Hydrogel for repairing peripheral nerve injury as well as preparation method and application of hydrogel

Abstract

The invention provides hydrogel for repairing peripheral nerve injury as well as a preparation method and application of the hydrogel, and belongs to the field of biomedical engineering materials. The hydrogel disclosed by the invention is good in biocompatibility, has degradability, contains a double-layer cross-linked network structure, has the wetting characteristic of common hydrogel, and also has the advantages of collagen antigenicity, biodegradability, high biocompatibility, cell adaptability, tissue regeneration promotion and the like. The hydrogel can effectively promote regeneration and remyelination of nerve fibers, can promote gastrocnemius muscle recovery and promote motor function recovery, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering materials, and particularly relates to a hydrogel for repairing peripheral nerve injury, a preparation method thereof and uses thereof. Background Art

[0002] Peripheral nerve injury (PIN) is damage to the structure and function of the peripheral nervous system (i.e., nerves outside the brain and spinal cord) caused by various reasons such as trauma, disease, metabolic disorders, poisoning, compression, ischemia, etc. In recent years, the incidence of peripheral nerve injury has shown an upward trend and has become a common traumatic disease. In severe cases of injury, due to the lack of effective treatment means, irreversible body damage and loss of function may occur, and even disability may result, seriously affecting the quality of life of patients. Traditional treatment methods such as autologous nerve transplantation, nerve reconstruction, and biophysical stimulation have problems such as insufficient donors, loss of donor area function, and inaccurate stimulation control, and the treatment effects are not ideal. With the development of tissue engineering, bioengineering technologies and new materials have been widely used in nerve repair. Among them, tissue-engineered nerve conduits have become a research hotspot due to their biomimetic characteristics. As a key component of nerve conduits, scaffold materials and fillers are also constantly updated to improve their performance to meet clinical needs. Hydrogel materials have attracted much attention in the field of nerve repair due to their good biocompatibility, tunability, and multifunctionality.

[0003] Hydrogels, as a kind of polymer, owe their multifunctionality to their three-dimensional network structure. They contain hydrophilic and hydrophobic residues and can absorb water and swell while maintaining their structure. Hydrogel materials are divided into two major categories: natural (such as collagen, chitosan, hyaluronic acid, sodium alginate, etc.) and synthetic (such as polyethylene oxide, polyvinyl alcohol, etc.). In tissue engineering and regenerative medicine, hydrogels support cell attachment, migration, and tissue regeneration, and promote nerve repair. Their internal network structure is conducive to nutrient and metabolic exchange and regulates the repair environment of damaged tissues. Hydrogels can also change their shape and structure with changes in pH, temperature, and other stimuli, so they are widely used in tissue medicine, sensing, and other fields. In addition, hydrogels can also be used in drug delivery systems. Due to their three-dimensional structure and biocompatibility, they can regulate the precise release of drugs through characteristics such as deformation and sensing response, enabling them to continuously play a role in the body.

[0004] Single-network hydrogels have a simple structure but lack tensile strength and toughness. Double-network hydrogels composed of two interpenetrating and intertwined cross-linked networks have shown balanced mechanical properties combining high strength and toughness. However, how to provide a natural double-crosslinked composite hydrogel for PIN treatment requires further research. Summary of the Invention

[0005] The object of the present invention is to provide a hydrogel for repairing peripheral nerve injuries, its preparation method and uses.

[0006] The present invention provides a hydrogel precursor for repairing peripheral nerve injuries, which is characterized in that it is formed by mixing the following components in parts by volume:

[0007] 1 - 5 parts of dopamine - modified hyaluronic acid solution, 1 - 5 parts of aminophenylboronic acid - modified sodium alginate solution, and 5 parts of methacrylated gelatin.

[0008] Further, the foregoing hydrogel precursor is formed by mixing the following components in parts by volume:

[0009] 1 part of dopamine - modified hyaluronic acid solution, 1 part of aminophenylboronic acid - modified sodium alginate solution, and 2.5 parts of methacrylated gelatin;

[0010] Preferably,

[0011] the concentration of the dopamine - modified hyaluronic acid solution is 10 - 50 mg / mL; and / or, the concentration of the aminophenylboronic acid - modified sodium alginate solution is 5 - 20 mg / mL;

[0012] More preferably,

[0013] the dopamine - modified hyaluronic acid solution is a PBS solution of dopamine - modified hyaluronic acid; and / or, the aminophenylboronic acid - modified sodium alginate solution is a PBS solution of aminophenylboronic acid - modified sodium alginate.

[0014] Further preferably, the concentration of the dopamine - modified hyaluronic acid solution is 25 mg / mL; and / or, the concentration of the aminophenylboronic acid - modified sodium alginate solution is 10 mg / mL.

[0015] Further, the preparation method of the dopamine - modified hyaluronic acid includes the following steps:

[0016] (1) Dissolve hyaluronic acid in a solvent, add EDC and NHS, and react in the dark;

[0017] (2) Add dopamine isothiocyanate to the reaction solution obtained in step (1) and react;

[0018] (3) Dialyze and lyophilize the reaction solution obtained in step (2) to obtain the product;

[0019] Preferably,

[0020] the mass - to - volume ratio of the hyaluronic acid, solvent, EDC, NHS, and dopamine isothiocyanate is 1 - 10 g: 100 - 500 mL: 1 - 3 g: 1 - 3 g: 1 - 5 g;

[0021] And / or, in the preparation method of the dopamine-modified hyaluronic acid, the pH value is maintained at 3-7 throughout the reaction process using hydrochloric acid or an aqueous NaOH solution;

[0022] More preferably,

[0023] In step (1), the solvent is a MES buffer solution;

[0024] And / or, in step (1), the reaction time is 30-120 min;

[0025] And / or, in step (2), the reaction is carried out under the protection of an inert gas;

[0026] And / or, in step (2), the reaction time is 12-48 h;

[0027] And / or, in step (3), the cut-off molecular weight of the dialysis is 3000-4000 Da;

[0028] And / or, in step (3), the dialysis solution for dialysis is water.

