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

Abstract

A tissue adhesion preparation system based on transglutaminase comprises an enzyme carrier unit which is a controlled release carrier composed of a biocompatible metal organic framework or microspheres and encapsulates TGase; a matrix unit selected from at least one curable matrix of a photosensitive hydrogel, a temperature-sensitive polymer or a pH-responsive gel; and an auxiliary crosslinking unit comprising a biopolymer having an active functional group or a derivative thereof. Through verification, the system disclosed by the invention shows excellent biocompatibility, immediate adhesion, hemostasis capability and therapeutic healing effect, and unification of mechanical strength, biological functions and traceless healing is realized in wound treatment without suture. When a wound occurs, the adhesive has adhesion to moist tissues, plays a role in bonding the wound, has a broad-spectrum antibacterial effect and a function of promoting tissue regeneration, promotes subsequent healing of the tissues and improves the rehabilitation process of a patient during wound healing, and is also beneficial to postoperative nursing and treatment.

Description

Technical Field

[0001] The present invention relates to a biomaterial, in particular to a transglutaminase-based biomaterial, which is used to prepare a tissue adhesive for wound adhesion and promoting tissue healing. Background Art

[0002] Currently, clinical wound closure relies primarily on surgical sutures and staples to achieve hemostasis, seal incisions, provide an antimicrobial barrier, and promote tissue healing (https: / / doi.org / 10.1016 / j.actbio.2021.09.054). However, suturing can damage delicate tissues, increase the risk of bacterial infection, and lead to excessive scar tissue production. Furthermore, suturing is a time-consuming process that requires high technical proficiency (https: / / doi.org / 10.1021 / acs.biomac.7b00969). To overcome these limitations, bioadhesives have been developed as alternatives to traditional sutures. The use of bioadhesives offers numerous benefits to patients and surgeons, such as shortened tissue healing time, reduced likelihood of infection, and fewer surgical complications (https: / / doi.org / 10.1039 / c9mh00157c).

[0003] First-generation bioadhesives, such as fibrin sealants and cyanoacrylate sealants, were initially introduced to achieve hemostasis and provide auxiliary support for sutures. Fibrin sealants exhibit soft tissue-like compliance but suffer from weak adhesive strength, while cyanoacrylate sealants, while possessing strong adhesion, are cytotoxic and possess unnatural hardness (https: / / doi.org / 10.1016 / j.cis.2020.102280). Therefore, there is an urgent need to develop advanced, multifunctional bioadhesives that can meet these critical requirements.

[0004] An ideal bioadhesive must possess several key properties, such as excellent tissue adhesion, high biodegradability, compatibility with the mechanical properties of the substrate, effective tissue integration and hemostasis, low toxicity to cells, and low inflammatory response. Although various biomaterials have been developed as bioadhesives for wound sealing and tissue adhesion, satisfactory properties have not yet been achieved.

[0005] Hydrogels, with their exceptional three-dimensional structure, high water absorption capacity, biocompatibility, and tissue-like mechanical properties, have become one of the most competitive materials in the field of wound healing. Compared to traditional dressings, hydrogels provide a moist environment during wound repair, offer physical protection, enhance exudate absorption, promote adhesion and remodeling of damaged tissue, facilitate targeted molecular delivery, and possess antibacterial, antioxidant, and anti-inflammatory properties. Furthermore, their excellent biodegradability reduces the risk of secondary injury caused by dressing changes, but their tensile strength still needs to be improved for clinical application.

[0006] Other studies have shown that metal-organic frameworks (MOFs) can achieve synergistic optimization of structure and function (https: / / doi.org / 10.1002 / adma.202007356). Polyphenols are a class of naturally occurring organic compounds that have been widely studied for their inherent anti-inflammatory, antibacterial, anti-aging, anti-cancer, and antioxidant properties. They also possess ultraviolet (UV) protection and free radical scavenging capabilities (https: / / doi.org / 10.1002 / advs.202101101). These supramolecular structures, known as metal-organic frameworks (MOFs), are primarily formed through the coordination of metal ions with polyphenol ligands. These metal ions impart diverse properties to metal-organic frameworks (MOFs), such as magnetic, radioactive, and catalytic properties (https: / / doi.org / 10.3390 / molecules28031186, https: / / doi.org / 10.1021 / acsnano.3c04556), making them highly promising for biomedical applications and the subject of extensive research. Metal polyphenol networks (MPNs) offer a rapid and facile approach to constructing multifunctional nanoplatforms, while the pH-responsiveness of MOFs facilitates targeted drug delivery and controlled release, enabling precise treatment of diseases. Furthermore, they possess excellent physical and chemical properties and good biocompatibility.

[0007] Composed of gallic acid and calcium ions (Ca 2+ ) can gradually degrade in an acidic microenvironment to achieve controlled release of calcium ions. Moreover, the released gallic acid exhibits a variety of biological properties, such as antioxidant, anti-inflammatory, anti-cancer, antibacterial, and anti-fibrin formation properties. Gong et al. prepared a hydrogel (Cur-Mg@PP) that can absorb exudate and has antibacterial properties, which incorporates a magnesium-polyphenol network containing curcumin to promote the healing process of burn wounds. Similarly, Ji et al. used lanthanide-based metal-organic frameworks as potential fluorescent agents for fluorescence-computed tomography (CT) dual-modality imaging, which helps to accurately locate the target site. Summary of the Invention

[0008] One object of the present invention is to provide a tissue adhesive preparation system based on transglutaminase (TGase), comprising:

[0009] (a) Enzyme carrier unit: a controlled-release carrier composed of a biocompatible metal-organic framework (MOF) or microspheres, encapsulating TGase;

[0010] (b) matrix unit: at least one curable matrix selected from a photosensitive hydrogel, a temperature-sensitive polymer, or a pH-responsive gel;

[0011] (c) Auxiliary cross-linking unit: comprising a biopolymer or a derivative thereof having an active functional group.

