Nanofiber membrane structured to have multifunctional double layer for preventing post-surgical leakage

Abstract

The present invention relates to: a hemostatic double layer membrane comprising alginate, gelatin and polycaprolactone; and a manufacturing method therefor. A multifunctional AG-thrombin (TH) / PG-mitomycin-C (MC) membrane comprising TH and MC, of the present invention, has the properties of effectively protecting suture lines from being ruptured and effectively preventing post-srugical pancreatic leakage, and thus the multifunctional nanofiber double layer membrane can be used as a suitable substitute during pancreatic surgery.

Description

A multifunctional double-layer nanofiber membrane for postoperative leakage prevention.

[0001] The present invention relates to a double-layer membrane comprising alginate, gelatin and polycaprolactone having a blood or intestinal fluid leakage prevention function, a hemostatic function and a tissue adhesion prevention function, and a method for producing the same, and more particularly, to a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function and a tissue adhesion prevention function around a surgical site after surgery, and a method for producing the same.

[0002] Tissue fluid leakage resulting from iatrogenic trauma, accidents, and surgical procedures can lead to serious consequences, including acute necrotizing pancreatitis, bleeding, infection, and even death. Furthermore, wound closure procedures such as suturing and ligation cause additional tissue damage, increasing capillary fragility. Therefore, commercially available waterproofing materials, such as fibrin glue and polyglycolic acid, are widely used in these situations. However, a standard technique for preventing pancreatic leakage, which frequently occurs after open surgery, remains unclear. Controlling pancreatic leakage is challenging due to factors such as low elasticity, inability to adhere to irregularly shaped organs, and inability to promote pancreatic regeneration. Materials used to prevent pancreatic leakage should enhance tissue regeneration, wound healing, flexibility, and adhesion to the pancreatic margins.

[0003] To address the existing problems, the present invention fabricated a novel double-layer (AG-TH / PG-MC) multifunctional membrane using electrospinning technology. The inner layer was fabricated from a combination of sodium alginate (A), gelatin (G), and thrombin (TH0). Alginate is a natural polymer derived mainly from brown algae, and has biocompatibility, low toxicity, low price, and Ca 2+ It has been studied and used in many medical fields due to its characteristics such as rapid gelation when a double-hybridized state such as this exists.

[0004] Gelatin, a natural water-soluble polymer derived from collagen, is one of the most actively studied materials as a tissue adhesive due to its advantageous natural properties. It is a relatively inexpensive and widely available structural protein with inherent properties such as biodegradability, bioactivity, and biocompatibility, making it suitable for use in the medical field.

[0005] Thrombin and gelatin-based coagulants can be used together to effectively maintain blood coagulation while achieving excellent biocompatibility and ease of manufacture. Therefore, the present invention developed an inner layer (AG-TH) that provides effective adhesion, blood coagulation, and a natural wound healing membrane. Thrombin is a coagulation factor and enzyme that functions as a key factor in blood coagulation.

[0006] This substance activates platelets, regulates fibroblast proliferation and fibrin deposition, and controls cell migration through protease activation. Furthermore, the outer layer (PG-MC) was developed using a combination of polycaprolactone (P), gelatin (Gel), and mitomycin-C (MC). A nanofiber membrane was developed using an electrospinning process using a biodegradable polymer called PCL. PCL has relatively high mechanical properties and is actively used in tissue engineering. However, the slow degradation of PCL can lead to fibrosis due to tissue adhesion to other organs during wound healing. Therefore, to enhance the antifibrotic properties of the membrane, the anticancer drug mitomycin-C was incorporated into the PG-MC layer. MC possesses antibiotic properties and can suppress fibrosis and angiogenesis for several weeks, thereby reducing tissue adhesion. MC can also inhibit the proliferation of TGF-beta receptors and fibroblasts. These mechanisms help prevent tissue adhesion to other organs. However, most research on these common combinations of substances has focused on wound healing, anti-adhesion, or blood coagulation. To date, research on multifunctional bilayer membranes that simultaneously prevent pancreatic leakage and restore normal pancreatic function is insufficient.

[0007] Therefore, the present invention developed a bilayer multifunctional membrane using electrospinning technology to prevent postoperative pancreatic leakage. It was hypothesized that cross-linking the AG-TH layer with a calcium chloride solution would increase the mechanical strength and adhesive properties of the membrane. Furthermore, the cumulative release of MC from the PG-MC layer was designed to exhibit anti-adhesive properties. The explosive release of TH is expected to have a multi-axis effect on blood clotting time, and TH added with gelatin is expected to significantly increase collagen activity, thereby promoting fibroblast proliferation. Therefore, to evaluate the multifunctionality of the scaffold, cytocompatibility, animal testing, hemocompatibility, and adhesion tests were performed. In silico molecular simulation studies verified the antifibrotic activity of the membrane, confirming that the bilayer multifunctional membrane represents a promising approach for preventing postoperative pancreatic leakage.

[0008] The purpose of the present invention is to provide a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, including an inner layer comprising alginate and gelatin; and an outer layer comprising polycaprolactone and gelatin.

[0009] Another object of the present invention is to provide a method for producing a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, the method comprising the steps of: adding alginate and gelatin to a first solution to produce an inner layer comprising alginate and gelatin; adding polycaprolactone and gelatin to a second solution to produce an outer layer comprising polycaprolactone and gelatin; and spraying an electrospinning solution onto the inner layer comprising alginate and gelatin.

[0010] Another object of the present invention is to provide an osteoinductive bilayer membrane comprising polycaprolactone and gelatin manufactured by the above manufacturing method.

[0011] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0012] Pancreatic leakage due to postoperative pancreatitis is a dangerous surgical complication that can lead to bleeding, infection, and even death. In this invention, we fabricated a multifunctional bilayer nanofibrous membrane loaded with mitomycin-C and thrombin, consisting of a combination of alginate, PCL, and gelatin, to address suture rupture-induced leakage, promote blood coagulation, accelerate wound healing, and prevent postoperative tissue adhesion. The inner and outer layers were formed using electrospinning. The inner layer was cross-linked with a CaCl2 solution to enhance adhesive properties. The developed bilayer nanofibrous membrane demonstrated high biodegradability and adherence to tissue surfaces due to the reduced fiber diameter and water contact angle caused by the gelatin and alginate in the inner layer, making the membrane more hydrophilic. This resulted in a highly biodegradable membrane and a strong adhesion to tissue surfaces. Biocompatibility and hemostatic properties were evaluated, demonstrating the bilayer's effective hemostatic properties, demonstrating higher cell proliferation. Cytocompatibility testing confirmed that the bilayer covered the wound, preventing suture rupture and leakage, suppressing bleeding, and reducing the incidence of postoperative tissue adhesion. Therefore, it was confirmed that the double-layer multifunctional nanofiber membrane has suitable properties for preventing postoperative pancreatic leakage.

[0013]

[0014] Hereinafter, the present invention will be described in more detail.

[0015]

[0016] The present invention provides a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, including an inner layer comprising alginate and gelatin; and an outer layer comprising polycaprolactone and gelatin.

[0017] In the present invention, the double-layer membrane has a function of preventing blood or serous fluid leakage, a hemostatic function, and a function of preventing tissue adhesion. Preferably, the double-layer membrane has a function of preventing and preventing blood or serous fluid leakage after surgery, a function of hemostasis of blood or serous fluid, and a function of preventing adhesion of tissues around the surgical site.

[0018] Pancreatic fluid leakage after pancreatic cancer surgery is a complication that can lead to bleeding, infection, and even life-threatening complications in severe cases. Therefore, the present invention aims to resolve intestinal anastomotic leakage by developing a multifunctional double-layer nanofiber membrane with a unique composition, promoting antibacterial, hemostasis, and tissue regeneration, and preventing tissue / organ adhesion, a challenging surgical problem. Therefore, in organ transplantation, severe inflammation and adhesion caused by intestinal fluid leakage in anastomoses of all organs in the human body, such as vascular anastomoses, entero-biliary anastomoses, esophageal, small-bowel, and ureteral anastomoses, have a direct negative impact on the patient's prognosis, and the present invention aims to resolve these issues.

[0019] Meanwhile, conventionally used leak-preventing therapeutic materials such as fibrin glue and polyglycolic acid have poor adhesion and low elasticity due to the irregular surface and shape of human tissues / organs, making it difficult to prevent leakage of blood or intestinal fluid. In addition, these therapeutic materials require not only excellent elasticity and flexibility, but also adhesiveness to living tissues for convenient clinical application and improved efficacy, and rapid hemostasis and rapid tissue regeneration for post-surgical recovery. Therefore, the present invention has produced a novel double-layer membrane composed of inner and outer nanofibers using electrospinning technology to solve the above-mentioned existing clinical problems.

[0020] In the present invention, the double-layer membrane can be used for preventing leakage, hemostasis, and adhesion after surgical operations.

[0021] In the present invention, the double-layer membrane can be manufactured into a double-layer structured nanofiber membrane using an electrospinning process.

[0022] In the present invention, the inner layer, which is attached to the anastomotic sites of various organs and the resected cancer tissues constituting the bilayer membrane, is ionically cross-linked with alginate-gelatin components loaded with thrombin. This maximizes hydrophilicity, implements a nanofiber structure, and has mechanical properties sufficiently applicable to clinical applications. Specifically, the inner layer, which comes into direct contact with human tissues or organs, is composed of alginate extracted from brown algae and gelatin, a water-soluble natural polymer, and is ionically cross-linked to increase hydrophilicity and maximize adhesiveness to the surgical site. In addition, tissue regeneration and hemostatic ability are significantly improved due to cell adhesion and migration in the inner layer and rapid release of thrombin. It was also demonstrated that the bilayer membrane covers the surgical site well, effectively preventing suture rupture and pancreatic fluid leakage after surgery.

[0023] In the present invention, the outer layer constituting the double-layer membrane is a nanofiber composed of polycaprolactone and gelatin components, and is loaded with mitomycin-C, thereby preventing antibacterial activity through sustained release and tissue / long-term adhesion.

[0024] In the present invention, the double-layer membrane is a technology that completely blocks the peeling phenomenon between the inner and outer layers through optimization of the composition between the double layers described above, and optimizes physical, chemical, and mechanical properties as well as in vivo properties. According to an embodiment of the present invention, the double-layer membrane of the present invention was proven to be a multifunctional surgical treatment material such as antibacterial, hemostasis, tissue regeneration, and adhesion prevention through in-vitro experiments, in-vivo experiments, and molecular dynamics simulation results.

[0025] According to one embodiment of the present invention, the bilayer membrane of the present invention showed excellent cell proliferation as a result of cytocompatibility and hemostasis evaluation and confirmed excellent hemostatic properties, and in an animal transplantation test evaluation, it was confirmed that the bilayer membrane was well attached to the site of organ resection, was effective in preventing pancreatic fluid leakage and hemostasis, and innovatively reduced postoperative adhesion phenomenon.

