Mussel bionic hemostatic material as well as preparation method and application thereof

Abstract

The invention relates to the field of hemostatic materials, and discloses a mussel bionic hemostatic material and a preparation method and application thereof, and the preparation method comprises the following steps: dissolving chitosan in an acidic aqueous solution to obtain a chitosan solution; the method comprises the following steps: mixing 3, 5-dihydroxybenzoic acid with a catalyst which is EDC and NHS, and carrying out reaction activation at room temperature to obtain activated 3, 5-dihydroxybenzoic acid; slowly titrating the activated 3, 5-dihydroxybenzoic acid into the chitosan solution, and stirring and reacting at normal temperature to obtain a reaction product; loading the reaction product into a dialysis bag for dialysis, and then freeze-drying to obtain the mussel bionic hemostatic material. The problems that in the prior art, the bonding capacity is weak, active coagulation promoting and anti-inflammatory functions are lacked, tissue damage is easily caused, and the biological activity function is single are solved.

Description

Technical Field

[0001] This invention relates to the field of hemostatic materials, and more particularly to a mussel-inspired hemostatic material, its preparation method, and its application. Background Technology

[0002] Hemostasis is a crucial aspect of trauma management in clinical medicine, especially for complex soft tissue injuries in the oral and maxillofacial region. This area is richly vascularized, moist, and frequently moving, often resulting in irregular, deep, or perforated wounds. This places extremely high demands on the wet adhesion, speed of hemostasis, biocompatibility, and multifunctional synergy of hemostatic materials. Rapid and effective hemostasis not only reduces blood loss and the risk of shock but also creates a favorable microenvironment for wound healing, preventing complications such as infection and tissue necrosis.

[0003] Currently, commonly used hemostatic materials in clinical practice are mainly divided into three categories, but all of them have significant technical limitations and are difficult to meet the hemostatic needs of complex wounds: The first category is physical occlusion hemostatic materials, typical examples of which include gelatin sponges and cellulose dressings. These materials mainly block blood loss by absorbing blood, concentrating clotting factors, or constructing physical scaffolds, but their core defect is their weak wet adhesion, making them prone to detachment on moist tissue surfaces or active areas, and unable to form a stable sealing barrier; at the same time, their function is singular, relying solely on physical action, lacking active procoagulant and anti-inflammatory activity, resulting in limited hemostatic efficiency, and poor adaptability to deep or irregular wounds.

[0004] The second category consists of chemically procoagulant hemostatic materials, such as zeolite and unmodified chitosan. Their mechanism of action is to accelerate thrombus formation by activating platelets or providing a surface for coagulation reactions. However, most of these materials have insufficient adhesion to moist tissues and are difficult to adhere firmly to wounds. Some materials (such as zeolite) generate a large amount of heat during water absorption, which may lead to burns to surrounding tissues, so their safety needs to be improved. They also lack auxiliary functions such as anti-inflammatory and healing-promoting effects.

[0005] The third category consists of mussel-inspired adhesive materials, mostly prepared based on compounds containing catechol structures such as polydopamine and tannic acid. These materials utilize the wet-state adhesive properties of the catechol groups to achieve tissue adhesion. While this type of material solves the wet adhesion problem, it has significant drawbacks: some materials have a long adhesion formation time, failing to meet the requirements for emergency hemostasis in clinical practice; furthermore, their bioactivity is limited, focusing only on adhesive function and lacking key properties such as synergistic coagulation, scavenging reactive oxygen species (ROS), and reducing inflammatory responses, making it difficult to simultaneously address the dual needs of hemostasis and promoting healing.

[0006] In addition, the dosage forms of existing hemostatic materials are mostly fixed, and a single dosage form is difficult to adapt to different types of trauma scenarios such as irregular surface wounds, deep wounds, penetrating wounds, and specific cavities, which limits their clinical application.

[0007] Therefore, developing a comprehensive hemostatic material that combines strong wet adhesion, rapid physical sealing and synergistic chemical coagulation, antioxidant and anti-inflammatory activity, and can be flexibly prepared into various dosage forms according to clinical needs, has become an urgent technical problem to be solved in this field. It is of great significance for improving the hemostatic effect and healing quality of complex soft tissue injuries. Summary of the Invention

[0008] The present invention aims to provide a mussel-inspired hemostatic material, its preparation method and application, in order to solve the problems of weak adhesion, lack of active coagulation and anti-inflammatory functions, easy tissue damage and single bioactive function in the prior art.

