Body fluid-resistant tissue adhesive

Abstract

A tissue adhesive material that provides fast and robust adhesion even on tissue surfaces covered with body fluids. [Solution] The tissue adhesive material is formed from a hydrophobic matrix and a plurality of bioadhesive microparticles dispersed within the hydrophobic matrix, and is configured such that disposing the adhesive material directly on a fluid-covered surface and applying pressure to the adhesive material causes (a) the hydrophobic matrix to repel the fluid, (b) the bioadhesive particles to compress and form an adhesive layer, and (c) the bioadhesive particles to form temporary crosslinks and then covalent crosslinks with the surface.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 942,874, filed December 3, 2019, entitled “Body Fluid Resistant Tissue Adhesives” (which is incorporated herein by reference in its entirety).

[0002] Description of government support This invention was made with government support under grant number CMMI1661627 from the National Science Foundation (NSF), grant number W911NF-13D-0001 from the Army Research Office (ARO), and grant number N00014-17-1-2920 from the Office of Naval Research (ONR). The government reserves certain rights to this invention.

[0003] Field of Invention The present invention generally relates to adhesive materials and methods for bonding tissues, wherein the adhesive material has the ability to rapidly and robustly bond fluid-covered tissues. The adhesive material comprises a hydrophobic matrix material in which dry bio-adhesive microparticles are dispersed. [Background technology]

[0004] Background of the Invention Traumatic injuries to tissues and organs are life-threatening and can be difficult to treat because they are quite time-constrained and have a complex nature (R. R. Rodrigues, M. J. C. Carmona, J. O. C. Junior, Bleeding and damage control surgery. Current Opinion in Anesthesiology 29, 229-233 (2016)). For example, uncontrolled bleeding after trauma is one of the leading causes of mortality worldwide, claiming over two million lives annually (R. Pfeifer, I. S. Tarkin, B. Rocos, H.-C. Pape, Patterns of mortality and causes of death in polytrauma patients - has anything changed? Injury 40, 907-911 (2009), M. El Sayad, H. Noureddine, Recent advances of hemorrhage management in severe trauma. Emergency Medicine International 2014, (2014)). Surgical suturing of traumatic injuries is most commonly performed with sutures or staples after hemostasis, but it is difficult to promptly and effectively address the implementation of this process at the scene after traumatic injury.

[0005] Tissue adhesives offer a promising alternative to sutures and staples for wound closure and tissue repair (TB Reece, TS Maxey, IL Kron, A prospectus on tissue adhesives. The American Journal of Surgery 182, S40-S44 (2001), P. Coulthard et al., Tissue adhesives for closure of surgical incisions. Cochrane Database of Systematic Reviews 5, CD004287 (2010), B. Sharma et al., Human cartilage repair with a photoreactive adhesive-hydrogel composite. Science Translational Medicine 5, 167ral66-167ral66 (2013), N. Annabi, K. Yue, A. Tamayol, A. Khademhosseini, Elastic sealants for surgical applications. European Journal of Pharmaceutics and Biopharmaceutics 95, 27-39 (2015), ET Roche et al. al., A light-reflecting balloon catheter for atraumatic tissue defect repair. Science Translational Medicine 7, 306ral49-306ral49 (2015)), existing tissue adhesives have several limitations.Commercially available tissue adhesives do not provide adhesion to tissue surfaces covered by body fluids such as blood and mucus, or only provide very weak and brittle adhesion (N. Lang et al., A blood-resistant surgical glue for minimally invasive repair of vessels and heart defects. Science Translational Medicine 6, 218ra216-218ra216 (2014), Y. Hong et al., A strongly adhesive hemostatic hydrogel for the repair of arterial and heart bleeds. Nature Communications 10, 2060 (2019)). Although a few blood-resistant tissue adhesives with improved adhesion performance have been developed, their usefulness in practical applications is substantially limited because ultraviolet (UV) irradiation and / or long-term constant pressure application (e.g., 5 minutes) are required for the formation of adhesion (Lang et al; Y. Hong et al; N. Annabi et al., Engineering a highly elastic human protein-based sealant for surgical applications. Science Translational Medicine 9, eaai7466 (2017), J. Li et al., Tough adhesives for diverse wet surfaces. Science 357, 378-381 (2017)). Summary of the Invention Problems to be Solved by the Invention

[0006] Therefore, further improvements in both adhesive materials and usage methods are highly needed. Means for Solving the Problems

[0007] Summary of the Invention This invention provides a tissue adhesive material that is particularly useful in humid environments. Because the tissue adhesive material provides rapid and robust adhesion even on tissue surfaces covered with bodily fluids, it offers significant advantages in a variety of applications, including traumatic injuries requiring rapid and reliable wound closure and tissue repair in the field.

[0008] According to one embodiment, the present invention provides an adhesive material for bonding one or more fluid-covered surfaces, comprising a hydrophobic matrix and a plurality of bio-adhesive microparticles dispersed within the hydrophobic matrix. The bio-adhesive microparticles comprise (i) one or more hydrophilic polymers or copolymers, (ii) one or more amine coupling groups, and (iii) one or more crosslinking agents. The hydrophobic matrix exists around the dispersed bio-adhesive microparticles in the form of a protective matrix that protects the bio-adhesive microparticles from the fluid. The adhesive material is structured such that direct placement of the adhesive material on a fluid-covered surface and application of pressure to the adhesive material causes (a) the hydrophobic matrix to repel the fluid, (b) the bio-adhesive particles to compress and form an adhesive layer, and (c) the bio-adhesive particles to form temporary crosslinks, and subsequently covalent crosslinks with the surface.

[0009] Embodiments relating to these aspects may include one or more of the following features: The adhesive material is in the form of an injectable adhesive material. One or more hydrophilic polymers or copolymers are selected from hydrophilic polymers or copolymers that absorb water in a dry state. One or more hydrophilic polymers or copolymers are selected from polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, polyurethane, casein, albumin, gelatin, chitosan, hyaluronic acid, alginate, oxidized alginate, cellulose, oxidized cellulose, polyvinylpyrrolidone, polystyrene sulfonate, collagen, alginic acid, pectin, and combinations thereof. One or more amine coupling groups are selected from N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, aldehydes, imide esters, epoxides, isocyanates, catechols, and combinations thereof. One or more crosslinking agents are selected from gelatin methacrylate, hyaluronic acid methacrylate, oxidized methacrylate-based alginates, polycaprolactone diacrylate, N,N'-bis(acryloyl)cystamine, N,N'-methylenebis(acrylamide), polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and combinations thereof. The hydrophobic matrix is ​​selected from silicone oil, mineral oil, essential oil, perfluoropolyether oil, lanolin oil, and combinations thereof. The adhesive material comprises a plurality of bio-adhesive microparticles prepared from (i) poly(acrylic acid), (ii) N-hydroxysuccinimide ester grafted onto it (PAAc-co-NHS ester), (iii) crosslinked with biodegradable gelatin methacrylate, and (iv) biodegradable chitosan, dispersed in a silicone oil hydrophobic matrix. The adhesive material is biocompatible. The adhesive material has a minimum capacity of about 100 Jm -2The adhesive adheres with interfacial toughness, a shear strength of at least about 30 kPa, and a tensile strength of at least about 10 kPa. The bio-adhesive microparticles contain carboxylic acid groups that form temporary crosslinks by intermolecular bonding, and amine coupling groups that form covalent crosslinks with the surface. The bio-adhesive microparticles have a particle size in the range of about 10 μm to about 200 μm. The ratio of bio-adhesive microparticles to the hydrophobic matrix is ​​in the range of about 1:3 to about 1:0.5. One or more fluids are physiological body fluids selected from blood, saliva, gastrointestinal fluid, mucus, juices, and combinations thereof. The adhesive material is biodegradable and configured to allow cell penetration into the crosslinked bio-adhesive microparticles and healing of underlying tissue injury. Healing of underlying tissue injury involves tissue cells replacing the biodegradable bio-adhesive microparticles to heal the underlying tissue injury.

[0010] In another embodiment, the present invention provides a method for bonding one or more tissue surfaces covered with one or more fluids, comprising: (a) directly applying an adhesive material to one or more of the fluid-covered tissue surfaces, the adhesive material comprising a hydrophobic matrix and a plurality of bio-adhesive microparticles dispersed within the hydrophobic matrix, the bio-adhesive microparticles comprising (i) one or more hydrophilic polymers or copolymers, (ii) one or more amine coupling groups, and (iii) one or more crosslinking agents; (b) applying a pressure to the adhesive material in the range of about 1 kPa to 50 kPa; (c) enabling the hydrophobic matrix to repel one or more fluids and remove them from the tissue surface; (d) enabling physical bonding groups in the bio-adhesive microparticles to form temporary crosslinks by intermolecular bonding; and (e) enabling the amine coupling groups in the bio-adhesive microparticles to form covalent crosslinks with the tissue surface.

[0011] Embodiments relating to these aspects may include one or more of the following features: Pressure is applied for approximately 5 seconds to approximately 30 seconds. The adhesive material is an injectable adhesive material, and the adhesive material is applied using a syringe. One or more hydrophilic polymers or copolymers are selected from hydrophilic polymers or copolymers that absorb water in a dry state. One or more hydrophilic polymers or copolymers are selected from polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, polyurethane, casein, albumin, gelatin, chitosan, hyaluronic acid, alginate, oxidized alginate, cellulose, oxidized cellulose, polyvinylpyrrolidone, polystyrene sulfonate, collagen, alginic acid, pectin, and combinations thereof. One or more amine coupling groups are selected from N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, aldehydes, imide esters, epoxides, isocyanates, catechol, and combinations thereof. One or more crosslinking agents are selected from gelatin methacrylate, hyaluronic acid methacrylate, oxidized methacrylate-based alginates, polycaprolactone diacrylate, N,N'-bis(acryloyl)cystamine, N,N'-methylenebis(acrylamide), polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and combinations thereof. The hydrophobic matrix is ​​selected from silicone oil, mineral oil, essential oil, perfluoropolyether oil, lanolin oil, and combinations thereof. The adhesive comprises a plurality of bio-adhesive microparticles dispersed in a silicone oil hydrophobic matrix, each made from (i) poly(acrylic acid) grafted with (ii) N-hydroxysuccinimide ester (PAAc-co-NHS ester), (iii) crosslinked with biodegradable gelatin methacrylate, and (iv) biodegradable chitosan. The adhesive material has a strength of at least about 100 Jm -2The adhesive adheres with interfacial toughness, a shear strength of at least about 30 kPa, and a tensile strength of at least about 10 kPa. The physical bond-forming groups in the bio-adhesive microparticles are carboxylic acid groups that form temporary crosslinks by intermolecular bonds. The bio-adhesive microparticles have a particle size in the range of about 10 μm to about 200 μm. The adhesive material includes a ratio of bio-adhesive microparticles to a hydrophobic matrix in the range of about 1:3 to about 1:0.5. One or more fluids are physiological body fluids selected from plasma, interstitial fluid, lymph, cerebrospinal fluid, gastrointestinal fluid, and combinations thereof. (a) After directly applying the adhesive material to one or more tissue surfaces covered with the fluid, and (b) before applying pressure, the method further includes applying a backing material to the adhesive material, where (b) applying pressure includes applying pressure to the adhesive material through the backing material. The backing material is made from a biocompatible material that does not adhere to wet surfaces. The backing material is made from oxidized cellulose, silicone elastomer, polyurethane, hydrogel, any other biocompatible material that does not adhere to wet tissue, or a combination thereof. One or more tissue surfaces may contain tissue damage, and the method further comprises enabling cell penetration into crosslinked bioadhesive microparticles and healing of the underlying tissue damage.

[0012] In another embodiment, the present invention provides a method for healing tissue injury, comprising: (a) directly applying an adhesive material to the tissue injury, the tissue injury comprising one or more tissue surfaces covered with a fluid, the adhesive material comprising a hydrophobic matrix and a plurality of bioadhesive microparticles dispersed within the hydrophobic matrix, the bioadhesive microparticles comprising (i) one or more hydrophilic polymers or copolymers, (ii) one or more amine coupling groups, and (iii) one or more crosslinking agents; (b) applying a pressure to the adhesive material in the range of about 1 kPa to 50 kPa; (c) enabling the hydrophobic matrix to repel one or more fluids and remove them from the tissue surface; (d) enabling physical bonding groups in the bioadhesive microparticles to form temporary crosslinks by intermolecular bonding; (e) enabling amine coupling groups in the bioadhesive microparticles to form covalent crosslinks with the tissue surface; and (f) enabling cell penetration into the crosslinked bioadhesive microparticles and healing of the underlying tissue injury.