[0026] According to one embodiment of the present invention, the double-layer membrane of the present invention exhibited anti-adhesion properties, which are a difficult problem in surgical operations, and was confirmed to have a superior effect in a pancreatic resection animal model compared to Seprafilm, an American product most widely used in existing clinical settings.

[0027] In the present invention, the inner layer including alginate and gelatin may further include thrombin.

[0028] In the present invention, the outer layer comprising polycaprolactone and gelatin may further comprise mitomycin-C.

[0029] In the present invention, the inner layer may contain 1% (w / v) alginate and 10% (w / v) gelatin.

[0030] In the present invention, the outer layer may contain 10% (w / v) polycaprolactone and 10% (w / v) gelatin.

[0031] In the present invention, the inner layer including alginate and gelatin may be cross-linked with calcium chloride.

[0032] In the present invention, the double-layer film may have a contact angle of 60 to 70°.

[0033] In the present invention, the double-layer membrane may have a swelling ratio of 500 to 600%.

[0034] In the present invention, the double-layer membrane may have a tensile strength of 4 to 15 MPa.

[0035] In the present invention, the double-layer membrane may have antibacterial activity. The antibacterial activity may be against Staphylococcus aureus and Escherichia coli.

[0036] In the present invention, the double-layer membrane may have anti-adhesion properties.

[0037] In the present invention, the double-layer membrane may be a scaffold.

[0038] In addition, the present invention provides a method for manufacturing a hemostatic double-layer membrane, comprising the steps of: adding alginate and gelatin to a first solution to manufacture an inner layer comprising alginate and gelatin; adding polycaprolactone and gelatin to a second solution to manufacture an outer layer comprising polycaprolactone and gelatin; and spraying an electrospinning solution onto the inner layer comprising alginate and gelatin.

[0039] In addition, the present invention provides an osteoinductive bilayer membrane comprising polycaprolactone and gelatin manufactured by the above manufacturing method.

[0040] Meanwhile, the corresponding features in the above details can be replaced in the above-described part, so their description is omitted.

[0041] In the present invention, a multifunctional bilayer (AG-TH / PG-MC) membrane for preventing postoperative pancreatic leakage was developed. The AG-TH / PG-MC scaffold effectively functions as an adhesive membrane, promoting blood coagulation, aiding wound healing, and preventing postoperative adhesion. Through various characterizations and ex vivo biocompatibility evaluations, it was confirmed that cross-linking of calcium chloride and sodium alginate altered the surface and physico-mechanical properties of the AG-TH layer, thereby enhancing adhesion and mechanical strength. The above AG-TH / PG-MC membrane showed excellent biocompatibility, and the release of thrombin from the AG-TH layer was confirmed to be excellent. In addition, the cytocompatibility evaluation and in silico molecular simulation evaluation showed that the cumulative release of mitomycin-C (MC0) from the PG-MC layer effectively controlled the TGF-beta receptor, thereby reducing fibronectin expression and exhibiting antifibrotic properties. The AG-TH / PG-MC membrane was confirmed to exhibit excellent wound healing and rapid blood clotting. Therefore, the multifunctional AG-TH / PG-MC scaffold containing thrombin (TH) and mitomycin-C (MC) is a membrane with effective properties for effectively protecting suture lines from rupture and preventing postoperative pancreatic leakage, and can be utilized as a suitable substitute for the multifunctional nanofiber bilayer membrane in pancreatic surgery.

[0042] In addition, the present invention is expected to be useful in securing post-surgical safety in various cancer surgeries, organ transplants, and surgical fields, which are becoming increasingly important due to the aging society, and has the advantage of being able to create high added value through the use of various therapeutic materials through commercialization in the future.

[0043] In addition, the present invention has the advantage of being applicable to most surgical cancer surgeries and living and brain-dead organ transplants due to terminal organ failure as well as to leakage caused by pancreatic cancer surgery as a multifunctional nanofiber membrane therapeutic material.

[0044] In addition, the present invention has the advantage of being able to improve the prognosis of patients by shortening the surgical time through a single application of a double-layered membrane, whereas two types of therapeutic materials, a hemostatic agent and an anti-adhesion agent, are used during surgery in existing clinical surgeries, and can be conveniently applied by medical staff.

[0045] Figure 1 shows scanning electron micrographs of the surface morphologies of (a) AG-TH and (b) PG-MC membranes fabricated. (c) Fiber diameter distribution. (d) Cross-linking mechanism of the AG-TH layer: sodium alginate is physically cross-linked with calcium chloride by an ionic reaction to form calcium alginate. (e) Surface morphology of AG-TH after ionic cross-linking. (f) Cross-sectional SEM images of the bilayer (AG-TH / PG-MC), (g) inner layer (AG-TH), and (h) outer layer (PG-MC). (i) FTIR spectrum of the raw material. (j) FTIR spectra of the outer and inner layer membranes before and after loading mitomycin-c and thrombin, respectively. Data are expressed as mean ± SD.

[0046] Figure 2 shows the adhesion properties of the inner layers with (AG-TH(Cr)) crosslinking and without (AG-TH(NCr)) crosslinking, and a commercial sample (Tachosil). (a) Quantitative shear strength (b) Quantitative interfacial toughness (c) Ex vivo tissue adhesion tests by (i,v) stretching, (ii,vi) twisting, (iii,vii) pressing, and (iv,viii) immersion in PBS for 8 h. (d) Tissue adhesion mechanism of the AG / TH layer. After crosslinking, the membrane strongly binds to the tissue surface through covalent bonds and non-covalent interactions such as hydrogen bonding and electrostatic interactions. (e) Water contact angle characteristics of different layers of the membrane for hydrophilicity. Data are expressed as mean ± SD.

[0047] Figure 3 shows optical image results for the degradation rates of various samples after culturing in SBF solution for 1, 7, and 14 days.

[0048] Figure 4 shows the results of the degradation rates of various samples after culturing in SBF solution for 1, 4, 7, 10, and 14 days.

[0049] Figure 5 shows the physical properties of the membranes: (a) degradation rate of the sample in PBS after 14 days; (b) swelling ratio after 24 hours; mechanical properties of the membranes: (c) stress-strain curve; (d) tensile strength; (e) Young's modulus obtained from the slope of the stress-strain curve; (f) thrombin release activity in the AG-TH layer; (g) cumulative release of mitomycin-c in the PG-MC layer; (h) schematic diagram of the burst release of thrombin in the AG-TH layer and (i) cumulative release of mitomycin-c in the PG-MC layer. Data are expressed as mean ± SD.

[0050] Figure 6 shows (a) sample extract preparation procedure and cell culture (b) fluorescence microscopy images of L929 fibroblast proliferation after 1, 3, and 7 days of culture. (c) F-actin assay for L929 fibroblasts after 7 days of culture. (d) MTT cytotoxicity assay after 1, 3, and 7 days of culture. (e) Digital images of hemolytic activity of composite membranes using PBS as a negative control and Triton X-100 as a positive control. (f) Digital images of clot formation in the samples. (g) Comparative hemolysis rate analysis. (h) Blood coagulation index (BCI) of the samples after 35 minutes. (i) Comparative bleeding time and (j) blood loss analysis. (k) Optical images of the zones of inhibition of various sample groups against Gram-positive [(+)ve] and Gram-negative [(-)ve] bacteria. (l) Quantitative analysis of the zones of inhibition of various sample groups. Scale bar: 100 μm. Data are presented as mean ± SD.

[0051] Figure 7 shows (a) a survival / death assay of L929 cells after 24 hours. (b) quantitative results of cell viability analysis after 24 hours. (Scale bar: 100 μm)

[0052] Figure 8 (a) shows an in vitro wound healing scratch test of L929 cells in various sample extracts. The wound healing interval was observed for 24 hours. (b) shows the results of a quantitative analysis of the wound healing scratch test to determine wound closure.

[0053] Figure 9 shows SEM images of (a) the valves attached to the AG and AG-TH membrane surfaces. In vivo hemostasis evaluation in a rat tail amputation model and comparison with (b) the commercial product Tachosil as a control and (c) the AG-TH membrane.

[0054] Figure 10. Preparation of a rat model of pancreatic fistula after surgery. (a) Schematic diagram of the rat pancreas and resection site. (b) Optical image of the rat pancreatic anatomy and pancreatic duct. (c) Rat pancreatic resection site. In vivo images of the control group (d) suture only, and (e) sutured resection site with a double-layer membrane applied. Representative optical images of the resection site at (f) 2 weeks and (g) 4 weeks after transplantation to observe the prevention of pancreatic leakage, adhesion, and adhesion to other organs. Representative H&E staining images of tissue samples treated with AG-TH / PG-MC membranes at 2 weeks and (i) 4 weeks after transplantation. Immunohistochemical staining images show Collagen-1 (j) expression in samples at 2 and 4 weeks after transplantation, and α-SMA (k) expression in samples at 2 and 4 weeks after transplantation. IOD analysis of (l) Col1A2 and (m) α-SMA. Scale bars: 50 μm and 10 μm, respectively. S: suture site, M: bilayer membrane. Data are expressed as mean ± SD.

[0055] Figure 11 shows histological findings of collagen accumulation for wound healing. Masson's trichrome staining images for total collagen detection 2 weeks after transplantation. Scale bars: 500 μm, 200 μm.

[0056] Figure 12 shows histological findings of collagen accumulation for wound healing. Masson's trichrome staining images for total collagen detection 4 weeks after transplantation. Scale bars: 500 μm, 200 μm.

[0057] Figure 13 shows the results of immunofluorescence studies on anti-adhesion properties. Staining images show the expression of fibronectin markers after (a) 2 weeks and (b) 4 weeks. Quantitative analysis of fibronectin-positive staining areas after (c) 2 weeks and (d) 4 weeks of implantation. In silico evaluation of MMC for antifibrotic activity. (e) Possible binding sites of the receptor TGF-β and TGF-β with IM412 and MMC, and various interactions between amino acids and atoms in the active site of the protein / receptor (3D and 2D views). Binding energy predictions compared to IM412 and MMC, (f) TGF-β, and (g) TGF-β. Scale bar: 100 μm. Data are expressed as mean ± SD.

[0058] Figure 14 shows the results of an anti-adhesion efficacy evaluation of the AG-TH / PG-MC membrane using a rat abdominal sidewall-cecal abrasion model. Adhesions were observed in the negative control group (untreated) on days 7 and 14. No adhesions were observed in either the AG-TH / PG-MC or positive control groups.

