Chitosan electrospinning liquid and preparation method thereof

Abstract

The application relates to the technical field of biological materials, and discloses a chitosan electrostatic spinning liquid and a preparation method thereof, which comprises the following steps: preparing chitosan through a homogeneous deacetylation method; and preparing the chitosan electrostatic spinning liquid. The preparation method of the chitosan electrostatic spinning liquid is simple and mild, the crystallinity and the intermolecular force are reduced through the mode of reducing the deacetylation degree and the molecular weight of chitosan, the concentration and the spinnability of the pure chitosan spinning stock solution can be improved, and the pure chitosan nanofiber can be successfully prepared.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, and mainly to a chitosan electrospinning solution and its preparation method. Background Technology

[0002] Difficult-to-heal wounds are characterized by high incidence, difficulty in treatment, and high risk. They mainly include traumatic ulcers, diabetic foot ulcers, and radiation injuries, requiring intensive and long-term treatment with expensive products. This not only significantly increases the medical burden but also reduces patients' quality of life. Therefore, the development of functional dressings for difficult-to-heal wounds has received widespread research attention. Wound dressings can greatly improve the wound healing microenvironment, which is crucial for the healing process. However, existing wound dressings suffer from limitations such as limited functionality and inadequate antibacterial and wound-healing-promoting properties.

[0003] In recent years, electrospun nanofiber dressings have attracted widespread attention in the biomedical field due to their unique microstructure and biodegradability. Electrospun nanofibers possess high porosity and high specific surface area; their interconnected three-dimensional porous structure is similar to the natural extracellular matrix. This allows them to effectively absorb wound exudate, enhance the permeability of nutrients and oxygen, effectively block bacterial infection, and promote epidermal cell proliferation and migration, thus showing broad application prospects in wound dressings.

[0004] Chitosan, a product of chitin deacetylation, possesses excellent biocompatibility and biodegradability. It also exhibits antibacterial, antioxidant, hemostatic, and wound-healing-promoting biological activities, making it widely used in medical dressings. Due to the numerous intramolecular and intermolecular hydrogen bonds, chitosan is difficult to dissolve alone in conventional solvents, often requiring dilute acid solutions such as acetic acid, trifluoroacetic acid, hydrochloric acid, or phosphoric acid. However, chitosan's low solubility, high solution viscosity, and the mutual repulsion caused by its polycationic properties under an electric field lead to jet instability during electrospinning, significantly limiting its preparation process. Reports on chitosan electrospinning primarily focus on blending with other flexible, water-soluble polymers, such as polyethylene oxide, polyvinyl alcohol, or polyvinylpyrrolidone (CN 113445155 A). CN110295401 A proposes using an ionic liquid with 1-ethyl-3-methylimidazolium acetate as a solvent, and adjusting the viscosity of the spinning solution with the organic solvent N,N'-dimethylformamide (or N,N'-dimethylacetamide) to prepare pure chitosan ultrafine fibers. However, ionic liquids are expensive and the preparation process is complex. CN 117535825 A proposes adding a dilute alkaline aqueous solution to a dilute acidic chitosan solution to form a hydrogel, reducing the crystallinity of chitosan and hydrogen bonding interactions. Then, after thorough washing with water, the solution is dissolved in hexafluoroisopropanol for electrospinning. This method has high process requirements and is complex.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this application is to provide a chitosan electrospinning solution and its preparation method, which aims to solve the problem of the complex preparation process of existing pure chitosan fiber membranes.

[0007] The technical solution of this application is as follows:

[0008] A method for preparing a chitosan electrospinning solution, comprising the following steps:

[0009] Chitosan was prepared by a homogeneous deacetylation method: chitin powder was uniformly dispersed in sodium hydroxide crushed ice, stirred until the sodium hydroxide crushed ice was completely melted, and after ultrasonic removal of air bubbles, it was frozen at -15℃ to -30℃ for 12 to 48 hours, and then thawed naturally; a homogeneous deacetylation reaction was carried out by heating and stirring in a water bath at 25 to 60℃ for 6 to 48 hours to obtain a deacetylated chitosan solution; the deacetylated chitosan solution was poured into anhydrous ethanol to precipitate, washed with 80% ethanol solution until neutral, and freeze-dried to obtain chitosan with a degree of deacetylation of 20 to 70%;

[0010] Preparation of the chitosan electrospinning solution: The chitosan is dissolved in an acetic acid solution with a mass concentration of 90%, wherein the mass concentration of the chitosan in the acetic acid solution is 2~12 wt%, to obtain the chitosan electrospinning solution.

