Tissue sealant, preparation method therefor, and hemostatic material

Abstract

The present application relates to the technical field of medical biomaterials, and in particular, to a tissue sealant, a preparation method therefor, and a hemostatic material. The tissue sealant comprises a biocompatible substrate and an adhesive layer located on the substrate. The adhesive layer is prepared from polyethylene glycol succinimidyl ester in a dry state and amino polyethylene glycol in a dry state, which imparts extremely strong adhesion between tissue sealants and between the tissue sealant and a tissue wound surface, and also enhances the mechanical properties of the tissue sealant itself, achieving a rapid and efficient hemostatic effect, and meeting the needs of clinical massive hemorrhage. In addition, the tissue sealant has good biocompatibility, biodegradability, and flexible adjustability of formulation ingredients, and thus can be customized to meet the hemostatic needs of short-term or long-term implantation in vivo. Furthermore, the formulation can be adjusted as needed for application in a clinical massive hemorrhage model.

Description

A tissue sealant, its preparation method, and a hemostatic material.

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510126659.X, filed on January 27, 2025, entitled "A Tissue Sealant and Its Preparation Method and Hemostatic Material", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of medical biomaterials technology, specifically to a tissue sealant, its preparation method, and a hemostatic material. Background Technology

[0004] External trauma caused by disasters and emergencies, resulting in massive blood loss, is the leading cause of pre-hospital mortality. Blood loss exceeding one-third of the body's volume can trigger a series of systemic reactions, such as deep coma and shock. Studies show that if bleeding can be controlled within 60 minutes, the survival rate of injured individuals can increase by 20-30%. On the other hand, bleeding is a common complication of surgery; massive hemorrhage severely impacts surgical outcomes and can even endanger the patient's life, placing an additional burden on the healthcare system. According to data from the *China Health Statistics Yearbook*, more than 80 million inpatients undergo surgery annually in my country, of which at least 40 million require surgical hemostasis. Because uncontrolled bleeding in surgical and trauma environments often leads to complications and adverse outcomes, controlling bleeding is a crucial measure to reduce complications and mortality, and improve patient prognosis.

[0005] Currently, clinical practice still primarily relies on traditional methods such as surgical sutures and staples to achieve hemostasis and wound closure. However, surgical sutures can cause secondary damage to fragile tissues, and the procedures are time-consuming and require high technical skills. Several tissue sealants have been developed to assist in controlling bleeding. Conventional tissue sealants include fibrin sealants, cyanoacrylate-based sealants, and other synthetic sealants and polymerizable monomers. These tissue adhesives have many drawbacks, limiting their suitability to specific applications. These drawbacks include the release of toxic degradation products, high cost, the need for refrigerated storage, slow curing, limited mechanical strength, high risk of infection, and difficulty in removal. Therefore, hydrogel tissue adhesives based on reactive polyethylene glycol (PEG) precursors have been developed. However, these hydrogel tissue adhesives swell or dissolve too quickly and lack sufficient adhesion, thus reducing their effectiveness as surgical adhesives and making them unsuitable for situations involving massive bleeding.

[0006] Tissue sealants used in cases of massive bleeding must not only have hemostatic properties but also meet the requirement of rapid sealing and hemostasis. Currently, new types of tissue sealants on the market include... Absorbable hemostatic gauze, Combat gauze, hemostatic powder Sealing hemostatic pad, Fluid gelatin, etc., are available. However, they all have significant limitations. For example, cellulose-based hemostatic gauze, represented by Surgicel, relies on water absorption and barrier action, making it difficult to form a strong interaction with the wet tissue interface, thus rendering it unsuitable for uncontrollable massive bleeding scenarios. Existing products, such as hemostatic sealing patches (HEMOPATCH), contain only a single component, ethylene glycol glutarate tetrasuccinimide, resulting in limited hemostatic ability. Furthermore, the self-adhesive biological patch provided by CN 115814163 A suffers from insufficient tissue adhesion due to premature gelation during the preparation of the PEG two-component system, making it unsuitable for handling massive bleeding situations. Therefore, there is an urgent need for a novel tissue sealant and hemostatic material with strong tissue adhesion to meet the requirements for rapid sealing and hemostasis in massive bleeding scenarios, thus satisfying the clinical needs for massive bleeding. Summary of the Invention

[0007] To overcome the problems of poor tissue adhesion and limited hemostatic effect of existing tissue sealants and hemostatic materials, this application provides a series of tissue sealants with high adhesion that can quickly stop bleeding, immediately seal wounds, and their preparation methods and hemostatic materials. These require no additional preparation and can be directly applied to adherent tissues to achieve rapid hemostasis.

[0008] Therefore, this application provides a tissue sealant comprising a biocompatible substrate and an adhesive layer located on the substrate, the adhesive layer being made of dried polyethylene glycol succinimide ester and dried amino polyethylene glycol.

[0009] Furthermore, the water content of the dried polyethylene glycol succinimide ester and the dried amino polyethylene glycol is less than 0.5 wt%; preferably less than 0.25 wt%.

[0010] Furthermore, the tissue sealant is prepared by applying dried polyethylene glycol succinimide ester and dried amino polyethylene glycol simultaneously or separately to the substrate.

[0011] Furthermore, the polyethylene glycol succinimide ester has n succinimide ester groups, n≥2; the amino polyethylene glycol has m amino groups, m≥2; and m+n≥5, where m and n are both integers.

[0012] Furthermore, in the adhesive layer, both polyethylene glycol succinimide and amino polyethylene glycol exist in particulate form or in a homogeneous system; or, amino polyethylene glycol is distributed in particulate form in polyethylene glycol succinimide, exhibiting an island structure.

[0013] Furthermore, the particle size of the island phase in the island structure is below 40 mesh, preferably below 60 mesh; more preferably below 80 mesh.

[0014] Furthermore, when both polyethylene glycol succinimide and amino polyethylene glycol exist in particulate form or in a homogeneous system, the molar ratio of succinimide groups to amino groups in the adhesive layer is 4:1 to 1:2; preferably 2:1 to 1:1.

[0015] Furthermore, when forming an island structure, the molar ratio of succinimide ester groups to amino groups in the adhesive layer is 4:1 to 1:4; preferably 2:1 to 1:2.

[0016] Furthermore, the number average molecular weights of the polyethylene glycol succinimide ester and the amino polyethylene glycol are independently 2,000 Da to 200,000 Da, preferably 10,000 Da to 40,000 Da.

[0017] Further, the succinimide ester in the polyethylene glycol succinimide ester is selected from one or more combinations of succinimide carbonate, succinimide acetate, succinimide oxalate, succinimide malonate, succinimide succinimide glutarate, succinimide adipate, succinimide carbamate, sulfosuccinimide carbonate, sulfosuccinimide acetate, sulfosuccinimide acetate, and sulfosuccinimide oxalate; and / or, the amino group in the amino polyethylene glycol is a primary amino group.

[0018] Furthermore, in the adhesive layer, the loading amounts of polyethylene glycol succinimide ester and amino polyethylene glycol are independently 2 mg / cm³. 2 -20mg / cm 2 Preferably 2mg / cm 2 -15mg / cm 2 .

[0019] Furthermore, the polyethylene glycol succinimide ester and amino polyethylene glycol have multiple arms; preferably, the multiple arms of the polyethylene glycol succinimide ester and the multiple arms of the amino polyethylene glycol are independently 2-10, and the polyethylene glycol succinimide ester and amino polyethylene glycol are not simultaneously a two-arm structure.

[0020] Further, the polyethylene glycol succinimide ester is a four-arm polyethylene glycol succinimide glycoside, and the amino polyethylene glycol is a four-arm polyethylene glycol amino; preferably, the amino polyethylene glycol is a four-arm polyethylene glycol primary amino.

[0021] Furthermore, the substrate is a biocompatible protein or a derivative thereof;

[0022] Preferably, the protein or its derivative comprises collagen, gelatin or its derivative;

[0023] Preferably, the thickness of the substrate is 0.2cm to 10cm, more preferably 0.2cm to 2cm.

[0024] Secondly, this application also provides a method for preparing a tissue sealant, the preparation method comprising one of method 1 or method 2:

[0025] Method 1: The mixture formed by mixing polyethylene glycol succinimide ester and amino polyethylene glycol is coated onto the substrate surface to obtain the product;

[0026] Method 2: Polyethylene glycol succinimide ester is coated onto the substrate surface to form a substrate containing polyethylene glycol succinimide ester melt, amino polyethylene glycol particles are dispersed into the polyethylene glycol succinimide ester melt, and the substrate is obtained after cooling and solidification.

[0027] Furthermore, in method 1 or method 2, the coating includes one or more of the following: powder coating, dip coating, spray coating, doctor blade coating, and roll coating; and / or, the coating is followed by a heating and melting step and a cooling and curing step.

[0028] Furthermore, method 1 or method 2 also satisfies one or more of the following ACs:

[0029] A. After coating, the tissue sealant is dried and / or sterilized. Optionally, the sterilization includes one or a combination of ethylene oxide sterilization, dry heat sterilization, ultraviolet sterilization, irradiation sterilization, and filtration sterilization. Optionally, the drying includes freeze drying, vacuum heating drying, and atmospheric pressure heating drying.

[0030] B. In method 1 or method 2, the entire preparation process is carried out under dry conditions; preferably, the water content of the obtained tissue sealant is less than 0.2 wt%.

