Anticoagulant nanocellulose tube and preparation and artificial blood vessel application thereof

Abstract

The invention relates to an anticoagulant nanocellulose tube and preparation and artificial blood vessel application thereof, a sulfating agent reacts with a pretreated nanocellulose tube, and after the reaction is terminated, the anticoagulant nanocellulose tube with two heterogeneous surfaces is obtained. The preparation process is simple, easy to operate and short in reaction time, and has a wide application prospect in blood contact medical substitutes such as linings of small-caliber arteriovenous and hemodialysis fistulization tubes and medical instruments. In addition, the material also has huge potential when being used as a surfactant, a humectant and an antiviral material.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and specifically relates to an anticoagulant cellulose nanotube, its preparation, and its application in artificial blood vessels. Background Technology

[0002] The high incidence of cardiovascular and other diseases has led to a surge in demand for small-diameter vascular grafts (inner diameter <6 mm). However, existing artificial blood vessel materials, such as expanded polytetrafluoroethylene (ePTFE) and polyester PET, are prone to triggering a coagulation cascade reaction upon blood contact, resulting in early thrombosis and long-term intimal hyperplasia. More importantly, there are currently no small-diameter artificial blood vessels available on the market for clinical use in coronary artery bypass grafting. Bacterial nanocellulose (BNC) possesses excellent biocompatibility, an extracellular matrix-like 3D nanofiber network structure, and shape plasticity, showing broad application prospects in biomedical materials such as artificial blood vessels, heart valves, medical catheters, corneas, cartilage, hemodialysis membranes, ECMO oxygenation membranes, and wound dressings.

[0003] Although the highly hydrophilic surface of BNC can induce short-term anticoagulation through the interfacial hydration layer, experimental data indicate that it still promotes complement activation, such as the formation of C3a and sC5b-9, which can potentially exacerbate platelet and protein adhesion, further inducing thrombosis and inflammatory responses. Furthermore, pure BNC hydrogel tubes, as artificial blood vessels, lack sufficient compliance and elasticity. Moreover, pure BNC hydrogel tubes have poor lubricity and transparency, hindering blood flow and observation within the vessels. More importantly, blood contact with medical materials requires tubes with different anticoagulant properties for different locations to meet various application scenarios. For example, some implanted catheter materials that remain in the body or have a certain duration of action, such as central venous catheters (CVC), peripherally inserted central venous catheters (PICC), dialysis catheters, and other implanted or interventional catheters, may be difficult to remove due to thrombus or fibrin sheath formation on the outer surface of the implant, exacerbating the inflammatory response. For catheters that are in contact with blood for a long time or have a long-term effect, such as extracorporeal circulation catheters, extracorporeal membrane oxygenation (ECMO) catheters, cardiopulmonary bypass (CPB) and dialysis membranes / devices, infusion / transfusion vessels, and implanted artificial blood vessels, intracatheter embolism often occurs due to the lack of anticoagulation in the lumen. Common cardiovascular materials, such as artificial blood vessels and valves, primarily rely on surface passivation and biofunctionalization for anticoagulation modification. However, these methods generally require the introduction of specific molecules, resulting in drawbacks such as poor stability, lack of persistence, easy loss of activity, and the risk of detachment. Alternatively, encapsulation of anticoagulants, such as heparin, can be used to achieve sustained release and long-term anticoagulation effects, but current technologies have not yet achieved this. Therefore, directly modifying the functional groups of the material itself would allow for self-integrated anticoagulation functionalization, circumventing these problems. Furthermore, developing bifacially heterogeneous self-anticoagulating BNC-based catheters is crucial for various application scenarios. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide an anticoagulant cellulose nanotube, its preparation, and its application in artificial blood vessels, thereby overcoming the bottleneck of poor long-term anticoagulation in small-diameter BNC-based blood vessels. More importantly, this invention innovatively prepares anticoagulant BNC tubes with different sulfation sites (outer wall only, inner lumen only, and the entire tube) to meet diverse clinical application needs and effectively solve the problem of targeted anticoagulation. Simultaneously, the prepared catheters possess good transparency, elasticity, strength, and lubricity, meeting corresponding functional requirements.

[0005] This invention is the first to modify BNC hydrogel tubes with biomimetic glycosaminoglycans, aiming to apply them to the lining of medical devices that are in long-term anticoagulation artificial blood vessels or in long-term contact with blood. It breaks through multiple technical bottlenecks such as local thrombosis and elasticity mismatch, opening up new directions for the treatment of end-stage cardiovascular diseases and the development of small-diameter vascular grafts.

[0006] This invention provides a method for preparing anticoagulant cellulose nanotubes, comprising:

[0007] (1) The nanocellulose tubes are pretreated to obtain pretreated nanocellulose tubes;

[0008] (2) The sulfate esterification agent reacts with the nanocellulose tubes pretreated in step (1), and after the reaction is terminated, anticoagulant nanocellulose tubes are obtained.

[0009] The preferred embodiment of the above preparation method is as follows:

[0010] In step (1), the nanocellulose is obtained and purified from cultured microorganisms.

[0011] The pretreatment in step (1) includes treating the nanocellulose tubes with N,N-dimethylformamide (DMF) and / or N,N-dimethylacetamide (DMAc).

[0012] The volume ratio of the nanocellulose tubes to N,N-dimethylacetamide (DMAc) and / or N,N-dimethylformamide (DMF) is 1:0.8-3.

