Bacterial nanocellulose composite tube as well as preparation method and application thereof

Abstract

The invention relates to a bacterial nanocellulose composite tube and a preparation method and application thereof, a three-dimensional network of bacterial nanocellulose is reserved, and the three-dimensional network is fully utilized as a dispersion phase to disperse introduced other macromolecules to form a homogeneous system; drugs such as heparin are encapsulated in situ or bioactive substances such as heparin are chemically grafted in the wall of the bacterial nanocellulose tube. Proteins such as silk fibroin, collagen, gelatin and fibronectin support adhesion and proliferation of cells, and the composite tube loaded with heparin has excellent anticoagulant activity and an effect of promoting endothelial cell function expression. The preparation process of the composite tube is simple, and the composite tube has a good application prospect in biomedical application of tubular artificial organs such as artificial blood vessels, artificial nerve conduits, artificial esophagus, artificial tracheas, bile ducts and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a bacterial nanocellulose composite tube and a preparation method and application thereof. Background Art

[0002] With the aging of the population and the irregular diet and lifestyle of many young people, the number of patients with cardiovascular diseases is getting younger and younger, and the number of patients is increasing year by year. Atherosclerosis and peripheral vascular disease are the main types of cardiovascular diseases, and clinical symptoms include arterial stenosis, embolism, chronic ischemia of the lower limbs, etc. Surgical treatment includes percutaneous transluminal angioplasty, bypass surgery, etc. There are commercial products for large-caliber blood vessels for vascular replacement, including expanded polytetrafluoroethylene and polyethylene terephthalate. These two materials are highly hydrophobic and have poor tissue compatibility, which is not conducive to rapid endothelialization of the inner surface and has a low long-term patency rate. Due to the small inner diameter of small-caliber blood vessels (<6mm) and the slow blood flow rate, thrombosis and intimal hyperplasia stenosis are easily formed in the lumen of small-caliber artificial blood vessels. At present, there are no small-caliber vascular products available for clinical use.

[0003] Bacterial Nano-Cellulose (BNC) is the most widely distributed natural polysaccharide in nature. It is a product produced by bacteria and secreted outside the cell. BNC has a three-dimensional nano-network structure similar to the natural extracellular matrix ECM, which provides a three-dimensional microenvironment for cell adhesion and proliferation. BNC contains a large number of hydroxyl groups and is a hydrophilic polymer, which is not conducive to platelet adhesion and thrombosis. Due to the high Young's modulus, good tensile properties and good biocompatibility in vivo of BNC tubes, they can be used as the base material for small-caliber artificial blood vessels. However, the low long-term patency rate of BNC tubes, weak long-term anticoagulation performance and slow endothelialization rate are the problems that need to be solved as small-caliber artificial blood vessels.

[0004] Silk fibroin (SF) is a natural polymer composed of heavy chain polypeptides, light chain polypeptides and glycoprotein P25. It has excellent properties such as low immunogenicity, cell compatibility and tissue affinity. It has been widely studied in the field of tissue engineering and can be used to prepare artificial blood vessels, wound dressings, cartilage, nerve conduits, etc., and SF sutures are FDA-approved safe materials that can be used in vivo. It has been reported in the literature that silk fibroin can promote the proliferation and adhesion of endothelial cells and smooth muscle cells and is a high-quality material for preparing artificial blood vessels. Common methods for preparing SF artificial blood vessels include weaving, freeze drying, frozen phase separation, 3D printing and electrospinning. However, SF lacks anticoagulant properties. Due to the small diameter of the lumen of small-caliber blood vessels and the slow blood flow rate, SF is prone to early thrombosis when used as a small-caliber artificial blood vessel, which reduces the long-term patency of SF blood vessels; in addition, SF has insufficient mechanical properties and a too fast degradation rate in vivo.

[0005] Gelatin (Gel) is a partial hydrolysis product of collagen. It exists in the form of collagen-like helical conformation and coiled conformation. The amino acid side chain contains multiple functional groups such as -NH, -OH, and -COOH. It has good blood compatibility, cell compatibility, and low immunogenicity. Recombinant human collagen protein (RHC) has a complete triple helical structure, and its performance is better than that of natural human collagen. It has higher biological activity and hydrophilicity. Fibronectin (Fn) is one of the components of the extracellular matrix ECM. It is a high molecular weight glycoprotein. Its RGD polypeptide chain affects cell adhesion and migration, and other adhesion sites of fibronectin molecules can bind to heparin. However, gelatin, recombinant human collagen, and fibronectin do not have anticoagulant properties. When used as small-caliber artificial blood vessels, acute coagulation is prone to occur, which is not conducive to the smooth blood flow of small-caliber blood vessels. In addition, its mechanical properties are weak, and its degradation rate in vivo is too fast, so it is not suitable as an artificial blood vessel material alone.

[0006] Heparin (Hep) is a commonly used anticoagulant in clinical practice. It contains hydroxyl, carboxyl, amino and sulfonic acid active groups, which can bind to antithrombin III, reduce platelet aggregation and activation, and reduce inflammatory response.

[0007] If a technology can be developed that can maintain the high activity of heparin while slowly releasing it to exert its anticoagulant effect for a long time, it will effectively promote the application of anticoagulant devices such as small-caliber artificial blood vessels. Summary of the invention

[0008] In view of the defects of the prior art, the technical problem to be solved by the present invention is to provide a bacterial nanocellulose composite tube and a preparation method and application thereof, and in particular to a protein / bacterial nanocellulose composite tube and a composite tube loaded with active pharmaceutical ingredients and their preparation and application in medical devices such as blood vessels.

[0009] The present invention introduces bacterial nanocellulose with good biocompatibility, natural protein (such as silk protein, fibronectin, gelatin) or human recombinant collagen and heparin with anticoagulant properties to obtain protein / bacterial nanocellulose composite tubes and heparin-loaded protein / bacterial nanocellulose composite tubes.

[0010] The invention provides a bacterial nanocellulose composite tube. The bacterial nanocellulose composite tube is a protein / bacterial nanocellulose composite tube or a medicine / protein / bacterial nanocellulose composite tube.

