Biological adhesive as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a biological adhesive as well as a preparation method and application thereof, and the biological adhesive is prepared by loading transglutaminase on a Ca-MOF material, adding HANB hydrogel and uniformly dispersing; the Ca-MOF material is a calcium-containing metal organic framework formed by carrying out coordination crosslinking on calcium ions and catechol groups of gallic acid. The biological adhesive provided by the invention shows good biocompatibility, immediate adhesive hemostasis and therapeutic healing effects, and can be used as a medicine for stopping bleeding and promoting wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bioadhesive and a preparation method and application thereof. Background Art

[0002] Currently, surgical sutures / staples are primarily used clinically to seal wounds and surgical incisions to achieve hemostasis, wound closure, antimicrobial barrier, and tissue healing. However, sutures can damage delicate tissues, increase the risk of bacterial infection, and promote scarring. They are time-consuming and technically sensitive. To overcome these limitations, bioadhesives have been developed as alternatives to traditional sutures. The use of bioadhesives offers significant benefits for patients and surgeons, such as reducing tissue healing time, infection, and other surgical complications. Various biomaterials have been developed as bioadhesives to seal wounds or bond tissues. First-generation bioadhesives, such as fibrin glue and cyanoacrylate glue, were developed for hemostasis and as auxiliary support for surgical sutures. Fiber sealants are soft like soft tissue but have weak adhesion strength. Cyanoacrylates, while having high adhesion strength, also exhibit significant cytotoxicity and unnatural hardness. Therefore, there is an urgent need to develop safe and effective bioadhesives. Summary of the Invention

[0003] To develop a safe bioadhesive, the present invention provides a bioadhesive, its preparation method, and application. The bioadhesive provided by the present invention exhibits good biocompatibility, immediate adhesive hemostasis, and therapeutic healing effects, and can be used as a drug for hemostasis and wound healing.

[0004] The present invention provides a bioadhesive, which is prepared by loading transglutaminase onto a Ca-MOF material and then adding HANB hydrogel for uniform dispersion; The Ca-MOF material is a calcium-containing metal organic framework formed by coordination cross-linking of calcium ions and catechol groups of gallic acid.

[0005] The bioadhesive provided by the present invention exhibits good biocompatibility, immediate adhesive hemostasis and therapeutic healing effects, and can be used as a drug for hemostasis and wound healing. The present invention forms a calcium-containing metal organic framework by cross-linking calcium ions with the catechol groups of gallic acid, which maintains the biological properties of gallic acid and loads TG enzyme onto the Ca-MOF to slowly release Ca. 2+It promotes platelet activation and aggregation. The AN hydrogel provides a platform for the Ca-MOF loaded with transglutaminase, allowing for its gradual release, enabling timely gelation and wound closure. The regenerative hydrogel based on enzyme catalysis and photocrosslinking exhibits excellent elasticity and flexibility, and demonstrates good biocompatibility, degradability, and negligible inflammatory response in a mouse skin incision adhesion model.

[0006] Furthermore, the calcium ions are derived from CaCl2.

[0007] Furthermore, the HANB hydrogel contains 0.25% to 4.7% by mass of LAP photoinitiator.

[0008] The present invention also provides a method for preparing the bioadhesive, comprising the following steps: CaCl2, gallic acid and ultrapure water are mixed uniformly to obtain a mixture, the pH of the mixture is adjusted to 8-12, and then heated at 100°C-120°C for 20-24 hours, and the precipitate is collected by centrifugation to obtain a Ca-MOF material; After Ca-MOF was dispersed in pure water, transglutaminase was added and ultrasonic cross-linking was performed to obtain a mixed solution. HANB hydrogel was dissolved in the mixed solution and uniformly dispersed to obtain HANB-MOF-TG composite hydrogel, i.e., bioadhesive.

[0009] Furthermore, the usage ratio of CaCl2, gallic acid and ultrapure water is 18g~10g:30g~38g:500mL.

[0010] Furthermore, the added amount of the transglutaminase is 100 U / g to 200 U / g.

[0011] Furthermore, HANB hydrogel contained 0.25% LAP.

[0012] Furthermore, the ratio of Ca-MOF to pure water is 300 μg to 500 μg: 8 mL to 12 mL.

[0013] The present invention also provides an application of the bioadhesive in preparing materials for stopping bleeding and promoting wound healing.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention constructs a Ca-based 2+ and gallic acid metal polyphenol network (Ca-MOF) to maintain the biological properties of gallic acid and achieve Ca 2+ The controlled release of gallic acid and gallic acid enhances its application value in the biomedical field. Figure 1As shown: Due to the controlled release effect of the MOF structure, the present invention loads the TG enzyme and slowly releases the Ca 2+ Gallic acid not only promotes platelet activation and aggregation but also regulates and activates TGase, leading to an open conformation. Gallic acid also creates conditions for TGase to convert to its reduced form. The regenerative hydrogel, based on enzyme catalysis and photocrosslinking, exhibited good elasticity and flexibility, as well as good biocompatibility, biodegradability, and negligible inflammatory response in a mouse skin incision adhesion model. This provides a foundation for the development of a bioadhesive for seamless sutures.

