Neurophilic peptide enhanced double-fiber network structure hydrogel and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials. The invention particularly relates to a neurophilic peptide enhanced double-fiber network structure hydrogel and a preparation method thereof. The hydrogel is prepared from the following raw materials: peptide nanofiber hydrogel, a cellulose nanofiber solution and neurophilic peptide, and the mass concentration ratio of the peptide nanofiber hydrogel to the cellulose nanofiber solution to the neurophilic peptide is (1-4): (10-20): 5. The preparation method is simple, conditions are easy to realize, the use amount of expensive RADA16 is reduced, and the method is suitable for biomedicine application, especially application in spinal cord injury repair. The prepared RCP hydrogel has excellent stability and neuropathic property, has a three-dimensional space suitable for loading stem cells or drug molecules, and is used as a carrier for cell culture and stem cell transplantation to support cell attachment and growth; drugs or growth factors can be loaded and released, and tissue repair and regeneration are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to a neurophilic peptide-enhanced double-fiber network structure hydrogel and a preparation method thereof. Background Art

[0002] Specifically designed peptides are the most promising "candidates" for making bioactive multifunctional hydrogels, which can be used in various biomedical and tissue engineering applications. The synthetic peptide RADA16 sequence (arginine-alanine-aspartic acid-alanine)4 can self-assemble into β-sheet peptide nanofibers (PNFs) in an acidic water environment to form a transparent hydrogel with a relatively stable three-dimensional structure. The structure of PNFs hydrogel is very similar to the three-dimensional structure of natural extracellular matrix. It can be used as a substitute for extracellular matrix to support cell behavior and interaction, which is essential for wound healing and tissue regeneration. The bioactive multifunctional PNFs hydrogel can reshape the microenvironmental homeostasis, support nerve regeneration, and promote functional recovery at the site of spinal cord injury, making significant progress. However, the neurotropism, insufficient stability and high price of PNFs hydrogel cannot fully meet the needs of clinical treatment.

[0003] Cellulose is a polysaccharide existing in nature, with excellent biocompatibility, forming a gel after absorbing water, and low price. Cellulose molecules contain a large number of hydroxyl and carboxyl groups, which can form abundant hydrogen bonds and van der Waals forces between and within chains; intermolecular hydrogen bonds form fibrous structures and semi-crystalline stacking, which make cellulose nanofibers (CNFs) have high strength, flexibility and stability, and can be used to make double fiber network structure hydrogels. Therefore, the design adds CNFs (second fiber network) to PNFs hydrogel (first fiber network), which can form a physically cross-linked hydrogel through ionic electrostatic interactions and / or hydrogen bond non-covalent interactions, with a more stable, flexible, self-healing double fiber network three-dimensional structure, reducing the use of expensive RADA16, which is beneficial to the repair of spinal cord injuries.

[0004] Peptide (PPFLMLLKGSTR, PP) has the effect of promoting nerve growth. It can load nerve growth factor and retinoic acid to induce pluripotent stem cells to differentiate into neurons. PP-modified manganese dioxide nanoparticle dot hydrogel promotes the adhesion growth of mesenchymal stem cells and nerve tissue bridges, guiding the efficient regeneration of spinal cord tissue. Adding PP to the double fiber network hydrogel with a stable three-dimensional spatial structure can further promote the repair of spinal cord injury. Therefore, it is very necessary to develop a method to improve the stability and neurotropism of hydrogel materials to promote the repair of spinal cord injury. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a neurophilic peptide-enhanced double-fiber network structure hydrogel and a preparation method thereof. The hydrogel provided and prepared by the present invention has biomimetic properties, injectability, self-healing properties, biodegradability and good biocompatibility, can significantly improve the stability and neurophilicity of the hydrogel, can realize in situ loading of cells / active molecules and in situ injection in vivo, and has very broad application prospects and clinical application value in the fields of three-dimensional cell culture, tissue engineering biological scaffold materials and drug carriers.

[0006] The technical solutions adopted are:

[0007] A neuropeptide-enhanced double fiber network structure hydrogel, the raw materials include PNFs hydrogel, CNFs solution, and neuropeptide PP, wherein the mass concentration ratio of PP, CNFs, and PNFs is 1-4:10-20:5-10. The preferred mass concentration ratio of PP, CNFs, and PNFs is 1-4:10-20:5.

