An injectable modified hyaluronic acid / functional short peptide composite hydrogel and its preparation method and application

Abstract

The present invention provides an injectable modified hyaluronic acid / functional short peptide composite hydrogel and a preparation method and application thereof, wherein the composite hydrogel comprises modified hyaluronic acid and a functional short peptide, wherein the modified hyaluronic acid is a hyaluronic acid functionalized by dopamine grafting; the functional short peptide is a functional amino acid sequence connected with Fmoc or a short peptide connected with an amino acid sequence containing a benzene ring and an imidazole group on one side and a functional amino acid sequence on the other side. The present invention utilizes the self-polymerization of the catechol group on dopamine and its reversible non-covalent bond with the functional short peptide to obtain an injectable hydrogel with better mechanical properties and mechanical stability, and the hydrogel can be used as a cell culture substrate for 3D cell culture and as a tissue engineering scaffold; it can be injected into the spinal cord injury site alone or in combination with cell transplantation and drugs / active factors, which can promote the repair of spinal cord injury and has a very broad application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an injectable modified hyaluronic acid / functional short peptide composite hydrogel and a preparation method and application thereof. Background Art

[0002] Spinal cord injury can easily cause lifelong disability in patients. There is no effective clinical treatment so far, which brings extremely serious economic and mental burdens to society and families. Traumatic injury destroys the anatomical structure of the spinal cord, triggering a series of pathophysiological cascade reactions, and finally forming a cystic cavity wrapped by glial-fibrous scars, making nerve regeneration difficult and limiting functional recovery. Therefore, trying to improve the adverse regeneration environment after spinal cord injury is the only way to overcome the stubborn disease of spinal cord injury. So far, scientists have done a lot of work in constructing a suitable environment for spinal nerve regeneration. Bridging the biomaterial scaffold to the injury site can provide support for the regenerated axons and guide them to grow toward the distal end of the injury. Almost all tissue cells grow under three-dimensional conditions in the body, and the cells are wrapped in nanofiber hydrogels composed of collagen fibers as the main component. Three-dimensional porous nanoscaffolds can mimic the natural extracellular matrix structure, provide support for cell growth and tissue regeneration, and are widely used in research fields such as biomedicine and tissue engineering. To date, a series of biomaterials including natural materials and synthetic biodegradable polyesters have been processed into 3D (three-dimensional) porous scaffolds or single / multi-channel nerve conduits to repair spinal cord injuries.

[0003] However, clinically, spinal cord injuries are mostly incomplete fractures caused by impact or sharp injuries, resulting in irregular wounds. Open surgery to transplant prefabricated scaffolds has the problem of being unable to effectively fuse with the host spinal cord, making it difficult for regenerated nerves to cross the injured area. Spinal cord tissue is structurally a hydrogel-like "material" with a hydrogel that matches the elastic modulus of the spinal cord. In particular, injectable hydrogels have the advantages of non-invasive or minimally invasive implantation, while avoiding the risks of major surgery, and in situ gelation is very suitable for the irregular shape of the spinal cord injury site in clinical practice, obtaining an excellent implantation host spinal cord interface. Chinese invention patent CN106046340A discloses a hydrogel containing short peptides and hyaluronic acid, which can be used to repair spinal cord injuries after loading neural stem cells, but the preparation process of the hydrogel requires the addition of initiators, catalysts and other additives, and there is a risk of toxic additives remaining. For spinal cord injury repair, it is necessary to develop a new material with injectable properties, higher biosafety, and the ability to fill the injured area intact, connect the host tissues at both ends of the injury without causing additional tissue damage. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing spinal cord repair materials, and to provide an injectable modified hyaluronic acid / functional short peptide composite hydrogel and its preparation method and application. The present invention successfully grafts dopamine on hyaluronic acid molecules through a chemical synthesis method, and composites it with functional short peptides. The dopamine oxidation self-polymerization characteristics on the modified hyaluronic acid molecules are combined with the dynamic and reversible π-π conjugation between the benzene ring of the dopa group and the benzene ring of the short peptide to form an injectable hydrogel. Since the hyaluronic acid has a large molecular weight, the hydrogel structure formed after it is combined with the functional short peptide is more stable, and the mechanical properties and mechanical properties are stronger, so that the hydrogel can improve the local microenvironment, and can provide extracellular matrix support for cell 3D growth and nerve regeneration, which can effectively improve the repair ability after spinal cord injury.