[0012] The metal ion of the enzyme carrier unit is selected from Ca 2+ 、Zn 2+ or Fe 2+ ;

[0013] The controlled-release carrier comprises an organic ligand, which has anti-oxidation or reducing properties.

[0014] In the present invention, the curable matrix satisfies at least one of the following conditions:

[0015] Cures within 30 seconds under UV / visible light;

[0016] Forms gel within 5 minutes at body temperature;

[0017] In situ cross-linking is initiated upon contact with physiological tissue.

[0018] In the present invention, the auxiliary cross-linking unit comprises:

[0019] At least one selected from hyaluronic acid, collagen, fibrin or their derivatives;

[0020] The derivatives are modified with amino, carboxyl, thiol or aldehyde groups.

[0021] Another object of the present invention is to provide a method for preparing the system of the present invention, comprising the steps of:

[0022] (i) preparing the enzyme carrier unit by coordination self-assembly or emulsification method;

[0023] (ii) compounding the enzyme carrier unit with the solidifiable matrix; and

[0024] (iii) Regulating the curing triggering conditions enables the system to obtain gradient adhesion properties.

[0025] After testing, the bonding system of the present invention satisfies any of the following conditions:

[0026] Wet adhesion strength ≥20kPa;

[0027] The in vitro degradation time can be controlled to 7-90 days;

[0028] Supports cell migration and proliferation.

[0029] Application of the system of the present invention in any of the following fields:

[0030] Surgical incision closure;

[0031] Emergency hemostasis of trauma;

[0032] tissue engineering scaffold fixation;

[0033] Wearable medical device fitting.

[0034] The system provided by the present invention integrates transglutaminase, calcium ions, gallic acid, and the healing properties of HANB to promote wound adhesion and healing. The therapeutic effects of the HANB-MOF-TG hydrogel are achieved through the following mechanisms: (i) Calcium ions bind and interact with gallic acid to form a porous calcium-based metal-organic framework (Ca-MOF) with excellent biocompatibility; (ii) the metal-organic framework (MOF) enables the sustained release of calcium ions, gallic acid, and transglutaminase, maintaining a reducing environment for enzyme activity; (iii) TG enzyme promotes the re-crosslinking of proteins in the body, promoting tissue regeneration; and (iv) the HANB hydrogel forms a physical barrier, enhancing adhesion and preventing secondary damage.

[0035] Mechanism studies have shown that the sustained release of calcium ions (Ca) from the calcium-based metal organic framework (MOF) structure 2+ ) synergistically with enzymatic cross-linking to activate fibroblast migration and angiogenesis.

[0036] In a full-thickness skin wound model in mice, the hydrogel of the present invention exhibited accelerated wound healing kinetics (on day 14, the wound healing rate was 30% higher than that of the control group) through tissue transglutaminase-mediated collagen fiber arrangement and extracellular matrix remodeling, while significantly inhibiting the formation of scar tissue (the expression of α-smooth muscle actin (α-SMA) was reduced by 60%).

[0037] Both in vitro and in vivo studies have demonstrated that HANB-MOF-TG hydrogel exhibits excellent biocompatibility, immediate adhesion, hemostasis, and therapeutic healing effects, achieving a unified combination of mechanical strength, biological functionality, and scarless healing in suture-free wound treatment. This makes it a suture-free wound closure material suitable for use in medical devices such as adhesives, bioglue, suture gels, or liquid bandages, meeting the needs of clinical applications. Its adhesion to moist tissues acts as a wound adhesive during wound healing, and its broad-spectrum antibacterial efficacy and ability to promote tissue regeneration during wound healing promote subsequent tissue healing, improve the patient's recovery process, and also facilitate postoperative care and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure 2 is a diagram of the synthesis and characterization of a calcium-based metal-organic framework; wherein: a is a schematic diagram of the synthesis process of Ca-MOF, b is a scanning electron microscope (SEM) image of Ca-MOF; c is an enlarged SEM image of the selected red area in Figure b; d is an SEM image of Ca-MOF and the corresponding elemental mapping of C, O and Ca; e is an X-ray diffraction (XRD) pattern of Ca-MOF powder, f is the UV-visible absorption spectrum of Ca-MOF, TG and TG-loaded metal-organic framework (MOF-TG), g is a statistical graph of cell viability after L929 cells were incubated with calcium-containing polyphenol nanocomposites (CaPN) for 48 hours;

[0039] Figure 2 Characterization diagram of HANB-MOF-TG hydrogel; wherein: a is a schematic diagram of the interaction between HANB-MOF-TG hydrogel and skin wound, b is a bright field photograph of the cross-linking effect of transglutaminase on gelatin and pork tissue, c is a photograph of in vitro adhesion test on pig skin, d is a statistical graph of the effect of calcium ions and metal organic framework (MOF) on transglutaminase activity, e is a graph of the tensile stress-strain test results of various hydrogels applied to pig skin, and f is the release curve of calcium ions in HANB-MOF-TG hydrogel;

[0040] Figure 3Figure 2 is a graph verifying the in vivo hemostasis and coagulation properties of Ca-MOF and HANB-MOF-TG; wherein, a is a photograph of the tail-cut site treated with various hemostatic agents, b is a photograph of the liver incision site treated with various hemostatic agents, c is a statistical graph of the hemostasis time of the rat liver trauma and tail-cutting experimental model (n=5), d is a statistical graph of the blood loss of the rat liver injury and tail-cutting experimental model (n=5), e is a photograph of blood samples incubated with phosphate buffered saline (PBS), Ca-MOF, MOF-TG, HANB-MOF-TG and deionized water (ddH2O), f is a statistical graph of the hemolysis rate of each sample (n=3), g is a photograph of the sample of blood clot formation after 5 minutes, and h is a statistical graph of the coagulation index of each material (n=3);