[0008] As a further preference, the mass concentration ratio of PP, CNFs and PNFs is 1:15:5.

[0009] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0010] (1) Self-assembly of PNFs hydrogel: RADA16 was added to deionized water, the pH value was adjusted to 7, and ultrasonic vibration was applied to fully dissolve it. After standing, it self-assembled to form PNFs hydrogel;

[0011] (2) Preparation of CNFs solution: weigh cellulose, add it into deionized water, and mix thoroughly by ultrasonic vibration to form a CNFs solution;

[0012] (3) Synthesis of dual-fiber network hydrogel: PNFs hydrogel and CNFs solution were mixed in equal volumes and heated in a water bath to form physical cross-links between the two fiber networks through non-covalent hydrogen bonding to form a stable dual-fiber network three-dimensional hydrogel;

[0013] (4) Adding neuropeptides: adding neuropeptide-enhanced PP to the synthesized double-fiber network hydrogel and mixing them to form a bioactive neuropeptide-enhanced double-fiber network structure hydrogel.

[0014] Preferably, in step (1), the ultrasonic oscillation time is 2 to 10 minutes; and the standing time at room temperature is 2 to 3 hours.

[0015] Preferably, in step (1), the mass ratio of RADA16 to deionized water is 10 to 20 (mg / mL).

[0016] Preferably, in step (2), the volume ratio of cellulose mass to deionized water is 20 to 40 (mg / mL); and the ultrasonic oscillation time is 2 to 10 min.

[0017] Preferably, in step (3), the temperature of the water bath heating after the PNFs hydrogel and the CNFs solution are mixed is 30 to 37°C.

[0018] Preferably, the heating time is 30 to 40 minutes.

[0019] Preferably, in step (4), the mixing treatment of PP into the synthesized double-fiber network hydrogel is carried out by stirring, and the stirring method is mechanical stirring or ultrasonic vibration.

[0020] Preferably, the stirring temperature is room temperature, and the stirring time is 5 to 60 minutes, until the mixture is fully mixed.

[0021] The invention is loaded with PP having a neurotropic effect, which further enhances the effect of promoting tissue and wound healing, especially promoting spinal nerve healing. The invention is injectable, simple to operate, and self-healing, and is an ideal biomaterial for promoting tissue healing.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The method for preparing the RCP hydrogel with a neurophilic peptide-enhanced double-fiber network structure described in the present invention has excellent stability and neurophilicity, reduces the amount of expensive RADA16 used, and is suitable for biomedical applications, especially for applications in spinal cord injury repair.

[0024] The preparation method of the invention is simple, the conditions are easy to achieve, and the production cost is reduced.

[0025] The RCP hydrogel of the present invention has a three-dimensional space suitable for loading stem cells or drug molecules, and serves as a carrier for cell culture and stem cell transplantation, supporting cell attachment and growth; it can load and release drugs or growth factors to promote tissue repair and regeneration. The RCP hydrogel of the present invention can be implanted into the site of spinal cord injury in rats, and supports the regeneration and growth of nerve cells through its three-dimensional structure; the addition of neurophilic peptides helps promote nerve regeneration and functional recovery. Therefore, the bioactive RCP hydrogel prepared by the present invention can be used as a biomaterial in the field of tissue engineering and regenerative medicine, and can support tissue regeneration and functional recovery, for example, it can effectively promote the repair of spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a flow chart of a method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel.

[0027] Figure 2 Atomic force microscope scans of PNFs (a), CNFs (b) and PNFs / CNF dual fibers (c), where the left images of (a), (b) and (c) are atomic force microscope scans of PNFs, CNFs and PNFs / CNF dual fibers, respectively, and the right images are their respective measurement analyses.

[0028] Figure 3 Electron microscope scanning images of double fiber network hydrogels with equal volume ratios and different concentrations: PNFs (10 mg / ml) + equal volumes of CNFs (20, 25, 30, 35 mg / ml), where a, b, c, and d correspond to electron microscope scanning images of double fiber network hydrogels formed when the CNFs concentrations are 20, 25, 30, and 35 mg / ml, respectively.