[0029] Furthermore, the preparation method of the 3-aminophenylboronic acid-modified sodium alginate includes the following steps:

[0030] (A) Dissolve sodium alginate in a solvent, add EDC and NHS, and react;

[0031] (B) Add 3-aminophenylboronic acid to the reaction solution obtained in step (A), and react;

[0032] (C) Dialyze the reaction solution obtained in step (B), and lyophilize to obtain the product;

[0033] Preferably,

[0034] The mass-volume ratio of the sodium alginate, solvent, EDC, NHS, and 3-aminophenylboronic acid is 1-5 g: 100-500 mL: 1-3 g: 1-3 g: 0.5-2 g;

[0035] More preferably,

[0036] In step (A), the solvent is a MES buffer solution;

[0037] And / or, in step (A), the reaction time is 30-120 min;

[0038] And / or, in step (B), the reaction is carried out under the protection of an inert gas;

[0039] And / or, in step (B), the reaction time is 12-48 h;

[0040] And / or, in step (C), the cut-off molecular weight of the dialysis is 3000-4000 Da;

[0041] And / or, in step (C), the dialysis solution for the dialysis is water.

[0042] Furthermore,

[0043] The temperature during mixing is 35-40 °C, and the pH is adjusted to 6-10 using hydrochloric acid or an aqueous NaOH solution;

[0044] And / or, the method for preparing the methacrylated gelatin includes the following steps: dissolving gelatin in a solvent, and then adding methacrylic anhydride and reacting to obtain it;

[0045] Preferably,

[0046] The mass-volume ratio of the gelatin, the solvent, and the methacrylic anhydride is 5-50 g: 100-500 mL: 1-20 mL;

[0047] And / or, the temperature for adding the methacrylic anhydride and reacting is 30-70 °C;

[0048] And / or, the time for adding the methacrylic anhydride and reacting is 3-12 h;

[0049] More preferably,

[0050] The solvent is PBS buffer solution.

[0051] The present invention also provides the use of the aforementioned hydrogel precursor in the preparation of a hydrogel carrier for peripheral nerve injury repair.

[0052] The present invention also provides a hydrogel carrier for peripheral nerve injury repair, which is obtained by adjusting the pH value of the aforementioned hydrogel precursor to 7.2-7.4 with hydrochloric acid or an aqueous NaOH solution and then performing ultraviolet irradiation;

[0053] Preferably, the temperature of the ultraviolet irradiation is 30-40 °C, and the time is 1-30 min.

[0054] The present invention also provides a hydrogel for peripheral nerve injury repair, which is obtained by adding a docosahexaenoic acid micelle solution and a fibroblast growth factor to the aforementioned hydrogel precursor, adjusting the pH value to 7.2-7.4, and then performing ultraviolet irradiation;

[0055] Preferably, the mass-volume ratio of the aforementioned hydrogel precursor, the docosahexaenoic acid micelle solution, and the fibroblast growth factor is 1 mL: (1-10) mL: (1-50) mg;

[0056] And / or, the temperature of the ultraviolet irradiation is 30 to 40 °C, and the time is 1 to 30 min.

[0057] Furthermore, the preparation method of the docosahexaenoic acid micellar solution comprises the following steps:

[0058] (a) Dissolve hyaluronic acid and cholesterol in a solvent, add N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, react, dialyze after the reaction, and lyophilize to obtain HA-CHOL;

[0059] (b) Dissolve docosahexaenoic acid in a solvent, then mix it with HA-CHOL, stir evenly and add water for reaction;

[0060] (c) Dialyze the reaction solution obtained from the reaction to obtain the docosahexaenoic acid micellar solution;

[0061] Preferably,

[0062] In step (a), the mass-volume ratio of the hyaluronic acid, cholesterol, solvent, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1 to 5 g: 1 to 5 g: 10 to 50 mL: 0.5 to 2 g: 0.1 to 0.5 g;

[0063] And / or, in step (b), the mass-volume ratio of the docosahexaenoic acid, solvent, HA-CHOL and water is 5 to 20 mg: 5 to 10 mL: 10 to 100 mg: 5 to 20 mL;

[0064] More preferably,

[0065] In step (a), the solvent is dimethyl sulfoxide;

[0066] And / or, in step (a), the temperature of the reaction is 80 to 100 °C, and the time of the reaction is 40 to 60 h;

[0067] And / or, in step (a), the cut-off molecular weight of the dialysis is 3000 to 4000 Da;

[0068] And / or, in step (b), the solvent is dimethyl sulfoxide;

[0069] And / or, in step (b), the temperature of the reaction is 20 to 30 °C; the time of the reaction is 60 to 120 min;

[0070] And / or, in step (c), the cut-off molecular weight of the dialysis is 3000 to 4000 Da.

[0071] The present invention also provides the use of the aforementioned hydrogel in the preparation of a drug for preventing and / or treating peripheral nerve injury.

[0072] Preferably, the drug is a drug that promotes nerve fiber regeneration and / or remyelination;

[0073] and / or, the drug is a drug that promotes the recovery of the gastrocnemius muscle;

[0074] and / or, the drug is a drug that promotes the recovery of motor function.

[0075] In the present invention, the hydrogel precursor refers to a gel solution before crosslinking to obtain a gel.