[0011] Furthermore, the bacterial nanocellulose composite tube is a protein / bacterial nanocellulose composite tube, or a protein / bacterial nanocellulose composite tube with drugs (such as heparin, etc.) grafted or encapsulated on the surface; specifically, it is a protein-filled nanocellulose network structure composite tube, or a protein / bacterial nanocellulose composite tube with drugs (such as heparin, etc.) grafted on the surface, or a protein / bacterial nanocellulose composite tube with drugs (such as heparin, etc.) encapsulated.

[0012] Preferably, the protein is a biocompatible protein; wherein the biocompatible protein includes one or more of silk fibroin, fibronectin, gelatin, and human recombinant collagen; the drug includes one or more of heparin, VEGF, and adhesion peptide;

[0013] Preferably, in the protein / bacterial nanocellulose composite tube, the protein encapsulates the bacterial nanocellulose and / or the protein particles are embedded in the three-dimensional network of the bacterial cellulose; further, the protein particles are protein microspheres;

[0014] Preferably, the drug in the drug / protein / bacterial nanocellulose composite tube is grafted onto the surface of the protein-filled bacterial nanocellulose tube, or the protein microsphere particles encapsulating the drug are embedded in the three-dimensional network of bacterial cellulose, wherein the protein-filled bacterial nanocellulose tube is protein-wrapped bacterial nanocellulose and / or protein particles are embedded in the three-dimensional network of bacterial cellulose; further, the protein particles are protein microspheres.

[0015] The present invention provides a method for preparing a bacterial nanocellulose composite tube, including method 1, method 2, method 3 or method 4;

[0016] Method 1: Fill the mixed solution into the tube wall of bacterial nanocellulose tube by liquid pressure, then fix the protein or protein / drug in the tube wall of bacterial nanocellulose by freezing phase separation, and wash to obtain bacterial nanocellulose composite tube; wherein the mixed solution is a mixed solution of protein solution and ethanol, or a mixed solution of protein / drug solution and ethanol;

[0017] Method 2: The mixed solution of protein solution and ethanol is filled into the tube wall of bacterial nanocellulose tube by liquid pressure, and then the protein is fixed in the tube wall of bacterial nanocellulose by frozen phase separation technology, and the residual reagents such as ethanol are washed away, and then the tube wall is immersed in a drug solution and reacted to obtain a bacterial nanocellulose composite tube;

[0018] Method 3: Fill the tube wall of bacterial nanocellulose tube with protein solution or a mixed solution of protein and drug, then place it in genipin solution for cross-linking, wash (deionized water to remove uncomplexed substances and cross-linking agent), and obtain bacterial nanocellulose composite tube;

[0019] Method 4: Fill the tube wall of bacterial nanocellulose tube with protein solution, then place it in genipin solution for cross-linking to fix the protein in the tube wall of bacterial nanocellulose, wash (deionized water to remove uncomplexed protein and cross-linking agent), and then immerse it in drug solution to react to obtain bacterial nanocellulose composite tube.

[0020] In the method 1-4, the bacterial nanocellulose tubes are prepared by fermenting with a bioreactor, acetobacter and culture medium, and the bacterial nanocellulose tubes with an endotoxin content of ≤0.25EU / mL are obtained after purification.

[0021] The purification is as follows: the bacterial nanocellulose tubes are immersed in a 0.1-0.2M NaOH solution and boiled at 80°C for 4-8 hours, then replaced with deionized water and heated at 80°C for 4-8 hours, and new deionized water is replaced every 2 hours to remove a large amount of culture medium and bacteria; 1% (v / v, mL / mL) acetic acid solution is added for neutralization, and ultrapure water is repeatedly rinsed until the pH is neutral. The fully washed bacterial nanocellulose tubes are placed in ultrapure water and sterilized at 115-121°C for 8-10 times (new ultrapure water is replaced before each sterilization) to remove endotoxins in the bacterial nanocellulose tubes.

[0022] The bioreactor includes three components, the first component is two cylindrical hollow silicone plugs, the second component is a cylindrical rod, and the third component is a breathable and waterproof silicone tube; the cylindrical hollow silicone plugs are respectively arranged at the upper and lower ends of the silicone tube, and the cylindrical rod is inserted into the cylindrical hollow silicone plugs.

[0023] The type of bacterial nanocellulose tube in the method 1-4 is a G-BNC tube, which is prepared by fermentation in an outer silicone tube reactor. The inner layer of the outer silicone tube reactor is an oxygen-impermeable rod, and the outer layer is an oxygen-permeable silicone tube. The oxygen-impermeable rod is fixed in the middle of the oxygen-permeable silicone tube through a cylindrical hollow silicone plug, and the bacterial nanocellulose tube bioreactor can be obtained after high temperature and high pressure sterilization at 115-121°C.

[0024] Preferably, in methods 1 to 4, the inner diameter of the bacterial nanocellulose tube is 2-6 mm, the outer diameter is 3-9 mm, and the wall thickness is 0.5-2.5 mm; the bacterial nanocellulose tube is pretreated to remove some free water in the tube wall, wherein the pretreatment is high temperature drying, room temperature air drying or filter paper water absorption.

[0025] Furthermore, the pretreatment in methods 1 to 4 is drying at 40-60° C. for 1-3 h, air drying at room temperature for 6-24 h, or absorbing water on filter paper for 0.5-5 min.

[0026] The flow rate of the solution filled in the tube wall of the bacterial nanocellulose tube by liquid pressure technology described in Method 1 and Method 2 is 0.05-0.5 mL / s, and the flow rate is 0.1-0.5 mL / cm.

[0027] In the method 1 and the method 2, the bacterial nanocellulose tube is sleeved on a rod-shaped material for frozen phase separation, wherein the diameter of the rod-shaped material is 2-6 mm; the rod-shaped material is one or more of glass, stainless steel, plastic, etc.

[0028] The frozen phase separation in the method 1 and the method 2 is first freezing and then thawing, wherein the freezing is freezing at -20°C to -196°C for 12-24 hours, and the thawing is thawing at room temperature for 1-4 hours.

[0029] The mixed solution of protein solution and ethanol in method 1 and method 2, wherein the concentration of the protein solution is 0.1%-5% (m / v, g / mL); the ratio of the protein solution to ethanol is 10:1-1:1 (v:v, mL:mL), wherein the preferred ratio is 4:1;

[0030] In the method 1, the protein / drug solution and the mixed solution of ethanol, wherein the concentration of protein in the protein / drug mixed solution is 0.1%-5% (m / v, g / mL), and the concentration of the drug is 1-20g / L (m / v); the ratio of the protein and drug mixed solution to ethanol is 10:1-1:1 (v:v, mL:mL), wherein the preferred ratio is 4:1.

