promote wound healing, belong to biomaterial field. BACKGROUND

[0002] After tissue injury, wound infection and continuous bleeding are the main causes of death, although suture and surgical stapling nail are currently used closure means in clinical treatment, but its operation is complicated and high technical requirements, and cause secondary tissue damage, tissue adhesive as the effective alternative way of simple operation arouses extensive interest, however, commercially available adhesives such as fibrin glue has weak adhesion to skin tissue (5-10 kPa), cyanoacrylate has high adhesive strength, has been approved for use in skin wound healing, however, cyanoacrylate is difficult to degrade, and will cause secondary damage to wound healing. In addition, these adhesives lack sufficient antibacterial ability and active ingredients to promote wound healing, therefore, it is urgent to develop an adhesive material that can achieve instant adhesion to natural wet tissue, has antibacterial properties to avoid wound infection and biocompatibility.

[0003] The rapid development of genetic engineering has produced multifunctional structural proteins for various purposes, especially inspired by the excellent adhesion ability of natural marine organisms such as mussels, barnacles, sand castle worms underwater, these organisms use a large number of highly diverse proteins to achieve strong underwater adhesion, therefore, recombinant adhesive proteins with good biocompatibility and biodegradability can be produced on a large scale by microbial fermentation, but how to use these recombinant adhesive proteins as biological adhesives is unknown. Therefore, the present application simulates the main proteoglycan components of extracellular matrix by genetic engineering means in order to obtain a biological material with enhanced instant adhesion performance. SUMMARY

[0004] In view of the above shortcomings of the prior art, the present application provides a kind of protein glycosaminoglycan colloidal material preparation and application of fast hemostasis and promote wound healing, the purpose is to lack the instant adhesion to wet tissue in the prior art, adhesive material with antibacterial properties to avoid wound infection and biocompatibility.

[0005] The present application provides a method for preparing a tissue adhesive, which utilizes hyaluronic acid and basic protein electrostatic self-assembly to form a protein glycosaminoglycan material with antibacterial properties, and adds a recombinant mucin modified by tyrosinase to endow the tissue adhesive material with excellent instant adhesion.

[0006] In one embodiment of the present application, in the electrostatic self-assembly method, the mass ratio of hyaluronic acid to basic protein is 1: (0.2-2); the tissue adhesive comprises hyaluronic acid, basic protein and mucin in a mass ratio of 1: (0.2-2): (0.1-2).

[0007] In one embodiment of the present application, the basic protein comprises one or more of protamine, elastin K32 and R36; the protamine has an arginine content of about 70%, and an amino acid sequence of PRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR (SEQ ID NO. 10); the amino acid sequence of K32 protein is [GVG(VPGKG)8VP]4; and the amino acid sequence of R36 protein is [GVG(VPGRG)9VP]4.

[0008] In one embodiment of the present application, the mucin is fused from two parts, namely the leucine zipper protein ASP with self-assembly ability and the mussel byssal protein MFP. The recombinant leucine protein comprises a leucine zipper domain (A), an unstructured polyelectrolyte domain (S), and a helix terminal domain (P). The amino acid sequence of the A domain is SGDLENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKAE (SEQ ID NO. 11), the amino acid sequence of the S domain is (AGAGPEG) n, and the amino acid sequence of the P domain is APQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDAS (SEQ ID NO. 12). 10 The mussel byssal protein is a tyrosine-rich protein, and comprises one or more of Mefp-3, Mgfp-5, and Mcofp-3.

[0009] The amino acid sequence of the Mefp-3 protein is ADYYGPNYGPPRRYGGGNYNRYNRYGRRYGGYKGWNNGWNRGRRGKYW (SEQ ID NO. 13), The amino acid sequence of the Mgfp-5 protein is SSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYY (SEQ ID NO. 14), The amino acid sequence of the Mcofp-3 protein is GYGYYPGYNALWPYNNGYYGCNGYNGYHGRYGWNKGWNSGPWGGSYYGNKGYLY (SEQ ID NO. 15). The position of the mussel byssal protein inserted into the leucine zipper domain comprises one or more of 3 / 5-A-S-P, A-3 / 5-S-P, A-S-3 / 5-P, and A-S-P-3 / 5.​​​​

[0010] In one embodiment of the present invention, the tyrosinases are VsTYR and BmTYR, respectively, and their sources are Polyrhachis microorganisms (VsTYR and BmTYR). Verrucomicrobium spinosum ) and Bacillus megaterium ( Bacillus megaterium Their GenBank numbers are MK550618.1 and ACC86108, respectively.