[0076] The hydrogel matrix adopted in the present invention is a modified natural polymer, which is characterized by good biocompatibility, degradability, containing a double crosslinked network, crosslinking of a hyaluronic acid derivative and an alginate derivative, and a collagen crosslinked network. This hydrogel utilizes the pH responsiveness of the PBA group. Under alkaline conditions, the adjacent hydroxyl groups of APBA and HA-DOPA can form reversible dynamic borate ester bonds to achieve the sol-gel transition. Collagen is introduced into the APBA / HA-DOPA hydrogel network. When in a neutral solvent close to physiological temperature and irradiated with an ultraviolet lamp, collagen can self-assemble into a hydrogel through intermolecular interactions. The collagen hydrogel not only has the wetting properties of ordinary hydrogels, but also has the advantages of collagen resistance to antigenicity, biodegradability, high biocompatibility, cell adaptability, and promoting tissue regeneration.

[0077] The present invention has achieved the following beneficial effects:

[0078] The present invention provides a hydrogel for repairing peripheral nerve injuries. This hydrogel has good biocompatibility, degradability, contains a double crosslinked network structure, has the wetting properties of ordinary hydrogels, and also has the advantages of collagen resistance to antigenicity, biodegradability, high biocompatibility, cell adaptability, and promoting tissue regeneration. In addition, the hydrogel of the present invention can effectively promote the regeneration and remyelination of nerve fibers, and can promote the recovery of the gastrocnemius muscle and the recovery of motor function, and has good application prospects.

[0079] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0080] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0081] Figure 1Schematic diagram of the synthesis process of HA-DOPA and APBA prepared in Examples 1-2 of the present invention: A is the schematic diagram of the synthesis process of HA-DOPA; B is the schematic diagram of the synthesis process of APBA.

[0082] Figure 2 Fourier transform infrared spectra of the raw material HA used in Examples 1-2 of the present invention and the prepared HA-DOPA.

[0083] Figure 3 Fourier transform infrared spectra of the raw material SA and PBA used in Examples 1-2 of the present invention and the prepared APBA.

[0084] Figure 4 Proton nuclear magnetic resonance spectra of the raw material HA used in Examples 1-2 of the present invention and the prepared HA-DOPA.

[0085] Figure 5 Proton nuclear magnetic resonance spectra of the raw material SA used in Examples 1-2 of the present invention and the prepared APBA.

[0086] Figure 6 Schematic diagram of the synthesis process of GelMA prepared in Examples 1-2 of the present invention.

[0087] Figure 7 Proton nuclear magnetic resonance spectra of GelMA prepared in Examples 1-2 of the present invention.

[0088] Figure 8 Schematic diagram of the gelation of the HD-B-GM hydrogel precursor solution prepared in Example 1 of the present invention at 37°C.

[0089] Figure 9 Schematic diagram of the synthesis process of HA-CHOL prepared in Example 2 of the present invention.

[0090] Figure 10 Fourier transform infrared spectra of the raw material HA used in Example 2 of the present invention and the prepared HA-CHOL.

[0091] Figure 11 Proton nuclear magnetic resonance spectra of the raw material HA used in Example 2 of the present invention and the prepared HA-CHOL.

[0092] Figure 12 Particle size distribution result diagram of MIC@DHA prepared in Example 2 of the present invention.

[0093] Figure 13 Transmission electron micrograph of MIC@DHA prepared in Example 2 of the present invention.

[0094] Figure 14This is a diagram showing the drug release process in vivo simulated by the HD-B-GM-D hydrogel prepared in Example 2 of the present invention.

[0095] Figure 15 This is the rheological characterization result of the hydrogel prepared in Example 1 of the present invention: a is the frequency sweep result diagram of HD-B and HD-B-GM hydrogels; b is the diagram of the changes of G’ and G” of HD-B and HD-B-GM hydrogels with stress.

[0096] Figure 16 This is the microstructural diagram of the hydrogel prepared in Example 1 of the present invention: A and B are the scanning electron microscope results of the HD-B hydrogel; C and D are the scanning electron microscope results of the HD-B-GM hydrogel.

[0097] Figure 17 This is a schematic diagram of the experimental process of using the hydrogels prepared in Examples 1-2 of the present invention for peripheral nerve in vivo repair: i is a schematic diagram of exposing the peripheral nerve; ii is a schematic diagram of damaging the peripheral nerve with a hemostat; iii is a schematic diagram of the damaged peripheral nerve; iv is a schematic diagram of the damaged peripheral nerve wrapped with the hydrogel.

[0098] Figure 18 This is the result of the hydrogels prepared in Examples 1-2 of the present invention promoting nerve regeneration. The last row in the figure is the enlarged local Merge view.

[0099] Figure 19 This is a diagram showing the result of the hydrogels prepared in Examples 1-2 of the present invention promoting the recovery of the gastrocnemius muscle: In Figure A, a is the gross observation result of the gastrocnemius muscle and the soleus muscle, and b is the HE staining result of the cross-section of the gastrocnemius muscle; In Figure B, a is the percentage of muscle fiber area in four groups, b is the percentage of collagen fiber area, and c is the percentage of wet weight (n = 5); ns P > 0.05 indicates no significant difference, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0100] Figure 20 This is a diagram showing the evaluation result of the lower limb motor function of rats 14 days after surgery: A is the footprint photo of each group of rats; B is the SFI index of each group of rats (n = 5); *P < 0.05, ****P < 0.0001. Detailed implementation manners

[0101] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0102] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0103] NHS: N-Hydroxysuccinimide;

[0104] DOPA: Dopamine isothiocyanate.

[0105] In the present invention, if the reaction temperature is not specifically mentioned, the reaction is carried out at room temperature.

[0106] Example 1. A preparation method of HD-B-GM hydrogel

[0107] (1) Dissolve hyaluronic acid (3.00 g) in MES buffer solution (300 mL, pH = 5.0), then add 1.936 g (10 mmol) of EDC and 1.164 g (10 mmol) of NHS. Adjust the pH value of the solution to 5.0 with 4M NaOH aqueous solution, and react for 30 minutes in the dark. Then, add DOPA (2.25 g) to the above solution, and protect the reaction with nitrogen for 24 hours. During the whole process, use 0.1M hydrochloric acid and NaOH aqueous solution to keep the pH value of the reaction solution around 5.0. The final reaction solution is dialyzed in dialysis tube MD44 (cut-off molecular weight: 3500 Da) (the dialysate is pure water), and then freeze-dried to obtain dopamine-modified hyaluronic acid (HA-DOPA).

