Preparation method of polypeptide-chitosan conjugate film nerve conduit

Abstract

The invention provides a preparation method of a polypeptide-chitosan conjugate film nerve conduit, which comprises the following steps: preparing an alkali / urea aqueous solution in a container according to a certain proportion, and adding gelatin into the prepared alkali / urea aqueous solution to prepare a gelatin hydrolyzed polypeptide solution; chitosan is added into the gelatin hydrolyzed polypeptide solution to be fully dissolved; putting the solution dissolved with the chitosan into a cold trap, dropwise adding 1, 4-butanediol diglycidyl ether to carry out polypeptide-chitosan coupling reaction, and carrying out low-temperature centrifugation to remove bubbles for later use; preparing a polypeptide-chitosan conjugate film by adopting a tape casting method and solution-gel transformation; coating one side of the strip-shaped polypeptide-chitosan conjugate film with a dilute acetic acid solution, and winding the strip-shaped polypeptide-chitosan conjugate film around a rod-shaped object to form a tube shape, so as to prepare the nerve conduit; through controllable chemical conjugation and structural design, the preparation method has high biological activity and bionic mechanical property, the preparation process is simple, the safety is high, and green and efficient large-scale production can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of tissue engineering, in particular to a method for preparing a polypeptide-chitosan conjugate thin film nerve conduit. Background Art

[0002] Peripheral nerve injury is a common traumatic condition in clinical practice, which can lead to permanent loss of sensory or motor function in severe cases. Currently, autologous nerve transplantation remains the gold standard for repairing long-segment nerve defects, but it suffers from drawbacks such as donor site damage, limited nerve sources, and size mismatch. Artificial nerve conduits, as an alternative, have become a research hotspot in the field of tissue engineering by creating a biomimetic microenvironment to guide axonal regeneration.

[0003] Chitosan is widely used in the development of nerve conduit materials due to its excellent biocompatibility, degradability, and antibacterial properties. However, pure chitosan-based materials still have the following limitations: 1. The lack of bioactive signals that promote nerve cell adhesion and proliferation leads to insufficient axon regeneration efficiency; 2. Mechanical properties (such as flexibility and tensile strength) differ from those of the natural epineurium, which may cause local inflammation or mechanical mismatch; 3. The degradation rate and nerve regeneration speed are difficult to precisely control, which can easily lead to conduit collapse or hinder the formation of a regenerative microenvironment. In addition, traditional nerve conduits mostly rely on macroscopic tubular structures and lack microscopic topological structures (such as nanofiber orientation and surface patterning) to guide the directional migration of Schwann cells and axon extension.

[0004] In recent years, researchers have attempted to enhance the bioactivity of chitosan catheters through surface modification (e.g., grafting RGD peptides) or co-modification (e.g., with collagen or gelatin). However, physically adsorbed or simply co-blended peptides are prone to shedding, making it difficult to maintain long-term activity. Chemical modification, if carried out under harsh reaction conditions, can disrupt the integrity of the chitosan molecular chain and affect the mechanical properties of the material. Furthermore, existing preparation processes (e.g., electrospinning and solvent casting) lack precise control over the catheter microstructure and may contain toxic solvent residues, limiting their potential for clinical application.

[0005] Based on the above background, the chitosan-based nerve conduits and their preparation methods in the existing technology still have the following key problems that need to be solved urgently: 1. Insufficient biological activity and stability: Physically modified polypeptides are easy to fall off, and the chemical modification process may destroy the chitosan structure, resulting in low density and uneven distribution of bioactive molecules on the conduit surface, making it difficult to continuously activate signal pathways related to nerve cell adhesion and axon growth; 2. Mismatch between mechanical properties and nerve regeneration needs: The elastic modulus and tensile strength of traditional chitosan films are significantly different from those of natural nerve tissue, which can easily cause mechanical stress imbalance after implantation and hinder the homeostasis of the regenerative microenvironment; 3. Complexity of the preparation process and safety defects: Traditional methods require the use of toxic cross-linking agents (such as glutaraldehyde) or multi-step reactions, resulting in poor process repeatability, high risk of residual toxicity, and difficulty in large-scale production of conduits with complex structures. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of the above-mentioned prior art and provide a method for preparing a polypeptide-chitosan conjugate film and a nerve conduit with high biological activity, biomimetic mechanical properties and safety.

