A method for the preparation of a hemostatic protein sponge

Abstract

The application relates to the field of medical product preparation, and particularly discloses a preparation method of a hemostatic protein sponge, which comprises the following steps: S1, mixing and stirring elastin with water to prepare an elastin solution; S2, adding sodium alginate, carrier biological cellulose and fucoidan into the elastin solution, and uniformly mixing to prepare a mixture; the sodium alginate accounts for 0.1-1% of the elastin solution, and the mass ratio of the sodium alginate, the carrier biological cellulose and the fucoidan is 1:1-2:0.5-1; S3, the mixture is subjected to freeze-drying, tabletting and post-treatment to prepare a finished product; the finished product has the advantages of rapid hemostasis, high mechanical strength, good adhesion, applicability to people with blood coagulation mechanism disorders and difficulty in producing rejection reactions.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical product preparation, and more particularly to a preparation method of a hemostatic protein sponge. BACKGROUND

[0002] Currently, the commonly used hemostatic sponges on the market mainly include polyvinyl alcohol hemostatic sponge, absorbable gelatin sponge, collagen sponge and the like. However, the polyvinyl alcohol hemostatic sponge has poor biocompatibility and degradability and is not absorbable. Although the absorbable gelatin sponge is absorbable, it has poor mechanical properties and is easily broken when encountering blood, and has weak pulling capacity on blood cells and platelets, so it is difficult to stop bleeding for large wounds, and has poor adhesion and is easily detached. In addition, the gelatin sponge needs the activation of blood coagulation factors in the body to promote blood coagulation, so the hemostatic effect is poor for people with coagulation mechanism disorders. The collagen in the collagen sponge is originally derived from the collagen of cattle and pigs, which has cross antigenicity with human collagen, and some patients may have rejection reactions, which may cause adverse reactions such as redness, itching and pain.

[0003] Therefore, how to prepare a hemostatic protein sponge which has the advantages of rapid hemostasis, high mechanical strength, good adhesion, can be used by people with coagulation mechanism disorders and is not prone to rejection is a problem to be solved. SUMMARY

[0004] In order to prepare a hemostatic protein sponge which has the advantages of rapid hemostasis, high mechanical strength, good adhesion, can be used by people with coagulation mechanism disorders and is not prone to rejection, the present application provides a preparation method of a hemostatic protein sponge.

[0005] The present application provides a preparation method of a hemostatic protein sponge, which adopts the following technical scheme:

[0006] A preparation method of a hemostatic protein sponge, comprising the following steps:

[0007] S1, mixing and stirring elastin with water uniformly to prepare an elastin solution;

[0008] S2, adding sodium alginate, carrier biological cellulose and fucoidan to the elastin solution and mixing uniformly to prepare a mixture; the sodium alginate accounts for 0.1-1% of the elastin solution, and the mass ratio of the sodium alginate, the carrier biological cellulose and the fucoidan is 1:1-2:0.5-1;

[0009] S3, freeze-drying, tabletting and post-treatment of the mixture to prepare a finished product.

[0010] By adopting the above technical scheme, the sodium alginate is dispersed in the elastin-like protein solution, the solubility viscosity of the sodium alginate is utilized to improve the cross-linking and bonding effect of the sodium alginate and the elastin-like protein, and the freeze-drying treatment is cooperated, so that the cross-linked network structure has high porosity, and after tabletting, the protein sponge with good adsorption is obtained; the protein sponge has the advantages of rapid hemostasis, high mechanical strength, good adhesion, can be used by people with coagulation mechanism disorder, and is not easy to produce rejection reaction.

[0011] Compared with animal collagen, the elastin-like protein as an engineering recombinant protein has lower rejection in the human body, higher matching degree with the human body, fewer sensitization sources, and is more stable, can be applied in the human body and on the body surface, and cooperates with the good biocompatibility of the elastin-like protein, can be degraded into polypeptides and amino acids in the human body, so as to be absorbed by the human body, and is not easy to produce rejection reaction in the human body, so that the protein sponge has the advantage of not easy to produce adverse reaction.