[0015] The HA in the HANB hydrogel of this invention, due to its hydrophilic groups such as carboxyl and hydroxyl groups, can absorb wound exudate and enhance cell adhesion. The introduction of NB groups into HA allows them to react with amino groups (-NH2) in skin tissue under ultraviolet light, forming a stable imine structure containing a carbon-nitrogen double bond. Furthermore, the invention utilizes TG enzyme to catalyze the acyl transfer reaction between the γ-carboxyamide group of glutamine residues and the ε-amino group of lysine or other primary amines. Through intermolecular or intramolecular crosslinking, stable isopeptide bonds are formed, enhancing the crosslinking strength of HA. Together, these two effects enable strong adhesion to skin tissue. The MOF structure allows for the sustained release of Ca²⁺ and gallic acid, providing continuously optimized reaction conditions for enzymatic crosslinking, thereby promoting wound healing.

[0016] The present invention compares the experimental results of gelatin alone and the sequential addition of TG and calcium ions. The addition of TGase and calcium ions significantly promotes the gelatin cross-linking reaction. The addition of TGase alone promotes gelatin cross-linking within 1 minute, while the addition of calcium ions increases cross-linking within 30 seconds, significantly accelerating the cross-linking speed. This indicates that TGase plays a key role in the cross-linking process, and the addition of calcium ions further enhances this effect.

[0017] HANB-MOF-TG hydrogel combines TG, Ca 2+ The therapeutic effects of HANB-MOF-TG hydrogel are achieved through the following aspects: (1) through Ca 2 The coordination and complexation of calcium ions (Ca) and gallic acid produces a Ca-MOF with a porous structure and good biocompatibility; (2) the MOF continuously releases calcium ions, gallic acid, and TG enzyme, and continuously provides reducing conditions for TG; (3) TG enzyme promotes protein re-crosslinking in vivo, promoting tissue regeneration; (4) the HANB hydrogel forms a physical barrier to enhance adhesion and prevent secondary damage. In vitro and in vivo experiments showed that the HANB-MOF-TG hydrogel exhibits good biocompatibility, immediate adhesive hemostasis, and therapeutic healing effects, and is expected to provide a valuable option for suture-free wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Schematic diagram of the working principle of the bioadhesive Ca-MOF prepared in the present invention in promoting wound adhesion and post-wound healing.

[0020] Figure 2 Synthesis and characterization of the bioadhesive Ca-MOF prepared for the present invention; In the figure, a is a schematic diagram of the synthesis of Ca-MOF-TG; b is the SEM image of Ca-MOF with a scale bar of 10 μm; c is a local magnified SEM image of the red area in b; d is the SEM image of Ca-MOF with a scale bar of 5 μm; e is the distribution diagram of carbon (C) element in Ca-MOF; f is the distribution diagram of oxygen (O) element in Ca-MOF; g is the distribution diagram of calcium (Ca) element in Ca-MOF; h is the X-ray diffraction (XRD) spectrum of Ca-MOF powder; i is the absorption spectra of Ca-MOF, TG, and MOF-TG; j: L929 cell viability after incubation with Ca-MOF and CaPN for 48 h.

[0021] Figure 3 To test the performance of HANB-MOF-TG hydrogel; In the figure, a is a schematic diagram of the effect of HANB-MOF-TG hydrogel on skin wounds; b shows the effect of TGase on the cross-linking degree of gelatin and pork; c shows the in vitro pig skin adhesion test of different groups; d is the effect of calcium ions and MOF on TG enzyme activity; e is the tensile stress-strain test of each group of hydrogels on pig skin; f is the cumulative amount of calcium ions released over time.

[0022] Figure 4 To test the in vivo hemostatic and coagulation properties of Ca-MOF and HANB-MOF-TG; Figure a: Photos of tail amputation after application of different hemostatic materials; b is a photo of the liver incision after application of different hemostatic materials; c is the hemostasis time of rat liver injury model and rat tail amputation model (n=5); d is the blood loss in the rat liver injury model and rat tail docking model (n=5); e is a photograph of blood after incubation with PBS, Ca-MOF, MOF-TG, HANB-MOF-TG, and ddH2O; f is the hemolysis rate of different samples (n=3); g is a photo of blood coagulation with different materials at 5 min; h is the coagulation index of different materials (n=3); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0023] Figure 5 To study the biocompatibility, antioxidant efficacy and in vitro cell migration behavior of HANB-MOF-TG; In the figure, a is a fluorescence microscopy image of live / dead cell double staining of endothelial cells and fibroblasts after co-culture with TG, MOF-TG, HANB, HANB-TG, and HANB-MOF-TG hydrogels for 48 hours, respectively; b is the ROS fluorescence staining image of each group of materials after LPS induction of RAW264.7 cells for 24 h; c is the proliferation and migration ability of fibroblasts within 24 h of co-culture of TG, MOF-TG, HANB, HANB-TG and HANB-MOF-TG hydrogels with fibroblasts; d is the cell viability of fibroblasts after co-culture with each group of materials for 48 h (n=3); e is the quantification of ROS fluorescence staining images (n = 3); f is the quantification of gap closure rate among different groups (n=3), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 6 The wound adhesion effect of HANB-MOF-TG hydrogel in vivo; In the figure, a is a photograph of the wound skin of mice treated with sutures and medical glue as positive controls, and the experimental groups were treated with TG, MOF-TG, HANB, HANB-TG, and HANB-MOF-TG, respectively; b is a schematic diagram of the dynamic wound healing process on day 0, 1, 3, 7, and 14; c, H&E staining of the wound site on days 7 and 14; d, Masson staining of the wound sites on days 7 and 14 (n=3).