[0009] To achieve the above objectives, the present invention provides the following method:

[0010] The present invention provides a method for preparing a mussel-inspired hemostatic material:

[0011] S1: Dissolve chitosan in an acidic aqueous solution to obtain a chitosan solution;

[0012] S2: Mix 3,5-dihydroxybenzoic acid with a catalyst, wherein the catalyst is EDC or NHS, and activate the mixture at room temperature to obtain activated 3,5-dihydroxybenzoic acid;

[0013] S3: The activated 3,5-dihydroxybenzoic acid is slowly titrated into the chitosan solution, and the reaction is stirred at room temperature to obtain the reaction product;

[0014] S4: The reaction product is placed in a dialysis bag for dialysis, and then freeze-dried to obtain a mussel-inspired hemostatic material.

[0015] Preferably, the acidic aqueous solution is a 1% glacial acetic acid solution, and the concentration of the chitosan solution is 20-100 mg / ml.

[0016] Preferably, the molar ratio of 3,5-dihydroxybenzoic acid, EDC and NHS is 1:1.5:2~3:4:5, and the activation time is 60-360 minutes.

[0017] Preferably, the activated 3,5-dihydroxybenzoic acid is slowly titrated into the chitosan solution, and the mixture is stirred at room temperature at a speed of 400-1000 rpm for 24-48 hours to obtain the reaction product.

[0018] Preferably, the dialysis bag has a molecular weight cutoff of 3500 Da and a dialysis time of 2 days.

[0019] Preferably, step S4 is followed by a product verification step: by detecting nuclear magnetic resonance spectroscopy and ultraviolet-visible spectroscopy, it is confirmed that the 3,5-dihydroxybenzoic acid has been successfully grafted onto the chitosan molecular chain, thus obtaining a mussel-inspired hemostatic material.

[0020] The present invention provides a mussel-inspired hemostatic material prepared according to the preparation method of a mussel-inspired hemostatic material described above.

[0021] The present invention provides the application of a mussel-inspired hemostatic material as described above in the fields of rapid hemostasis and promoting healing of complex soft tissue injuries of the oral and maxillofacial region, as well as irregular surface wounds, deep wounds, penetrating wounds, wound cavity filling, superficial bleeding, or hemostasis of specific cavities.

[0022] The beneficial effects of this invention are reflected in:

[0023] 1. Strong wet adhesion and physical sealing: 3,5-Dihydroxybenzoic acid (DHBA) is a key adhesive unit of mussel byssal proteins. Through esterification, it is covalently grafted onto the chitosan backbone, giving the material strong wet adhesion properties similar to those of mussels. When applied to wounds, the material adheres tightly to moist tissue surfaces, rapidly forming a physical barrier to seal the wound and prevent blood loss.

[0024] 2. Synergistic chemical coagulation effect:

[0025] Positive charge of chitosan: Positively charged chitosan can attract negatively charged red blood cells and platelets, promoting their aggregation.

[0026] The catechol groups of DHBA can form hydrogen bonds and π-π stacking interactions with proteins in the blood (such as fibrinogen), further enriching clotting factors and platelets.

[0027] The synergistic effect of the positive charge and the catechol group significantly accelerates thrombus formation, achieving a "dual hemostatic mechanism" of physical blockage and chemical coagulation.

[0028] Antioxidant and anti-inflammatory effects: The catechol structure of DHBA is an excellent scavenger of reactive oxygen species (ROS). During the inflammatory phase of a wound, the material can effectively remove excess ROS, reduce oxidative stress and inflammatory response, and create a favorable microenvironment for wound healing.

[0029] 3. Flexible and adjustable dosage forms: Based on the above-mentioned grafted products, they can be prepared into the following formulations through simple processing techniques (such as freeze-drying, molding, and cross-linking):

[0030] Quick-adhesive type: hydrogel or injectable gel, suitable for irregular surfaces and deep wounds.

[0031] Filler type: porous sponge or powder, suitable for penetrating wounds or wound cavities requiring large amounts of filling.