[0059] Figure 15 shows hematoxylin and eosin (H&E) stained images showing anti-adhesion properties in AG-TH / PG-MC membranes and positive control groups 7 days after transplantation. AW: abdominal wall, CE: cecal wall, Me: mesothelial layer, SA: sample. Scale bar: 200 μm.

[0060] Figure 16 shows hematoxylin and eosin (H&E) stained images showing anti-adhesion properties in AG-TH / PG-MC membranes and positive control 14 days after transplantation. AW: abdominal wall, CE: cecal wall, Me: mesothelial layer, SA: sample. Scale bar: 200 μm.

[0061] Figure 17 is a schematic illustration of the antifibrotic, hemostatic and healing mechanisms of the bilayer (AG / TH-PG / MC) membrane.

[0062] Figure 18 shows a schematic illustration of the fabrication of (A) an electrospun AG-TH / PG-MC bilayer membrane of the present invention, (B) its application in a rat pancreatic leak model, and (C) its multifunctional capabilities.

[0063] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0064]

[0065] 1. Experimental method

[0066]

[0067] 1.1. Preparation of electrospinning solution

[0068]

[0069] The electrospinning solution was prepared before fabricating the double-layer electrospun membrane. First, a 1% (w / v) sodium alginate (A) solution (Junsei, Japan) was dissolved in deionized water and stirred continuously until the powder was completely dissolved. Next, 0.5 mg / ml thrombin (TH) extracted from bovine plasma solution (Sigma-Aldrich, USA) was dissolved in the 1% A solution at room temperature. Next, 10% (w / v) gelatin (extracted from porcine skin, Sigma-Aldrich, USA) was dissolved in 2,2,2,-trifluoroethanol (Sigma-Aldrich, USA) with continuous stirring until a homogeneous solution was formed.

[0070] 10% (w / v) polycaprolactone (P) (Sigma-Aldrich, USA) was dissolved separately in 2,2,2-trifluoroethanol (Sigma-Aldrich, USA) solution. For the AG-TH layer, 1% A, TH, and 10% G solutions were mixed in a 1:1 ratio. Afterwards, 50-100 μl of the nonionic surfactant Span 80 (Sigma-Aldrich, USA) was added to the AG solution to stabilize the oil-in-water emulsion phase of the mixed solution. In addition, for the PG-MC layer, 10% G and 10% P solutions were mixed in a 1:1 ratio, and then 0.4 μg / 100 μl MC (Roche, Germany) was dissolved in the PG mixed solution. The various compositions of the electrospun solutions are reported in Table 1.

[0071]

[0072] [Table 1]

[0073]

[0074]

[0075] 1.2. Fabrication of electrospun membranes

[0076]

[0077] The AG-TH layer was electrospun at a voltage of 15 kV at a feed rate of 1.23 ml / hr. A 21-gauge (ID: 0.50 mm) needle tip was used for the electrospinning process, and 8 ml of the electrospinning solution was used. The fabricated AG-TH layer membrane was air-dried at room temperature under a laminar flow hood. After drying, the membrane was immersed in a 100 mM CaCl2 solution for 10 min for cross-linking, and the air-drying process was performed again. Subsequently, the PG-MC layer was fabricated at a voltage of 18 kV at a feed rate of 1 ml / hr. A 25-gauge (ID: 0.26 mm) needle tip was used for the PG-MC layer, and 4 ml of the electrospinning solution was sprayed onto the AG-TH layer. After the final electrospinning process, the air-drying process was performed at room temperature under a laminar flow hood. The gap between the membrane collector and the needle tip of the AG-TH and PG-MC layers was approximately 10 cm.

[0078]

[0079] 1.3. Surface morphology and fiber diameter distribution

[0080]

[0081] The surface morphology of the scaffolds was observed using a scanning electron microscope (SEM, JEOL, JSM-6701F, 197 Tokyo, Japan). The dried electrospun membrane was cut into small pieces and placed on a sample holder. The samples were then coated with platinum using a sputter coater (Cressington Scientific Instrument, Watford, UK). Images were taken at an accelerating voltage of 10 kV. Next, the diameters of 50 randomly selected fibers were analyzed using Fiji software (National Institutes of Health (NIH), USA) to evaluate the fiber diameter distribution.

[0082]

[0083] 1.4. Fourier transform infrared spectroscopy (FT-IR)

[0084]

[0085] Fourier transform infrared spectroscopy (FT-IR) was performed to identify the functional groups of the raw materials and various electrospun films. The chemical compositions of the AG-TH and PG-MC layers were determined by Fourier transform infrared spectroscopy on a Nicolet spectrometer (Nicolet Ia10, Thermo Scientific). Samples were analyzed in the 4000-1000 cm range using OMNIC 7.3 spectral software. -1 was analyzed in .

[0086]

[0087] 1.5. Adhesion analysis

[0088]

[0089] Adhesion tests were performed on pig skins according to the American Society of Testing Materials (ASTM). Fresh pig skins were collected, degreased, and analyzed for shear strength according to ASTM F2255. First, all pig skins were cut into small pieces (4 cm x 2.5 cm). Then, a 4 cm x 1.5 cm AG-TH layer membrane was attached to the surface of one of the pig skins and the other skin was attached from the opposite direction. The two pieces attached in opposite directions were stored at room temperature for 2 hours. After 2 hours, a shear strength test was performed using a universal testing machine at a strain rate of 3 mm min. -1 The force-displacement curve was analyzed along the shear direction at a speed of . The shear strength was calculated using the following equation (1).

[0090]

[0091] Shear strength (KPa) = F max (N) / area (cm) 2 ) (1)

[0092]

[0093] The interfacial toughness test was performed according to ASTM F2256. After cutting the pig skin into 4 cm × 2.5 cm, the AG-TH layer film (4 cm × 1.5 cm) was attached to the leather surface. At the same time, another piece of pig skin was attached in the same direction. The two pieces attached in the same direction were left at room temperature for 2 hours. After 2 hours, the interfacial toughness test was performed using a universal testing machine at a rate of 3 mm min. -1 The force-displacement curve and shear direction were analyzed at a speed of . The interfacial toughness was calculated using the following equation (2).

[0094]

[0095] Interfacial toughness (Jcm) -2 ) = 2 × F max (N) / width (cm) (2)

[0096]

[0097] 1.6. Water contact angle analysis

[0098]

[0099] The hydrophilic and hydrophobic properties of different electrospun membranes were analyzed using a Drop Shape Analyst (DSA) 100 (KRUSS, Germany). 0.5 μl of distilled water was dropped onto the electrospun membrane surface using a stainless steel needle. The contact angle of the electrospun membrane was then measured by video recording the angular deflection every 20 seconds. The contact angle was calculated as the average of three readings from different areas of each sample.

[0100]

[0101] 1.7. Evaluation of swelling and biodegradation behavior

[0102]

[0103] The swelling behavior of the electrospun membranes was evaluated by a conventional gravimetric method. All electrospun membranes were cut into square pieces of approximately 2 × 2 cm in size. The dried samples were then weighed (W o) were measured and soaked in 2 ml of PBS to make them wet. After that, all wet samples were placed in a 37°C incubator. At specific time intervals (1, 3, 7, 12, and 24 h), the membranes were taken out of the incubator and excess water was wiped with filter paper. After that, the wet weight (W) of the membranes t ) was measured. The percentage of expansion ratio was calculated using the following equation (3).

[0104]

[0105] Expansion ratio (%) = ((W t -W o )) / W o ×100 (3)

[0106] Equation (3) is W o with initial dry weight, W t The wet weight of each sample at different times is expressed as the wet weight of each sample. Each sample was independently repeated three times.

[0107] The in vitro degradation behavior of the membrane was examined for 14 days. First, all membranes were cut into square shapes (2 × 2 cm). Afterwards, the membranes were weighed (W o ) were separately immersed in 5 ml PBS and SBF solutions and placed in a 37°C incubator. The solutions were changed every 3 days. After each specific time interval ((1, 4, 7, 10, 14 days)), the samples were removed from the solutions and freeze-dried for 24 h. After this, the samples were re-weighed (W t ) was measured and the dry weight loss ratio (W%) was measured according to the following equation (4).

[0108]

[0109] Dry weight loss (W%) = ((W o -W t )) / W o ×100 (4)

[0110] Equation (4) is W o with initial dry weight, W t The weight of the dried membrane was measured at each time point. Each sample was independently repeated three times.

[0111]

[0112] 1.8. Tensile strength study

[0113]

[0114] The tensile properties of the electrospun membranes were measured using a universal mechanical testing machine (UTM, Qmesys, QM100T). The membranes were carefully cut into rectangular shapes with a width of 10 mm and a length of 30 mm and mounted vertically on two mechanical gripping devices. The thickness was measured using a micrometer with an accuracy of 0.01 mm. The tensile test of the membranes was performed at a tension of 3 mm min. -1 The tests were performed with a 5 N load cell at a cross-head speed of 1000 rpm until failure. The tensile stress-strain curves were calculated based on the apparent cross-sectional area of ​​the membrane. The elastic modulus was calculated from the slope of the strain-stress curve. Each sample was independently repeated three times.

[0115]

[0116] 1.9. Release rate of MC from electrospun membrane

[0117]

[0118] The release rate of MC from the electrospun membrane was measured by extracting the drug from the membrane. Membranes of known weight were immersed in 5 ml of PBS solution. At specific time points, 2 ml of PBS was collected and replenished with 2 ml of fresh PBS solution. The amount of drug released from the electrospun membrane was analyzed using a nanodrop spectroscopy at 364 nm. The concentration of the released drug was calculated using a standard calibration curve of MC. The percentage of MC released from the membrane was calculated based on the initial weight of MC incorporated into the electrospun fibrous membrane.

[0119]

[0120] 1.10. Quantitative TH release activity

[0121]

[0122] The electrospun membrane loaded with thrombin was immersed in 5 ml of PBS. After a specified time interval, the 2 ml PBS solution was removed and replaced with an equal volume of fresh PBS solution. TH activity at various time points was determined using a thrombin activity test kit (Abcam, ab197006, USA) according to the manufacturer's protocol. A 2.5 ng / μl standard was prepared by diluting 5 μl of a 50 ng / μl thrombin standard with 95 μl of thrombin dilution buffer. A standard curve was then prepared using serial dilutions of the 2.5 ng / μl thrombin standard. Next, 50 μl of the reaction mixture was prepared for each well by mixing 45 μl of thrombin test buffer and 5 μl of thrombin substrate. The 50 μl reaction mixture and 50 μl of each sample extract were mixed in a 96-well plate. Fluorescence measurements were performed at 2-min intervals for 30 min at 37°C using an Elisa microplate reader (Biotek, Synergy H1, USA) at Ex / Em = 350 / 450 nm. Two time points were selected to measure relative fluorescence units (RFU) in kinetic mode. For statistical reasons, each sample was analyzed in duplicate. Thrombin activity was calculated according to the following equation (5, 6):

[0123]

[0124] RFU=RFU2-RFU1 (5)

[0125] Thrombin activity (ng / ml) = B / V×D (6)

[0126] B represents the amount of thrombin (ng) in the sample well, and B ng thrombin was calculated using RFU. V represents the sample volume (ml) added to the reaction well, and D is the sample dilution factor.