[0013] The treatment conditions are 25~50℃ air bath or water bath for 3-48 h; the treatment is repeated 1 to 5 times; the treatment method is one or more of the following: static setting, oscillation, and ultrasonic treatment, and the oscillation includes circular, horizontal or figure-eight oscillation forms.

[0014] The preparation of the sulfate esterification agent in step (2) includes: adding dimethylformamide (DMF) to the reaction apparatus, precooling it under ice bath and nitrogen conditions for 20-100 min, then adding chlorosulfonic acid (ClSO3H) dropwise to the stirred dimethylformamide (DMF) until it is stable and free of smoke, and then heating it to 20-30℃ to obtain the sulfate esterification agent.

[0015] The stirring speed is 500-1000 r / min.

[0016] The chlorosulfonic acid ClSO3H was added dropwise over a period of 0.5-2 hours.

[0017] The ratio of dimethylformamide (DMF) (concentration >99.8%) to chlorosulfonic acid (ClSO3H) is 20-200 mL: 30-100 mol.

[0018] The molar amount of chlorosulfonic acid is 10-100 times the molar amount of nanocellulose (molar ratio).

[0019] Further, the preparation of the sulfation esterification agent: A dry three / four-necked flask is connected to a condenser and a separatory funnel. The condenser is connected to a three-way valve with a nitrogen balloon and sealed with a rubber stopper. DMF is added to the flask and placed in an ice bath. The flask is evacuated to a vacuum using a vacuum pump and pre-cooled for 20-100 min. Chlorosulfonic acid is injected into the separatory funnel by piercing the rubber stopper with a glass syringe with a long needle. Stirred DMF is then slowly added dropwise over 0.5-2 h to prepare the sulfation esterification agent until it is stable and free of fumes. The temperature is then raised to 20-30℃ to obtain the sulfation esterification agent.

[0020] In step (2), the sulfate esterification agent reacts with the outer wall or the entire nanofiber tube after pretreatment in step (1): Under nitrogen protection, 1-30 pretreated nanofiber tubes are added to the sulfate esterification agent and stirred for 0.2-2 h.

[0021] Or the sulfuric acid esterifying agent in step (2) reacts with the inner cavity of the nanocellulose tube after pretreatment in step (1): the sulfuric acid esterifying agent is injected into the inner cavity of the treated nanocellulose tube and placed in DMF solution under nitrogen protection for 0.2-2 h. During the reaction, the BNC tube shows obvious shrinkage.

[0022] The outer wall and inner cavity reactions are described, wherein the two ends of the pretreated nanocellulose tube are closed; the whole tube reaction is described, wherein the two ends of the pretreated nanocellulose tube are not closed.

[0023] For the reactions of the outer wall and inner cavity, corrosion-resistant material ropes are used to close both ends of the pretreated nanocellulose tube.

[0024] In step (2), the reaction is terminated by using a 2-6% (w / v) weak base salt-short chain alcohol solution; where w / v is g / mL.

[0025] The weak base salt is one or more of acetate, bicarbonate, and citrate; the short-chain alcohol is one or more of methanol, ethanol, isopropanol, or n-butanol.

[0026] After the reaction is terminated, the mixture is washed with water until no reagent residue remains.

[0027] This invention provides an anticoagulant cellulose nanotube prepared by the method described above.

[0028] This invention provides the application of anticoagulant nanocellulose tubes in blood-contact medical blood vessels and their composite materials, surfactants, humectants, and antiviral materials, such as small-diameter arteries and veins, hemodialysis fistula tubes, and medical device linings.

[0029] The anticoagulant catheter is a high-molecular polymer based on cellulose monomers. After dehydration by organic solvent replacement, specific areas of the BNC catheter are heparinized using a sulfation esterification technique. The catheter is then terminated and cleaned to remove impurities. The catheter exhibits good blood compatibility, transparency, elasticity, lubricity, and bifacial heterogeneity.

[0030] This invention provides an anticoagulant nanocellulose catheter, wherein the nanocellulose is bacterial nanocellulose (BNC) or a cellulose analog with a fiber diameter of 10-100 nm. The anticoagulant catheter improves transparency, elasticity, and lubricity by controlling the amount of sulfation agent added, reaction time, and reaction site. The anticoagulant catheter can generate a bi-dimensional heterogeneous anticoagulant catheter by controlling the reaction contact surface, including only the outer wall, only the lumen, and the entire catheter. The anticoagulant catheter can delay clotting time and plasma recalcification time, inhibit platelet activation and aggregation, has a hemolysis rate of less than 1%, and exhibits good blood compatibility. Furthermore, this catheter also has great potential as a surfactant, humectant, and antiviral material.

[0031] Beneficial effects

[0032] (1) After the surface of BNC is modified by sulfation, a heparin-like structure is formed, which exhibits anti-platelet aggregation and adhesion. Even if a small amount of platelets adhere, their activation is inhibited, which significantly improves blood compatibility, reduces the risk of systemic coagulation activation and inflammatory response, and improves the overall safety of treatment.

[0033] (2) After the BNC surface is modified by sulfation, the larger sulfuric acid groups replace the hydroxyl groups and give it high electronegativity, which further improves the self-support performance and electrostatic effect of BNC tube, as well as better hydrophilicity and transparency.

[0034] (3) BNC-based anticoagulant catheters meet the anticoagulant requirements of different parts (such as the outer wall, inner lumen and the whole), adapt to the market demand of different blood contact products, and expand the application range of medical blood contact materials.