[0031] C. Before coating, the process further includes drying and / or grinding polyethylene glycol succinimide and amino polyethylene glycol; optionally, the drying includes one or a combination of freeze drying, vacuum heating drying, and atmospheric pressure heating drying; optionally, the grinding process is followed by a sieving process.

[0032] Furthermore, Method 1 includes the following steps:

[0033] (1) Polyethylene glycol succinimide ester and amino polyethylene glycol were subjected to vacuum heating and drying, grinding and sieving respectively;

[0034] (2) The polyethylene glycol succinimide ester and amino polyethylene glycol obtained in step (1) are mixed and coated onto the substrate surface by powder spraying process. After drying and sterilization, the product is obtained. Optionally, after coating, the product is further heated to melt and then cooled to solidify.

[0035] Furthermore, method 2 includes the following steps:

[0036] (1) Polyethylene glycol succinimide ester and amino polyethylene glycol were subjected to vacuum heating and drying treatment respectively;

[0037] (2) Spray polyethylene glycol succinimide onto the substrate at 55-65℃ to form a substrate containing polyethylene glycol succinimide melt; disperse amino polyethylene glycol particles with a particle size of less than 80 mesh into the polyethylene glycol succinimide melt and allow them to settle naturally for 10-20 minutes; cool and solidify to form an adhesive layer with an island structure on the substrate surface;

[0038] (3) Dry and sterilize to obtain the product.

[0039] Thirdly, this application also provides the use of the tissue sealant described in any of the first aspects or the tissue sealant prepared by any of the preparation methods described in any of the second aspects in the preparation of hemostatic materials.

[0040] Fourthly, this application also provides a hemostatic material, which includes the tissue sealant described in any of the first aspects or the tissue sealant prepared by any of the preparation methods described in any of the second aspects, and optionally includes one or more of medically or pharmaceutically acceptable carriers, other drugs or contrast agents.

[0041] In some alternative embodiments, the other drugs include one or more of the following: growth factors, antifibrotic drugs, nonsteroidal anti-inflammatory drugs, glucocorticoids, and analgesics.

[0042] In some alternative embodiments, the developer is a biocompatible material and is located on the substrate on the side opposite to the adhesive layer.

[0043] In some alternative embodiments, the medically or pharmaceutically acceptable carrier includes an anti-adhesion agent located on the substrate on a side opposite to the adhesive layer.

[0044] The technical solution of this application has the following advantages:

[0045] This application provides a tissue sealant comprising a substrate and an adhesive layer situated on the substrate. The adhesive layer is made from dried polyethylene glycol succinimidyl ester and dried amino polyethylene glycol. The adhesive layer is made from dried polyethylene glycol succinimidyl ester and dried amino polyethylene glycol, ensuring that the polyethylene glycol succinimidyl ester and amino polyethylene glycol are not cross-linked before use. Once the tissue sealant is applied to a patient (to a wound), the uncross-linked polyethylene glycol succinimidyl ester and amino polyethylene glycol undergo a hydrophilic cross-linking reaction upon contact with body fluids. A covalent bond reaction occurs between the succinimidyl ester of the polyethylene glycol succinimidyl ester and the amino group of the amino polyethylene glycol, forming a three-dimensional network polymer adhesive layer. Covalent or hydrogen bonding reactions can occur between the succinimide ester of polyethylene glycol and the amino groups of amino-polyethylene glycol (PEG) and the amino and carboxyl groups of proteins exposed in blood or tissue, as well as between the succinimide ester of PEG and the amino-polyethylene glycol and the amino and carboxyl groups of the substrate. This imparts extremely strong adhesion between tissue sealants and between the tissue sealant and the wound surface, while simultaneously enhancing the mechanical properties of the tissue sealant itself. This allows for rapid and efficient hemostasis, meeting the needs of clinical massive hemorrhage. Furthermore, the tissue sealant exhibits good biocompatibility and biodegradability, promoting wound healing. The flexible and adjustable formulation allows for customization to meet short-term or long-term hemostasis requirements in vivo, and the formulation can be adjusted as needed for clinical massive hemorrhage models.

[0046] Additional aspects and advantages of the embodiments of this application will be described and shown in part in the following description, or illustrated by practice of the embodiments of this application. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 shows the adhesive strength results in Experiment Example 1;

[0049] Figure 2 shows the results of the in vitro cytotoxicity test in Experiment Example 2, where the vertical axis vability rate represents the percentage of cell viability.

[0050] Figure 3 shows the staining patterns of cells in each group in Experiment Example 3;

[0051] Figure 4 shows pictures of the hemostatic patches used in each group in Experiment Example 4;

[0052] Figure 5 shows pictures of the hemostatic patches used in each group in Experiment Example 5;

[0053] Figure 6 shows HE staining images of each group of hemostatic patches implanted one week after the implantation in Experiment 6 (total length of the scale is 2.5 mm, and the scale is 0.5 mm).

[0054] Figure 7 shows HE staining images of each group of hemostatic patches implanted 2 weeks after the implantation in Experiment 7 (total length of the scale is 2.5 mm, and the scale is 0.5 mm). Detailed Implementation

[0055] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0056] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0057] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0058] Given the current high demand for hemostatic materials, but the shortcomings of existing products such as insufficient adhesion, limited hemostatic effect, and inability to cope with massive bleeding situations.

[0059] In a first aspect, this application provides a tissue sealant comprising a substrate and an adhesive layer situated on the substrate, the adhesive layer being made of dried polyethylene glycol succinimide and dried amino polyethylene glycol. The adhesive layer is made of dried polyethylene glycol succinimide and dried amino polyethylene glycol such that, before use, the polyethylene glycol succinimide and amino polyethylene glycol in the adhesive layer are not cross-linked. Once the tissue sealant is applied to a patient (to a wound), the uncross-linked polyethylene glycol succinimide and amino polyethylene glycol undergo a hydrophilic cross-linking reaction upon contact with body fluids. Covalent bonds are formed between the succinimide of the polyethylene glycol succinimide and the amino groups of the amino polyethylene glycol, forming an adhesive layer of a three-dimensional network polymer. The succinimide ester of polyethylene glycol succinimide and the amino group of amino polyethylene glycol can form covalent bonds or hydrogen bonds with the amino and carboxyl groups of proteins exposed in blood or tissue, as well as with the amino and carboxyl groups of the substrate. This gives the tissue sealant extremely strong adhesion between itself and between the tissue sealant and the tissue wound, while also enhancing the mechanical properties of the tissue sealant itself. This allows for rapid and efficient hemostasis, meeting the needs of clinical massive hemorrhage.

[0060] In this application, the succinimide ester of the polyethylene glycol succinimide ester and the amino group of the amino polyethylene glycol are non-reactive in dry environments, aprotic solutions, and acidic protic solutions (pH ≤ 5.5), but become reactive when exposed to neutral or alkaline protic solutions (pH > 5.5). Thus, the two components can react with each other in an aqueous environment to form a three-dimensional network polymer. In other words, when polyethylene glycol succinimide ester and amino polyethylene glycol are simultaneously in contact with water or an aqueous liquid, hydrophilic covalent crosslinking occurs. The term "non-crosslinked" means that the polyethylene glycol succinimide ester and amino polyethylene glycol have not undergone the aforementioned expected crosslinking reaction.

[0061] In some alternative embodiments, the tissue sealant is prepared by simultaneously or separately applying dried polyethylene glycol succinimide and dried amino polyethylene glycol to a substrate. For the purposes of this application, it is important to ensure that the reactivity of the polyethylene glycol succinimide and amino polyethylene glycol is retained (i.e., uncrosslinked) before application to a patient. Therefore, in manufacturing the tissue sealant according to this application, it is essential to maintain the dryness of the polyethylene glycol succinimide and amino polyethylene glycol during the manufacturing process so that once the sponge is applied to the wound, the reactive groups in the polyethylene glycol succinimide and amino polyethylene glycol immediately undergo a hydrophilic crosslinking reaction, forming a three-dimensional network polymer.

[0062] The terms "dryness" and "dry state" have the same meaning, referring to controlling the water content of polyethylene glycol succinimide or amino polyethylene glycol to be less than or equal to 0.5 wt%, more preferably less than or equal to 0.25 wt%.

[0063] In some alternative embodiments, the tissue sealant may use pre-dehydrated polyethylene glycol succinimide ester and amino polyethylene glycol as raw materials, and both shall be kept dry during the preparation process.

[0064] In some optional embodiments, the polyethylene glycol succinimide ester has n succinimide ester groups, n≥2; the amino polyethylene glycol has m amino groups, m≥2; and m+n≥5, where m and n are both integers. For example, n is an integer between 2 and 10, and m is an integer between 2 and 10.

[0065] In some alternative embodiments, both polyethylene glycol succinimide and amino polyethylene glycol exist in particulate form or as a homogeneous system in the adhesive layer; or, amino polyethylene glycol is distributed in particulate form within polyethylene glycol succinimide, exhibiting an island structure.

[0066] In this application, "island structure" refers to a structure in which two incompatible polymers undergo phase separation, with a dispersed phase (island phase) containing the other polymer dispersed in a continuous phase (ocean phase) containing one polymer.