[0127]

[0128] 1.11. In vitro biocompatibility of electrospun membranes

[0129]

[0130] 1.11.1. Cell culture of L929 fibroblasts

[0131]

[0132] L929 fibroblast cells were used to evaluate in vitro cell biocompatibility. L929 cells were cultured in primary medium containing alpha-DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (100 U / mL) in a humidified incubator at 37°C and 5% CO2. The medium was changed three times a week. When the cell density reached 80% to 90%, adherent cells were trypsinized, harvested, and subcultured on another plate. In this study, L929 fibroblast cells passage 7 were used for in vitro experiments.

[0133]

[0134] 1.11.2. Cytotoxicity and Viability / Death Tests

[0135]

[0136] The in vitro cytotoxicity of various membranes was investigated by MTT test using sample extracts according to the standard test protocol. According to Fig. 6a, the sterilized electrospun membranes were cut into square shapes (1 cm 2 ) were cut into pieces and separately immersed in culture medium at 37°C for 24 hours. Afterwards, 500 μl of each sample condition medium was collected and placed in a 48-well plate (n=3 per group). Typically, 1 x 10 4 L929 cell suspension (200 μl) was seeded into each well at a density of 100 μl and then cultured at 37°C with 5% CO2 for 1, 3, and 7 days. On specific days, MTT solution (1:10 ratio) was added to each well of the sample and incubated at 37°C for 4 h. The solution was removed, and 400 μl of DMSO was added to each well and incubated for 30 min to dissolve the formazan crystals. The absorbance of the solution (n=3 per group) was measured at a wavelength of 570 nm using a microplate reader (Biotek, Synergy H1, USA). The cell culture medium was changed every 2 days.

[0137] The survival / death cell assay was performed using a survival / death viability / cytotoxicity kit (Thermofisher Scientific, USA) according to the manufacturer's protocol. L929 cell suspension (1 × 10 5 ) were inoculated onto the sample extracts in 48-well plates (n=3 per group). The samples were then cultured for 24 h at 37°C with 5% CO2. The staining solution was prepared by adding 5 μL of calcein AM and 20 μL of ethidium homodimer-1 to 10 mL of PBS. After removing the medium at a specific time, 100–200 μL of the staining solution was added directly to the cells in each well and incubated for 30 min at 20–25°C. Afterwards, live / dead cells were visualized and analyzed using a confocal fluorescence microscope (Olympus, FV10i-W, Tokyo, Japan). Live / dead cells were counted from fluorescence images after 24 h of culture using Fiji software (National Institutes of Health (NIH), USA). Cell viability was calculated using the following equation (7).

[0138]

[0139] Cell viability (%) = (total number of cells - number of dead cells) / (total number of cells) × 100 (7)

[0140]

[0141] 1.11.3. Cell proliferation test

[0142]

[0143] For cell proliferation assays, conditioned media were collected and L929 cell suspension (1 × 10 4) were seeded into each membrane well group (n = 3 per group). The samples were then cultured for 1, 3, and 7 days at 37°C with 5% CO2. The sample wells were washed with PBS at specific time points and fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, USA) for 10–15 min. After fixation, the sample wells were washed three times with PBS and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, USA) for 10 min. Afterwards, 2.5% bovine serum albumin (BSA) was used as a blocking reagent for 1 h. Immunostaining was performed overnight at 4°C in the dark using fluorescein isothiocyanate (FITC)-conjugated phalloidin (25 μg / mL; Sigma-Aldrich, USA). Next, nuclei were washed three times with PBS and stained with Hoechst 33342 (Invitrogen, USA) for 5 min. Scaffolds were visualized and analyzed using a confocal fluorescence microscope (Olympus, FV10i-W, Tokyo, Japan).

[0144]

[0145] 1.11.4. Wound healing and antibacterial testing

[0146]

[0147] Wound healing assay was performed with L929 cells (1 × 10 4Cells / well) were seeded and cultured at 37°C in 5% CO2. Conditioned media was collected from different sample groups by soaking the sample membrane in culture media for 24 h at 37°C. After L929 cells reached confluence, a liner wound was created by slowly scraping the L929 cells from the center of the well with a 200-μl pipette tip. After washing with PBS to remove detached cells and other cell debris, the conditioned media was added to each well of the different groups. Scratch images were captured for 0–24 h to determine the wound healing rate. The wound area was analyzed in the images using scratch analysis software (TScratch, CSElab). Each experiment was performed three times independently.

[0148] Antibacterial activity was determined on AG-TH, PG-MC, and AG-TH / PG-MC membranes containing Gram-positive and Gram-negative bacteria. The disk diffusion protocol was used to evaluate the antibacterial activity of AG-TH, PG-MC, and AG-TH / PG-MC. Gram-positive bacteria, Staphylococcus aureus (NCCP14402), and Gram-negative bacteria, Escherichia coli (NCCP14762), were incubated in lysogeny medium for 4 h. The temperature was maintained at 37°C. To prepare the lawn, the strains were inoculated onto Muller-Hinton agar plates. Samples were prepared by cutting into disc-sized (8 mm) pieces using a biopsy punch. The cut samples were placed on agar plates containing bacteria. The system was incubated at 37°C for 18–24 h. The antibacterial effect of drug release was determined based on the zone of inhibition on the plates. Images were acquired using a digital microscope (Sony, Thailand).

[0149]

[0150] 1.12. In vitro blood compatibility assessment

[0151]

[0152] Whole blood samples from healthy Sprague-Dawley rats were collected in tubes containing a 1:9 ratio of acid citrate dextrose (ACD) solution. The whole blood was used to assess the blood coagulation index (BCI). Additionally, the whole blood was centrifuged to separate platelet-rich plasma (PRP) and red blood cells, and the precipitate was used to assess hemolysis. All tests were performed in triplicate.

[0153]

[0154] 1.12.1. In vitro hemolysis test

[0155]

[0156] ACD whole blood was first centrifuged at 3000 rpm for 10 minutes to separate blood and plasma. After aspirating the plasma from the supernatant, the cells were washed twice with PBS and centrifuged again. The erythrocyte sediment was diluted to 2% erythrocytes in PBS and tested. The entire assay was performed using sample extracts (AG-TH and AG) prepared in PBS and incubated at 37°C for 24 hours. A 0.2% erythrocyte suspension was mixed with 10 ml of each sample extract and 0.1% Triton-X100 and PBS as positive and negative controls, respectively, and incubated at 37°C for 3 hours. The erythrocyte suspension mixture was centrifuged at 3000 rpm for 10 minutes, and the hemolysis rate was measured by the absorbance of the solution at 540 nm using a microplate reader. The hemolysis rate was calculated using the following equation (8):

[0157]

[0158] Hemolysis rate = (As -An) / (Ap -An) ×100 (8)

[0159] In Equation (8), As, Ap, and An represent the absorbance of the sample, positive control, and negative control, respectively.

[0160]

[0161] 1.12.2. Whole blood clotting capacity

[0162]

[0163] Whole blood coagulation capacity was measured and calculated as a percentage of the blood coagulation index (BCI). Thrombin-containing and thrombin-free inner membranes (AG-TH and AG) were cut into squares (1 cm2). Coagulation was activated by adding a 10% 0.1 M calcium chloride solution to citrated whole blood. Samples were then placed in 15 ml conical tubes, and 100 μl of activated blood was added. All samples were incubated at 37°C for 30 min. After incubation, 3 ml of deionized water was gently added to each sample tube. Each sample was collected in triplicate, and the supernatant was placed in a 96-well plate. The absorbance was observed at 540 nm using a microplate reader. Whole blood recalcified with deionized water was used as a negative control and analyzed for baseline values. The blood coagulation index (BCI) of the samples was calculated using the following equation (9):

[0164]

[0165] BCI(%) = I s / I r ×100 (9)

[0166] Is and Ir represent the absorbance of the sample and the reference value, respectively.

[0167]

[0168] 1.12.3. Platelet activation and adhesion

[0169]

[0170] Platelet activation and adhesion were performed by introducing the samples into PRP and then observed using SEM. PRP was isolated from whole blood by centrifugation at 2,500 rpm for 5 minutes. 200 μl of PRP was then added to each sample and incubated at 37°C for 1 hour. After incubation, unattached PRP was removed by washing three times with 1× PBS and fixed with a 2.5% glutaraldehyde solution for 2 hours. For SEM observation, the samples were gradually dehydrated in an ethanol series and air-dried for 24 hours in a fume hood at room temperature. For SEM observation, the samples were coated with a thin layer of platinum and observed at an accelerating voltage of 10 kV.

[0171]

[0172] 1.13. In vivo blood compatibility, leakage prevention, and adhesion prevention evaluation

[0173]

[0174] Twelve adult Sprague-Dawley rats (250–300 g, male, 9 weeks old) were selected for in vivo experiments in the pancreas and cecum models. All procedures were approved by the Animal Ethics Committee of Soonchunhyang University, Korea (Ethics Number: SCH22-0089). Animals were housed in standard cages with equal access to food and water. All in vivo experiments involving animals complied with the guidelines and regulations of the institution.

[0175]

[0176] 1.13.1. In vivo hemostatic performance evaluation

[0177]

[0178] The hemostatic properties of the AG-TH layer were evaluated in a rat tail amputation model and compared with Tachosil, a commercially available product composed of fibrinogen, thrombin, and collagen, as a control. For the hemostasis evaluation in the rat tail model, six animals were divided into two groups: control (n = 3) and AG-TH (n = 3). The surgical procedure was performed under isoflurane anesthesia with sufficient oxygen supply. Afterward, the tails of the animals were cut 50% with surgical scissors, and bleeding was allowed to proceed for 10-15 seconds. The sample and control were applied to the bleeding site to stop the bleeding, and the time was recorded. The blood secreted from the sample and control was collected and weighed. The total blood loss at the injury site was analyzed using a pre-weighed gauge and sample to assess the total blood loss in both groups.