[0013] Embodiments relating to these aspects may include one or more of the following features: The adhesive material is biodegradable, and cells heal underlying tissue damage by replacing it with biodegradable bioadhesive microparticles. Pressure is applied for approximately 5 to 30 seconds. The adhesive material is an injectable adhesive material, and the adhesive material is applied using a syringe. One or more hydrophilic polymers or copolymers are selected from hydrophilic polymers or copolymers that absorb water in a dry state. One or more hydrophilic polymers or copolymers are selected from polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, polyurethane, casein, albumin, gelatin, chitosan, hyaluronic acid, alginate, oxidized alginate, cellulose, oxidized cellulose, polyvinylpyrrolidone, polystyrene sulfonate, collagen, alginic acid, pectin, and combinations thereof. One or more amine coupling groups are selected from N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, aldehydes, imide esters, epoxides, isocyanates, catechols, and combinations thereof. One or more crosslinking agents are selected from gelatin methacrylate, hyaluronic acid methacrylate, oxidized methacrylate-based alginates, polycaprolactone diacrylate, N,N'-bis(acryloyl)cystamine, N,N'-methylenebis(acrylamide), polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and combinations thereof. The hydrophobic matrix is ​​selected from silicone oil, mineral oil, essential oil, perfluoropolyether oil, lanolin oil, and combinations thereof. The adhesive contains multiple bio-adhesive microparticles, each made from (i) poly(acrylic acid) grafted with (ii) N-hydroxysuccinimide ester (PAAc-co-NHS ester), (iii) crosslinked with biodegradable gelatin methacrylate, and (iv) biodegradable chitosan, dispersed in a silicone oil hydrophobic matrix. The adhesive material has a capacity of at least approximately 100 Jm -2The adhesive adheres with interfacial toughness, a shear strength of at least about 30 kPa, and a tensile strength of at least about 10 kPa. The physical bond-forming groups in the bio-adhesive microparticles are carboxylic acid groups that form temporary crosslinks by intermolecular bonds. The bio-adhesive microparticles have a particle size in the range of about 10 μm to about 200 μm. The adhesive material includes a ratio of bio-adhesive microparticles to a hydrophobic matrix in the range of about 1:3 to about 1:0.5. One or more fluids are physiological body fluids selected from plasma, interstitial fluid, lymph, cerebrospinal fluid, gastrointestinal fluid, and combinations thereof. (a) After directly applying the adhesive material to one or more tissue surfaces covered with the fluid, and (b) before applying pressure, the method further includes applying a backing material to the adhesive material, where (b) applying pressure includes applying pressure to the adhesive material through the backing material. The backing material is made from a biocompatible material that does not adhere to wet surfaces. The backing material is made from oxidized cellulose, silicone elastomer, polyurethane, hydrogel, any other biocompatible material that does not adhere to wet tissue, or combinations thereof.

[0014] Other systems, methods, and features of the present invention will be apparent to those skilled in the art by examining the drawings and detailed description below. All such additional systems, methods, and features are incorporated herein, within the scope of the present invention, and intended to be protected by the appended claims.

[0015] Brief explanation of the drawing The accompanying drawings are included to provide a further understanding of the present invention and are incorporated herein by reference and constitute part thereof. The elements of the drawings are not necessarily to scale, and instead the focus is on clearly illustrating the principles of the present invention. The drawings illustrate embodiments of the present invention and, together with the description, serve to illustrate the main parts of the present invention. [Brief explanation of the drawing]