[0007] The technical solution of the present invention is to provide a method for preparing a polypeptide-chitosan conjugate thin film nerve conduit, comprising the following steps: S1. Preparation of polypeptide-chitosan coupling solution: S1.1. Add lithium hydroxide monohydrate, potassium hydroxide, urea, and water in a certain proportion to a container and stir to dissolve at room temperature to prepare an alkali / urea aqueous solution; S1.2. Add gelatin to the prepared alkali / urea aqueous solution and conduct a reaction under magnetic stirring at room temperature to prepare a gelatin hydrolyzed polypeptide solution; S1.3. Add chitosan to the above-mentioned gelatin hydrolyzed polypeptide solution, stir and disperse it evenly, place it in a freezer and freeze it at low temperature, then take it out and thaw it at room temperature. When it starts to melt, mechanically stir it to accelerate the dissolution of the chitosan; repeat the above freeze-thaw cycle multiple times to fully dissolve the chitosan; S1.4. Place the chitosan solution in a cold trap, then slowly add an appropriate amount of 1,4-butanediol diglycidyl ether while mechanically stirring to carry out a polypeptide-chitosan coupling reaction. After the reaction continues for a period of time, stirring is stopped to obtain a polypeptide-chitosan coupling solution. The bubbles in the polypeptide-chitosan coupling solution are then removed by low-temperature centrifugation for later use; S2. Preparation of polypeptide-chitosan conjugate film: S2.1. Use a casting method to evenly spread the degassed polypeptide-chitosan coupling solution on a glass plate through a glass rod. The thickness of the spread solution can be controlled by the thickness of the plastic ties tightly clamped at both ends of the glass rod. S2.2. Transfer the glass plate to an oven and bake. The polypeptide-chitosan coupling solution will undergo a solution-gel transition at high temperature. After the polypeptide-chitosan coupling solution solidifies, remove the glass plate and place it in pure water. The hydrogel film formed by the polypeptide-chitosan conjugate will detach from the glass plate. Continue to wash the hydrogel film with pure water to remove the alkali and urea therein. S2.3. Attach the hydrogel film to a polypropylene plastic plate and secure it with tape on all sides. Place the film in a sterile room and air-dry it at room temperature to obtain a dry polypeptide-chitosan conjugate film. S3. Nerve catheter preparation: S3.1. Since the polypeptide-chitosan conjugate can be dissolved in an acidic aqueous solution, an appropriate amount of dilute acetic acid solution is used as a dissolving adhesive and evenly coated on one side of a strip of polypeptide-chitosan conjugate film. The polypeptide-chitosan conjugate film is then wound around a rod into a tube with the side coated with the dissolving adhesive facing outward. After bonding, the tube is removed and allowed to stand in a sterile room to evaporate and remove residual acetic acid, thereby producing a nerve conduit.

[0008] Preferably, in the alkali / urea aqueous solution prepared in step S1.1, the mass ratio of lithium hydroxide monohydrate, potassium hydroxide, urea and water is 9:14:16:161.

[0009] Preferably, the mass of the alkali / urea aqueous solution in step S1.2 is 100 g, the molecular weight of gelatin is 50,000-100,000, and the mass is 0.01-6 g.

[0010] Preferably, the magnetic stirring reaction time at room temperature in step S1.2 is 0.5 to 3 hours.

[0011] Preferably, the mass of chitosan in step S1.3 is 1-6 g, and the degree of deacetylation of chitosan is 50-90%.

[0012] Preferably, the temperature range of the freezing equipment in step S1.3 is set to -80~-20°C, and the number of freeze-thaw cycles is repeated 2~3 times.

[0013] Preferably, the reaction temperature of the cold trap in step S1.4 is set to -10~4°C; the amount of 1,4-butanediol diglycidyl ether added is 0.1~1 mL, and the reaction duration is 1~3 hours; the temperature during low-temperature centrifugation to remove bubbles is -10~4°C.

[0014] Preferably, in step S2.1, the thickness of the paving solution is 0.5-2 mm.

[0015] Preferably, the temperature of the oven in step S2.2 is 20-60° C., and the baking time is 5-20 minutes.

[0016] Preferably, the concentration of the dilute acetic acid solution in step S3.1 is 0.1-2 wt %.