[0067] When amine polyethylene glycol (PEG) is distributed in particulate form within PEG succinimidyl ester, exhibiting an island-like structure, PEG succinimidyl ester forms the ocean phase, and amine PEG forms the island phase. The nucleophilic groups (amino groups) are embedded within the ocean phase, effectively protecting their reactivity and preventing moisture absorption and oxidation. This also effectively prevents premature hydrophilic covalent crosslinking with the electrophilic groups (PEG succinimidyl ester). Conversely, when PEG succinimidyl ester is distributed in particulate form within amine PEG, exhibiting an island-like structure, amine PEG forms the ocean phase, and PEG succinimidyl ester forms the island phase. The electrophilic groups are embedded within the ocean phase, while the nucleophilic groups form a molten flowing phase on the outside. This also protects the electrophilic groups and prevents moisture absorption and oxidation.

[0068] This study found that when amino polyethylene glycol is distributed in particulate form within polyethylene glycol succinimide ester, exhibiting an island-like structure, an unexpectedly short gelation time was achieved in the tissue sealant coating, resulting in rapid sealing of bleeding wounds. This can be attributed to two main factors: firstly, the island-like structure significantly enhances the adhesion between the tissue sealant coating and the biological matrix material and wound, shortening the physical distance between electrophilic and nucleophilic groups and improving reaction efficiency. Secondly, the introduction of the island-like structure into the coating enhances the mechanical properties of the tissue sealant. This may stem from the fact that after contact with blood, the particles can form uniformly distributed physical cross-linking points within the gel, effectively transferring stress and preventing stress concentration, thereby improving the mechanical properties of the gel layer.

[0069] For example, the tissue sealant of the aforementioned island structure can seal bleeding wounds within 100s, 80s, 60s, further, within 40s, 20s, 10s, or even 5s.

[0070] In some optional embodiments, the particle size of the island phase in the island structure is below 40 mesh (particle size ≤ 425 μm), preferably below 60 mesh (particle size ≤ 250 μm); more preferably below 80 mesh (particle size ≤ 178 μm). By controlling the particle size of the island phase in the island structure to below 40 mesh, preferably below 60 mesh, and more preferably below 80 mesh, the uniformity of coating distribution can be improved, product quality and stability can be enhanced, and the hemostatic effect can be better achieved.

[0071] In some optional embodiments, when both polyethylene glycol succinimide and amino polyethylene glycol exist in particulate form or as a homogeneous system, the molar ratio of succinimide groups to amino groups in the adhesive layer is 4:1 to 1:2; preferably 2:1 to 1:1. By controlling the molar ratio of succinimide groups to amino groups within the above range, especially within the preferred range, the degree of crosslinking is more suitable, the mechanical properties are stronger, and the interfacial adhesion is better.

[0072] In some alternative embodiments, when forming an island structure, the molar ratio of succinimide ester groups to amino groups in the adhesive layer is 4:1 to 1:4; preferably 2:1 to 1:2. By controlling the molar ratio of succinimide ester groups to amino groups within the above range, especially within the preferred range, the gelation speed is extremely fast, and the amino polyethylene glycol of the island phase toughens the adhesive layer, enabling the tissue sealant to stop bleeding more quickly to cope with massive bleeding wounds.

[0073] In some alternative embodiments, the number-average molecular weights of the polyethylene glycol succinimide and amino polyethylene glycol are independently 2,000 Da to 200,000 Da, preferably 10,000 Da to 40,000 Da. For example, the number-average molecular weights of the polyethylene glycol succinimide and amino polyethylene glycol are independently 2,000 Da, 5,000 Da, 10,000 Da, 20,000 Da, 50,000 Da, 200,000 Da, etc.

[0074] In some optional embodiments, the succinimide ester in the polyethylene glycol succinimide ester is selected from one or more combinations of succinimide carbonate, succinimide acetate, succinimide glycosyl ester, succinimide malonate, succinimide succinimide glutarate, succinimide adipate, succinimide carbamate, sulfosuccinimide carbonate, sulfosuccinimide acetate, sulfosuccinimide acetate, and sulfosuccinimide glycosyl ester; and / or, the amino group in the amino polyethylene glycol is a primary amino group.

[0075] In some alternative embodiments, the loading of polyethylene glycol succinimide ester and amino polyethylene glycol in the adhesive layer is independently 2 mg / cm³. 2 -20mg / cm 2 Preferably 2mg / cm 2 -15mg / cm 2 By controlling the loading amounts of polyethylene glycol succinimide and amino polyethylene glycol within the aforementioned range, and especially within the preferred range, on the one hand, 30–60 vol.% of the pores on the substrate surface remain uncovered by polyethylene glycol succinimide and amino polyethylene glycol, allowing the substrate to absorb bodily fluids from the wound; on the other hand, this loading amount results in the strongest tissue adhesion of the adhesive layer, further enhancing the hemostatic effect. The loading amounts of polyethylene glycol succinimide and amino polyethylene glycol can be the same or different.

[0076] In some optional embodiments, the polyethylene glycol succinimide ester and the polyethylene glycol in the amino-polyethylene glycol have multiple arms; preferably, the multiple arms of the polyethylene glycol succinimide ester and the multiple arms of the polyethylene glycol in the amino-polyethylene glycol are independently 2-10, and the polyethylene glycol succinimide ester and the amino-polyethylene glycol are not simultaneously a two-arm structure. For example, two-arm, four-arm, six-arm, eight-arm, ten-arm, etc.

[0077] In some alternative embodiments, the polyethylene glycol succinimide ester is a four-arm polyethylene glycol succinimide oxalate, and the amino polyethylene glycol is a four-arm polyethylene glycol amino; preferably, the amino polyethylene glycol is a four-arm polyethylene glycol primary amino. Studies have found that using four-arm polyethylene glycol succinimide oxalate and four-arm polyethylene glycol amino, especially four-arm polyethylene glycol primary amino, can further improve hemostatic efficiency and better treat or alleviate the symptoms of massive bleeding.

[0078] In some alternative embodiments, the substrate is a biocompatible protein or a derivative thereof;

[0079] Optionally, the protein or its derivative may comprise collagen, gelatin, or a derivative thereof.

[0080] In this application, the substrate is a biocompatible protein or its derivative (e.g., gelatin, collagen, albumin, hemoglobin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, and laminin, and their derivatives and compositions), which can be naturally derived, artificially synthesized, or modified or cross-linked protein derivatives. Collagen, gelatin, or their derivatives are preferred, including but not limited to those derived from cattle, pigs, horses, sheep, etc. The collagen or gelatin component can trigger platelet recruitment and the body's coagulation cascade mechanism, exerting a hemostatic effect. The collagen or gelatin matrix can be commercially available absorbable collagen or gelatin sponges, including but not limited to... Medical collagen sponge Collagen sponge, Collagen sponge, Absorbent gelatin sponge, Gelatin sponge.

[0081] The absorbable collagen or gelatin sponges purchased above can be further modified or cross-linked as needed, including but not limited to grafting of reactive groups, ultraviolet cross-linking, vacuum heating cross-linking, and a series or combination thereof.

[0082] The absorbable collagen or gelatin sponge described above is further modified or cross-linked and then dried, including but not limited to freeze drying, vacuum heating drying, atmospheric pressure heating drying (not exceeding 90°C), or a combination of both.

[0083] In this application, the biomaterial matrix is ​​a porous solid matrix that has the function of absorbing and containing body fluids (such as blood).

[0084] In some alternative embodiments, the thickness of the substrate is 0.2 cm to 10 cm, preferably 0.2 cm to 2 cm. For example, the thickness of the substrate is 0.2 cm, 0.5 cm, or 1 cm.

[0085] According to this application, the adhesive layer may be a continuous or discontinuous layer on at least one surface of the biological matrix. In this application, discontinuity means that the adhesive layer may be mesh-like or porous.

[0086] Secondly, this application also provides a method for preparing a tissue sealant, the preparation method comprising one of method 1 or method 2:

[0087] Method 1: The mixture formed by mixing polyethylene glycol succinimide ester and amino polyethylene glycol is coated onto the substrate surface to obtain the product;

[0088] Method 2: Polyethylene glycol succinimide ester is coated onto the substrate surface to form a substrate containing polyethylene glycol succinimide ester melt, amino polyethylene glycol particles are dispersed into the polyethylene glycol succinimide ester melt, and the substrate is obtained after cooling and solidification.

[0089] Method 1 can be used to prepare an adhesive layer in which both polyethylene glycol succinimide ester and amino polyethylene glycol exist in particulate form or in a homogeneous system.

[0090] Method 2 can produce an adhesive layer exhibiting an island structure. Polyethylene glycol succinimide forms the marine phase, while amino polyethylene glycol forms the island phase.

[0091] In this application, "coating" and "applying" have the same meaning and are known and commonly used in the art. Coating refers to a process in which a liquid, paste, or powdered substance is uniformly applied to the surface of a solid substrate to form a thin film or coating. This process can be applied to various materials, such as porous matrices, sponges, pads, fiber webs, paper, plastics, metals, and glass, to modify or enhance their surface properties, such as appearance, abrasion resistance, corrosion resistance, and conductivity. It is typically achieved using specialized coating equipment, such as blade coaters, roller coaters, spraying equipment, dip coating, hot melt spraying, and powder coating.

[0092] In some alternative embodiments, in method 1 or method 2, the coating includes one or more of the following: powder coating, dip coating, spray coating, doctor blade coating, and roll coating; and / or, the coating is followed by a heating and melting step and a cooling and curing step.