[0031] The method for removing the residual reagent in the method 1 and the method 2 is to wash with deionized water or ultrapure water for 3-5 times by oscillation or ultrasonic washing for 3-5 times.

[0032] In the method 3 and the method 4, the tube wall filled with the bacterial nanocellulose tube is made by oscillating immersion or liquid pressure technology, wherein the oscillating immersion temperature is 4°C-25°C, and the time is 12-24h; the flow rate of the solution in the liquid pressure technology is 0.05-0.5mL / s, and the flow rate is 0.1-0.5mL / cm;

[0033] In the method 3 and the method 4, the cross-linking in the genipin solution is performed by static cross-linking for 1-12 hours.

[0034] The concentration of the protein solution in methods 3 and 4 is 0.1%-5% (m / v, g / mL); the concentration of the genipin solution is 0.5%-2% (m / v, g / mL);

[0035] In the method 3, the concentration of the protein in the mixed solution of the protein and the drug is 0.1%-5% (m / v, g / mL), and the concentration of the drug is 1-20 g / L.

[0036] In the method 2 and the method 4, the drug-containing solution is immersed and reacted for 12-24 hours; the drug-containing solution is a drug-containing EDC / NHS cross-linking system, wherein the concentration of the drug is 1-20 g / L, the concentration of EDC is 0.06-0.12 M, and the concentration of NHS is 0.03-0.06 M.

[0037] Furthermore, the drug-containing solution in Method 2 and Method 4 is an EDC / NHS cross-linking system containing heparin, wherein the concentrations of the components are as follows: the concentration of heparin is 1-20 g / L (m / v, g / mL), the concentration of NaCl is 0.25-0.5 M, the concentration of MES is 0.05-0.1 M, the concentration of EDC is 0.06-0.12 M, and the concentration of NHS is 0.03-0.06 M.

[0038] In the method 2 and the method 4, the reaction temperature is 25-37° C., and the oscillation reaction time is 12-24 hours. After the reaction is completed, the residual reagent is removed by repeated washing, wherein the method for repeatedly washing to remove the residual reagent is oscillating washing with deionized water or ultrapure water for 3-5 times or ultrasonic washing for 3-5 times.

[0039] The present invention provides an application of any bacterial nanocellulose composite tube in the preparation of tubular artificial organs, such as artificial blood vessels, artificial nerve conduits, artificial esophagus, artificial trachea, bile duct and other tubular artificial organs.

[0040] The present invention provides the preparation of the protein / bacterial nanocellulose composite tube and the heparin-loaded protein / bacterial nanocellulose composite tube and their application in medical devices such as artificial blood vessels, neural tissue engineering scaffolds, neural catheters, tracheal scaffolds, artificial esophagus, etc.

[0041] The present invention relates to the preparation and application of protein / bacterial nanocellulose composite tubes and heparin-loaded protein / bacterial nanocellulose composite tubes. The macroscopic morphology of the silk fibroin / bacterial nanocellulose composite tubes and the heparin-loaded silk fibroin bacterial nanocellulose composite tubes is white opaque tubular, and the microscopic morphology is that the silk fibroin is wrapped on the bacterial nanofiber, and the silk fibroin or silk fibroin / heparin is embedded in the three-dimensional network pores in the shape of microsphere particles. The preparation method comprises: preparation and pretreatment of bacterial nanocellulose tubes; removal of part of the water in the bacterial nanocellulose tube wall; preparation of silk fibroin / ethanol solution or silk fibroin / heparin / ethanol solution; injection of the solution into the BNC tube wall, and freezing phase separation.

[0042] The present invention utilizes an efficient, green, environmentally friendly and fully utilized liquid pressure technology to inject a silk fibroin / ethanol solution or a silk fibroin / heparin / ethanol solution into the bacterial nanocellulose tube wall, and then utilizes a frozen phase separation method to in-situ fix the silk fibroin or the silk fibroin and heparin in the bacterial nanocellulose tube, and washes the residual ethanol or the unfixed silk fibroin and heparin with deionized water to obtain a silk fibroin / bacterial nanocellulose composite tube and a silk fibroin / bacterial nanocellulose composite tube encapsulating heparin. The silk fibroin / bacterial nanocellulose composite tube is immersed in a heparin solution, and the heparin is chemically grafted on the surface of the silk fibroin / bacterial nanocellulose composite tube using an EDC / NHS cross-linking system to obtain a surface-grafted heparin / silk fibroin / bacterial nanocellulose composite tube.

[0043] The inner surface of the composite tube prepared by the present invention supports the adhesion and proliferation of endothelial cells and smooth muscle cells, has good blood compatibility, and the platelet adhesion of the composite tube loaded with heparin is lower than that of the bacterial nanocellulose tube surface, and the whole blood coagulation time and plasma recalcification time are significantly prolonged. It has good application prospects in the biomedical applications of tubular artificial organs such as artificial blood vessels, artificial nerve conduits, artificial esophagus, artificial trachea, bile ducts, etc.

[0044] Although BNC tubes have hydrophilicity and good cell compatibility, their long-term patency rate is low when implanted in animals, and intimal hyperplasia is easily formed in the lumen. BNC has fewer active groups and weak electronegativity, making it difficult to add bioactive factors by chemical or physical methods. The present invention uses proteins with excellent biocompatibility such as silk fibroin as carriers to graft or encapsulate bioactive factors such as heparin, VEGF, and adhesion peptides to improve the anti-coagulation performance of BNC tubes and the proliferation rate of endothelial cells, thereby achieving the purpose of rapid endothelialization and long-term patency of BNC composite tubes in vivo.

[0045] The present invention retains the three-dimensional network of bacterial nanocellulose, and makes full use of the three-dimensional network to disperse other polymers introduced as the dispersed phase to form a homogeneous system, and in situ encapsulates heparin or chemically grafts heparin and other bioactive substances in the bacterial nanocellulose tube wall. Proteins such as silk fibroin, collagen, gelatin and fibronectin support cell adhesion and proliferation, and the heparin-loaded composite tube has excellent anticoagulant properties and the effect of promoting the expression of endothelial cell function. The preparation process of the composite tube is simple, and it has good application prospects in the biomedical applications of tubular artificial organs such as artificial blood vessels, artificial nerve conduits, artificial esophagus, artificial trachea, bile ducts, etc.