[0029] Figure 4 This is a graph showing the change in viscosity of double-fiber network hydrogel containing CNFs (10, 12.5, 15, 17.5 mg / mL) with shear rate.

[0030] Figure 5 The injectability and self-healing properties of RCP hydrogel (5mg / ml PNFs+15mg / ml CNFs+1mg / ml PP).

[0031] Figure 6 These are live / dead cell staining and CCK-8 experimental analysis graphs after culturing neural stem cells with RCP hydrogels of different concentrations. Figure a is a representative graph of the live / dead cell staining experiment after culturing neural stem cells with RCP hydrogels of different concentrations, Figure b is the survival rate of neural stem cells in the live / dead cell staining experiment, and Figure c is the CCK-8 statistical analysis graph of neural stem cells. DETAILED DESCRIPTION

[0032] The accompanying drawings are only used for exemplary description. The technical solution of the present invention is described in detail below through embodiments in combination with the accompanying drawings. For those skilled in the art, some well-known structures and their descriptions in the accompanying drawings may be omitted, and therefore, they should not be understood as limitations on the present invention.

[0033] The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed. The raw materials, instruments and equipment used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Other technical methods not described herein adopt existing technologies.

[0034] Example 1

[0035] like Figure 1As shown, a method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0036] (1) Self-assembled PNFs hydrogel: 40 mg of RADA16 was added to 4 ml of deionized water, pH 7, and sonicated for 5 minutes to fully dissolve it. The mixture was allowed to stand at room temperature (20-26°C) for 3 hours to self-assemble into PNFs hydrogel with a concentration of 10 mg / mL.

[0037] (2) Preparation of CNFs solution: Prepare the cellulose concentration, weigh 20 mg of cellulose, add 1 mL of deionized water to dissolve, and oscillate by ultrasound for 5 minutes to fully mix to obtain a CNFs solution with a concentration of 20 mg / mL.

[0038] (3) Synthesis of dual fiber network hydrogel: PNFs hydrogel and CNFs solution were mixed in equal volumes (1:1) and incubated at 37°C for 30 minutes. The two fiber networks were physically cross-linked through non-covalent hydrogen bonding to form a stable dual fiber network three-dimensional hydrogel, and a hydrogel with a concentration of 10 mg / mL CNFs + 5 mg / mL PNFs was obtained.

[0039] (4) Adding neuropeptides: Add neuropeptide PP to the synthesized double fiber network hydrogel and mix thoroughly to form a bioactive neuropeptide-enhanced double fiber network structure hydrogel (RCP), wherein the concentration of each raw material in RCP is 1 mg / mL PP+10 mg / mL CNFs+5 mg / mL PNFs.

[0040] Example 2

[0041] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0042] The difference from Example 1 is that the CNFs solution is prepared by weighing 25 mg of cellulose, adding 1 mL of deionized water to dissolve, ultrasonically shaking for 5 minutes, and fully mixing to obtain a CNFs solution with a concentration of 20 mg / mL. Then, a CNFs hydrogel with a concentration of 12.5 mg / mL + 5 mg / mL PNFs (mixed in equal volumes) is obtained. The concentration of each raw material in the formed RCP is 1 mg / mL PP + 12.5 mg / mL CNFs + 5 mg / mL PNFs.

[0043] Other unmentioned contents are the same as those in Example 1.

[0044] Example 3

[0045] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0046] The difference from Example 1 is that the CNFs solution is prepared by weighing 30 mg of cellulose, adding 1 mL of deionized water to dissolve, ultrasonically shaking for 5 minutes, and fully mixing to obtain a CNFs solution with a concentration of 30 mg / mL. Then, a 15 mg / mL CNFs+5 mg / mL PNFs hydrogel (mixed in equal volumes) is obtained. The concentration of each raw material in the formed RCP is 1 mg / mL PP+15 mg / mL CNFs+5 mg / mL PNFs.

[0047] Other unmentioned contents are the same as those in Example 1.