[0035] (4) BNC-based anticoagulant medical materials are essential for maintaining blood flow, reducing the risk of tubing blockage, reducing the need for treatment interruption, device replacement, thrombolysis or thrombectomy due to coagulation, improving treatment efficiency and patient safety, and improving tubing patency and long-term safety. They include, but are not limited to, small-diameter vascular grafts, and can also be used in dialysis membranes and tubing, extracorporeal circulation tubing, indwelling catheters, heart valves, extracorporeal membrane oxygenation (ECMO) and other medical materials.

[0036] (5) This material also has great potential as a surfactant, humectant, and antiviral material. The modified sulfated BNC exhibits significantly improved surface slipperiness and hydrophilicity due to its saponification effect, thanks to the formation of sulfate groups. Furthermore, sulfated nanocellulose possesses antiviral properties, which greatly benefits its application in medical materials, including but not limited to artificial blood vessels.

[0037] (6) The present invention uses N,N-dimethylacetamide (DMAc) to replace and remove water from bacterial nanocellulose (BNC) hydrogel tubes. Its toxicity is lower than that of DMF and its thermal stability is higher than that of DMF. Then, specific parts of the BNC tube (only the outer wall, only the inner lumen, or the entire tube) are heparinized by sulfation technology. Further purification by termination and impurity removal yields a bulk anticoagulant catheter with complete nanofiber 3D structure and good mechanical properties. This method can maintain the three-dimensional morphology of the BNC tube without dissolving, control the degree of substitution and the location of sulfation, and is suitable for cellulose tubes with different parts, inner diameters, and wall thicknesses. Attached Figure Description

[0038] Figure 1 The images show the appearance (a) and transmittance (b) of the BNC tube in Example 1 and the sulfated BNC tube with different ClSO3H addition amounts.

[0039] Figure 2 The Fourier transform attenuated total reflectance infrared spectroscopy (ATR-FTIR) (a), Zeta potential (b), X-ray photoelectron spectroscopy (XPS) (c), and X-ray diffraction (XRD) (d) of the BNC tube and the sulfated BNC tube with different ClSO3H addition amounts in Example 1 are shown.

[0040] Figure 3 The whole blood coagulation time (a), hemolysis rate (b), and plasma recalcification curve (c) and time (d) of the BNC tube and BNC tubes with different sulfation times in Example 2 are shown.

[0041] Figure 4 The images show SEM images of the BNC tube in Example 2 and BNC tubes with different sulfation times.

[0042] Figure 5 The images show the inner and outer cavities of the BNC tube in Example 2 and BNC tubes with different sulfation times, measured by an Attenuated Total Reflectance Fourier Transform Infrared Spectrometer (ATR-FTIR). The left image shows the inner cavity, and the right image shows the outer surface.

[0043] Figure 6 The full X-ray photoelectron spectroscopy (XPS) spectra of the BNC tube in Example 2 and BNC tubes with different sulfation times, along with their peak fitting diagrams, are shown.

[0044] Figure 7 The whole blood coagulation time (a), hemolysis rate (b), and plasma recalcification curve (c) and time (d) of the BNC tube and BNC tubes with different sulfation times in Example 2 are shown.

[0045] Figure 8 Platelet adhesion in BNC tubes and sulfated BNC tubes in Example 2.

[0046] Figure 9 This is a comparison of the mechanical appearance of the BNC tube and the sulfated BNC tube in Example 3.

[0047] Figure 10 The time-strain curve (a) and force-strain curve (b) are used to compare the compressive elasticity of BNC tube and sulfated BNC tube in Example 3.

[0048] Figure 11 The whole blood coagulation time (a), hemolysis rate (b), and plasma recalcification curve (c) and time (d) of the BNC tube and sulfated BNC tube in Example 3 are shown.

[0049] Figure 12 The X-ray photoelectron spectroscopy (XPS) full spectrum (a) and the degree of substitution of sulfate ester groups (b) for groups A, B, and C in Example 4 are shown. Detailed Implementation

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0051] The BNC used in this experiment is a high-molecular-weight polysaccharide biosynthesized by the Komagataeibacter xylinus ATCC 23770 strain, distinct from non-nanofibers such as microcrystalline cellulose and lignocellulose. The tubular mold structure consists of a central glass or metal core (≤6 mm), an outer wall of silica gel, and both ends sealed with polymeric stoppers. The nanocellulose tubes were obtained by culturing and purifying the microorganism Komagataeibacter xylinus ATCC 23770. The ATCC23770 strain was first inoculated into a 250 mL wide-mouth conical flask containing 50 mL of activation medium and cultured for 7 days until a BNC membrane formed. The activation medium contained 40 g / L glucose, 5 g / L tryptone, and 3 g / L yeast extract, with a pH of 5.0-5.2. A quarter of the activated membrane was cut and shredded to 5 mm size, then inoculated into the tube-forming medium and cultured at 30℃ and 160 rpm for 12-36 h. The culture medium for tube preparation contained 100 g / L fructose, 5 g / L tryptone, and 3 g / L yeast extract, with a pH of 5.0-5.2. The culture medium was injected into the cavity of a sterile tubular mold (a glass rod with an inner diameter of 3 mm, a silicone tube with an inner diameter of 8 mm × outer diameter of 9 mm, and silicone plugs of 8-10 mm at both ends) using a sterile syringe in a laminar flow hood. The mold was then placed in a culture flask and incubated at 30°C for 10-14 days, with oxygenation every one day. The resulting BNC tubes were then immersed in a 0.25 M NaOH aqueous solution at 80°C to remove endotoxins, repeated 5-7 times. Next, they were immersed in deionized DI water at 80°C, repeated 5-7 times. Finally, they were sterilized by steam sterilization at 121°C and 15 psi for 20 min, repeated 5 times, to obtain clean BNC tubes for use in the following examples.