[0012] When the sodium alginate cooperates with the elastin-like protein and acts on the surface of the wound, the hygroscopicity of the protein sponge is utilized to facilitate the absorption of water in the blood and tissue fluid, and the sodium alginate is dissolved in water to produce viscosity, the high bonding effect of the sodium alginate is utilized to facilitate the stable adhesion of the protein sponge at the wound; not only no stimulation, but also the calcium ions in the wound exudate and blood can exchange with the sodium ions in the sodium alginate, forming a stable gel thin layer on the surface of the wound, increasing the adhesion effect, and releasing the calcium ions to accelerate the formation of blood clots in the end of the capillary, achieving the effect of rapid hemostasis, and the hemostasis does not need to activate the coagulation factor, so that people with coagulation mechanism disorder can also use it without rejection reaction.

[0013] The cross-linking of the elastin-like protein and the sodium alginate makes the tensile strength of the protein sponge higher than that of the collagen sponge and the polyvinyl alcohol hemostatic sponge, so that the protein sponge has a wider application range and the wound is not easy to tear.

[0014] The carrier biological cellulose and fucoidan cooperate with each other, the hydroxyl groups on the surface of the biological cellulose and fucoidan are cross-linked with the carboxyl groups in the sodium alginate and the elastin-like protein to form a network structure, and the biological cellulose itself has a porous adsorption property, which facilitates the adsorption of tissue fluid, blood and the like exuded from the wound, and achieves the effect of rapid hemostasis.

[0015] The high elasticity and flexibility of the biological cellulose and the elasticity and toughness of the cross-linked network further improve the tensile strength of the protein sponge; and the biological cellulose and fucoidan can be degraded in the human body, have no rejection reaction in the human body, and can be used in the human body.

[0016] When the biological cellulose and fucoidan gradually absorb blood and tissue fluid, the biological cellulose is used to absorb water but not to dissolve in water, and the fucoidan is dispersed in water and prone to be in a cluster phenomenon, so as to reduce the molecular spacing and increase the intermolecular force, so as to facilitate the formation of a gel film layer with high cross-linking degree and good adhesion stability on the surface of the wound, thereby promoting wound healing and improving the tensile strength of the protein sponge, so as to prevent the wound from being infected and torn.

[0017] Preferably, the elastin in the elastin solution is 10-650 g / L.

[0018] By adopting the above technical solution, the protein content is limited, so that the protein sponge has the advantages of high mechanical strength, good hemostatic effect and promotion of wound healing.

[0019] Preferably, the carrier biological cellulose is prepared by adhering chitosan micro powder to the biological cellulose loaded with polydopamine solution.

[0020] By adopting the above technical solution, the biological cellulose uses its high adsorbability to adsorb the polydopamine solution, and then uses the adhesion effect of the polydopamine solution to adhere the chitosan micro powder to the surface of the biological cellulose.

[0021] When the carrier cellulose is dispersed in the elastin solution and sodium alginate, the amino groups on the surface of the polydopamine are used to cross-link with the amino groups and carboxyl groups on the surface of the chitosan micro powder, so as to form a relatively dense network structure, increase the adsorption amount of the protein sponge to blood and tissue fluid, and improve the tensile strength of the protein sponge.

[0022] When the protein sponge acts on the wound, the water absorption and swelling effect of the chitosan micro powder is used to expand the occupied area, and the water absorption and swelling property of the biological cellulose is used to facilitate the contact with the surrounding carrier biological cellulose and sodium alginate, so as to fill the pores in the protein sponge by swelling, promote the contact between the adjacent carrier biological cellulose, fucoidan and sodium alginate, and form a gel network film layer with high cross-linking degree and good structure stability on the surface of the wound, thereby improving the tensile strength of the protein sponge in the affected area while improving the adhesion stability.

[0023] Preferably, the post-processing specific steps are as follows:

[0024] The semi-finished product is cut, and the polyortho ester micro powder is adhered to one side of the semi-finished product to obtain the finished product.

[0025] Preferably, the average particle size of the polyortho ester micro powder is 1-3 μm.

[0026] By adopting the technical scheme, the hydrophobic effect of the polyolide micro powder is utilized to realize the hydrophobic effect on the protein sponge side, when the protein sponge acts on the human body, the hydrophilic side can absorb the water in the blood and tissue fluid to play a bonding effect, so that the protein sponge is adhered to the surface of the wound, but the side of the protein sponge away from the wound utilizes the hydrophobicity to prevent the membrane layer in the body from being adhered to the surface of the wound membrane layer, by preventing the adhesion between the membrane layers, it can be applied in the field of internal organ hemostasis to ensure the wound healing and the health of the body.

