Preparation method of fibrous protein / bacterial cellulose efficient hemostatic patch

Abstract

The invention discloses a preparation method of a fibrous protein / bacterial cellulose efficient hemostatic patch. The preparation method comprises the following steps: (1) in-situ preparation of large-aperture bacterial cellulose; (2) infiltration of fibrinogen in the large-aperture bacterial cellulose; and (3) enzyme digestion and freeze-drying treatment of fibrinogen. The problems that fibrinogen is fast in degradation and poor in stability and mechanical property are solved, the prepared fibrous protein / bacterial cellulose efficient hemostatic patch can prevent secondary bleeding and avoid thrombus to a certain extent, the preparation process is simple and easy to implement, and large-scale preparation and storage can be achieved.

Description

Technical Field

[0001] This invention relates to a hemostatic dressing and its preparation method, specifically to a method for preparing a highly effective fibrin / bacterial cellulose hemostatic patch. It belongs to the fields of biomedical engineering and materials science and technology. Background Technology

[0002] Excessive bleeding, as well as massive bleeding caused by surgery, traumatic wounds, or certain congenital or disease-related conditions (such as clotting factor deficiency and platelet dysfunction), often leads to serious complications and even death, and has always been a serious problem that needs to be addressed.

[0003] Fibrinogen is an endogenous hemostatic factor composed of six paired polypeptide chains (Aα, Bβ, γ)² linked by 29 disulfide bonds. Thrombin can cleave the fibrin peptide fragments of the Aα and Bβ chains into fibrin, producing fibrin monomers (α, β, γ)², which ultimately polymerize into fibrin clots, playing a dominant role in effective hemostasis. Currently, fibrin is considered a highly effective hemostatic material for emergency treatment of large wound bleeding. However, its rapid degradation and poor mechanical properties have limited its application development.

[0004] Bacterial cellulose is a biomedical material composed of three-dimensionally cross-linked fibers secreted by bacteria, possessing excellent mechanical properties, high porosity, and good biocompatibility. Its cross-linking degree can be controlled by methods such as freeze-drying or using pore-forming agents to obtain specific porosities to meet different performance requirements. In this invention, a pore-forming agent is added to a fermentation culture medium to synthesize large-pore bacterial cellulose in situ. Then, a fibrin / bacterial cellulose high-efficiency hemostatic patch is prepared through physical impregnation and enzymatic cleavage to improve the stability and mechanical properties of fibrin. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a highly effective hemostatic patch made of fibrin / bacterial cellulose.

[0006] The objective of this invention is achieved through the following method: a method for preparing a highly effective fibrin / bacterial cellulose hemostatic patch, comprising the following steps: (1) In-situ preparation of macroporous bacterial cellulose: Add an appropriate amount of bioporation agent to the Acetobacter xylinum fermentation medium (5g yeast powder, 5g peptone, 2.7g disodium hydrogen phosphate, 20g glucose, 1.15g citric acid, deionized water to a final volume of 1L, pH=5-7), and sterilize at 115℃ for 30min; then inoculate Acetobacter xylinum into the above medium and ferment for 2-7 days; after the culture is completed, collect the membrane on the liquid surface, wash away the residual bacteria and poration agent in the membrane, and freeze-dry to obtain macroporous bacterial cellulose.

[0007] (2) Infiltration of fibrinogen into macroporous bacterial cellulose: The lyophilized powder of fibrinogen from mammals was dissolved in physiological saline at 37°C to form a homogeneous solution; the above-mentioned large-pore bacterial cellulose was immersed in the fibrinogen solution and left to stand at 4°C for a period of time to allow the fibrinogen solution to fully penetrate the cellulose network. (3) Enzymatic digestion and lyophilization of fibrinogen: The obtained wet film was quickly rinsed with physiological saline to remove the fibrinogen that was wetted on the surface. After being treated with thrombin at 37°C for a certain period of time, it was transferred to -80°C for freezing and then vacuum dried.

[0008] Preferably, the biopore-forming agent in step (1) is starch, paraffin, agar microspheres, xanthan gum, etc.

[0009] Preferably, the concentration of the bioporation agent in step (1) is 0.5-30% (w / v).

[0010] Preferably, the mammal from which the fibrinogen is derived in step (2) is one of the following: human, cow, sheep, horse, or pig.

[0011] Preferably, the concentration of fibrinogen in step (2) is 0.1 to 1000 mg / ml.

[0012] Preferably, the concentration of thrombin in step (3) is 0.2 to 1000 IU / ml.

[0013] Preferably, the thrombin action time in step (3) is 0 to 24 hours.

[0014] This invention not only solves the problems of rapid fibrinogen degradation, poor stability and mechanical properties, but also the prepared fibrin / bacterial cellulose high-efficiency hemostatic patch can prevent secondary bleeding and avoid thrombosis to a certain extent. The preparation process is simple and easy to implement, and it can be prepared and stored in large quantities.