[0011] In one embodiment of the present invention, the specific steps are as follows: Hyaluronic acid, basic protein and mucin are mixed, an appropriate amount of deionized water is added, the supernatant is removed by centrifugation, and the tissue adhesive is obtained by freeze-drying.

[0012] In one embodiment of the present invention, the concentration of hyaluronic acid is 20-100 mg / mL, the concentration of basic protein is 50-100 mg / mL, and the concentration of mucin is 50-100 mg / mL.

[0013] In one embodiment of the present invention, the centrifugation speed is 10,000 rpm, the temperature is 4°C, and the time is 10-20 min.

[0014] In one embodiment of the present invention, the freeze-drying time is 5-10 min, the sample temperature is -20℃, the cold trap temperature is -60℃, and the vacuum degree is less than 10 Pa.

[0015] The second technical solution provided by the present invention is a tissue adhesive prepared using the first technical solution. The protein adhesive includes hyaluronic acid, basic protein and mucin, and the mass ratio of hyaluronic acid, basic protein and mucin is 1:(0.2~2):(0.1~2).

[0016] In one embodiment of the present invention, the basic protein that electrostatically self-assembles with hyaluronic acid includes one or more of protamine, elastin K32, or R36; the mussel byssal protein is one or more of Mefp-3, Mgfp-5, and Mcofp-3; the position of the mussel byssal protein inserted into the leucine zipper domain includes one or more of 3 / 5-ASP, A-3 / 5-SP, AS-3 / 5-P, and ASP-3 / 5; and the tyrosinase is one or more of VsTYR and BmTYR.

[0017] The second technical solution provided by the present invention describes the application of the tissue adhesive in the preparation of antibacterial materials, wound hemostasis, cell culture, tissue adhesion and / or wound healing products.

[0018] In one embodiment of the present invention, the antibacterial type includes Gram-positive bacteria and / or Gram-negative bacteria.

[0019] In one embodiment of the present application, the tissue adhesion includes one or more of skin tissue, internal organ tissue, muscle tissue, connective tissue, and mucosal tissue.

[0020] Compared with the prior art, the present application has the following advantages: The present application expresses basic amino acids rich in lysine or arginine through molecular engineering design, and then prepares a proteoglycan material with antibacterial ability by combining the basic protein with negatively charged hyaluronic acid through a supramolecular assembly method; subsequently, a recombinant mucin rich in tyrosine is designed to be expressed, and a DOPA adhesion group is formed by tyrosinase modification to enhance the immediate adhesion of the tissue adhesive material. The amino acids of the basic protein involved in the present application significantly enhance the various molecular interactions of the proteoglycan, and at the same time, the basic amino acids endow the tissue adhesive with excellent antibacterial ability. The tissue adhesive provided by the present application has immediate tissue adhesion performance, excellent biocompatibility, and outstanding antibacterial ability and hemostatic performance, which is conducive to dealing with different types of bleeding scenarios and irregular wound healing.

[0021] The present application provides a gel material with antibacterial ability by electrostatic assembly of hyaluronic acid and basic protein using a supramolecular assembly method. The method is simple to prepare, and a series of adhesive proteins are designed, and a DOPA group is formed by tyrosinase modification, which greatly improves the adhesion performance of the tissue adhesive. Through cell compatibility and blood compatibility tests, the results show that the material has excellent biocompatibility, and the basic protein has super strong antibacterial ability, avoids the invasion of external bacteria during wound repair, and has strong healing ability, greatly shortening the healing period of the wound. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is the protein expression of basic protein K32, recombinant mucin, and tyrosinase.

[0023] Figure 2 Figure 5 is the shear adhesion change of the tissue adhesive to different substrates.

[0024] Figure 3 Figure 8 is the blood compatibility of the tissue adhesive.

[0025] Figure 4 Figure 10 is the effect of the tissue adhesive on the growth of mouse fibroblasts L929.

[0026] Figure 5 Figure 12 is the antibacterial performance of the tissue adhesive on E. coli and S. aureus.

[0027] Figure 6 Figure 14 is the liver hemostatic effect of the tissue adhesive.

[0028] Figure 7 The tail-off hemostatic effect of the tissue adhesive.

[0029] Figure 8 The rat visceral adhesion effect of the tissue adhesive.

[0030] Figure 9 The healing of linear wounds by the tissue adhesive.

[0031] Figure 10 H&E staining and Masson staining after repair of linear wounds.

[0032] Figure 11 Immunofluorescence staining after repair of linear wounds. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better illustrate the present application, and are not used to limit the present application.

