Sponge for stopping bleeding and promoting wound healing based on giant salamander mucus as well as preparation method and application of sponge

Abstract

The invention discloses a hemostatic and wound healing promoting sponge based on giant salamander mucus and a preparation method and application thereof, and relates to the technical field of biomedical materials. The method comprises the following steps: uniformly mixing a giant salamander mucus acetic acid solution with a polylysine hydrochloride aqueous solution to obtain a giant salamander mucus / polylysine hydrochloride mixed solution; centrifuging the giant salamander mucus / polylysine hydrochloride mixed solution, collecting precipitates, adding the precipitates into a mold, pre-cooling the mold at-85 to-75 DEG C, and freeze-drying the mold in a vacuum freeze-drying machine to obtain the hemostatic wound healing promoting sponge based on the giant salamander mucus. The prepared sponge has good hemostatic performance, the preparation process is simple, the production cost is low, the sponge has wide application potential in the aspect of wound hemostasis, and a new thought is provided for giant salamander resource utilization.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a hemostatic and wound healing-promoting sponge based on giant salamander mucus, and a preparation method and application thereof. Background Art

[0002] Uncontrollable massive bleeding is one of the main causes of death in surgery, natural disasters, and accidents. Most patients die from bleeding within the first few hours after injury, usually before the injured patient arrives at the hospital. The golden rescue time after a hemorrhagic trauma is only a few minutes. If the bleeding is quickly controlled after the injury (<10 minutes), about 90% of lives can be saved. Therefore, it is crucial to stop bleeding effectively as soon as possible after the trauma occurs. However, the commonly used hemostatic materials on the market, such as active hemostatic materials such as fibrin glue and thrombin preparations, have problems such as high cost and easy entry into blood vessels to cause thrombosis during hemostasis. Passive hemostatic materials such as gauze and tissue adhesives have problems such as non-degradability, poor biocompatibility, and easy to cause secondary injuries.

[0003] The giant salamander, commonly known as the baby fish, has many glands on its skin that can secrete a white mucus. The main components of this mucus are glycoproteins and peptides with special structures and diverse functions. It has the effect of promoting wound healing and has good biocompatibility. Since the giant salamander mucus protein can be collected by stimulating the giant salamander's skin to secrete mucus, and this process does not affect the giant salamander's normal survival, the giant salamander mucus protein is a renewable high-quality resource.

[0004] Due to the above-mentioned special properties of giant salamander mucus protein (excellent biocompatibility and the effect of promoting wound healing), it has great application value in the biomedical field. The prior art discloses a hemostatic sponge technology using giant salamander mucus, which uses giant salamander mucus, cellulose nanofibers and cellulose nanocrystals as main components to prepare hemostatic materials for non-pressable wounds. However, the products prepared by the existing technology have a low proportion of giant salamander mucus, and it is not easy to give full play to the efficacy of giant salamander mucus protein. In addition, giant salamander mucus protein itself is easy to aggregate and easily wrap some bacteria and other microorganisms. Conventional disinfection and sterilization methods easily cause the protein to lose activity, or fail to kill bacteria wrapped in mucus clusters. Therefore, the shelf life of giant salamander mucus sponge materials manufactured by the prior art is short, and it is difficult to prepare products on a large scale and in a standardized manner. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned background technology, the present invention proposes a new preparation technology of giant salamander mucus protein sponge, including special processes such as acetic acid dispersion, centrifugation, and freeze-drying, which can prepare a hemostatic sponge material with giant salamander mucus protein as the main component or the single component, which can be used for hemostasis of human skin or organs. It can not only give full play to the efficacy of giant salamander mucus protein (excellent biocompatibility, can be completely degraded in the body, and significantly promote wound healing), but also can completely solve the problems of difficulty in disinfection and sterilization of the product, short shelf life, and difficulty in storage.