[0108] (2) Add sodium alginate (2.00 g) to MES solution (200 mL, pH = 5.5) and stir well. After sodium alginate is completely dissolved, add 1.164 g (10 mmol) of NHS and 1.936 g (10 mmol) of EDC, and activate the reaction for 45 minutes. Then add 3-aminophenylboronic acid (PBA, 1.00 g), and react at room temperature under nitrogen protection for 1 day. The final reaction solution is dialyzed in MD44 dialysis tube (cut-off molecular weight: 3500 Da) (the dialysate is pure water), and then freeze-dried to obtain 3-aminophenylboronic acid-modified sodium alginate (APBA).

[0109] (3) Completely dissolve gelatin (10.00 g) in PBS (90 mL, pH = 7.2 - 7.4) at 50 °C, and then slowly add methacrylic anhydride (10 mL) dropwise to the gelatin solution. React with stirring at 50 °C for 3 hours to obtain methacryloylated gelatin (GelMA).

[0110] (4) Dissolve the dopamine-modified hyaluronic acid obtained above in PBS to obtain an HA-DOPA solution with a concentration of 25 mg / mL, and dissolve the aminophenylboronic acid-modified sodium alginate obtained above in PBS to obtain an APBA solution with a concentration of 10 mg / mL. The HA-DOPA solution, the APBA solution, and methacrylated gelatin are mixed in a volume ratio of 1:1:2.5 to obtain a hydrogel precursor solution, and the pH of the hydrogel precursor solution is adjusted to an alkaline state (pH = 10.0) with hydrochloric acid or an aqueous NaOH solution during the preparation process. Irradiate the hydrogel precursor solution with a UV lamp for 10 min at 37 °C and pH = 7.2 - 7.4 (adjust the pH with hydrochloric acid or an aqueous NaOH solution) to obtain the HD-B-GM hydrogel.

[0111] The synthesis process of HA-DOPA and APBA prepared in this example is as Figure 1 shown.

[0112] The infrared spectra of the raw material hyaluronic acid (HA) and the prepared HA-DOPA in this example are as Figure 2 shown. In FT-IR, HA has a strong stretching vibration peak of -OH at 3000 - 3750 cm -1 , which is the O-H absorption band in the side chain of HA; there is an amide I band absorption peak near 1648 cm -1 ; there is a stretching vibration peak of C=O near 1645 cm -1 ; there is an N-H absorption peak of the amide II band near 1560 cm -1 , indicating the presence of the acetamido structure; there is a C-O absorption peak near 1042 cm -1 . The modified HA-DOPA still retains these characteristic absorption peaks of HA, indicating that the main structure of the modified HA has not changed. However, the intensity of the O-H absorption peak at 3000 - 3750 cm -1 weakens; a =C-H absorption peak on the dopamine benzene ring appears near 3102 cm -1 ; the intensity of the C=O absorption peak near 1645 cm -1 weakens. It is proved that an amidation reaction occurs between HA and dopamine, and dopamine is successfully grafted onto the hyaluronic acid molecular chain.

[0113] The infrared spectra of the raw materials SA, PBA, and the prepared APBA in this example are as Figure 3 shown. It can be seen that the spectrum of APBA has an obvious absorption peak at 711 cm -1 , which may be related to the vibration of the borate group. This peak does not exist in the spectrum of SA but exists in the spectrum of PBA, indicating that PBA is successfully connected to SA. The spectrum of PBA is in the range of 3300 - 3500 cm -1Characteristic absorption peaks related to amino groups (-NH2) exist within a certain range. Peaks in this region appear in the spectrum of APBA and show changes compared to the spectrum of SA, indicating that the amino group of PBA has reacted with the carboxyl group of SA. In the range of 1500 - 1600 cm -1 Vibration peaks of the aromatic ring of PBA exist within a certain range. In the spectrum of APBA, the intensity of the existing peaks in this region increases, indicating the connection between the aromatic ring of PBA and SA.

[0114] The 1H NMR spectra of the raw material HA and the prepared HA - DOPA in this example are as Figure 4 shown. The characteristic peak of the solvent D2O is at δ 4.790 ppm; the characteristic peak of the 3 methyl H in -NHCOCH3 is at δ 1.93 ppm; the characteristic peaks of the HA polysaccharide backbone are in the range of δ 3.25 - 3.85 ppm. By comparing with the HA spectrum, it can be seen that three new coupling peaks appear in HA - DOPA at δ 6.5 - 7.5 ppm. By observing Figure 1 the reaction process of A, it is speculated that the three new coupling peaks at δ 6.5 - 7.5 ppm are the characteristic peaks (a, b, c) of the H on the DOPA benzene ring. This can indicate that dopamine has been successfully grafted onto the hyaluronic acid molecular chain.

[0115] The 1H NMR spectra of the raw material SA and the prepared APBA in this example are as Figure 5 shown. It can be seen that the D2O solvent peak is at δ 4.70 ppm, the characteristic peaks of SA are in the range of δ 3.5 - 4 ppm, and the characteristic peak of the hydrogen atoms on the PBA aromatic ring appears at about δ 7.5 ppm in the APBA curve below, indicating that PBA has been connected to SA, proving the successful connection.

[0116] The synthesis process of the GelMA prepared in this example is as Figure 6 shown.

[0117] The 1H NMR spectrum of the GelMA prepared in this example is as Figure 7 shown. It can be seen that the D2O solvent peak is at δ 4.70 ppm. Due to the reaction between MA and gelatin (Gel), the chemical shift of the vinyl hydrogen atoms results in a peak corresponding to the -CH=CH2 structure in methacrylic anhydride (MA) appearing at about δ 5.5 ppm in the 1H NMR spectrum of GelMA, indicating the successful connection between Gel and MA.