[0034] Test method: 1. H&E staining: Paraffin section deparaffinization to water: put the paraffin section into xylene I 15 min-xylene II 15 min-anhydrous ethanol I 10 min-anhydrous ethanol II 10 min-95% alcohol 10 min-85% alcohol 10 min in sequence for gradient deparaffinization.

[0035] HE staining: put the paraffin section into hematoxylin for 5-10 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for several seconds, rinse with tap water, then return to blue with saturated lithium carbonate aqueous solution for 1 min, rinse with running water for several seconds, and then put into eosin staining solution for several seconds and rinse with running water.

[0036] Dehydration and mounting: put the paraffin section into 75% ethanol 2 min-85% ethanol 2 min-anhydrous ethanol 2 min-anhydrous ethanol 2 min-xylene 2 min in sequence for transparency, and then take the section out of xylene and mount with neutral balsam.

[0037] Use upright fluorescence microscope for microscopic examination and collection and analysis.

[0038] 2. Masson staining Paraffin section deparaffinization to water: put the section into xylene I 15 min-xylene II 15 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water.

[0039] Bouin fixation: put the section into Bouin solution, and perform dyeing at room temperature overnight or in a 37°C incubator for 2 h, and then rinse with running water until the yellow color on the section disappears.

[0040] Nuclear staining: Weigert's iron hematoxylin (mix Weigert's iron hematoxylin solution A and B in equal proportion) staining for 5-10 min, and rinse with water slightly.

[0041] Differentiation: 1% hydrochloric acid alcohol differentiation for several seconds, and rinse with water for several minutes.

[0042] Stain: Eosin Y staining solution drop staining for 5-10 min, and rinse with water slightly.

[0043] Acid treatment: Phosphomolybdic acid solution treatment for about 5 min. Pour off the phosphomolybdic acid solution on the slide (without washing with water).

[0044] Aniline blue: re-stain with aniline blue staining solution for 3-5 min. Pour off the staining solution on the slide (without washing with water).

[0045] Decolorization: rinse the section with 1% glacial acetic acid aqueous solution (1% glacial acetic acid aqueous solution is prepared by mixing 99 ml of distilled water with 1 ml of glacial acetic acid) until the section is free of blue color (if necessary, control under the microscope) Dehydration and mounting: 95% ethanol, dehydrate with anhydrous ethanol, xylene transparent, and neutral resin fixation.

[0046] 3. Immunofluorescence staining Paraffin section deparaffinization to water: sequentially place the section in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water.

[0047] Antigen repair: place the tissue section in a microwave oven filled with citric acid antigen repair buffer (PH 6.0) or EDTA antigen repair buffer (PH 9.0) for antigen repair, with medium heat, medium-high heat, and high heat for 5 min each. During this process, prevent the buffer from evaporating too much, and do not dry the slide. After natural cooling, place the slide in PBS (PH 7.4) and shake, wash, and soak for 3 times, 5 min each time. (For more difficult repair, use a pressure cooker for antigen repair. Start timing when the gas begins to escape for 3 min.) The repair solution and repair conditions are determined according to the antibody.

[0048] Circle self-fluorescence quenching: after the section is slightly shaken dry, draw a circle around the tissue with a histological pen (to prevent the antibody from flowing away), and add a self-fluorescence quencher in the circle for 5 min, and rinse with water for 10 min.

[0049] Permeation: after the section is slightly shaken dry, add Triton X-100 permeation solution in the circle, and permeate for 30 min.

[0050] Blocking: add 1% BSA in the circle, and incubate for 30 min.

[0051] Add primary antibody: Shake off the blocking solution, add the same proportion of primary antibody on the slice, and incubate the slice in a wet box at 4°C overnight (add a small amount of water to the wet box to prevent the antibody from evaporating).

[0052] Add secondary antibody: Place the slide in PBS (pH 7.4) and shake on a shaker for 3 times, 5 min each time. After the slice is slightly shaken dry, add the same proportion of secondary antibody corresponding to the species of the primary antibody to cover the tissue in the circle, and incubate at room temperature for 50 min in the dark.

[0053] DAPI restain the nucleus: Place the slide in PBS (pH 7.4) and shake on a shaker for 3 times, 5 min each time. After the slice is slightly shaken dry, add DAPI staining solution in the circle, and incubate at room temperature for 10 min in the dark.

[0054] Mounting: Place the slide in PBS (pH 7.4) and shake on a shaker for 3 times, 5 min each time. After the slice is slightly shaken dry, mount it with an anti-fluorescence quenching mounting agent, and store it at 4°C in the dark after mounting.