[0032] Pre-formed instruments: hemostatic plugs or hemostatic stoppers, suitable for precise hemostasis in specific locations (such as tooth extraction sockets, sinuses).

[0033] Dressing type: A hemostatic dressing is made by coating a material solution onto a substrate (such as non-woven fabric). Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0035] Figure 1 The image shows the characterization of the powder self-gelling properties provided in the embodiments of the present invention.

[0036] Figure 2 This is a schematic diagram of NMR results provided in an embodiment of the present invention;

[0037] Figure 3 A schematic diagram showing the self-gelling properties and liquid absorption rate provided in an embodiment of the present invention;

[0038] Figure 4 This is a schematic diagram comparing the material bonding performance of the present invention with that of foreign commercial products;

[0039] Figure 5 The coagulation time comparison chart and hemolysis rate comparison chart provided in the embodiments of the present invention;

[0040] Figure 6 This is a comparison diagram of the biocompatibility of materials provided in the embodiments of the present invention;

[0041] Figure 7 A comparative diagram showing the hemostatic effect of the rat liver injury model provided in this embodiment of the invention. Detailed Implementation

[0042] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or end that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or ends.

[0044] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0045] Currently, commonly used hemostatic materials in clinical practice are mainly divided into three categories, but all of them have significant technical limitations and are difficult to meet the hemostatic needs of complex wounds: The first category is physical occlusion hemostatic materials, typical examples of which include gelatin sponges and cellulose dressings. These materials mainly block blood loss by absorbing blood, concentrating clotting factors, or constructing physical scaffolds, but their core defect is their weak wet adhesion, making them prone to detachment on moist tissue surfaces or active areas, and unable to form a stable sealing barrier; at the same time, their function is singular, relying solely on physical action, lacking active procoagulant and anti-inflammatory activity, resulting in limited hemostatic efficiency, and poor adaptability to deep or irregular wounds.

[0046] The second category consists of chemically procoagulant hemostatic materials, such as zeolite and unmodified chitosan. Their mechanism of action is to accelerate thrombus formation by activating platelets or providing a surface for coagulation reactions. However, most of these materials have insufficient adhesion to moist tissues and are difficult to adhere firmly to wounds. Some materials (such as zeolite) generate a large amount of heat during water absorption, which may lead to burns to surrounding tissues, so their safety needs to be improved. They also lack auxiliary functions such as anti-inflammatory and healing-promoting effects.

[0047] The third category consists of mussel-inspired adhesive materials, mostly prepared based on compounds containing catechol structures such as polydopamine and tannic acid. These materials utilize the wet-state adhesive properties of the catechol groups to achieve tissue adhesion. While this type of material solves the wet adhesion problem, it has significant drawbacks: some materials have a long adhesion formation time, failing to meet the requirements for emergency hemostasis in clinical practice; furthermore, their bioactivity is limited, focusing only on adhesive function and lacking key properties such as synergistic coagulation, scavenging reactive oxygen species (ROS), and reducing inflammatory responses, making it difficult to simultaneously address the dual needs of hemostasis and promoting healing.

[0048] In addition, the dosage forms of existing hemostatic materials are mostly fixed, and a single dosage form is difficult to adapt to different types of trauma scenarios such as irregular surface wounds, deep wounds, penetrating wounds, and specific cavities, which limits their clinical application.

[0049] Therefore, developing a comprehensive hemostatic material that combines strong wet adhesion, rapid physical sealing and synergistic chemical coagulation, antioxidant and anti-inflammatory activity, and can be flexibly prepared into various dosage forms according to clinical needs, has become an urgent technical problem to be solved in this field. It is of great significance for improving the hemostatic effect and healing quality of complex soft tissue injuries.

[0050] The present invention aims to provide a mussel-inspired hemostatic material, its preparation method and application, in order to solve the problems of weak adhesion, lack of active coagulation and anti-inflammatory functions, easy tissue damage and single bioactive function in the prior art.

[0051] This invention provides a method for preparing a mussel-inspired hemostatic material, comprising the following steps:

[0052] S1: Dissolve chitosan in an acidic aqueous solution to obtain a chitosan solution.