[0118] The schematic diagram of the gel formation of the HD - B - GM hydrogel precursor solution prepared in this example at 37 °C is as Figure 8 shown. It can be seen that the HD - B - GM hydrogel precursor solution can rapidly form a stable gel at 37 °C.

[0119] Example 2. Preparation method of an HD-B-GM-D hydrogel

[0120] (1) Preparation of the HD-B-GM hydrogel precursor solution: The preparation method of the HD-B-GM hydrogel precursor solution is the same as that in Example 1.

[0121] (2) Dissolve hyaluronic acid (HA, 1.50 g) and cholesterol (CHOL, 1.00 g, 2.59 mmol) in dimethyl sulfoxide (DMSO, 25 mL) at 80 °C. Add N,N'-dicyclohexylcarbodiimide (DCC, 0.75 g, 3.63 mmol) and 4-dimethylaminopyridine (DMAP, 0.40 g, 3.28 mmol) to the solution, and continuously stir the mixture at 80 °C for 48 hours. Then dialyze the reacted mixture in water for 2 days (the cut-off molecular weight is 3500 Da), and use centrifugation to remove the excess cholesterol. Finally, lyophilize the obtained product to get HA-CHOL for use.

[0122] (3) Dissolve docosahexaenoic acid (10.00 mg) in dimethyl sulfoxide (DMSO, 10 mL), mix HA-CHOL (50.0 mg) with the solution, and slowly stir at 80 °C until evenly mixed. Drop in water (H2O, 15 mL) for further reaction (the reaction temperature is 25 °C, and the reaction time is 60 - 120 min). Finally, purify the product by dialysis in water (the cut-off molecular weight is 3500 Da) to obtain a micelle (MIC@DHA) solution encapsulating docosahexaenoic acid.

[0123] (4) Take 1 mL of the above-obtained micelle solution encapsulating docosahexaenoic acid (DHA) and 50 mg of fibroblast growth factor and add them to 1 mL of the HD-B-GM hydrogel solution simultaneously to obtain an HD-B-GM-D hydrogel precursor solution encapsulating DHA and fibroblast growth factor. Irradiate the HD-B-GM-D hydrogel precursor solution with ultraviolet light for 10 min at 37 °C and pH = 7.2 - 7.4 (adjust the pH with hydrochloric acid or NaOH aqueous solution) to obtain the HD-B-GM-D hydrogel.

[0124] The synthesis process of HA-CHOL prepared in this example is as Figure 9 shown.

[0125] The infrared spectra of the raw material HA and the prepared HA-CHOL in this example are as Figure 10 shown. It can be seen that in the range of 1000 - 1300 cm -1 , new absorption peaks appear in the spectrum of HA-CHOL, which is due to the reaction between the carboxyl group of HA and the hydroxyl group of CHOL to form a C-O-C bond.

[0126] The proton nuclear magnetic resonance spectra of the raw material HA and the prepared HA-CHOL in this example are as Figure 11 shown. It can be seen that in the spectrum of HA-CHOL, signals related to the hydrogen atoms on the alkyl chain of cholesterol appear in the range of δ 0.5-2 ppm, and signals related to the hydrogen atoms in the cyclic structure appear in the range of δ 3-5 ppm. The appearance of these new signals, as well as possible chemical shift changes and signal intensity changes, can indicate the successful connection of HA and CHOL.

[0127] The particle size distribution of the prepared MIC@DHA in this example is as Figure 12 shown. It can be seen that the particle size of the micelles is mainly concentrated in the range of 100-200 nm. The transmission electron micrograph of the prepared MIC@DHA in this example is as Figure 13 shown. It can be seen that the morphology of the micelles presents an approximately spherical structure, and the size of the micelles ranges from dozens of nanometers to hundreds of nanometers, which is consistent with the information provided by the particle size distribution diagram.

[0128] The drug release process in vivo of the prepared HD-B-GM-D hydrogel in this example is as Figure 14 shown. It can be seen that the initially implanted hydrogel tightly encapsulates many MIC@DHA. As the hydrogel decomposes in vivo, MIC@DHA is released. Under physiological conditions, the micelles disintegrate, and the DHA therein is released accordingly, thereby exerting a pharmacological effect.

[0129] The beneficial effects of the present invention are demonstrated by the following specific test examples.

[0130] Test Example 1, Rheological Characterization

[0131] Preparation method of HD-B hydrogel: Take the dopamine-modified hyaluronic acid obtained in Example 1 and dissolve it in PBS to obtain an HA-DOPA solution with a concentration of 25 mg / mL. Take the 4-aminophenylboronic acid-modified sodium alginate obtained in Example 1 and dissolve it in PBS to obtain an APBA solution with a concentration of 10 mg / mL. The HA-DOPA solution and the APBA solution are mixed in a volume ratio of 1:1 to obtain a hydrogel precursor solution, and the pH of the hydrogel precursor solution is adjusted to an alkaline state (pH = 10.0) with hydrochloric acid or NaOH aqueous solution during the preparation process. Under the conditions of 37 °C and pH = 7.2-7.4 (adjust the pH with hydrochloric acid or NaOH aqueous solution) and irradiated with an ultraviolet lamp for 10 min, the HD-B hydrogel is obtained.

[0132] Rheological tests were carried out on the HD-B hydrogel and the HD-B-GM hydrogel prepared in Example 1. The hydrogels were rheologically analyzed by a rotational rheometer. The strain sweep (0.1%-100% strain range) was measured at 25 °C to determine the linear viscoelastic region, and the storage modulus (G') and loss modulus (G") of the HD-B-GM hydrogel were determined under the frequency sweep (0.1-100 rad / s angular frequency range). The rheological experiments were performed in triplicate.