[0016] [Figure 1A]A schematic example of a tissue adhesive material according to one embodiment of the present invention is shown. The adhesive material is formed from dried bio-adhesive microparticles and a hydrophobic oil matrix deposited on tissue covered with bodily fluids. [Figure 1B] A schematic example of a tissue adhesive material according to one embodiment of the present invention is provided. The adhesive material exhibits a repulsive-crosslinking mechanism that repels bodily fluids. [Figure 1C] A schematic example of a tissue adhesive material according to one embodiment of the present invention is provided. The repulsion-crosslinking mechanism by which the adhesive material forms robust adhesion is shown. [Figure 1D] A photograph shows adhesive material injected onto blood-covered pig skin tissue. This corresponds to panel 1A. [Figure 1E] A photograph shows adhesive material pressed against a gelatin-coated glass substrate. This corresponds to the panel in Figure 1B. [Figure 1F] Photograph the adhesive material bonded to the tissue surface. This corresponds to panel 1C. [Figure 2A] The photograph shows two blood-covered pig heart tissues being rapidly and robustly bonded together after applying gentle pressure for 5 seconds using a tissue adhesive according to one embodiment of the present invention. [Figure 2B] The photograph shows two blood-covered pig heart tissues being rapidly and robustly bonded together after applying gentle pressure for 5 seconds using a tissue adhesive according to one embodiment of the present invention. [Figure 2C] The photograph shows two blood-covered pig heart tissues being rapidly and robustly bonded together after applying gentle pressure for 5 seconds using a tissue adhesive according to one embodiment of the present invention. [Figure 2D] The photograph shows two blood-covered pig heart tissues being rapidly and robustly bonded together after applying gentle pressure for 5 seconds using a tissue adhesive according to one embodiment of the present invention. [Figure 3A] The following photograph illustrates the preparation of a tissue adhesive material according to one embodiment of the present invention. A small section of the dried bio-adhesive is shown. [Figure 3B]The following photograph illustrates the preparation of a tissue adhesive material according to one embodiment of the present invention. It shows the addition of dried bio-adhesive to a stainless steel container containing stainless steel balls. [Figure 3C] The following photograph illustrates the preparation of a tissue adhesive material according to one embodiment of the present invention. It shows the use of a cryogenic ball mill for grinding the dried bio-adhesive. [Figure 3D] A photograph illustrating the preparation of a tissue adhesive material according to one embodiment of the present invention is shown. The resulting dried bio-adhesive microparticles are shown. [Figure 3E] The following photograph illustrates the preparation of a tissue adhesive material according to one embodiment of the present invention. It shows the tissue adhesive material (a mixture of dried bio-adhesive microparticles and a hydrophobic matrix) delivered via a syringe. [Figure 4] The entire process of applying a tissue adhesive according to one embodiment of the present invention is schematically illustrated. The adhesive is applied directly to a tissue surface covered with bodily fluids, and then by applying gentle pressure, (i) the hydrophobic matrix repels the bodily fluids and removes them from the tissue surface, while (ii) the physical bonding groups in the bio-adhesive microparticles temporarily crosslink them by intermolecular bonds, followed by covalent crosslinking, to form a thin layer of hydrogel with the swollen and crosslinked tissue adhesive material, providing robust adhesion between tissues. [Figure 5] The image shows photographs of cross-sections of two blood-covered pig heart tissues bonded with a tissue adhesive material according to one embodiment of the present invention, 5 minutes and 24 hours after application. [Figure 6] The images show the swelling and crosslinked adhesive layer between two gelatin hydrogels 30 minutes after application of a tissue adhesive according to one embodiment of the present invention. [Figure 7] Using whiskers corresponding to the upper and lower poles, the sizes of dried bio-adhesive microparticles under various cryogenic grinding frequencies are plotted graphically. The line represents the median, and the error bars represent the upper and lower quartiles (N=3). [Figure 8A]This section illustrates SEM images of various dry bio-adhesive microparticle sizes obtained by 2-minute cryogenic grinding at different grinding frequencies. The image corresponds to 10Hz. [Figure 8B] This section illustrates the injection properties of tissue adhesives using various dry bio-adhesive microparticle sizes via 2.5 mm or 1.2 mm diameter syringes. The 2.5 mm diameter syringe is used for this purpose. [Figure 8C] This section illustrates SEM images of various dry bio-adhesive microparticle sizes obtained by 2-minute cryogenic grinding at different grinding frequencies. The image corresponds to 15Hz. [Figure 8D] This section illustrates the injection properties of tissue adhesives using various dry bio-adhesive microparticle sizes via 2.5 mm or 1.2 mm diameter syringes. The 1.2 mm diameter syringe is used for this purpose. [Figure 8E] This section illustrates SEM images of various dry bio-adhesive microparticle sizes obtained by 2-minute cryogenic grinding at different grinding frequencies. The image corresponds to 20Hz. [Figure 8F] This section illustrates the injection properties of tissue adhesives using various dry bio-adhesive microparticle sizes via 2.5 mm or 1.2 mm diameter syringes. The 1.2 mm diameter syringe is used for this purpose. [Figure 8G] This section illustrates SEM images of various dry bio-adhesive microparticle sizes obtained by 2-minute cryogenic grinding at different grinding frequencies. The image corresponds to 25Hz. [Figure 8H] This section illustrates the injection properties of tissue adhesives using various dry bio-adhesive microparticle sizes via 2.5 mm or 1.2 mm diameter syringes. The 1.2 mm diameter syringe is used for this purpose. [Figure 8I] This section illustrates SEM images of various dry bio-adhesive microparticle sizes obtained by 2-minute cryogenic grinding at different grinding frequencies. The image corresponds to 30Hz. [Figure 8J] This section illustrates the injection properties of tissue adhesives using various dry bio-adhesive microparticle sizes via 2.5 mm or 1.2 mm diameter syringes. The 1.2 mm diameter syringe is used for this purpose. [Figure 9A]Photographs of various mixing ratios of tissue adhesives according to embodiments of the present invention are shown. The mass ratio of dry bio-adhesive microparticles to silicone oil matrix is ​​1:3. [Figure 9B] The images show photographs of various mixing ratios of tissue adhesives according to embodiments of the present invention. The mass ratio of dried bio-adhesive microparticles to silicone oil matrix is ​​1:2. [Figure 9C] The images show photographs of various mixing ratios of tissue adhesives according to embodiments of the present invention. The mass ratio of dried bio-adhesive microparticles to silicone oil matrix is ​​1:1. [Figure 9D] Photographs of various tissue adhesives with different mixing ratios according to embodiments of the present invention are shown. The mass ratio of dry bio-adhesive microparticles to silicone oil matrix is ​​1:0.5. [Figure 9E] A photograph of injection onto a vertical substrate is shown to illustrate the fluidity. [Figure 10A] This section provides a schematic illustration of the effects of matrix materials in tissue adhesives. It also provides a schematic illustration of setup and pull-off test procedures. [Figure 10B] The effect of the matrix material in tissue adhesives is illustrated in the graph. The graph shows the pull-off force using dried bio-adhesive microparticles measured in a PBS bath with and without a hydrophobic matrix (silicone oil). [Figure 10C] The effect of the matrix material in tissue adhesives is illustrated in the graph. The pull-off force using dried bio-adhesive microparticles, measured in a pig blood bath with and without a silicone oil matrix, is illustrated in the graph. [Figure 10D] The influence of the matrix material in the tissue adhesive is schematically illustrated. The composition of the tissue adhesive and the corresponding total surface energy are schematically illustrated. [Figure 10E]The effect of matrix material in tissue adhesives is illustrated in the graph. The graph illustrates the pull-off force against pressure applied between two tissues bonded by dried bio-adhesive microparticles, measured in a pig blood bath using silicone oil matrices of various viscosities (ηm = 5 cSt or 100 cSt). The vertical dashed line represents the threshold pressure applied. Error bars represent standard deviation (SD) (N=5). The p-value is determined by Student's t-test (***p ≤ 0.001). [Figure 11] This example illustrates the use of dry bio-adhesive microparticles without a hydrophobic oil matrix. Because the infiltrating blood swells and inactivates the bio-adhesive microparticles, adhesion between the gelatin hydrogel and the porcine aorta was not formed. [Figure 12] This illustrates a blood repulsion process using a tissue adhesive according to one embodiment of the present invention. Blood on the tissue surface is repelled and removed by the tissue adhesive, which forms a robust bond between the gelatin hydrogel and the porcine aorta. [Figure 13A] A schematic example of a mechanical test setup is provided (for measuring adhesive performance). The 180-degree peel test standard (ASTM F2256) is used as an example. [Figure 13B] A schematic example of a mechanical test setup is provided (for measuring adhesive performance). An example of a test setup for measuring shear strength in accordance with the lap shear test standard (ASTM F2255) is provided. [Figure 13C] A schematic example of a mechanical test setup is provided (for measuring adhesive performance). An example of a test setup for measuring tensile strength in accordance with the tensile test standard (ASTM F2258) is provided, along with graphs and photographs showing the results. [Figure 14A] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. The interfacial toughness versus pressing time in pig skin tissue covered with adhered blood is plotted on a graph. Error bars represent standard deviation (SD) (N=3~5). Statistical significance and p-values ​​are determined by one-way ANOVA and Tukey's multiple comparison test (ns are not significant). [Figure 14B]The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. The interfacial toughness versus storage time in pig skin tissue covered with adhered blood is plotted in a graph. Error bars represent standard deviation (SD) (N=3~5). Statistical significance and p-values ​​are determined by one-way ANOVA and Tukey's multiple comparison test (ns are not significant). [Figure 14C] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. The adhesive performance of the tissue adhesive on blood-covered pig skin tissue is compared graphically with various commercially available tissue adhesives and glues. Error bars represent standard deviation (SD) (N=3~5). Statistical significance and p-values ​​are determined by one-way ANOVA and Tukey's multiple comparison test (ns are not significant). [Figure 14D] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Interfacial toughness, shear strength, and tensile strength are compared graphically between various tissues covered with blood or mucus and bonded with the tissue adhesive. Error bars represent standard deviation (SD) (N=3-5). Statistical significance and p-values ​​are determined by one-way ANOVA and Tukey's multiple comparison test (ns indicates non-significant values). [Figure 14E] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Various tissues covered with blood or mucus, bonded with the tissue adhesive, are illustrated in photographs. All samples were stored in a humid environment before mechanical testing. [Figure 14F] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Various tissues covered with blood or mucus, bonded with the tissue adhesive, are illustrated in photographs. All samples were stored in a humid environment before mechanical testing. [Figure 14G] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Various tissues covered with blood or mucus, bonded with the tissue adhesive, are illustrated in photographs. All samples were stored in a humid environment before mechanical testing. [Figure 14H] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Various tissues covered with blood or mucus, bonded with the tissue adhesive, are illustrated in photographs. All samples were stored in a humid environment before mechanical testing. [Figure 14I] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Various tissues covered with blood or mucus, bonded with the tissue adhesive, are illustrated in photographs. All samples were stored in a humid environment before mechanical testing. [Figure 14J] The adhesive performance of a tissue adhesive according to one embodiment of the present invention is illustrated. Various tissues covered with blood or mucus, bonded with the tissue adhesive, are illustrated in photographs. All samples were stored in a humid environment before mechanical testing. [Figure 15A] The following photographs illustrate the adhesion of a hydrogel to blood-covered pig skin using a tissue adhesive according to one embodiment of the present invention. All samples were stored in a humid environment before mechanical testing. [Figure 15B] The graph illustrates the interfacial toughness, shear strength, and tensile strength between hydrogel bonded with tissue adhesive and porcine skin covered with blood. All samples were stored in a humid environment before mechanical testing. Error bars represent standard deviation (SD) (N=4). [Figure 16A] The present invention illustrates potential applications of a tissue adhesive according to one embodiment. [Figure 16B] This example demonstrates instantaneous hemostatic sealing of a bleeding exvivota aorta using tissue adhesive with Surgicel® backing. [Figure 16C] The rupture pressure of blood-covered pig aortas treated with tissue adhesive, sutures, Surgiflo®, and Tisseel is compared graphically. Error bars represent standard deviation (SD) (N=3). [Figure 17] A photograph illustrates a porcine blood bath filtered through a 100 μm mesh after 6 hours of continuous flow through an exvivobuta aorta sealed with a tissue adhesive according to one embodiment of the present invention. No bio-adhesive microparticles were observed filtered onto the mesh. [Figure 18A]This illustrates the biocompatibility of a tissue adhesive according to one embodiment of the present invention. The in vitro cell viability of rat cardiomyocytes based on a LIVE / DEAD assay after 24 hours of incubation in control medium (DMEM), Coseal incubation medium, and the tissue adhesive incubation medium of the present invention is shown (lower right panel). Representative confocal images of the LIVE / DEAD assay in control (upper left panel), Coseal (upper right panel), and tissue glue (lower left panel). DMEM, Dulbecco's modified Eagle medium. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-sided Student's t-5 test (ns are not significant, *p≦0.05). [Figure 18B] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Representative histological images of Coseal and tissue glue stained with hematoxylin and eosin (H&E) after 1 day of subcutaneous implantation in rats are shown. Four independent experiments were conducted and obtained similar results. [Figure 18C] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Representative histological images of Coseal and tissue glue stained with hematoxylin and eosin (H&E) after 2 weeks of subcutaneous implantation in rats are shown. Four independent experiments were conducted and obtained similar results. [Figure 18D] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. The degree of inflammation, as assessed by a blinded pathologist, is illustrated (0, normal, 1, very mild, 2, mild, 3, severe, 4, very severe). The values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-5 test (ns are not significant, *p≦0.05). [Figure 18E] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. A representative immunofluorescence image of Coseal after 1 day of subcutaneous implantation in rats is shown (cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue), and green fluorescence corresponds to the expression of fibroblasts (αSMA), type I collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively). [Figure 18F] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. A representative immunofluorescence image of the tissue adhesive of the present invention after 1 day of subcutaneous implantation in rats is shown (cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue), and green fluorescence corresponds to the expression of fibroblasts (αSMA), type I collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively). [Figure 18G] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Representative immunofluorescence images of Coseal after 2 weeks of subcutaneous implantation in rats are shown (cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue), and green fluorescence corresponds to the expression of fibroblasts (αSMA), type I collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively). [Figure 18H] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Representative immunofluorescence images of the tissue adhesive of the present invention after 2 weeks of subcutaneous implantation in rats are shown (cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue), and green fluorescence corresponds to the expression of fibroblasts (αSMA), type I collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively). [Figure 18I] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Normalized fluorescence intensities from immunofluorescence images of Coseal and tissue glue after 1 day of subcutaneous implantation in rats are illustrated. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-sided Student's t-5 test (ns are not significant, *p≦0.05). [Figure 18J] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Normalized fluorescence intensities from immunofluorescence imaging of Coseal and tissue glue after 3 days of subcutaneous implantation in rats are illustrated. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-sided Student's t-5 test (ns are not significant, *p≦0.05). [Figure 18K] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Normalized fluorescence intensities from immunofluorescence images of Coseal and tissue glue after 1 week of subcutaneous implantation in rats are illustrated. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-sided Student's t-5 test (ns are not significant, *p ≤ 0.05). [Figure 18L] The biocompatibility of a tissue adhesive according to one embodiment of the present invention is illustrated. Normalized fluorescence intensities from immunofluorescence imaging of Coseal and tissue glue after 2 weeks of subcutaneous implantation in rats are illustrated. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-sided Student's t-5 test (ns are not significant, *p≦0.05). [Figure 19A] The following are illustrative histological immunofluorescence images of subcutaneous implants. Representative histological images stained with hematoxylin and eosin (H&E) using Coseal and the tissue adhesive of the present invention after 3 days of subcutaneous implantation in rats are shown. Four independent experiments were conducted and similar results were obtained. [Figure 19B] The following are illustrative histological immunofluorescence images of subcutaneous implants. Representative histological images stained with hematoxylin and eosin (H&E) using Coseal and the tissue adhesive of the present invention, one week after subcutaneous implantation in rats, are shown. Four independent experiments were conducted, yielding similar results. [Figure 19C] Histological immunofluorescence images of subcutaneous implants are illustrated. Representative immunofluorescence images of Coseal after 3 days of subcutaneous implantation in rats are shown. Cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Green fluorescence corresponds to the expression of 15 fibroblasts (αSMA), type 1 collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively. [Figure 19D]Histological immunofluorescence images of subcutaneous implants are illustrated. Representative immunofluorescence images of tissue glue after 3 days of subcutaneous implantation in rats are shown. Cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Green fluorescence corresponds to the expression of 15 fibroblasts (αSMA), type 1 collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively. [Figure 19E] Histological immunofluorescence images of subcutaneous implants are illustrated. Representative immunofluorescence images of Coseal implants one week after subcutaneous implantation in rats are shown. Cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Green fluorescence corresponds to the expression of 15 fibroblasts (αSMA), type 1 collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively. [Figure 19F] Histological immunofluorescence images of subcutaneous implants are illustrated. Representative immunofluorescence images of tissue glue one week after subcutaneous implantation in rats are also illustrated. Cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Green fluorescence corresponds to the expression of 15 fibroblasts (αSMA), type 1 collagen (collagen I), T cells (CD3), and macrophages (CD68), respectively. [Figure 20A] This document illustrates the in vivo degradation of the tissue adhesive of the present invention. Representative histological images of the tissue adhesive stained with hematoxylin and eosin (H&E) after 2 weeks of subcutaneous implantation in rats are shown. Four independent experiments were conducted and similar results were obtained. [Figure 20B] This document illustrates the in vivo degradation of the tissue adhesive of the present invention. Representative histological images of the tissue adhesive stained with hematoxylin and eosin (H&E) after 4 weeks of subcutaneous implantation in rats are shown. Four independent experiments were conducted and similar results were obtained. [Figure 20C] This document illustrates the in vivo degradation of the tissue adhesive of the present invention. Representative histological images of the tissue adhesive stained with hematoxylin and eosin (H&E) after 8 weeks of subcutaneous implantation in rats are shown. Four independent experiments were conducted and similar results were obtained. [Figure 20D]This document illustrates the in vivo degradation of the tissue adhesive of the present invention. Representative histological images of gelatin-based tissue adhesive stained with hematoxylin and eosin (H&E) after 2 weeks of subcutaneous implantation in rats are shown. Four independent experiments were conducted and similar results were obtained. [Figure 21A] An example of instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention, according to an embodiment of the present invention, is shown. Instantaneous hemostatic sealing of a bleeding rat liver in vivo using the tissue adhesive is demonstrated. [Figure 21B] An example of instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention is shown according to an embodiment of the present invention. The image shows a resected rat liver two weeks after hemostatic sealing with the tissue adhesive. [Figure 21C] This document illustrates the instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention according to embodiments of the present invention. It shows the hemostatic time for liver bleeding with no treatment (injury), Surgicel, Coseal, and the tissue adhesive. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​were determined by a two-tailed Student's t-test (ns were not significant, *p≦0.05, **p≦0.01, ***p≦0.001). [Figure 21D] This document illustrates the instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention according to embodiments of the present invention. It shows the blood loss until hemostasis of liver bleeding with no treatment (injury), Surgicel, Coseal, and the tissue adhesive. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​were determined by a two-tailed Student's t-test (ns were not significant, *p≦0.05, **p≦0.01, ***p≦0.001). [Figure 21E] This document illustrates the instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention according to embodiments of the present invention. Representative histological images stained with hematoxylin and eosin (H&E) are shown of an untreated injured liver (injury) and hemostatic seals formed with Surgicel, Coseal, and the tissue adhesive of the present invention two weeks after hemostasis. Four independent experiments were conducted and similar results were obtained. [Figure 21F]This document illustrates the instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention according to an embodiment of the present invention. Representative immunofluorescence images are shown of an untreated injured liver (injury) and hemostatic sealing with Surgicel, Coseal, and the tissue adhesive of the present invention two weeks after hemostasis (cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Green fluorescence corresponds to T cell (CD3) expression). [Figure 21G] An example of instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention, according to an embodiment of the present invention, is illustrated. Representative immunofluorescence images are shown of an untreated injured liver (injury) and hemostatic sealing with Surgicel, Coseal, and the tissue adhesive of the present invention two weeks after hemostasis (cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Green fluorescence corresponds to the expression of macrophages (CD68)). [Figure 21H] An example of instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention, according to an embodiment of the present invention, is illustrated. Normalized fluorescence intensity from immunofluorescence imaging of CD3 is shown. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​were determined by a two-sided Student's t-test (ns were not significant, *p≦0.05, **p≦0.01, ***p≦0.001). [Figure 21I] An example of instantaneous hemostatic sealing of the liver using the tissue adhesive of the present invention, according to an embodiment of the present invention, is illustrated. Normalized fluorescence intensity from immunofluorescence imaging of CD68 is shown. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​were determined by a two-sided Student's t-test (ns were not significant, *p≦0.05, **p≦0.01, ***p≦0.001). [Figure 22A] An example of instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention, according to an embodiment of the present invention, is provided. An example of instantaneous hemostatic sealing of a bleeding rat heart in vivo using the tissue adhesive is provided. [Figure 22B] An example of instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention, according to an embodiment of the present invention, is shown. The image shows a resected rat heart two weeks after hemostatic sealing with the tissue adhesive. [Figure 22C] This document illustrates instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention according to embodiments of the present invention. It shows the hemostatic time for cardiac bleeding in the untreated (injured) state, with Surgicel, Coseal, and with the tissue adhesive of the present invention. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by one-way ANOVA and Tukey's multiple comparison test (**p≦0.01, ***p≦0.001). [Figure 22D] This document illustrates instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention according to embodiments of the present invention. It shows blood loss to hemostasis of cardiac bleeding in untreated (injured), Surgicel, Coseal, and with the tissue adhesive of the present invention. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by one-way ANOVA and Tukey's multiple comparison test (**p≦0.01, ***p≦0.001). [Figure 22E] An example of instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention, according to an embodiment of the present invention, is shown. The intraventricular blood pressure and heart rate of a rat heart before injury, after injury (2 mm biopsy punch), and after hemostatic sealing with the tissue adhesive of the present invention are shown. [Figure 22F] An example of instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention, according to an embodiment of the present invention, is illustrated. Representative histological images stained with Masson's trichrome are shown of an injured heart with hemostatic sealing using the tissue adhesive of the present invention immediately after hemostasis. Four independent experiments were conducted and similar results were obtained. [Figure 22G] An example of instantaneous hemostatic sealing of the heart using the tissue adhesive of the present invention, according to an embodiment of the present invention, is illustrated. Representative histological images stained with Masson's trichrome are shown of an injured heart with hemostatic sealing using the tissue adhesive of the present invention two weeks after hemostasis. Four independent experiments were conducted and similar results were obtained. [Figure 23A] This example illustrates hemostatic sealing of the liver using commercially available products. It shows in vivo hemostatic sealing of bleeding rat livers using Surgicel. [Figure 23B]This example illustrates hemostatic sealing of the liver using a commercially available product. The image shows a resected rat liver two weeks after hemostatic sealing with Surgicel. [Figure 23C] This example illustrates hemostatic sealing of the liver using commercially available products. It shows in vivo hemostatic sealing of bleeding rat livers using Coseal. [Figure 23D] This example illustrates hemostatic sealing of the liver using a commercially available product. The image shows a resected rat liver two weeks after hemostatic sealing with Coseal. [Figure 24A] This section provides examples of cardiac hemostatic sealing using commercially available products. It demonstrates in vivo hemostatic sealing of bleeding rat hearts using Surgicel. [Figure 24B] This example illustrates cardiac hemostatic sealing using commercially available products. It shows in vivo hemostatic sealing of a bleeding rat heart using Coseal. [Figure 25A] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows the total blood cell count (CBC) of healthy animals and animals two weeks after hemostatic sealing of the liver, for white blood cells (WBC). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25B] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows the total blood cell count (CBC) of neutrophils (NE) in healthy animals and animals two weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25C] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows the total blood cell count (CBC) for monocytes (MO) in healthy animals and animals two weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25D]This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows the total blood cell count (CBC) of lymphocytes (LYMPH) in healthy animals and animals two weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25E] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows the total blood cell count (CBC) for healthy animals and animals two weeks after hemostatic sealing of the liver, with respect to red blood cells (RBCs). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p ≤ 10 = 0.05). [Figure 25F] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows hemoglobin (HGB) levels in healthy animals and animals two weeks after hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p ≤ 10 = 0.05). [Figure 25G] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows hematocrit (HCT) values ​​for healthy animals and animals two weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25H] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows platelet (PLT) levels for healthy animals and animals two weeks after hemostatic sealing, along with total blood cell count (CBC). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25I]This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows blood chemistry for alkaline phosphatase (ALP) in healthy animals and animals two weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-sided Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25J] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows blood chemistry for aspartate transaminase (AST) in healthy animals and animals 2 weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns are not significant, *p≦10 0.05). [Figure 25K] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows blood chemistry for globulin (GB) in healthy animals and animals 2 weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p ≤ 10 = 0.05). [Figure 25L] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows blood chemistry for healthy animals and animals two weeks after hemostatic sealing, with respect to blood urea nitrogen (BUN). Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25M] This graph illustrates the blood analysis of animals with hemostatic sealing of the liver. It shows the blood chemistry of healthy animals and animals two weeks after hemostatic sealing of the liver, with respect to albumin (ALB). Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25N]This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows blood chemistry for amylase (AMY) levels in healthy animals and animals two weeks after hemostatic sealing of the liver. Values ​​represent the mean and standard deviation (n=4 independent samples). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p≦10 0.05). [Figure 25O] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows the blood chemistry of healthy animals and animals two weeks after hemostatic sealing of the liver, with respect to lipase (LIP). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p ≤ 10 = 0.05). [Figure 25P] This graph illustrates blood analysis of animals with hemostatic sealing of the liver. It shows blood chemistry for healthy animals and animals two weeks after hemostatic sealing of the liver, with respect to glucose (GLU). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant, *p ≤ 10 = 0.05). [Figure 26A] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows the total blood cell count (CBC) for healthy animals and animals two weeks after cardiac hemostatic sealing, for white blood cells (WBC). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant). [Figure 26B] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows neutrophil (NE) counts (CBC) for healthy animals and animals 2 weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns are not significant). [Figure 26C]This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows the total blood cell count (CBC) for monocytes (MO) in healthy animals and animals 2 weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns indicates non-significant). [Figure 26D] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows the total blood cell count (CBC) for lymphocytes (LYMPH) in healthy animals and animals two weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns are not significant). [Figure 26E] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows the total blood cell count (CBC) for healthy animals and animals two weeks after cardiac hemostatic sealing, with respect to red blood cells (RBCs). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns is not significant). [Figure 26F] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows hemoglobin (HGB) levels in healthy animals and animals two weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns are not significant). [Figure 26G] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows hematocrit (HCT) values ​​for healthy animals and animals two weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns are not significant). [Figure 26H]This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows platelet (PLT) levels for healthy animals and animals 2 weeks after cardiac hemostatic sealing, along with total blood cell count (CBC). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-tailed Student's t-test (ns are not significant). [Figure 26I] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows blood chemistry for alkaline phosphatase (ALP) levels in healthy animals and animals two weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-sided Student's t-test (ns are not significant). [Figure 26J] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows blood chemistry for aspartate transaminase (AST) in healthy animals and animals 2 weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-sided Student's t-test (ns are not significant). [Figure 26K] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows blood chemistry for globulin (GB) in healthy animals and animals 2 weeks after cardiac hemostatic sealing. Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-sided Student's t-test (ns are not significant). [Figure 26L] This graph illustrates blood analysis of animals with cardiac hemostatic sealing. It shows blood chemistry for healthy animals and animals two weeks after cardiac hemostatic sealing, with respect to blood urea nitrogen (BUN). Values ​​represent the mean and standard deviation (n=4 independent sample). Statistical significance and p-values ​​are determined by a two-sided Student's t-test (ns are not significant). [Figure 26M] The blood analysis of animals with cardiac hemostatic sealing is illustrated in a graph. The blood chemistry of healthy animals and animals two weeks after cardiac hemostatic sealing is shown for albumin (ALB). [Figure 26N]Graphically illustrate the blood analysis of animals having a cardiac hemostatic seal. Show the blood chemistry of healthy animals and animals two weeks after cardiac hemostatic seal for amylase (AMY). Values represent mean and standard deviation (n = 4 independent samples). Statistical significance and p-values are determined by two-sided Student's t-test (ns is not significant). [Figure 26O] Graphically illustrate the blood analysis of animals having a cardiac hemostatic seal. Show the blood chemistry of healthy animals and animals two weeks after cardiac hemostatic seal for lipase (LIP). Values represent mean and standard deviation (n = 4 independent samples). Statistical significance and p-values are determined by two-sided Student's t-test (ns is not significant). [Figure 26P] Graphically illustrate the blood analysis of animals having a cardiac hemostatic seal. Show the blood chemistry of healthy animals and animals two weeks after cardiac hemostatic seal for glucose (GLU). Values represent mean and standard deviation (n = 4 independent samples). Statistical significance and p-values are determined by two-sided Student's t-test (ns is not significant).