[0041] Figure 4 Figure 3 is a graph showing the experimental results of biocompatibility, antioxidant activity and in vitro cell migration behavior of HANB-MOF-TG hydrogel; wherein, a is a fluorescence microscopy image showing the live / dead staining of endothelial cells and fibroblasts after co-culture with TG, MOF-TG, HANB, HANB-TG and HANB-MOF-TG hydrogels for 48 hours; b is a fluorescence staining image of reactive oxygen species (ROS) in RAW264.7 cells (pretreated with lipopolysaccharide (LPS) for 24 hours before contact with various materials); c is a characterization image of the proliferation and migration ability of fibroblasts after co-culture with TG, MOF-TG, HANB, HANB-TG and HANB-MOF-TG hydrogels for 24 hours; d is a statistical graph of cell viability after co-culture of fibroblasts with different materials for 48 hours (n=3); e is a statistical graph of the quantitative evaluation of ROS fluorescence intensity (n=3); f is a quantitative statistical graph of the wound closure rate among each group (n=3);

[0042] Figure 5 Figure 2 is a graph showing the in vivo wound adhesion performance verification of HANB-MOF-TG hydrogel; wherein, a is a photograph of mouse skin wounds treated with TG, MOF-TG, HANB, HANB-TG, and HANB-MOF-TG, with sutures and medical glue as positive controls; b is a schematic diagram of the wound healing timeline at days 0, 1, 3, 7, and 14; c is a hematoxylin-eosin (H&E) staining of the wound area at days 7 and 14; d is a Masson's trichrome staining of the wound area at days 7 and 14 (n=3);

[0043] Figure 6Figure 1 shows the in vivo validation results of tissue regeneration and angiogenesis induced by TG, MOF-TG, HANB, HANB-TG, and HANB-MOF-TG. Immunofluorescence images show anti-CD31 (green) and anti-α-smooth muscle actin (α-SMA, red) staining in different groups on days 7 and 14. 4',6-diamidino-2-phenylindole (DAPI, blue) was used to counterstain cell nuclei.

[0044] Figure 7 Schematic diagram of the technical pathway of the enzyme-catalyzed double-cross-linked regenerative hydrogel used in the present invention to enhance wound adhesion and post-injury healing. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are intended only to illustrate the technical solution of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or equivalents should be included in the scope of the claims of the present invention.

[0046] Structurally, transglutaminase consists of an N-terminal β-pleated domain, a catalytic and regulatory core domain, and two C-terminal β-barrel domains. Upon activation, the two β-barrel domains extend outward, causing the entire protein structure to open and promote cross-linking of polymers containing amino and glutamic acid functional groups. This conformational change is induced by calcium ions (Ca 2+ ) binding and a reducing environment. Under low calcium concentrations, transglutaminase 2 (TG2) primarily plays a role in GTP / GDP binding signal transduction, while higher calcium levels enhance its transamidation activity (switch-on). Under oxidative stress (ROS), disulfide bonds formed between cysteine ​​(Cys) residues inactivate its transamidase activity, inhibiting TG2-mediated catalysis (switch-off) and halting cross-linking of polymers containing amine and glutamate functional groups.

[0047] The technical approach of the enzyme-catalyzed double-crosslinked regenerative hydrogel used in this embodiment to enhance wound adhesion and post-injury healing is as follows: Figure 7 As shown. Due to the controlled release characteristics of the metal organic framework structure, in this embodiment, TG is loaded onto the framework. Calcium ions (Ca 2+The sustained release of gallic acid not only promotes platelet activation and aggregation but also regulates and activates transglutaminase, prompting it to open its conformation. Gallic acid also creates a reducing environment for transglutaminase. This enzyme-catalyzed and photocrosslinked regenerative hydrogel demonstrated excellent elasticity and flexibility in a mouse skin incision adhesion model, as well as good biocompatibility, biodegradability, and minimal inflammatory response. This paves the way for the development of suture-free bioadhesives.

[0048] The various test methods used in the following examples of the present invention are specifically described as follows:

[0049] 1) Materials and cell culture

[0050] Gallic acid was purchased from Shanghai McLean Biochemical Technology Co., Ltd., China. Transglutaminase was purchased from Shanghai Qingrui Food Co., Ltd., China. HANB hydrogel was purchased from Shanghai Lingjiu Medical Device Co., Ltd. High-glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were provided by Shanghai Yuanpei Co., Ltd., China. Cell lines, including fibroblasts, human umbilical vein endothelial cells (HUVECs), and Raw264.7 cells, were cultured in a humidified incubator set at 37°C and 5% carbon dioxide using complete medium consisting of high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0051] 2) Preparation of calcium-based metal-organic framework (Ca-MOF) microspheres

[0052] Ca-MOF was synthesized using the method described in a previous paper (https: / / doi.org / 10.1021 / acs.accounts.0c00832). First, 1 gram of calcium chloride (CaCl2), 3.8 grams of gallic acid, and 50 milliliters of deionized water were mixed in a beaker and stirred for 10 minutes. Subsequently, a 10 mol / L sodium hydroxide (NaOH) solution was added to adjust the pH to 8, and the mixture was placed in a muffle furnace and heated at 120°C for 24 hours. Finally, the gray-black solid was collected by centrifugation (10,000 rpm, 10 minutes) and rinsed twice with ethanol and deionized water.

[0053] 3) Preparation of HANB-MOF-TG (composite) hydrogel

[0054] First, 40 mg of Ca-MOF was ultrasonically dispersed in pure water. Then, 200 units / gram of transglutaminase was added, and the resulting mixture was ultrasonically crosslinked for 2 hours. Next, 2.5 weight percent of HANB hydrogel (containing 0.25% photoinitiator LAP) was dissolved in the above solution and stirred at 37°C for 1 hour to obtain a uniformly dispersed hydrogel. HANB is a hyaluronic acid (HA) with an o-nitrobenzyl group (NB).