[0055] Raw materials used in the examples: 1. Escherichia coli BL21 (DE3), pET28a (+), and pETduet-1 were preserved in the laboratory.

[0056] 2. Plasmid construction reagents and sequencing verification were purchased and completed by Shanghai Biomics Biotech Co., Ltd.

[0057] 3. Various analytical reagents were purchased from National Pharmaceutical Group, and hyaluronic acid was purchased from Huaxi Biological.

[0058] 4. Culture medium: LB medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder.

[0059] TB medium, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, 12 g / L tryptone, 24 g / L yeast powder, 4 mL / L glycerol.

[0060] Example 1: Heterologous expression and purification of basic protein and mucin Heterologous expression of basic protein: The genes of protamine PS, elastin K32, and R36 (nucleotide sequences are shown in SEQ ID NO. 1-3, respectively) were synthesized by Tianlin Biological Technology Co., Ltd. after codon optimization, and the sequences were connected with the vector pET-28a (+). The insertion site of the target fragment was located at the enzyme cutting site Bam HI and Hibetween Nde I and Xho I, to obtain recombinant plasmids pET28a-PS, pET28a-K32, pET28a-R36, respectively Escherichia coli BL21(DE3) to obtain recombinant strains expressing the target proteins E. coli BL21 / pET28a-PS, E. coli BL21 / pET28a-K32, E. coli BL21 / pET28a-R36.

[0061] Heterologous expression of mucin: The genes (nucleotide sequences are shown in SEQ ID NO. 4-7, respectively) of leucine zipper protein ASP, mussel byssal proteins Mefp-3, Mgfp-5, Mcofp-3 were synthesized after codon optimization by Tianlin Biotechnology Co., Ltd. The sequences were connected with the vector pET-28a(+), and the target fragment was inserted into the site between the enzyme cutting sites Bam HI and Hi Xho I, to obtain recombinant plasmids pET28a-ASP, pET28a-Mefp-3, pET28a-Mgfp-5, pET28a-Mcofp-3.

[0062] The target fragments were amplified from pET28a-Mefp-3 and pET28a-ASP using primers Mefp-3-F / Mefp-3-R, A-S-Mefp-3-P-F / A-S-Mefp-3-P-R, respectively, and Gibson assembly was performed to construct the recombinant vector A-S-Mefp-3-P.

[0063] The target fragments were amplified from pET28a-Mgfp-5 and pET28a-ASP using primers Mgfp-5-F / Mgfp-5-R, A-S-Mgfp-5-P-F / A-S-Mgfp-5-P-R, respectively, and Gibson assembly was performed to construct the recombinant vector A-S-Mgfp-5-P.

[0064] The target fragments were amplified from pET28a-Mcofp-3 and pET28a-ASP using primers Mcofp-3-F / Mcofp-3-R, A-S-Mcofp-3-P-F / A-S-Mcofp-3-P-R, respectively, and Gibson assembly was performed to construct the recombinant vector A-S-Mcofp-3-P.

[0065] The above recombinant plasmids were transformed Escherichia coli BL21(DE3) to obtain recombinant strains expressing the target proteins E. coliBL21 / AS-Mefp-3-P E. coli BL21 / AS-Mgfp-5-P, E. coli BL21 / AS-Mcofp-3-P.

[0066] Table 1 Primer sequences for constructing recombinant mucin

[0067] Shake-flask fermentation: The recombinant *E. coli* strain was streaked onto plates containing kanamycin (50 μg / L). Single colonies were picked and inoculated into LB medium. After incubation at 37°C for 8–12 h, the seed culture was transferred to 50 mL of TB medium at a volume fraction of 1 mL / 50 mL. The initial inoculum size was 2 × 10⁻⁶. 9 After incubating for 2 h, add 1 mM IPTG and induce culture at 30℃ for 12 h. Collect bacterial cells in 50 mL centrifuge tubes, centrifuge at 8000 rpm for 10 min at 4℃, discard the supernatant, resuspend the cells in 50 mL of pH 7.5, 20 mM Tris-HCl buffer, and sonicate for approximately 25 min. Centrifuge the lysate at 10000 rpm for 20 min at 4℃, and discard the precipitate.

[0068] The supernatant was filtered through a 0.45 µM filter membrane and set aside as the sample to be purified.