[0025] Figure 7 In vivo study of the tissue regeneration and angiogenesis efficacy of TG, MOF-TG, HANB, HANB-TG and HANB-MOF-TG. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0027] Example 1: A bioadhesive and its preparation method and application.

[0028] 1. Experimental Materials and Methods 1. Experimental materials and sources Gallic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS number: 149-91-7. Transglutaminase (TGase) was purchased from Shanghai Qingrui Food Co., Ltd. HANB hydrogel was purchased from Shanghai Lingjiu Medical Co., Ltd. The photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), was purchased from EFL. All other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Nanjing, China) and used as received without further purification. High-glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Shanghai Yuanpei (China). Fibroblasts (L929), HUVECs, and a mouse macrophage cell line (Raw264.7) were cultured in complete medium (high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) at 37°C in a 5% CO2 incubator. HUVECs are a normal primary human umbilical vein endothelial cell line purchased from the American Type Culture Collection.

[0029] 2. Preparation of Ca-MOF materials Ca-MOF was synthesized using the method described in the literature [P. Zhang, Y. Gong, Q. Pan, Z. Fan, G. Li, M. Pei, J. Zhang, T. Wang, G. Zhou, X. Wang]. The specific steps are as follows: First, 10 g of CaCl₂, 38 g of gallic acid, and 500 mL of ultrapure water (18.2 MΩ, Millipore Co., Burlington, MA, USA) were added to a beaker and mixed with a magnetic stirrer for 10 minutes to obtain a mixture. Subsequently, the pH of the mixture was adjusted to 12 with 10 M aqueous NaOH and heated at 120°C in a muffle furnace for 24 hours. Finally, the mixture was centrifuged at 10,000 rpm at 4°C for 15 minutes, and the gray-brown precipitate was collected to obtain the Ca-MOF material. The Ca-MOF was washed twice with ethanol and then deionized water before use. In this method, calcium ions (Ca⁺) coordinate and crosslink with the catechol groups of gallic acid to form a calcium-containing metal-organic framework (Ca-MOF).

[0030] 3. Preparation of HANB-MOF-TG composite hydrogel 400 μg of the Ca-MOF prepared in the previous steps was dispersed in 10 mL of pure water by ultrasonication. Subsequently, transglutaminase was added at a rate of 200 U / g and ultrasonicated for 2 h to load the transglutaminase into the Ca-MOF to obtain a MOF-TG mixed solution. HANB hydrogel (containing 0.25% by mass of photoinitiator LAP) with a final concentration of 2.5 wt% was added and dissolved. The mixture was stirred at 37°C for 1 h to prepare a uniformly dispersed hydrogel, namely, the HANB-MOF-TG composite hydrogel.

[0031] 4. Cytotoxicity test Mouse fibroblasts (L929) were seeded at a density of 5,000 cells per well in 96-well plates. L929 cells were treated with Ca-MOF at various concentrations (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL) for 48 hours, with triplicate replicates per group. A control group received no Ca-MOF. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA). CCK-8 assays were performed for different materials. Extracts were prepared by placing 200 μL of HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel in 10 mL of complete culture medium for 24 hours. Each 5,000 fibroblast was seeded into one well of a 96-well plate and co-cultured with either no material (control group) or the extracts from each group for 48 hours. Other experimental procedures were the same as described above. Finally, cell viability was analyzed using a microplate reader.

[0032] The preparation steps of HANB-TG hydrogel are as follows: A 2.5% HANB hydrogel (0.25% LAP) was prepared using pure water as the solvent. Transglutaminase was added at 200 U / g and stirred at 37°C for 1 hour to prepare a uniformly dispersed hydrogel (HANB-TG). LAP decomposes under irradiation with light at a wavelength of approximately 400 nm to generate reactive free radicals (such as phosphorus-centered free radicals and benzoyl radicals). These reactive free radicals initiate cross-linking and polymerization of hydrogel precursors (such as acrylamide and polyethylene glycol diacrylate (PEGDA)), forming a three-dimensional network structure to obtain the HANB-TG hydrogel.

[0033] 5. Live / dead cell double staining Each 2×10 5 Fibroblasts (L929) or HUVEC cells were seeded in one well of a 12-well plate. Each cell type was divided into six groups and co-cultured with TG enzyme solution, MOF-TG mixed solution, HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel, respectively. The co-culture conditions were: 37°C, 5% CO2. Cells were cultured alone as a control. The calcein-AM / PI assay (Yeasen, Shanghai, China) was used. The culture medium was replaced with 400 μL of staining solution. The 12-well plate was then placed in an incubator at 37°C, 5% CO2 for 15 minutes, and the supernatant was removed. The stained cells were then imaged using a fluorescence microscope.

[0034] The TG enzyme solution was obtained by dissolving TG enzyme at 200 U / g in pure water.

[0035] 6. In vitro migration analysis First, L929 fibroblasts were cultured at 4 × 10 5 Cells were seeded at a density of 100 μg / mL in 6-well plates. When the cell confluency approached 90%, the cells were starved for 6 hours. A straight line was drawn in the center of each well of the plates using a 200 μL sterile pipette tip. The lined area was then washed twice with PBS. The extracted solution prepared in low-serum (serum containing 1% FBS) high-glucose medium was added to the wells of each group. Fibroblast growth at the scratch edge was observed and images were acquired after treatment for 0, 6, 12, and 24 hours.