[0179]

[0180] 1.13.2. In vivo transplantation procedure

[0181]

[0182] In this study, a total of 12 animals were divided into two groups: a control group (n = 6) and a group with a double-layer (AG-TH / PG-MC) membrane suture (n = 6). Before surgery, the membranes were sterilized overnight with UV (254 nm) and then prepared for transplantation. The surgical procedure was performed with the rats anesthetized with isoflurane and placed in the supine position with sufficient oxygen supply. The resection site for the experiment was the division of the gastric and splenic tracts (Figures 10a and 10b). Initially, the hair on the rats' abdomens was thoroughly shaved, and the designated area was cleaned with 70% ethanol and povidone-iodine. A midline abdominal incision was then made. The stomach was grasped with forceps, the pancreas was exposed, and the duodenum and spleen were removed. After determining the resection site of the pancreas (left portal vein) (Figure 10c), the vessels were ligated with absorbable sutures (4-0) and resected (Figure 10d). Afterwards, AG-TH / PG-MC membranes were attached to the resection site (Fig. 10e). All ducts were divided, and all mice were monitored for 2 and 4 weeks.

[0183] Twelve adult Sprague-Dawley rats (250-300 g, male, 9 weeks old) were selected for in vivo experiments in the cecal model. All procedures were approved by the Animal Ethics Committee of Soonchunhyang University, Korea (Ethics Number: SCH22-0089). Animals were housed in standard cages with equal access to food and water. All in vivo experiments involving animals complied with the guidelines and regulations of the institution. A total of 18 animals were divided into three groups: negative control (untreated) (n = 6), AG-TH / PG-MC (bilayer) membrane (n = 6), and positive control (commercial: Seprafilm adhesive barrier) (n = 6). Before surgery, the membranes were sterilized overnight under UV light (254 nm) and then prepared for implantation. The surgical procedure was performed with the rats anesthetized with isoflurane and placed in the supine position with sufficient oxygen supply. The areas of application for the experiment were as shown in Figure 14. Initially, the hair on the abdomen of the mice was thoroughly shaved, and the designated area was cleaned with 70% ethanol and povidone-iodine. Using aseptic technique, a 5-cm midline incision was made along the linea alba of the abdominal wall of the mice. The surfaces of the abdominal wall (1.5 x 1.5 cm2) and the cecum (1 x 1 cm2) were carefully scraped with sandpaper, but without perforation. Afterwards, (1.5 x 1.5 cm) of AG-TH / PG-MC membrane and commercially available products were applied to the injured area of ​​the cecum. The abdomen and peritoneum were closed with sutures. For the negative control group, the abdomen and peritoneum were closed with sutures without any treatment. After 1 and 2 weeks, the mice were euthanized with diethyl ether, and the incision site was reopened. Histological staining with hematoxylin and eosin (H&E) was performed to examine adhesions, inflammation, fibrosis, and angiogenesis.

[0184]

[0185] 1.13.3. Histological analysis

[0186]

[0187] Two and four weeks after transplantation, the mice were sacrificed, and the surgical site was immediately fixed in 4% PFA for 4 days. The samples were then washed with water and immersed in a 30% sucrose solution at 4°C until all samples were adequately dehydrated. After dehydration, the samples were embedded in OCT compound for cryosectioning. Sectioning was performed on 10-μm-thick transverse sections. The sections were then stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT) for immunohistological analysis of wound healing and adhesive properties. Tissue sections were evaluated using a light microscope (Olympus, Japan), and images were analyzed using cellSens software.

[0188]

[0189] 1.13.4. Immunohistochemistry (IHC)

[0190]

[0191] The expression of specific markers, collagen type 1 alpha 2 (Col1A2) and smooth muscle cell marker α-SMA, was observed using immunohistochemistry. 3,3'-diaminobenzidine (DAB) staining was performed using a horseradish peroxidase (HRP) / DAB detection immunohistochemistry (IHC) kit (Abcam, UK) according to the manufacturer's protocol. Tissue sections were incubated with hydrogen peroxide block for 10 minutes and then with protein block for 10 minutes. Afterwards, tissue sections were incubated overnight at 4°C with primary antibodies, Col1A2 (Santa Cruz, USA) and α-SMA (Abcam, UK), in 2.5% BSA (Bovogen, Australia) and 0.3% Triton X-100 (Sigma-Aldrich, USA, USA) in phosphate-buffered saline (PBS). After incubation, biotinylated goat anti-multivalent antibody and streptavidin peroxidase were applied to the tissue sections for 10 minutes. The sections were washed with DAB chromogen and DAB substrate. Furthermore, the tissue sections were counterstained with hematoxylin (H&E Stain Kit, Abcam, UK). After staining, the sections were observed under a light microscope (Olympus, Japan). The integrated optical density (IOD) of the expressed markers was analyzed using Fiji software (National Institutes of Health (NIH), USA).

[0192]

[0193] 1.13.5. Immunofluorescence analysis

[0194]

[0195] Fibronectin expression was observed using immunofluorescence staining. Cryosection samples were permeabilized with 0.5% Triton X-100. After washing the samples three times with PBS, they were blocked with 10% goat serum for 50 minutes at room temperature. The sections were then incubated overnight at 4°C with a primary antibody against fibronectin (ab2413, Abcam, UK). After incubation, the samples were washed three times with PBS, and the sections were incubated with Alexa 488 anti-mouse (A12379, Thermo Fisher, US) in a dark chamber at 37°C for 1 hour. Nuclei were stained with DAPI. After staining, the tissue sections were observed under a light microscope (Olympus, Japan). The positive areas of the expressed markers were analyzed using Fiji software (National Institutes of Health (NIH), USA).

[0196]

[0197] 1.14. In silico molecular simulation (MS)

[0198]

[0199] 1.14.1. Ligand Preparation

[0200]

[0201] Mitomycin C (PubChem CID: 5746) and the control IM-412 (PubChem CID: 16616198) were downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ). Next, all ligands were optimized using the same program, Avogadro (Avogadro: an open-source molecule builder and visualizer. Version 1.2.0 http: / / avogadro.cc / ), using the UFF force field. The ligands were saved in .pdb (Protein Data Bank) format and were ready for docking.

[0202]

[0203] 1.14.2. Protein Preparation

[0204]

[0205] The TGF-B1 receptor (PDB ID: 1KLC) and TGF-B2 receptor (PDB ID: 2TGI) were obtained from the Protein Data Bank (http: / / www.rcsb.org / ). The downloaded proteins were organized with PyMOL (PyMOL Molecular Graphics System, version 2.0 Schrodinger, LLC) and optimized with Swiss-PdbViewer (Swiss-PdbViewer / DeepView, v4.1 by Nicolas Guex, Alexandre Diemand, Manuel C. Peitsch, & Torsten Schwede).

[0206]

[0207] 1.14.3. Molecular Docking and Visualization

[0208]

[0209] Molecular docking between receptors and ligands (individually) was performed using the 'Vina Wizard' program in PyRx - Python Prescription 0.8. The ligands (individually) and receptors were loaded into the program along with the appropriate compound (i.e., ligand or macromolecule) declarations. The docked ligands (individually) and receptors were then combined using PyMOL (PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC). The combined structures were opened for visualization in Discovery Studio (BIOVIA, Dassault Systemes, Discovery Studio Visualizer, v4.5.0.15071, San Diego: Dassault Systemes, ©2005-15). Ligand interactions were observed, and snapshots of the best poses were taken.

[0210]

[0211] 1.15. Statistical Analysis

[0212]

[0213] Statistical analyses were performed using GraphPad Prism 8.0 using t tests, one-way analysis of variance (ANOVA), and two-way analysis of variance (ANOVA, Tukey's test). All experiments were repeated three times, and the results are expressed as the mean ± standard deviation (SD). Statistical significance was set at ns, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0214]

[0215] 2. Experimental Results

[0216]

[0217] 2.1. Surface morphology and characterization of multifunctional nanofiber membranes

[0218]

[0219] The detailed morphologies of AG-TH and PG-MC were observed by SEM, as shown in Figures 1a and 1b. The electrospinning process is highly dependent on the ionic strength, viscoelastic properties, concentration, and flow rate of the solution. Several studies have shown that the parameters mentioned above affect the amount of solute extruded from the needle during the electrospinning process. Therefore, all of these factors are important for controlling the appropriate fiber size. The SEM images in Figures 1a and 1b show that the fibers in the inner and outer layers are uniform and regular. The high gelatin content, which was achieved by mixing 1% A and 10% G in a 1:1 ratio, reduced the fiber diameter of the AG-TH layer. In contrast, a 1:1 ratio of 10% P and 10% G formed fibers in the PG-MC layer with thicker diameters. However, due to the viscoelastic properties of the electrospinning solution in the AG-TH layer, some microbeads were initially formed during the electrospinning process. Beads were formed due to improper entanglement of alginate caused by the rigidity of the sugar chains and the strong repulsive force between the polyanions. The shape of the beads was controlled by adjusting the voltage and injection flow rate. The results in Figure 1c showed that the average fiber diameter of the AG-TH layer was 0.13 ± 45 μm, and that of the PG-MC layer was 0.45 ± 61 μm. Nanofiber membranes are an excellent choice as wound healing matrices, but maintaining homeostasis is difficult to control. Furthermore, improving the hemostatic effect and membrane adhesiveness is necessary to prevent postoperative pancreatic leakage. It is also important to prevent membrane adhesion to other organs and tissues after surgery. Therefore, the approach is to fabricate a multifunctional bilayer membrane to overcome these limitations.

[0220] Co-electrospinning of gelatin and alginate has been attempted in several studies. However, the resulting fibrous membranes exhibit poor water integrity and poor mechanical properties. Therefore, in the present invention, the AG-TH layer was crosslinked with a CaCl2 solution to improve the mechanical and adhesive properties with the tissue surface. After crosslinking and air-drying, the rapid gelation of the calcium ions and alginate significantly increased the mechanical properties and water integrity. In the present invention, the crosslinking process was a physical crosslinking process. During crosslinking, the water-soluble and charged polymers were crosslinked with oppositely charged ions, transforming the liquid polymer into a gel-like solid structure. Therefore, Figure 1e shows that the surface morphology of the fibrous AG-TH layer after crosslinking transformed into a gel-like solid structure. However, some fibers remained in the deformation phase. When sodium alginate was exposed to calcium ions, various ionic interactions were established to form calcium alginate (Figure 1d). The ionic interactions between sodium alginate and calcium chloride significantly altered the surface morphology of the inner layer. Additionally, in Fig. 1f, the cross-sectional SEM image of AG-TH / PG-MC showed that the fibers were transformed into a gel-like shape in the AG-TH layer (Fig. 1g) after cross-linking. In contrast, a fibrous structure was observed in the PG-MC layer (Fig. 1h).