[0046] Beneficial Effects

[0047] (1) The preparation process of the present invention is simple, efficient, environmentally friendly, and saves reagents.

[0048] (2) The inner surface of the silk fibroin / bacterial nanocellulose composite tube (BNC / SF composite tube) prepared by the present invention supports the adhesion and proliferation of endothelial cells and smooth muscle cells and has good blood compatibility.

[0049] (3) The heparin-encapsulated composite tube of the present invention has a heparin release effect of at least 2 weeks, and its inner surface promotes the proliferation and spreading of endothelial cells and smooth muscle cells, and promotes the expression of endothelial cell function; the amount of platelets adhered to the inner surface is less than that of the bacterial nanocellulose tube, and the whole blood coagulation time and plasma recalcification time are significantly prolonged.

[0050] (4) The present invention introduces silk fibroin and human recombinant collagen into bacterial nanocellulose tubes, forms microspheres in situ and encapsulates bioactive molecules such as heparin and VEGF, and the introduced proteins can provide more grafting sites for the chemical grafting sites of heparin. Compared with bacterial nanocellulose tubes, the heparin / silk fibroin / bacterial nanocellulose composite tubes using the chemical grafting method have lower platelet adhesion, significantly prolonged whole blood coagulation time and plasma recalcification time.

[0051] (5) The protein / bacterial nanocellulose composite tube and the heparin-loaded protein / bacterial nanocellulose composite tube prepared by the present invention also have good application prospects in vascular repair stents, nerve repair stents, and tracheal stents. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A bioreactor according to an embodiment of the present invention;

[0053] Figure 2 The microscopic morphology of the silk fibroin / bacterial nanocellulose composite tube at different silk fibroin concentrations described in Example 1;

[0054] Figure 3 The microscopic morphology of the silk fibroin / bacterial nanocellulose composite tube when the ratio of silk fibroin to ethanol is 10:1-1:1 (v:v, mL:mL) in Example 2;

[0055] Figure 4 The results of endothelial cell proliferation on the inner surface of the BNC / SFMPs-Hep composite tube described in Example 3;

[0056] Figure 5 The FE-SEM image and live cell staining image of the endothelial cells on the inner surface of the BNC / SFMPs-Hep composite tube described in Example 3;

[0057] Figure 6 The whole blood coagulation curve (A), plasma recalcification curve (B), hemolysis rate (C) and platelet adhesion FE-SEM image (D) of the BNC / SFMPs-Hep composite tube described in Example 3;

[0058] Figure 7 This is a fluorescence staining image of the rat abdominal aorta replaced by the BNC / SFMPs-Hep composite tube described in Example 3 for 5 months;

[0059] Figure 8 The microscopic morphology of the inner and outer surfaces and cross-section of the BNC / Hep@SFMPs ​​composite tube described in Example 4;

[0060] Fig. 9 The whole blood coagulation curve (A), plasma recalcification curve (B), plasma recalcification time (C), hemolysis rate (D) and platelet adhesion FE-SEM image (E) of the BNC / Hep@SFMPs ​​composite tube described in Example 4;

[0061] Fig.10 The cytotoxicity results of the BNC / Hep@SFMPs ​​composite tube described in Example 4;

[0062] Fig.11 The live cell staining image and FE-SEM image of human umbilical vein endothelial cells on the inner surface of the BNC / Hep@SFMPs ​​composite tube described in Example 4;

[0063] Fig.12 is the expression level of VEGFA and IL-6 genes of human umbilical vein endothelial cells on the inner surface of the BNC / Hep@SFMPs ​​composite tube described in Example 4;

[0064] Fig.13 1 and 2 show the heparin encapsulation amount and heparin release curve of the BNC / Hep@SFMPs ​​composite tube at different SF concentrations described in Example 5.

[0065] Fig.14 The macroscopic morphology and FTIR images of the BNC / RHC composite tube described in Example 7;

[0066] Fig.15 is a SEM image of the BNC / RHC composite tube described in Example 7;

[0067] Fig.16 This is a macroscopic morphology of the BNC / Gel / Hep composite tube described in Example 9;

[0068] Fig.17 The toluidine blue staining image and FTIR image of the BNC / Gel / Hep composite tube described in Example 9;

[0069] Fig.18 The SEM image and fiber diameter of the BNC / Gel / Hep composite tube described in Example 9;

[0070] Fig.19is the surface heparin grafting amount of the BNC / Gel / Hep composite tube and the BNC / RHC / Hep composite tube described in Example 9 and Example 10;

[0071] Fig. 20 The macroscopic morphology and FTIR images of the BNC / Gel composite tube described in Example 11;

[0072] Fig.21 This is the SEM image of the BNC / Gel composite tube described in Example 11. DETAILED DESCRIPTION

[0073] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0074] Example 1

[0075] (1) Preparation and pretreatment of bacterial nanocellulose tubes

[0076] Use Figure 1 The bioreactor shown uses Komagataeibacterxylinus ATCC 23770 as the strain, fructose medium as the fermentation medium, and ferments at 30°C for 10 days to obtain bacterial nanocellulose tubes with an inner diameter of 3mm, an outer diameter of 8mm, and a length of 120mm. The bacterial nanocellulose tubes are placed in a 1% (m / v, g / mL) NaOH solution at 80°C for heating for 6 hours, replaced with deionized water and heated at 80°C for 8 hours, and replaced with new deionized water every 2 hours to remove a large amount of culture medium and bacteria; 1% (v / v, mL / mL) acetic acid solution is added for neutralization, and ultrapure water is repeatedly rinsed until the pH is neutral. The fully washed bacterial nanocellulose tubes are placed in ultrapure water and placed in an autoclave (121°C, 20min sterilization 10 times, and new ultrapure water is replaced before each sterilization) until the endotoxin content is below 0.25EU / mL.

[0077] (2) Preparation of silk fibroin / ethanol solution

[0078] Weigh a certain amount of silk fibroin into a wide-mouth container, add a certain amount of deionized water, and slowly shake or stand to dissolve to obtain silk fibroin solutions with concentrations of 0.5%, 1%, 2%, and 3% (m / v, g / mL). Add ethanol solution, wherein the silk fibroin solution: ethanol = 4:1 (v:v, mL:mL).