[0036] TG (0.1%), MOF-TG (80 μg / mL), HANB (200 μL), HANB-TG (200 μL), and HANB-MOF-TG (200 μL) were immersed in DMEM for 24 h to obtain the extracts of each group for in vitro migration analysis.

[0037] 7. In vitro antioxidant test In order to evaluate the antioxidant effect of the experimental groups, RAW cells were cultured at 4 × 10 5 Cells were evenly seeded at a density of 100 μg / mL in six-well plates. After 24 hours, all groups except the blank control group were induced with 250 ng / mL of LPS for 24 hours. A reactive oxygen species detection kit (Beyotime) was used according to the manufacturer's instructions, with 1 mL of 10 μmol / L DCFH-DA probe added to each well. The stained cells were imaged using a fluorescence microscope.

[0038] 8. In vitro hemolysis test Fresh whole blood was drawn from 200 g SD rats and immediately collected in tubes containing sodium heparin according to the method described in the previous literature [T. Deng, D. Gao, X. Song, Z. Zhou, L. Zhou, M. Tao, Z. Jiang, L. Yang, L. Luo, A. Zhou, L. Hu, H. Qin, M. Wu, A natural biological adhesive from snail mucus for wound repair, Nat. Commun. 14(1) (2023) 396.]. The whole blood was diluted with PBS to prepare a red blood cell (RBC) suspension (5% by volume). 0.5 mL of the RBC suspension was incubated with 0.5 mL of Ca-MOF (0.5 mg / mL dissolved in PBS), MOF-TG, and 0.5 mL of the hydrogel, and blood compatibility was assessed using a microplate reader. After incubation at 37.0°C for 1.0 h, the samples were centrifuged at 116.0 × g for 10 min, and the absorbance of the supernatant was measured at 540 nm using a microplate reader. Blood diluted to 5.0% with deionized water and PBS was used as a positive control and a negative control, respectively. The percentage of hemolysis was calculated as follows:

[0039] Hemolysis rate (%) = A sample - A control / A positive - A control × 100%; Among them, Asample, Acontrol and Apositive represent the absorbance of sample, negative control and positive control, respectively.

[0040] 9. Blood coagulation (BCI) test Whole blood was collected from healthy SD rats and anticoagulated. 5 mg of Ca-MOF and MOF-TG were collected, and the HANB-MOF-TG hydrogel was cut into square slices 5 mm long and 1 mm high. Gauze was used as a positive control and cut into the same size as the hydrogel slices. 10 µL of whole blood concentrated with 10 μM CaCl2 was incubated with each group of materials at 37°C for 5 minutes to induce coagulation. Thereafter, 2 mL of deionized water was added to the centrifuge tube to break / dissolve uncoagulated red blood cells. After centrifugation (116.0 × g, 1 min), the absorbance of hemoglobin in the supernatant was measured at 540.0 nm using a microplate reader. BCI was calculated using the following formula:

[0041] BCI (%) = A sample / A control × 100%; Where Asample and Acontrol represent the absorbance of sample and control (10 μL purified whole blood dissolved in 1 mL deionized water), respectively.

[0042] 10. In vivo hemostasis study To evaluate the in vivo hemostatic potential of Ca-MOF, MOF-TG, and HANB-MOF-TG composite hydrogels, a rat model of liver incision and tail amputation was established.

[0043] Seven-week-old female SD rats were anesthetized and immobilized. To calculate blood loss, a weighed gauze was pre-placed under the liver, and a 5 mm (deep) and 10 mm (long) incision was made with a sterile surgical blade to injure the liver. In the experimental groups, the bleeding livers were randomly coated with Ca-MOF, MOF-TG, and HANB-MOF-TG hydrogels at a dose of 100 mg kg −1 The control group received no treatment after hepatic hemorrhage, and the amount of bleeding and the duration of hemostasis were recorded. A 20 μL HANB-MOF-TG hydrogel was applied and immediately irradiated with UV light for 1 minute.

[0044] The rat tail was cut off 5 cm away from the tail using sterile scissors and placed in air for 5 s. Then, Ca-MOF, MOF-TG and HANB-MOF-TG hydrogels were used at a dose of 100 mg kg −1 HANB-MOF-TG hydrogel was applied to 5 rats. Immediately after application, the rats were irradiated with UV light for 1 minute. A control group of 5 rats received no treatment. Hemostasis time and total blood loss were recorded.

[0045] 11. Tissue adhesion in vivo The in vivo tissue adhesion of the composite hydrogel was evaluated using a mouse dorsal skin incision model. Sutures and medical glue served as positive controls, while saline treatment served as a negative control. Four mice were included in each group. Seven-week-old male mice were injected with a 5% chloral hydrate solution, and the hair on the waist and abdomen was shaved. After surgery, a 1.5 cm incision was made, and TG enzyme solution, MOF-TG solution, HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel were injected into the incision, serving as experimental groups. In the suture group, wounds were fixed with sutures, which were removed after 5 days of care. The blank control group received no treatment. Optical images of the wounds were captured at predetermined intervals (0, 1, 3, 5, 7, 10, and 14 days), and wound sections were removed for histological sectioning and staining.