[0011] The method for preparing the chitosan electrospinning solution in this application is simple and mild, and can improve the concentration and spinnability of the pure chitosan spinning solution, which is beneficial for the successful preparation of pure chitosan nanofibers.

[0012] The method for preparing the chitosan electrospinning solution, wherein the amount of chitin powder added is 3-6 g of chitin powder per 600 mL of sodium hydroxide crushed ice;

[0013] The sodium hydroxide in the sodium hydroxide crushed ice has a mass concentration of 5-40 wt%.

[0014] This application optimizes the steps for dissolving chitosan powder. The sodium hydroxide solution is frozen into sodium hydroxide crushed ice, which is then further crushed into smaller pieces. The chitosan powder is then directly dispersed in the sodium hydroxide crushed ice. This ensures a uniform temperature throughout the reaction system, guaranteeing even dissolution of the chitosan. Using this ratio ensures complete dissolution of the chitosan after one freeze-thaw cycle with NaOH crushed ice. If too much chitosan is added, the number of subsequent freeze-thaw cycles needs to be increased, requiring more time.

[0015] In the method for preparing the chitosan electrospinning solution, the amount of anhydrous ethanol used is four times the volume of the deacetylated chitosan solution.

[0016] The method for preparing the chitosan electrospinning solution, wherein the ultrasonic bubble removal process has an ultrasonic power of 500W and an ultrasonic time of 5 min.

[0017] The method for preparing the chitosan electrospinning solution further includes the following steps before the step of preparing the chitosan electrospinning solution:

[0018] The chitosan is subjected to enzymatic hydrolysis to reduce its molecular weight to 200-700 kDa.

[0019] The method for preparing the chitosan electrospinning solution, wherein the step of enzymatic hydrolysis of the chitosan specifically includes the following steps:

[0020] The chitosan was dissolved in an acetic acid solution with a mass concentration of 1-2%, chitosanase was added, and the mixture was stirred and hydrolyzed in a water bath at 25-60°C for 0.5-4 h.

[0021] The method for preparing the chitosan electrospinning solution involves dissolving 0.5-2g of the chitosan in 100mL of an acetic acid solution with a mass concentration of 1-2%.

[0022] The method for preparing the chitosan electrospinning solution includes adding 2-10 mg of chitosanase per 1 g of chitosan.

[0023] The method for preparing the chitosan electrospinning solution, wherein, in the step of preparing the chitosan electrospinning solution, when the molecular weight of the chitosan is 690 kDa, the mass concentration of the chitosan in the acetic acid solution is 4-5 wt%, and when the molecular weight of the chitosan is 340 kDa, the mass concentration of the chitosan in the acetic acid solution is 9-11 wt%.

[0024] A chitosan electrospinning solution, wherein the chitosan electrospinning solution is prepared by the method described above.

[0025] Beneficial effects: The preparation method of chitosan electrospinning solution in this application is simple and mild. By reducing the degree of deacetylation and molecular weight of chitosan, the crystallinity and intermolecular forces are reduced, which can improve the concentration and spinnability of pure chitosan spinning solution, and facilitate the successful preparation of pure chitosan nanofibers. Attached Figure Description

[0026] Figure 1 This is a scanning electron microscope image of the chitosan electrospun fiber membrane prepared in Example 1 of this application.

[0027] Figure 2 This is a scanning electron microscope image of the chitosan electrospun fiber membrane prepared in Example 2 of this application.

[0028] Figure 3 This is a scanning electron microscope image of the chitosan electrospun fiber membrane prepared in Example 3 of this application.

[0029] Figure 4 This is a scanning electron microscope image of the chitosan electrospun fiber membrane prepared in Example 4 of this application.

[0030] Figure 5 The figures show the cytotoxicity test results for Examples 1-4. Detailed Implementation

[0031] This application provides a chitosan electrospinning solution and its preparation method. To make the objectives, technical solutions, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit the scope of this application.