[0069] Ni-column affinity chromatography was performed using the AKTA protein purification system. First, the Ni-His Trap FF column was equilibrated with 50 mL of solution A (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Then, the sample to be purified was loaded. Finally, the protein was purified and eluted using solution B of the appropriate concentration (50 mM Tris-HCl, 150 mM NaCl, 500 mM Imidazole, pH 7.4).

[0070] The purified proteins protamine (PS), elastin (K32), and R36, as well as mucins AS-Mefp-3-P, AS-Mgfp-5-P, and AS-Mcofp-3-P were obtained. The SDS-PAGE results of the proteins are shown in [Figure number missing]. Figure 1 .

[0071] The eluted protein was dialyzed in ultrapure water for 2 days. After dialyzing, it was frozen at -20°C and then freeze-dried using a vacuum freeze dryer. It was then stored at -20°C for further use.

[0072] Example 2: Tyrosinase Modification of Recombinant Mucin The genes encoding tyrosinase, VsTYR, BmTYR (nucleotide sequences are shown in SEQ ID NO. 8~9, respectively) were ligated with plasmid pETduet-1, and tyrosinase expression plasmids pETduet-1-VsTYR, pETduet-1-BmTYR were constructed by GenScript Biotech (Shanghai) Co., Ltd. The tyrosinase expression was performed using a similar culture method as in Example 1, with the different operation procedures as follows: 1 mM IPTG was added, and the fermentation was induced at 37°C, 220 rpm for 6 h, and then the fermentation was ended. After the fermentation was ended, the bacterial solution was centrifuged at 4°C, 6000 rpm for 8 min, and the supernatant was discarded, and the bacterial cells were reserved. The bacterial cells were washed with 20 mM Tris-HCl (pH 7.4) for 1~2 times, and then the bacterial solution was adjusted to OD600 of 10.0, and then the bacterial solution was broken by high-pressure homogenization, and then the solution was centrifuged at 4°C, 10000 rpm for 20 min, and the supernatant was reserved, which was the crude enzyme solution. The tyrosinase SDS-PAGE results are shown in Figure 1 .

[0073] The broken wall supernatant of mucin A-S-Mefp-3-P, A-S-Mgfp-5-P, A-S-Mcofp-3-P was loaded onto a gravity column. The gravity column was washed with a washing solution (60 mM imidazole, 50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Subsequently, 5 mL tyrosinase solution (20 mM sodium borate, 100 mM PBS, 0.2 mM copper sulfate, pH 7.0) was added into the column for three times, and the reaction was oscillated at room temperature for 3 hours. Then, 10 mL buffer solution (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) was used for washing twice to remove the tyrosinase. Finally, the modified protein was eluted from the gravity column using an elution buffer (300 mM imidazole, 50 mM Tris-HCl, 150 mM NaCl, pH 7.4).

[0074] The eluted protein was dialyzed in an ultrapure water environment for 2 days, and then was frozen at -20°C, and then was freeze-dried using a vacuum freeze dryer, and was stored at -20°C for further use.

[0075] Example 3: Preparation of tissue adhesives and adhesion strength test experiment Hyaluronic acid, basic protein (one of PS, K32, R36, K32 in this example) were mixed at a ratio of 1:1 to prepare HP hydrogel. Hyaluronic acid, basic protein (one of PS, K32, R36), mucin (one of modified A-S-Mefp-3-P, modified A-S-Mgfp-5-P, modified A-S-Mcofp-3-P, modified A-S-Mcofp-3-P in this example) were mixed at a ratio of 1:1:1 to prepare HPA hydrogel.

[0076] The shear adhesive strength of HP hydrogel, HPA hydrogel to natural wet tissue (such as pig skin) was determined. The pig skin was prepared into a rectangle of 10 mm x 50 mm, and further immersed in 1x PBS buffer before use. After adding tissue adhesive to the surface of a piece of pig skin with an area of 10 mm x 50 mm, another piece of pig skin was pressed onto the glued position with an overlapping area of 10 mm x 10 mm, clamped using a dovetail clamp, and kept for 2 h. Finally, a universal testing machine was used for lap shear test, with a fixed crosshead speed of 60 mm / min -1 . The maximum force divided by the bonding area determined the shear adhesive strength, and all experiments were independently repeated 3 times.