[0046] 12. Histological Analysis and Immunofluorescence Staining For histopathological analysis, wound tissues were collected from mice on days 7 and 14, fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and cut into 5-μm-thick sections. Tissue sections attached to slides were dewaxed, rehydrated, and stained with hematoxylin and eosin and Masson's trichrome. For immunofluorescence staining, heat-induced antigen retrieval was performed on dewaxed and rehydrated sections in citrate buffer (10 mM, pH 6.0) at 98°C for 10 minutes. After permeabilization for 10 minutes, nonspecific binding was blocked with goat serum (10%) for 1 hour. To evaluate angiogenesis and tissue repair, primary antibodies against CD31 and α-SMA and corresponding secondary antibodies were used. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Servicebio, China) for 10 minutes. Immunofluorescence images were acquired using Carl Zeiss Microscopy GmbH (Axio Scan Z1, Germany) and quantified using ImageJ software.

[0047] 13. Statistical analysis All experiments were performed in triplicate, and quantitative data were expressed as mean ± standard deviation. Unpaired Student's t-test was used for comparisons between two groups, and one-way ANOVA with Tukey's post hoc test was used for comparisons between more than two groups. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Boston, MA, USA). Tests were considered statistically significant when the p-values ​​were as follows: *p < 0.05, **p < 0.01, *p < 0.001, and *p < 0.0001.

[0048] 2. Experimental Results 1. Synthesis and characterization of Ca-MOF like Figure 2 As shown, the Ca-MOF composite was prepared using a hydrothermal method. The Ca-MOF composite is a porous material with gallic acid as an organic ligand and calcium ions as metal junctions. Using Ca-MOF as a carrier, the MOF-TG composite was loaded with TG enzyme, enabling targeted drug delivery with potential biomedical applications.

[0049] Scanning electron microscope (SEM) images show that Ca²⁺ and gallic acid form a metal organic framework (MOF) through metal coordination, while the π-π stacking effect promotes the stability of the material. It exhibits a uniform spherical structure with a diameter of about 10μm ( Figure 2 b and Figure 2c). The material's surface is uneven, with significant gaps between the rectangular crystals, indicating the material's excellent porous structure, which enhances the Ca-MOF's water absorption. Ultimately, the MOF precipitated as a gray powder, exhibiting spherical particles, a porous structure, and good stability and biocompatibility.

[0050] In order to verify the synthesis and composition of Ca-MOF, elemental energy spectrum analysis was performed. Figure 2 As shown, carbon (C), calcium (Ca) and oxygen (O) are uniformly distributed in Ca-MOF ( Figure 2 d~ Figure 2 g). The X-ray diffraction (XRD) pattern of Ca-MOF powder produced obvious characteristic peaks ( Figure 2 h), indicating the successful synthesis of Ca-MOF. In addition, spectral analysis showed that TG enzyme and Ca-MOF showed characteristic absorption peaks at 265 nm and 290 nm, respectively, and Ca-MOF had a characteristic absorption peak at 280 nm, indicating that TG enzyme was successfully loaded onto Ca-MOF ( Figure 2 i). To verify the cytotoxicity of Ca-MOF, the present invention measured the cytotoxicity of Ca-MOF in the mouse fibroblast cell line L929. CCK-8 assay was performed. When the concentration reached 120 μg mL −1 When the Ca-MOF solution has no obvious cytotoxicity to fibroblasts ( Figure 2 The optimal concentration of Ca-MOF for fibroblast proliferation was 80 μg / mL. Therefore, unless otherwise stated, 80 μg mL was used in subsequent experiments. −1 The above results demonstrate the strong structural integrity and multifunctionality of Ca-MOF, which provides hope for the development of drug delivery systems and biomedical materials.

[0051] 2. Performance of HANB-MOF-TG hydrogel TG enzyme is used to catalyze the acyl transfer reaction between the γ-carboxyamide group of glutamine residues and the ε-amino group of lysine or other primary amines, forming a stable isopeptide bond through intermolecular or intramolecular cross-linking, thereby enhancing the cross-linking strength of HA. Under the combined action of the two, a strong adhesion effect can be formed with skin tissue. Figure 3 a).

[0052] In order to more intuitively see the catalytic effect of TG enzyme, the present invention compares the experimental results of pure gelatin and the results of adding TG and calcium ions in sequence ( Figure 3b) After adding TG enzyme and calcium ions, the gelatin cross-linking reaction was significantly promoted. After adding TG enzyme alone, gelatin cross-linking was promoted within 1 minute, and after adding calcium ions, cross-linking was achieved within 30 seconds, greatly accelerating the speed of cross-linking. In order to further observe the effect of TG on skin in vitro, the present invention selected pig skin containing a large amount of collagen similar to human tissue to demonstrate the adhesion effect ( Figure 3 c).