[0032] This application provides a simple method for preparing a pure chitosan electrospinning solution that requires no spinning aids and can be applied to the repair of refractory wounds. The method uses low-deacetylated chitosan as the raw material, reducing the number of protonated amino groups in the acid solution system, thereby minimizing the impact of intermolecular electrostatic repulsion on the spinning process. For chitosan with a deacetylation degree of approximately 50% prepared by a homogeneous deacetylation process, the random distribution and minimal deviation of N-acetyl groups maximize the disruption of the secondary structure of chitosan molecules, weakening crystallinity and intermolecular forces, and enhancing spinnability. Furthermore, by reducing the molecular weight of chitosan, its intermolecular forces can be further reduced, while simultaneously decreasing the viscosity of the spinning solution and increasing its concentration.

[0033] Chitosan possesses excellent biocompatibility and biodegradability, along with antibacterial, anti-inflammatory, hemostatic, and wound-healing-promoting bioactivities. Chitosan nanofiber membranes constructed using electrospinning technology not only retain these excellent bioactivities but also exhibit a structure similar to the extracellular matrix, promoting epidermal cell proliferation and migration. The antibacterial properties of chitosan itself and the physical barrier effect provided by the nanofiber membrane offer dual protection for the wound while ensuring the breathability of the wound dressing. Furthermore, chitosan with a degree of deacetylation of approximately 50%, due to its low crystallinity and interchain hydrogen bonds, significantly increases the hydrophilicity of the nanofibers, further facilitating the absorption of wound exudate and accelerating epidermal cell migration, making it an ideal wound dressing material.

[0034] Specifically, the method for preparing the chitosan electrospinning solution of this application includes the following steps:

[0035] (1) Chitosan was prepared by homogeneous deacetylation:

[0036] Chitosan powder was evenly dispersed in sodium hydroxide crushed ice (NaOH mass concentration of 5~40 wt%), stirred until the sodium hydroxide crushed ice was completely melted, and after removing air bubbles by ultrasonication, it was frozen at -15℃~-30℃ for 12~48 h, and then thawed naturally.

[0037] The homogeneous deacetylation reaction was carried out by heating and stirring in a water bath at 25~60℃ for 6~48 h to obtain a deacetylated chitosan solution.

[0038] The deacetylated chitosan solution was poured into 4 times its volume of anhydrous ethanol to precipitate, washed with 80% ethanol solution until neutral, and lyophilized to obtain chitosan with a degree of deacetylation of 20-70%.

[0039] In step (1), the amount of chitin powder added can be 3-6 g of chitin powder per 600 mL of sodium hydroxide crushed ice. Through multiple experiments, it has been verified that the above ratio can ensure that the chitin is completely dissolved after NaOH crushed ice and one subsequent freeze-thaw cycle. If too much chitin is added, the number of subsequent freeze-thaw cycles needs to be increased, which will take more time.

[0040] Furthermore, in existing technologies, chitosan powder and sodium hydroxide solution are typically mixed in an ice bath. However, this method is not suitable for large-scale production. If the volume of solution used in production is large, mixing chitosan powder and sodium hydroxide solution in an ice bath will result in a temperature difference between the edge and center of the container, making it difficult for the center temperature to reach approximately 0°C, leading to uneven dissolution of chitosan. Therefore, this application optimizes the step of dissolving chitosan powder by freezing the sodium hydroxide solution into sodium hydroxide crushed ice, then breaking it into crushed ice, and directly dispersing the chitosan powder in the sodium hydroxide crushed ice. This ensures a uniform temperature throughout the reaction system, guaranteeing uniform dissolution of chitosan.

[0041] In step (1), freezing at -15℃ to -30℃ for 12 to 48 hours is to allow water molecules and NaOH molecules to form ice crystals. The mechanical force generated during the formation and growth of ice crystals can destroy the dense crystalline structure inside chitin, thereby dissolving the chitin. The required freezing temperature is -15℃ to -30℃. Within this temperature range, the ice crystal formation and growth process is slower, and the ice crystals are larger, which can destroy the crystalline structure to a greater extent. However, when the temperature is below -30℃, the ice crystal formation process is faster and the ice crystals are smaller, which is insufficient to destroy the crystalline structure of chitin, resulting in poor solubility.