[0055] 4) Cytotoxicity assay

[0056] Mouse fibroblasts (L929) were seeded in 96-well plates at a density of 5000 cells per well. L929 cells were treated with different concentrations of Ca-MOF (20, 40, 60, 80, 100 and 120 μg / ml) for 48 hours, with five replicates for each concentration. The control group was not treated with Ca-MOF. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8). For CCK-8 detection of different materials, 200 microliters of HANB hydrogel, HANB-TG hydrogel or HANB-MOF-TG hydrogel were incubated in 10 ml of complete medium for 24 hours to prepare extracts. Fibroblasts (5000 cells per well) were then seeded into 96-well plates and co-cultured with extracts or a control group without any material for 48 hours. All other experimental steps were performed as previously described. Finally, cell viability was measured with the aid of a microplate reader.

[0057] 5) Live / dead cell double staining

[0058] Fibroblasts or human umbilical vein endothelial cells (HUVECs, 2×10 per well) 5 Cells were seeded in 12-well plates. The cells were co-cultured with tannic acid (TG), tannic acid-loaded metal-organic frameworks (MOF-TG), HANB hydrogels, HANB-TG hydrogels, HANB-MOF-TG hydrogels, or nothing. The medium was replaced with 400 μL of live / dead staining solution, and the cells were incubated in an incubator for 15 minutes. The supernatant was then carefully aspirated and photographed under a fluorescence microscope.

[0059] 6) In vitro migration assay

[0060] Fibroblasts (4 × 10 per well) 5Cells (100 μg / ml) were seeded in 6-well plates. When cells reached approximately 90% confluence, they were starved for 6 hours. A straight line was scratched across the center of the well using a sterile 200 μl pipette tip. The wells were then washed twice with phosphate-buffered saline (PBS), and the corresponding extracts (prepared in low-serum medium containing 1% fetal bovine serum) were added to the wells. Fibroblast migration at the scratch edge was observed and photographed after 0, 6, 12, and 24 hours of treatment.

[0061] 7) In vitro antioxidant test

[0062] To evaluate the antioxidant activity of the experimental groups, RAW cells (4 × 10 5 Cells (cells) were seeded in 6-well plates. Twenty-four hours later, all groups except the blank control group were treated with 250 ng / mL lipopolysaccharide (LPS) for 24 hours to induce oxidative stress. Reactive oxygen species (ROS) were quantified using the Bio-Tech Reactive Oxygen Species (ROS) Detection Kit. Following the manufacturer's instructions, 1 mL of 10 μmol / L DCFH-DA probe was added to each well. Images were taken under a fluorescence microscope.

[0063] 8) In vitro hemolysis test

[0064] Fresh whole blood was collected from 6-8 week old C57 mice and immediately transferred to heparinized anticoagulant tubes as described previously (https: / / doi.org / 10.1038 / s41467-023-35907-4). Whole blood was diluted with PBS to prepare a 5% (vol / vol) red blood cell (RBC) suspension. 0.5 ml of the RBC suspension was incubated with 0.5 ml of Ca-MOF (dissolved in PBS at a concentration of 0.5 mg / ml), MOF-TG, and 0.5 ml of hydrogel. Hemolytic activity was assessed using a microplate reader. After incubation of the sample at 37°C for 1 hour, the mixture was centrifuged (116 × g, 10 minutes), and the absorbance of the resulting supernatant at 540 nm was recorded. Deionized water and PBS diluted with 5.0% blood were used as positive and negative control samples, respectively. The hemolysis rate was calculated according to the following specified formula:

[0065] Hemolysis rate (%) = [(A sample - A negative control) / (A positive control - A negative control)] × 100%

[0066] The absorbance values ​​of the sample, negative control, and positive control are represented by: A sample (A sample), A control (A control), and A positive (A positive), respectively.

[0067] 9) Blood coagulation (coagulation index, BCI) test

[0068] Whole blood was collected from C57 mice and anticoagulated. 5 mg of Ca-MOF and MOF-TG samples were prepared, and the HANB-MOF-TG hydrogel was cut into square pieces (5 mm × 1 mm). Gauze was used as a positive control and its size was adjusted to be consistent with the hydrogel pieces. 10 μL of concentrated whole blood was incubated with each material group at 37°C for 5 minutes to initiate the coagulation process. After incubation, 2 ml of deionized water was carefully added to the centrifuge tube to lyse the uncoagulated red blood cells. The absorbance at 540 nm was recorded. The coagulation index (BCI) was then calculated according to the following specified formula:

[0069] BCI (%) = (A sample / A control) × 100%

[0070] The absorbance values ​​of the sample and control are indicated as "A sample" and "A control," respectively. In this case, the control was composed of 10 μl of purified whole blood mixed with 1.0 ml of deionized water.

[0071] 10) In vivo hemostasis studies

[0072] To evaluate the hemostatic ability of Ca-MOF, MOF-TG and HANB-MOF-TG composite hydrogels in vivo, experiments were conducted using rat liver incision and tail amputation models. Briefly, the experiment used SD rats aged 6-8 weeks. The amount of blood loss was measured by weighing the gauze, which was placed under the liver and a 1 cm long and 0.5 cm deep incision was made with a sterile scalpel. In the experimental group, the bleeding liver was treated with drugs coated with Ca-MOF, MOF-TG and HANB-MOF-TG (100 mg / kg, n=5), respectively. In the control group, no treatment was performed after liver bleeding, and the amount of blood loss and hemostasis time were recorded throughout the hemostasis process. In the tail amputation model, the tail was cut 5 cm from the tail with sterile scissors and exposed to air for 5 seconds. Subsequently, Ca-MOF, MOF-TG and HANB-MOF-TG (100 mg / kg, n=5) were applied to the wound. The control group (n=5) did not receive any treatment. The hemostasis time and total blood loss were recorded according to the above method.

