A hemostatic wound healing sponge based on andrias davidianus mucus and a preparation method and application thereof

Abstract

The application discloses a androcambarus slime-based hemostatic and wound healing sponge, a preparation method and application thereof, and relates to the technical field of biomedical materials. The method comprises the following steps: uniformly mixing androcambarus slime acetic acid solution and polylysine hydrochloride aqueous solution to obtain androcambarus slime / polylysine hydrochloride mixed solution; collecting the precipitate after centrifugation of the androcambarus slime / polylysine hydrochloride mixed solution, and adding the precipitate into a mold; then, the mold is pre-cooled at-85 to-75 DEG C, and is placed in a vacuum freeze dryer for freeze drying, so that the androcambarus slime-based hemostatic and wound healing sponge is obtained. The sponge prepared by the application has good hemostatic performance, and has the advantages of simple preparation process, low production cost, wide application potential in wound hemostasis, and new ideas for utilization of androcambarus resources.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a hemostatic and wound-healing sponge based on giant salamander mucus, its preparation method, and its application. Background Technology

[0002] Uncontrollable massive bleeding is one of the leading causes of death during surgery, natural disasters, and accidents. Most patients die from bleeding within the first few hours after injury, usually before reaching a hospital. The golden window for rescue after a hemorrhagic trauma is only a few minutes; if bleeding is controlled quickly (within 10 minutes), approximately 90% of lives can be saved. Therefore, effective hemostasis immediately after trauma is crucial. However, commonly used hemostatic materials on the market, such as fibrin glue and thrombin preparations, have problems such as high cost and the risk of thrombosis during the hemostasis process. Passive hemostatic materials, such as gauze and tissue adhesives, have problems such as non-degradability, poor biocompatibility, and the potential for secondary injury.

[0003] The giant salamander, also known as the Chinese giant salamander, has numerous glands on its skin that secrete a white mucus. This mucus is primarily composed of structurally unique and functionally diverse glycoproteins and peptides, which promote wound healing and exhibit good biocompatibility. Because the giant salamander's mucus protein can be collected by stimulating the skin to secrete mucus, and this process does not affect the salamander's normal survival, it is considered a high-quality, renewable resource.

[0004] Due to the unique properties of giant salamander mucin (excellent biocompatibility and wound-healing effects), it has significant application value in the biomedical field. Existing technology discloses a hemostatic sponge technology utilizing giant salamander mucin. This technology uses giant salamander mucin, cellulose nanofibers, and cellulose nanocrystals as the main components to prepare hemostatic materials for wounds that cannot be pressed. However, products prepared using existing technologies have a low proportion of giant salamander mucin, making it difficult to fully utilize the efficacy of the giant salamander mucin protein. Furthermore, giant salamander mucin protein itself is prone to agglomeration, easily encapsulating bacteria and other microorganisms. Conventional sterilization methods can easily cause the protein to lose its activity or fail to kill the bacteria encapsulated within the mucin clusters. Therefore, giant salamander mucin protein sponge materials manufactured using existing technologies have a short shelf life, and large-scale, standardized production is difficult. Summary of the Invention

[0005] To address the shortcomings of the aforementioned background technologies, this invention proposes a novel technology for preparing giant salamander mucin sponges, including special processes such as acetic acid dispersion, centrifugation, and freeze-drying. This technology can produce hemostatic sponge materials with giant salamander mucin as the main component or a single component, for use in hemostasis of human skin or organs. It not only fully utilizes the efficacy of giant salamander mucin (excellent biocompatibility, complete degradation in the body, and significant wound healing effects), but also completely solves the problems of difficult product sterilization, short shelf life, and difficulty in storage.

[0006] This invention provides a method for preparing a giant salamander mucin hemostatic sponge, comprising the following steps:

[0007] The acetic acid solution of giant salamander mucus and the aqueous solution of polylysine hydrochloride were thoroughly and uniformly mixed on a rotary mixer to obtain a giant salamander mucus / polylysine hydrochloride mixed solution.

[0008] The mixture of giant salamander mucus and polylysine hydrochloride was centrifuged and the precipitate was collected and added to a mold. The mold was then pre-cooled at -85 to -75°C. After it was completely frozen, it was freeze-dried in a vacuum freeze dryer to obtain a hemostatic and wound-healing sponge based on giant salamander mucus.