[0005] The purpose of the invention is to provide an injectable modified hyaluronic acid / functional short peptide composite hydrogel.

[0006] Another object of the present invention is to provide a method for preparing an injectable modified hyaluronic acid / functional short peptide composite hydrogel.

[0007] Another object of the present invention is to provide an injectable modified hyaluronic acid / functional short peptide composite hydrogel for use in 3D cell culture and / or repair of spinal cord injury.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] An injectable modified hyaluronic acid / functional short peptide composite hydrogel is obtained by compounding modified hyaluronic acid and a functional short peptide, wherein the modified hyaluronic acid is dopamine-grafted functionalized hyaluronic acid; the functional short peptide is a functional amino acid sequence connected to Fmoc or a short peptide connected to an amino acid sequence containing a benzene ring and an imidazole group on one side and a functional amino acid sequence on the other side.

[0010] The present invention utilizes the oxidative self-polymerization characteristics of dopamine combined with the dynamic and reversible π-π conjugation between the benzene ring of the dopa group and the benzene ring of the short peptide to obtain an injectable hydrogel. This π-π conjugation is a non-covalent bond, which makes the cross-linking points of the composite hydrogel in a dynamic "binding-dissociation" state, giving the hydrogel injectability; at the same time, the oxidation between the catechols in the dopamine-grafted functionalized hyaluronic acid forms a covalent bond, ensuring the integrity and stability of the hydrogel.

[0011] Preferably, the functional short peptide is a short peptide having an amino acid sequence containing a benzene ring and an imidazole group connected on one side of the (arginine-alanine-aspartic acid-alanine)4-I(RADA16-I) sequence and a functional amino acid sequence connected on the other side.

[0012] Further preferably, there are 1 to 3 positively charged amino acids between the RADA16-I sequence and the functional amino acid sequence.

[0013] More preferably, the amino acid sequence containing a benzene ring and an imidazole group is histidine-glycine-phenylalanine (HGF).

[0014] Further preferably, the positively charged amino acid is lysine and / or arginine.

[0015] More preferably, the positively charged amino acid is arginine.

[0016] Preferably, the functional amino acid sequence connected with Fmoc contains 1 to 3 negatively charged amino acids between the Fmoc and the functional amino acid sequence.

[0017] Further preferably, the negatively charged amino acid is aspartic acid and / or glutamic acid.

[0018] Further preferably, the functional amino acid sequence is an amino acid that can promote nerve cell adhesion, including but not limited to arginine-glycine-aspartic acid (RGD), tyrosine-isoleucine-glycine-serine-arginine (YIGSR), phenylalanine-aspartic acid-glycine-aspartic acid-arginine-glycine-aspartic acid-serine (FDGDRGDS) or a functional amino acid sequence that can promote axon growth, including isoleucine-lysine-valine-alanine-valine (IKVAV).

[0019] Preferably, the pH of the composite hydrogel is 6.5-7.5.

[0020] Most preferably, the composite hydrogel has a pH of 7.

[0021] A method for preparing an injectable modified hyaluronic acid / functional short peptide composite hydrogel, characterized in that it comprises the following steps:

[0022] S1. Preparation of modified hyaluronic acid

[0023] After the hyaluronic acid is dissolved, the pH is adjusted to 5.0-6.5; sodium N-hydroxysuccinimide (NHS) is added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is added after 12-15 minutes to obtain a mixed solution; dopamine (DA) is dissolved, and the pH is adjusted to 5.0-6.5 to obtain a DA solution, and then the DA solution is mixed with the mixed solution, and the reaction is carried out at 20-30° C. for 24 hours to obtain a reactant, and the reaction system is maintained in an inert gas atmosphere during the whole process; the reactant is dialyzed for 1-3 days, and the retentate is freeze-dried to obtain a dopamine-hyaluronic acid composite bio-gel, that is, dopamine-grafted functionalized hyaluronic acid, that is, modified hyaluronic acid;

[0024] S2. Preparation of functional short peptides

[0025] An amino acid sequence containing a benzene ring and an imidazole group is connected to one side of the short peptide, and a functional amino acid sequence is connected to the other side, or Fmoc is connected to the functional amino acid sequence to obtain a functional short peptide;

[0026] S3. Preparation of modified hyaluronic acid / functional short peptide composite hydrogel

[0027] The dopamine-grafted functionalized hyaluronic acid described in step S1 and the functional short peptide described in step S2 are dissolved and mixed, and the pH is adjusted to 6.5-7.5 to obtain a composite aqueous solution, which is then injected into a buffer solution or a cell culture medium to obtain a composite hydrogel.