[0073] 11) In vivo tissue adhesion studies

[0074] The in vivo tissue adhesion of the composite hydrogel was evaluated using a mouse dorsal skin incision model, using sutures and medical adhesives as positive controls

[40] . The backs and abdomens of 6-8 week-old male mice (n=4) were shaved and firmly fixed after anesthesia. After surgery, a 1.5 cm incision was made, and TG solution, MOF-TG solution, HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel were injected into the incision of the experimental group. In the suture group, the wound was sutured and removed after five days. The blank control group did not receive any treatment. Optical images of the wound were taken at predetermined time intervals (0 / 1 / 3 / 5 / 7 / 10 / 14 days), and tissue sections were collected for histological examination and staining.

[0075] 12) Histology and immunofluorescence staining

[0076] On days 7 and 14, wound tissue was harvested from the dorsal region of mice, fixed with 4% paraformaldehyde for at least 24 hours, embedded in paraffin, and sectioned at a minimum thickness of 5 μm. After dewaxing and rehydration, tissue sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome. To further assess angiogenesis and tissue repair, immunofluorescence staining was performed. After dewaxing and rehydration, sections underwent heat-induced antigen retrieval in phosphate-buffered saline at 95°C for 15 minutes, followed by application of blocking solution to reduce nonspecific antibody binding. Antibodies against CD31 (1:500) and α-smooth muscle actin (α-SMA, 1:500) were used, followed by corresponding secondary antibodies. Images were captured using a fluorescence microscope, and quantitative analysis was performed using ImageJ software.

[0077] 13) Statistical analysis

[0078] All experiments were repeated three times, and quantitative data are expressed as mean ± standard deviation. Comparisons between two groups were performed using an unpaired Student's t-test; comparisons of more than two groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Data were analyzed using GraphPad Prism 8.0 software. Statistical significance was determined based on the following p-values. "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001.

[0079] Example 1 Synthesis and Characterization of Calcium-based Metal-Organic Framework (Ca-MOF)

[0080] Gallic acid is a naturally occurring polyphenolic compound recognized for its anti-inflammatory, antioxidant, antibacterial, anticancer and hemostatic properties. Increasing its porosity can further optimize its hemostatic function. This embodiment aims to utilize calcium ions (Ca 2+) and gallic acid to construct a calcium-based metal-polyphenol network (Ca-MOF). This approach aims to retain the biological properties of gallic acid while promoting the controlled release of calcium ions and gallic acid, thereby enhancing its application in the biomedical field. The Ca-MOF composite was synthesized and characterized by a hydrothermal method. This porous material uses gallic acid as an organic ligand and calcium ions as metal nodes ( Figure 1 a). To further explore its biomedical potential, Ca-MOF was used as a carrier to load tannic acid (TG), forming a composite MOF-TG for targeted drug delivery applications. Scanning electron microscopy (SEM) images confirmed the formation of a well-defined metal-organic framework (MOF) structure through metal coordination between calcium ions and gallic acid, and additional π-π stacking interactions enhanced the stability of the material. The resulting structure exhibited a uniform spherical shape with an average diameter of approximately 10 microns ( Figure 1 b and Figure 1 c). Its surface is irregular, with obvious gaps between the rectangular crystal regions, indicating a highly porous structure that enhances the water absorption capacity of Ca-MOF. The final MOF precipitated in the form of gray powder, forming spherical microparticles with a porous structure, showing excellent stability and biocompatibility. In order to verify the synthesis process and composition of Ca-MOF, energy dispersive X-ray spectroscopy (EDS) analysis was performed, confirming that carbon (C), calcium ions (Ca 2+ ) and oxygen (O) are uniformly distributed in the Ca-MOF structure ( Figure 1 d). X-ray diffraction (XRD) analysis further revealed obvious characteristic peaks ( Figure 1 e), confirming the successful synthesis of Ca-MOF. Spectral analysis showed that tannic acid (TG) and Ca-MOF showed obvious absorption peaks at 265 nm and 290 nm, respectively. Ca-MOF also had an absorption peak at 280 nm, indicating that tannic acid was successfully loaded on Ca-MOF ( Figure 1 f).

[0081] To evaluate the cytotoxicity of Ca-MOF, CCK-8 assay was performed on the mouse fibroblast cell line L929. At a concentration of 120 μg / mL, the Ca-MOF solution showed no significant cytotoxicity to the fibroblasts ( Figure 1 g). The optimal concentration of Ca-MOF for promoting fibroblast proliferation was determined to be 80 μg / mL.

[0082] Therefore, unless otherwise stated, subsequent experiments used a concentration of 80 μg / mL of Ca-MOF. These results highlight the structural integrity and multifunctionality of Ca-MOF, providing promising prospects for the development of drug-loaded sustained-release systems and advanced biomedical materials.

[0083] Example 2 Properties of HANB-MOF-TG Hydrogel

[0084] Hyaluronic acid (HA) contains hydrophilic groups such as carboxyl and hydroxyl groups in its molecular structure, which enables it to absorb wound exudate and enhance cell adhesion. Modification of the HA polymer backbone by introducing various functional groups can not only control its degradation rate but also enhance its mechanical strength, rheological behavior, and swelling properties.

[0085] In order to further enhance the adhesion of hyaluronic acid to the skin, this embodiment introduces a group - o-nitrobenzyl (NB) into hyaluronic acid, so that it can react with the amino group (-NH2) in the skin tissue under ultraviolet irradiation to form a stable imine structure with a C=N bond. In addition, transglutaminase (TG) can promote the acyl transfer reaction between the γ-carboxamide group of the glutamine residue and the ε-amino group of lysine and other primary amines, forming a stable isopeptide bond through intermolecular or intramolecular cross-linking. This enhances the cross-linking strength of hyaluronic acid and, combined with the ultraviolet-induced imine bond, gives it strong adhesion to skin tissue ( Figure 2 a).

[0086] Using metal organic framework (MOF) structure to realize the synthesis of gallic acid and calcium ion (Ca 2+ ) is continuously released, providing optimized reaction conditions for enzymatic cross-linking, thereby promoting wound healing.