[0042] In step (1), a homogeneous deacetylation reaction is carried out by heating and stirring in a water bath at 25~60℃ for 6~48h. The degree of deacetylation is controlled by controlling different reaction times. Generally speaking, 8h of reaction corresponds to a degree of deacetylation of 28%, 16h of reaction corresponds to a degree of deacetylation of 51%, and 24h of reaction corresponds to a degree of deacetylation of 63%.

[0043] In step (1), the deacetylated chitosan is poured into 4 times its volume of anhydrous ethanol to precipitate. Using 4 times its volume of anhydrous ethanol is the minimum amount required to completely settle the chitosan. Further increasing the ethanol content will not increase the yield, but it is not recommended to reduce the amount of anhydrous ethanol, as this will reduce the yield.

[0044] In step (1), during the process of removing air bubbles by ultrasound, the ultrasonic power can be 500W and the ultrasonic time can be 5min.

[0045] (2) Enzymatic hydrolysis of chitosan:

[0046] The chitosan prepared in step (1) was dissolved in an acetic acid solution with a mass concentration of 1-2%, and then chitosanase was added. The mixture was stirred and hydrolyzed in a water bath at 25-60℃ for 0.5-4 h to obtain chitosan with a molecular weight range (weight average molecular weight) of 200-700 kDa.

[0047] In step (2), by optimizing the concentration of chitosan in the acetic acid solution and the amount of chitosanase added, the viscosity and reaction rate of the reaction system are moderate, resulting in high molecular weight controllability.

[0048] Specifically, 0.5-2g of chitosan can be dissolved in 100mL of acetic acid solution with a mass concentration of 1-2%. The advantage of using the above ratio is that the viscosity of the dissolved chitosan is moderate, which can be easily stirred in the early stage of enzyme degradation, thus helping to ensure the uniformity of the reaction system. If the viscosity is too high, it will be more difficult to stir, which will make the reaction system more likely to be uneven, resulting in a wider molecular weight distribution of chitosan.

[0049] Specifically, 2-10 mg of chitosanase can be added per 1g of chitosan. The amount of chitosanase added is based on considerations of the reaction rate. Using this addition ratio makes the reaction relatively mild and allows for the acquisition of different molecular weight parameters in a short time. If the amount added is too small, the degradation rate will be slow, which will greatly prolong the reaction time. If the amount added is too large, the degradation rate will be too fast, the uniformity of the reaction will decrease, the reaction time will be short, and it will be difficult to collect chitosan of different molecular weights.

[0050] In step (2), during the enzymatic hydrolysis in a water bath at 25~60℃ for 0.5~4h, if the temperature is too low, the reaction rate will slow down; if the temperature is increased, the reaction rate will speed up. However, chitosan raw material itself is prone to browning when heated for a long time, so it is not recommended to exceed 60℃. In this application, the molecular weight of the prepared chitosan is mainly controlled by controlling the enzymatic hydrolysis time; the longer the reaction time, the lower the molecular weight.

[0051] (3) Preparation of chitosan electrospinning solution:

[0052] Chitosan was dissolved in a 90% acetic acid solution, with a chitosan concentration of 2-12 wt%, to obtain a chitosan electrospinning solution.

[0053] For chitosan raw materials with the above-mentioned molecular weight and degree of deacetylation ranges, a chitosan mass concentration of 2-12 wt% in the chitosan electrospinning solution can ensure the spinnability of the chitosan electrospinning solution. Different molecular weights of chitosan correspond to different optimal concentrations; the smaller the molecular weight, the higher the optimal concentration. For example, for chitosan raw materials with a molecular weight of 690 kDa, a preferred concentration is 4-5 wt%, and for chitosan raw materials with a molecular weight of 340 kDa, a preferred concentration is 9-11 wt%.