[0006] The present invention provides a method for preparing a giant salamander mucus protein hemostatic sponge, comprising the following steps: The giant salamander mucus acetic acid solution and the polylysine hydrochloride aqueous solution are fully and evenly mixed on a rotary mixer to obtain a giant salamander mucus / polylysine hydrochloride mixed solution; The mixed solution of giant salamander mucus / polylysine hydrochloride is placed in a centrifuge to collect the precipitate and add it to a mold, and then the mold is pre-cooled at -85~-75℃, and after it is completely frozen, it is placed in a vacuum freeze dryer for freeze drying to obtain a hemostatic and wound healing sponge based on giant salamander mucus.

[0007] Preferably, the giant salamander mucus acetic acid solution is prepared by preparing giant salamander mucus freeze-dried powder and acetic acid solution, wherein the concentration of the acetic acid solution is 0.5-1 mol / L; the concentration of the giant salamander mucus acetic acid solution is 10-15 mg / mL.

[0008] Preferably, in the giant salamander mucus / polylysine hydrochloride mixed solution, the mass ratio of giant salamander mucus freeze-dried powder to polylysine hydrochloride is 10:1-3.

[0009] 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.

[0010] Preferably, the molecular weight of the polylysine hydrochloride is 2000-5000.

[0011] Preferably, the mold precooling temperature is -85~-75°C, and the precooling time is 12 to 24 hours.

[0012] Preferably, the freeze-drying time is 48 to 72 hours.

[0013] Preferably, when collecting the precipitate by centrifugation, the centrifugal speed is 4000-8000 r / min and the time is 10-20 min.

[0014] The second object of the present invention is to provide a hemostatic and wound healing sponge based on giant salamander mucus.

[0015] The third object of the present invention is to provide an application of a sponge in preparing a medical material for hemostasis and promoting wound healing.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a hemostatic wound healing sponge based on giant salamander mucus, and a preparation method and application thereof. The invention prepares a composite sponge material by dispersing giant salamander mucus with an acetic acid solution, simply mixing with polylysine hydrochloride, centrifugally collecting precipitation, and then freeze-drying. Only acetic acid reagent is used in the preparation process, wherein giant salamander mucus is used as the main skeleton of the sponge, and polylysine hydrochloride is interspersed in the giant salamander mucus skeleton through mutual entanglement between peptide chains, giving the sponge excellent antibacterial activity. The preparation process is simple, the preparation cycle is short, the required raw material cost is low, the product performance is stable, and it is easy to enter actual large-scale production.

[0017] The main component of the invention is giant salamander mucus, which is easy to extract, has a natural source, and can be obtained periodically and sustainably, which can not only make full use of existing biological resources, but also conform to my country's concept of sustainable development.

[0018] The invention can be prepared without a cross-linking agent, can avoid the cytotoxicity caused by the cross-linking agent, and has good biocompatibility. The invention also has good liquid absorption capacity, good hemostatic effect, low hemolysis rate, and has a good effect on rapid hemostasis of superficial wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a scanning electron microscope (SEM) image of the hemostatic sponge prepared in the present invention.

[0020] Figure 2 The water absorption test result of the hemostatic sponge prepared by the present invention is shown.

[0021] Figure 3 The results are the in vitro coagulation index test results of the hemostatic sponge prepared by the present invention.

[0022] Figure 4 The figure shows the hemolysis rate test result of the hemostatic sponge prepared by the present invention.

[0023] Figure 5 The invention discloses the antibacterial effect of the hemostatic sponge prepared by the invention. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0025] The purpose of the present invention is to provide a composite hemostatic sponge material made from giant salamander mucus as a main raw material, which can be used for hemostasis of human skin or organs. The preparation method of the hemostatic sponge provided by the present invention comprises giant salamander mucus freeze-dried powder and polylysine hydrochloride. The present invention solves the problems of complex preparation process, high cost and disadvantageous for large-scale production of existing hemostatic materials.