[0087] In order to visually observe the catalytic effect of transglutaminase (TG), this example compares the experimental results of using gelatin alone and the experimental results of adding transglutaminase and calcium ions in sequence ( Figure 2 b) The addition of transglutaminase and calcium ions significantly enhanced the cross-linking reaction of gelatin. When transglutaminase alone was added, gelatin cross-linked within one minute, while the subsequent addition of calcium ions further accelerated the process, causing cross-linking to occur within 30 seconds, significantly increasing the cross-linking speed. Similar results were observed in pork tissue, which is rich in myofibrils and collagen, further confirming the key role of transglutaminase in the cross-linking process and the additional enhancing effect of calcium ions.

[0088] In order to further study the effect of transglutaminase on the adhesion of ex vivo skin, this example selected pig skin, which is rich in collagen and very similar to human tissue, to evaluate its adhesion properties ( Figure 2c) Transglutaminase enzymatic activity can be measured by introducing appropriately labeled primary amines into protein substrates containing γ-glutamine groups. Previous studies have shown that transglutaminase activity is allosterically activated at calcium concentrations of 100 micromolar or higher but is inhibited by guanine nucleotides. At low calcium concentrations, transglutaminase in the cytoplasm is generally believed to exist in a closed conformation that is inactive.

[0089] This example also quantitatively evaluated the activity of transglutaminase under various conditions and found that calcium ions effectively enhanced the activity of transglutaminase. In addition, the metal organic framework structure synthesized with gallic acid did not weaken the activity of transglutaminase. On the contrary, it was able to continuously release gallic acid and calcium ions, thereby maintaining a reducing environment at the site of inflammatory wounds and continuously enhancing enzyme activity ( Figure 2 d).

[0090] To evaluate adhesion properties, 2 cm incisions were made in pig skin and treated with various agents: transglutaminase (TG), tannic acid-loaded metal-organic framework (MOF-TG), hyaluronic acid with NB groups (HANB), HANB-TG, and HANB-MOF-TG. An untreated control group was also included. After 30 minutes, only slight adhesion was observed in the transglutaminase and MOF-TG groups. In contrast, the HANB, HANB-TG, and HANB-MOF-TG groups achieved rapid crosslinking and adhesion within 30 seconds, demonstrating excellent adhesion properties with virtually invisible incision lines.

[0091] Tensile tests were performed on pig skin treated with HANB, HANB-TG, HANB-MOF, and HANB-MOF-TG to evaluate the adhesion strength of the hydrogels ( Figure 2 e). The results show that the group containing HANB-MOF-TG has the strongest resistance to tensile force.

[0092] The release curve of calcium ions in HANB-MOF-TG hydrogel is shown in Figure 2. Figure 2 As shown in (f), calcium ions were stably released from day 1 to day 14, ensuring the long-term optimization of the cross-linking process.

[0093] In summary, HANB-MOF-TG hydrogel utilizes the controlled release properties of metal-organic frameworks to enhance adhesion while maintaining a stable enzymatic cross-linking reaction, providing an advanced biomaterial solution for tissue repair.

[0094] Example 3 Hemolysis and in vitro hemostasis evaluation

[0095] Bleeding is a major problem caused by injuries, trauma, and surgical procedures. Uncontrolled bleeding is one of the leading causes of death in traumatic events. Therefore, bioadhesive materials with hemostatic properties are crucial to prevent excessive bleeding and promote wound healing.

[0096] This example verifies the hemostatic ability of HANB-MOF-TG, including its hemostatic effect in vitro and in vivo, coagulation time, blood compatibility, and potential hemostatic mechanism.

[0097] The in vivo hemostatic effects of each material were evaluated using rat tail amputation and liver injury models. Previous research has shown that Ca-MOF exhibits excellent hemostatic effects by forming blood clots within the microparticles and at the wound site. Figure 3 As shown in Figures 3a and 3b, after application of the hydrogel and 30 seconds of UV irradiation, the wound was effectively sealed. In both injury models, a significant reduction in blood loss was observed, demonstrating its excellent hemostatic properties.

[0098] In addition, blood coagulation index (BCI) tests were performed to measure the effect of these materials on blood coagulation, while hemolysis tests were used to assess blood compatibility. Figure 3 c and Figure 3 d) shows differences among the control, Ca-MOF, MOF-TG, and HANB-MOF-TG groups in the liver injury and tail amputation models. In the liver injury model, the Ca-MOF group (0.857±0.085 g), MOF-TG group (0.677±0.095 g), and HANB-MOF-TG group (0.283±0.047 g) showed significantly reduced blood loss compared to the control group (1.897±0.10 g) and Celox group (1.03±0.133 g), with the HANB-MOF-TG group demonstrating the most effective hemostatic ability. Similarly, in the tail amputation model, the HANB-MOF-TG group had the lowest blood loss (0.496 ± 0.055 g) compared with the control group (2.85 ± 0.18 g), the Ca-MOF group (1.717 ± 0.076 g), and the MOF-TG group (1.423 ± 0.068 g).

[0099] The coagulation index is a quantitative measure of the clotting ability of different materials. Higher coagulation index values ​​indicate slower clotting. The ability of blood to form clots is assessed by measuring the absorbance at 540 nm and determining the coagulation index value. Figure 3 g and Figure 3h shows that the relative coagulation index of the Ca-MOF group was approximately 46.3%, while that of the MOF-TG group was 42.7%, similar to the 42% of gauze. The coagulation index of the HANB-MOF-TG group was significantly lower (26%), indicating that it has the most effective hemostatic ability among all the tested materials.

[0100] Blood compatibility is a prerequisite for the in vivo application of biomaterials. Hemolysis tests were performed using fresh anticoagulated blood, with deionized water (dd-H2O) as a positive control. As expected, hemolysis was observed in the deionized water control group, while no hemolysis was detected in the PBS group, Ca-MOF group, MOF-TG group, or HANB-MOF-TG group ( Figure 3 e). Quantitative analysis further confirmed that the hemolysis rate of the HANB-MOF-TG group was far below the safe range (<5%) ( Figure 3 f). Although the inherent color of the Ca-MOF powder may have potentially interfered with the results, the overall results indicate that HANB-MOF-TG has excellent blood compatibility and efficient hemostatic potential.