[0079] (3) Preparation of silk fibroin microspheres / bacterial nanocellulose composite tubes (BNC / SFMPs ​​composite tubes)

[0080] Sandwich the bacterial nanocellulose tube between the filter paper and absorb the water in its tube wall (1min). Use raw tape to connect one end of the bacterial nanocellulose tube to the syringe and seal the other end with a hemostatic clamp (the effective length of the bacterial nanocellulose tube is 10cm). Inject the silk fibroin / ethanol solution into the bacterial nanocellulose tube, with a total injection volume of 0.5mL / cm and a flow rate of 0.1mL / s. Put the bacterial nanocellulose tube containing silk fibroin / ethanol on the outside of a glass rod with a diameter of 3mm, freeze it at -20℃ for 12h, thaw it at 25℃ for 2h, and wash it with ultrapure water 3 times.

[0081] (4) Characteristics and potential applications

[0082] After in-situ phase separation of silk fibroin solutions with different concentrations in BNC tubes, the microstructures of the obtained BNC / SFMPs ​​composite tubes were different (e.g. Figure 2 As shown). The size and number of SF particles on the inner surface of the BNC / SFMPs ​​composite tube at the same SF concentration are greater than those on the outer surface. This is the result of the difference in SF content on the inner and outer surfaces caused by the liquid pressure technology. The BNC / SFMPs ​​composite tube retains the three-dimensional nano-network structure of BNC, providing a spatial structure for cell adhesion and proliferation, and providing a channel for the transmission of nutrients; the microspherical SF embedded in the BNC network can be used as a carrier of bioactive substances, providing the possibility of sustained drug release; and the preparation process of the BNC / SFMPs ​​composite tube is simple and efficient, without the need for additional cross-linking agents. The preparation technology and products of the BNC / SFMPs ​​composite tube have great application potential in the field of small-caliber blood vessels.

[0083] Example 2

[0084] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0085] (2) Preparation of silk fibroin / ethanol solution: Same as step (2) in Example 1, wherein the concentration of the silk fibroin solution is 2% (m / v, g / mL), and the ratio of silk fibroin solution to ethanol is 10:1, 8:1, 4:1, 2:1, 1:1 (v:v, mL:mL).

[0086] (3) Preparation of silk fibroin / bacterial nanocellulose composite tube (BNC / SF composite tube): The same as step (3) in Example 1 was performed to obtain a BNC / SF composite tube.

[0087] (4) Characteristics and potential applications

[0088] The ratio of silk fibroin to ethanol will affect the morphology of silk fibroin in BNC tubes (e.g. Figure 3As shown in the figure, when the ratio of silk fibroin to ethanol is 10:1, the microspheres formed by SF are connected into sheets; when the ratio of the two is 8:1 and 4:1, SF forms particles of uniform size embedded in the BNC network; when the ratio of ethanol continues to increase, SF no longer forms microspheres, but directly adheres to the surface of BNC fibers and completely covers the pores in the BNC network. The above experimental results show that changing the ratio of silk fibroin to ethanol can regulate the micromorphology of SF, thereby affecting the micromorphology of BNC / SF composite tubes, and then changing the mechanical properties and cell compatibility of BNC / SF composite tubes, providing new ideas and methods for preparing tubular materials and regulating their various properties.

[0089] Example 3

[0090] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0091] (2) Preparation of silk fibroin / ethanol solution: Same as step (2) in Example 1, wherein the concentration of the silk fibroin solution is 2% (m / v, g / mL), and the silk fibroin solution: ethanol = 4:1 (v:v, mL:mL).

[0092] (3) Preparation of silk fibroin / bacterial nanocellulose composite tube (BNC / SFMPs ​​composite tube): The same as step (3) in Example 1 was performed to obtain a BNC / SFMPs ​​composite tube.

[0093] (4) Preparation of heparin-grafted silk fibroin / bacterial nanocellulose composite tube (BNC / SFMPs-Hep composite tube): The BNC / SFMPs ​​composite tube obtained in step (3) was immersed in a MES buffer solution with a pH of 5.5 and shaken in a water bath for 2 h. The composite tube was then transferred to an EDC / NHS solution with a heparin concentration of 1 g / L (m / v, g / mL) (wherein the concentration of EDC was 0.06 M and the concentration of NHS was 0.03 M), and the reaction was carried out in a water bath with shaking for 24 h at 30°C in the dark. After being taken out, the tube was washed in ultrapure water for 5 times to obtain a BNC / SFMPs-Hep composite tube.

[0094] (5) Characteristics and potential applications

[0095] The inner wall of the BNC / SFMPs-Hep tube supports the adhesion and proliferation of human umbilical vein endothelial cells (e.g. Figure 4 and Figure 5As shown). The blood compatibility of the materials was evaluated with reference to ISO 10993-4:2017. From the whole blood coagulation curve, it can be observed that the OD value of the BNC / SFMPs-Hep tube decreased the least in the first 5 minutes of contact with blood, the absorbance value within 5-20 minutes remained almost unchanged, and the OD value at the 40th minute was the largest, which means that the coagulation rate of the BNC / SFMPs-Hep tube was the slowest and had an anticoagulant effect; the plasma recalcification time was significantly prolonged, and blood coagulation was not complete within the 60 minutes of the test; the hemolysis rate of all materials was less than 0.5% (as shown in Figure 2). Figure 6 The results of rat abdominal aorta replacement (as shown) reached the hemolysis rate standard of Class III medical devices (hemolysis rate less than 5%). Figure 7 Figure 3 (shown in Figure 4) showed that the BNC / SFMPs-Hep tubes maintained smooth blood flow 5 months after implantation, and the endothelial layer and smooth muscle layer were generated in situ in vivo, but hyperplasia occurred in the middle section of the blood vessel.

[0096] Example 4

[0097] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0098] (2) Preparation of heparin / silk fibroin / ethanol solution: Weigh 0.1 g of silk fibroin into a wide-mouth container, add 5 mL of deionized water, slowly shake or let stand to dissolve, then add 50 mg of heparin (Sinopharm Chemical Reagent Co., Ltd.), after the heparin is completely dissolved, add 1.25 mL of ethanol, and gently shake to mix.