[0221] FTIR spectra were obtained to determine the functional groups of biomaterials using specific absorption peaks that may be required for specific chemical reactions. FT-IR was performed using individual raw materials in the present invention (Fig. 1i). The FTIR spectrum of sodium alginate showed a peak at 3400 cm corresponding to the stretching of OH. -1 , 2925cm, seen from CH's stretching -1 , 1603 cm corresponding to the asymmetric stretching of the carboxylate group. -1 A specific peak at 1412 cm corresponding to the symmetric stretching of the carboxylate group. -1 It showed a characteristic absorption band of 1033cm -1The peaks represent the stretching of the COC group. The gelatin absorption bands are 3300 cm corresponding to OH and amide-A, amide I, amide II and amide III, respectively. -1 , 1640cm -1 , 1536cm -1 and 1243cm -1 It showed a broad peak at 1450cm -1 The peak of PCL showed the bending vibration of the methyl group. PCL showed the bending vibration of 1724 cm corresponding to carbonyl stretching. -1 It has a characteristic broad absorption peak at 2950cm -1 The peak of the sample membrane showed asymmetric CH2 stretching. As shown in Fig. 1j, the functional groups of the individual raw materials were detected and the functional groups of the sample membrane were analyzed. The FTIR spectrum of the AG layer without TH showed a peak at 3296 cm -1 , 1641cm -1 , 1541cm -1 , 1451cm -1 In the AG-TH layer, the spectral bands were observed, which correspond to the OH and amide-A, amide I, amide II, and CH vibration frequencies, respectively. In the AG-TH layer, the cross-linking with thrombin was integrated, and the carboxyl group of alginate cooperated with calcium ions to produce a 1639 cm -1 The absorption peak of the symmetric stretching vibration peak is weakened. Moreover, the PG layer without MC has an absorption peak at 3302 cm corresponding to the amide group, CH stretching, and carbonyl stretching. -1 , 2948cm -1 , 1729cm -1 The band was shown in . After loading MC, the carbonyl group showed 1727 cm -1 An increase in the absorption peak was observed at 3250–3310 cm . The presence of high gelatin content in all sample groups was observed at 3250–3310 cm . -1 showed a broad and sharp peak in the sample group. In contrast, PCL and alginate showed slightly lower intensities, indicating proper mixing of raw materials in the sample group.

[0222] The primary goal of the present invention was to improve the adhesive properties of the membrane as a sealant patch to prevent leakage. Crosslinking increases the adhesive properties between the AG-TH layer and the pancreatic tissue surface. Two different approaches were used to evaluate the mechanical adhesion of the AG-TH membrane. Crosslinked (AG-TH(Cr)) and non-crosslinked (AG-TH(NCr)) samples were compared with a commercial adhesive product (Tachosil). First, the shear strength was analyzed by lap shear measurements (Fig. 1a), and second, the interfacial toughness was examined by peel measurements (Fig. 2b). The results showed that the shear strength and interfacial toughness of the AG-TH(Cr) membrane increased after crosslinking with a CaCl2 solution. The uncrosslinked AG-TH(NCr) layer showed a shear strength of 1.34 ± 2 KPa, Tachosil showed a shear strength of 1.88 ± 2.1 KPa, and the crosslinked AG-TH(Cr) layer showed a shear strength of 2.62 ± 5 KPa. The results of the interfacial toughness also showed that the adhesive toughness after crosslinking increased from 10.7 to 22.30 Jcm -2 showed an increase of 16.83 Jcm, and Tachosil showed an increase of 16.83 Jcm -2 . Furthermore, the cross-linked membranes AG-TH and AG-TH / PG-MC were subjected to various situations to further confirm their ex vivo tissue adhesion, such as stretching, twisting, and compressing tests (Fig. 2c(i-viii)). All results showed that both AG-TH and AG-TH / PG-MC were firmly attached to the tissue surface due to the cross-linking. Moreover, pancreatic tissue is located with the surrounding liquid. Therefore, the membrane may be covered and affected by water penetration. Therefore, an additional immersion test in PBS for 8 h was performed. The results (Fig. 2c(iv, viii)) showed that the AG-TH and AG-TH / PG-MC membranes were firmly attached to the porcine skin while continuously immersed and washed in PBS for 8 h.

[0223] The adhesion mechanisms of the AG-TH layer include mechanical interlocking and chemical adsorption. Mechanical interlocking refers to adhesion that occurs when the adhesive penetrates the irregular surface of the tissue. In contrast, chemical adsorption occurs through primary intramolecular (ionic and covalent bonds) or secondary intermolecular (van der Waals and hydrogen bonds) interactions between the molecules on the adhesive surface and the adhesive. In the present invention, cross-linking the alginate-gelatin layer with calcium chloride transformed the layer into a gel-like structure, enhancing its ability to adhere to irregular tissue surfaces. Furthermore, the amide groups of the gelatin reacted with covalent interactions, such as hydrogen bonds, and electrostatic interactions with the tissue surface. Therefore, the layer adhered firmly to the tissue surface (Fig. 2d), which acted as a sealing patch to prevent pancreatic leakage.

[0224] The surface of a hydrophilic material exhibits a contact angle less than 90°, whereas a material with a surface contact angle exceeding 90° is considered hydrophobic. In the present invention, the contact angle results (Fig. 2e) showed that the AG-TH layer was more hydrophilic than the PG-MC layer. The high gelatin and alginate contents of the AG-TH layer led to a decrease in the fiber diameter. After crosslinking, the water contact angle decreased and the hydrophilicity of the AG-TH layer increased. The AG-TH layer showed a water contact angle of 42 ± 5°. Due to the hydrophobicity of PCL, the PG-MC layer was less hydrophilic than the AG-TH layer, with a contact angle of 67 ± 8°, and the AG-TH / PG-MC layer showed a contact angle of 64 ± 4°.

[0225] In the present invention, gelatin, which is a water-soluble material and hydrophilic through its hydroxyl and carboxyl groups, was combined with alginate. As a result, the AG-TH layer exhibited rapid degradation characteristics by decomposing the hydroxyl groups upon contact with moisture, accelerating the degradation rate (Fig. 5a). After 1 day of incubation in PBS, the AG-TH membrane showed a weight loss of more than 60%. After 14 days of incubation, an 85% decrease in the total membrane weight was observed. In contrast, the PG-MC layer showed a slow degradation rate due to its low hydrophilicity and the presence of synthetic polymers such as PCL. Therefore, after 14 days of incubation, the total membrane weight decreased by only 47%. The AG-TH / PG-MC layer membrane showed a weight loss of 65% after 14 days of incubation. The higher degradation rate of the AG-TH layer and the slower degradation rate of the PG-MC layer maintained the controlled degradation rate of the AG-TH / PG-MC membrane. Additionally, degradation studies were further investigated in simulated body fluid (SBF) solution (Figures 3 and 4), which has an ionic concentration close to human plasma. Compared to the PBS solution, the degradation rate increased in the SBF solution. The AG-TH membrane was found to be nearly 90% degraded after 14 days of culture. In contrast, the PG-MC and AG-TH / PG-MC layers were found to be more than 50% and 68% degraded, respectively. However, the swelling property is also related to the degradation rate of the scaffold. A higher swelling ratio is due to a higher degradation rate when in contact with a fluid. Therefore, the more hydrophilic groups in a sample, the more fluid it absorbs when in contact with water, loosening molecular interactions and increasing the degradation rate. As shown in Figure 5b, the swelling ratios after 24 h were higher for the AG-TH and AG-TH / PG-MC layer membranes than for the PG-MC layer membrane. According to the results, the AG-TH layer showed an increased degradation rate, reaching a swelling ratio of 637% after 24 hours. The PG-MC layer showed a swelling ratio of 406%, and the AG-TH / PG-MC layer showed a maximum swelling ratio of 562%.

[0226] The stress-strain curves and maximum tensile strength of the membranes are shown in Figures 5c and 3d. According to the results, cross-linking the AG-TH layer significantly increased the tensile strength of the membrane. Cross-linking allowed calcium ions to be incorporated into the alginate-based structure, forming a stable network, thereby enhancing the mechanical properties of the membrane. Furthermore, the combination of PCL and gelatin with the PG-MC layer did not improve the tensile strength. However, higher strength and higher elongation at break were observed, indicating the excellent flexibility and deformability of the PG-MC layer. The AG-TH / PG-MC layer membrane exhibited the highest tensile strength (9.4 ± 5 ​​MPa), with the AG-TH layer exhibiting 7.4 ± 21.2 MPa and the PG-MC layer exhibiting 3.2 ± 6 MPa. The elastic modulus represents the elastic properties of the sample membrane. In the present invention, Figure 5e shows that mixing PCL and gelatin increases the elastic properties of the PG-MC layer. This is very important for the study because the membrane adheres to the appropriate implantation site due to elastic behavior without damage due to muscle movement. The PG-MC layer showed a young elastic modulus of 0.33 ± 13.4 MPa, while the AG-TH layer and the AG-TH / PG-MC layer showed 3.05 ± 51.2 MPa and 1.84 ± 40.1 MPa, respectively. All results showed that crosslinking increased the mechanical strength of the AG-TH layer when mixed with PCL-gelatin, and the PG-MC layer membrane exhibited more elastic properties. Therefore, when used with AG-TH as the inner layer and PG-MC as the outer layer, the bilayer membrane (AG-TH / PG-MC) can significantly increase the tensile strength and be suitable as an effective sealant to prevent pancreatic leakage.

[0227] TH plays a crucial role in coagulation, particularly in the final stage of blood coagulation by converting fibrinogen to fibrin and promoting platelet aggregation. Because suture lines can break off at the tissue surface after surgery, rapid hemostatic activity is essential to prevent bleeding. Therefore, a material that is rapidly degradable, easy to handle, and directly applicable would be an excellent choice as a scaffold capable of incorporating the enzymatic activity of TH. In the present invention, TH loaded with an AG-TH layer released TH activity explosively (21.8 ng / ml) after 50 minutes of incubation (Fig. 5f). This explosive release occurred due to the rapid decomposition of the hydroxyl groups in the AG-TH layer (Fig. 5h). Upon contact with fluid, the gel-like intermolecular structure of the AG-TH layer readily decomposes, resulting in the explosive release of TH from the AG-TH layer. Conversely, several studies have demonstrated that MC can prevent tissue adhesion after surgery. The anti-tissue adhesion mechanism occurred by inhibiting fibroblast proliferation and apoptosis. Furthermore, it has been reported that collagen synthesis is inhibited in normal skin fibroblasts. The release profile of MC (Fig. 5g) showed cumulative release according to the degradation profile of the outer layer. A potential role of MC is to reduce tissue adhesion through fibrosis and vascular inhibition over several weeks. Accordingly, after 21 days of culture, MC showed a release efficiency of 75%. The cumulative release profile of MC from the PG-MC layer membrane appeared to be slowed due to the slow degradation of the PG-MC layer (Fig. 5i).