[0054] In this application, the chitosan obtained in step (1) can also be used directly to prepare chitosan electrospinning solution without enzymatic hydrolysis in step (2). While chitosan electrospinning solution without enzymatic hydrolysis can also be used for electrospinning, due to the large molecular weight of chitosan, the concentration of the chitosan electrospinning solution is low but the viscosity is high, requiring a high critical voltage for spinning. This leads to droplet formation during spinning, resulting in poor nanofiber morphology. Therefore, in this application, it is preferable to further reduce the molecular weight of chitosan through enzymatic hydrolysis. This increases the concentration and viscosity of the chitosan electrospinning solution, which is beneficial for improving the entanglement of molecular chains during spinning, enhancing spinnability, and resulting in uniform nanofiber morphology while improving spinning efficiency. However, the molecular weight of chitosan cannot be too low; otherwise, the polymer jet will break during spinning, causing point spraying.

[0055] The method for preparing the chitosan electrospinning solution in this application has the following advantages:

[0056] (1) This application reduces the crystallinity and intermolecular interactions of chitosan by lowering its degree of deacetylation and molecular weight, thereby improving the spinnability of pure chitosan without the need for any additives;

[0057] (2) The spinning solution prepared by the above preparation method can be used to prepare fiber membranes through a simple and mild process, which is conducive to ensuring the bioactivity of the fiber membranes and making the prepared fiber membranes a potential wound repair material.

[0058] This application also provides a chitosan electrospinning solution, which is prepared by the above-described method for preparing chitosan electrospinning solution.

[0059] The chitosan electrospinning solution of this application can be used for electrospinning to prepare chitosan electrospinned fiber membranes.

[0060] The chitosan electrospinning solution of this application can be spun using conventional electrospinning processes.

[0061] This application also provides a method for preparing a chitosan electrospun fiber membrane, comprising the following steps:

[0062] (1) Preparation of electrospun fiber membranes:

[0063] Using the above-mentioned chitosan electrospinning solution as the spinning solution, electrospun fiber membranes were prepared by electrospinning. The spinning parameters were set as follows: positive electrode voltage of 15~20 kV, negative electrode voltage of -3 kV, spinning solution flow rate of 0.2~3 mL / h, receiving distance of 10~25 cm, and air humidity of 30~60%.

[0064] In step (1), the air humidity is preferably 30%, and the electrospun fiber membrane produced under this air humidity has a better fiber morphology.

[0065] (2) Post-treatment of electrospun fiber membranes:

[0066] Preparation of curing solution: The curing solution is prepared by dissolving sodium hydroxide in an aqueous ethanol solution with a volume concentration of 75% and the mass concentration of sodium hydroxide in the aqueous ethanol solution is 5 wt%.

[0067] The electrospun fiber membrane was immersed in a mixture of ethanol and sodium hydroxide for at least 4 hours, then washed with water until neutral, and dried at room temperature to obtain a chitosan electrospun fiber membrane.

[0068] In step (2), the immersion treatment in the ethanol-sodium hydroxide mixture for at least 4 hours is to solidify the water-soluble electrospun fiber membrane, stabilize its nanofiber structure, and render it insoluble in water. This solidification method should also be applicable to other chitosan-based fiber membranes.

[0069] This application also provides a pure chitosan electrospun fiber membrane, prepared using the aforementioned method for preparing chitosan electrospun fiber membranes. In this application, pure chitosan refers to chitosan free of other flexible water-soluble polymers. However, in the aforementioned method for preparing the chitosan electrospun solution, chitosanase is essentially removed during the sedimentation and washing process of the chitosan raw material. Similarly, in the aforementioned method for preparing the chitosan electrospun fiber membrane, acetic acid is also removed during the post-treatment of the electrospun membrane. Therefore, the pure chitosan electrospun fiber membrane of this application, in addition to being free of other flexible water-soluble polymers, is also essentially free of chitosanase and acetic acid, allowing the prepared pure chitosan electrospun fiber membrane to better exert the bioactivity of chitosan.

[0070] The present application will be further described below through specific embodiments.

[0071] Example 1:

[0072] (1) Preparation of spinning solution: 4.5 g of chitin powder was uniformly dispersed in 600 mL of sodium hydroxide crushed ice (NaOH mass concentration of 15 wt%), stirred until the sodium hydroxide crushed ice was completely melted, and ultrasonically removed air bubbles (ultrasonic power of 500 W, ultrasonic time of 5 min). Then it was frozen at -20℃ for 24 h and thawed naturally. Then it was heated and stirred in a 40℃ water bath to carry out a homogeneous deacetylation reaction for 16 h. Then the deacetylated chitosan solution was poured into 4 times the volume of anhydrous ethanol to precipitate, washed with 80% ethanol solution until neutral, and freeze-dried to obtain chitosan with a degree of deacetylation of 51%. The weight average molecular weight of the undegraded chitosan was tested to be 690 kDa. The above chitosan was dissolved in 90% acetic acid solution with a mass concentration of 4.5 wt%.