[0133] The rheological characterization of the hydrogel prepared in Example 1 is as Figure 15 shown, where Figure 15 A shows the trend that the storage modulus (G') and loss modulus (G") of the HD-B hydrogel and the HD-B-GM hydrogel increase with the increase of frequency. The storage modulus (G') represents the elasticity of the material, while the loss modulus (G") represents the viscosity of the material. In the whole test range, the G' of each group of hydrogels is much greater than G", which confirms the elastic response characteristics of the hydrogels. Among them, the G' of the HD-B-GM hydrogel is higher than that of the HD-B hydrogel, indicating that the HD-B-GM hydrogel has a higher crosslinking density and stronger corresponding mechanical properties. The strain sweep was performed on the HD-B hydrogel and the HD-B-GM hydrogel, and the results are as Figure 15 shown in B. At low strain, the G' of the hydrogel sample is greater than G", showing the elastic properties of the gel. When the strain increases, the G' of the HD-B-GM hydrogel rapidly decreases and is lower than G", indicating that the crosslinking structure inside the HD-B-GM hydrogel is damaged under high strain, reflecting the transition strain of the hydrogel from elastic-dominated to viscosity-dominated.

[0134] Test Example 2. Microstructure

[0135] The preparation method of the HD-B hydrogel is the same as that in Test Example 1.

[0136] The HD-B hydrogel and the HD-B-GM hydrogel prepared in Example 1 were freeze-dried and then broken, and the cross-sectional morphology was observed by scanning electron microscopy (SEM).

[0137] The microstructural diagram of the hydrogel prepared in Example 1 is as Figure 16 shown. By observing the internal morphology of the HD-B hydrogel and the HD-B-GM hydrogel by scanning electron microscopy, typical cavities and uniform porous structures can be observed. And as Figure 16 shown in C and 16D, the spatial grid structure of the HD-B-GM hydrogel is more compact and the pore size is relatively smaller, indicating that its crosslinking degree is stronger than that of the HD-GM hydrogel and has a more compact and dense structure.

[0138] Test Example 3. Promote nerve regeneration and gastrocnemius muscle recovery

[0139] The hydrogels prepared in Examples 1 and 2 were subjected to experiments on promoting nerve regeneration and gastrocnemius muscle recovery.

[0140] In this study, healthy adult Sprague-Dawley rats (purchased from Chengdu Enesville Biotechnology Co., Ltd.) weighing 200 - 250 g were selected. The rats were placed in a SPF-class animal room for adaptive feeding for 1 week, maintaining a 12-hour light / dark cycle and having free access to food and water. All experimental operations and care regarding the animals were approved by the Ethics Committee of West China Hospital, Sichuan University, with the batch number 20240627010.

[0141] This test example was divided into a sham operation group (Sham), a model group (Model), and a drug administration group. The drug administration group included an HD-B-GM hydrogel group (HD-B-GM) and an HD-B-GM-D hydrogel group (HD-B-GM-D).

[0142] All rats in each group were first anesthetized by inhaling isoflurane to ensure complete anesthesia. After anesthesia, the rats were fixed on the operating table, and the hair in the surgical area of the hind limb was shaved off. The surgical area was alternately disinfected with iodophor and 75% ethanol. A longitudinal incision (about 1.5 - 2 cm) was made on the outer side of the rat's hind limb, and the muscle tissue was separated layer by layer to expose the sciatic nerve. The connective tissue around the nerve was carefully separated to avoid damaging the nerve. In the model group and the drug administration groups (HD-B-GM group and HD-B-GM-D group), a hemostat was used to apply a constant pressure on the middle segment of the sciatic nerve for 2 minutes, with an interval of 15 seconds, for eight cycles. The sham operation group (Sham group) did not perform this operation. Then, after applying a constant pressure with a hemostat in the drug administration groups, the same volume of HD-B-GM hydrogel and HD-B-GM-D hydrogel were administered to the middle segment of the sciatic nerve respectively. The surgical areas of the rats were rinsed with normal saline, and the muscle and skin were sutured layer by layer. The rats were put back into the cage, kept warm until they woke up, had free access to food and water, and their recovery was observed daily. On the 14th day after surgery, the rats were sacrificed, the sciatic nerve tissue was taken for histological staining (HE staining, myelin basic protein staining, and neurofilament staining); at the same time, on the 14th day after surgery, the rats were sacrificed, the gastrocnemius muscle and soleus muscle were taken for observation, the cross-section of the gastrocnemius muscle was subjected to HE staining, and the sciatic nerve tissue was subjected to Masson staining. The cross-sectional area percentage of muscle fibers, the collagen fiber area percentage, and the wet weight percentage of the gastrocnemius muscle in each group were calculated. n = 5.

[0143] Schematic diagram of the use of the hydrogels prepared in Examples 1 - 2 for the repair of peripheral nerve injury in rats is as Figure 17 shown, Figure 17 (iv) It can be seen that the hydrogel spontaneously wraps into a tight semi-transparent thick film around the nerve injury site.

[0144] The results of promoting nerve regeneration of the hydrogels prepared in Examples 1 - 2 are as Figure 18 shown, from Figure 18It can be seen that: the sciatic nerve tissue structure in the Sham group is intact and the nerve fiber distribution is normal; the sciatic nerve tissue structure in the Model group is damaged and the nerve fiber distribution is reduced; the sciatic nerve tissue structure in the HD-B-GM group is somewhat restored and the nerve fiber distribution is increased; the sciatic nerve tissue structure in the HD-B-GM-D group is further restored and the nerve fiber distribution is close to normal. Specifically: 14 days after surgery, the HE results of the sciatic nerve showed that: the nerve fiber continuity in the HD-B-GM-D group was more complete than that in the model group and the HD-B-GM group, and there was no massive infiltration of inflammatory cells. The MBP fluorescence results showed that: the number of myelin sheaths in the HD-B-GM-D group was more than that in the model group and the HD-B-GM group, the arrangement was relatively neat, the distribution was also relatively uniform, and most of them were continuously distributed. The NF fluorescence results showed that: compared with the model group, more continuous and dense NF-positive fiber infiltration could be observed in the HD-B-GM-D group, and the defect length was significantly smaller than that in the model group. In addition, the results of the three groups of sections all showed that the nerve fibers in the HD-B-GM group were more complete than those in the model group. The above results indicate that the sciatic nerve injury wrapped by the HD-B-GM-D hydrogel promoted the regeneration and remyelination of nerve fibers in two weeks, and the effect was significantly better than that of the HD-B-GM hydrogel.