Mode for Carrying Out the Invention

[0017] Detailed Description The following definitions are useful for interpreting terms applied to the features of the embodiments disclosed herein and are meant to simply define elements within the scope of this disclosure.

[0018] As used herein, the term "toughness" when describing an adhesion formed by a tissue adhesive is at least about 100 Jm -2 , 120 Jm -2 , 140 Jm -2 , 160 Jm -2 , 180 Jm -2 , 200 Jm -2 , 220 Jm -2 , 240 Jm -2 , at least about 250 Jm -2 , at least about 260 Jm -2 , at least about 270 Jm -2, at least about 280 Jm -2 , at least about 290 Jm -2 Furthermore, at least approximately 300 Jm -2 This value represents the interfacial toughness.

[0019] As used herein, the term “strong” when describing adhesion formed by tissue adhesives means a shear strength or tensile strength of at least about 10 kPa, at least about 20 kPa, at least about 30 kPa, at least about 40 kPa, at least about 50 kPa, at least about 60 kPa, and at least about 70 kPa.

[0020] As used herein, the term "robust" when describing adhesion formed by tissue adhesives means 100 Jm -2 It exhibits superior interfacial toughness, shear strength exceeding 30 kPa, and tensile strength exceeding 10 kPa, with a preferred embodiment being at least about 240 Jm. -2 The toughness and strength of the adhesive are collectively referred to as interfacial toughness, shear strength of at least approximately 70 kPa, and tensile strength of at least approximately 50 kPa.

[0021] As used herein, the terms “instantaneous” and “fast” when used to describe the instantaneous / fast adhesion provided by tissue adhesives mean a time of 30 seconds or less, more preferably 25 seconds or less, more preferably 20 seconds or less, more preferably 15 seconds or less, more preferably 10 seconds or less, more preferably 9 seconds or less, more preferably 8 seconds or less, more preferably 7 seconds or less, more preferably 6 seconds or less, and even more preferably 5 seconds or less. This time is measured from the moment the tissue adhesive is applied to the tissue surface and gentle pressure is applied to the time it takes for the bio-adhesive microparticles in the tissue adhesive to crosslink with the surface and form a robust bond. The formation of the bond can be experimentally determined by a simple tensile test and visual inspection, by the absence of separation of the bonded tissue when pulled.

[0022] As used herein, the term “mild” when used to describe the pressure applied to the adhesive material means a pressure of about 50 kPa or less, for example, a pressure in the range of about 1 kPa to about 50 kPa. For example, mild pressure would mean a low pressure of about 45 kPa or less, about 40 kPa or less, about 35 kPa or less, about 30 kPa or less, about 25 kPa or less, about 20 kPa or less, about 15 kPa or less, about 10 kPa or less, about 8 kPa or less, about 6 kPa or less, about 5 kPa or less, about 4 kPa or less, about 3 kPa or less, about 2 kPa or less, and even about 1 kPa. According to an exemplary embodiment, a preferred mild pressure is about 10 kPa.

[0023] As used herein, the term “covered” when used to describe a surface to which adhesive material is applied as “covered” with a fluid means a surface that is partially or completely covered with a fluid. Therefore, “covered” may include configurations in which the entire fluid layer is disposed on the surface to which the adhesive material is applied such that the fluid layer separates the entire adhesive material from the surface when the adhesive material is applied and before the fluid recoils. “Covered” may also include configurations in which only a portion of the surface to which the adhesive material is applied (less than 100% but more than 50%) has a fluid layer disposed on it such that one or more portions of the adhesive material are separated from the surface by the fluid, and one or more portions of the adhesive material are in direct contact with the surface before the fluid recoils.

[0024] As used herein, the term "dry" when describing the bio-adhesive microparticles of the present invention means a material with a moisture content below the equilibrium moisture content of the material at the time of use. Therefore, when the dry bio-adhesive microparticles of the present invention are placed in contact with the moist tissue or other moist or humidified (e.g., moistened with physiological saline) surface to which they are to be adhered, the material will absorb water, physiological saline, moisture, interstitial fluid, and intracellular fluid from the moist or humidified surface. Generally, the dry bio-adhesive microparticles will have less than about 50% by weight of liquid components based on the total weight of the dry bio-adhesive microparticles.

[0025] As used herein, the term “body fluid” means aqueous physiological fluids, including blood, saliva, gastrointestinal fluid, mucus, and juices.

[0026] As used herein, the term "moist tissue" means biological tissue containing an aqueous solvent, including water, saline solution, interstitial fluid, and intracellular fluid.

[0027] As used herein, the term “absorption” when describing the mechanism by which dry bio-adhesive microparticles absorb aqueous media, including water, saline, moisture, interstitial fluid, and intracellular fluid, from a moist tissue surface to which they are in contact, means that atoms or molecules from the liquid on the moist surface cross the surface of the dry bio-adhesive microparticles and enter them.

[0028] As used herein, the term “bio-adhesive” when used to describe dry bio-adhesive microparticles means the ability of a material to form adhesion to the surface of biological tissue.

[0029] As used herein, the term "microparticles" when used to describe dry bio-adhesive microparticles means particulate material having an average diameter of about 200 μm or less, for example, any value in the range of about 5 μm to about 200 μm. For example, the term microparticles may mean particulate material having an average diameter of about 180 μm or less, about 160 μm or less, about 140 μm or less, about 120 μm or less, about 100 μm or less, about 80 μm or less, about 60 μm or less, about 40 μm or less, about 20 μm or less, and about 10 μm or less. However, depending on the ultimate use of the adhesive material and other factors such as the desired rheological properties of the adhesive material, any particle size in the range of about 5 μm to about 200 μm can be suitably selected. According to an exemplary embodiment, suitable microparticles have a size of about 10 μm.