[0228]

[0229] 2.2. In vitro biocompatibility and wound healing tests

[0230]

[0231] In vitro cell proliferation studies of L929 fibroblasts using AG-TH, PG-MC, and AG-TH / PG-MC (Fig. 6b) were performed for 1, 3, and 7 days. After 7 days of culture, the AG-TH / PG-MC membrane induced the highest cell proliferation rate compared to the PG-MC and AG-TH membranes. The PG-MC membrane is composed of MC, an anticancer agent that inhibits fibroblast collagen synthesis by inhibiting DNA-dependent RNA synthesis and fibroblast proliferation. Therefore, the outer layer, PG-MC, showed a low cell proliferation rate. However, the AG-TH layer showed a cell proliferation rate almost similar to the control and AG-TH / PG-MC due to appropriate cell adhesion to the hydrophilic membrane. The results of quantifying the f-actin area of ​​fibroblasts in the proliferation images after 7 days of culture also showed similar results (Fig. 6c).

[0232] Additionally, an MTT cytotoxicity test (Fig. 6d) was performed to evaluate the biocompatibility of the composite membrane. After 7 days of in vitro culture of L929 cells, the AG-TH / PG-MC and AG-TH layer membranes showed the highest absorbance values ​​compared to the PG-MC layer. In contrast, as MC was cumulatively released from the PG-MC layer, L929 cells were apoptotic, showing low absorbance values ​​after 7 days of culture. The visual examination of the cell viability of the membrane was further observed using a live / dead test (Fig. 7a). Fluorescent staining images of live and dead cells showed that the cells had a healthy morphology. However, compared with the control, AG-TH, and AG-TH / PG-MC layers, the PG-MC layer showed more cell death.

[0233] Furthermore, quantification of cell viability results (Fig. 7b) showed that the AG-TH / PG-MC layer showed the highest cell viability (<91%) after 24 h, while the AG-TH layer showed <82% cell viability and the PG-MC layer showed lower cell viability (<67%). In vitro biocompatibility results demonstrated that the hydrophilicity of the AG-TH layer increased cell adhesion and cell proliferation. In addition, the burst release of thrombin plays an important role in the proliferation and migration of fibroblasts through the activation of protease-activated receptors (PARs). On the other hand, the continuous release of MC from the PG-MC layer caused fibroblast apoptosis and decreased cell proliferation.

[0234] Additionally, in vitro wound healing tests (Figures 8a and 8b) were performed to investigate the wound healing ability and fibroblast migration characteristics of the fabricated membranes. Results showed that the width of the wound (30%) was identical in all membrane groups at 0 h. After 12 h of incubation with the extraction medium, the scratches of the control, AG-TH, and AG-TH / PG-MC groups narrowed, and the wound gap decreased to 11.7 ± 1.40%, 7.94 ± 0.67%, and 6.68 ± 0.34%, respectively. However, wound closure of the PG-MC layer was slightly reduced (14.64 ± 0.637%) after 24 h. The culture results showed that the percentage of the wound gap was significantly closed in both the AG-TH (0.67±0.2%) and AG-TH / PG-MC (0.51±0.16%) membranes, but the wound gap scratch was not completely closed in the PG-MC membrane, and 5.157±0.319% of the wound gap was not yet healed. The possible reason was the remarkable antifibrotic activity of MC released from the PG-MC layer. Fibroblasts always migrate to the wound site and proliferate during the wound healing process. In the present invention, we found that MC significantly inhibited the migration of fibroblasts into the scratched wound site. This mechanism of action is important for achieving the anti-adhesion property of the PG-MC layer.

[0235]

[0236] 2.3. In vitro and in vivo blood compatibility assessment

[0237]

[0238] One of the key issues related to hemostatic graft materials is analyzing the efficacy of hemolysis rates upon contact with blood. These interaction results demonstrated that red blood cell lysis releases intracellular hemoglobin, and the amount of released hemoglobin ensures that the graft membrane is toxic. A high amount of released hemoglobin indicates toxicity, while a low amount of released hemoglobin ensures no toxicity. Figure 6e shows optical images of hemolysis results in the inner layer supernatant with thrombin (AG-TH), the inner layer supernatant without thrombin (AG), PBS as a negative control, and Triton X-100 as a positive control. Compared to the positive control, which was red in the sample group, the AG-TH membrane exhibited a more vivid color than the negative control. Quantitative results showed that the degree of hemolysis was reduced in the AG-TH sample group due to the presence of thrombin in the sample. The results (Figure 6g) showed hemolysis rates of 1.33% and 1.6% for the AG-TH and AG membranes, respectively.

[0239] Furthermore, the blood coagulation effect of a material, as assessed by a blood coagulation assay, is also an important factor in relation to the thrombogenicity and coagulation activity of the biomaterial. Coagulation activity occurs immediately when an excellent hemostatic biomaterial comes into direct contact with blood. In Figure 6f, an optical image of the blood coagulation assay shows that coagulation activity occurred immediately in the thrombin-loaded group AG-TH. In contrast, coagulation activity did not occur properly in the AG group, and blood was still mixed with the supernatant after 35 minutes of incubation. The results demonstrated that blood cells in the AG-TH membrane were trapped within the clot and did not burst, releasing hemoglobin from the sample even after the addition of deionized water. Furthermore, the anticoagulant exhibited a higher absorbance value, while the coagulant exhibited a lower absorbance value, indicating a rapid clotting rate of the biomaterial. As shown in Figure 6h, the AG-TH membrane exhibited a BCI index of 7.5%, while the AG membrane exhibited a BCI of 17.98%.

[0240] Additionally, the coagulation properties of the biomaterial lead to the adsorption of plasma proteins, either through the activation of blood coagulation factors or through the activation of platelet adhesion. This mechanism forms an insoluble network of fibrin or thrombus. Therefore, SEM images were taken to observe platelet adhesion and activation in vitro. In Figure 9a, excessive platelet adhesion and activated platelets were observed on the AG-TH membrane in the thrombin-loaded group. In contrast, less activated platelet adhesion was observed in the AG membrane group.

[0241] In addition, an in vivo hemostasis evaluation was performed to analyze the degree of activation of coagulation factors and clotting time affected by the AG-TH group, and compared them with the commercially available product Tachosil (control group). The hemostatic ability was observed in a rat tail amputation model using the control group (Fig. 9b) and the AG-TH membrane (Fig. 9c). According to the results (Fig. 6i), the bleeding time in the AG-TH group was significantly reduced, stopping after 140 ± 9 seconds. In contrast, the bleeding time in the control group stopped after 190 ± 25 seconds. The shorter the blood coagulation and cessation time, the better the material coagulation. In addition, the blood loss was also reduced in the AG-TH group (2.8 ± 0.4 g) compared to the control group (3.35 ± 0.5 g) (Fig. 6j). In the present invention, the AG-TH membrane is used as a hemostatic agent to activate the protease-activated receptor of the platelet cell membrane, thereby promoting platelet activation and aggregation, thereby converting fibrinogen into fibrin and regulating coagulation-activated protein C.

[0242]

[0243] 2.4. Antibacterial evaluation

[0244]

[0245] Infection is a common cause of all postoperative complications. Most postoperative infections become chronic. In cases of pancreatic leakage, infectious complications are a major cause of postoperative morbidity. However, despite the increase in postoperative pancreatic infections, postoperative mortality has recently decreased. Anticancer agents with antimicrobial activity are promising options for preventing infection, especially those that affect the endogenous basal system. Some anticancer agents have significant antimicrobial properties due to the similarities between bacterial infections and growing tumors, such as high replication rates, high dissemination potential, high resistance to the immune system, and a tendency to be insensitive to treatment. Therefore, mitomycin-C (MC), a potent anticancer agent with antimicrobial properties, was used in the present invention to prevent postoperative infections. MC has remarkable antimicrobial properties against various bacterial pathogens, including E. coli and S. aureus. It is also used clinically in the treatment of gastric and pancreatic cancers.

[0246] In the present invention, antibacterial tests were performed against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli using the AH-TH, PG-MC, and AG-TH / PG-MC membranes. The results showed that both the outer layer (PG-MC) and the double-layer membrane (AG-TH / PG-MC) exhibited potent antibacterial activity against all types of bacteria used in the present invention (Fig. 6k). The cumulative release of MC from the outer layer revealed antibacterial properties. Furthermore, PG-MC and AG-TH / PG-MC exhibited inhibition zones against both types of bacteria (Fig. 6l).

[0247]

[0248] 2.5. Evaluation of in vivo and in silico molecular simulation studies

[0249]

[0250] In vivo studies divided the suture-only group into a control group (Fig. 10d) and a sample group using the AG-TH / PG-MC membrane (Fig. 10e). Animal experiments were performed for 2 and 4 weeks to assess the possibility of leakage at the resection site, wound healing, and the adhesive properties of the membrane. Results at 2 and 4 weeks post-implantation (Figs. 10f and 5g) showed that the suture group had suture line breakage, resulting in pancreatic leakage at the resection site. In contrast, the AG-TH / PG-MC group had no possibility of leakage, as the double-layer membrane attached to the resection site protected the suture line from breakage. The suture line broke after 2 weeks in the control group and deteriorated after 4 weeks. In addition, no adhesions were found with other organs (stomach, intestine, kidney, and tissue surfaces) surrounding the pancreas in the AG-TH / PG-MC group due to the anti-adhesive properties of the PG-MC layer. However, tissue adhesions with other organs near the pancreas occurred in the control group.

[0251] Additional histological staining was performed to evaluate the hemostasis, wound healing, and adhesion properties of the membrane. H&E staining (Figs. 10h and 5i) demonstrated that the AG-TH / PG-MC membrane adhered adequately to the resection site for up to 4 weeks after transplantation in the sample group. Controlled release of MMC from the outer layer of the membrane prevented tissue adhesion between the membrane and the surrounding pancreatic organs. However, the control group developed severe inflammation due to leakage at the suture site, with a large number of neutrophils and hemorrhage observed at the suture site. Two weeks after transplantation, the AG-TH layer degraded, and some neutrophils and monocytes were observed at the interface due to inflammation, while a layer of fibroblasts formed in the inner zone. After 4 weeks, fibroblasts penetrated the membrane and adhered properly to the resection surface. No inflammation was observed at the interface or inner zone. However, suture line damage was noticeable in the control group 4 weeks after transplantation. These results indicated that the wound healing process was slowed in the control group due to pancreatic leakage.