[0099] (3) Preparation of heparin-encapsulated silk fibroin / bacterial nanocellulose composite tubes (BNC / Hep@SFMPs ​​composite tubes): The operation steps are the same as step (3) in Example 1, wherein the solution injected into the bacterial nanocellulose tube is a silk fibroin / heparin / ethanol mixed solution.

[0100] (4) Characteristics and potential applications

[0101] The microscopic morphology of the above-mentioned small-caliber artificial blood vessels is as follows Figure 8 As shown in Figure 2, SF and Hep were embedded in the pores of the BNC network or adhered to the cellulose fibers in the form of microspheres, and the BNC / Hep@SFMPs ​​composite tubes had no cytotoxicity (e.g. Fig.10 The inner surface supports the proliferation and adhesion of endothelial cells and smooth muscle cells (as shown in Fig.11 ), significantly up-regulated the expression of VEGFA gene and down-regulated the expression of IL-6 gene (as shown in Fig.12 In the whole blood coagulation curve ( Fig. 9A), the initial OD value of the BNC / Hep@SFMPs ​​composite tube is lower than that of the BNC tube, and the curve decline rate is also lower; within 60 minutes of the plasma recalcification experiment, the plasma did not coagulate on the inner surface of the BNC / Hep@SFMPs ​​composite tube, while the BNC tube completely coagulated within 10 minutes after the platelet-poor plasma contacted its inner surface ( Fig. 9 B and 9C); the hemolysis rate of all materials tested was less than 0.3% ( Fig. 9 D), meeting the requirements of Class III medical devices; fewer platelets adhered to the inner surface of the BNC / Hep@SFMPs ​​composite tube than those on the inner surface of commercial ePTFE large-caliber blood vessels, and the degree of platelet activation was lower ( Fig. 9 E). The above experimental results show that the BNC / Hep@SFMPs ​​composite tube has excellent blood compatibility, no cytotoxicity, promotes the expression of VEGFA, and has the effect of reducing inflammation. In summary, the BNC / Hep@SFMPs ​​composite tube has great potential as a small-caliber artificial blood vessel.

[0102] Example 5

[0103] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0104] (2) Preparation of silk fibroin / heparin / ethanol solution: The concentration of silk fibroin solution was 0.5%, 1%, 2%, and 3% (m / v, g / mL), the concentration of heparin was 10 mg / mL, and the ratio of silk fibroin to heparin mixed solution: ethanol = 4:1 (v:v, mL:mL). (3) Preparation of heparin-encapsulated silk fibroin / bacterial nanocellulose composite tube (BNC / Hep@SFMPs ​​composite tube): The same as step (3) in Examples 1 and 4 was used to obtain a BNC / Hep@SFMPs ​​composite tube.

[0105] (4) Characteristics and potential applications

[0106] The encapsulation rate of heparin in the above BNC / Hep@SFMPs ​​composite tube is 65%-80%, and it has a long-term release effect of heparin in PBS (pH 7.0, 37°C) (such as Fig.13The heparin release in the first 24 hours was the largest, and the heparin release was relatively stable during the 24-72 hours. The heparin release rate decreased significantly after 3 days. The total heparin release of BNC / Hep@1%SFMPs ​​and BNC / Hep@2%SFMPs ​​composite tubes was 1.54μg / cm2 from 7d to 15d, 1.4μg / cm2 from BNC / Hep@3%SFMPs, and 0.75μg / mL from BNC / Hep@0.5%SFMPs. The heparin release of BNC / Hep@3%SFMPs ​​was the largest in the first 48h. The heparin release rate was related to the silk fibroin concentration and increased with the increase of silk fibroin concentration. From the 3rd day onwards, there was no difference in the heparin release of BNC / Hep@SFMPs ​​composite tubes under different SF concentrations. The long-term sustained release of heparin by silk fibroin microspheres helped the artificial blood vessels to exert long-term anticoagulant effect and reduce the probability of acute thrombosis of artificial blood vessels.

[0107] Example 6

[0108] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0109] (2) Preparation of SF / Hep / ethanol solution and VEGF165A solution: SF / Hep mixed solution: ethanol = 4:1 (v:v, mL:mL), the concentration of silk fibroin solution was 1% (m / v, g / mL), the concentration of heparin was 10 mg / mL, the concentration of VEGF165A was 100 ng / mL, and VEGF165A was purchased from Abcam.

[0110] (3) Preparation of silk fibroin / bacterial nanocellulose composite tubes loaded with VEGF and heparin (BNC / Hep@SFMPs / VEGF composite tubes): The BNC / Hep@SFMPs ​​composite tubes were prepared in the same manner as step (3) in Examples 1 and 4. The obtained BNC / Hep@SFMPs ​​composite tubes were then placed in a 0.1% BSA (m / v, g / mL) aqueous solution containing VEGF for 12 h (4°C). After washing with PBS, the BNC / Hep@SFMPs / VEGF composite tubes were obtained.

[0111] (4) Potential Applications

[0112] The above-mentioned BNC / Hep@SFMPs / VEGF composite tube has good in vitro blood compatibility, supports cell growth, and has great application potential in the field of small-caliber artificial blood vessels.

[0113] Example 7

[0114] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0115] (2) Preparation of recombinant human collagen / ethanol solution: Same as step (2) in Example 1, wherein the recombinant human collagen was purchased from Hubei Langbowan Biotechnology Co., Ltd., the concentration of the recombinant human collagen solution was 1% (m / v, g / mL), and the recombinant human collagen solution: ethanol = 8:1, 4:1, 2:1, 1:2, 1:4 (v:v, mL:mL).

[0116] (3) Potential applications

[0117] When the ratio of recombinant human collagen (RHC) to ethanol is 1:2, the solution becomes turbid and the mixed solution cannot be evenly distributed in the BNC tube wall. Only a small amount of solution is adsorbed in the lumen of the BNC tube. The FTIR image of the inner surface of the bacterial cellulose / recombinant human collagen composite tube (BNC / RHC composite tube) shows (e.g. Fig.14 When the ratio of RHC to ethanol is 4:1 and 2:1, the wavelength is 1620 cm -1 There is an absorption peak (carbonyl) at Fig.15 As shown in the figure, there are protein particle aggregates on the inner surface of the composite tube. When the volume ratio of recombinant human collagen to ethanol is 4:1 and 2:1, there are more protein particles, and when the volume ratio of recombinant human collagen to ethanol is 4:1 and 2:1, there are fewer protein particles on the inner surface of the composite tube with other ratios. This may be the reason why FTIR failed to detect the characteristic absorption peak of recombinant human collagen in the BNC tube. Human recombinant collagen makes the BNC tube have amino groups, which can be further grafted with bioactive substances such as heparin, and has the potential to be used as small-caliber artificial blood vessels.