[0030] As used herein, the term “temporary” when used to describe temporary crosslinking formed by bioadhesive microparticles means both a physical bond-forming group, such as a carboxylic acid group, in the bioadhesive microparticles that forms temporary crosslinking by intermolecular bonds, and a time range between the time it takes for instantaneous temporary crosslinking to occur and the time it takes for stable covalent crosslinking to occur between an amine coupling group, such as an NHS ester group, and primary amine groups of the adhesive surface and itself.

[0031] As used herein, "swelling" when used to describe the absorption of an aqueous solvent by dry bio-adhesive microparticles, and swelling upon contact with one or more moist tissue surfaces, generally means an increase in the size of the dry bio-adhesive microparticles.

[0032] As used herein, “biodegradable” when used to describe dry bio-adhesive microparticles means the partial or total degradation and / or subsequent removal of the implant material within a living animal by endogenous enzymes and / or water within the animal.

[0033] The present invention provides an adhesive material that can form instantaneous, tough, and strong adhesion with various materials, even in the presence of fluids. Specifically, the adhesive material has the ability to adhere to and integrate with various tissue surfaces, even in the presence of fluids. Such fluids, but are not limited to, include water, saline solution, moisture, and physiological bodily fluids such as blood, saliva, gastrointestinal fluid, mucus, and juices.

[0034] The adhesive material is made from a hydrophobic oil matrix containing dispersed bio-adhesive microparticles to provide a repulsive-crosslinking mechanism. When the adhesive material is applied to a fluid-covered surface and gentle pressure is applied, the hydrophobic oil matrix repels the fluid and cleans the surface, and subsequently the bio-adhesive microparticles form crosslinks with each other and with the underlying cleaned, moist tissue surface. When gentle pressure (less than about 50 kPa, e.g., pressure in the range of about 1 kPa to about 50 kPa) is applied to a wide area of ​​a fluid-covered surface for a time ranging from about 5 seconds to about 30 seconds, the adhesive material exhibits toughness (i.e., at least about 100 Jm). -2 Furthermore, at least approximately 240 Jm -2 The adhesive material of the present invention provides a high degree of interfacial toughness and strong adhesion (i.e., high shear strength of at least about 30 kPa, and even at least about 70 kPa, and high tensile strength of at least about 10 kPa, and even at least about 50 kPa). For this reason, the adhesive material of the present invention is particularly useful in a variety of applications, including emergency trauma situations where rapid and robust sealing / adhesion (e.g., hemostatic sealing of severely bleeding aorta) is required. The adhesive material of the present invention overcomes the limitations of existing adhesive materials because it does not require UV irradiation or prolonged application of steady pressure for adhesion formation.

[0035] Embodiments of the present invention will now be referenced in detail. Examples are shown in the accompanying drawings. Where possible, the same reference numerals will be used in the drawings and description to refer to identical or similar parts.

[0036] According to one embodiment, the present invention provides an adhesive material 1 comprising a combination of a hydrophobic matrix 2, particularly a hydrophobic oil matrix, and dried bio-adhesive microparticles 3. The dried bio-adhesive microparticles 3 are uniformly dispersed within the hydrophobic matrix 2 so that the hydrophobic matrix 2 acts as a protective matrix (see Figure 1A). Before use, preferably, the adhesive material 1 will be ensured to be a homogeneous mixture of the dried bio-adhesive microparticles 3 dispersed within the hydrophobic matrix 2 by vigorously shaking or stirring. When the adhesive material 1 is applied to a fluid-covered tissue surface 100 (in this case, blood-covered pig skin tissue) and gentle pressure is applied (pressed with a gelatin-coated glass substrate, as shown in Figures 1A-C), the hydrophobic matrix 2 clarifies the surface by protecting the dried bio-adhesive microparticles 3 from the bodily fluids and repelling them (Figure 1B). This allows the dried bio-adhesive microparticles 3 to come into contact with each other and with the moist tissue surface 100. Therefore, the adhesive material 1 of the present invention provides fluid resistance that achieves instantaneous robust adhesion of tissues covered with fluids (for example, water, physiological saline, moisture, interstitial fluid, and body fluids, such as blood, saliva, gastrointestinal fluid, mucus, and juices).

[0037] According to embodiments of the present invention, the adhesive material 1 is in the form of an injectable material comprising a hydrophobic matrix 2 containing dispersed dried bio-adhesive microparticles 3 (see, for example, Figures 1A, 3E, 4, 8B, 8D, 8F, 8H, and 8J). Upon application of gentle pressure (e.g., 10 kPa), the hydrophobic matrix 2 repels bodily fluids, allowing the dried bio-adhesive microparticles 3 to come into contact with each other and with a clean, moist tissue surface (see, for example, Figures 1B, 1E, and 4). Subsequently, the dried bio-adhesive microparticles 3 crosslink with each other and with the moist tissue surface 100 to form a robust bond within approximately 5 seconds without requiring additional stimulation such as UV irradiation (see, for example, Figures 1C, 1F, and 2). For example, as shown in Figures 2A-D, two pig heart tissues were covered with blood (Figure 2A), the tissue adhesive of the present invention was applied to the blood-covered tissue (Figure 2B), and then gentle pressure was applied for 5 seconds (Figure 5C), resulting in the formation of a robust bond between the two blood-covered tissue surfaces (Figure 2D).

[0038] According to one embodiment, the dried bio-adhesive microparticles 3 are formed from a dried bio-adhesive material comprising a combination of (i) one or more hydrophilic polymers or copolymers, (ii) one or more amine coupling groups, and (iii) one or more crosslinking agents.

[0039] According to embodiments of the present invention, (i) the hydrophilic polymer or copolymer is selected from any conventional hydrophilic polymer or copolymer that absorbs water in a dry state. Suitable hydrophilic polymers or copolymers include, but are not limited to, polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, polyvinylpyrrolidone, polystyrene sulfonate, polyurethane, casein, albumin, collagen, gelatin, chitosan, hyaluronic acid, alginate, oxidized alginate, pectin, cellulose, and oxidized cellulose, as well as combinations thereof. Since the adhesive material can be used in a wide variety of biomedical applications, the polymers used in the present invention are preferably biocompatible (however, for non-biomedic applications, it may not be necessary to use only biocompatible polymer materials).

[0040] According to embodiments of the present invention, (i) one or more hydrophobic polymers or copolymers are (ii) grafted with one or more amine coupling groups. Preferred amine coupling groups are, but are not limited, selected from conventional amine coupling groups including N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, aldehydes, imide esters, epoxides, isocyanates, catechols and combinations thereof. Since the adhesive material can be used in a wide variety of biomedical applications, the amine coupling groups used in the present invention are preferably biocompatible (however, in non-biomedic applications, it may not be necessary to use only biocompatible amine coupling groups). The amine coupling groups are configured such that one or more amine coupling groups can be grafted onto one or more hydrophilic polymers or copolymers, and then one or more amine coupling groups form covalent crosslinks with the surface to which the adhesive material is bonded.

[0041] According to embodiments of the present invention, a hydrophilic polymer or copolymer is preferably crosslinked with (iii) one or more crosslinking agents selected from conventional crosslinking agents. Such crosslinking agents include, but are not limited to, gelatin methacrylate, hyaluronic acid methacrylate, methacrylate-based oxidized alginates, polycaprolactone diacrylate, N,N'-bis(acryloyl)cystamine, N,N'-methylenebis(acrylamide), polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and combinations thereof. Since this adhesive material can be used in a wide variety of biomedical applications, the crosslinking agents used in the present invention are preferably biocompatible (however, in non-biomedic applications, it may not be necessary to use only biocompatible crosslinking agents).

[0042] According to a preferred embodiment, the dried bio-adhesive microparticles 3 are prepared by first preparing a bio-adhesive material made from a combination of (i) one or more hydrophilic polymers or copolymers, (ii) one or more amine coupling groups, (iii) one or more crosslinking agents, and deionized water. According to one embodiment of the present invention, suitable amounts of the various components used in preparing the bio-adhesive material are, in their as-prepared (pre-dried) form, within the range of (i) about 20 w / w% to about 55 w / w% of one or more hydrophilic polymers, (ii) about 0.5 w / w% to about 1.5 w / w% of one or more amine coupling groups, and (iii) about 0.05 w / w% to about 0.15 w / w% of one or more crosslinking agents, with the remainder being deionized water.

[0043] According to an exemplary embodiment, the bio-adhesive material, in its as-prepared (pre-dried) form, comprises about 30 w / w% poly(acrylic acid), about 2 w / w% chitosan, about 1 w / w% PAAc-NHS ester, about 0.1 w / w% gelatin methacrylate, and the remainder being deionized water.

[0044] Next, the as-prepared bio-adhesive material is dehydrated, and the dehydrated bio-adhesive material is subjected to cryogenic grinding to produce dry bio-adhesive microparticles 3 of a desired average particle size. For example, as depicted in Figures 3A-E, the dry bio-adhesive is first cut into small pieces (Figure 3A), then added to a stainless steel container with stainless steel balls (Figure 3B), and then ground under cryogenic conditions using a cryogenic ball mill (Figure 3C) to produce dry bio-adhesive microparticles 3 (Figure 3D). The thus formed dry bio-adhesive microparticles 3 are then mixed with a desired hydrophobic matrix 2 in a desired ratio to prepare adhesive material 1, which is depicted as being injected via syringe onto a blood-covered pig heart (Figure 3E). When adhesive material 1 is used as a tissue adhesive, the hydrophobic matrix 2 is a biocompatible matrix material. Suitable hydrophobic matrix 2 materials include, but are not limited to, silicone oil, mineral oil, essential oil, perfluoropolyether oil, and / or lanolin oil.

[0045] According to an exemplary embodiment, adhesive material 1 comprises (i) poly(acrylic acid) grafted with N-hydroxysuccinimide ester (PAAc-co-NHS ester) and crosslinked with biodegradable gelatin methacrylate, and (ii) biodegradable chitosan dispersed in a medical-grade silicone oil hydrophobic matrix, which comprises dried bioadhesive microparticles.

[0046] According to embodiments of the present invention, the average size of the dried bio-adhesive microparticles 3 can be controlled by cryogenic grinding conditions. Specifically, as shown in Figure 7, the grinding time was fixed at 2 minutes, and the grinding frequency was varied from 10 Hz to 30 Hz (particularly 10 Hz, 15 Hz, 20 Hz, 25 Hz, and 30 Hz). As demonstrated in Figure 7, the higher the grinding frequency, the smaller the average size of the dried bio-adhesive microparticles (approximately 200 μm at 10 Hz and approximately 10 μm at 30 Hz). Therefore, the desired average size of the bio-adhesive microparticles 3 can be achieved depending on the use and specifications of the adhesive material 1.

[0047] As shown in Figures 8A-J, the obtained cryogenically ground bio-adhesive microparticles 3 were used to prepare adhesive material 1 by adding these bio-adhesive microparticles 3 to a hydrophobic matrix material 2, and the injectability of the obtained adhesive material 1 was demonstrated by injection via syringe. Specifically, examples include SEM images of dried bio-adhesive microparticles 3 at a cryogenic grinding frequency of 10 Hz (Figure 8A) and the corresponding adhesive material 1 pressurized via a 2.5 mm diameter syringe (Figure 8B), SEM images of dried bio-adhesive microparticles at a cryogenic grinding frequency of 15 Hz (Figure 8C) and the corresponding adhesive material 1 pressurized via a 1.2 mm diameter nozzle (Figure 8D), SEM images of dried bio-adhesive microparticles at a cryogenic grinding frequency of 20 Hz (Figure 8E) and the corresponding adhesive material 1 pressurized via a 1.2 mm diameter nozzle (Figure 8F), SEM images of dried bio-adhesive microparticles at a cryogenic grinding frequency of 25 Hz (Figure 8G) and the corresponding adhesive material 1 pressurized via a 1.2 mm diameter nozzle (Figure 8H), and SEM images of dried bio-adhesive microparticles at a cryogenic grinding frequency of 30 Hz (Figure 8I) and the corresponding adhesive material 1 pressurized via a 1.2 mm diameter nozzle (Figure 8J). In all cases, the cryogenic grinding time for each of the adhesive materials 1 was set to 2 minutes, and the same hydrophobic matrix material 2 was used for all adhesive materials 1.