[0093] The term "powdering method" refers to dispersing powder onto a substrate. In this application, it specifically refers to dispersing polyethylene glycol succinimide powder and amino polyethylene glycol powder simultaneously or separately onto a substrate to form an adhesive layer.

[0094] "Immersion coating" refers to immersing a substrate in a solution of polyethylene glycol succinimide and amino polyethylene glycol, allowing the substrate to absorb the polyethylene glycol succinimide and amino polyethylene glycol and form an adhesive layer on its surface. The solution is, for example, an aqueous solution with a pH below 5, preferably about 3, such as an acetic acid solution; or an anhydrous organic solvent solution, such as an organic solvent based on ethanol, acetone, dichloromethane, etc., ensuring that the polymer components containing polyethylene glycol succinimide and amino polyethylene glycol do not react. The substrate is brought into contact with the solution so that the substrate is impregnated with the solution containing polyethylene glycol succinimide and amino polyethylene glycol. The solution is allowed to penetrate the substrate for a sufficient period of time for absorption to occur, for example from about 5 minutes to about 3 hours, preferably 60 minutes.

[0095] The spraying methods include air spraying, aerosol spraying, ultrasonic spraying, electrostatic spray gun spraying, 3D printing, and other methods or combinations thereof.

[0096] The heating and melting method involves uniformly melting polyethylene glycol succinimide and amino polyethylene glycol onto a substrate in an oven preheated at a temperature between 50°C and 90°C, preferably between 60°C and 90°C, and maintaining the mixture in a fixed state for a period of time, such as 5 to 60 minutes, preferably 10 to 40 minutes.

[0097] Both electrostatic spraying and powder coating can directly apply powder to the substrate without first dispersing it in a solvent to prepare a slurry. After electrostatic spraying or powder coating, the powder can be melted by heating and then cooled and solidified to form an adhesive layer, or the adhesive layer can be formed directly without the heating and solidification process.

[0098] In some optional embodiments, method 1 or method 2 further includes drying and / or sterilizing the tissue sealant after coating. Optionally, the sterilization includes one or more of the following: ethylene oxide sterilization, dry heat sterilization, ultraviolet sterilization, irradiation sterilization, and filtration sterilization. Optionally, the drying includes freeze drying, vacuum heating drying, and atmospheric pressure heating drying.

[0099] In some alternative embodiments, in method 1 or method 2, the entire preparation process is carried out under dry conditions; preferably, the resulting tissue sealant has a water content of less than 0.2 wt%.

[0100] In some optional embodiments, prior to coating, the process includes drying and / or grinding polyethylene glycol succinimide and amino polyethylene glycol; optionally, the drying includes one or a combination of freeze drying, vacuum heating drying, and atmospheric pressure heating drying; optionally, the drying temperature is no more than 90°C (e.g., 60–90°C) and the time is 4–12 hours; optionally, the grinding process is followed by a sieving step.

[0101] In some alternative implementations, method 1 includes the following steps:

[0102] (1) Polyethylene glycol succinimide ester and amino polyethylene glycol were subjected to vacuum heating and drying, grinding and sieving respectively;

[0103] (2) The polyethylene glycol succinimide ester and amino polyethylene glycol obtained in step (1) are mixed and coated onto the substrate surface by powder spraying process. After drying and sterilization, the product is obtained. Optionally, after coating, the product is further heated to melt and then cooled to solidify.

[0104] In some optional implementations, method 2 includes the following steps:

[0105] (1) Polyethylene glycol succinimide ester and amino polyethylene glycol were subjected to vacuum heating and drying treatment respectively;

[0106] (2) Spray polyethylene glycol succinimide onto the substrate at 55-65℃ to form a substrate containing polyethylene glycol succinimide melt; disperse 20-200μm amino polyethylene glycol particles into the polyethylene glycol succinimide melt and allow them to settle naturally for 10-20 minutes; cool and solidify to form an adhesive layer with an island structure on the substrate surface;

[0107] (3) Dry and sterilize to obtain the product.

[0108] The melting point of the four-armed polyethylene glycol amino is between 70-75℃, and the polyethylene glycol succinimide ester forms a melt at 55-65℃. In method 2, by controlling the spraying temperature, an adhesive layer with an island structure is obtained, and the polyethylene glycol succinimide ester forms the sea phase and the amino polyethylene glycol forms the island phase. The hemostatic effect of the tissue sealant with this structure is significantly improved.

[0109] Thirdly, this application also provides the use of the tissue sealant described in any of the first aspects or the tissue sealant prepared by any of the preparation methods described in any of the second aspects in the preparation of hemostatic materials.

[0110] Fourthly, this application also provides a hemostatic material, which includes the tissue sealant described in any of the first aspects or the tissue sealant prepared by any of the preparation methods described in any of the second aspects, and optionally includes one or more of medically or pharmaceutically acceptable carriers, other drugs or contrast agents.

[0111] In some alternative embodiments, the other drugs include one or more of growth factors, antifibrotic drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and analgesics. For example, the other drugs may include one or more of granulocyte colony-stimulating factor (G-CSF), recombinant human interleukin-11 (IL-11), recombinant human platelet-stimulating factor (rhTPO), and recombinant human erythropoietin (EPO). Antifibrotic drugs include interferon, pirfenidone, etc. NSAIDs may include one or more of indomethacin, diclofenac, ibuprofen, naproxen, loxoprofen, nimesulide, celecoxib, and etoricoxib. Glucocorticoids include one or more of methylprednisolone and prednisone, dexamethasone, and betamethasone. Analgesics include one or more of codeine, tramadol, morphine, oxycodone, hydromorphone, methadone, and fentanyl.

[0112] In some alternative embodiments, the developer is a biocompatible material and is located on the substrate on a side opposite to the adhesive layer. For example, the developer comprises one or more of barium sulfate and iodine preparations.

[0113] In some alternative embodiments, the medically or pharmaceutically acceptable carrier comprises an anti-blocking agent located on a side of the substrate opposite to the adhesive layer. For example, the anti-blocking agent comprises one or more of diatomaceous earth, talc, calcium carbonate, polylactic acid (PLA), gelatin, and sodium hyaluronate.

[0114] The term "optional" means that it may or may not be included.

[0115] Fifthly, this application also provides a method for stopping bleeding, comprising applying the hemostatic material, including the tissue sealant described in any of the first aspects, the tissue sealant prepared by any of the preparation methods described in any of the second aspects, or the hemostatic material described in any of the fourth aspects, to the bleeding site.

[0116] In some optional embodiments, when used, the tissue sealant or hemostatic material achieves hemostasis within 30 seconds for tissue bleeding with a wound length of 1 cm or less, further within 20 seconds or 10 seconds, and even further within 5 seconds; for tissue bleeding with a wound length of 10 cm or less, hemostasis is achieved within 100 seconds, further within 90 seconds, and even further within 60 seconds. For tissue bleeding from large wounds, hemostasis can be further accelerated by increasing the size of the tissue sealant or hemostatic material, for example, achieving hemostasis within 10 seconds.

[0117] The requirements for hemostatic materials, such as adhesion, flexibility, and biodegradability, vary depending on the bleeding site, including the gastrointestinal tract, blood vessels, lymphatic vessels, or internal organs. For example, venous bleeding is relatively minor, requiring hemostatic materials that can degrade quickly to avoid prolonged placement. The anatomical and physiological characteristics of liver tissue dictate that liver surgery is prone to bleeding, necessitating rapid and immediate hemostatic materials. Arterial bleeding is substantial, requiring extremely strong adhesion to quickly close the wound and extremely high strength to withstand arterial blood pressure, thus placing higher demands on tissue sealants. The tissue sealant or hemostatic material of this application has a wide range of applications, meeting the requirements for rapid hemostasis, strong adhesion, and high mechanical strength in cases of large-scale bleeding such as arterial bleeding, as well as the need for rapid and immediate application in cases of large tissue bleeding. It is also an absorbable material with excellent biocompatibility and can promote wound healing.

[0118] In some alternative embodiments, the application method includes placing a tissue sealant or tissue sealing material on the bleeding site and pressing for 1 to 50 seconds.

[0119] In this application, "biodegradable", "biodegradable", "bioabsorbable", and "absorbable" have the same meaning and have the known and common meaning in the art. That is, the hemostatic material of this application can fully function in the body for a period of time, and after a period of time it begins to degrade and lose its original function. Its degradation products are absorbed or excreted from the body after metabolism, and there are no residues in the body.

[0120] The tissue sealant of this application is biodegradable and absorbable. This means that the tissue sealant can fully function in the body for a period of time, after which it begins to degrade and loses its original function. Its degradation products are absorbed or excreted through metabolism, leaving no residue in the body. In a series of optional specific material embodiments, the tissue sealant can generally be absorbed by the body in less than 6 months, less than 3 months, less than 1 month, or less than 2 weeks.

[0121] The tissue sealant or hemostatic material of this application exists in the form of a patch, or the patch can be cut into pieces and applied to irregular wound surfaces.

[0122] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0123] Example 1

[0124] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, including the following steps:

[0125] (1) Take four-arm polyethylene glycol succinimide glycosyl ester (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 15000 Da, 4-arm-PEG-NHS) and four-arm polyethylene glycol primary amino (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 20000 Da, 4-arm-PEG-NH2), and then dry the two components in a vacuum drying oven (-0.1 MPa, room temperature) for 6 hours. The water content of both components is less than 0.25 wt%.