[0053] In this embodiment of the invention, the acidic aqueous solution is a 1% glacial acetic acid solution, and the concentration of the chitosan solution is 20-100 mg / ml.

[0054] S2: Mix 3,5-dihydroxybenzoic acid with a catalyst, EDC and NHS, and activate the reaction at room temperature to obtain activated 3,5-dihydroxybenzoic acid.

[0055] In this embodiment of the invention, the molar ratio of 3,5-dihydroxybenzoic acid, EDC and NHS is 1:1.5:2~3:4:5, and the activation time is 60-360 minutes.

[0056] S3: The activated 3,5-dihydroxybenzoic acid was slowly titrated into the chitosan solution, and the reaction was stirred at room temperature to obtain the reaction product.

[0057] In this embodiment of the invention, activated 3,5-dihydroxybenzoic acid was slowly titrated into a chitosan solution, and the mixture was stirred at room temperature at a speed of 400-1000 rpm for 24-48 hours to obtain the reaction product.

[0058] S4: The reaction product was placed in a dialysis bag for dialysis and then freeze-dried to obtain mussel-inspired hemostatic material.

[0059] In this embodiment of the invention, the molecular weight cutoff of the dialysis bag is 3500 Da, and the dialysis time is 2 days; it also includes a product verification step: by nuclear magnetic resonance spectroscopy and ultraviolet-visible spectroscopy, it is confirmed that 3,5-dihydroxybenzoic acid has been successfully grafted onto the chitosan molecular chain to obtain a mussel biomimetic hemostatic material.

[0060] The present invention provides a mussel-inspired hemostatic material prepared according to the preparation method of a mussel-inspired hemostatic material described above.

[0061] The present invention provides the application of a mussel-inspired hemostatic material as described above in the fields of rapid hemostasis and promoting healing of complex soft tissue injuries of the oral and maxillofacial region, as well as irregular surface wounds, deep wounds, penetrating wounds, wound cavity filling, superficial bleeding, or hemostasis of specific cavities.

[0062] according to Figure 1 The provided characterization results of the powder's self-gelling properties indicate that CS-DHBA has good ability to absorb blood and moisture and rapidly self-gel.

[0063] according to Figure 2 The provided NMR results diagram shows that DHBA was successfully grafted onto the CS chain.

[0064] according to Figure 3 The provided diagram showing the self-gelling properties and liquid absorption rate indicates that the synthesized material has a higher liquid absorption rate than chitosan alone and the commercial product Yunnan Baiyao, with 10mg of powder achieving a liquid absorption of 478mg.

[0065] according to Figure 4 The provided diagram comparing the adhesive performance of the material with that of foreign commercial adhesives shows that the material's adhesive performance in lap shear and tensile tests is superior to that of many foreign commercial adhesives: the lap shear force is 8.2 times that of commercial materials, and in tensile tests it is 2.7 times that of the commercially available Tegadem and 10.1 times that of Surgiflo.

[0066] according to Figure 5 The provided comparison charts of clotting time and hemolysis rate show that the clotting time of the material group is significantly better than that of chitosan-based hemostatic materials and commercial Yunnan Baiyao products. The clotting time is only 35 seconds. Meanwhile, the hemolysis rate experiment shows that the material's hemolysis rate is less than 2%.

[0067] according to Figure 6 The provided biocompatibility comparison chart shows that the CCK8 data indicates that the material group has good biocompatibility, and the cell survival rate reaches over 90% at multiple time points such as 24 hours, 48 ​​hours, and 72 hours.

[0068] according to Figure 7The comparison chart of hemostatic effects in the provided rat liver injury model shows that: in the rat liver injury model, the material group is superior to the imported Celox hemostatic powder, with a hemostasis time of only 35 seconds, and blood loss 3.87 times less than the gauze treatment group and 2.08 times less than the commercial Celox hemostatic powder.

[0069] The beneficial effects of this invention are reflected in:

[0070] 1. Strong wet adhesion and physical sealing: 3,5-Dihydroxybenzoic acid (DHBA) is a key adhesive unit of mussel byssal proteins. Through esterification, it is covalently grafted onto the chitosan backbone, giving the material strong wet adhesion properties similar to those of mussels. When applied to wounds, the material adheres tightly to moist tissue surfaces, rapidly forming a physical barrier to seal the wound and prevent blood loss.