[0027] Preferably, the S3 and the mixture are freeze-dried, tabletted, cut to obtain a semi-finished product, the semi-finished product is uniformly sprayed with a bonding liquid on one side, and then uniformly sprayed with polyolide micro powder, the mass ratio of the semi-finished product to the bonding liquid and the polyolide micro powder is 100:10-20:15-30, and the finished product is obtained after drying.

[0028] By adopting the technical scheme, the sodium alginate, the carrier biological cellulose and the fucoidan are matched to form a cross-linked network, after the semi-finished product is obtained by freeze-drying and tabletting, the bonding liquid is sprayed on the surface to utilize the adhesion effect of the bonding liquid to adhere the polyolide micro powder to one side of the semi-finished product, so that the finished product is hydrophilic on one side and hydrophobic on the other side, the hydrophilic side acts on the surface of the wound to play a bonding effect and promote wound healing, and the hydrophobic side prevents the adhesion between the membrane layer and the membrane layer where the wound is located, thereby ensuring the health of the human body.

[0029] Preferably, the bonding liquid is composed of a carboxymethyl chitosan aqueous solution and a rhamnolipid solution with a mass ratio of 1:0.05-0.1.

[0030] By adopting the technical scheme, the carboxymethyl chitosan aqueous solution and the rhamnolipid solution are matched, the rhamnolipid solution is hydrophilic on one end and hydrophobic on the other end, the hydrophilic end is adhered to the carboxyl and amino groups in the carboxymethyl chitosan, and the hydrophobic end is adhered to the polyolide micro powder, the adhesion of the carboxymethyl chitosan is matched to realize the adhesion of the polyolide micro powder to the surface of the semi-finished product, so that the finished product protein sponge is hydrophilic on one end and hydrophobic on the other end, the wound healing is ensured, and the adhesion problem of the membrane layer in the body is avoided, thereby protecting the health of the human body.

[0031] Preferably, the freeze-drying temperature is-5 to-25℃.

[0032] Preferably, the freeze-drying time is 1-4h.

[0033] By adopting the technical scheme, the freeze-drying temperature and time are limited, the protein sponge has high porosity, can absorb more exuded blood and tissue fluid, can maintain moisture, is not prone to dryness under long-time use conditions, prolongs the action time of the protein sponge on the wound, and promotes wound healing.

[0034] In summary, this application has the following beneficial effects:

[0035] 1. Elastin-like proteins have high biocompatibility and are less likely to cause rejection reactions in the body; the high adhesion effect of sodium alginate after dissolving in water improves the adhesion stability of the protein sponge on the wound surface; at the same time, the release of calcium ions accelerates the formation of blood clots in the capillary terminals, achieving rapid hemostasis. Furthermore, hemostasis does not require activation of coagulation factors, allowing it to be used by individuals with coagulation disorders without causing rejection reactions; the cross-linking of elastin-like proteins and sodium alginate makes the tensile strength of the protein sponge higher than that of collagen sponges and polyvinyl alcohol hemostatic sponges. Therefore, the protein sponge simultaneously possesses the advantages of rapid hemostasis, high mechanical strength, good adhesion, usability by individuals with coagulation disorders, and low risk of rejection reactions.

[0036] 2. After freeze-drying, the protein sponge develops a high porosity, and chitosan micropowder and bio-cellulose are also easy to prevent contact between each other. As the protein sponge is used on the wound surface and absorbs tissue fluid or blood, the chitosan micropowder can absorb water and swell, and the bio-cellulose can also absorb water and swell. After swelling, it not only promotes contact between each other, but also cross-links and bonds with substances such as sodium alginate and elastin to form a gel film layer. This ensures the stable adhesion of the protein sponge on the wound surface, while improving the adsorption stability of tissue fluid and blood, and making it less prone to backflow problems.

[0037] 3. Chitosan micro powder, carboxymethyl chitosan solution, biological cellulose, and Dunaliella salina polysaccharide all have antibacterial and bactericidal effects, which can minimize bacterial infection at the wound site and thus promote wound healing.

[0038] 4. Limiting the amount of sodium alginate added ensures that the protein sponge will not become too sticky after absorbing blood and tissue fluid, thus affecting wound healing, and will not affect the adhesion stability of the protein sponge at the wound site due to low viscosity; in addition, the cross-linking network of elastin and sodium alginate can also improve water absorption rate, while also quickly stopping bleeding and promoting wound healing. Implementation

[0039] The present application will be further described in detail below with reference to the embodiments.