[0101] Example 4 In vitro cytotoxicity, antioxidant and anti-inflammatory effects

[0102] The applicability of biomaterials in tissue repair depends largely on their cytocompatibility, antioxidant properties, and ability to promote wound healing. This study aimed to evaluate the cytotoxicity, biocompatibility, and antioxidant properties of tannic acid (TG), tannic acid-loaded metal-organic frameworks (MOF-TG), hyaluronic acid with NB groups (HANB), HANB-TG, and HANB-MOF-TG, and further investigate their effects on cell proliferation and migration to validate their potential applications in wound healing.

[0103] Tannic acid (0.1%), MOF-TG (80 μg / ml), HANB (200 μl), HANB-TG (200 μl), and HANB-MOF-TG (200 μl) were immersed in Dulbecco's modified Eagle's medium (DMEM) for 24 hours to prepare extracts for in vitro experiments. The cytotoxicity of these extracts was then evaluated by the CCK-8 method. The results showed that compared with the control group, tannic acid, MOF-TG, HANB, HANB-TG, and HANB-MOF-TG did not show obvious cytotoxicity, and the cell viability of all experimental groups was comparable to that of the control group, showing excellent biocompatibility ( Figure 4 d).

[0104] In addition, fibroblasts and human umbilical vein endothelial cells (HUVECs) were co-cultured with the hydrogel for 48 hours, and then live / dead cell staining was performed to further verify the biocompatibility. The results showed that the cells in all experimental groups remained viable and no obvious cell death was observed ( Figure 4 a).

[0105] The antioxidant potential of these materials was verified by evaluating the reactive oxygen species (ROS) levels using a lipopolysaccharide (LPS)-induced oxidative stress model. Fluorescence imaging and quantitative analysis of fluorescence intensity were used to evaluate the effects of these materials on the ROS levels. Figure 4 b, Compared with the lipopolysaccharide group, the experimental group containing metal-organic frameworks and HANB hydrogels showed significantly reduced reactive oxygen species levels, indicating significant antioxidant and anti-inflammatory properties. This suggests that these materials can reduce oxidative stress in the cellular microenvironment and alleviate inflammation.

[0106] Quantitative analysis of fluorescence intensity further confirmed this trend. Figure 4 e The results showed that the reactive oxygen levels in the lipopolysaccharide + MOF-TG group, lipopolysaccharide + HANB group, lipopolysaccharide + HANB-TG group and lipopolysaccharide + HANB-MOF-TG group were significantly lower than those in the lipopolysaccharide group (p<0.0001).

[0107] The results of the scratch test showed that all experimental groups showed varying degrees of cell migration, and the wounds gradually closed over time ( Figure 4 c). Compared with the control group and other experimental groups, the cell migration improvement in the HANB-TG group and HANB-MOF-TG group was the most significant.

[0108] Quantitative analysis of wound healing rates further supported these findings. Figure 4 fThe results showed that compared with the control group, the HANB-TG group and HANB-MOF-TG group significantly accelerated wound healing at 6 hours, 12 hours and 24 hours (p < 0.01), indicating their effectiveness in promoting wound repair.

[0109] These experimental results demonstrate that HANB-MOF-TG possesses excellent biocompatibility, antioxidant capacity, and potential to promote cell migration. Under lipopolysaccharide-induced oxidative stress, the material effectively reduced reactive oxygen species (ROS) levels, demonstrating its protective effect within the cellular microenvironment. Furthermore, scratch assay results further support HANB-MOF-TG's remarkable ability to promote cell migration, highlighting its potential applications in tissue repair and wound healing.

[0110] Example 5 In vivo skin adhesion and wound healing assessment

[0111] In addition to hemostatic properties, tissue adhesion is also crucial for wound repair. An ideal wound closure material should have strong adhesion, biocompatibility, and be able to effectively promote tissue regeneration.

[0112] In this example, the adhesion ability of the composite hydrogels was evaluated in vivo using a mouse skin incision model and compared with surgical sutures and commercially available medical adhesives (based on cyanoacrylate) to assess their potential as suture-free wound closure materials.

[0113] A 1.5 cm incision was made in the dorsal skin of mice. 30 μL of tannic acid (TG) solution, tannic acid-loaded metal-organic framework (MOF-TG) solution, hyaluronic acid (HANB) hydrogel with NB groups, HANB-TG hydrogel, and HANB-MOF-TG hydrogel were injected into the incision site as experimental groups. A surgical suture group, a commercially available adhesive group, and a control group were also used for comparison.

[0114] Figure 5 aThe results showed that HANB hydrogel, HANB-TG hydrogel, and HANB-MOF-TG hydrogel exhibited good adhesion to moist wound tissue. After 7 days of treatment, the wounds in the experimental groups closed well, while the wounds in the control group healed more slowly, with significant inflammation and incomplete wound closure. Although the suture group effectively closed the wound, it left noticeable suture marks and scars. The commercially available adhesive group exhibited some adhesion but still showed signs of inflammation. In contrast, the HANB-TG and HANB-MOF-TG groups achieved the best wound closure and minimal scarring, indicating higher healing efficiency. The TG and MOF-TG groups also showed improvement compared to the control group, but the results were not as good as those of the HANB-TG and HANB-MOF-TG groups.

[0115] By day 14, the HANB-MOF-TG group achieved almost scar-free healing, outperforming the other experimental groups. Quantitative analysis of wound contraction and closure rate ( Figure 5 b) shows that the wound contraction in the HANB-TG and HANB-MOF-TG groups was the most significant, with statistically significant differences over time compared to the control and other experimental groups. This suggests that the hydrogel formulation not only adheres to the wound but also promotes tissue remodeling and accelerates the healing process.