[0077] The shear adhesive strength of HP hydrogel, HPA hydrogel to different substrates (stainless steel, aluminum sheet, PTFE, PVC) was determined. Different substrates were cut into rectangles of 10 mm x 60 mm, washed with deionized water, and then placed in a 50°C oven for drying, and further dried overnight at room temperature. The tissue adhesive was evenly coated on the first substrate, and then another substrate was pressed onto the glued position with an overlapping area of 10 mm x 10 mm, clamped using a dovetail clamp, and kept for 2 h. A universal testing machine was used for lap shear test, with a fixed crosshead speed of 60 mm / min -1 . The maximum force divided by the bonding area determined the shear adhesive strength, and all experiments were independently repeated 3 times. The adhesive strength of HP, HPA hydrogel to different media is shown in Figure 2 , where HPA obtained an adhesive strength of more than 60 kPa using PVC as the substrate.

[0078] Example 4: Antibacterial properties and biocompatibility of tissue adhesive 1. Blood compatibility Anti-coagulated blood was centrifuged at 1000 rpm for 10 min to obtain red blood cells. The cells were then washed repeatedly with sterile PBS buffer until the supernatant became clear, and then the supernatant was removed. Subsequently, the red blood cells were diluted with PBS buffer to a concentration of 5% (v / v) as a red blood cell suspension. 0.1 mL of HPA hydrogel (20%wt, 10%wt, 5%wt PBS) was added to 0.5 mL of whole blood diluent (5.0% PBS), and then 0.4 mL of PBS buffer was added. After incubation at 37°C for 2 hours, centrifugation was performed at 1000 rpm for 10 minutes. The supernatant was collected, and its absorbance at 540 nm was determined using a microplate reader. Deionized water and PBS buffer were used as positive and negative controls, respectively. The blood compatibility results of HPA hydrogel are shown in Figure 3 , and the hemolysis rate was calculated according to the following formula, where sample, control and positive represent the absorbance of the sample, negative control and positive control, respectively. The hemolysis rate results are shown in Figure 3 , and the hemolysis rate of HPA hydrogel at different concentrations was within the acceptable range of biomaterials (<5%).

[0079]

[0080] 2. Cell compatibility MTT method and FDA live / dead staining method were used to evaluate the cell compatibility of HPA hydrogel. HPA hydrogel extract preparation: 1 mL of HPA hydrogel was extracted with 10 mL of DMEM medium for 24 hours to obtain a 100% concentration extract, which was filtered with a 0.22 μm membrane. Subsequently, 50%, 25%, 12.5%, 6.25%, 3.12% and 1.56% extracts were obtained by dilution. At the same time, 1 mL of HPA hydrogel was extracted with 5 mL of DMEM medium for 24 hours to obtain a 200% concentration extract.

[0081] L929 cells were cultured to the logarithmic growth phase, and after trypsinization of the cells, they were inoculated in a 96-well plate at a density of 2x10 5 cells / mL, 100 μL per well (5 replicates for each treatment concentration), and incubated at 37°C for 24 h. The culture medium was replaced with normal culture medium or mixed culture medium (culture medium with hydrogel extract added), and incubated at 37°C for 24, 48 and 72 h. The culture medium was discarded, 100 μL of MTT solution (5 mg / mL) was added, and the plate was incubated in a 37°C, 5% CO2 incubator for 4 h. After removing the supernatant, 100 μL of DMSO was added to each well, and the plate was shaken on a shaker until the purple crystals were completely dissolved in DMSO. The absorbance of each group at 570 nm was measured using a microplate reader, and the relative survival rate of the cells was calculated. The cell compatibility results are shown inFigure 4 The extract had no significant effect on cell viability after 24 h, 48 h and 72 h of treatment. Even at the highest concentration of 200%, the cell survival rate remained above 70%. In addition, the FDA staining results further confirmed that there was no significant difference between the 50% and 100% extract concentrations and the control group.

[0082] 3. Antibacterial performance Antibacterial performance of hydrogel: E. coli ATCC25922 and S. aureus ATCC6538 were cultured overnight, and the bacterial solution was diluted with sterile saline to the appropriate multiple. 400 μL of the diluted bacterial suspension was mixed with 100 μL of HPA hydrogel, followed by the addition of 500 μL of LB liquid, and incubation at 37°C for 12 h. After incubation, the bacterial suspension was further diluted 10 times with sterile saline. 100 μL of the diluted bacterial suspension was spread on LB agar plates. Incubation was carried out at 37°C for 24 hours, and photographs were taken. The number of colonies was counted, and the experiment was repeated three times.