[0040] All of the following raw materials are medical grade.

[0041] Example of preparation of loaded biocellulose

[0042] Preparation Example 1: Loaded biocellulose was prepared by the following method:

[0043] Weigh out polydopamine and dissolve it in water by stirring to obtain a 5% polydopamine solution by mass fraction.

[0044] Weigh 1 kg of biocellulose and soak and disperse it in 10 kg of polydopamine solution. The average length of the biocellulose is 10-13 μm. Stir at 1000 r / min for 30 min, filter out the biocellulose, and uniformly spray 0.3 kg of chitosan micro powder on its surface. The average particle size of the chitosan micro powder is 0.5-1 μm. After drying and dispersing until the biocellulose does not stick together and agglomerate, the finished product carrier biocellulose is obtained.

[0045] Example of adhesive preparation

[0046] The rhamnolipid solution in the following raw materials was purchased from a medical device manufactured by Wuhan Kanos Technology Co., Ltd.

[0047] Rhamnose lipolipase solution was used; the solid content was 40%; other raw materials and equipment were all commercially available.

[0048] Preparation Example 2: The adhesive liquid was prepared by the following method:

[0049] Weigh out carboxymethyl chitosan and place it in water. Stir until the carboxymethyl chitosan is completely dissolved to obtain a 1% (w / w) carboxymethyl chitosan aqueous solution.

[0050] Add 0.08 kg of rhamnolipin solution to 1 kg of carboxymethyl chitosan aqueous solution, stir at 500 r / min for 20 min, mix evenly, and the finished adhesive liquid is obtained.

[0051] Preparation Example 3: The difference between this preparation example and Preparation Example 2 is that:

[0052] Add 0.05g of rhamnolipin solution to 1kg of carboxymethyl chitosan aqueous solution, stir at 500r / min for 20min, mix evenly, and the finished adhesive liquid is obtained.

[0053] Preparation Example 4: The difference between this preparation example and Preparation Example 2 is that:

[0054] Add 0.1g of rhamnolipin solution to 1kg of carboxymethyl chitosan aqueous solution, stir at 500r / min for 20min, mix evenly, and the finished adhesive liquid is obtained. Example

[0055] The elastin-like protein in the following raw materials was purchased from Beijing Shunxing Biotechnology Co., Ltd.; other raw materials and equipment are commercially available.

[0056] Example 1: A hemostatic protein sponge was prepared using the following method:

[0057] S1. Elastin-like protein is mixed with water and stirred until homogeneous to obtain an elastin-like protein solution; the elastin-like protein content in the elastin-like protein solution is 250 g / L.

[0058] S2. Sodium alginate, the carrier biocellulose and fucoidan prepared in Preparation Example 1 are added to the elastin-like solution. Sodium alginate accounts for 0.5% of the elastin-like solution. The mass ratio of sodium alginate, carrier biocellulose and fucoidan is 1:1.6:0.8. The mixture is mixed evenly to obtain a mixture.

[0059] S3. The mixture is freeze-dried at -40℃ for 36 hours, then pressed into tablets and cut to obtain a semi-finished product. 15g of the adhesive liquid prepared in Preparation Example 2 is evenly sprayed onto one side of 100g of the semi-finished product, and then 20g of polyorthoester micro powder is evenly sprayed on. The average particle size of the polyorthoester micro powder is 1-3μm. After drying, the finished product is obtained.

[0060] The elastin-like sequence number is: VPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGYPGKGVPGKGVPGKGVPGKGVPGKGVPGKKGVPGKGVPGKGVPG KGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPCGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG.

[0061] Example 2: The difference between this example and Example 1 is that:

[0062] S2. Sodium alginate, the carrier biocellulose and fucoidan prepared in Preparation Example 1 are added to the elastin-like solution. Sodium alginate accounts for 0.1% of the elastin-like solution. The mass ratio of sodium alginate, carrier biocellulose and fucoidan is 1:1:0.5. The mixture is mixed evenly to obtain a mixture.

[0063] S3. The mixture is freeze-dried at -40℃ for 36 hours, then pressed into tablets and cut to obtain a semi-finished product. 10g of the adhesive liquid prepared in Preparation Example 3 is evenly sprayed onto one side of 100g of the semi-finished product, and then 15g of polyorthoester micro powder is evenly sprayed on. The average particle size of the polyorthoester micro powder is 1-3μm. After drying, the finished product is obtained.