[0052] Example 1

[0053] I. Effects of different ClSO3H additions on the sulfation of BNC.

[0054] Step (1) In this embodiment, a BNC tube with an inner diameter of 3 mm and an outer diameter of 8 mm is used as an example. After the BNC tube is freeze-dried and dehydrated, its dry weight is calculated using a 0.01% balance, and the molar mass is calculated. DMAc is used to replace the water in the BNC tube, with a volume ratio of BNC tube to organic solvent of 1:2. The treatment conditions are: 30°C air bath, circular shaking at 150 rpm; total time: 12 h; repeated 3 times, and finally impregnated with N,N-dimethylformamide (DMF).

[0055] Step (2): Connect a dry three-necked flask to a condenser and a separatory funnel. Connect the condenser to a three-way valve with a nitrogen balloon and seal it with a rubber stopper. Fill the flask with 100 mL of DMF and place it in an ice bath. Evacuate the flask to a vacuum using a vacuum pump. Pre-cool the flask for 30 min. Using a glass syringe with a long needle, inject the amounts of chlorosulfonic acid (SBNC-1, 3, 6, 9, 10) determined according to 10, 30, 60, 90, and 100 times the molar amount of BNC into the separatory funnel via a rubber stopper. Slowly add the slowly stirred DMF dropwise over 1 h to prepare the sulfation agent until it is stable and free of fumes. Then, heat the mixture to room temperature to obtain the sulfation agent for use.

[0056] Step (3) Add 10 dehydrated BNC tubes (dry weight 0.1 g) to a flask and stir magnetically at 900 r / m. React for 2 h under nitrogen protection and terminate with 2-6% (w / v) sodium bicarbonate-n-butanol solution. Then wash with DI water until no reagent residue is left to obtain sulfated BNC tubes.

[0057] II. Testing Methods

[0058] Transparency measurement: BNC tubes and sulfated BNC tubes were cut along the axis and cut into 15 mm discs, which were then placed at the bottom of a 24-well plate. The transmittance was scanned at 350-900 nm using a UV-Vis-NIR spectrometer, with an empty plate as a control group.

[0059] Infrared measurement: After freeze-drying the inner surface of BNC tubes and sulfated BNC tubes, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed at 4000-400 cm⁻¹. -1 scanning.

[0060] Potentiometric determination: BNC tubes and sulfated BNC tubes were lyophilized and dehydrated using a freeze dryer, then shredded and dissolved in 10 mM NaCl (aq.) at pH 6.425. The solution was then sonicated and shaken at 37°C and 150 rpm to obtain a 0.01% (w / v) dispersion. The supernatant was used to determine the potential using a Zeta-potential analyzer and particle size analyzer (NANO ZS).

[0061] X-ray photoelectron spectroscopy (XPS) determination: Thermo SCIENTIFIC K-ALPHA was used to determine the full and fine spectra of C, O, and S in a sulfated BNC tube. Type: Monochromatic Al target (E = 1486.68 eV); Voltage: 12000 V; Current: 0.006 A; Vacuum: P < 10 -9 mBar; Bandpass energy: 100 eV (full spectrum), 50 eV (high resolution); Work function: 4.2 eV.

[0062] X-ray wide-angle diffraction (XRD) measurement: After freeze-drying the inner surface of the BNC tube and the sulfated BNC tube, the flat surface is facing upwards for measurement. The working voltage is 40 kV, the working current is 100 mA, the 2θ range is 5-40°, and the scanning speed is 10° / min.

[0063] Blood compatibility testing

[0064] Whole blood from rabbits was purchased from Nanjing Senbeijia Company. The rabbit whole blood was centrifuged at 4℃ and 2000 ×g for 5 min; the supernatant was obtained as platelet-poor plasma (PPP). The precipitate was diluted with physiological saline to obtain red blood cells. Before use, the material was replaced with physiological saline at 37℃ and incubated for 4 h.

[0065] Determination of whole blood clotting time: BNC and sulfated BNC tubes were cut along the axis into 15 mm discs and placed in a 24-well plate. A control group was prepared by adding blood only without any other materials. 5 mL of rabbit whole blood (sodium citrate) was added to 10% of a 0.025 mol / L CaCl2 solution and quickly and gently mixed. 100 μL of blood was added to each well and incubated at 37°C. At 5, 15, 25, 35, 45, and 55 min, 2 mL of DI water was added, followed by another 5 min of incubation. 200 μL of the solution was then transferred to a 96-well plate to determine the clotting time (A). 540 .

[0066] Determination of plasma recalcification time: BNC and sulfated BNC tubes were cut along the axis into 15 mm discs, placed in 24-well plates, and incubated with 500 μL of PPP for 1 h. Then, 100 μL of the incubation solution was transferred to a 96-well plate, and 100 μL of 0.01 mol / L CaCl2 solution was added. Kinetics were measured at 405 nm for a total time of 45 min, with 30 s intervals. The positive control consisted of 100 μL of PPP with 100 μL of 0.01 mol / L CaCl2 solution, while the negative control consisted of 100 μL of PPP with 100 μL of physiological saline. When A... 405 The time corresponding to reaching half of the maximum value is recorded as the plasma recalcification time.