[0145] The results of promoting the recovery of the gastrocnemius muscle by the hydrogels prepared in Examples 1 to 2 are as Figure 19 shown, among which, Figure 19 A(a) It can be seen that 14 days after surgery, the gastrocnemius muscle in the normal group (Sham group) was plump, while in the model group and the HD-B-GM group, atrophy of the affected gastrocnemius muscle occurred after sciatic nerve injury. According to Figure 19 B(a) The results of the cross-sectional area of gastrocnemius muscle fibers showed that there was no significant difference between the model group and the HD-B-GM group (P>0.05); the cross-sectional area of muscle fibers in the HD-B-GM-D group was larger than that in the model group (P<0.0001) and was close to that in the normal group (Sham group). Figure 19 A(b) The HE results of the cross-section of the gastrocnemius muscle showed that the diameter of muscle fiber cells in the HD-B-GM-D group was smaller than that in the normal group (Sham group), larger than that in the model group and the HD-B-GM group, the gastrocnemius muscle fibers were arranged tightly, the swollen and ruptured cells were greatly reduced, and the cell boundaries were relatively clear. Figure 19 B(b) The results of the collagen fiber area after Masson staining of the sciatic nerve showed that the proportion of the collagen fiber area of the sciatic nerve in the HD-B-GM-D group and the HD-B-GM group was lower than that in the model group (P<0.001). Figure 19 B(c) The wet weight of the gastrocnemius muscle showed that: the wet weight of the gastrocnemius muscle in the normal group (Sham group) was significantly higher than that in the model group, the HD-B-GM group and the HD-B-GM-D group. There was no significant difference in the wet weight of the gastrocnemius muscle between the model group and the HD-B-GM group (P>0.05), but there was a difference compared with the HD-B-GM-D group (P<0.05).

[0146] The above results indicate that the HD-B-GM-D hydrogel can promote nerve regeneration and gastrocnemius recovery, while the HD-B-GM hydrogel has no such effect.

[0147] Experimental Example 4: Promotion of motor function recovery

[0148] Animal experiments were conducted according to the method of Experimental Example 3, and the lower limb motor function of rats in each group was evaluated. Fourteen days after surgery, a gait analysis system (CatWalk) was used. The walking platform was wiped with a wet towel to evenly cover it with a layer of liquid, and then the rats were placed on the walking platform to freely pass through it, and the gait parameters during their free walking were recorded, including step length, step width, swing time, support time, and step sequence pattern, etc. Before each test, the rats were allowed to adapt to the walking platform to ensure data accuracy; each experiment in each group was repeated at least 3 times, and the average value was taken. During data analysis, the changes in gait parameters between the model group and the control group (Sham group) were compared to evaluate the nerve repair effect. During the experiment, it was necessary to keep the environment quiet to avoid interfering with the behavior of the rats, and at the same time ensure the cleanliness of the walking platform to prevent odor from interfering with the experimental results. Combining with the analysis of the sciatic nerve function index (SFI), the calculation formula is as follows:

[0149] Sciatic nerve function index = -38.3(LExp - LCtrl) / (LCtrl) + 109.5(MExp - MCtrl) / (MCtrl) + 13.3(SExp - SCtrl) / (SCtrl) - 8.8

[0150] Among them, M is the distance between the first and fifth toes, L is the length of the toes, S is the distance between the second and fourth toes, Exp is the experimental toe, and Ctrl is the normal toe. The measurement was performed by researchers who were unaware of the experimental group.

[0151] The evaluation of the lower limb motor function of rats 14 days after surgery is as Figure 20 shown. Figure 20 A are the footprint photos of the 4 groups of rats. It can be seen that the distances between the toes on the left side of the surgery in the model group and the HD-B-GM group are unclear, with severe flexion contracture, and the extension and abduction of each toe of the foot are limited. While the clarity of the footprints in the HD-B-GM-D group and the Sham group is significantly better than the above two groups. As Figure 20 shown in B, there are significant differences in the results between the HD-B-GM-D group and the model group (P < 0.0001). The results indicate that the application of the HD-B-GM-D hydrogel is helpful for the functional recovery of the sciatic nerve after 2 weeks of treatment.

[0152] The above results indicate that the HD-B-GM-D hydrogel can promote motor function recovery.

[0153] In summary, the present invention provides a hydrogel for repairing peripheral nerve injuries. This hydrogel has good biocompatibility, is degradable, contains a double-crosslinked network structure, has the wetting properties of ordinary hydrogels, and also has the advantages of collagen's resistance to antigenicity, biodegradability, high biocompatibility, cell adaptability, and promotion of tissue regeneration. The hydrogel of the present invention can effectively promote the regeneration and remyelination of nerve fibers, can promote the recovery of the gastrocnemius muscle, and promote the recovery of motor function, and has good application prospects.

Claims

1. A hydrogel precursor for repairing peripheral nerve injuries, characterized in that: It is prepared by mixing the following components in parts by volume: 1 - 5 parts of dopamine - modified hyaluronic acid solution, 1 - 5 parts of 3 - aminophenylboronic acid - modified sodium alginate solution, and 5 parts of methacrylated gelatin.