[0053] The activity of TGase was quantitatively determined under different conditions. The results showed that Ca 2+ It can improve the activity of TG. In the MOF structure prepared with gallic acid, it does not affect the activity of TG enzyme. On the contrary, MOF can slowly release Ca 2+ and gallic acid, thus providing the reduced form of TG at the inflammatory wound surface and promoting the activity of the enzyme ( Figure 3 d). At the same time, a 2 cm incision was made on each piece of pigskin, and TG, MOF-TG, HANB, HANB-TG, and HANB-MOF-TG were applied respectively. The control group did not receive any treatment. The TG and MOF-TG groups showed only a slight adhesion sign after 30 minutes. However, the HANB, HANB-TG, and HANB-MOF-TG groups quickly achieved cross-linking adhesion within 30 seconds after addition and showed good adhesion properties, with almost no visible incision. To characterize the viscosity of the hydrogel, tensile tests were performed on pigskin treated with HANB, HANB-TG, HANB-MOF, and HANB-MOF-TG ( Figure 3 e), the results show that the HANB-MOF-TG group can resist a large tensile force. In order to observe the sustained release of Ca 2+ The ability to conduct Ca 2+ The release curves of HANB-MOF-TG hydrogels showed that Ca 2+ The stable release from day 1 to day 14 ensures the long-term optimization of the cross-linking process. Figure 3 Based on the above experimental results, the HANB-MOF-TG system, through the controlled release of MOF, enhances adhesion while maintaining a stable enzymatic cross-linking reaction, providing a better biomaterial solution for tissue repair.

[0054] 3. Hemolysis and in vitro hemostasis evaluation To evaluate the hemostatic properties of HANB-MOF-TG materials, including their in vitro and in vivo hemostatic effects, coagulation time, blood compatibility, and hemostatic mechanism.

[0055] The present invention evaluated the in vivo hemostatic effects of different materials using rat tail amputation and liver bleeding experiments. The experimental results showed that Ca-MOF exhibited excellent hemostatic effects and formed blood clots within the particles and wounds. Furthermore, the addition of hydrogel sealed the wound within 30 seconds of UV irradiation. Blood loss was significantly reduced in both injury models, demonstrating excellent hemostatic properties. Figure 4 a and Figure 4 b).

[0056] In addition, the blood coagulation index (BCI) test was used to quantify the effect of the material on blood coagulation ability, and the hemolysis test was used to evaluate its blood compatibility. The present invention quantified the coagulation time and blood loss of rats ( Figure 4 c and Figure 4 Figure d) shows the blood loss in the control, Ca-MOF, MOF-TG, and HANB-MOF-TG groups during the liver bleeding and tail amputation hemostasis experiments. In the liver bleeding model, blood loss in the Ca-MOF group (0.857±0.085g), MOF-TG group (0.677±0.095g), and HANB-MOF-TG group (0.283±0.047g) was significantly lower than that in the control group (1.897±0.10g), with HANB-MOF-TG showing the best effect. In the tail amputation model, the blood loss of the control group, Ca-MOF group, MOF-TG group, and HANB-MOF-TG group showed that the blood loss of the HANB-MOF-TG group was the lowest (0.496±0.055g) compared with the control group (2.85±0.18g), Ca-MOF group (1.717±0.076g), and MOF-TG group (1.423±0.068g). As a quantitative indicator for evaluating the blood coagulation energy of different materials, the larger the BCI value, the slower the coagulation rate. In addition, the blood coagulation ability of different materials was quantified by measuring the absorbance at 540 nm and calculating the BCI. Figure 4 As shown in h, the relative BCI of the Ca-MOF group was about 46.3%, and the relative BCI of the MOF-TG group was about 42.7%, which were similar to the relative BCI of gauze of about 42%. The relative BCI of the HANB-MOF-TG group was about 26%, which was significantly lower than that of gauze. From the perspective of BCI, HANB-MOF-TG had the best hemostatic ability. The blood compatibility of biomaterials is a prerequisite for their in vivo application. Fresh anticoagulated blood was used to test the blood compatibility of Ca-MOF, and dd-H2O was used as a positive control. A hemolytic reaction of dd-H2O in the control group was expected, however, no hemolytic reaction was observed in the PBS, Ca-MOF, MOF-TG, and HANB-MOF-TG groups ( Figure 4 Quantitative results showed that the hemolysis rate of HANB-MOF-TG group was lower than the safety range (5%) ( Figure 4f), but the Ca-MOF powder itself will have color precipitation, which will have a certain impact on the results. But overall, HANB-MOF-TG shows good blood compatibility and has strong hemostatic potential.