[0126] (2) After thoroughly grinding and classifying both, they were mixed evenly at a mass ratio of 4-arm-PEG-NHS to 4-arm-PEG-NH2 of 4:3. Then, they were applied to the gelatin using an electrostatic spray gun (at room temperature, the powder was directly sprayed onto the substrate). Absorbent gelatin sponge (0.5 cm thick, water content less than 0.2 wt%) was coated on a bio-based substrate with a 4-arm PEG-NHS loading of 4 mg / cm³. 2 The loading of 4-arm PEG-NH2 on the substrate was 3 mg / cm³. 2 A tissue sealant was prepared, comprising a substrate and an adhesive layer on the substrate, wherein both the four-arm polyethylene glycol succinimide oxoester and the four-arm polyethylene glycol primary amino group exist in particulate form in the adhesive layer, and the molar ratio of succinimide oxoester to amino group in the adhesive layer is 1.78:1.

[0127] (3) The above tissue sealant was placed in a pharmaceutical grade vial for freeze-drying (the water content of the tissue sealant after freeze-drying was less than 0.2 wt%). After freeze-drying, it was sealed in a light-proof vacuum aluminum foil bag and sterilized by β-irradiation with an intensity of 25 kGy to obtain a hemostatic patch.

[0128] Example 2

[0129] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Embodiment 1, except that the mixing mass ratio in step (2) is different and the loading of the two components is different. In this embodiment, the mass ratio of 4-arm-PEG-NHS to 4-arm-PEG-NH2 is 3:4 and they are mixed evenly. The loading of 4-arm-PEG-NHS on the substrate is 3 mg / cm². 2The loading of 4-arm PEG-NH2 coated on the substrate was 4 mg / cm³. 2 This results in a 1:1 molar ratio of succinimide glycoester to amino group in the adhesive layer.

[0130] Example 3

[0131] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Embodiment 1, except that the mixing mass ratio in step (2) is different and the loading of the two components is different. In this embodiment, the mass ratio of 4-arm-PEG-NHS to 4-arm-PEG-NH2 is 2:5, and the loading of 4-arm-PEG-NHS on the substrate is 2 mg / cm². 2 The loading of 4-arm PEG-NH2 on the substrate was 5 mg / cm³. 2 This results in a molar ratio of succinimide oxalate to amino group in the adhesive layer of 0.53:1.

[0132] Example 4

[0133] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, including the following steps:

[0134] (1) Take four-arm polyethylene glycol succinimide glycoside (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 15000 Da, 4-arm-PEG-NHS) and four-arm polyethylene glycol primary amino (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 20000 Da, 4-arm-PEG-NH2), and coat the substrate with 4 mg / cm³ of 4-arm-PEG-NHS. 2 The loading of 4-arm PEG-NH2 on the substrate was 3 mg / cm³. 2 The two components were accurately weighed using an analytical balance, and then dried separately in a vacuum drying oven (-0.1 MPa, room temperature) for 6 hours. The moisture content of both components was less than 0.25 wt%.

[0135] (2) After thoroughly grinding and classifying both, mix them evenly, and then use a powder spreader to apply them to the gelatin ( Absorbent gelatin sponge (0.5 cm thick, water content less than 0.2 wt%) was placed on a bio-based substrate (substrate). The oven was preheated to 70°C, and the gelatin sponge coated with the adhesive layer mixed powder was placed in the oven and heated to melt for 20 minutes. Subsequently, it was placed in a dry and clean environment to cool and cure (room temperature for 20 minutes) to obtain a tissue sealant. The tissue sealant includes a substrate and an adhesive layer located on the substrate. In the adhesive layer, both the four-arm polyethylene glycol succinimidyl oxalate and the four-arm polyethylene glycol primary amino group exist in homogeneous solid form, and the molar ratio of succinimidyl oxalate to amino group in the adhesive layer is 1.78:1.

[0136] (3) The above tissue sealant was placed in a pharmaceutical grade vial for freeze-drying (the water content of the tissue sealant after freeze-drying was less than 0.2 wt%). After freeze-drying, it was sealed in a light-proof vacuum aluminum foil bag and sterilized by β-irradiation with an intensity of 25 kGy to obtain a hemostatic patch.

[0137] Example 5

[0138] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, including the following steps:

[0139] (1) Take four-arm polyethylene glycol succinimide glycoside (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 15000 Da, 4-arm-PEG-NHS) and four-arm polyethylene glycol primary amino (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 20000 Da, 4-arm-PEG-NH2), and coat the substrate with 4 mg / cm³ of 4-arm-PEG-NHS. 2 The loading of 4-arm PEG-NH2 on the substrate was 3 mg / cm³. 2 The two components were accurately weighed using an analytical balance, and then dried separately in a vacuum drying oven (-0.1 MPa, room temperature) for 6 hours. The moisture content of both components was less than 0.25 wt%.

[0140] (2) Preheat the ultrasonic pyrolysis sprayer to 55°C, and use the ultrasonic pyrolysis sprayer to spray the 4-arm PEG-NHS onto the gelatin ( Absorbent gelatin sponge (0.5 cm, water content less than 0.2 wt%) was applied to a bio-based substrate. Then, using a fully automated high-voltage electrostatic powder sprayer, 4-arm PEG-NH2 particles with a particle size less than 80 mesh (≤178 μm) were uniformly dispersed into a molten 4-arm PEG-NHS continuous phase and naturally settled at 55°C for 20 minutes. Subsequently, it was placed in a dry, clean environment to cool and solidify (room temperature for 20 minutes) to obtain a tissue sealant. This tissue sealant includes a substrate and an adhesive layer located on the substrate. In the adhesive layer, the four-arm polyethylene glycol primary amino groups are distributed in particulate form within the four-arm polyethylene glycol succinimidyl oxalate, exhibiting an island structure. The molar ratio of succinimidyl oxalate to amino groups in the adhesive layer is 1.78:1.

[0141] (3) The above tissue sealant was placed in a pharmaceutical grade vial for freeze-drying (the water content of the tissue sealant after freeze-drying was less than 0.2 wt%). After freeze-drying, it was sealed in a light-proof vacuum aluminum foil bag and sterilized by β-irradiation with an intensity of 25 kGy to obtain a hemostatic patch.

[0142] Example 6

[0143] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that the loading amounts of the two components are different. In this embodiment, the loading amount of 4-arm-PEG-NHS coated on the substrate is 3 mg / cm². 2 The loading of 4-arm PEG-NH2 coated on the substrate was 4 mg / cm³. 2 In the adhesive layer of the formed tissue sealant, the four-arm polyethylene glycol primary amino group is distributed in the form of particles in the four-arm polyethylene glycol succinimide glycosyl ester, exhibiting an island structure. The molar ratio of succinimide glycosyl ester to amino group in the adhesive layer is 1:1.

[0144] Example 7

[0145] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that the loading amounts of the two components are different. In this embodiment, the loading amount of 4-arm-PEG-NHS coated on the substrate is 2 mg / cm². 2 The loading of 4-arm PEG-NH2 on the substrate was 5 mg / cm³. 2 In the adhesive layer of the formed tissue sealant, the four-arm polyethylene glycol primary amino group is distributed in the form of particles in the four-arm polyethylene glycol succinimide oxalate, exhibiting an island structure. The molar ratio of succinimide oxalate to amino group in the adhesive layer is 0.53:1.

[0146] Example 8

[0147] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that a four-armed polyethylene glycol succinimide glutarate (number average molecular weight 15000 Da) is used instead of a four-armed polyethylene glycol succinimide ethylene glycol glutarate.

[0148] Example 9

[0149] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that the loading amounts of the two components are different. In this embodiment, the loading amount of 4-arm-PEG-NHS coated on the substrate is 5 mg / cm². 2 The loading of 4-arm PEG-NH2 coated on the substrate was 2 mg / cm³. 2 This results in a molar ratio of succinimide glycoester to amino group in the adhesive layer of 3.3:1.

[0150] Example 10

[0151] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that the loading amounts of the two components are different. In this embodiment, the loading amount of 4-arm-PEG-NHS coated on the substrate is 1.5 mg / cm². 2 The loading of 4-arm PEG-NH2 coated on the substrate was 5.5 mg / cm³. 2 This results in a molar ratio of succinimide oxalate to amino group in the adhesive layer of 0.36:1.

[0152] Example 11

[0153] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that the number of arms and number-average molecular weight of the polyethylene glycol primary amino group are different. This embodiment uses a six-arm polyethylene glycol primary amino group (4-arm-PEG-NH2) with a number-average molecular weight of 40,000 Da. In this embodiment, the loading amount of the four-arm-PEG-NH2 coated on the substrate is 3.5 mg / cm³. 2 The loading of the six-arm PEG-NH2 coating on the substrate was 3.5 mg / cm³. 2 This results in a molar ratio of succinimide oxalate to amino group in the adhesive layer of 1.78:1.

[0154] Example 12

[0155] This embodiment provides a method for preparing a tissue sealant and a hemostatic patch, which is basically the same as that in Example 5, except that the 4-arm-PEG-NH2 particles with a particle size of less than 40 mesh (less than or equal to 425 μm) obtained after grinding and grading by a 40-mesh sieve are uniformly dispersed in a molten 4-arm-PEG-NHS continuous phase.

[0156] Comparative Example 1

[0157] Direct procurement Absorbable gelatin sponge (0.5 cm) was used as a tissue sealant and sterilized by β-irradiation (25 kGy intensity) to prepare a hemostatic patch.