[0009] Preferably, the acetic acid solution of giant salamander mucus is prepared by mixing giant salamander mucus freeze-dried powder and acetic acid solution, wherein the concentration of the acetic acid solution is 0.5-1 mol / L and the concentration of the acetic acid solution of giant salamander mucus is 10-15 mg / mL.

[0010] Preferably, in the mixed solution of giant salamander mucus / polylysine hydrochloride, the mass ratio of giant salamander mucus freeze-dried powder to polylysine hydrochloride is 10:1 to 3.

[0011] Preferably, the polylysine hydrochloride aqueous solution is prepared by dispersing polylysine hydrochloride in an aqueous solvent, wherein the concentration of the polylysine hydrochloride aqueous solution is 8~12 mg / mL.

[0012] Preferably, the polylysine hydrochloride has a molecular weight of 2000 to 5000.

[0013] Preferably, the mold pre-cooling temperature is -85~-75℃, and the pre-cooling time is 12~24h.

[0014] Preferably, the freeze-drying time is 48–72 hours.

[0015] Preferably, when collecting the precipitate by centrifugation, the centrifugation speed is 4000~8000 r / min and the time is 10~20 min.

[0016] The second objective of this invention is to provide a hemostatic and wound-healing sponge based on giant salamander mucus.

[0017] The third objective of this invention is to provide an application of a sponge in the preparation of medical materials for hemostasis and wound healing.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention provides a hemostatic and wound-healing sponge based on giant salamander mucus, its preparation method, and its application. The invention prepares a composite sponge material by dispersing giant salamander mucus in acetic acid solution, simply mixing it with polylysine hydrochloride, centrifuging to collect the precipitate, and then freeze-drying. Only acetic acid is used in the preparation process. The giant salamander mucus serves as the main framework of the sponge, and the polylysine hydrochloride, through the entanglement of peptide chains within the giant salamander mucus framework, endows the sponge with excellent antibacterial activity. The preparation process is simple, has a short preparation cycle, requires low-cost raw materials, produces stable product performance, and is easily applicable to large-scale production.

[0020] The main component of this invention is giant salamander mucus. Giant salamander mucus is easy to extract, comes from natural sources, and can be obtained sustainably and periodically. This not only makes full use of existing biological resources, but also aligns with my country's concept of sustainable development.

[0021] This invention can be prepared without a cross-linking agent, thus avoiding the cytotoxicity associated with cross-linking agents and exhibiting good biocompatibility. This invention also possesses good liquid absorption capacity, good hemostatic effect, and low hemolysis rate, making it effective for rapid hemostasis of superficial wounds. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope (SEM) image of the hemostatic sponge prepared according to the present invention.

[0023] Figure 2 The results of the water absorption rate test for the hemostatic sponge prepared according to the present invention are shown.

[0024] Figure 3 The results of in vitro coagulation index testing of the hemostatic sponge prepared in this invention are shown.

[0025] Figure 4 The results of the hemolysis rate test of the hemostatic sponge prepared in this invention are shown.

[0026] Figure 5 The antibacterial effect of the hemostatic sponge prepared in this invention. Detailed Implementation

[0027] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0028] The purpose of this invention is to provide a composite hemostatic sponge material made primarily from the mucus of the giant salamander, which can be used for hemostasis of human skin or organs. The preparation method of the hemostatic sponge provided by this invention includes freeze-dried giant salamander mucus powder and polylysine hydrochloride as raw materials. This solves the problems of complex preparation processes, high costs, and unfavorable large-scale production of existing hemostatic materials.

[0029] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a hemostatic and wound-healing sponge based on giant salamander mucus, comprising the following steps:

[0030] The acetic acid solution of giant salamander mucus and the aqueous solution of polylysine hydrochloride were thoroughly and uniformly mixed on a rotary mixer to obtain a giant salamander mucus / polylysine hydrochloride mixed solution.

[0031] After centrifuging the mixed solution of giant salamander mucus and polylysine hydrochloride, the precipitate was collected and added to a mold. The mold was then placed in a -80°C freezer for pre-cooling. After it was completely frozen, it was placed in a vacuum freeze dryer for freeze drying to obtain a hemostatic and wound-healing sponge based on giant salamander mucus.

[0032] The hemostatic sponge provided by this invention can be prepared without cross-linking agents, thus solving the problem that existing hemostatic materials have certain cytotoxicity when using cross-linking agents.