[0067] Determination of hemolysis rate: BNC and sulfated BNC tubes were cut along the axis into 15 mm discs, laid on a 24-well plate, and 1.5 × 10⁻⁶ ppm was added. 8 One red blood cell and 2 mL of physiological saline were incubated at 37°C for 1 h, followed by centrifugation at 750×g for 5 min. The supernatant was then used to determine A. 540 Dissolved water was used as a positive control instead of physiological saline. The hemolysis rate was calculated using the following formula.

[0068]

[0069] III. Test Results

[0070] Depend on Figure 1 As can be seen from a, compared to BNC, the mechanical strength increases with the increase of chlorosulfonic acid content, and the gel tube becomes straighter, smoother, and more transparent. For example... Figure 1 As can be seen from b, the transparency of BNC tubes is similar to that of SBNC-1 and SBNC-3, while the transparency of other sulfated BNC tubes gradually increases with the increase of chlorosulfonic acid addition. However, when the amount of chlorosulfonic acid added is 100 times that of cellulose, the surface of the sulfated BNC tubes shows damage.

[0071] Depend on Figure 2 As can be seen from a, when the amount of chlorosulfonic acid added is ≥60 times, the inner lumen of the sulfated BNC tube is 1228-1232 cm. -1 and 815-819 cm -1 Characteristic absorption peaks for COC and COS were detected respectively. Meanwhile, by... Figure 2 c shows that the XPS full spectrum reveals the presence of S2p at 168.9 eV, and its trend is consistent with that of the infrared spectrum. Furthermore, from Figure 2 b shows that the potential increases continuously with the increase of chlorosulfonic acid addition, while... Figure 2 XRD results showed that the crystallinity of sulfated cellulose gradually decreased, specifically, the peaks at 14.6°, 16.8°, and 22.7° corresponding to the crystal planes (110), (10ī), and (200) of cellulose I gradually decreased. These results indicate that when the amount of chlorosulfonic acid added is ≥60 times, -OH groups of BNC are successfully grafted with -SO3H, meaning that BNC is successfully sulfated, and the crystallinity gradually decreases.

[0072] Further blood compatibility testing was conducted on the samples. Figure 3 As shown in b, the hemolysis rate of both BNC and sulfated BNC tubes is <1%, classifying them as non-hemolytic materials, far exceeding the requirements of ISO 10993-4 for blood contact materials. When the amount of chlorosulfonic acid added is ≥60 times, the whole blood clotting time of the SBNC-9 sample in the sulfated BNC tube (…) Figure 3 a) The blood had not clotted after 25 minutes. Meanwhile, by Figure 3 c. Plasma recalcification curve and Figure 3 The plasma recalcification time for SBNC-9 was observed to be delayed to 7–8 minutes, significantly longer than that of BNC (3 minutes). These results indicate that the sulfated BNC prepared at this dosage exhibits good anticoagulant properties.

[0073] Example 2

[0074] I. Preparation of sulfated BNC at different sulfation times.

[0075] In this embodiment, the preparation method is the same as in Example 1, but the amount of chlorosulfonic acid added in step (2) is adjusted to 90 times the molar amount of nanocellulose, the reaction time of sulfation in step (3) is adjusted to 40 min and 80 min, and the concentration of sodium bicarbonate-n-butanol solution is changed to 4% (w / v). Finally, they are recorded as SBNC-40 and SBNC-80, respectively.

[0076] II. Testing Methods

[0077] Scanning electron microscopy (SEM) observation: After freeze-drying the BNC and sulfated BNC tubes, gold sputtering was performed, and the microstructure of the sulfated contact surface tubes was photographed using field emission scanning electron microscopy.

[0078] Infrared measurement: After freeze-drying the inner and outer surfaces of BNC tubes and sulfated BNC tubes, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed at 4000-400 cm⁻¹. -1 scanning.

[0079] X-ray photoelectron spectroscopy (XPS) determination: Thermo SCIENTIFIC K-ALPHA was used to determine the full and fine spectra of C, O, and S in a sulfated BNC tube. Type: Monochromatic Al target (E = 1486.68 eV); Voltage: 12000 V; Current: 0.006 A; Vacuum: P < 10 -9 mBar; Bandpass energy: 100 eV (full spectrum), 50 eV (high resolution); Work function: 4.2 eV. Calculate the degree of sulfation substitution according to the following formula.

[0080]

[0081] The determination of blood compatibility is the same as in Example 1.

[0082] Platelet adhesion: BNC and sulfated BNC tubes were cut along the axis and cut into 15 mm round pieces, which were then placed in 24-well plates. 500 μL of PRP was added to each well and incubated at 37°C for 2 h. The plates were then washed three times with physiological saline, and 2 mL of 4% paraformaldehyde (PFA) tissue fixative was added. After fixation overnight, the plates were washed three times with ultrapure water and eluted with 25%, 50%, 75%, and 100% ethanol. The plates were then replaced and impregnated with tert-butanol, freeze-dried, and SEM images were taken to observe platelet morphology and adhesion.

[0083] III. Test Results

[0084] Depend on Figure 4It can be seen that after BNC sulfation treatment, its fibers become more disordered and interwoven, with SBNC-80 being significantly more disordered than SBNC-40. This is due to the introduction of sulfate groups on the surface of bacterial nanocellulose and the longer reaction time leading to cellulose degradation, which breaks the hydrogen bond between crystalline regions, thereby increasing the amorphous and disordered degree of the fiber network.