[0158] Comparative Example 2

[0159] This comparative example provides a method for preparing a tissue sealant and a hemostatic patch, comprising the following steps:

[0160] (1) The four-arm polyethylene glycol succinimide ethylene glycol ester (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 15000 Da) was accurately weighed using an analytical balance, ground, and dried in a vacuum drying oven (-0.1 MPa, room temperature) for 6 hours. Then, it was coated onto gelatin using an electrostatic spray gun. The loading of 4-arm PEG-NHS coating on an absorbent gelatin sponge (0.5 cm) bio-based substrate was 7 mg / cm². 2 A tissue sealant was prepared.

[0161] (2) The above tissue sealant was placed in a pharmaceutical grade vial for freeze-drying. After freeze-drying, it was sealed in a light-proof vacuum aluminum foil bag and sterilized by β-irradiation with an intensity of 25 kGy to obtain a hemostatic patch.

[0162] Comparative Example 3

[0163] This comparative example provides a method for preparing a tissue sealant and a hemostatic patch, comprising the following steps:

[0164] (1) The four-armed polyethylene glycol primary amino group (Shanghai Pengshuo Biotechnology Co., Ltd., number average molecular weight 20000 Da, 4-arm -PEG-NH2) was accurately weighed using an analytical balance, ground, and dried in a vacuum drying oven (-0.1 MPa, room temperature) for 6 hours. Then, it was coated onto gelatin using an electrostatic spray gun. The loading of 4-arm PEG-NHS coating on an absorbent gelatin sponge (0.5 cm) bio-based substrate was 7 mg / cm². 2 A tissue sealant was prepared.

[0165] (2) The above tissue sealant was placed in a pharmaceutical grade vial for freeze-drying. After freeze-drying, it was sealed in a light-proof vacuum aluminum foil bag and sterilized by β-irradiation with an intensity of 25 kGy to obtain a hemostatic patch.

[0166] To verify the performance of the tissue sealants prepared in each embodiment and the tissue sealants of Comparative Examples 1-3, the sterilized hemostatic patches were subjected to adhesive strength tests, in vitro cytotoxicity tests, animal hemostasis experiments, animal back implantation experiments, and long-term in vivo implantation toxicity experiments.

[0167] Experimental Example 1

[0168] Overlap shear bond performance test: Referencing the American Society for Testing and Materials standard (ASTM F2255-05), this test is used to compare the bond strength of tissue sealants on soft tissue as surgical adhesives or sealants, or both; it can also be used for quality control in the production of tissue sealant medical devices, provided the substrate is appropriately selected.

[0169] Hemostatic patches (sample size 2.5cm × 1cm × 0.5cm) prepared by the methods of Examples 1-12 and Comparative Examples 1-3 were bonded to the surface of fresh pigskin (pigskin size 2.5cm × 8cm × 0.5cm) using cyanoacrylate (cyanoacrylate was evenly applied without overflow). Hemostatic patches prepared by the methods of Examples 1-12 and Comparative Examples 1-3 were bonded to fresh pigskin using the same operation. The two samples were placed with one end aligned, and 0.2mL of fresh pig plasma was evenly dropped between the two samples, with an overlap contact length of 1cm. The adhesive properties of the samples were tested using a universal testing machine at a strain rate of 5mm / min. The test endpoint was recorded, and the adhesive strength (kPa) was obtained. The results are shown in Figure 1. The test conditions were 24.2℃ and 41% RH. As shown in Figure 1, the overlap bonding strength of the tissue sealants in Examples 1 and 4 of this application is basically consistent, proving that the mixing of the two components, 4-arm-PEG-NHS and 4-arm-PEG-NH2, has little impact on the bonding strength, whether the powder is directly coated or the powder is coated and then heated to melt. The bonding strength of Example 3 is lower than that of Examples 1 and 2, and the bonding strength of Examples 2 and 3 is lower than that of Example 1, proving that controlling the molar ratio of succinimide glycoside to amino group within the preferred range is beneficial to cross-linking with porcine plasma and enhancing adhesion. The bonding strength of Example 5 is greater than that of Examples 1 and 4. Surprisingly, the bonding strength of Examples 6 and 7 not only did not decrease, but also increased to a certain extent. This may be attributed to the uniform distribution of 4-arm-PEG-NH2 microparticles in the 4-arm-PEG-NHS in the island structure, which improves the mechanical properties of the tissue sealant.

[0170] In Example 8, the gelation speed of four-arm polyethylene glycol succinimide glutarate was around 30s (gelation time tested by the inverted bottle method), which was inferior to that of four-arm polyethylene glycol succinimide ethylene glycol (gelation within 10s). This was because the elongation of the active ester groups and the PEG backbone reduced the nucleophilic and electrophilic reactivity, resulting in a decrease in adhesive strength. In Examples 9 and 10, the excessively high or low molar ratio of polyethylene glycol succinimide ethylene glycol to amino polyethylene glycol led to low crosslinking between four-arm PEG-NHS and four-arm PEG-NH2, resulting in a significant decrease in adhesive strength, but it was still higher than that of Comparative Examples 1-3. In Example 12, the occasional presence of large four-arm PEG-NH2 particles caused uneven distribution of the adhesive coating, localized stress concentration, and premature breakage, resulting in a significant decrease in adhesive strength, but it was still higher than that of Comparative Examples 1-3.

[0171] Experimental Example 2: In vitro cytotoxicity test

[0172] To evaluate the cytotoxicity of the tissue sealant in this application, this experiment was conducted in accordance with GB / T16886.5—2017 Medical Device Biological Evaluation—Part 5: In Vitro Cytotoxicity Assays. Sample extracts were prepared according to ISO10993-12, and L929 cells (mouse fibroblasts) were used for testing. Relative cell viability was assessed using the CCK-8 assay kit (purchased from Beyotime), and cell viability and morphology were further evaluated using the Calcein AM / PI (propidium iodide) dual-fluorescence assay kit for cell viability and cytotoxicity (purchased from Beyotime).

[0173] Cell relative viability assessment: L929 cells were seeded into 96-well plates at 4000 cells per well and cultured for 24 hours (~1 cycle) until a semi-confluent monolayer was formed. The original culture medium was then removed and replaced with a series of extracts.

[0174] The series of extract groups were set up as follows: ① Positive control group (0.67% phenol group): 0.67% phenol obtained by dissolving phenol in complete culture medium was used to replace the original culture medium; ② 100% negative control group (3cm 2 / mL (Comparative Example 1): Comparative Example 1 Absorbent gelatin sponge, at 3cm 2 / mL blank coating area was extracted in complete culture medium and incubated at 37℃ on a shaker for 24h. The resulting 100% negative control group extract was then replaced with the original culture medium; ③ 50% negative control group (1.5cm) 2 / mL Comparative Example 1): The above 100% negative control group extract was diluted twice with complete culture medium to obtain a 50% negative control group extract. The resulting 50% negative control group extract was then used to replace the original culture medium; ④ Control group: Blank group cells using only complete culture medium, without any treatment; ⑤ 100% experimental group (3cm 2 / mL Example 4): The hemostatic patch obtained in Example 4 was applied at a 3cm... 2 / mL coating area was extracted in complete culture medium and incubated at 37℃ on a shaker for 24h. The resulting 100% experimental group extract was then replaced with the original culture medium; ⑥ 50% experimental group (1.5cm 2 / mL Example 4): The 100% experimental group extract was diluted twice with complete culture medium to a 50% extract, replacing the original culture medium. After culturing with extracts of various concentrations for 24 hours, the absorbance value of each group was measured using a CCK-8 assay kit and compared with the absorbance value of the blank group cells to obtain the relative cell viability, as shown in Figure 2. The results in Figure 2 show that the hemostatic patch of Example 4, 3cm 2 / mL, 1.5cm 2The cell activity of the extract group with a coating area of ​​ / mL was higher than that of the blank group, indicating that the hemostatic patch of this application not only has no cytotoxicity but also promotes cell proliferation. The negative control group of Comparative Example 1 also showed the effect of promoting cell survival and proliferation.