[0048] According to embodiments of the present invention, the rheological properties (i.e., flow behavior, viscosity, and shear yield stress) of the adhesive material 1 were adjusted by controlling the mixing ratio of the dried bio-adhesive microparticles 3 and the hydrophobic matrix 2. As demonstrated in Figures 8B, 8D, 8F, 8H, 8J, and 9A-E, the adhesive material 1 ranged from a viscous fluid to a stable thixotropic paste. To visualize the fluidity of the adhesive material 1, photographs were taken of adhesive material 1 at various mixing ratios, with the mass ratio of dried bio-adhesive microparticles 3 to the hydrophobic silicone oil matrix 2 set to 1:3 (Figure 9A), 1:2 (Figure 9B), 1:1 (Figure 9C), and 1:0.5 (Figure 9D). To further demonstrate the fluidity, photographs of adhesive material 1 at mixing ratios of 1:1, 1:2, and 1:3 were taken by injection onto a vertical substrate, as shown in Figure 9E.

[0049] Dry bio-adhesive microparticles 3 are formed and dispersed within a hydrophobic matrix 2 so that instantaneous strong adhesion is formed on and / or between themselves on the desired surface when used as described herein. For example, as schematically depicted in Figure 4, the adhesive material 1 can be applied directly to a desired fluid-covered surface (e.g., a tissue surface covered with bodily fluids) without any other preparation or cleaning process (steps 1-2 in Figure 4). Upon application of gentle pressure (steps 3-4 in Figure 4), the hydrophobic matrix repels the bodily fluids and removes them from the tissue surface (in this case, the adhesive material is placed between two tissue surfaces covered with bodily fluids). Simultaneously, physical bond-forming groups in the bio-adhesive microparticles, such as carboxylic acid groups, form temporary crosslinks by intermolecular bonds (step 5 in Figure 4), and subsequently form stable covalent crosslinks with amine coupling groups, such as NHS ester groups, and with primary amine groups of their own and the cleaned, moist tissue surface (step 6 in Figure 4). After adhesion to / between the moist tissue surface, the swollen and crosslinked adhesive material forms a hydrogel thin layer that provides robust adhesion between tissues (step 7 in Figure 4).

[0050] Photographs of cross-sections of two blood-covered pig heart tissues bonded with adhesive material 1 of the present invention are shown in Figure 5, the left photograph showing adhesion after 5 minutes, and the right photograph showing adhesion after 24 hours (samples were stored in a humid environment throughout the experiment). As depicted in Figure 6, the confocal microscope image shows the swelling of the adhesive material and the crosslinked adhesive layer between the two gelatin hydrogels after 30 minutes of application. Specifically, as described herein, the adhesive material is in the form of dry bio-adhesive microparticles, which swell upon contact with a humid tissue surface by absorbing an aqueous solvent from the humid biological tissue. This absorption of aqueous solvent and swelling of the dry bio-adhesive microparticles provides instantaneous transient crosslinking between the adhesive material and the humid surface, and further enables subsequent rapid covalent coupling or crosslinking between the adhesive material and the humid surface.

[0051] To demonstrate the structure and function of the components of adhesive material 1 in tissue adhesion, a pull-off test was performed using the setup and procedure illustrated in Figure 10A. The surface area of ​​the bonded porcine heart tissue was 1 cm². 2 The pull-off force between porcine heart tissues bonded with dry bio-adhesive microparticles 3, both without and with a hydrophobic silicone oil matrix, was tested. The pull-off tests conducted in a phosphate-buffered saline (PBS) bath showed similarly high pull-off forces (p=0.48) between dry bio-adhesive microparticles 3 without and with a silicone oil matrix 2 (Figure 10B). These results suggest that dry bio-adhesive microparticles alone can provide instantaneous strong adhesion between moist tissues in the absence of body fluids. On the other hand, pull-off tests conducted in a porcine blood bath showed high pull-off forces with dry bio-adhesive microparticles mixed with a silicone oil matrix, but significantly lower pull-off forces with dry bio-adhesive microparticles without a silicone oil matrix (Figure 10C).

[0052] To illustrate the role of applied pressure and the properties of the silicone oil matrix in adhesion to tissue surfaces, the pull-off force between porcine heart tissues bonded with the adhesive material of the present invention was tested using various applied pressures and the viscosity of the silicone oil matrix (Figure 10E). As shown, the pull-off force initially increased with increasing applied pressure, and then plateaued when the applied pressure exceeded a certain threshold (e.g., 10 kPa for silicone oil with a kinematic viscosity of 5 cSt). Furthermore, silicone oils with higher viscosity showed a higher threshold for applied pressure (e.g., 16 kPa for silicone oil with a kinematic viscosity of 100 cSt), which is consistent with the granular suspension theory in the literature (E. Guazzelli, O. Pouliquen, Rheology of dense granular suspensions. Journal of Fluid Mechanics 852, (2018)).

[0053] Furthermore, as shown in Figure 10D, the role of the hydrophobic matrix 2 as a protective matrix for the dry bio-adhesive microparticles 3 against body fluids on the tissue surface was demonstrated using the total surface energy of three configurations: i) configuration 1 (protected state E1) where a layer of body fluids faces downward and the dry bio-adhesive microparticles are completely wetted with silicone oil, ii) configuration 2 (unprotected state E2) where a layer of silicone oil faces downward and the dry bio-adhesive microparticles are completely wetted with body fluids, and iii) configuration 3 (repulsive state E3) where there is no body fluid and the dry bio-adhesive microparticles are completely wetted with silicone oil. To ensure energetically stable protection of the dry bio-adhesive microparticles and repulsion of body fluids by the silicone oil matrix, ΔE A =E2-E1>0 and ΔE B The condition =E1-E3>0 must be satisfied. This can be expressed as follows: R(γ oil / air cosθ oil / ad -γ bf / air cosθ bf / ad )+γ bf / air cosθ bf / tissue-γ oil / air cosθ oil / issue >0 (1) γ oil / bf +γ oil / air cosθ oil / tissue -γ bf / air cosθ bf / tissue >0 (2) In the formula, R is a roughness coefficient representing the ratio of the actual surface area to the projected surface area of ​​the dried bio-adhesive microparticles, and γ A / B θ represents the interfacial energy between A and B, and θ represents the interfacial energy between A and B. A / B This represents the contact angle of A on B (the subscript "ad" represents bio-adhesive microparticles, and "bf" represents bodily fluids). Note that R is set to π for dry bio-adhesive microparticles in the adhesive material, based on a first-order approximation of densely arranged spherical particles of the same diameter. Substituting the corresponding values ​​into equations (1-2) gives (R=π, γ oil / air = 20.9 mNm -1 gamma bf / air = 72.0 mNm -1 gamma oil / bf = 40 mNm -1 θ oil / ad =4.5°, θ bf / ad =96°, θ oil / tissue =4.2°, θ bf / tissue (=84°), it is clear that adhesive materials satisfy these inequalities. Therefore, the silicone oil matrix protects the dry bio-adhesive microparticles from bodily fluids and repels bodily fluids from the tissue surface, thus demonstrating the design and mechanism of the adhesive material of the present invention.

[0054] Therefore, in the absence of the protective hydrophobic matrix 2, bodily fluids can easily penetrate and interact with the dry bio-adhesive microparticles 3, thus hindering the formation of robust adhesion between microparticles and / or with the tissue surface. These results suggest that the hydrophobic matrix 2 of the adhesive material 1 of the present invention effectively protects and preserves the dry bio-adhesive microparticles 3 in the presence of bodily fluids. Specifically, as illustrated in Figure 11, (i) a blood-covered gelatin hydrogel was placed on a pig aorta covered with a layer of dry bio-adhesive microparticles without the hydrophobic matrix, and then (ii) the blood-covered gelatin hydrogel was pressed onto the tissue, (iii) the blood began to penetrate into the bio-adhesive microparticles, (iv-v) the penetrating blood swelled and inactivated the bio-adhesive microparticles, and (vi) no adhesion by the dry bio-adhesive microparticles was formed between the gelatin hydrogel and the pig aorta.

[0055] On the other hand, the blood repulsion process by the adhesive material of the present invention, which contains dried bio-adhesive microparticles 3 dispersed within a hydrophobic matrix 2, is demonstrated in Figure 12. As shown, (i) a blood-covered gelatin hydrogel is placed on a porcine aorta covered with a layer of the adhesive material of the present invention, and then (ii) the blood-covered gelatin hydrogel is pressed onto the tissue, (iii) the blood begins to be repelled by the adhesive material, (iv) the blood on the tissue surface is removed from the tissue surface by the adhesive material, and (v) a robust adhesion is formed between the gelatin hydrogel and the cleaned porcine aorta by the adhesive material. Specifically, when gentle pressure is applied, the dried bio-adhesive microparticles 3 in the adhesive material 1 are compacted densely, forming a non-flowing, packed adhesive layer. In this compaction process, the hydrophobic matrix 2 is pushed out from the adhesive material 1 and repelled from the tissue surface by bodily fluids (Figure 12). Thus, the applied pressure and the hydrophobicity of the silicone oil matrix enable the adhesion formation of the adhesive material by the bio-adhesive microparticles 3.

[0056] To further evaluate the adhesive performance of adhesive material 1, three different types of mechanical tests were performed: (i) interfacial toughness was measured using a test setup compliant with the 180 peel test standard (ASTM F2256) (Figure 13A), (ii) shear strength was measured using a test setup compliant with the lap shear test standard (ASTM F2255) (Figure 13B), and (iii) tensile strength was measured using a test setup compliant with the tensile test standard (ASTM F2258) (Figure 13C). The adhesive performance of adhesive material 1 was then plotted graphically by plotting interfacial toughness against pressing time on blood-covered pig skin tissue (Figure 14A) and by plotting interfacial toughness against storage time on blood-covered pig skin tissue (Figure 14B). When adhesive material 1 of the present invention was brought into contact with blood-covered pig skin tissue and subjected to a gentle pressure (10 kPa) for less than 5 seconds, it exhibited an adhesion of 240 Jm -2 The ability to form robust adhesion with interfacial toughness exceeding [value missing] demonstrates rapid fluid-resistant bonding capability (Figure 14A). Tissue bonded with adhesive material 1 showed no significant deterioration in interfacial toughness (p=0.78) measured over 48 hours of storage after initial pressing (Figure 14B). Furthermore, a comparison of the bonding performance of adhesive material 1 of the present invention with various commercially available tissue adhesives and glues on blood-covered pig skin tissue (Figure 14C) shows that adhesive material 1 of the present invention has superior bonding performance compared to existing commercially available tissue adhesives and glues, including gelatin-based hemostatic sealants (e.g., Surgiflo®), fibrin-based hemostatic sealants (e.g., Tisseel, TachoSil®), albumin-based adhesives (e.g., BioGlue®), PEG-based adhesives (e.g., Coseal), and cyanoacrylate adhesives (Histoacryl®). As shown in Figure 14C, these commercially available tissue adhesives and glues exhibit relatively slow adhesion formation (longer than 1 minute) and limited adhesion performance (30 Jm) on blood-covered pig skin tissue. -2 It exhibits interfacial toughness of less than 20 kPa and shear / tensile strength of less than 20 kPa. In contrast, adhesive material 1 of the present invention exhibits 240 Jm in less than 5 s. -2To demonstrate robust adhesion with interfacial toughness exceeding [a certain level], shear strength exceeding 70 kPa, and tensile strength exceeding 50 kPa, it is significantly superior to commercially available tissue adhesives and glues.

[0057] As further demonstrated in Figures 14D-E and 15, the adhesive material of the present invention exhibits high interfacial toughness (240 Jm against skin) in less than 5 seconds. -2 150 Jm against the aorta -2 , 140 Jm to the heart -2 330 Jm for muscle -2 And 170 Jm against a hydrogel covered with blood -2 It exceeds 120 Jm to the stomach. -2 Furthermore, 100 Jm for the small intestine covered with mucus and gastric juice. -2 It is applicable to a wide range of fluid-covered tissues and hydrogels to form robust adhesion with shear and tensile strength (exceeding 70 kPa for skin, 55 kPa for the aorta, 45 kPa for the heart, 50 kPa for muscle, and 45 kPa for blood-covered hydrogels, 30 kPa for the stomach, and 35 kPa for the small intestine covered with mucus and gastric juice).

[0058] The unique ability of adhesive material 1 to form instantaneous, strong adhesion on bodily fluid-covered tissues and organs without requiring additional equipment (e.g., UV) or the initial removal of fluid from the surface before application of adhesive material 1 will benefit a variety of clinical and biomedical applications. To explore the potential applications of adhesive material, hemostatic sealing of ex vivo porcine aorta and in vivo rat cardiac models was investigated using adhesive material 1. As demonstrated in Figure 16A, adhesive material 1, combined with a commercially available oxidized cellulose backing (Surgicel®, 2.5 cm × 2.5 cm size), formed a robust hemostatic seal on a bleeding porcine aorta (3 mm hole, 150 mmHg blood flow pressure) in less than 5 seconds. The aorta sealed with adhesive material 1 maintained adhesion under continuous blood flow and withstood high physiological pressure (250 mmHg) without leakage for 6 hours after initial application of adhesive material 1. Furthermore, after the test, blood was collected from the hemorrhagic pig aorta and filtered through a mesh (100 μm mesh size) (closed-loop flow over 6 hours). As shown in Figure 17, no bio-adhesive microparticles were observed on the mesh, demonstrating the robustness of the hemostatic seal formed by adhesive material 1 against the potential risk of embolism. To quantitatively evaluate the strength of the hemostatic seal formed using adhesive material 1, the burst pressure of adhesive material 1 was measured using pig aortic tissue (ASTM F2392-04) (Figure 16C). Adhesive material 1 demonstrated a high burst pressure of over 350 mmHg, significantly exceeding the performance of normal human systolic arterial blood pressure (approximately 120 mmHg) and sutures and commercially available hemostatic sealants (e.g., Surgiflo® and Tisseel).