[0083] Antibacterial performance of hydrogel extract: The prepared hydrogel 100 μL was added to 900 μL of LB broth and stirred to completely dissolve, to prepare the hydrogel extract. After 24 h of extraction at room temperature, 500 μL of the extract was mixed with 100 μL of the diluted bacterial suspension. After 12 hours of incubation, 100 μL of the diluted bacterial suspension was spread on LB agar plates. Incubation was carried out at 37°C for 24 hours, and photographs were taken. The number of colonies was counted, and the experiment was repeated three times. The results of the antibacterial experiment are shown in Figure 5 The antibacterial activity of HPA hydrogel and its extract was close to 100% for both E. coli and S. aureus.

[0084] Example 5: Hemostatic effect of tissue adhesive and organ adhesion 1. Liver hemostasis Healthy SD rats (n=3) weighing 280-300 g were randomly selected and anesthetized with isoflurane. A nose cone was used to maintain anesthesia. After 10 minutes of complete anesthesia, the abdominal and thoracic cavities were disinfected with 75% ethanol. Then the thoracic cavity was opened to expose the surface of the liver. Filter paper was weighed and placed under the liver. A needle with a diameter of 0.5 mm was used to puncture the rat liver, and HPA hydrogel patches (diameter 10 mm, thickness 2 mm) were applied to the puncture site. After the experiment, the filter paper was weighed to calculate the amount of blood loss. Commercial cyanoacrylate tissue glue was used as a control. The hemostasis process was recorded using a camera. The blank control group, commercial cyanoacrylate group and hydrogel group were all performed in accordance with the animal ethics guidelines. The liver hemostasis effect is shown in Figure 6The amount of blood loss in the HPA hydrogel patch group (≈44 mg) was significantly lower than that in the untreated group (≈261.7 mg) and the medical cyanoacrylate group (≈176.7 mg).

[0085] 2. Tail amputation hemostasis Healthy SD rats (n=3) weighing 280 ~ 300 g were randomly selected and anesthetized with isoflurane. The nasal cone maintained anesthesia. After complete anesthesia for 10 minutes, the rat tail was measured with a vernier caliper, and a mark was made at a diameter of 10 mm. The pre-weighed filter paper was placed under the tail. Then the tail was transected at the marked position with surgical scissors, and the tail surface was covered with an HPA hydrogel patch. After the experiment, the filter paper was weighed, and the amount of blood loss was calculated. Commercial cyanoacrylate tissue glue was used as a control. The hemostasis process was recorded with a video camera. The blank control group, commercial cyanoacrylate group and hydrogel group, all experiments were carried out in accordance with the animal ethics guidelines. The tail amputation hemostasis effect is shown in Figure 7 The untreated group bled significantly within 150 s after transection, with a blood loss of 442 mg, while the medical cyanoacrylate still had a large amount of bleeding (≈200 mg). In sharp contrast, the HPA hydrogel showed a significant hemostatic effect, immediately stopping bleeding at the wound site after transection of the tail by strong interfacial adhesion.

[0086] 3. Organ adhesion The rat viscera (heart, liver, spleen, lung, kidney) were removed, and the viscera were placed on a flat plate. The HPA hydrogel was placed close to the viscera, and the viscera were lifted by adhesion force. The adhesion effect was observed, and the results are shown in Figure 8 Only 0.05 g of hydrogel could successfully adhere to the viscera tissue without external pressure, and the weight of the rat liver was 15.39 g, indicating that the hydrogel could adhere to a weight 300 times its own weight.

[0087] Example 6: Wound healing of tissue adhesive To evaluate the sealing and healing effect of HPA hydrogel on linear wounds, we made a 2 cm long linear incision with a surgical blade. All experiments were carried out in accordance with the animal ethics guidelines. Healthy rats weighing 280 ~ 300 g were randomly divided into 3 groups (n=6) and anesthetized with isoflurane. After complete anesthesia, the back hair was removed and disinfected with 75% ethanol. Except for the untreated control group, the rest of the wound was treated with medical cyanoacrylate or hydrogel dressing, and each group was placed separately. The wound was photographed regularly with a digital camera. On the 9th day, fresh tissue around the wound of each rat was quickly collected, then fixed with 4% paraformaldehyde for more than 24 h, and entrusted to Shanghai Fanke Wei for H&E staining, Masson staining and immunofluorescence staining.

[0088] The results of the wound healing process are shown in Figure 9In the control group, the wound failed to close properly, resulting in severe inflammation and pus accumulation throughout the healing process, even leading to extensive crusting by day 8. HPA hydrogel demonstrated excellent adhesion. While medical cyanoacrylate achieved tight wound closure, the residual medical adhesive at the incision site was difficult to degrade, severely hindering healing. In contrast, HPA hydrogel maintained secure wound closure, prevented secondary dehiscence, and achieved complete skin regeneration by day 5.