[0064] Example 3: The difference between this example and Example 1 is that:

[0065] S2. Add sodium alginate, the carrier biocellulose and fucoidan prepared in Preparation Example 1 to the elastin-like solution. Sodium alginate accounts for 1% of the elastin-like solution. The mass ratio of sodium alginate, carrier biocellulose and fucoidan is 1:2:1. Mix evenly to obtain a mixture.

[0066] S3. The mixture is freeze-dried at -40℃ for 36 hours, then pressed into tablets and cut to obtain a semi-finished product. 20g of the adhesive liquid prepared in Preparation Example 4 is evenly sprayed onto one side of 100g of the semi-finished product, and then 30g of polyorthoester micro powder is evenly sprayed on. The average particle size of the polyorthoester micro powder is 1-3μm. After drying, the finished product is obtained.

[0067] Example 4: The difference between this example and Example 1 is that:

[0068] Protein sponges replace the carrier biocellulose with an equal mass of biocellulose.

[0069] Example 5: The difference between this example and Example 1 is that:

[0070] No chitosan powder was added during the preparation of the biocellulose load in the protein sponge.

[0071] Example 6: The difference between this example and Example 1 is that:

[0072] No fucoidan was added to the protein sponge.

[0073] Example 7: The difference between this example and Example 1 is that:

[0074] S3. The mixture is freeze-dried at -40℃ for 36 hours, then compressed and cut to obtain the finished product.

[0075] Example 8: The difference between this example and Example 1 is that:

[0076] The polyorthoester powder was replaced with an equal mass of sodium alginate in the raw materials.

[0077] Example 9: The difference between this example and Example 1 is that:

[0078] No rhamnolipid solution was added to the binder of the raw material. Comparative Example

[0079] Comparative Example 1: The difference between this comparative example and Example 1 is that:

[0080] The elastin solution was replaced with an equal mass of gelatin solution in the raw materials; the gelatin solution was prepared by stirring 250g of gelatin in 750g of warm water until the gelatin was completely dissolved, and the temperature of the warm water was 70℃.

[0081] Comparative Example 2: This comparative example differs from Example 1 in that:

[0082] The elastin solution was replaced with an equal mass of polyvinyl alcohol solution in the raw materials. The polyvinyl alcohol solution was prepared by stirring 250g of polyvinyl alcohol in 750g of water until all the polyvinyl alcohol was dissolved.

[0083] Performance testing

[0084] 1. Hemostatic effect test

[0085] The finished protein sponges were prepared using the methods described in Examples 1-3 and Comparative Examples 1-2, respectively. Experimental pigs weighing 60-70 kg were anesthetized. After anesthesia, the skin of the leg was incised, and the femoral artery was located. The muscle was quickly incised perpendicular to the femoral artery's course using a scalpel, severing the blood vessel and allowing free bleeding for 10 seconds. The protein sponge was then placed on the wound to completely cover it, and pressure was applied to stop the bleeding. After 100 seconds, the protein sponge was turned over from one side to observe the hemostasis. If bleeding continued, the sponge was continued to cover and apply pressure, with observations every 10 seconds. If bleeding stopped, observation continued for 120 seconds to see if bleeding would resume. The time to cessation of bleeding was recorded.

[0086] 2. Mechanical testing

[0087] Finished protein sponges were prepared using the preparation methods of Examples 1-9 and Comparative Examples 1-2, respectively. The tensile strength of collagen sponges was tested according to YY / T1511—2017, and the data were recorded. Samples of the same size were placed in beakers, and test solutions (pure water) preheated to (37±1)℃ were added. The test solution was 50 times the weight of the sample. After the sample was soaked for 1 minute, the tensile strength was tested again, and the data were recorded. The test speed was 100 mm / min.

[0088] 3. Adhesion testing

[0089] The finished protein sponges were prepared using the preparation methods of Examples 1-6 and Comparative Examples 1-2, respectively. The sample size was 5cm×5cm. The sample was placed in a beaker and a test solution (pure water) preheated to (37±1)℃ was added. The test solution was 50 times the weight of the sample. After the sample was soaked for 1 minute, it was applied to the surface of pigskin (the side of the sample with polyorthoester powder was facing away from the pigskin, and the side without polyorthoester powder was applied to the surface of the pigskin). After standing for 5 minutes, the peel strength was tested using a tensile testing machine, and the force (N) when the sample was peeled from the surface of the pigskin was recorded. The average value of 5 samples was recorded.