[0057] 4. In vitro cytotoxicity, antioxidant and anti-inflammatory effects of TG, MOF-TG, HANB, HANB-TG and HANB-MOF-TG The cell compatibility, antioxidant capacity and ability to promote wound healing of biomaterials are crucial for their application in tissue repair. The purpose of this invention is to evaluate the cytotoxicity, biocompatibility and antioxidant properties of TG, MOF-TG, HANB, HANB-TG and HANB-MOF-TG, and further explore their effects on cell proliferation and migration to verify their potential application value in promoting wound healing. In order to evaluate the cell compatibility of each group of materials, TG (0.1%), MOF-TG (80μg / mL), HANB (200μL), HANB-TG (200μL) and HANB-MOF-TG (200μL) were soaked in DMEM for 24 hours to obtain extracts for in vitro experiments. The cytotoxicity was evaluated using the CCK-8 assay. The results showed that the cell viability of all experimental groups was comparable to that of the control group, and all showed obvious cytotoxicity, proving that the materials have good biocompatibility ( Figure 5 In addition, the biocompatibility of the material was further verified by live / dead cell double staining experiments. After fibroblasts and HUVEC were co-cultured with each group of hydrogels for 48 hours, live / dead cell double staining was performed. The figure shows that the cells in each experimental group survived well and no obvious cell death was observed ( Figure 5 In order to study the antioxidant potential of the material, the present invention adopted the lipopolysaccharide (LPS)-induced oxidative stress model and evaluated the level of reactive oxygen species (ROS) ( Figure 5 (b) The effects of each material group on ROS levels were evaluated by fluorescence imaging and quantitative analysis of fluorescence intensity. Compared with the LPS group, the ROS levels of the experimental groups containing MOF and HANB hydrogels were significantly reduced, showing significant antioxidant and anti-inflammatory properties. This suggests that these materials can reduce oxidative stress in the cellular microenvironment and alleviate inflammation. Quantification of fluorescence intensity ( Figure 5 e) further confirmed this trend. Compared with the LPS group, the ROS levels in the LPS+MOF-TG group, LPS+HANB group, LPS+HANB-TG group, and LPS+HANB-MOF-TG group were significantly reduced (p<0.0001). In addition, to study the effects of the materials on cell migration and proliferation, the present invention used a scratch test and quantitatively analyzed the wound healing rate. The cell scratch results showed that ( Figure 5c), cells in all experimental groups had varying degrees of migration ability, and wound healing in all experimental groups improved over time. Compared with the control group and other groups, the HANB-TG group and HANB-MOF-TG group showed the most significant improvement in cell migration. Quantitative analysis of wound healing rate ( Figure 5 These findings were further confirmed by the results of a study (f), which showed that the HANB-TG and HANB-MOF-TG groups significantly accelerated wound healing at 6, 12, and 24 hours compared to the control group (p < 0.01), indicating that they can effectively promote wound healing. Taken together, the present invention demonstrates that HANB-MOF-TG has good biocompatibility, antioxidant capacity, and the potential to promote cell migration. Under LPS-induced oxidative stress, the material can effectively reduce ROS levels, indicating that it has a protective effect on the cellular microenvironment. In addition, the scratch test results further support the superiority of HANB-MOF-TG in promoting cell migration, suggesting that it has potential application value in wound healing and tissue repair.

[0058] 5. In vivo evaluation of skin adhesion and wound healing In addition to hemostatic properties, tissue adhesion is also crucial for wound repair. An ideal wound closure material should possess excellent adhesion, biocompatibility, and effectively promote tissue regeneration. Therefore, the present invention evaluated the in vivo adhesion ability of the composite hydrogel using a mouse skin incision model and compared it with surgical sutures and a commonly used commercially available medical adhesive (containing cyanoacrylate) to verify its potential application as a seamless wound closure material.

[0059] The present invention created a 1.5 cm incision on the back of mice and injected TG solution, MOF-TG solution, HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel (30 μL) into the incision as experimental groups. A suture group, a commercial glue group, and a control group were also established for comparative analysis. The experimental results showed that HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel all exhibited good adhesion to moist wound tissue ( Figure 6a). After 7 days of treatment, the wounds in the experimental group were well closed, while the wounds in the control group were closed more slowly, with obvious inflammatory reactions and incomplete healing still observed. Although the suture group was able to effectively close the wounds, obvious suture marks and scars were left. Although the commercial glue group showed a certain degree of adhesion, there were still signs of inflammation. In contrast, the HANB-TG group and the HANB-MOF-TG group had the best wound closure effect and the smallest scars, indicating that their healing efficiency was higher. The healing of the TG and MOF-TG groups was also improved compared with the control group, but not as good as the HANB-TG and HANB-MOF-TG groups. By the 14th day, the wounds in the HANB-MOF-TG group were basically scar-free, and the healing effect was better than that of the other experimental groups. Wound contraction and closure rate were quantitatively analyzed ( Figure 6 (b) The results showed that the HANB-TG and HANB-MOF-TG groups had the highest degree of wound contraction, and over time, there was a significant difference compared with the control group and other experimental groups. This shows that the hydrogel formula can not only adhere to the wound, but also effectively promote tissue remodeling and accelerate the healing process. To further explore the tissue regeneration, tissue sections at the wound site were stained with hematoxylin and eosin (H&E). Figure 6 c). On the 7th day, the epidermal structure of the control group and the commercial glue group was still incomplete, and there was a large amount of inflammatory cell infiltration, while the epidermal structure of the HANB-TG group and the HANB-MOF-TG group was relatively complete, and the inflammatory response was significantly reduced. On the 14th day, the HANB-TG group and the HANB-MOF-TG group had formed a relatively complete epidermal layer, and the wound gap was significantly reduced, while the suture group and the commercial glue group were still not completely healed. In the glue group, there was a wide cavity in the wound due to undegraded residues, while the suture group had relatively good healing except that the dermis was not completely healed. These results indicate that the HANB-TG and HANB-MOF-TG groups can effectively promote the epithelialization process and reduce the inflammatory response, thereby accelerating wound repair. Collagen deposition is an important indicator of wound healing, so Masson trichrome staining was used to evaluate the collagen fiber formation in each group ( Figure 6d). On day 7, the collagen fibers in the control group were sparse and disorganized, while the collagen deposition in the HANB-TG and HANB-MOF-TG groups was denser. By day 14, the collagen in the HANB-TG and HANB-MOF-TG groups was more densely arranged and organized, demonstrating enhanced tissue remodeling. In contrast, the collagen fibers in the control and suture groups were still less uniform and incompletely remodeled. This result suggests that the HANB-TG and HANB-MOF-TG groups effectively promote extracellular matrix (ECM) remodeling, thereby accelerating the repair process of skin tissue. In summary, the HANB-TG and HANB-MOF-TG groups demonstrated excellent results in wound closure, tissue regeneration, and collagen deposition. Compared with traditional sutures and commercial glues, they possessed superior biocompatibility and adhesion properties, effectively promoting skin healing, and accelerating the wound healing process. These results suggest that this type of hydrogel has potential clinical application as a sutureless wound closure material.