2. The hydrogel precursor according to claim 1, wherein: It is prepared by mixing the following components in parts by volume: 1 part of dopamine - modified hyaluronic acid solution, 1 part of 3 - aminophenylboronic acid - modified sodium alginate solution, and 2.5 parts of methacrylated gelatin; Preferably, the concentration of the dopamine - modified hyaluronic acid solution is 10 - 50 mg / mL; and / or, the concentration of the 3 - aminophenylboronic acid - modified sodium alginate solution is 5 - 20 mg / mL; More preferably, the dopamine - modified hyaluronic acid solution is a PBS solution of dopamine - modified hyaluronic acid; and / or, the 3 - aminophenylboronic acid - modified sodium alginate solution is a PBS solution of 3 - aminophenylboronic acid - modified sodium alginate.

3. The hydrogel precursor according to claim 2, wherein: The preparation method of the dopamine - modified hyaluronic acid comprises the following steps: (1) Dissolve hyaluronic acid in a solvent, add EDC and NHS, and react in the dark; (2) Add dopamine isothiocyanate to the reaction solution obtained in step (1) and react; (3) Dialyze the reaction solution obtained in step (2), and freeze - dry to obtain it; Preferably, the mass - to - volume ratio of the hyaluronic acid, solvent, EDC, NHS, and dopamine thiocyanate is 1 - 10 g: 100 - 500 mL: 1 - 3 g: 1 - 3 g: 1 - 5 g; and / or, in the preparation method of the dopamine - modified hyaluronic acid, the pH value is maintained at 3 - 7 throughout the reaction process using hydrochloric acid or an aqueous NaOH solution; More preferably, in step (1), the solvent is a MES buffer solution; and / or, in step (1), the reaction time is 30 - 120 min; and / or, in step (2), the reaction is carried out under the protection of an inert gas; and / or, in step (2), the reaction time is 12 - 48 h; and / or, in step (3), the cut - off molecular weight of the dialysis is 3000 - 4000 Da; and / or, in step (3), the dialysis solution for dialysis is water.

4. The hydrogel precursor according to claim 2, wherein: The preparation method of the 3 - aminophenylboronic acid - modified sodium alginate comprises the following steps: [[ID= ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5. The hydrogel precursor according to claim 1, wherein: During mixing, the temperature is 35-40°C, and the pH is adjusted to 6-10 using hydrochloric acid or an aqueous NaOH solution; and / or, the method for preparing the methacrylated gelatin comprises the following steps: dissolving gelatin in a solvent, then adding methacrylic anhydride and reacting to obtain the product; Preferably, the mass-volume ratio of the gelatin, solvent and methacrylic anhydride is 5-50 g: 100-500 mL: 1-20 mL; and / or, the temperature for adding methacrylic anhydride and reacting is 30-70°C; and / or, the time for adding methacrylic anhydride and reacting is 3-12 h; More preferably, the solvent is PBS buffer.

6. Use of the hydrogel precursor according to any one of claims 1-5 in the preparation of a hydrogel carrier for peripheral nerve injury repair.

7. A hydrogel carrier for repairing peripheral nerve injuries, characterized in that: It is obtained by adjusting the pH value of the hydrogel precursor according to any one of claims 1-5 to 7.2-7.4 using hydrochloric acid or an aqueous NaOH solution and then performing ultraviolet irradiation; Preferably, the temperature of the ultraviolet irradiation is 30-40°C and the time is 1-30 min.

8. A hydrogel for repairing peripheral nerve injuries, characterized in that: It is obtained by adding a docosahexaenoic acid micelle solution and a fibroblast growth factor to the hydrogel precursor according to any one of claims 1-5, adjusting the pH value to 7.2-7.4, and then performing ultraviolet irradiation; Preferably, the mass-volume ratio of the hydrogel precursor according to any one of claims 1-5, the docosahexaenoic acid micelle solution and the fibroblast growth factor is 1 mL: (1-10) mL: (1-50) mg; and / or, the temperature of the ultraviolet irradiation is 30-40°C and the time is 1-30 min.

9. The hydrogel according to claim 8, wherein: The method for preparing the docosahexaenoic acid micelle solution comprises the following steps: (a) Dissolving hyaluronic acid and cholesterol in a solvent, adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, reacting, and after the reaction, performing dialysis and freeze-drying to obtain HA-CHOL; (b) Dissolving docosahexaenoic acid in a solvent, then mixing it with HA-CHOL, stirring evenly, and adding water for reaction; (c) Dialyzing the reaction solution obtained from the reaction to obtain the docosahexaenoic acid micelle solution; Preferably, in step (a), the mass-volume ratio of the hyaluronic acid, cholesterol, solvent, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1-5 g: 1-5 g: 10-50 mL: 0.5-2 g: 0.1-0.5 g; and / or, in step (b), the mass-volume ratio of the docosahexaenoic acid, solvent, HA-CHOL and water is 5-20 mg: 5-10 mL: 10-100 mg: 5-20 mL; More preferably, in step (a), the solvent is dimethyl sulfoxide; and / or, in step (a), the temperature of the reaction is 80-100°C and the time of the reaction is 40-60 h; and / or, in step (a), the cut-off molecular weight of the dialysis is 3000-4000 Da; and / or, in step (b), the solvent is dimethyl sulfoxide; And / or, in step (b), the temperature of the reaction is 20 to 30 °C; the reaction time is 60 to 120 min; And / or, in step (c), the cut-off molecular weight of the dialysis is 3000 to 4000 Da.

10. Use of the hydrogel according to claim 8 or 9 in the preparation of a drug for preventing and / or treating peripheral nerve injury; Preferably, the drug is a drug for promoting nerve fiber regeneration and / or remyelination; And / or, the drug is a drug for promoting the recovery of the gastrocnemius muscle; And / or, the drug is a drug for promoting the recovery of motor function.

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