[0090] 4. Anti-adhesion test

[0091] Finished protein sponges were prepared using the methods described in Examples 1-3, 7-8, and Comparative Examples 1-2, respectively. Experimental pigs weighing 60-70 kg were anesthetized. After anesthesia, the leg skin was incised, and the femoral artery was located. The muscle was quickly incised perpendicular to the femoral artery's direction using a scalpel, severing the blood vessel and allowing free bleeding for 10 seconds. The protein sponge was then placed on the wound to completely cover it, and pressure was applied to stop the bleeding (the side containing polyorthoester micropowder was away from the wound surface, ensuring contact between the polyorthoester micropowder side and the non-invasive membrane layer). All incisional membrane layers were then sutured. After two weeks, the original wound was reopened, and the adhesion effect of the protein sponge to the non-invasive membrane layer was observed and scored. The scoring criteria were as follows: 10 points for no adhesion to the non-invasive membrane layer → 0 points for severe adhesion to the non-invasive membrane layer, making peeling impossible. The scores were recorded.

[0092] 5. Rejection testing

[0093] The finished protein sponges were prepared using the preparation methods of Examples 1-3 and Comparative Example 2, respectively. Experimental pigs weighing 60-70 kg were anesthetized. After anesthesia, the skin of the leg was cut open, and then the femoral artery was located. The muscle was quickly cut open with a scalpel perpendicular to the direction of the femoral artery, and the blood vessel was severed. Free bleeding was allowed for 10 seconds. The protein sponge was placed on the surface of the blood vessel at the wound site, and then the epidermis was sutured. The experimental pigs were observed to see if they showed any rejection reaction.

[0094] Note: In the above experiments, the testing conditions for each indicator were the same except for the sample, following a single variable; " / " in Table 1 indicates that the corresponding sample was not tested for this item, meaning there is no data.

[0095] Table 1 Performance Test Table

[0096]

[0097] As can be seen from Examples 1-3 and Table 1, the protein sponge prepared in this application has good hemostatic effect, high tensile strength, and high tensile strength even after absorbing water. It also has high peel force and no rejection. This indicates that the protein sponge has the advantages of rapid hemostasis, high mechanical strength, good adhesion, and can be used by patients with coagulation disorders without easily causing rejection.

[0098] Combining Examples 1 and 4-9 with Table 1, it can be seen that in Example 4, the protein sponge replaced the carrier biocellulose with an equal mass of biocellulose. In Example 5, no chitosan powder was added during the preparation of the carrier biocellulose. Compared with Example 1, the protein sponges prepared in Examples 4 and 5 had lower tensile strength and peel force than those in Example 1. This indicates that the combination of carrier biocellulose and fucoidan, along with the combination of biocellulose, chitosan powder, and polydopamine solution, utilizes the amino groups on the surface of polydopamine to combine with the amino and carboxyl groups on the surface of chitosan powder, facilitating cross-linking with the carboxyl groups in elastin-like proteins and sodium alginate, thereby increasing the tensile strength of the protein sponge. When the protein sponge is applied to the wound, the water-absorbing and swelling effect of chitosan powder expands its own occupied area. Combined with the water-absorbing and swelling properties of biocellulose, it promotes the contact between adjacent carrier biocellulose, fucoidan, and sodium alginate, forming a gel network film layer with high cross-linking degree and good structural stability on the wound surface. This improves the tensile strength of the protein sponge at the affected area while also enhancing its adhesive stability.

[0099] In Example 6, no fucoidan was added to the protein sponge. Compared to Example 1, the protein sponge prepared in Example 6 had lower tensile strength and peel force than that of Example 1. This indicates that the swelling effect of bio-cellulose, which absorbs water but is insoluble in water, combined with the fact that fucoidan disperses in water and easily clumps together upon contact with water, reduces the molecular spacing and increases intermolecular forces. This facilitates the formation of a highly cross-linked gel film with good adhesion stability on the surface of the wound, thereby promoting wound healing while improving the tensile strength of the protein sponge. It not only reduces the risk of wound infection but also prevents tearing after the wound is stretched.