[0015] This invention not only solves the problems of rapid degradation, poor stability, and poor mechanical properties of fibrinogen, but also has the following advantages compared with existing technologies: 1. Combining bacterial cellulose with fibrinogen can improve the mechanical properties of the bandage and, to some extent, prevent thrombosis caused by excessively high fibrinogen concentration at the site of blood loss. 2. The intermolecular forces between bacterial cellulose and fibrinogen can slowly and continuously release fibrin peptides, preventing secondary bleeding; 3. Adding the bioporation agent directly to the bacterial cellulose culture medium can prepare large-pore bacterial cellulose in situ, improve the infiltration degree of fibrinogen, reduce the accumulation of fibrinogen on the cellulose membrane surface, maintain the activity of fibrinogen, and improve hemostasis efficiency. 4. Compared with pure fibrin hemostatic patches, the fibrin / bacterial cellulose high-efficiency hemostatic patch of the present invention has low preparation cost, simple and easy process, and can be prepared and stored in large quantities. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 Macroscopic morphology of the fibrin / bacterial cellulose high-efficiency hemostatic patch before and after contact with blood; Figure 2 The results of in vitro coagulation time testing for fibrin / bacterial cellulose high-efficiency hemostatic patch. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0018] Example 1 A highly effective hemostatic patch made of fibrin / bacterial cellulose is prepared according to the following steps: (1) In-situ preparation of macroporous bacterial cellulose: 1) Starch was used as a pore-forming agent. 2.5g of starch was weighed and sterilized under ultraviolet light for 30min, and then dissolved in 10mL of sterile deionized water at 80℃ for 20min to obtain starch microsphere solution. 2) Preparation of Acetobacter xylinum culture medium: Weigh 0.5g yeast powder, 0.5g peptone, 0.27g Na2HPO4, 2g glucose, and 0.115g citric acid. Add deionized water to a final volume of 100mL, adjust the pH of the solution to 6.8, and then transfer it to an Erlenmeyer flask. Seal the flask and sterilize it at 121℃ for 20min. After inoculating the above starch microsphere solution and Acetobacter xylinum (8% inoculum) into the above culture medium, fermentation culture was carried out for 2-7 days; After the culture was completed, the membrane on the liquid surface was collected, placed in 0.1M NaOH solution, and allowed to stand at room temperature for 24 hours. It was then rinsed with a large amount of deionized water until the fiber surface was neutral to remove residual bacteria from the membrane. 2% amylase was added to dissolve the residual starch at 65℃. After repeated rinsing with a large amount of deionized water, the membrane was freeze-dried to obtain large-pore bacterial cellulose. (2) Infiltration of fibrinogen into macroporous bacterial cellulose: Weigh out human fibrinogen powder and dissolve it in physiological saline at 37°C to obtain a solution of 0.1 mg / mL; immerse lyophilized macroporous bacterial cellulose in the fibrinogen solution and place it at 4°C overnight to allow the fibrinogen solution to fully penetrate the cellulose network and obtain a wet film; (3) Enzymatic digestion and lyophilization of fibrinogen: The obtained wet film was quickly rinsed with physiological saline and then immersed in 10 U / mL thrombin and placed at 37°C for 2 hours. The resulting product was rinsed clean with physiological saline at 37°C, transferred to -80°C for freezing, and then vacuum dried to obtain a hemostatic patch.