[0017] Compared with the prior art, the preparation method of the polypeptide-chitosan conjugate thin film nerve conduit of the present invention has the following advantages and beneficial effects: The present invention prepares a polypeptide-chitosan nerve conduit film with high biological activity, biomimetic mechanical properties, directional topological structure and adaptive degradation behavior through controllable chemical conjugation and structural design. The preparation process is simple and safe. A dilute acetic acid solution is used as a dissolving adhesive to roll the polypeptide-chitosan nerve conduit film into a nerve conduit. The acetic acid can be volatilized and removed, and there is no risk of toxic residues, which can achieve green and efficient large-scale production. DETAILED DESCRIPTION

[0018] The following is a detailed description of an embodiment of the present invention: This embodiment is implemented under the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments. Example

[0019] The present invention provides a method for preparing a polypeptide-chitosan conjugate thin film nerve conduit, comprising the following steps: Prepare an alkali / urea aqueous solution according to the mass ratio. For example, for 100 g of the solution, add 4.5 g of lithium hydroxide monohydrate (LiOH·H2O), 7 g of potassium hydroxide (KOH), 8 g of urea, and 80.5 g of water into a plastic beaker and stir to dissolve at room temperature.

[0020] 0.01-6 g of gelatin (molecular weight 50,000-100,000) was added to 100 g of alkali / urea aqueous solution, and then reacted under magnetic stirring at room temperature for 1 hour to obtain a gelatin hydrolyzed polypeptide solution.

[0021] Add 1-6 g of chitosan (90% deacetylation degree) to the above-mentioned gelatin hydrolyzed polypeptide solution and stir to disperse evenly. Then place it in a freezing device at low temperature (-80°C) to freeze it solid, then take it out and thaw it at room temperature. When the "ice cube" (frozen mixed solution) becomes loose, accelerate the dissolution of chitosan by mechanical stirring. Repeat the freeze-thaw cycle 2-3 times to fully dissolve the chitosan.

[0022] Place the solution containing dissolved chitosan in a cold trap and set the reaction temperature to 0°C. Then, slowly add an appropriate amount (0.1~1 mL) of 1,4-butanediol diglycidyl ether (BDDE) dropwise while mechanically stirring. Continue the reaction for 1~3 hours, then stop stirring. Take out the solution and remove the bubbles in the solution by low-temperature (0°C) centrifugation before setting aside.

[0023] By regulating the mass ratio of gelatin to chitosan, a series of polypeptide-chitosan conjugates with different polypeptide / chitosan mass ratios can be synthesized. Therefore, the specific amount of gelatin and chitosan can be added within a range according to the needs of the user.

[0024] The peptide-chitosan conjugate film was prepared by casting and solution-gel transition. The degassed peptide-chitosan conjugate solution was first evenly spread on a glass plate using a casting method. The thickness of the spread solution was controlled by the thickness of the plastic ties tightly wrapped around the glass rod, which was 1 mm thick. The glass plate was then transferred to an oven and heated at 50°C for 5-20 minutes. The peptide-chitosan conjugate solution underwent a solution-gel transition under the high temperature of the oven. After the peptide-chitosan conjugate solution solidified, the glass plate was removed and placed in pure water. After more than 10 minutes, the hydrogel film formed by the peptide-chitosan conjugate detached from the glass plate. The hydrogel film was then washed with pure water to remove small molecules such as alkali and urea. The hydrogel film was attached to a polypropylene plastic plate and fixed with tape on all sides. The film was then placed in a sterile room and air-dried at room temperature to obtain a dry peptide-chitosan conjugate film.

[0025] A polypeptide-chitosan conjugate film with a thickness of 30 μm was selected and cut into 12×20 mm strips. Then 30 μL of dilute acetic acid aqueous solution (0.5wt%) was evenly applied to one side of the strip polypeptide-chitosan conjugate film. Then, with the side coated with dilute acetic acid aqueous solution facing outward, it was wrapped around a small round rod with a diameter of about 3 mm to form a tube. After bonding, the tube was removed and allowed to stand in a sterile room to evaporate and remove residual acetic acid. Finally, a catheter with a length of 12 mm, an inner diameter of 3 mm, and a wall thickness of about 60 μm was obtained.

[0026] The present invention discloses a method for preparing a polypeptide-chitosan conjugate thin film nerve conduit. The method uses controlled chemical conjugation and structural design to prepare a polypeptide-chitosan nerve conduit film having high biological activity, biomimetic mechanical properties, a directional topological structure, and adaptive degradation behavior. The preparation process is simple and highly safe. A dilute acetic acid solution is used as a dissolving adhesive to roll the polypeptide-chitosan nerve conduit film into a nerve conduit. The acetic acid can be volatilized and removed, and there is no risk of toxic residues, thereby achieving green and efficient large-scale production.