[0059] It should be noted that in this example, an oxidized cellulose backing material was introduced to provide a non-tissue adhesive cover for the injected adhesive material 1. In this example, the adhesive material 1 was provided to the target tissue site in the form of an injectable paste or glue, and then the backing was placed on top of the deposited adhesive material 1 to facilitate pressing onto the target biological tissue. Similarly, the backing material can also be used for other methods of depositing the adhesive material 1, e.g., for coating or spreading the adhesive material 1 onto a tissue site. Similarly, various other backing material compositions can be suitably used. Suitable backing material compositions include oxidized cellulose, silicone elastomers, polyurethanes, hydrogels, any other biocompatible material that does not adhere to moist tissue, and combinations thereof. In embodiments in which the adhesive material 1 is placed between two surfaces (e.g., two tissue surfaces) and sandwiched between them to bond and integrate the surfaces, the backing material would not be necessary.

[0060] Therefore, without the need for pre-cleaning the surface, using additional equipment (e.g., UV), and / or applying steady pressure for extended periods to form adhesion, adhesive materials offer the unique ability to form instantaneous, strong adhesion on surfaces such as fluid-covered tissues and organs. For this reason, adhesive materials may be beneficial for use in a variety of clinical and biomedical applications. The ability of adhesive materials to bond rapidly, accurately, and robustly to fluid-covered surfaces can further address the need for on-site treatment of life-threatening traumatic injuries to inherently complex tissues and organs, where time constraints are quite significant. The unique capabilities offered by adhesive materials can address long-standing challenges of a group of existing tissue adhesives and may provide new opportunities for future developments in tissue engineering, drug delivery, and biointegration devices. Novel repulsion-crosslinking mechanisms for wet adhesion may further inspire the design of future adhesives in wet and aquatic environments. [Examples]

[0061] Experimental data material Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich and used without further purification. Acrylic acid, gelatin methacrylate (gelMA, type A bloom 90-100 derived from porcine skin with 60% substitution), N-hydroxysuccinimide acrylate (AAc-NHS ester), α-ketoglutaric acid, and chitosan (75-85% deacetylated) were used to prepare the dry bio-adhesive. Silicone oils with different viscosities (5 cSt and 100 cSt) were used as the matrix for the dry bio-adhesive microparticles. FITC-chitosan (KITO-8, PolySciTech®) was used for confocal microscopy imaging to visualize the adhesive material. Acrylamide, gelatin (type A bloom 300 derived from porcine skin), gelMA, and Irgacure 2959 were used to prepare the hydrogel. Pig blood was purchased from Lampire Biological Laboratories, Inc. All porcine tissue used for ex vivo experiments was purchased from a research-grade porcine tissue vendor (Sierra Medical Inc.).

[0062] method Preparation of a bodily fluid-resistant adhesive material. To prepare the bio-adhesive, 30 w / w% acrylic acid, 2 w / w% chitosan, 1 w / w% AAc-NHS ester, 0.1 w / w% gelMA, and 0.5 w / w% α-ketoglutaric acid were dissolved in deionized water. The mixture was then filtered through a 0.4 μm sterile syringe filter and poured onto a glass mold equipped with a 500 μm spacer. The bio-adhesive was cured in a UV chamber (284 nm, 10 W power) for 60 minutes and completely dried under a nitrogen flow for 24 hours. The dried bio-adhesive was sealed in a plastic bag and stored at -20°C before use. To aid in the visualization of the adhesive material for confocal microscopy imaging, 0.2 w / w% FITC-chitosan was further added to the precursor solution before curing.

[0063] To prepare the dried bio-adhesive microparticles, the dried bio-adhesive was cut into small pieces and added to the container of a cryogenic grinder (CryoMill, Retsch), followed by a cryogenic grinding process (frequency 30 Hz, 2 minutes). The adhesive material was prepared by thoroughly mixing the dried bio-adhesive microparticles with a silicone oil matrix. The prepared adhesive material was sealed in a plastic bag containing a desiccant (silica gel packet) and stored at -20°C before use. Unless otherwise specified, silicone oil with a viscosity of 5 cSt and a mass ratio of 1:1 (equivalent to a volume fraction of 0.4) of dried bio-adhesive microparticles to silicone oil was used.

[0064] Mechanical testing. Tissue samples stored for more than 10 minutes prior to mechanical testing were covered with a large spray of 0.01 w / v% sodium azide solution (in PBS) and sealed in a plastic bag to prevent tissue degradation and dehydration. Unless otherwise specified, all tissues and hydrogels were covered with body fluid (blood or gastric juice), pressed for 5 seconds (10 kPa pressure applied by either a mechanical testing machine or an equivalent weight), and bonded with adhesive material. Unless otherwise specified, all mechanical testing on bonded samples was performed 6 hours after initial pressing to ensure equilibrium swelling of the bonded adhesive material in a moist physiological environment. The application of commercially available tissue adhesives and glues followed the manual provided for each product.

[0065] In the pull-off test, pig heart tissue is removed by 1 cm 2The tissue was cut to a surface area and thickness of 5 mm. Separately, porcine heart tissue was bonded to a glass container filled with PBS or porcine blood bath using cyanoacrylate glue (Krazy Glue®). Alternatively, porcine heart tissue was bonded to an aluminum fixture using cyanoacrylate glue, and the surface of the tissue was covered with dry bio-adhesive microparticles, with or without a silicone oil matrix of different viscosities (5 cSt or 100 cSt). Using a mechanical testing machine (2.5 kN load cell, Zwick / Roell Z2.5), the adhesive-covered porcine heart tissue was pressed against the immersed tissue in the bath at different pressures for 5 seconds. The bonded tissue was then pulled by lifting the aluminum fixture, and the maximum tensile force was measured as the pull-off force.

[0066] To measure interfacial toughness, bonded samples 2.5 cm wide were prepared and tested using a mechanical testing machine according to the 180-degree peel test standard (ASTM F2256). All tests were performed over a 50 mm period. -1 The test was performed at a constant peeling speed. The measured force reached a plateau when the peeling process entered a steady state. Interfacial toughness was determined by dividing twice the plateau force (180-degree peel test) by the width of the tissue sample (Figure 13A). A poly(methyl methacrylate) film (50 μm thick, Goodfellow) was applied as a rigid backing to the tissue and hydrogel using cyanoacrylate glue.

[0067] To measure shear strength, bonded samples with a bonded area of ​​2.5 cm in width and 1 cm in length were prepared and tested using a mechanical testing machine according to the lap shear test standard (ASTM F2255) (Figure 13B). All tests were performed over a 50 mm period. -1 The test was performed at a constant tensile speed. Shear strength was determined by dividing the maximum force by the adhesive area. A poly(methyl methacrylate) film was applied as a rigid backing to the tissue and hydrogel using cyanoacrylate glue.

[0068] To measure tensile strength, bonded samples with a bonded area of ​​2.5 cm in width and 2.5 cm in length were prepared and tested using a mechanical testing machine according to the tensile test standard (ASTM F2258) (Figure 13C). All tests were performed over a 50 mm period. -1 The test was performed at a constant tensile speed. Tensile strength was determined by dividing the maximum force by the adhesive area. Aluminum fixtures were applied using cyanoacrylate glue to provide a grip for the tensile test.

[0069] Preparation of hydrogel. To prepare a hydrogel for adhesion testing, 20 w / w% acrylamide, 10 w / w% gelatin, 0.2 w / w% gelMA, and 0.2 w / w% Irgacure 2959 were dissolved in deionized water. The mixture was then filtered through a 0.4 μm sterile syringe filter and poured onto a glass mold equipped with a 3 mm spacer. The hydrogel was cured in a UV chamber (284 nm, 10 W power) for 60 minutes.

[0070] Microscopic imaging. Scanning electron microscope (SEM) images of cryogenically pulverized and dried bio-adhesive microparticles were obtained using a SEM (JSM-6010LA, JEOL) after applying 5nm gold sputtering to enhance image contrast. Confocal microscope images of the adhesive material were obtained using an upright confocal microscope (SP8, Leica) with a 490nm excitation wavelength for FITC.

[0071] Contact angle measurement. Dry bio-adhesive or porcine skin tissue was bonded to a glass substrate, and the contact angles of silicone oil and porcine blood were measured using a contact angle device (Rame-Hart). The contact angle measurements were performed at a relative humidity of 35% and room temperature (23-26°C).

[0072] Ex vivo study. All ex vivo experiments were reviewed and approved by the Committee on Animal Care at the Massachusetts Institute of Technology. To hemostatic seal the bleeding aorta, the porcine aorta was connected to a porcine blood bath and pump via a silicone tube to generate a closed-loop blood flow at a pressure of 150 mmHg (Figure 16A). A 3 mm diameter hole was made in the porcine aorta using a biopsy punch (Dynarex). To form a hemostatic seal, 1 mL of adhesive material was injected onto a commercially available biodegradable surgical gauze (Surgicel®, Ethicon) measuring 2.5 cm wide and 2.5 cm long, and gently pressed over the puncture site for 5 seconds. The sealed porcine aorta was stored at room temperature for 6 hours with continuous blood flow, and the hemostatic seal created by the adhesive material was monitored. To avoid tissue dehydration and degradation, a 0.01 w / v% sodium azide solution (in PBS) was sprayed onto the porcine aorta. After the test, the blood bath was filtered using a 100 μm mesh to check for the presence of free-floating bio-adhesive microparticles in the blood.

[0073] To measure the burst pressure, a porcine aorta with an area of ​​2.5 cm in width and 2.5 cm in length was prepared and tested according to the burst pressure test standard (ASTM F2392-04) (Figure 16B). A 3 mm hole was introduced into the porcine aorta using a biopsy punch (Dynarex). The punctured porcine aorta was then covered with porcine blood and sealed using various adhesives (adhesive material in combination with Surgicel® backing, sutures (Ethicon 4-0 vicryl), Surgiflo®, and Tisseel). Thirty minutes after sealing, pressure was applied to the sealed porcine aorta by pumping PBS at a flow rate of 2 ml / min using a syringe pump. The maximum pressure was recorded as the burst pressure using a pressure gauge (Omega).

[0074] Biocompatibility and biodegradability To evaluate the biocompatibility and biodegradability of the tissue glue, in vitro and in vivo characterization was performed based on a rat model (Figures 18A-L). Cell culture medium (DMEM) adapted with the tissue glue exhibited in vitro cytotoxicity of rat cardiomyocytes to a similar degree to that of the control (original DMEM) after 24 hours of culture (Figure 18A). The in vivo biocompatibility of the tissue glue was evaluated based on dorsal subcutaneous implantation of the rat model at various time points from 1 day to 2 weeks (Figures 18B-C and 19A-B). Histological assessment by blinded pathologists demonstrated that the tissue glue induced very mild to mild inflammation at all time points, comparable to that of commercially available US Food and Drug Administration (FDA) approved tissue adhesive Coseal (p=0.39 at 1 day, p=0.54 at 3 days, p=0.67 at 1 week, p=0.21 at 2 weeks, Figure 18D). To further investigate the in vivo biocompatibility of the tissue glue, immunofluorescence staining was performed for various markers related to inflammatory and xenobiotic responses, including fibroblasts (αSMA), type I collagen (collagen I), T cells (CD3), and macrophages (CD68) (Figures 18E-H and 19C-F). Normalized immunofluorescence intensity analysis demonstrated that the tissue glue did not show significant differences in αSMA, collagen I, CD3, and CD68 expression at any time point compared to Coseal (Figures 18I-L). In particular, the tissue glue exhibited gradual biodegradation over longer implantation periods via macrophage reabsorption (Figures 20A-C), and the rate of biodegradation could be accelerated by replacing the chitosan in the bio-adhesive microparticles with a faster-degrading material such as gelatin (Figure 20D).