[0071] 2. Synergistic chemical coagulation effect:

[0072] Positive charge of chitosan: Positively charged chitosan can attract negatively charged red blood cells and platelets, promoting their aggregation.

[0073] The catechol groups of DHBA can form hydrogen bonds and π-π stacking interactions with proteins in the blood (such as fibrinogen), further enriching clotting factors and platelets.

[0074] The synergistic effect of the positive charge and the catechol group significantly accelerates thrombus formation, achieving a "dual hemostatic mechanism" of physical blockage and chemical coagulation.

[0075] Antioxidant and anti-inflammatory effects: The catechol structure of DHBA is an excellent scavenger of reactive oxygen species (ROS). During the inflammatory phase of a wound, the material can effectively remove excess ROS, reduce oxidative stress and inflammatory response, and create a favorable microenvironment for wound healing.

[0076] 3. Flexible and adjustable dosage forms: Based on the above-mentioned grafted products, they can be prepared into the following formulations through simple processing techniques (such as freeze-drying, molding, and cross-linking):

[0077] Quick-adhesive type: hydrogel or injectable gel, suitable for irregular surfaces and deep wounds.

[0078] Filler type: porous sponge or powder, suitable for penetrating wounds or wound cavities requiring large amounts of filling.

[0079] Pre-formed instruments: hemostatic plugs or hemostatic stoppers, suitable for precise hemostasis in specific locations (such as tooth extraction sockets, sinuses).

[0080] Dressing type: A hemostatic dressing is made by coating a material solution onto a substrate (such as non-woven fabric).

[0081] The above descriptions are merely embodiments of the present invention. Commonly known technical solutions or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the scope of the present invention, and these should also be considered within the protection scope of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a mussel-inspired hemostatic material, characterized in that, The method includes: S1: Dissolve chitosan in an acidic aqueous solution to obtain a chitosan solution; S2: Mix 3,5-dihydroxybenzoic acid with a catalyst, wherein the catalyst is EDC or NHS, and activate the mixture at room temperature to obtain activated 3,5-dihydroxybenzoic acid; S3: The activated 3,5-dihydroxybenzoic acid is slowly titrated into the chitosan solution, and the reaction is stirred at room temperature to obtain the reaction product; S4: The reaction product is placed in a dialysis bag for dialysis, and then freeze-dried to obtain a mussel-inspired hemostatic material.

2. The method for preparing a mussel-inspired hemostatic material according to claim 1, characterized in that: The acidic aqueous solution is a 1% glacial acetic acid solution, and the concentration of the chitosan solution is 20-100 mg / ml.

3. The method for preparing a mussel-inspired hemostatic material according to claim 1, characterized in that: The molar ratio of 3,5-dihydroxybenzoic acid, EDC and NHS is 1:1.5:2~3:4:5, and the activation time is 60-360 minutes.

4. The method for preparing a mussel-inspired hemostatic material according to claim 1, characterized in that: The activated 3,5-dihydroxybenzoic acid was slowly titrated into the chitosan solution, and the mixture was stirred at room temperature at a speed of 400-1000 rpm for 24-48 hours to obtain the reaction product.

5. The method for preparing a mussel-inspired hemostatic material according to claim 1, characterized in that: The dialysis bag has a molecular weight cutoff of 3500 Da, and the dialysis time is 2 days.

6. The method for preparing a mussel-inspired hemostatic material according to claim 1, characterized in that: Step S4 is followed by a product verification step: by detecting nuclear magnetic resonance spectroscopy and ultraviolet-visible spectroscopy, it is confirmed that the 3,5-dihydroxybenzoic acid has been successfully grafted onto the chitosan molecular chain, thus obtaining a mussel-inspired hemostatic material.

7. A mussel-inspired hemostatic material prepared according to the preparation method of a mussel-inspired hemostatic material according to claims 1-6.

8. The application of the mussel biomimetic hemostatic material according to claim 7 in the field of rapid hemostasis and healing promotion of complex soft tissue injuries of the oral and maxillofacial region, irregular surface wounds, deep wounds, penetrating wounds, wound cavity filling, superficial bleeding or hemostasis of specific cavities.

Images