[0026] In order to achieve the above object, the first aspect of the present invention provides a method for preparing a hemostatic wound healing sponge based on giant salamander mucus, comprising the following steps: The giant salamander mucus acetic acid solution and the polylysine hydrochloride aqueous solution are fully and evenly mixed on a rotary mixer to obtain a giant salamander mucus / polylysine hydrochloride mixed solution; After centrifuging the mixed solution of giant salamander mucus / polylysine hydrochloride, the precipitate is collected and added to a mold, and then the mold is placed in a -80°C refrigerator for precooling. After it is completely frozen, it is placed in a vacuum freeze dryer for freeze drying to obtain a hemostatic and wound healing sponge based on giant salamander mucus.

[0027] The hemostatic sponge provided by the invention can be prepared without a cross-linking agent, thereby solving the problem that the cross-linking agent used in the existing hemostatic material has certain cytotoxicity.

[0028] The giant salamander mucus acetic acid solution 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; the concentration of the giant salamander mucus acetic acid solution is 10-15 mg / mL.

[0029] 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.

[0030] The molecular weight of the polylysine hydrochloride is 2000-5000.

[0031] In the giant salamander mucus / polylysine hydrochloride mixed solution, the mass ratio of giant salamander mucus to polylysine hydrochloride is 10:1-3.

[0032] The centrifugal speed of the giant salamander mucus / polylysine hydrochloride mixed solution is 4000-8000 r / min, and the time is 10-20 min.

[0033] The mold precooling time is 12 to 24 hours. After the mold is precooled, the freeze drying time is 48 to 72 hours.

[0034] In one embodiment, a method for preparing a hemostatic wound healing sponge based on giant salamander mucus comprises: The giant salamander mucus freeze-dried powder is added to the acetic acid solution to prepare a 10-15 mg / mL solution, and the giant salamander mucus acetic acid solution is obtained by dissolving the solution with the assistance of a vortex oscillator for 30-60 minutes.

[0035] After dissolving polylysine hydrochloride in water, add it into the giant salamander mucus acetic acid solution in a certain proportion and mix evenly on a rotary mixer.

[0036] The mass ratio of giant salamander mucus to polylysine hydrochloride in the mixed solution is 10:1-3.

[0037] The second aspect of the present invention provides a hemostatic and wound healing-promoting sponge based on giant salamander mucus.

[0038] The third aspect of the present invention provides an application of a sponge in preparing a medical material for hemostasis and promoting wound healing.

[0039] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0040] Example 1 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 giant salamander mucus freeze-dried powder to the acetic acid solution, and shake on a vortex oscillator to dissolve for 30 minutes to obtain a 10 mg / mL giant salamander mucus acetic acid solution.

[0041] Preparation of giant salamander mucus / polylysine hydrochloride solution: First, prepare a 10 mg / mL polylysine hydrochloride aqueous solution, then, fully and evenly 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 a giant salamander mucus / polylysine hydrochloride solution.

[0042] Preparation of the finished hemostatic sponge: The prepared giant salamander mucus / polylysine hydrochloride solution was placed in a centrifuge and centrifuged at 6000r / min for 10min, the precipitate was collected and injected into a mold, and the mold was placed in a -80°C pre-freeze for 12h. After it was completely frozen, it was placed in a vacuum freeze dryer and freeze-dried for 48h to obtain the finished hemostatic sponge, named SSAD-EPL1.

[0043] Example 2 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 giant salamander mucus freeze-dried powder to the acetic acid solution, and shake on a vortex oscillator to dissolve for 30 minutes to obtain a 10 mg / mL giant salamander mucus acetic acid solution.

[0044] Preparation of giant salamander mucus / polylysine hydrochloride solution: First, prepare a 10 mg / mL polylysine hydrochloride aqueous solution, then, fully and evenly 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 a giant salamander mucus / polylysine hydrochloride solution.

[0045] Preparation of the finished hemostatic sponge: The prepared giant salamander mucus / polylysine hydrochloride solution was placed in a centrifuge and centrifuged at 6000r / min for 10min, the precipitate was collected and injected into a mold, and the mold was placed in a -80°C pre-freeze for 24h. After it was completely frozen, it was placed in a vacuum freeze dryer and freeze-dried for 72h to obtain the finished hemostatic sponge, named SSAD-EPL2.