[0060] 6. In vivo study of tissue regeneration and angiogenesis efficacy of composite hydrogels Angiogenesis is a crucial biological process in the wound repair process, which can provide oxygen and nutrients and promote tissue regeneration and healing. Therefore, the present invention detects angiogenesis markers CD31 and α-SMA by immunofluorescence staining to evaluate the effects of different hydrogel groups in promoting angiogenesis and tissue regeneration. The wound tissue was stained for CD31 and α-SMA on the 7th and 14th days respectively. CD31 is a marker of endothelial cells, reflecting the formation of new capillaries, while α-SMA is mainly expressed in smooth muscle cells and is a sign of vascular maturation. From the results ( Figure 7Representative images of anti-CD31 (green) and anti-α-SMA (red) immunofluorescence staining in different groups at days 7 and 14. Cell nuclei were stained with DAPI (blue). On day 7, CD31 and α-SMA expression was weak in all groups, indicating that angiogenesis was still in the early stages. Vascular marker expression was low in the control, suture, and commercial glue groups, suggesting that these treatments had limited effects on promoting early angiogenesis. However, CD31 and α-SMA expression was relatively enhanced in the HANB-TG and HANB-MOF-TG groups, demonstrating their ability to promote angiogenesis. On day 14, CD31 and α-SMA expression levels increased in all experimental groups. Vascular formation was still limited in the control, suture, and commercial glue groups, while CD31 and α-SMA expression was significantly enhanced in the HANB-TG and HANB-MOF-TG groups, indicating that they effectively promoted the formation and maturation of new blood vessels. The TG and MOF-TG groups also promoted angiogenesis to a certain extent, but the effect was not as great as that of the HANB-TG and HANB-MOF-TG groups. In summary, the HANB-TG and HANB-MOF-TG groups significantly promoted the formation and maturation of new blood vessels, providing a better blood supply for wound repair, thereby accelerating tissue regeneration and healing. This further demonstrates the excellent performance of this type of hydrogel in promoting angiogenesis and its potential clinical application value.

[0061] In summary, HANB-MOF-TG hydrogel combines TG, Ca 2+ The therapeutic effects of HANB-MOF-TG hydrogel are achieved through the following aspects: (1) through Ca 2 The coordination and complexation of calcium ions (Ca) and gallic acid produces a Ca-MOF with a porous structure and good biocompatibility; (2) the MOF continuously releases calcium ions, gallic acid, and TG enzyme, and continuously provides reducing conditions for TG; (3) TG enzyme promotes protein re-crosslinking in vivo, promoting tissue regeneration; (4) the HANB hydrogel forms a physical barrier to enhance adhesion and prevent secondary damage. In vitro and in vivo experiments showed that the HANB-MOF-TG hydrogel exhibits good biocompatibility, immediate adhesive hemostasis, and therapeutic healing effects, and is expected to provide a valuable option for suture-free wounds.

[0062] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0063] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A bioadhesive, characterized in that The bioadhesive is prepared by loading transglutaminase on Ca-MOF material and then adding HANB hydrogel to uniformly disperse the material. The Ca-MOF material is a calcium-containing metal organic framework formed by coordination cross-linking of calcium ions and catechol groups of gallic acid.

2. The bioadhesive according to claim 1, wherein The calcium ions are derived from CaCl2.

3. The bioadhesive according to claim 1, wherein The HANB hydrogel contains 0.25% to 4.7% by mass of LAP photoinitiator.

4. A method for preparing the bioadhesive according to claim 1, characterized in that: The steps include: CaCl2, gallic acid and ultrapure water are mixed uniformly to obtain a mixture, the pH of the mixture is adjusted to 8-12, and then heated at 100°C-120°C for 20-24 hours, and the precipitate is collected by centrifugation to obtain a Ca-MOF material; After Ca-MOF was dispersed in pure water, transglutaminase was added and ultrasonic cross-linking was performed to obtain a mixed solution. HANB hydrogel was dissolved in the mixed solution and uniformly dispersed to obtain HANB-MOF-TG composite hydrogel, i.e., bioadhesive.

5. The method for preparing the bioadhesive according to claim 4, characterized in that: The usage ratio of the CaCl2, gallic acid and ultrapure water is 18g-10g:30g-38g:500mL.

6. The method for preparing the bioadhesive according to claim 4, characterized in that: The added amount of the transglutaminase is 100 U / g to 200 U / g.

7. The method for preparing the bioadhesive according to claim 4, wherein: HANB hydrogel contains 0.25% LAP.

8. The method for preparing the bioadhesive according to claim 4, characterized in that: The ratio of Ca-MOF to pure water is 300 μg~500 μg:8 mL~12 mL.

9. Use of the bioadhesive according to any one of claims 1 to 3 in preparing materials for stopping bleeding and promoting wound healing.

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