[0089] The results of H&E staining and Masson staining are shown in the figure. Figure 10 H&E staining results showed that the HPA hydrogel-treated group had complete epidermal regeneration by day 8, with a large number of regenerated hair follicles and sebaceous glands at the incision site. In contrast, the control group and the cyanoacrylate group both showed obvious wound defects, with no hair follicle or sebaceous gland regeneration. Masson staining showed that the collagen arrangement in the incision after HPA hydrogel treatment was almost identical to that of normal tissue, and collagen deposition was significantly enhanced.

[0090] Immunofluorescence staining results are shown in Figure 11 Compared with the control and cyanoacrylate groups, CD31 expression at the wound site was significantly reduced in the HPA hydrogel group on day 8. Combined with H&E staining results, this indicates that the HPA-treated wound had entered the vascular remodeling phase, while the control and cyanoacrylate groups remained in the angiogenesis proliferation phase. These findings provide evidence that HPA hydrogel actively promotes angiogenesis and accelerates wound healing. Furthermore, red fluorescence imaging showed a significant reduction in TNF-α secretion in the HPA hydrogel group, demonstrating its anti-inflammatory properties.

[0091] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a tissue adhesive, characterized in that, Hyaluronic acid and basic protein are electrostatically self-assembled to form an antibacterial proteoglycan material, and recombinant mucin modified by tyrosinase is added to the proteoglycan material. The basic proteins include one or more of protamine, elastin K32, and R36; the amino acid sequence of the protamine is shown in SEQ ID NO.10, the amino acid sequence of the K32 protein is [GVG(VPGKG)8VP]4, and the amino acid sequence of the R36 protein is [GVG(VPGRG)9VP]4. The mucin is formed by the fusion of leucine zipper protein ASP and mussel foot filament protein MFP. The recombinant leucine protein includes a leucine zipper domain (A), an unstructured polyelectrolyte domain (S), and a helical terminal domain (P). The amino acid sequence of the A domain is shown in SEQ ID NO.11, and the amino acid sequence of the S domain is (AGAGPEG). 10 The amino acid sequence of the P domain is shown in SEQ ID NO.

12. The mussel byssal protein includes one or more of Mefp-3, Mgfp-5, and Mcofp-3. The amino acid sequences of Mefp-3, Mgfp-5, and Mcofp-3 are shown in SEQ ID NO.13~15, respectively. The position of the mussel byssal protein inserted into the leucine zipper domain includes one or more of 3 / 5-ASP, A-3 / 5-SP, AS-3 / 5-P, and ASP-3 / 5.

2. The method according to claim 1, characterized in that, The method includes the following steps: hyaluronic acid, basic protein, and recombinant mucin are mixed, deionized water is added, the supernatant is removed by centrifugation, and the tissue adhesive is obtained by freeze-drying.

3. The method according to claim 1 or 2, characterized in that, In the proteoglycan material, the mass ratio of hyaluronic acid to basic protein is 1:(0.2~2); in the tissue adhesive, the mass ratio of hyaluronic acid, basic protein, and mucin is 1:(0.2~2):(0.1~2).

4. The method according to claim 2, characterized in that, The concentration of hyaluronic acid is 20-100 mg / mL, the concentration of basic protein is 50-100 mg / mL, and the concentration of mucin is 50-100 mg / mL.

5. The method according to claim 2, characterized in that, The centrifugation speed is 10000 rpm, the temperature is 4℃, and the time is 10-20 min; the freeze-drying time is 5-10 min, the sample temperature is -20℃, the cold trap temperature is -60℃, and the vacuum degree is less than 10 Pa.

6. The method according to claim 1, characterized in that, The tyrosinases mentioned are VsTYR and BmTYR, respectively, and their sources are Polyrhiza spinosa microorganisms (VsTYR and BmTYR). Verrucomicrobium spinosum ) and Bacillus megaterium ( Bacillus megaterium Their GenBank numbers are MK550618.1 and ACC86108, respectively.

7. A tissue adhesive prepared by the method according to any one of claims 1 to 6.

8. The use of the tissue adhesive of claim 7 in the preparation of products for antibacterial materials, wound hemostasis, cell culture, tissue adhesion and / or wound healing.

9. The application according to claim 8, characterized in that, The types of bacteria to be treated include Gram-positive and / or Gram-negative bacteria.

10. The application according to claim 8, characterized in that, The tissue adhesion includes one or more of the following: skin tissue, visceral tissue, muscle tissue, connective tissue, and mucous membrane tissue.

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