[0085] Depend on Figure 5 It can be seen that during the sulfation of the entire tube, the functional groups on the outer wall (right figure) are 1228-1232 cm⁻¹ more numerous than those in the inner lumen (left figure). -1 and 815-819 cm -1 The characteristic absorption peaks of COC and COS were detected more clearly. Therefore, this reaction is a gradual heterogeneous reaction, and it is feasible to obtain differentiated sulfation effects by controlling the reaction contact surface, such as only the outer wall or only the inner wall.

[0086] Depend on Figure 6 It can be seen that the SBNC-40 and SBNC-80 samples obtained by further optimizing the sulfation time have calculated degrees of substitution of approximately 0.3 and 0.5, respectively. Peak fitting of the XPS full spectrum (left) and fine spectrum (right) shows that S2p was detected at 168.9 eV, with the intensity of SBNC-80 > SBNC-40, consistent with the degree of substitution.

[0087] Further blood compatibility testing was conducted on the samples. Figure 7 As shown in b, both BNC and sulfated BNC tubes have a hemolysis rate of <1%, indicating they are non-hemolytic materials, far exceeding the requirements of ISO 10993-4. Regarding whole blood clotting time, within the same timeframe, the sample reacting for 80 min resulted in slower blood clotting than the sample reacting for 40 min (see [reference needed]). Figure 7 a). Meanwhile, the plasma recalcification curve ( Figure 7 c) and plasma recalcification time ( Figure 7 d) The curves show a significant shift to the later stage and a time delay, indicating that it has good anticoagulant properties.

[0088] Depend on Figure 8 It is evident that platelets are more easily removed by "rolling," and sulfated BNC becomes even more slippery, exhibiting the characteristics of a surfactant. This inhibits platelet aggregation and activation, further demonstrating that sulfated BNC possesses excellent anti-platelet adhesion properties. Compared to BNC, both SBNC-40 and SBNC-80 exhibit good anti-platelet adhesion performance.

[0089] Example 3

[0090] I. Investigation into the elasticity of sulfated BNC.

[0091] In this embodiment, the preparation method is the same as in Example 2. The reaction time of sulfation in step (3) is further adjusted to 10, 20, 30, 40 and 60 min, respectively denoted as SBNC-10, SBNC-20, SBNC-30, SBNC-40 and SBNC-60.

[0092] II. Testing Methods

[0093] Compression elasticity determination: The inner and outer diameters of BNC tubes and sulfated BNC tubes (SBNC-10, 30, 60) were measured. The sample was placed vertically in the center of the test stage, and the compressive force was increased from 0 N to 0.5 N at a rate of 2 N / min. When the compressive force reached 0.5 N, it was held for 3 s, and then the compressive force was removed at -2 N / min and held for 10 s. This process was repeated 100 times.

[0094] The determination of blood compatibility is the same as in Example 1.

[0095] III. Test Results

[0096] like Figure 9 As shown, sulfated BNC tubing is more upright than BNC tubing, exhibiting improved mechanical strength. Its inner and outer surfaces are very smooth and slippery, making it less prone to gripping and possessing surfactant-like properties. Compression rebound tests demonstrate that sulfated BNC tubing exhibits superior compressive elasticity (e.g., ...) compared to BNC. Figure 10 (As shown in b), the strain distance is shorter. SBNC-30 and SBNC-60 have significantly better compressive elasticity and creep resistance than SBNC-10, and their compressive strain distance is much shorter than that of BNC and SBNC-10 (as shown in b). Figure 10 As shown in Figure a), this indicates that an appropriate sulfation time can regulate the mechanical strength of sulfated BNC.

[0097] Further determination of the blood compatibility of the sample, such as Figure 11 As shown in b, the hemolysis rate of both BNC and sulfated BNC tubing is <1%, indicating they are non-hemolytic materials, far exceeding the requirements of ISO 10993-4. Figure 11 As shown in Figure a, in the OD change curve at 540 nm hemoglobin, the whole blood clotting time of sulfated BNC is significantly longer than that of the BNC line and the control group line, indicating that it has better anticoagulation ability. Figure 11 The plasma recalcification curves for c show that the curves for sulfated BNC are all shifted later than those for BNC, indicating a delayed plasma recalcification time. Further calculations of the plasma recalcification time (...) Figure 11d) During sulfation for 10-30 min, the plasma recalcification time gradually increased, with SBNC-30 showing the best results and superior anticoagulant effect. Further, with prolonged sulfation, the plasma recalcification time decreased, but remained higher than that of BNC. Combined with the previous compression results, it can be concluded that sulfation-treated BNC tubes possess both high elasticity and anticoagulant properties.

[0098] Example 4

[0099] I. Investigation into the preparation method of sulfated BNC.

[0100] Step (1) In this embodiment, a BNC tube with an inner diameter of 3 mm and an outer diameter of 8 mm is used as an example. The BNC tube is freeze-dried and dehydrated, and its dry weight is calculated using a 0.01% balance to determine its molar mass. The BNC tube undergoes three different pretreatment methods:

[0101] Group A: Water in BNC tubes was replaced with N,N-dimethylformamide (DMF) at a length (cm) / volume (mL) ratio of 1:2 for BNC tubes to organic solvent. The treatment conditions were: 30°C air bath, circular shaking at 160 rpm; total time: 12 h; repeated 3 times; and finally immersed in N,N-dimethylformamide (DMF).