[0033] The acetic acid solution of giant salamander mucus is prepared by mixing giant salamander mucus freeze-dried powder and acetic acid solution, wherein the concentration of the acetic acid solution is 0.5-1 mol / L and the concentration of the acetic acid solution of giant salamander mucus is 10-15 mg / mL.

[0034] The polylysine hydrochloride aqueous solution is prepared by dispersing polylysine hydrochloride in an aqueous solvent, wherein the concentration of the polylysine hydrochloride aqueous solution is 8~12 mg / mL, preferably 10 mg / mL.

[0035] The polylysine hydrochloride has a molecular weight of 2000-5000.

[0036] In the mixed solution of giant salamander mucus and polylysine hydrochloride, the mass ratio of giant salamander mucus to polylysine hydrochloride is 10:1 to 3.

[0037] The giant salamander mucus / polylysine hydrochloride mixture was centrifuged at a speed of 4000~8000 r / min for 10~20 min.

[0038] The pre-cooling time for the mold is 12–24 hours. After pre-cooling, the freeze-drying time is 48–72 hours.

[0039] In one embodiment, a method for preparing a hemostatic and wound-healing sponge based on giant salamander mucus includes:

[0040] The freeze-dried giant salamander mucus powder was added to an acetic acid solution to prepare a solution of 10-15 mg / mL. The solution was then dissolved for 30-60 minutes using a vortex mixer to obtain the giant salamander mucus acetic acid solution.

[0041] After dissolving polylysine hydrochloride in water, it was added to the acetic acid solution of giant salamander mucus in a certain proportion and mixed evenly on a rotary mixer.

[0042] The mass ratio of giant salamander mucus to polylysine hydrochloride in the mixed solution is 10:1 to 3.

[0043] A second aspect of the present invention provides a hemostatic and wound-healing sponge based on giant salamander mucus.

[0044] The third aspect of this invention provides the application of a sponge in the preparation of medical materials for hemostasis and wound healing.

[0045] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods; and the reagents and materials used, unless otherwise specified, are all commercially available.

[0046] Example 1

[0047] To prepare a 10 mg / mL acetic acid solution of giant salamander mucus: First, prepare a 0.75 mol / L acetic acid solution. Add a certain amount of lyophilized giant salamander mucus powder to the acetic acid solution and shake it in a vortex mixer for 30 min to dissolve it, thus obtaining a 10 mg / mL acetic acid solution of giant salamander mucus.

[0048] Preparation of giant salamander mucus / polylysine hydrochloride solution: First, prepare a 10 mg / mL polylysine hydrochloride aqueous solution. Then, mix the giant salamander mucus acetic acid solution and the polylysine hydrochloride aqueous solution at a mass ratio of 10:1 on a rotary mixer to prepare the giant salamander mucus / polylysine hydrochloride solution.

[0049] Preparation of the hemostatic sponge: The prepared giant salamander mucus / polylysine hydrochloride solution was centrifuged at 6000 r / min for 10 min in a centrifuge. The precipitate was collected and poured into a mold. The mold was pre-frozen at -80°C for 12 h. After it was completely frozen, it was freeze-dried in a vacuum freeze dryer for 48 h to obtain the hemostatic sponge, named SSAD-EPL1.

[0050] Example 2

[0051] To prepare a 10 mg / mL acetic acid solution of giant salamander mucus: First, prepare a 0.75 mol / L acetic acid solution. Add a certain amount of lyophilized giant salamander mucus powder to the acetic acid solution and shake it in a vortex mixer for 30 min to dissolve it, thus obtaining a 10 mg / mL acetic acid solution of giant salamander mucus.

[0052] Preparation of giant salamander mucus / polylysine hydrochloride solution: First, prepare a 10 mg / mL polylysine hydrochloride aqueous solution. Then, mix the giant salamander mucus acetic acid solution and the polylysine hydrochloride aqueous solution at a mass ratio of 10:2 on a rotary mixer to prepare the giant salamander mucus / polylysine hydrochloride solution.

[0053] Preparation of the hemostatic sponge: The prepared giant salamander mucus / polylysine hydrochloride solution was centrifuged at 6000 r / min for 10 min in a centrifuge. The precipitate was collected and poured into a mold. The mold was pre-frozen at -80°C for 24 h. After it was completely frozen, it was freeze-dried in a vacuum freeze dryer for 72 h to obtain the hemostatic sponge, named SSAD-EPL2.