[0027] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing a polypeptide-chitosan conjugate thin film nerve conduit, characterized in that: The following steps are involved: S1. Preparation of polypeptide-chitosan coupling solution: S1.

1. Add lithium hydroxide monohydrate, potassium hydroxide, urea, and water in a certain proportion to a container and stir to dissolve at room temperature to prepare an alkali / urea aqueous solution; S1.

2. Add gelatin to the prepared alkali / urea aqueous solution and conduct a reaction under magnetic stirring at room temperature to prepare a gelatin hydrolyzed polypeptide solution; S1.

3. Add chitosan to the above-mentioned gelatin hydrolyzed polypeptide solution, stir and disperse it evenly, place it in a freezer and freeze it at low temperature, then take it out and thaw it at room temperature. When it starts to melt, mechanically stir it to accelerate the dissolution of the chitosan; repeat the above freeze-thaw cycle multiple times to fully dissolve the chitosan; S1.

4. Place the chitosan solution in a cold trap, then slowly add an appropriate amount of 1,4-butanediol diglycidyl ether while mechanically stirring to carry out a polypeptide-chitosan coupling reaction. After the reaction continues for a period of time, stirring is stopped to obtain a polypeptide-chitosan coupling solution. The bubbles in the polypeptide-chitosan coupling solution are then removed by low-temperature centrifugation for later use; S2. Preparation of polypeptide-chitosan conjugate film: S2.

1. Use a casting method to evenly spread the degassed polypeptide-chitosan coupling solution on a glass plate through a glass rod. The thickness of the spread solution can be controlled by the thickness of the plastic ties tightly clamped at both ends of the glass rod. S2.

2. Transfer the glass plate to an oven and bake. The polypeptide-chitosan coupling solution will undergo a solution-gel transition at high temperature. After the polypeptide-chitosan coupling solution solidifies, remove the glass plate and place it in pure water. The hydrogel film formed by the polypeptide-chitosan conjugate will detach from the glass plate. Continue to wash the hydrogel film with pure water to remove the alkali and urea therein. S2.

3. Attach the hydrogel film to a polypropylene plastic plate and secure it with tape on all sides. Place the film in a sterile room and air-dry it at room temperature to obtain a dry polypeptide-chitosan conjugate film. S3. Nerve catheter preparation: S3.

1. Since the polypeptide-chitosan conjugate can be dissolved in an acidic aqueous solution, an appropriate amount of dilute acetic acid solution is used as a dissolving adhesive and evenly coated on one side of a strip of polypeptide-chitosan conjugate film. The polypeptide-chitosan conjugate film is then wound around a rod into a tube with the side coated with the dissolving adhesive facing outward. After bonding, the tube is removed and allowed to stand in a sterile room to evaporate and remove residual acetic acid, thereby producing a nerve conduit.

2. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 1, characterized in that: In the alkali / urea aqueous solution prepared in step S1.1, the mass ratio of lithium hydroxide monohydrate, potassium hydroxide, urea and water is 9:14:16:

161.

3. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 2, characterized in that: The mass of the alkali / urea aqueous solution in step S1.2 is 100 g, the molecular weight of gelatin is 50,000-100,000, and the mass is 0.01-6 g.

4. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 3, characterized in that: The magnetic stirring reaction time at room temperature in step S1.2 is 0.5 to 3 hours.

5. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 3, characterized in that: The mass of chitosan in step S1.3 is 1-6 g, and the degree of deacetylation of chitosan is 50-90%.

6. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 5, characterized in that: The temperature range of the freezing equipment in step S1.3 is set to -80~-20℃, and the number of freeze-thaw cycles is repeated 2~3 times.

7. The method for preparing a polypeptide-chitosan conjugate thin film nerve guide according to claim 6, characterized in that: The reaction temperature of the cold trap in step S1.4 is set to -10~4°C; 0.1~1 mL of 1,4-butanediol diglycidyl ether is added, and the reaction duration is 1~3 hours; the temperature during low-temperature centrifugation to remove bubbles is -10~4°C.

8. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 1, characterized in that: In step S2.1, the thickness of the paving solution is 0.5-2 mm.

9. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 8, characterized in that: In step S2.2, the oven temperature is 20-60°C, and the baking time is 5-20 minutes.

10. The method for preparing a polypeptide-chitosan conjugate thin film nerve conduit according to claim 1, characterized in that: The concentration of the dilute acetic acid solution in step S3.1 is 0.1-2 wt%.