[0019] Example 2 A highly effective hemostatic patch made of fibrin / bacterial cellulose is prepared according to the following steps: (1) In-situ preparation of macroporous bacterial cellulose: 1) Using liquid paraffin as a pore-forming agent, 160 mL of solid paraffin was placed in a beaker and heated until melted to obtain liquid paraffin at 50°C. 200 mL of a 0.5% polyvinyl alcohol solution was prepared and heated and stirred to 75°C. The liquid paraffin was slowly added to the 200 mL polyvinyl alcohol solution, and stirred until homogeneous to form an oil-water emulsion. The oil-water emulsion was poured into ice-salt water (22.5 wt%, solute NaCl) at -20°C, causing the paraffin droplets in the oil-water emulsion to rapidly solidify into solid paraffin microspheres, thus obtaining a mixture. The mixture was then wet-sieved using a combination of 10-mesh and 20-mesh standard vibrating sieves at a vibration frequency of 150 rpm, while continuously rinsing with purified water at 4°C to accelerate the sieving of the paraffin microspheres and remove residual dispersant and ice-salt water. The paraffin microspheres obtained after wet sieving were filtered, pre-frozen in a -20°C freezer, and then freeze-dried in a freeze dryer. 2) Preparation of Acetobacter xylinum culture medium: Weigh out 5g yeast powder, 5g peptone, 2.7g Na₂HPO₄, 20g glucose, and 1.15g citric acid. Dilute to 1L with deionized water and adjust the pH to 6.8. Pour into an Erlenmeyer flask, seal, and sterilize at 121℃ for 20 minutes. 2g of paraffin microspheres and Acetobacter xylinum (8% inoculum) were inoculated into the above culture medium and fermented for 2-7 days. After the culture was completed, the membrane on the liquid surface was collected and washed repeatedly with 0.1M NaOH solution and 1% Berol EZ-1 surfactant at 80℃ to remove residual bacteria and paraffin. The macroporous bacterial cellulose was obtained by freeze drying. (2) Infiltration of fibrinogen into macroporous bacterial cellulose: Weigh out human fibrinogen powder and dissolve it in physiological saline at 37°C to obtain a solution of 0.02 mg / mL; immerse lyophilized macroporous bacterial cellulose in the fibrinogen solution and place it at 4°C overnight to allow the fibrinogen solution to fully penetrate the cellulose network, thus obtaining a wet membrane; (3) Enzymatic digestion and lyophilization of fibrinogen: The obtained wet film was quickly rinsed with physiological saline and then immersed in 5 U / mL thrombin at 37°C for 1 hour. The resulting product was rinsed clean with 37°C physiological saline, transferred to -80°C for freezing and vacuum drying to obtain a hemostatic patch.

[0020] Material characterization 1. Macroscopic morphology: Figure 1 The macroscopic morphology of the fibrin / bacterial cellulose high-efficiency hemostatic patch (a) is shown as fluffy, regular, and white. After the patch came into contact with anticoagulated whole blood for 30 minutes (b), obvious blood coagulation was observed.

[0021] 2. Coagulation test: Place the hemostatic patch into a centrifuge tube, add fresh SD rat citrate-anticoagulated whole blood, slightly tilt the centrifuge tube, and observe and record the liquid coagulation time. See [link to in vitro coagulation test results] for details. Figure 2 The hemostatic patch prepared in this invention (approximately 151 seconds) has a significantly improved hemostatic time compared to the control group (pure bacterial cellulose, approximately 302 seconds), indicating that this invention has a better hemostatic effect.

[0022] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a high-efficiency hemostatic patch of fibrin / bacterial cellulose, characterized in that, The method comprises the following steps: (1) In-situ preparation of large-pore bacterial cellulose: Preparation of fermentation medium of X. camphorium: 5 g of yeast powder, 5 g of peptone, 2.7 g of Na2HPO4, 20 g of glucose, 1.15 g of citric acid, and deionized water to 1 L, pH = 5-7; Add an appropriate amount of pore-forming agent to the fermentation medium of X. camphorium, sterilize at 115°C for 30 min, then, Inoculate X. camphorium into the above-mentioned medium and ferment for 2-7 days; After the culture ends, collect the membrane on the liquid surface, remove the residual bacteria and pore-forming agent after washing, and obtain large-pore bacterial cellulose through freeze-drying treatment; (2) Infiltration of fibrinogen in large-pore bacterial cellulose: Dissolve fibrinogen freeze-dried powder of mammalian origin into a uniform solution with saline at 37°C; immerse the above-mentioned large-pore bacterial cellulose into the fibrinogen solution and stand at 4°C for a period of time to allow the fibrinogen solution to fully penetrate the cellulose network; (3) Enzymatic digestion of fibrinogen and freeze-drying treatment: After the above-mentioned wet membrane is quickly washed with saline to remove the surface-infiltrated fibrinogen, transfer it to -80°C after the action of thrombin at 37°C for a certain period of time, and then vacuum dry.

2. The method of claim 1, wherein the fibrin / bacterial cellulose high- efficiency hemostatic patch is prepared by the steps of: The pore-forming agent is any one or a combination of starch, paraffin, agar microspheres, and xanthan gum.

3. The method of claim 1, wherein the fibrin / bacterial cellulose high- efficiency hemostatic patch is prepared by the steps of: The concentration of the biological pore-forming agent is 0.1-30% (w / v).

4. The method of claim 1, wherein the fibrin / bacterial cellulose high- efficiency hemostatic patch is prepared by the steps of: The mammalian source of fibrinogen is one of human, bovine, sheep, horse, and pig.

5. The method of claim 1, wherein the fibrin / bacterial cellulose high- efficiency hemostatic patch is prepared by the steps of: The concentration of fibrinogen is 0.1-1000 mg / ml.

6. The method of claim 1, wherein the fibrin / bacterial cellulose high- efficiency hemostatic patch is prepared by the steps of: The concentration of thrombin is 0.2-1000 IU / ml.

7. The method of claim 1, wherein the fibrin / bacterial cellulose high- efficiency hemostatic patch is prepared by the steps of: The thrombin action time is 0-24 h.

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