[0046] Example 3 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 giant salamander mucus freeze-dried powder to the acetic acid solution, and shake on a vortex oscillator to dissolve for 30 minutes to obtain a 10 mg / mL giant salamander mucus acetic acid solution.

[0047] Preparation of giant salamander mucus / polylysine hydrochloride solution: First, prepare a 10 mg / mL polylysine hydrochloride aqueous solution, then, fully and evenly 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 a giant salamander mucus / polylysine hydrochloride solution.

[0048] Preparation of the finished hemostatic sponge: The prepared giant salamander mucus / polylysine hydrochloride solution was placed in a centrifuge and centrifuged at 6000r / min for 10min, the precipitate was collected and injected into a mold, and the mold was placed in a -80°C pre-freeze for 12h. After it was completely frozen, it was placed in a vacuum freeze dryer and freeze-dried for 48h to obtain the finished hemostatic sponge, named SSAD-EPL3.

[0049] Example 4 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 giant salamander mucus freeze-dried powder to the acetic acid solution, and shake on a vortex oscillator to dissolve for 30 minutes to obtain a 10 mg / mL giant salamander mucus acetic acid solution.

[0050] Preparation of the finished hemostatic sponge: The prepared giant salamander mucus acetic acid solution was placed in a centrifuge and centrifuged at 6000r / min for 10min, the precipitate was collected and injected into a mold, and the mold was placed in a -80°C pre-freeze for 24h. After it was completely frozen, it was placed in a vacuum freeze dryer and freeze-dried for 72h to obtain the finished hemostatic sponge, named SSAD.

[0051] Comparative Example 1 Commercial gelatin hemostatic sponge, sourced from Guangzhou Kuaikang Medical Instrument Co., Ltd., is named CS in the attached figure.

[0052] In order to illustrate the relevant performance of the sponge provided by the present invention, it is described in conjunction with the accompanying drawings. The relevant performance tests of the hemostatic sponge prepared in the example are as follows.

[0053] Surface morphology test: The surface morphology of the hemostatic sponge materials of Examples 1 to 4 of the present invention was tested by cutting the hemostatic sponges of Examples 1 to 4, and observing them using a scanning electron microscope (SEM) after fully exposing the internal structure of the material. Figure 1 As shown, Examples 1 to 4 all exhibit a uniform and dense porous structure.

[0054] Water absorption test: The water absorption rate of the hemostatic sponge materials of Examples 1 to 4 of the present invention was tested. The testing method is as follows: weigh the initial mass of the hemostatic sponge materials of Examples 1 to 4, soak them completely in deionized water, take them out and weigh them after they have fully absorbed water, and calculate the water absorption rate. Figure 2 As shown, the hemostatic sponges of Examples 1 to 4 all exhibit excellent liquid absorption capacity, and the liquid absorption rate can reach 30 to 45 times of its own weight.

[0055] In vitro coagulation test: The hemostatic sponge materials of Examples 1 to 4 of the present invention were subjected to an in vitro coagulation test. The test method was as follows: 50 µL of fresh blood was dripped onto the surface of the hemostatic sponge of Examples 1 to 4, and after incubation at 37°C for 3 minutes, 10 mL of deionized water was slowly added, and after further incubation at 37°C for 3 minutes, the absorbance of the supernatant at 540 nm was measured, and the group without material addition was used as a blank control. The in vitro coagulation index was calculated. The lower the in vitro coagulation index, the better the hemostatic effect. Figure 3 As shown, the hemostatic sponges of Examples 1 to 4 all exhibited excellent in vitro coagulation ability, and the in vitro coagulation indexes were all between 20% and 30%, which were significantly lower than those of commercial hemostatic sponges.