[0116] The wound tissues were histologically examined by hematoxylin-eosin (H&E) staining. Figure 5c). On day 7, the epidermal structure of the control and commercial adhesive groups remained incomplete, with extensive inflammatory cell infiltration. In contrast, the epidermal structure of the HANB-TG and HANB-MOF-TG groups was more complete, and inflammation was significantly reduced. By day 14, the HANB-TG and HANB-MOF-TG groups had formed a more complete epidermal layer, and the wound gap was significantly reduced, while the suture and commercial adhesive groups had not yet fully healed. In the adhesive group, undegraded residues led to a wider wound gap, while the healing of the suture group was relatively improved, although the dermis layer remained partially unhealed. These results indicate that the HANB-TG and HANB-MOF-TG groups effectively promoted epithelialization and reduced inflammation, thereby accelerating wound healing.

[0117] Collagen deposition is an important indicator of wound healing. Masson's trichrome staining was used to assess the formation of collagen fibers ( Figure 5 d). On day 7, collagen fibers in the control group were sparse and disorganized, while collagen deposition in the HANB-TG and HANB-MOF-TG groups was denser. By day 14, collagen fibers in the HANB-TG and HANB-MOF-TG groups were more tightly packed and organized, indicating enhanced tissue remodeling. In contrast, collagen fibers in the control and suture groups were less uniformly arranged, indicating incomplete remodeling.

[0118] These results indicate that HANB-TG and HANB-MOF-TG hydrogels effectively promoted the reconstruction of the extracellular matrix (ECM), thereby accelerating the repair of skin tissue.

[0119] In summary, HANB-TG and HANB-MOF-TG hydrogels demonstrated excellent performance in wound closure, tissue regeneration, and collagen deposition. Compared with traditional sutures and commercially available adhesives, they exhibited improved biocompatibility and adhesion, effectively promoted skin healing, and accelerated the wound healing process. These findings suggest that these hydrogels have potential clinical applications as suture-free wound closure materials.

[0120] Example 6 In vivo tissue regeneration and angiogenesis assessment

[0121] Angiogenesis plays a crucial role in wound healing, promoting tissue regeneration and repair by providing essential oxygen and nutrients. Therefore, this study evaluated the angiogenesis-related markers CD31 and α-smooth muscle actin (α-SMA) expression by immunofluorescence staining to assess the potential of different hydrogel formulations to promote angiogenesis.

[0122] On days 7 and 14 after treatment, wound tissue samples were stained for CD31 and α-SMA. CD31 is a marker of endothelial cells, indicating newly formed capillaries, while α-SMA is primarily found in smooth muscle cells and serves as a marker of vascular maturation.

[0123] like Figure 6 As shown, on day 7, all groups showed relatively low expression levels of CD31 and α-SMA, indicating that angiogenesis was still in its early stages. The control, suture, and commercial adhesive groups showed minimal expression of vascular markers, suggesting that these treatments had limited stimulating effects on early angiogenesis. In contrast, the HANB-TG and HANB-MOF-TG groups showed relatively high expression of CD31 and α-SMA, suggesting their potential to promote angiogenesis.

[0124] By day 14, CD31 and α-SMA expression levels increased in all experimental groups. However, vascularization remained limited in the control, suture, and commercial adhesive groups. Notably, expression of these two markers was significantly enhanced in the HANB-TG and HANB-MOF-TG groups, suggesting their ability to promote angiogenesis and vascular maturation. While the TG and MOF-TG groups also contributed to angiogenesis to some extent, their effects were less pronounced than those in the HANB-TG and HANB-MOF-TG groups.

[0125] These results demonstrate that HANB-TG and HANB-MOF-TG hydrogels significantly promote angiogenesis and vascular maturation, providing improved blood supply to wound sites and accelerating tissue regeneration and healing. These results highlight the remarkable angiogenic capacity of these hydrogels and suggest their broad clinical application prospects in wound healing and tissue regeneration.

Claims

1. A tissue adhesive preparation system based on transglutaminase, characterized in that include Enzyme carrier unit: a controlled-release carrier composed of a biocompatible metal-organic framework or microspheres, encapsulating TGase; Matrix unit: at least one curable matrix selected from photosensitive hydrogel, temperature-sensitive polymer or pH-responsive gel; and Auxiliary cross-linking unit: contains biopolymers or their derivatives with active functional groups.

2. The system according to claim 1, characterized in that The metal ion of the enzyme carrier unit is selected from Ca 2+ 、Zn 2+ or Fe 2+ .

3. The system according to claim 1, characterized in that The controlled-release carrier contains an organic ligand, which has antioxidant or reducing properties.

4. The system according to claim 1, characterized in that The curable matrix satisfies at least one of the following conditions: Cures within 30 seconds under UV / visible light; Forms gel within 5 minutes at body temperature; In situ cross-linking is initiated upon contact with physiological tissue.

5. The system according to claim 4, characterized in that The auxiliary cross-linking unit comprises: At least one selected from hyaluronic acid, collagen, fibrin or their derivatives; The derivatives are modified with amino, carboxyl, thiol or aldehyde groups.

6. A method for producing the system according to any one of claims 1 to 4, characterized in that Including in order: i. Preparation of enzyme carrier units by coordination self-assembly or emulsification method; ii. compounding the enzyme carrier unit with the solidifiable matrix; and iii. Regulate the curing trigger conditions to enable the system to obtain gradient adhesion properties.

7. Application of the system according to any one of claims 1 to 4 in any of the following fields: Surgical incision closure; Emergency hemostasis of trauma; tissue engineering scaffold fixation; Wearable medical device fitting.

8. The use according to claim 7, characterized in that The system satisfies any of the following conditions: Wet adhesion strength ≥20kPa; The in vitro degradation time can be controlled to 7-90 days; Supports cell migration and proliferation.

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