[0175] Experiment 3: Cell morphology assessment

[0176] L929 cells were seeded into 48-well plates at 6000 cells per well and cultured at 37°C for 24 hours (approximately 1 cycle) until a semi-confluent monolayer formed. The original culture medium was then removed and replaced with a series of extraction solutions. The series of extraction solutions were set up as follows: ① Positive control group (0.67% phenol group): 0.67% phenol was obtained by dissolving phenol in complete culture medium, replacing the original culture medium; ② 100% gelatin sponge group (3cm... 2 / mL (Comparative Example 1): Comparative Example 1 Absorbent gelatin sponge, at 3cm 2 / mL blank coating area was extracted in complete culture medium and incubated at 37℃ on a shaker for 24h. The resulting 100% negative control group extract was then used to replace the original culture medium; ③ 50% gelatin sponge group (1.5cm 2 / mL Comparative Example 1): The above 100% negative control group extract was diluted twice with complete culture medium to obtain a 50% negative control group extract. The resulting 50% negative control group extract was used to replace the original culture medium. ④ Blank control group: The original culture medium was replaced with complete culture medium. That is, the blank control group only used the blank group cells with complete culture medium and did not undergo any treatment. ⑤ 100% coated sponge group (3cm 2 / mL Example 4): The hemostatic patch obtained in Example 4 was applied at a 3cm... 2 / mL coating area was extracted in complete culture medium and incubated at 37℃ on a shaker for 24h. The resulting 100% experimental group extract was then replaced with the original culture medium; ⑥ 50% coated sponge group (1.5cm) 2 / mL Example 4): The 100% extract of the above experimental group was diluted twice with complete culture medium to a 50% extract, and the original culture medium was replaced. After culturing with extracts of various concentrations for 24 h, cell viability and morphology were further evaluated using the Calcein AM / PI Cell Viability and Cytotoxicity Kit, as shown in Figure 3. The principle of this kit is that two probes can detect intracellular esterase activity and cell membrane integrity respectively, thereby reflecting cell viability and cytotoxicity; Calcein AM stains live cells and shows green fluorescence; while propidium iodide (PI) stains dead cells and shows red fluorescence. As shown in Figure 3, the hemostatic patch of Example 4, 3cm 2 / mL (Figure 3-E), 1.5cm 2The cells in the extract group with a coating area of ​​ / mL (Figure 3-F) showed good cell viability, and no dead cells exhibiting red fluorescence were observed in the field of view. Compared with the blank group, the cell number increased, indicating that the hemostatic patch of this application has excellent cell compatibility. The negative control group of Comparative Example 1 (Figure 3-C, Figure 3-D) also showed good cell compatibility.

[0177] Experiment Example 4: Animal Hemostasis Experiment

[0178] Hemostasis experiment of liver incision in New Zealand white rabbits: Male New Zealand white rabbits (2.5-3kg) were selected. After anesthesia, the rabbits were fixed on the operating table, the abdomen was exposed, and an incision was made in the middle of the abdomen to expose the liver. A 10mm long and 5mm deep incision was made on the surface of the liver, and free bleeding was allowed for 10 seconds to observe whether the bleeding was normal. The bleeding was then wiped clean with sterile gauze. The rabbits were randomly divided into groups of four. The hemostatic patches (cubic prisms of size 1.2cm×1cm×0.5cm) prepared by the methods of Examples 5-10 and Comparative Examples 1-3 were applied to the liver incision site (n=4). After pressing for 5 seconds, the hemostasis was recorded, including the hemostasis time (the time for bleeding to stop was observed, and the 5-second pressing time was included) and the amount of blood loss (after wiping the bleeding clean, the hemostatic patch was applied to the wound while a weighed filter paper was placed under the liver incision. The bleeding during the hemostasis process flowed onto the filter paper. The mass of the filter paper after absorbing the blood was weighed, and the amount of blood loss was calculated as: blood loss = weight after blood absorption - weight before blood absorption), as shown in Table 1; the bleeding after pressing is shown in Figure 4.

[0179] As shown in Table 1 and Figure 4, the hemostatic patches of Examples 5-7 prepared in this application have excellent hemostatic effects. After pressing for 5 seconds, the wound stopped bleeding, and the blood loss was minimal and essentially the same. In contrast, the hemostatic effects of Comparative Examples 1 and 3 were very poor, and Comparative Example 2 only had 7 mg / cm³ applied. 2 PEG-NHS While absorbent gelatin sponges offer some hemostatic effect, they are far less effective than the hemostatic patches described in Examples 5-7 of this application. Compared to Example 5, Example 8 exhibits a slightly weaker hemostatic effect due to the slower hydrogel formation rate of the adhesive layer. Examples 9 and 10 suffer from limited hemostatic and sealing effects due to excessively high or low molar ratios of polyethylene glycol succinimide glycosides and amino polyethylene glycol, resulting in insufficient cross-linking of the adhesive hydrogel layer.

[0180] Table 1 Hemostasis Results

[0181] Experiment Example 5: Animal Hemostasis Experiment

[0182] Hemostasis experiment during liver resection in New Zealand white rabbits: Male New Zealand white rabbits (2.5–3 kg) were selected. After anesthesia, the rabbits were fixed on the operating table, and the abdomen was exposed. A midline incision was made in the abdomen to expose the liver. The liver lobe was held with forceps, and a 5–6 cm long edge of the liver lobe was removed by scissors (transverse incision). Free bleeding was allowed for 10 seconds to observe whether the bleeding was normal. The bleeding was then wiped clean with sterile gauze. The rabbits were randomly divided into groups of 4, and hemostasis was carried out separately in each group.

[0183] Examples 1, 3, 5, and 7, and Comparative Example 2, used hemostatic patches (size: 6cm×2cm×0.5cm cuboids) prepared by the method. These patches were applied to the liver incision sites (n=4). After pressing for 20 seconds, the hemostasis status was recorded, including the hemostasis time (the 20-second pressing time was already included) and the amount of blood loss (after wiping away the bleeding, the hemostatic patch was applied to the wound while a weighed filter paper was placed under the liver incision. The bleeding during the hemostasis process flowed onto the filter paper. The mass of the filter paper after absorbing the blood was weighed, and the amount of blood loss was calculated as: amount of blood loss = weight after blood absorption - weight before blood absorption). See Table 2 and Figure 5. As shown in Table 2, the hemostatic patches of Examples 5 and 7 prepared in this application have excellent hemostatic effects; within 60 seconds, bleeding from the liver resection wound has essentially stopped. In contrast, the hemostatic effects of Examples 1 and 3 are slightly weaker; after applying pressure for 20 seconds, varying degrees of oozing can be observed around 60 seconds (some oozing from the gelatin matrix layer, and some from the edges). Comparative Example 2 only applied 7 mg / cm³ of the patch. 2 PEG-NHS While absorbent gelatin sponges have some hemostatic effect, they are far less effective than the hemostatic effects of embodiments 1, 3, 5, and 7 of this application.

[0184] Table 2 Hemostasis Results

[0185] Experiment 6: Evaluation of local response to implantation in vivo

[0186] This test was conducted in accordance with GB / T16886.6—2022 Biological Evaluation of Medical Devices—Part 6: Local Response Tests After Implantation. It is applicable to degradable and / or absorbable solid or non-solid materials, and also suitable for evaluating local tissue responses produced by medical devices intended for use on the surface or inner surface of injuries. Male SD rats weighing 300–350 g were used, with six rats in each parallel group at the same time point. Two subcutaneous sacs were prepared by making a skin incision on each side of the midline of the rat's back using blunt dissection. The bottom of each sac should be at least 10 mm from the skin incision. One implant material (a cube measuring 1 cm × 1 cm × 0.5 cm) was placed in each sac, ensuring that the implants did not contact each other. The wound was then sutured, iodine was applied, and antibiotics were injected. The hemostatic patches from Examples 5-7 and Comparative Example 1 of this application were used as implant materials in each group (n=6, 6 rats per sampling point per group). For absorbable materials, the testing period should be related to the estimated degradation time of the test product at the clinically relevant implantation site. The implantation testing periods for this study were set at 1 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks. At the end of each implantation period, rats were anesthetized, and blood samples were collected for complete blood count and biochemical analysis. Additionally, the implanted material and surrounding skin and tissue were removed from the rat's back for HE staining to assess the level of local inflammation at the implantation site.

[0187] In Comparative Example 1, the material was completely degraded two weeks after subcutaneous implantation. In Examples 5-7 of this application, degradation began at four weeks and was essentially complete by six weeks. Because the local biological response of the implanted material is related to material properties and surgical trauma response, changes in the tissue structure surrounding the implant after surgery vary over time. For absorbable materials, it is generally advisable to use samples taken one week after implantation to assess early tissue response. On the other hand, based on our experience, a severe immune storm occurs in animals one week after implantation; therefore, we used HE staining results at one week (see Figure 6) to assess the inflammatory response.

[0188] HE staining results showed that Examples 5-7 and Comparative Example 1 of this application only caused mild inflammatory reactions one week after implantation, which may also be due to surgical procedures. Overall, the HE staining results were no different from those of Comparative Example 1. Although fibrous cysts were formed, the number of polymorphonuclear leukocytes, giant cells, and plasma cells did not increase significantly. The number and content of white blood cells, lymphocytes, and neutrophils in the blood routine test were no different from those in normal mice. The above results demonstrate that the hemostatic patch of this application has excellent biocompatibility, and no abnormalities were observed in the local tissue after subcutaneous implantation.

[0189] Experiment 7 assesses systemic toxicity of implanted drugs.

[0190] This test was conducted in accordance with GB / T16886.11—2021 Biological Evaluation of Medical Devices—Part 11: Systemic Toxicity Testing. Systemic toxicity refers to a range of potential adverse effects that may occur during the use of medical devices. Leachable materials from devices or equipment can reach parts of the human body that do not come into direct contact with them through absorption, distribution, and metabolism, thereby producing general toxic effects as well as organ and organ system effects. Male SD rats weighing 300–350 g were selected, with six rats in each parallel group at the same time point. The anesthetized rats were fixed on the operating table, and the abdomen was exposed. A midline incision was made in the abdomen to expose the liver. A 10 mm long and 5 mm deep incision was made on the surface of the liver, and free bleeding was allowed for 10 seconds to observe whether the bleeding was normal. The bleeding was then wiped clean with sterile gauze. Hemostatic patches prepared by the methods of Examples 5 and 7 of this application and Comparative Example 2 were selected for each group and applied to the liver incision. The size of each patch was 1cm×1cm×0.5cm. A sterile gauze hemostasis group (n=6, 6 rats per sampling point in each group) was also set up, and hemostasis was achieved by applying sterile gauze to the incision.