[0056] Hemolysis rate test: The hemostatic sponge materials of Examples 1 to 4 of the present invention were subjected to an in vitro hemolysis test. The test method was as follows: the hemostatic sponges of Examples 1 to 4 were immersed in 1 mL of a 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 samples were 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 rates of the hemostatic sponges of Examples 1 to 4 are all lower than 5%, indicating that they are safe and have no risk of causing hemolysis.

[0057] In vitro antibacterial activity test: The hemostatic sponge materials of Examples 1 to 4 of the present invention were tested for in vitro antibacterial activity. The test method was as follows: the hemostatic sponges of Examples 1 to 4 were placed flat in LB solid culture medium coated with Escherichia coli and Staphylococcus aureus, incubated at 37°C for 12 hours, and the size of the inhibition zone was observed. Figure 5 As shown, Example 4, i.e., the sponge prepared without adding polylysine hydrochloride, had no obvious antibacterial effect, while Examples 1 to 3, i.e., the sponges prepared with adding polylysine salts, all showed obvious effects of inhibiting the growth of Escherichia coli and Staphylococcus aureus, and as the amount of polylysine hydrochloride added increased, the inhibition zone increased, indicating that polylysine hydrochloride gave the sponge an antibacterial effect.

[0058] In summary, the present invention provides a hemostatic sponge using giant salamander mucus as a raw material and a preparation method thereof, wherein the raw materials of the composite sponge include giant salamander mucus and polylysine hydrochloride, and the preparation method of the composite sponge is firstly dissolving giant salamander mucus freeze-dried powder in acetic acid to prepare a solution, then adding polylysine hydrochloride aqueous solution to giant salamander mucus acetic acid solution, and fully and evenly mixing on a rotary mixer, centrifuging the mixed solution, collecting the precipitate, pouring it into a mold for freeze drying, so as to obtain a composite hemostatic sponge. The sponge has good hemostatic performance, a simple preparation process, and low production cost, has broad application potential in trauma hemostasis, and provides a new idea for the utilization of giant salamander resources.

[0059] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a hemostatic and wound healing sponge based on giant salamander mucus, characterized in that: The following steps are involved: The giant salamander mucus acetic acid solution and the polylysine hydrochloride aqueous solution are fully and evenly mixed on a rotary mixer to obtain a giant salamander mucus / polylysine hydrochloride mixed solution; The mixed solution of giant salamander mucus / polylysine hydrochloride is placed in a centrifuge to collect the precipitate and add it to a mold. The mold is then pre-cooled and placed in a vacuum freeze dryer for freeze drying after it is completely frozen, thereby obtaining a hemostatic and wound healing sponge based on giant salamander mucus.

2. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 1, characterized in that: The giant salamander mucus acetic acid solution 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; the concentration of the giant salamander mucus acetic acid solution is 10-15 mg / mL.

3. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 2, characterized in that: In the giant salamander mucus / polylysine hydrochloride mixed solution, the mass ratio of giant salamander mucus freeze-dried powder to polylysine hydrochloride is 10:1-3.

4. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 1, characterized in that: The polylysine hydrochloride aqueous solution is prepared by dispersing polylysine hydrochloride in a water solvent, wherein the concentration of the polylysine hydrochloride aqueous solution is 8-12 mg / mL.

5. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 4, characterized in that: The molecular weight of the polylysine hydrochloride is 2000-5000.

6. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 1, characterized in that: The mold precooling temperature is -85~-75℃, and the precooling time is 12~24h.

7. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 1, characterized in that: The freeze-drying time is 48 to 72 hours.

8. The method for preparing the hemostatic and wound healing sponge based on giant salamander mucus according to claim 1, characterized in that: When collecting the precipitate by centrifugation, the centrifugal speed is 4000~8000r / min and the time is 10~20min.

9. A hemostatic and wound healing-promoting sponge based on giant salamander mucus prepared by the method according to any one of claims 1 to 8.

10. Use of the sponge according to claim 9 in preparing a medical material for hemostasis and wound healing.

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