[0073] (2) Preparation of chitosan electrospun fiber membrane: Chitosan electrospun fiber membrane was prepared by electrospinning. The spinning conditions were: positive electrode voltage 18 kV, negative electrode voltage -3 kV, spinning solution flow rate 0.8 ml / h, receiving distance 20 cm, and air humidity 50%.

[0074] (3) Post-treatment of electrospun fiber membrane: Sodium hydroxide was dissolved in an ethanol aqueous solution with a volume concentration of 75% to prepare a curing solution. The mass concentration of sodium hydroxide in the ethanol aqueous solution was 5 wt%. The electrospun fiber membrane was immersed in the curing solution for 4 h, then washed with water until neutral, and dried at room temperature to obtain chitosan electrospun fiber membrane.

[0075] Example 2: (The main difference from Example 1 is the degree of deacetylation)

[0076] (1) Preparation of spinning solution: 4.5 g of chitin powder was uniformly dispersed in 600 mL of sodium hydroxide crushed ice (NaOH mass concentration of 15 wt%), stirred until the sodium hydroxide crushed ice was completely melted, and ultrasonically removed air bubbles (ultrasonic power of 500 W, ultrasonic time of 5 min). Then it was frozen at -20℃ for 24 h and thawed naturally. Then it was heated and stirred in a 40℃ water bath to carry out a homogeneous deacetylation reaction for 24 h. Then the deacetylated chitosan solution was poured into 4 times the volume of anhydrous ethanol to precipitate, washed with 80% ethanol solution until neutral, and freeze-dried to obtain chitosan with a degree of deacetylation of 63%. The weight average molecular weight of the undegraded chitosan was tested to be 690 kDa. The above chitosan was dissolved in 90% acetic acid solution with a mass concentration of 4.5 wt%.

[0077] (2) Preparation of chitosan electrospun fiber membrane: Chitosan electrospun fiber membrane was prepared by electrospinning. The spinning conditions were: positive electrode voltage 18 kV, negative electrode voltage -3 kV, spinning solution flow rate 0.8 ml / h, receiving distance 20 cm, and air humidity 50%.

[0078] (3) Post-treatment of electrospun fiber membrane: Sodium hydroxide was dissolved in an ethanol aqueous solution with a volume concentration of 75% to prepare a curing solution. The mass concentration of sodium hydroxide in the ethanol aqueous solution was 5 wt%. The electrospun fiber membrane was immersed in the curing solution for 4 h, then washed with water until neutral, and dried at room temperature to obtain chitosan electrospun fiber membrane.

[0079] Example 3: (The main difference from Example 1 is the molecular weight)

[0080] (1) Preparation of spinning solution: 4.5 g of chitin powder was uniformly dispersed in 600 mL of sodium hydroxide crushed ice (NaOH mass concentration of 15 wt%), stirred until the sodium hydroxide crushed ice was completely melted, and ultrasonically removed air bubbles (ultrasonic power of 500 W, ultrasonic time of 5 min). The solution was then frozen at -20℃ for 24 h and thawed naturally. Then, a homogeneous deacetylation reaction was carried out by heating and stirring in a 40℃ water bath for 16 h. The deacetylated chitosan solution was then poured into 4 times the volume of anhydrous ethanol to precipitate the precipitate. The solution was washed with 80% ethanol solution until neutral and freeze-dried to obtain chitosan with a degree of deacetylation of 51%. 1 g of chitosan was dissolved in 100 mL of 1% acetic acid solution, and then 5 mg of chitosanase was added. The solution was stirred and hydrolyzed in a 40℃ water bath for 1.5 h. The weight-average molecular weight of the chitosan was 340 kDa. The chitosan was dissolved in a 90% acetic acid solution, with a mass concentration of 10 wt%.