[0102] Group B: Water in BNC tubes was replaced with dimethylacetamide (DMAc) at a length (cm) / volume (mL) ratio of 1:2 for BNC tubes to organic solvent. The treatment conditions were: 30°C air bath, circular shaking at 160 rpm; total time: 12 h; repeated 3 times; and finally immersed in N,N-dimethylacetamide (DMAc).

[0103] Group C: Water in BNC tubes was replaced with dimethylacetamide (DMAc), with a length (cm) / volume (mL) ratio of BNC tube to organic solvent of 1:2. The treatment conditions were: 30°C air bath, circular shaking at 160 rpm; total time: 12 h; repeated 3 times; and finally immersed in N,N-dimethylformamide (DMF).

[0104] Step (2): Connect a dry three-necked flask to a condenser and a separatory funnel. Connect the condenser to a three-way valve with a nitrogen balloon and seal it with a rubber stopper. Fill the flask with 100 mL of DMF, place it in an ice bath, and evacuate it to a vacuum using a vacuum pump. Pre-cool for 30 min. Using a glass syringe with a long needle, inject the amount of chlorosulfonic acid (90 times the molar amount of BNC) into the separatory funnel via a rubber stopper. Slowly add DMF with slow stirring over 1 h to prepare the sulfation agent until it is stable and free of fumes. Then, heat to room temperature to obtain the sulfation agent for use.

[0105] Step (3) Add 10 BNC tubes (dry weight 0.1 g) after water removal to a flask and stir magnetically at 900 r / m. React for 30 min under nitrogen protection and terminate with 4% (w / v) sodium bicarbonate-n-butanol solution. Then wash with DI water until no reagent residue is left to obtain the product.

[0106] II. Testing Methods

[0107] X-ray photoelectron spectroscopy (XPS) determination: Thermo SCIENTIFIC K-ALPHA was used to determine the full and fine spectra of C, O, and S in a sulfated BNC tube. Type: Monochromatic Al target (E = 1486.68 eV); Voltage: 12000 V; Current: 0.006 A; Vacuum: P < 10 -9 mBar; Bandpass energy: 100 eV (full spectrum), 50 eV (high resolution); Work function: 4.2 eV. Calculate the degree of sulfation substitution according to the following formula.

[0108]

[0109] III. Test Results

[0110] Depend on Figure 12 As shown in Figure a, X-ray photoelectron spectroscopy (XPS) detected S2p at 168.9 eV in groups A, B, and C, indicating that sulfation can occur under all three conditions. By adjusting the sulfation pretreatment method of the BNC tube, the degree of substitution of sulfation can be effectively controlled between 0.2 and 0.4, as seen in... Figure 12 b. The degree of substitution in this range is beneficial for maintaining the three-dimensional structure and imparting good anticoagulant effects to BNC tubes. Furthermore, DMAc, as a potential substitute for DMF, has the advantages of lower toxicity and higher thermal stability than DMF. Therefore, DMAc effectively inhibits the adverse effects of excessive DMF sulfation.

[0111] Comparative Example

[0112] The two patents, CN1176107C (Preparation Method of Sulfated Cellulose) and CN1431225A (Sulfated Cellulose and its Preparation Method and its Application in the Preparation of Anticoagulant Drugs), both use the ClSO3H-DMF method to prepare sulfated cellulose. However, it is particularly important to emphasize that the experimental conditions of the comparative patents are not suitable for the reaction requirements of the tubular cellulose in this patent. The concentration and volume of the sulfate esterifying agent added in this patent ensure the morphology of the BNC tubes and impart multiple performance enhancements, such as anticoagulation, transparency, and lubricity. Furthermore, the experimental raw material of this invention is nanocellulose tubes obtained and purified by microorganisms and used in medical materials, rather than microcrystalline cellulose powder obtained by acid hydrolysis of natural plant cellulose and used in drug synthesis. In addition, the DMF used in this patent does not require thorough drying with molecular sieves followed by vacuum distillation, simplifying the process and making it suitable for industrial production. The BNC tubes are dehydrated using displacement dehydration, ensuring morphological integrity, unlike the high-temperature drying of powder matrices. The two patents differ in the amount of sulfation agent added. This patent uses 30–90 mol chlorosulfonic acid per mol of cellulose, but the concentration is <5% (v / v), meaning the volume concentration is the volume of chlorosulfonic acid added divided by (the volume of chlorosulfonic acid added + the volume of DMF in the BNC tube + the volume of DMF). The comparative patent uses 3–4.5 mol chlorosulfonic acid per mol of cellulose, with a concentration of 10–30% (v / v, meaning the volume concentration is the volume of chlorosulfonic acid added / the volume of DMF). The BNC tubes produced by this patent can be used directly after cleaning and impurity removal, without further dissolution, dialysis, concentration, or lyophilization (drying). This patent emphasizes the novelty and innovation of this method in the preparation of anticoagulant cellulose nanotube biomaterials.

[0113] A method for preparing bacterial cellulose sulfate (CN102702362A) also uses the ClSO3H-DMF method to prepare sulfated nanocellulose, but this patent differs from the comparative patent in the following ways: ① The amount of reaction medium used is different; this patent uses 2-20 mL / g, compared to the comparative patent's 10-100 mL / g. ② The comparative patent uses 0.5-12 mol of esterification reagent per glucose residue, while this patent uses 30-100 mol, therefore the process conditions of the comparative patent are not applicable to this patent. ③ The optimized reaction time in this patent is 20-30 min, while the comparative patent's is >0.5 h, indicating that this patent shortens the reaction time. Furthermore, the patented product, BNC tubes, can be used directly after cleaning and impurity removal, maintaining the original structure of the material, and also requires no further dissolution, dialysis, concentration, or drying.