[0191] In Examples 5 and 7, hemostasis was achieved after 5 seconds of pressure with the hemostatic patch; in Comparative Example 2, hemostasis was achieved after approximately 2 minutes of pressure with the hemostatic patch; and in the sterile gauze group, hemostasis was achieved after approximately 3-4 minutes of pressure with the patch. Subsequently, the livers with the hemostatic patches (Examples 5 and 7, Comparative Example 2) were returned to their original position in the abdominal cavity. In the sterile gauze group, only the liver lobe was returned after hemostasis. The abdominal wall and skin were sutured; iodine was applied to the suture site, and antibiotics were injected. Rats were fed until the scheduled data collection date. For three days post-surgery, iodine was applied to the suture site daily, and antibiotics were injected intraperitoneally. This model simulates the long-term systemic toxicity of applying absorbable hemostatic patches to in vivo wounds. Data were collected at fixed times to assess toxicity and inflammatory responses.

[0192] For absorbable materials, the testing period should be related to the estimated degradation time of the test product at the clinically relevant implantation site. The implantation testing periods for this study were set at 1 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks. At the end of the implantation period, rats were anesthetized, and blood was collected for complete blood count and biochemical analysis. The rat liver and hemostatic patch were also removed and stained with hematoxylin and eosin (HE) to assess the level of local inflammation after implantation in the liver wound. Considering the wound recovery, HE staining results were selected 2 weeks after liver incision implantation, as shown in Figure 7. According to the HE staining results, no increase in lymphocytes, neutrophils, macrophages, etc., was observed in Examples 5 and 7, Comparative Example 2, and the blank gauze group; however, neovascularization and fibrosis were observed in Comparative Example 2 and the blank gauze group. Examples 5 and 7 of this application exhibited excellent adhesion, remaining firmly attached to the liver surface 4 weeks after implantation, gradually degrading only at 6 weeks, and completely degrading in approximately 8 weeks. In addition, it was observed that the liver wound scars treated with the hemostatic patches of Examples 5 and 7 of this application healed well within 1-2 weeks, with good recovery of the liver wound surface and virtually no visible wound or fibrosis; while in Comparative Example 2, the wound healed basically within about 3 weeks; the wound in the sterile gauze hemostatic group healed more slowly, with obvious fibrosis on the wound surface, and required 3-4 weeks to recover.

[0193] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. A tissue sealant, characterized in that, The tissue sealant comprises a biocompatible substrate and an adhesive layer located on the substrate, the adhesive layer being made of dried polyethylene glycol succinimide ester and dried amino polyethylene glycol.

2. The tissue sealant according to claim 1, characterized in that, The water content of the dried polyethylene glycol succinimide ester and the dried amino polyethylene glycol is less than 0.5 wt%; preferably less than 0.25 wt%.

3. The tissue sealant according to claim 1, characterized in that, The polyethylene glycol succinimide ester has n succinimide ester groups, n≥2; the amino polyethylene glycol has m amino groups, m≥2; and m+n≥5, where m and n are both integers.

4. The tissue sealant according to any one of claims 1-3, characterized in that, In the adhesive layer, both polyethylene glycol succinimide and amino polyethylene glycol exist in particulate form or in a homogeneous system; or, amino polyethylene glycol is distributed in particulate form in polyethylene glycol succinimide, exhibiting an island structure.

5. The tissue sealant according to claim 4, characterized in that, The island phase in the island structure has a particle size of less than 40 mesh, preferably less than 60 mesh, and more preferably less than 80 mesh.

6. The tissue sealant according to claim 4, characterized in that, When both polyethylene glycol succinimide and amino polyethylene glycol exist in particulate form or in a homogeneous system, the molar ratio of succinimide groups to amino groups in the adhesive layer is 4:1 to 1:2; preferably 2:1 to 1:

1.

7. The tissue sealant according to claim 4 or 5, characterized in that, When an island structure is formed, the molar ratio of succinimide ester groups to amino groups in the adhesive layer is 4:1 to 1:4; preferably 2:1 to 1:

2.

8. The tissue sealant according to any one of claims 1-7, characterized in that, The number average molecular weights of the polyethylene glycol succinimide ester and the amino polyethylene glycol are independently 2,000 Da to 200,000 Da, preferably 10,000 Da to 40,000 Da.

9. The tissue sealant according to any one of claims 1-8, characterized in that, The succinimide ester in the polyethylene glycol succinimide ester is selected from one or more combinations of succinimide carbonate, succinimide acetate, succinimide glycosyl ester, succinimide malonate, succinimide succinimide glutarate, succinimide adipate, succinimide carbamate, sulfosuccinimide carbonate, sulfosuccinimide acetate, sulfosuccinimide acetate, and sulfosuccinimide glycosyl ester; and / or, the amino group in the amino polyethylene glycol is a primary amino group.

10. The tissue sealant according to any one of claims 1-9, characterized in that, In the adhesive layer, the loading amounts of polyethylene glycol succinimide ester and amino polyethylene glycol are independently 1 mg / cm³. 2 -20mg / cm 2 Preferably 2 mg / cm 2 -15mg / cm 2 .

11. The tissue sealant according to any one of claims 1-10, characterized in that, The polyethylene glycol succinimide ester and amino polyethylene glycol have multiple arms; preferably, the multiple arms of the polyethylene glycol succinimide ester and the multiple arms of the amino polyethylene glycol are independently 2-10, and the polyethylene glycol succinimide ester and amino polyethylene glycol are not simultaneously a two-arm structure.

12. The tissue sealant according to claim 11, characterized in that, The polyethylene glycol succinimide ester is a four-arm polyethylene glycol succinimide glycoester, and the amino polyethylene glycol is a four-arm polyethylene glycol amino; preferably, the amino polyethylene glycol is a four-arm polyethylene glycol primary amino.

13. The tissue sealant according to any one of claims 1-12, characterized in that, The substrate is a biocompatible protein or its derivative; Preferably, the protein or its derivative comprises collagen, gelatin or its derivative; Preferably, the thickness of the substrate is 0.2cm to 10cm, more preferably 0.2cm to 2cm.

14. A method for preparing the tissue sealant according to any one of claims 1-13, characterized in that, The preparation method includes one of the following methods 1 or 2: Method 1: The mixture formed by mixing polyethylene glycol succinimide ester and amino polyethylene glycol is coated onto the substrate surface to obtain the product; Method 2: Polyethylene glycol succinimide ester is coated onto the substrate surface to form a substrate containing polyethylene glycol succinimide ester melt, amino polyethylene glycol particles are dispersed into the polyethylene glycol succinimide ester melt, and the substrate is obtained after cooling and solidification.

15. The method for preparing the tissue sealant according to claim 14, characterized in that, In method 1 or method 2, the coating includes one or more of the following: powder coating, dip coating, spray coating, doctor blade coating, and roll coating; and / or, the coating is followed by a heating and melting step and a cooling and curing step.

16. The method for preparing the tissue sealant according to claim 14 or 15, characterized in that, The method 1 or method 2 also satisfies one or more of the following AC: A. After coating, the tissue sealant is dried and / or sterilized. Optionally, the sterilization includes one or a combination of ethylene oxide sterilization, dry heat sterilization, ultraviolet sterilization, irradiation sterilization, and filtration sterilization. Optionally, the drying includes freeze drying, vacuum heating drying, and atmospheric pressure heating drying. B. In method 1 or method 2, the entire preparation process is carried out under dry conditions; preferably, the water content of the obtained tissue sealant is less than 0.2 wt%. C. Before coating, the process further includes drying and / or grinding polyethylene glycol succinimide and amino polyethylene glycol; optionally, the drying includes one or a combination of freeze drying, vacuum heating drying, and atmospheric pressure heating drying; optionally, the grinding process is followed by a sieving process.

17. The method for preparing the tissue sealant according to any one of claims 14-16, characterized in that, Method 1 includes the following steps: (1) Polyethylene glycol succinimide ester and amino polyethylene glycol were subjected to vacuum heating and drying, grinding and sieving respectively; (2) The polyethylene glycol succinimide ester and amino polyethylene glycol obtained in step (1) are mixed and coated onto the substrate surface by powder spraying process. After drying and sterilization, the product is obtained. Optionally, the coating process may include a step of heating to melt and then cooling to cure.

18. The method for preparing the tissue sealant according to any one of claims 14-16, characterized in that, Method 2 includes the following steps: (1) Polyethylene glycol succinimide ester and amino polyethylene glycol were subjected to vacuum heating and drying treatment respectively; (2) Spray polyethylene glycol succinimide onto the substrate at 55-65℃ to form a substrate containing polyethylene glycol succinimide melt; disperse amino polyethylene glycol particles with a particle size of less than 80 mesh into the polyethylene glycol succinimide melt and allow them to settle naturally for 10-20 minutes. Cooling and solidification form an adhesive layer with an island structure on the substrate surface; (3) Dry and sterilize to obtain the product.

19. Use of a tissue sealant according to any one of claims 1-13 or a tissue sealant prepared by any one of claims 14-18 in the preparation of hemostatic materials.

20. A hemostatic material, characterized in that, The hemostatic material comprises the tissue sealant according to any one of claims 1-13 or the tissue sealant prepared by the preparation method according to any one of claims 14-18, and optionally further comprises one or more of a medically or pharmaceutically acceptable carrier, other drugs or contrast agents.

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