[0048] like Figure 2 As shown in the figure, the atomic force microscope scanning and measurement analysis of PNFs, CNFs and PNFs / CNF dual fibers. A large number of PNFs fibers can be seen in the atomic force microscope image of PNFs, with a measured length of about 1-4μm. This suitable fiber can increase the number of fibers per unit volume of hydrogel and provide more loading sites for loading stem cells or drug molecules. A large number of CNFs fibers can be observed in the atomic force microscope image of CNFs, with a measured length of about 0.5-3μm. In the atomic force microscope image of PNFs / CNFs dual fiber network hydrogel, PNFs fibers (long) and CNFs fibers (short) can be seen at the same time. PNFs and CNFs fibers of unequal lengths are intertwined. The CNFs (second fiber) with the right fiber length can assist the PNFs hydrogel (first fiber) to form a dual fiber network with a stronger three-dimensional structure, reduce the use of RADA16, increase the number of fibers per unit volume of hydrogel, and provide more loading sites for loading stem cells or drug molecules.

[0049] Example 4

[0050] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0051] The difference from Example 1 is that the CNFs solution is prepared, 35 mg of cellulose is weighed, 1 mL of deionized water is added to dissolve, ultrasonic vibration is performed for 5 minutes, and sufficient mixing is performed to obtain a CNFs solution with a concentration of 35 mg / mL. Then, a 15 mg / mL CNFs+5 mg / mL PNFs hydrogel (equal volume mixing) is obtained. The concentration of each raw material in the formed RCP is 1 mg / mL PP+17.5 mg / mL CNFs+5 mg / mL PNFs.

[0052] Other unmentioned contents are the same as those in Example 1.

[0053] like Figure 3As shown in the electron microscope scanning images of the synthesized double fiber network hydrogel PNFs (10 mg / ml) + equal volume of CNFs (20, 25, 30, 35 mg / ml) in Example ad, the three-dimensional space test measured by electron microscope scanning showed that they all had a loose and uniform three-dimensional structure and were suitable for loading stem cells or drug molecules. Figure 4 As shown, the double fiber network hydrogel containing 15 mg / mL CNFs with moderate viscosity and smooth injection was selected as the optimal concentration.

[0054] like Figure 5 As shown, Figure a shows that the RCP hydrogel (5mg / ml PNFs+15mg / ml CNFs+1mg / ml PP) prepared by the present invention is injectable, Figure b shows that two drops of RCP hydrogel are injected separately with a 10ml syringe, and Figure c shows that after 3 seconds, the hydrogels at the two locations quickly merge into one and restore their structural stability.

[0055] Example 5

[0056] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0057] The difference from Example 3 is that the concentration of each raw material in the formed RCP is 2 mg / mL PP+17.5 mg / mL CNFs+5 mg / mL PNFs.

[0058] Other unmentioned contents are the same as those in Example 1.

[0059] Example 6

[0060] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0061] The difference from Example 3 is that the concentration of each raw material in the formed RCP is 3 mg / mL PP+17.5 mg / mL CNFs+5 mg / mL PNFs.

[0062] Other unmentioned contents are the same as those in Example 1.

[0063] Example 7

[0064] A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel comprises the following steps:

[0065] The difference from Example 3 is that the concentration of each raw material in the formed RCP is 4 mg / mL PP+17.5 mg / mL CNFs+5 mg / mL PNFs.

[0066] Other unmentioned contents are the same as those in Example 1.

[0067] like Figure 6 The figure shows the live / dead cell staining and CCK-8 experimental analysis of RCP hydrogels with different concentrations after culturing neural stem cells. (a) shows the live / dead cell staining results of RCP hydrogels with different concentrations of PP after culturing neural stem cells for 3 days. From left to right, the first is the control group: adding an equal amount of PBS; followed by the experimental group: adding PNFs (5mg / ml) + CNFs (15mg / ml), RCP with PP (1mg / ml), RCP with PP (2mg / ml), RCP with PP (3mg / ml), and RCP with PP (4mg / ml). The synthetic picture of neural stem cells (green is live cells, red is dead cells). The cells in RCP with PP (1mg / ml) survived the most.