[0054] Example 3

[0055] To prepare a 10 mg / mL acetic acid solution of giant salamander mucus: First, prepare a 0.75 mol / L acetic acid solution. Add a certain amount of lyophilized giant salamander mucus powder to the acetic acid solution and shake it in a vortex mixer for 30 min to dissolve it, thus obtaining a 10 mg / mL acetic acid solution of giant salamander mucus.

[0056] Preparation of giant salamander mucus / polylysine hydrochloride solution: First, prepare a 10 mg / mL polylysine hydrochloride aqueous solution. Then, mix the giant salamander mucus acetic acid solution and the polylysine hydrochloride aqueous solution at a mass ratio of 10:3 on a rotary mixer to prepare the giant salamander mucus / polylysine hydrochloride solution.

[0057] Preparation of the hemostatic sponge: The prepared giant salamander mucus / polylysine hydrochloride solution was centrifuged at 6000 r / min for 10 min in a centrifuge. The precipitate was collected and poured into a mold. The mold was pre-frozen at -80°C for 12 h. After it was completely frozen, it was freeze-dried in a vacuum freeze dryer for 48 h to obtain the hemostatic sponge, named SSAD-EPL3.

[0058] Example 4

[0059] To prepare a 10 mg / mL acetic acid solution of giant salamander mucus: First, prepare a 0.75 mol / L acetic acid solution. Add a certain amount of lyophilized giant salamander mucus powder to the acetic acid solution and shake it in a vortex mixer for 30 min to dissolve it, thus obtaining a 10 mg / mL acetic acid solution of giant salamander mucus.

[0060] Preparation of the hemostatic sponge: The prepared acetic acid solution of giant salamander mucus was centrifuged at 6000 r / min for 10 min in a centrifuge. The precipitate was collected and poured into a mold. The mold was pre-frozen at -80°C for 24 h. After it was completely frozen, it was freeze-dried in a vacuum freeze dryer for 72 h to obtain the hemostatic sponge, which was named SSAD.

[0061] Comparative Example 1

[0062] The commercial gelatin hemostatic sponge, sourced from Guangzhou Kuaikang Medical Equipment Co., Ltd., is named CS in the attached diagram.

[0063] To illustrate the properties of the sponge provided by this invention, the accompanying drawings are provided. The relevant performance tests on the hemostatic sponge prepared in the examples are as follows.

[0064] Surface morphology test:

[0065] The surface morphology of the hemostatic sponge materials of Examples 1-4 of the present invention was tested. The test method was as follows: the hemostatic sponges of Examples 1-4 were cut open to fully expose the internal structure of the material, and then observed using a scanning electron microscope (SEM). Figure 1 As shown, Examples 1 to 4 all exhibit a uniform and dense porous structure.

[0066] Water absorption rate test:

[0067] The water absorption rate of the hemostatic sponge materials of Examples 1-4 of the present invention was tested. The test method was as follows: the initial mass of the hemostatic sponge materials of Examples 1-4 was weighed, and they were completely immersed in deionized water. After they had fully absorbed water, they were taken out and weighed, and the water absorption rate was calculated. Figure 2 As shown, the hemostatic sponges in Examples 1 to 4 all exhibited excellent liquid absorption capacity, with an absorption rate of 30 to 45 times their own weight.

[0068] In vitro coagulation test:

[0069] In vitro coagulation tests were conducted on the hemostatic sponge materials of Examples 1-4 of this invention. The test method was as follows: 50 µL of fresh blood was dropped onto the surface of the hemostatic sponge of Examples 1-4, and incubated at 37°C for 3 minutes. Then, 10 mL of deionized water was slowly added, and incubation was continued at 37°C for another 3 minutes. The absorbance of the supernatant at 540 nm was measured, with the group without added material serving as a blank control. The in vitro coagulation index was calculated. A lower in vitro coagulation index indicates a better hemostatic effect. Figure 3 As shown, the hemostatic sponges of Examples 1 to 4 all exhibited excellent in vitro coagulation ability, with in vitro coagulation indices ranging from 20% to 30%, which were significantly lower than those of commercial hemostatic sponges.