[0118] Example 8

[0119] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0120] (2) Preparation of recombinant human collagen / heparin / ethanol solution: RHC / Hep mixed solution: ethanol = 4:1 (v:v, mL:mL), RHC concentration is 1% (m / v, g / mL), and heparin concentration is 10 mg / mL.

[0121] (3) Preparation of heparin-encapsulated recombinant human collagen / bacterial nanocellulose composite tubes (BNC / RHC / Hep@SFMPs ​​composite tubes): same as step (3) in Example 1.

[0122] (4) Potential Applications

[0123] The above-mentioned BNC / RHC / Hep@SFMPs ​​composite tubes have great potential as small-caliber blood vessels.

[0124] Example 9

[0125] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0126] (2) Preparation of gelatin solution, gelatin / heparin mixture and genipin solution: The gelatin concentration in the gelatin solution or gelatin / heparin mixture is 1% (m / v, g / mL), the heparin concentration in the mixture is 1 g / L (m / v, g / mL), and the concentration of the genipin solution is 0.5% (m / v, g / mL).

[0127] (3) Preparation of bacterial nanocellulose / gelatin composite tubes (BNC / Gel composite tubes) and bacterial nanocellulose / gelatin / heparin composite tubes (BNC / Gel / Hep composite tubes): Gelatin solution or gelatin / heparin mixture was injected into the wall of bacterial nanocellulose tubes using liquid pressure technology, and then placed in genipin solution for static cross-linking for 12 h, and then ultrasonically cleaned.

[0128] (4) Potential Applications

[0129] The above-mentioned BNC / Gel composite tube is translucent, and the BNC / Gel / Hep composite tube is translucent and light green (such as Fig.16 FTIR graph (as shown) Fig.17 The inner surfaces of BNC / Gel and BNC / Gel / Hep were shown in Figure 1,640 and 1,230 cm -1 There is a characteristic absorption peak of gelatin at the BNC tube, which proves that gelatin is successfully compounded on the BNC tube. Toluidine blue staining results show ( Fig.17 ), the BNC tube is blue, and the BNC / Gel / Hep composite tube is purple. This is because the BNC tube physically adsorbs a small amount of toluidine blue solution, and the heparin grafted on the surface of the BNC / Gel / Hep composite tube reacts with toluidine blue. From the SEM images of the inner surfaces of the BNC tube and the BNC composite tube, we can see that ( Fig.18 ), a small amount of gelatin forms particles on the fiber surface, and the fiber diameter of the composite tube is significantly larger than that of the BNC tube, indicating that gelatin is coated on the surface of the BNC fiber. The grafting amount of heparin is 0.23μg / cm 2 ( Fig.19 ), which is significantly greater than the heparin grafting amount of the BNC / Hep tube prepared by this method (0.11 μg / cm 2 ). The above method successfully grafted gelatin and heparin into the BNC tube to prepare a BNC / Gel / Hep composite tube, which has the potential to be used as a small-caliber blood vessel.

[0130] Example 10

[0131] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0132] (2) Preparation of recombinant human collagen / heparin mixed solution and genipin solution: wherein the concentration of recombinant human collagen is 1% (m / v, g / mL), the concentration of heparin is 1 g / L (m / v, g / mL), and the concentration of genipin solution is 0.5% (m / v, g / mL). (3) Preparation of bacterial nanocellulose / recombinant human collagen / heparin composite tube (BNC / RHC / Hep composite tube) and bacterial nanocellulose / heparin composite tube (BNC / Hep composite tube): same as step (3) in Example 9.

[0133] (4) Potential Applications

[0134] The heparin grafting amount of the above BNC / RHC / Hep composite tube is 0.27 μg / cm 2 ( Fig.19 ), which is significantly greater than the heparin grafting amount of the BNC / Hep tube prepared by this method (0.11 μg / cm 2 ). Heparin is compounded on the BNC tube using the "one-step method" of genipin to improve the anticoagulant property of the BNC tube, and the composite tube has the potential to be used as a small-caliber blood vessel.

[0135] Embodiment 11

[0136] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0137] (2) Preparation of gelatin / ethanol solution: The concentration of gelatin solution is 1% (m / v, g / mL), gelatin:ethanol = 8:1, 4:1, 2:1, 1:2, 1:4 (v:v, mL:mL).

[0138] (3) Preparation of gelatin / bacterial nanocellulose composite tubes (BNC / Gel composite tubes): same as step (3) in Example 1.

[0139] (4) Potential Applications

[0140] FTIR graph shows (such as Fig. 20 As shown in the figure, except for the composite tube with a gelatin and ethanol ratio of 1:4, which has no obvious characteristic peak on the inner surface, the other composite tubes have a wavelength of 1640cm -1 and 1520cm -1 There are characteristic absorption peaks at all locations, which means that gelatin is successfully compounded in the BNC tube through the freezing phase separation method. Fig.21As shown in the figure, the proportion of gelatin to ethanol is different, and the morphology of gelatin in the BNC tube is also different. When gelatin: ethanol = 8:1, gelatin adheres to the BNC fiber in sheets; when gelatin: ethanol = 4:1, gelatin is distributed in the BNC network in particles; as the gelatin ratio decreases, the number of gelatin particles decreases sharply, and more gelatin is coated on the surface of cellulose fibers. The frozen phase separation method provides a new method for the preparation of BNC / Gel composite tubes, which gives BNC amino groups and can obtain functional composite tubes by physical encapsulation or chemical grafting, providing a new idea for the preparation of BNC-based small-caliber blood vessels.

[0141] Example 12

[0142] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0143] (2) Preparation of recombinant human collagen solution, heparin solution and genipin solution: the concentration of recombinant human collagen is 3% (m / v, g / mL), the concentration of heparin is 5 g / L (m / v, g / mL), and the concentration of genipin solution is 1% (m / v, g / mL).