[0075] Methods for evaluating biocompatibility and biodegradability In vitro biocompatibility assessment. In vitro biocompatibility testing was performed by using tissue glue-conditioned medium for cell culture. To prepare tissue glue-conditioned medium or Coseal-conditioned medium, 0.5 ml of tissue glue or Coseal was incubated at 37°C for 24 hours in 10 mL of DMEM supplemented with 10 v / v% fetal bovine serum (FBS) and 100 U ml-1 penicillin-streptomycin. Supplemented DMEM without tissue glue incubation was used as a control. 0.5 × 10 5 Rat embryonic cardiomyocytes (H9c2(2-1), ATCC) were plated onto confocal dishes (20 mm in diameter) at a cell density (n=4 per group). The cells were then treated with tissue glue-conditioned medium and incubated at 37°C in 5% CO2 for 24 hours. Cell viability was determined using a LIVE / DEAD viability / cytotoxicity kit for mammalian cells (Thermo Fisher Scientific). Live cells were imaged using a laser confocal microscope (SP 8, Leica) at 495 nm / 515 nm excitation / emission, and dead cells at 495 nm / 635 nm. Cell viability was calculated by counting the number of live cells (green fluorescence) and dead cells (red fluorescence) using ImageJ (version 2.1.0).

[0076] Evaluation of in vivo biocompatibility and biodegradability. All animal surgeries were reviewed and approved by the Committee on Animal Care at the Massachusetts Institute of Technology. Female Sprague Dolly rats (225-250g, Charles River Laboratories) were used in all in vivo studies.

[0077] Prior to implantation, tissue glue was prepared using aseptic techniques and further sterilized under UV light for 3 hours. For implantation into the dorsal subcutaneous space, rats were anesthetized in an anesthesia chamber using isoflurane (1-2% isoflurane in oxygen). Anesthesia was maintained using a nose cone. Dorsal hair was removed, and the animals were placed on a heating pad for the duration of the procedure. The subcutaneous space was accessed through a 1-2 cm skin incision / implant in the center of the animal's back. To create a space for implant placement, an incision was made toward the animal's scapula, and blunt lymph node dissection was performed. Either 0.5 ml of tissue glue (n=4 for each endpoint) or a similar volume of commercially available tissue adhesive (Coseal, n=4 for each endpoint) was placed in the subcutaneous pocket formed above the incision. The incision was closed using intermittent sutures (4-0 Vicryl, Ethicon), and 3-6 ml of saline was subcutaneously injected. Up to four implants were placed per animal, ensuring that there was no overlap between the formed subcutaneous pockets. Animals were euthanized by CO2 inhalation one day, three days, one week, or two weeks after implantation. The subcutaneous target area was excised and fixed in 10% formalin for 24 hours for histological and immunofluorescence analysis.

[0078] Instant hemostasis and tissue sealing. The unique ability of the tissue adhesive of the present invention to form instantaneous robust adhesion on blood-covered tissue may be advantageous for rapid and coagulation-independent hemostatic sealing of various tissues in clinical and biomedical applications. To quantitatively evaluate the hemostatic sealing ability of the tissue glue in vivo, hemostasis time and blood loss to hemostasis were measured based on rat liver and cardiac hemostasis models (Figures 21 and 22). The tissue adhesive achieved hemostatic sealing of bleeding liver (5 mm diameter and 2 mm depth injury) and heart (2 mm diameter ventricular wall injury) within 5 seconds (Figures 21A and 22A), demonstrating significantly shorter hemostasis time (Figures 21C and 22C) and blood loss to hemostasis (Figures 21D and 22D) compared to the injury group (no hemostasis) and commercially available hemostatic agents Surgicel and Coseal (Figures 23 and 24). In particular, in the Surgicel and Coseal groups, there are injuries that prevent the formation of a hemostatic seal in the heart due to high pressure and massive bleeding caused by ventricular injury (Figures 22C-D and 24). On the other hand, the tissue adhesive of the present invention forms an instantaneous hemostatic seal for ventricular injury within 5 seconds and restores normal intraventricular blood pressure immediately after hemostasis (Figure 22E).

[0079] The tissue adhesive of the present invention was demonstrated to maintain seals over injured liver and injured heart two weeks after initial hemostasis application (Figures 21B and 22B). Furthermore, histological analysis of the sealed liver and cardiac tissues suggested that the tissue adhesive enabled cell penetration into the cross-linked bioadhesive microparticles and healing of the underlying injury (Figures 21E and 22F-G). Immunofluorescence analysis of inflammatory and xenobiotic markers (CD3 for T cells and CD68 for macrophages) showed that the tissue adhesive of the present invention induced CD3 and CD68 expression comparable to Coseal and significantly lower than that of Surgicel (Figures 22F-I). In addition, total blood cell count (CBC) and blood chemistry analysis of animals two weeks after hemostatic sealing demonstrated that the tissue adhesive did not show significant differences in inflammation-related blood cells and organ-specific disease markers compared to healthy animals (Figures 25 and 26).

[0080] In vivo hemostatic sealing of the liver. For hemostatic sealing of liver injury, animals were anesthetized in an anesthesia chamber using isoflurane (1-3% isoflurane in oxygen). Abdominal hair was removed, and the animals were placed on a heated pad for the duration of the procedure. The liver was exposed by laparotomy. A 5 mm diameter and 2 mm deep injury was created in the heart using a biopsy punch (Dynarex). To form a hemostatic seal, 0.5 ml of tissue glue was injected onto the bleeding site, and then gentle pressure was applied to the puncture site for 5 seconds using a surgical spatula (n=4). Commercially available products used were Surgicel (Ethicon) (n=4) with dimensions of 20 mm in length and 20 mm in width, or 2 ml of Coseal (Baxter) (n=4). Hemostasis was not performed in the injury group (n=4). Blood loss and hemostasis time were recorded for each group. After confirming hemostasis and sealing, the incision was closed using intermittent sutures (4-0 Vicryl, Ethicon), and 3-6 ml of physiological saline was injected subcutaneously. Two weeks after implantation, blood was collected for blood analysis, and the animals were euthanized by CO2 inhalation. The liver containing the implant was resected and fixed in 10% formalin for 24 hours for histological and immunofluorescence analysis.

[0081] In vivo hemostatic sealing of the heart. For hemostatic sealing of ventricular injury of sufficient thickness, animals were anesthetized in an anesthesia chamber using isoflurane (1-3% isoflurane in oxygen). Chest hair was removed. Endotracheal intubation was performed, and the animals were connected to a ventilator (Model 683, Harvard Apparatus) and placed on a heated pad for the duration of the procedure. The heart was exposed by thoracotomy, and the pericardium was removed using precision forceps. For intraventricular blood pressure measurement, a pressure-volume (PV) catheter (SPR-838, Millar) was inserted into the left ventricle (LV) via a tip-tipped stick, and LV blood pressure was monitored during the test. A 2 mm diameter injury was created in the left or right ventricular wall of the heart using a biopsy punch (Dynarex). To form a hemostatic seal, 0.25 ml of the tissue adhesive of the present invention was injected onto the bleeding site, and then gentle pressure was applied to the puncture site for 5 seconds using a surgical spatula (n=5). For commercially available implants, Surgicel 25 (Ethicon) (n=4) with dimensions of 20 mm in length and 20 mm in width, or 2 ml of Coseal (Baxter) (n=4) were used. In the injured group, hemostasis was not performed (n=4). For each group, the amount of blood loss and the time to hemostasis were recorded up to 300 seconds. After confirming hemostatic sealing, the incision was closed using intermittent sutures (4-0 Vicryl, Ethicon), and 3-6 ml of physiological saline was injected subcutaneously. In groups where hemostasis could not be achieved by 300 seconds, the animals were euthanized by bloodletting. Two weeks after implantation, blood was collected for blood analysis, and the animals were euthanized by CO2 inhalation. The heart with the implant was resected and fixed in 10% formalin for 24 hours for histological analysis and immunofluorescence analysis.

[0082] Immunofluorescence analysis. After immunofluorescence staining of collected tissue, the expression of target proteins (αSMA, collagen I, CD68, CD3) was analyzed. Prior to immunofluorescence analysis, paraffin-embedded tissue was sliced ​​and prepared as slides. The slides were deparaffinized and rehydrated with deionized water. Antigen recovery was performed using the steam method. During this process, the slides were steamed in IHC-Tek Epitope Retrieval Solution (IW-1100) for 35 minutes, followed by cooling for 20 minutes. Then, the slides were washed by changing PBS three times every 5 minutes for each cycle. After washing, the slides were incubated at room temperature for 1 hour in primary antibodies diluted with IHC-Tek Antibody Diluent (1:200 mouse anti-αSMA (ab7817, Abcam) for fibroblasts, 1:200 mouse anti-CD68 (ab201340, Abcam) for macrophages, 1:100 rabbit anti-CD3 (ab5690, Abcam) for T cells, and 1:200 rabbit anti-collagen-I (ab21286, Abcam) for collagen). The slides were then washed three times in PBS and incubated with Alexa Fluor 488-labeled anti-rabbit or anti-mouse secondary antibodies (1:200, Jackson Immunoresearch) for 30 minutes. The slides were washed in PBS and then counterstained with propidium iodide solution for 20 minutes. A laser confocal microscope (SP 8, Leica) was used for image acquisition. Fluorescence intensity of the expressed antibodies was quantified using ImageJ (version 2.1.0). All images were converted to 8-bit binary images, and fluorescence intensity was calculated using normalized analysis. All analyses were blinded to the experimental conditions.

Claims

1. An adhesive material for bonding one or more surfaces covered with a fluid, A hydrophobic matrix selected from silicone oil, mineral oil, essential oil, perfluoropolyether oil, lanolin oil, and combinations thereof, Multiple bio-adhesive microparticles dispersed within the hydrophobic matrix The bio-adhesive microparticles include, (i) one or more hydrophilic polymers or copolymers, (ii) One or more amine coupling groups, (iii) One or more crosslinking agents and Includes, The bio-adhesive microparticles further comprise (ii) poly(acrylic acid) grafted with N-hydroxysuccinimide ester, (i) biodegradable chitosan, and (iii) one or more crosslinking agents. The hydrophobic matrix forms a protective matrix around the dispersed bio-adhesive microparticles that protects the bio-adhesive microparticles from the fluid, and the adhesive material is directly placed on the fluid-covered surface and pressure is applied to the adhesive material, causing (a) the hydrophobic matrix to repel the fluid, (b) the bio-adhesive microparticles to compress and form an adhesive layer, and (c) the bio-adhesive microparticles to form temporary crosslinks and subsequently covalent crosslinks with the surface, wherein the adhesive material is configured such that

2. The adhesive material according to claim 1, in the form of an adhesive material for injection.

3. The adhesive material according to claim 1, wherein (i) one or more hydrophilic polymers or copolymers are selected from hydrophilic polymers or copolymers that absorb water in a dry state.

4. The adhesive material according to claim 1, wherein (i) one or more hydrophilic polymers or copolymers are selected from polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, polyurethane, casein, albumin, gelatin, chitosan, hyaluronic acid, alginate, oxidized alginate, cellulose, oxidized cellulose, polyvinylpyrrolidone, polystyrene sulfonate, collagen, alginic acid, pectin, and combinations thereof.

5. The adhesive material according to claim 1, wherein the (ii) one or more amine coupling groups are selected from N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, aldehydes, imide esters, epoxides, isocyanates, catechols, and combinations thereof.

6. The adhesive material according to claim 1, wherein the (iii) one or more crosslinking agents are selected from gelatin methacrylate, hyaluronic acid methacrylate, methacrylate-based oxidized alginate, polycaprolactone diacrylate, N,N'-bis(acryloyl)cystamine, N,N'-methylenebis(acrylamide), polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and combinations thereof.

7. The adhesive material according to claim 1, which is biocompatible.

8. At least approximately 100 Jm －２ The adhesive material according to claim 1, which adheres with interfacial toughness, a shear strength of at least about 30 kPa, and a tensile strength of at least about 10 kPa.

9. The adhesive material according to claim 1, wherein the bio-adhesive microparticles contain carboxylic acid groups that form the temporary crosslinks through intermolecular bonding, and the amine coupling groups form covalent crosslinks with the surface.

10. The adhesive material according to claim 1, wherein the bio-adhesive microparticles have a particle size in the range of about 10 μm to about 200 μm.

11. The adhesive material according to claim 1, comprising a ratio of the bio-adhesive microparticles to the hydrophobic matrix in the range of approximately 1:3 to approximately 1:0.

5.

12. The adhesive material according to claim 1, wherein the one or more fluids are physiological bodily fluids selected from plasma, interstitial fluid, lymph, cerebrospinal fluid, gastrointestinal fluid, and combinations thereof.

13. The adhesive material according to claim 1, which is biodegradable and configured to allow cell penetration into the crosslinked bio-adhesive microparticles and healing of underlying tissue damage.

14. The adhesive material according to claim 13, wherein the adhesive material is configured such that tissue cells replace the biodegradable bio-adhesive microparticles to heal the underlying tissue damage.

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