[0114] A simplified synthesis process for cellulose sulfate (CN103360499B) uses SO3 and 1,2-dichloromethane or dichloromethane as sulfation agents in a homogeneous reaction with aprotic solvents. This process requires strict control of the SO3 concentration, and the reaction product needs further dissolution, filtration, and concentration to obtain a crude product. In contrast, this patent describes a heterogeneous reaction, suitable for sulfation of specific parts of pipelines without completely dissolving the pipe body. The product post-processing is simple, and it is a purified product.

[0115] The method for manufacturing cellulose nanofibers modified by sulfate esterification (CN110446722A) prepares amorphous sulfate esterified cellulose using a fibrinolytic solution of acetic anhydride, propionic anhydride and sulfuric acid, but does not show an improvement in anticoagulant properties.

[0116] It should be further noted that chlorosulfonic acid (ClSO3H), as a very strong dehydrating and sulfonating agent, readily reacts with water vapor (H2O) in the air to produce sulfuric acid (H2SO4) and hydrochloric acid (HCl), which rapidly corrodes metals and carbonized rubber products. Common liquid transfer methods in anhydrous and oxygen-free operations include syringe methods and double-ended needle methods (negative pressure and positive pressure methods). This invention emphasizes the use of glass syringes and stainless steel long-needle syringes for liquid transfer. Although the amount of chlorosulfonic acid (ClSO3H) added can be accurately controlled, it is still impossible to completely avoid the inactivation of some chlorosulfonic acid. Therefore, it should be noted that results under the same experimental conditions may have some deviation, but the trend of the results remains unchanged. It should also be emphasized that when chlorosulfonic acid (ClSO3H) turns yellow or black, if the same degree of substitution and anticoagulant effect as BNC is required, the reaction time needs to be delayed. This invention emphasizes the importance, uniqueness, and distinctiveness of this experimental method to illustrate the uniqueness of this patent. The invention optimizes the amount of chlorosulfonic acid (ClSO3H) added and the reaction time, highlighting the consistency between the experimental method and the experimental results.

Claims

1. A method for preparing an anticoagulant cellulose nanotube, comprising: (1) The nanocellulose tubes are pretreated to obtain pretreated nanocellulose tubes; (2) The sulfate esterification agent reacts with the nanocellulose tubes pretreated in step (1), and after terminating the reaction, anticoagulant nanocellulose tubes are obtained.

2. The preparation method according to claim 1, characterized in that, The pretreatment includes treating the nanocellulose tubes with N,N-dimethylacetamide (DMF) and / or N,N-dimethylacetamide (DMAc).

3. The preparation method according to claim 2, characterized in that, The volume ratio of the nanocellulose tubes to N,N-dimethylacetamide (DMF) and / or N,N-dimethylacetamide (DMAc) is 1:0.8-3. The treatment conditions are 25~50℃ air bath or water bath for 3-48 h; the treatment is repeated 1 to 5 times, and the treatment method is one or more of the following: static setting, oscillation, and ultrasonic treatment. The oscillations include circular, horizontal, or figure-eight oscillations.

4. The preparation method according to claim 1, characterized in that, The preparation of the sulfate esterification agent in step (2) includes: adding dimethylformamide DMF to the reaction apparatus, precooling it under ice bath and nitrogen conditions for 20-100 min, then adding chlorosulfonic acid ClSO3H dropwise to the stirred dimethylformamide DMF until it is stable and smoke-free, and then heating it to 20-30℃ to obtain the sulfate esterification agent. The stirring speed is 500-1000 r / min; The chlorosulfonic acid ClSO3H was added dropwise over a period of 0.5-2 hours. The ratio of dimethylformamide (DMF) to chlorosulfonic acid (ClSO3H) is 20-200 mL: 30-100 mol.

5. The preparation method according to claim 4, characterized in that, The molar amount of chlorosulfonic acid is 10-100 times the molar amount of nanocellulose.

6. The preparation method according to claim 1, characterized in that, In step (2), the sulfate esterification agent reacts with the outer wall or the entire nanofiber tube after pretreatment in step (1): Under nitrogen protection, the pretreated nanofiber tube is added to the sulfate esterification agent and stirred for 0.2-2 h. Or the sulfuric acid esterifying agent in step (2) reacts with the inner cavity of the nanocellulose tube after pretreatment in step (1): the sulfuric acid esterifying agent is injected into the inner cavity of the treated nanocellulose tube and placed in DMF solution under nitrogen protection for 0.2-2 h.

7. The preparation method according to claim 6, characterized in that, The outer wall and inner cavity reactions are described, wherein the two ends of the pretreated nanocellulose tube are closed; the whole tube reaction is described, wherein the two ends of the pretreated nanocellulose tube are not closed.

8. The preparation method according to claim 1, characterized in that, In step (2), the reaction is terminated by using a 2-6% (w / v) weak base salt-short chain alcohol solution; The weak base salt is one or more of acetate, bicarbonate, and citrate; the short-chain alcohol is one or more of methanol, ethanol, isopropanol, or n-butanol. After the reaction is terminated, the mixture is washed with water until no reagent residue remains.

9. An anticoagulant cellulose nanotube prepared by the method of claim 1.

10. The use of the anticoagulant nanocellulose tube of claim 9 in blood contact medical blood vessels and their composite materials, surfactants, humectants and antiviral materials.

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