[0100] In Example 7, the protein sponge was prepared without the use of adhesive liquid and polyorthoester micropowder. Compared with Example 1, the tensile strength and anti-adhesion score of the protein sponge prepared in Example 7 were lower than those in Example 1. This indicates that the adhesive liquid adheres the polyorthoester micropowder to one side of the semi-finished product. Utilizing the hydrophobicity of the polyorthoester micropowder, a hydrophobic layer is easily formed on one side of the protein sponge, allowing the protein sponge to absorb water and adhere to the wound surface on one side, while the other side is less likely to adhere to other in vivo membrane layers. This ensures the health of the substrate while improving the mechanical strength of the protein sponge.

[0101] In Example 8, sodium alginate was used to replace polyorthoester powder in the same mass of raw materials. Compared with Example 1, the anti-adhesion score of the protein sponge prepared in Example 8 was lower than that in Example 1. This indicates that although sodium alginate has adhesive properties, sodium alginate solution causes adjacent membrane layers in the body to adhere, which affects the body's health.

[0102] In Example 9, no rhamnolipin solution was added to the binder of the raw materials. Compared with Example 1, the tensile strength of the protein sponge prepared in Example 9 was lower than that in Example 1. This indicates that the hydrophilic end of rhamnolipin is easy to adhere to the protein sponge, while the hydrophobic end is easy to adhere to the polyorthoester powder, thereby improving the adhesion stability of the polyorthoester powder on the surface of the protein sponge and thus improving the tensile strength.

[0103] Combining Example 1 and Comparative Examples 1-2 with Table 1, it can be seen that in Comparative Example 1, by replacing the elastin-like solution with an equal mass of gelatin solution in the raw materials, the sponge prepared in Comparative Example 1 had a longer hemostatic time, lower tensile strength, lower peel force, and poorer anti-adhesion effect compared to Example 1. This indicates that the hemostatic effect of gelatin sponge is worse than that of protein sponge, its mechanical strength is worse than that of protein sponge, and its adhesion stability on the wound surface and anti-adhesion effect on non-wound locations are worse than those of protein sponge.

[0104] In Comparative Example 2, the same mass of polyvinyl alcohol solution was used to replace the elastin solution. Compared with Example 1, the sponge prepared in Comparative Example 2 had a longer hemostatic time, lower tensile strength, lower peel force, and poorer anti-adhesion effect than Example 1, and exhibited rejection. This indicates that the polyvinyl alcohol sponge not only has poorer mechanical strength and hemostatic effect than the protein sponge, but also is prone to rejection.

[0105] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a hemostatic protein sponge, characterized in that, Includes the following steps: S1. Elastin-like protein is mixed with water and stirred evenly to obtain an elastin-like protein solution. S2. Add sodium alginate, loaded bio-cellulose, and fucoidan to the elastin-like solution and mix thoroughly to obtain a mixture; sodium alginate accounts for 0.1-1% of the elastin-like solution, and the mass ratio of sodium alginate, loaded bio-cellulose, and fucoidan is 1:1-2:0.5-1; the loaded bio-cellulose is prepared by loading bio-cellulose with polydopamine solution and then adhering chitosan micropowder; S3. The mixture is freeze-dried, tableted, and post-processed to obtain the finished product.

2. The method for preparing a hemostatic protein sponge according to claim 1, characterized in that: The elastin-like protein in the solution is 10-650 g / L.

3. The method for preparing a hemostatic protein sponge according to claim 1, characterized in that, The specific steps of the post-processing are as follows: After cutting, a semi-finished product is obtained. Polyorthoester micro powder is then adhered to one side of the semi-finished product to obtain the finished product.

4. The method for preparing a hemostatic protein sponge according to claim 3, characterized in that, The average particle size of the polyorthoester micro powder is 1-3 μm.

5. The method for preparing a hemostatic protein sponge according to claim 4, characterized in that, S3. The mixture is freeze-dried, pressed into tablets, and cut to obtain a semi-finished product. Adhesive liquid is evenly sprayed on one side of the semi-finished product, and then polyorthoester micro powder is evenly sprayed on it. The mass ratio of the semi-finished product to the adhesive liquid and polyorthoester micro powder is 100:10-20:15-30. After drying, the finished product is obtained.

6. The method for preparing a hemostatic protein sponge according to claim 5, characterized in that, The adhesive is composed of an aqueous solution of carboxymethyl chitosan and a solution of rhamnolipin in a mass ratio of 1:0.05-0.

1.

7. The method for preparing a hemostatic protein sponge according to claim 1, characterized in that, The freeze-drying temperature is -5 to -25℃.

8. The method for preparing a hemostatic protein sponge according to claim 1, characterized in that, The freeze-drying time is 1-4 hours.

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