[0070] Hemolysis rate test:

[0071] The hemostatic sponge materials of Examples 1-4 of this invention were subjected to an in vitro hemolysis test. The test method was as follows: the hemostatic sponge of Examples 1-4 was immersed in 1 mL of 5% red blood cell suspension and incubated at 37°C for 1 hour. Red blood cells diluted with deionized water and PBS solution were used as positive and negative controls. After incubation, the mixture was centrifuged at 3000 r / min for 10 minutes, and the absorbance of the supernatant at 540 nm was measured to calculate the hemolysis rate. Figure 4 As shown, the hemolysis rate of the hemostatic sponges in Examples 1-4 was all less than 5%, indicating that they are safe and do not pose a risk of hemolysis.

[0072] In vitro antibacterial activity test:

[0073] The hemostatic sponge materials of Examples 1-4 of the present invention were subjected to in vitro antibacterial activity tests. The test method was as follows: the hemostatic sponges of Examples 1-4 were placed flat in LB solid medium coated with Escherichia coli and Staphylococcus aureus, and incubated at 37°C for 12 hours. The size of the inhibition zone was then observed. Figure 5 As shown, the sponge prepared in Example 4 without the addition of polylysine hydrochloride did not show a significant antibacterial effect. However, the sponges prepared in Examples 1 to 3 with the addition of polylysine hydrochloride all showed a significant inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus. Furthermore, the inhibition zone increased with the increase of the amount of polylysine hydrochloride added, indicating that polylysine hydrochloride imparted an antibacterial effect to the sponge.

[0074] In summary, this invention provides a hemostatic sponge made from giant salamander mucus and its preparation method. The raw materials for the composite sponge include giant salamander mucus and polylysine hydrochloride. The preparation method involves first dissolving freeze-dried giant salamander mucus powder in acetic acid to form a solution, then adding an aqueous solution of polylysine hydrochloride to the acetic acid solution of giant salamander mucus, and mixing thoroughly and uniformly on a rotary mixer. After centrifugation, the precipitate is collected, poured into a mold, and freeze-dried to obtain the composite hemostatic sponge. This sponge exhibits good hemostatic properties, has a simple preparation process, and low production cost. It has broad application potential in wound hemostasis and provides a new approach for the utilization of giant salamander resources.

[0075] This invention describes preferred embodiments and their effects. However, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to include both the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0076] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a hemostatic pro-wound healing sponge based on Andrias davidianus mucus, characterized in that, The method comprises the following steps: The andrias slime acetic acid solution and the polylysine hydrochloride aqueous solution are uniformly mixed on a rotating mixer to obtain an andrias slime / polylysine hydrochloride mixed solution; The andrias slime / polylysine hydrochloride mixed solution is centrifuged to collect the precipitate, which is then added to a mold, and the mold is pre-cooled until it is completely frozen, and then the mold is placed in a vacuum freeze dryer for freeze drying, thereby obtaining the andrias slime-based hemostatic and wound healing sponge; The andrias slime acetic acid solution is prepared by mixing andrias slime freeze-dried powder and an acetic acid solution, wherein the concentration of the acetic acid solution is 0.5-1 mol / L, and the concentration of the andrias slime acetic acid solution is 10-15 mg / mL. In the andrias slime / polylysine hydrochloride mixed solution, the mass ratio of the andrias slime freeze-dried powder to the polylysine hydrochloride is 10:1-3.

2. The method of claim 1, wherein the preparation of the Andrias davidianus mucilage-based hemostatic and wound healing-promoting sponge is characterized by, The polylysine hydrochloride aqueous solution is prepared by dispersing polylysine hydrochloride in an aqueous solvent, and the concentration of the polylysine hydrochloride aqueous solution is 8-12 mg / mL.

3. The method of claim 2, wherein the preparation of the andrias slime-based hemostatic pro-wound healing sponge is characterized by, The molecular weight of the polylysine hydrochloride is 2000-5000.

4. The method of claim 1, wherein the preparation of the andrias slime-based hemostatic pro-wound healing sponge is characterized by, The pre-cooling temperature of the mold is -85 to -75°C, and the pre-cooling time is 12-24 h.

5. The method of claim 1, wherein the preparation of the andrias slime-based hemostatic pro-wound healing sponge is characterized by, The freeze drying time is 48-72 h.

6. The method of claim 1, wherein the preparation of the andrias slime-based hemostatic pro-wound healing sponge is characterized by, When the precipitate is collected by centrifugation, the centrifugation speed is 4000-8000 r / min, and the time is 10-20 min.

7. An andrias slime-based hemostatic and wound healing sponge prepared by the method of any one of claims 1-6.

8. Use of the sponge of claim 7 in the preparation of a hemostatic and wound healing medical material.

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