[0068] Figure 6 Figure (b) shows the survival rate of neural stem cells in the live / dead cell staining experiment. The survival rate of neural stem cells in each group was greater than 95%. There was no significant difference in the live / dead cell staining experiment between the RCP group with 1 mg / ml PP and the group with PBS. This shows that the RCP hydrogel containing (0, 1, 2, 3, 4 mg / ml) PP is non-cytotoxic, and the RCP hydrogel with 1 mg / ml PP has better biocompatibility. Figure (c) shows the CCK-8 statistical analysis of neural stem cells. After 1, 3, and 5 days of neural stem cell culture, there was a significant growth difference between the RCP group with 1 mg / ml PP and the group with PBS. This shows that the RCP hydrogel containing (0, 1, 2, 3, 4 mg / ml) PP is non-toxic to cells and can promote the growth of neural stem cells. The RCP hydrogel containing 1 mg / ml PP has the most obvious effect in promoting cell growth.

[0069] Verified by live and dead cell staining of neural stem cells and CCK-8 experiments, the optimal concentration of RCP hydrogel is 1mg / mLPP+15mg / mL CNFs+5mg / mL PNFs.

[0070] The meanings of the English abbreviations used in the present invention are:

[0071] RADA16: (Arginine-Alanine-Aspartic Acid-Alanine) 4 ;

[0072] PNFs: peptide nanofibers;

[0073] CNFs: cellulose nanofibers;

[0074] RCP: Bioactive hydrogel with neurophilic peptide-reinforced dual-fiber network structure;

[0075] PP: polypeptide (PPFLMLLKGSTR);

[0076] PBS: phosphate buffered saline;

[0077] CCK-8: Cell counting kit-8;

[0078] NSCs: neural stem cells.

[0079] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A neuropeptide-enhanced double-fiber network structure hydrogel, characterized in that: The raw materials include PNFs hydrogel, CNFs solution and PP, wherein the mass concentration ratio of PP, CNFs and PNFs is 1 to 4:10 to 20:

5.

2. The neuropeptide-enhanced double-fiber network structure hydrogel according to claim 1, characterized in that: The mass concentration ratio of PP, CNFs and PNFs is 1:15:

5.

3. A method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to any one of claims 1 or 2, characterized in that: The steps include: (1) Self-assembled PNFs hydrogel: RADA16 was added to deionized water, the pH value was adjusted to 7, ultrasonic vibration was performed to fully dissolve it, and self-assembled PNFs hydrogel was formed after standing; (2) Preparation of CNFs solution: weigh cellulose, add it into deionized water, and mix thoroughly by ultrasonic vibration to form a CNFs solution; (3) Synthesis of dual-fiber network hydrogel: PNFs hydrogel and CNFs solution were mixed in equal volumes and heated in a water bath to form physical cross-links between the two fiber networks through non-covalent hydrogen bonding to form a stable dual-fiber network three-dimensional hydrogel; (4) Adding neuropeptides: PP is added to the synthesized double-fiber network hydrogel and mixed to form a bioactive neuropeptide-enhanced double-fiber network structure hydrogel.

4. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 3, characterized in that: In the step (1), the ultrasonic oscillation time is 2 to 10 minutes; and the standing time at room temperature is 2 to 3 hours.

5. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 3, characterized in that: In the step (1), the mass ratio of RADA16 to deionized water is 10 to 20 (mg / mL).

6. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 3, characterized in that: In the step (2), the volume ratio of cellulose mass to deionized water is 20 to 40 (mg / mL); and the ultrasonic oscillation time is 2 to 10 minutes.

7. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 3, characterized in that: In the step (3), the temperature of the water bath heating after the PNFs hydrogel and the CNFs solution are mixed is 30 to 37°C.

8. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 7, characterized in that: The heating time is 30 to 40 minutes.

9. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 3, characterized in that: In the step (4), the PP is added to the synthesized double-fiber network hydrogel for mixing by stirring, and the stirring method is mechanical stirring or ultrasonic vibration.

10. The method for preparing a neuropeptide-enhanced double-fiber network structure hydrogel according to claim 9, characterized in that: The stirring temperature is room temperature, and the stirring time is 5 to 60 minutes.

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