[0252] Furthermore, collagen accumulation and fibroblast migration to the resection site are necessary for effective wound healing. Therefore, we performed immunohistochemical staining for collagen-1 and α-SMA markers to analyze the progress of wound healing. The results showed that the AG-TH / PG-MC membrane had higher contents of collagen-1 and α-SMA markers than the control group. The results in Figure 10j suggest that the AG-TH / PG-MC membrane has the ability to enhance the wound healing process. The AG-TH layer, composed of alginate, gelatin, and thrombin, was confirmed to play an important role in wound regeneration due to its excellent adhesive and hemostatic properties after cross-linking with calcium chloride. Therefore, collagen-1 accumulation was higher in the AG-TH / PG-MC membrane than in the control group. According to the statistical results, the relative positive staining area for collagen-1 was 62.730 ± 2.0% in the AG-TH / PG-MC group after 4 weeks, compared to 42.982 ± 2.5% in the control group (Figure 10l). Furthermore, Masson's trichrome staining was additionally performed to detect total collagen accumulation in the membrane (Figs. 11 and 12). The results showed positive expression 4 weeks after transplantation. Total collagen accumulation was higher in the AG-TH / PG-MC membrane than in the control group. Furthermore, the collagen content of rat pancreas is relatively low. However, thrombin activity in the AG-TH layer of the membrane increased collagen formation in the pancreas 4 weeks after transplantation. α-SMA plays an essential role in the function of myofibroblasts. Therefore, during wound healing, fibroblasts secrete the α-SMA factor, which promotes cell migration. Figure 10k shows that the expression of the α-SMA marker was almost similar in the AG-TH / PG-MC group and the suture-only (control) group after 4 weeks. Due to the healing process, a thin layer of new tissue was formed on the upper surface of the sample membrane after 2 weeks. However, the expression of α-SMA was slightly lower in the AG-TH / PG-MC group due to the antifibrotic activity of the PG-MC layer membrane, and the layer formed a single tissue layer with the membrane after 4 weeks.IOD analysis results showed that after 4 weeks, the positive expression areas of α-SMA were 56.920±1.38% and 51.59±32% in the control and AG-TH / PG-MC membranes, respectively (Fig. 10m).

[0253] Although the AG-TH membrane showed positive expression of collagen-1 and α-SMA markers due to the wound healing process, we predicted that MC released from the PG-MC layer would reduce fibroblast proliferation and thus fibronectin formation, which would further help reduce adhesion between the adventitia and other organs. Therefore, we performed immunofluorescence staining of the fibronectin marker. Figures 13a and 13b show that the expression of the fibronectin marker was significantly reduced in the PG-MC layer 2 and 4 weeks after transplantation compared to the control group. Release of MC from the PG-MC layer inhibits fibroblast migration and proliferation, thereby suppressing fibronectin formation in the adventitia. However, the AG-TH layer membrane showed positive expression of the fibronectin marker. The mechanism of fibroblast cell death occurs by inhibiting DNA-dependent RNA synthesis and cell proliferation. Therefore, the quantitative analysis results in Fig. 13c show that the expression of fibronectin-positive area was only 1.59% in AG-TH / PG-MC after 2 weeks, while it was 30.45% in the control group. Four weeks after transplantation, the positive area in AG-TH / PG-MC was 2.68%, while it was 53.80% in the control group (Fig. 13d).

[0254] Several studies have shown that MC reduces fibrosis by inhibiting transforming growth factor ββ. TGF-β is a multifactorial growth factor synthesized by cells throughout the body. It is a key fibrogenic mediator that plays a crucial role in the proliferation, activation, migration, and transformation of fibroblasts into myofibroblasts. Furthermore, TGF-β induces collagen production in fibroblasts, leading to their differentiation into myofibroblasts. Myofibroblasts actively produce collagen compared to normal fibroblasts. In the present invention, we hypothesized that cumulative release of MC from the PG-MC layer suppresses transforming growth factor β, thereby reducing cell proliferation and migration and lowering the expression of positive fibronectin markers in the outer layer. Therefore, when MC is released from the PG-MC layer, it acts as an antifibrotic agent, preventing adhesion of surrounding organs and membranes.

[0255] To demonstrate the antifibrotic activity of the PG-MC layer following MC release, in silico molecular simulation studies were performed. As a control to demonstrate the antifibrotic mechanism, 3-(2-chlorobenzyl)-1,7-dimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-412), a known inhibitor drug, was selected. Several studies have previously demonstrated that IM-412 significantly inhibits the expression of TGF-β receptor types I and II and downregulates fibronectin expression. IM-412 is an active compound with a marked inhibitory effect on TGF-β. Therefore, in silico molecular simulation studies were performed to identify potential binding sites of MC and the TGF-β receptor and TGF-β in the present invention, and compared the known inhibitor drug IM-412 with the TGF-β receptor and the possible binding sites of TGF-β. Figure 13e shows the possible binding sites of IM-412 and MC and the TGF-β receptor and TGF-β. The results showed that MC has a binding site, binding distance, binding type, and location similar to those of IM-412.

[0256] In addition, the binding energies of MC and IM-412 with TGF-β (Fig. 13f) were almost similar, 7.40 ± 0.52 and 8.73 ± 0.15, respectively. In Fig. 13g, the results showed that IM-412 and MC could bind to the receptor TGF-β, and the binding energies of MC and IM-412 to TGF-β were 7.467 ± 0.85 and 8.167 ± 0.83, respectively. The binding distance, binding type, binding category, and pose are described in more detail in Table 2, and the known inhibitor drugs were used as controls and the binding of MC to each protein or receptor was compared. These results demonstrated that the antifibrotic mechanisms of MC and IM-412 were the same, and both drugs had similar binding sites, binding types, and positions. The study results demonstrated that, like IM-412, MC significantly inhibited myofibroblast differentiation by inhibiting the TGF-β response. Therefore, it is clear that the PG-MC layer acts as a powerful antifibrotic membrane, suppressing the expression of fibronectin markers.

[0257]

[0258] [Table 2]

[0259] Table 2: Molecular interactions of IM412 and MC with TGF-β and TGF-β target proteins / receptors.

[0260]

[0261]

[0262]

[0263] The antifibrotic activity of the PG-MC layer, induced by MC release, was further confirmed through in vivo animal experiments in a rat cecum model. The in vivo study was divided into a negative control group (untreated), AG-TH / PG-MC (sample group), and a positive control group (commercial product: Seprafilm adhesive barrier). The animal experiments were observed for 7 and 14 days to assess the possible antifibrotic properties of the membrane. Figure 14 shows that the untreated negative control group exhibited severe adhesions to the abdominal side wall and cecal wall 7 and 14 days after implantation. However, the AG-TH / PG-MC membrane, similar to the positive control group, did not exhibit adhesions 7 and 14 days after implantation. The abrasion site healed on the abdominal wall, and sample degradation was observed after 14 days. Histological staining was also performed to evaluate the antifibrotic activity of the outer layer (Figures 15 and 16). At 7 and 14 days after transplantation, no fibrosis or vascularization was observed at the AG-TH / PG-MC membrane interface. As part of the healing process, some monocytes and neutrophils were observed at the basal portion of the membrane, where the cecal wall was located. Fibroblasts were observed at the abrasion site of the abdominal wall after 14 days, and the wound was almost completely healed in both the bilayer and positive control groups.

[0264] The mechanism of the multifunctional bilayer membrane is illustrated in Figure 17. The fabricated AG-TH / PG-MC membrane exhibited the adhesive, hemostatic, and wound healing properties of the AG-TH layer, and the anti-adhesion and antibacterial properties of the PG-MC layer. Furthermore, it was evident that cross-linking the AG-TH layer with a calcium chloride solution and imparting hydrophilicity enhanced the adhesive strength. The rapid degradation of the AG-TH layer induces the burst release of thrombin, which converts fibrinogen into insoluble fibrin clots and enhances platelet adhesion for effective hemostasis. Furthermore, the burst release of TH also increases collagen secretion and promotes fibroblast proliferation for wound regeneration. The sustained release of MC from the PG-MC layer was observed to suppress the expression of the TGF-β response. TGF-β and TGF-β are major profibrotic mediators known to stimulate fibrosis. The MC in the PG-MC layer suppresses the expression profile of TGF-β and reduces fibroblast migration, proliferation, and apoptosis. This significantly enhances the antifibrotic properties of the PG-MC layer. The multifunctional AG-TH / PG-MC membrane may help improve pancreatic function recovery while providing effective leak prevention.

[0265]

[0266] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0267]

[0268] The national research and development projects that supported this invention are as follows.

[0269] [Project ID] 1345284644

[0270] [Assignment Number] 2015R1A6A1A030325223

[0271] [Ministry Name] Ministry of Education

[0272] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0273] [Research Project Name] University Key Research Institute Support Project (Science and Engineering)

[0274] [Research Project Title] Development of Metal-Ceramic-Polymer Hybrid Biomaterials for Clinical Treatment

[0275] and tissue regeneration research

[0276] [Name of the project performing organization] Soonchunhyang University Industry-Academic Cooperation Foundation

[0277] [Research Institute] March 1, 2023 - February 28, 2024

Claims

1. A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, comprising an inner layer comprising alginate and gelatin; and an outer layer comprising polycaprolactone and gelatin.

2. In paragraph 1, A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the inner layer containing the above alginate and gelatin further contains thrombin.

3. In paragraph 1, A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the outer layer including the polycaprolactone and gelatin further includes mitomycin-C.

4. In paragraph 1, A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the inner layer contains 1% (w / v) alginate and 10% (w / v) gelatin.

5. In paragraph 1, A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the outer layer contains 10% (w / v) polycaprolactone and 10% (w / v) gelatin in a concentration.

6. In paragraph 1, A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the inner layer containing the above alginate and gelatin is cross-linked with calcium chloride.

7. In paragraph 1, The above double-layer membrane is a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the contact angle is 60 to 70°.

8. In paragraph 1, The above double-layer membrane is a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the double-layer membrane has a swelling ratio of 500 to 600%.

9. In paragraph 1, The above double-layer membrane is a double-layer membrane having a tensile strength of 4 to 15 MPa, a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function.

10. In paragraph 1, The above double-layer membrane is a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the double-layer membrane has antibacterial activity.

11. In paragraph 1, The above antibacterial activity is characterized by a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function against Staphylococcus aureus and Escherichia coli.

12. In paragraph 1, A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the double-layer membrane has anti-adhesion properties.

13. In paragraph 1, A bilayer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, characterized in that the bilayer membrane is a scaffold.

14. A step of manufacturing an inner layer containing alginate and gelatin by adding alginate and gelatin to the first solution; A step of manufacturing an outer layer including polycaprolactone and gelatin by adding polycaprolactone and gelatin to a second solution; and A method for producing a double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function, comprising the step of spraying an electrospinning solution onto an inner layer containing the alginate and gelatin.

15. A double-layer membrane having a blood or intestinal fluid leakage prevention function, a hemostatic function, and a tissue adhesion prevention function manufactured by the manufacturing method of Article 14.

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