[0081] (2) Preparation of chitosan electrospun fiber membrane: Chitosan electrospun fiber membrane was prepared by electrospinning. The spinning conditions were: positive electrode voltage 18 kV, negative electrode voltage -3 kV, spinning solution flow rate 0.8 ml / h, receiving distance 20 cm, and air humidity 50%.

[0082] (3) Post-treatment of electrospun fiber membrane: Sodium hydroxide was dissolved in an ethanol aqueous solution with a volume concentration of 75% to prepare a curing solution. The mass concentration of sodium hydroxide in the ethanol aqueous solution was 5 wt%. The electrospun fiber membrane was immersed in the curing solution for 4 h, then washed with water until neutral, and dried at room temperature to obtain chitosan electrospun fiber membrane.

[0083] Example 4: (The main difference from Example 3 is the air humidity)

[0084] (1) Preparation of spinning solution: 4.5 g of chitin powder was uniformly dispersed in 600 mL of sodium hydroxide crushed ice (NaOH mass concentration of 15 wt%), stirred until the sodium hydroxide crushed ice was completely melted, and ultrasonically removed air bubbles (ultrasonic power of 500 W, ultrasonic time of 5 min). The solution was then frozen at -20℃ for 24 h and thawed naturally. Then, a homogeneous deacetylation reaction was carried out by heating and stirring in a 40℃ water bath for 16 h. The deacetylated chitosan solution was then poured into 4 times the volume of anhydrous ethanol to precipitate the precipitate. The solution was washed with 80% ethanol solution until neutral and freeze-dried to obtain chitosan with a degree of deacetylation of 51%. 1 g of chitosan was dissolved in 100 mL of 1% acetic acid solution, and then 5 mg of chitosanase was added. The solution was stirred and hydrolyzed in a 40℃ water bath for 1.5 h. The weight-average molecular weight of the chitosan was 340 kDa. The chitosan was dissolved in a 90% acetic acid solution, with a mass concentration of 10 wt%.

[0085] (2) Preparation of chitosan electrospun fiber membrane: Chitosan electrospun fiber membrane was prepared by electrospinning. The spinning conditions were: positive electrode voltage 18 kV, negative electrode voltage -3 kV, spinning solution flow rate 0.8 ml / h, receiving distance 20 cm, and air humidity 30%.

[0086] (3) Post-treatment of electrospun fiber membrane: Sodium hydroxide was dissolved in an ethanol aqueous solution with a volume concentration of 75% to prepare a curing solution. The mass concentration of sodium hydroxide in the ethanol aqueous solution was 5 wt%. The electrospun fiber membrane was immersed in the curing solution for 4 h, then washed with water until neutral, and dried at room temperature to obtain chitosan electrospun fiber membrane.

[0087] Figure 1-3 The images shown are scanning electron microscope (SEM) images of the chitosan electrospun fiber membranes prepared in Examples 1-3, respectively. Figure 1-3This indicates that the prepared spinning solution is spinnable. However, the spinning solution concentrations in Examples 1 and 2 were low, resulting in some bead-like structures. Example 3, by reducing the molecular weight and increasing the concentration of the spinning solution, resulted in a more uniform nanofiber structure. The difference between Example 3 and Example 1 lies in the addition of an enzymatic hydrolysis step. As seen in Examples 1 and 3, spinning can be achieved without enzymatic hydrolysis, but droplet formation occurs during the spinning process in Example 1, leading to suboptimal nanofiber morphology. In Example 3, enzymatic hydrolysis reduces the molecular weight of chitosan and increases the concentration of the spinning solution, resulting in nanofibers with a uniform morphology.

[0088] Figure 4 This is a scanning electron microscope (SEM) image of the chitosan electrospun fiber membrane prepared in Example 4. Example 4 was prepared under low air humidity conditions. Figure 4 As can be seen, it has a clear nanofiber structure and no beaded structure, indicating that it has good spinning performance.