[0144] (3) Preparation of bacterial nanocellulose / recombinant human collagen / heparin composite tubes (BNC / RHC-Hep composite tubes): The recombinant human collagen solution was injected into the bacterial nanocellulose tubes using liquid pressure technology, or the BNC tubes were placed in the recombinant human collagen solution for 12 hours of oscillation and then placed in the genipin solution for 12 hours of static crosslinking, and the uncomplexed collagen was fully washed to remove. Then, the tubes were placed in the EDC / NHS crosslinking system containing heparin for 24 hours to obtain the BNC / RHC-Hep composite tubes.

[0145] Embodiment 13

[0146] (1) Preparation and pretreatment of bacterial nanocellulose tubes: same as step (1) in Example 1.

[0147] (2) Preparation of fibronectin (Fn) solution, heparin solution and genipin solution: the concentration of the fibronectin solution is 2% (m / v, g / mL), the concentration of heparin is 5 g / L (m / v, g / mL), and the concentration of the genipin solution is 1% (m / v, g / mL).

[0148] (3) Preparation of bacterial nanocellulose / fibronectin / heparin composite tubes (BNC / Fn-Hep composite tubes): Fibronectin solution was injected into bacterial nanocellulose tubes using liquid pressure technology, or BNC tubes were placed in fibronectin solution for 12 h of oscillation and then placed in genipin solution for 12 h of static crosslinking, and then fully washed to remove uncomplexed fibronectin. Then, the tubes were placed in EDC / NHS crosslinking system containing heparin for 24 h to obtain BNC / Fn-Hep composite tubes.

Claims

1. A bacterial nanocellulose composite tube, characterized in that: The bacterial nanocellulose composite tube is a protein / bacterial nanocellulose composite tube, or a drug / protein / bacterial nanocellulose composite tube.

2. The bacterial nanocellulose composite tube according to claim 1, characterized in that: The protein is a biocompatible protein; wherein the biocompatible protein includes one or more of silk fibroin, fibronectin, gelatin, and human recombinant collagen; the drug includes one or more of heparin, VEGF, and adhesion peptide; In the protein / bacterial nanocellulose composite tube, protein encapsulates bacterial nanocellulose and / or protein particles are embedded in a three-dimensional network of bacterial cellulose; The drug in the drug / protein / bacterial nanocellulose composite tube is grafted on the surface of the bacterial nanocellulose tube filled with protein, or the protein particles encapsulating the drug are embedded in the three-dimensional network of bacterial nanocellulose.

3. A method for preparing a bacterial nanocellulose composite tube, comprising method 1, method 2, method 3 or method 4; Method 1: Fill the mixed solution into the tube wall of the bacterial nanocellulose tube by liquid pressure, then freeze phase separation, wash, and obtain the bacterial nanocellulose composite tube; wherein the mixed solution is a mixed solution of protein solution and ethanol, or a mixed solution of protein / drug solution and ethanol; Method 2: The mixed solution of protein solution and ethanol is filled into the tube wall of bacterial nanocellulose tube by liquid pressure, and then frozen for phase separation, washed, and then immersed in drug solution for reaction to obtain bacterial nanocellulose composite tube; Method 3: Fill the tube wall of the bacterial nanocellulose tube with a protein solution or a mixed solution of protein and drug, then place it in a genipin solution for cross-linking, wash, and obtain a bacterial nanocellulose composite tube; Method 4: Fill the tube wall of the bacterial nanocellulose tube with a protein solution, then place it in a genipin solution for cross-linking, wash it, and then immerse it in a drug-containing solution for reaction to obtain a bacterial nanocellulose composite tube.

4. The preparation method according to claim 3, characterized in that: In methods 1 to 4, the inner diameter of the bacterial nanocellulose tube is 2-6 mm, the outer diameter is 3-9 mm, and the wall thickness is 0.5-2.5 mm; the bacterial nanocellulose tube is pretreated, wherein the pretreatment is high temperature drying, room temperature air drying or filter paper water absorption.

5. The preparation method according to claim 3, characterized in that: The flow rate of the solution filled in the tube wall of the bacterial nanocellulose tube by the liquid pressure technology in the method 1 and the method 2 is 0.05-0.5 mL / s, and the flow rate is 0.1-0.5 mL / cm; The frozen phase separation is first freezing and then thawing, wherein the freezing is freezing at -20°C to -196°C for 12-24 hours, and the thawing is thawing at room temperature for 1-4 hours.

6. The preparation method according to claim 3, characterized in that: The mixed solution of protein solution and ethanol in method 1 and method 2, wherein the concentration of the protein solution is 0.1%-5% (m / v); the volume ratio of the protein solution to ethanol is 10:1-1:1; the mixed solution of protein / drug solution and ethanol in method 1, wherein the concentration of protein in the protein / drug solution is 0.1%-5% (m / v), and the concentration of the drug is 1-20g / L; the volume ratio of the protein / drug solution to ethanol is 10:1-1:

1.

7. The preparation method according to claim 3, characterized in that: In the method 3 and the method 4, the tube wall filled with the bacterial nanocellulose tube is made by oscillating immersion or liquid pressure technology, wherein the oscillating immersion temperature is 4°C-25°C, and the time is 12-24h; the flow rate of the solution in the liquid pressure technology is 0.05-0.5mL / s, and the flow rate is 0.1-0.5mL / cm; The cross-linking in the genipin solution is performed by static cross-linking for 1-12 hours.

8. The preparation method according to claim 3, characterized in that: The concentration of the protein solution in methods 3 and 4 is 0.1%-5% (m / v, g / mL); the concentration of the genipin solution is 0.5%-2% (m / v, g / mL); In the method 3, the concentration of the protein in the mixed solution of the protein and the drug is 0.1%-5% (m / v, g / mL), and the concentration of the drug is 1-20 g / L.

9. The preparation method according to claim 3, characterized in that: In the method 2 and the method 4, the drug-containing solution is immersed and reacted for 12-24 hours; the drug-containing solution is a drug-containing EDC / NHS cross-linking system, wherein the concentration of the drug is 1-20 g / L, the concentration of EDC is 0.06-0.12 M, and the concentration of NHS is 0.03-0.06 M.

10. Use of the bacterial nanocellulose composite tube according to any one of claims 1 to 2 in the preparation of tubular artificial organs.

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