[0089] Figure 5 The graph shows the cytotoxicity test results of Examples 1-4. The cell activity of each group is >80%, indicating that the chitosan electrospun fiber membranes prepared in Examples 1-4 have excellent biocompatibility. The cytotoxicity test method described above was to test the cytotoxicity of the chitosan electrospun fiber membranes prepared in Examples 1-4 using a direct contact method: L929 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution; chitosan electrospun fiber membranes prepared in Examples 1-4 with a diameter of 1 cm were added to 24-well plates, and L929 cells were seeded onto the surface of the chitosan electrospun fiber membrane at a density of 80,000 cells per well. The 24-well plates were then cultured in a humidified atmosphere of 37°C and 5% CO2, with the control group using standard medium; after 24 hours of culture, medium supplemented with 10% CCK-8 reagent was added to each well, and the plates were cultured for another 2 hours. The absorbance of each well at 450 nm was recorded using a microplate reader (Bio-Tek ELx800, USA).

[0090] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A method for preparing a chitosan electrospinning solution, characterized by, The method comprises the following steps: The chitosan is prepared by a homogeneous deacetylation method: chitin powder is uniformly dispersed in sodium hydroxide ice, stirred until the sodium hydroxide ice is completely melted, frozen at -15°C to -30°C for 12 to 48 hours after ultrasonic removal of bubbles, and then naturally thawed; a homogeneous deacetylation reaction is carried out by heating and stirring in a water bath at 25 to 60°C, and the reaction time is 6 to 48 hours to obtain a deacetylated chitosan solution; the deacetylated chitosan solution is poured into anhydrous ethanol for precipitation, washed with an ethanol solution with a volume concentration of 80% until neutral, and then freeze-dried to obtain the chitosan with a deacetylation degree of 20 to 70%; The chitosan is subjected to enzymatic hydrolysis treatment to make the molecular weight of the chitosan in the range of 200 to 700 kDa; The chitosan electrospinning solution is prepared by dissolving the chitosan in an acetic acid solution with a mass concentration of 90%, and the mass concentration of the chitosan in the acetic acid solution is 2 to 12 wt% to obtain the chitosan electrospinning solution; The step of subjecting the chitosan to enzymatic hydrolysis treatment specifically comprises the following steps: The chitosan is dissolved in an acetic acid solution with a mass concentration of 1 to 2%, and chitosanase is added, and the enzymatic hydrolysis is carried out by stirring in a water bath at 25 to 60°C for 0.5 to 4 hours. 100 mL of the acetic acid solution with a mass concentration of 1 to 2% is used to dissolve 0.5 to 2 g of the chitosan.

2. The method of claim 1, wherein the chitosan electrospinning solution is prepared by dissolving chitosan in an acidic solution to form a chitosan solution, and adding a surfactant to the chitosan solution to form the chitosan electrospinning solution. The chitin powder is added in an amount of 3 to 6 g per 600 mL of the sodium hydroxide ice. The mass concentration of sodium hydroxide in the sodium hydroxide ice is 5 to 40 wt%.

3. The method of claim 1, wherein the chitosan electrospinning solution is prepared by dissolving chitosan in an acidic solution to form a chitosan solution, and adding a surfactant to the chitosan solution to form the chitosan electrospinning solution. The amount of anhydrous ethanol is 4 times the volume of the deacetylated chitosan solution.

4. The method of claim 1, wherein the chitosan electrospinning solution is prepared by dissolving chitosan in an acidic solution to form a chitosan solution, and adding a surfactant to the chitosan solution to form the chitosan electrospinning solution. In the process of ultrasonic removal of bubbles, the ultrasonic power is 500 W, and the ultrasonic time is 5 minutes.

5. The method of claim 1, wherein the chitosan electrospinning solution is prepared by dissolving chitosan in an acidic solution to form a chitosan solution, and adding a surfactant to the chitosan solution to form the chitosan electrospinning solution. 2 to 10 mg of chitosanase is added per 1 g of chitosan.

6. The method of claim 1, wherein the chitosan electrospinning solution is prepared by dissolving chitosan in an acidic solution to form a chitosan solution, and adding a surfactant to the chitosan solution to form the chitosan electrospinning solution. In the step of preparing the chitosan electrospinning solution, when the molecular weight of the chitosan is 690 kDa, the mass concentration of the chitosan in the acetic acid solution is 4 to 5 wt%, and when the molecular weight of the chitosan is 340 kDa, the mass concentration of the chitosan in the acetic acid solution is 9 to 11 wt%.

7. An electrospinning solution of chitosan, characterized in that, The chitosan electrospinning solution is prepared by the method according to any one of claims 1 to 6.

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