[0028] Preferably, the hyaluronic acid and dopamine in step S1 are dissolved in deionized water, and the final concentration of the hyaluronic acid solution is 1 to 20 mg / mL.

[0029] More preferably, the final concentration of the hyaluronic acid solution is 10 to 20 mg / mL.

[0030] Most preferably, the final concentration of the hyaluronic acid solution is 10 mg / mL.

[0031] Preferably, in step S1, the mass ratio of hyaluronic acid, NHS and EDC is 1:0.6-2:0.6-2; the mass ratio of hyaluronic acid to dopamine is 1:1-6.

[0032] Further preferably, in step S1, the mass ratio of hyaluronic acid, NHS and EDC is 1:1-2:1-2; and the mass ratio of hyaluronic acid to dopamine is 1:1-2.

[0033] Most preferably, in step S1, the mass ratio of hyaluronic acid, NHS and EDC is 1:1:1; and the mass ratio of hyaluronic acid to dopamine is 1:2.

[0034] Preferably, the freeze-drying conditions in step S1 are a pressure of 0 to 50 Pa, a temperature of -50 to -100° C., and a time of 24 to 72 h.

[0035] Further preferably, the freeze-drying conditions in step S1 are a pressure of 40 to 50 Pa, a temperature of -50 to -60°C, and a time of 36 to 72 hours.

[0036] Most preferably, the freeze-drying conditions in step S1 are a pressure of 50 Pa, a temperature of -50°C, and a time of 72 h.

[0037] Preferably, the inert gas in step S1 is nitrogen or helium.

[0038] Preferably, the dialysis in step S1 is performed using deionized water in a dialysis bag with a molecular weight cut-off of 3000 to 4000.

[0039] Preferably, the short peptide in step S2 is a (arginine-alanine-aspartic acid-alanine)4-I (RADA16-I) sequence.

[0040] More preferably, there are 1 to 3 positively charged amino acids between the RADA16-I sequence and the functional amino acid sequence.

[0041] More preferably, the amino acid sequence containing a benzene ring and an imidazole group is histidine-glycine-phenylalanine (HGF).

[0042] More preferably, the positively charged amino acid is lysine and / or arginine; and the negatively charged amino acid is aspartic acid and / or glutamic acid.

[0043] More preferably, the positively charged amino acid is arginine.

[0044] Further preferably, the functional amino acid sequence is an amino acid that can promote nerve cell adhesion, including but not limited to arginine-glycine-aspartic acid (RGD), tyrosine-isoleucine-glycine-serine-arginine (YIGSR), phenylalanine-aspartic acid-glycine-aspartic acid-arginine-glycine-aspartic acid-serine (FDGDRGDS) or a functional amino acid sequence that can promote axon growth, including isoleucine-lysine-valine-alanine-valine (IKVAV)

[0045] Further preferably, the functional short peptide in step S2 is HGF-(RADA)4-RIKVAV, HGF-(RADA)4-RYIGSR, HGF-(RADA)4-RRGD or Fmoc-FDGDRGDS.

[0046] Preferably, the dopamine-grafted functionalized hyaluronic acid and the functional short peptide in step S3 are dissolved in water and then mixed.

[0047] Preferably, in step S3, the volume ratio of the dopamine-grafted functionalized hyaluronic acid and the functional short peptide is 1-2:1-2.

[0048] Further preferably, in step S3, the volume ratio of the dopamine-grafted functionalized hyaluronic acid and the functional short peptide is 1:1.

[0049] Preferably, step S3 adjusts the pH to 7.

[0050] Preferably, the concentration of the modified hyaluronic acid / functional short peptide composite solution in step S3 is 5 to 100 mg / mL.

[0051] Further preferably, the concentration of the modified hyaluronic acid / functional short peptide composite solution in step S3 is 5 to 20 mg / mL.

[0052] Most preferably, the concentration of the modified hyaluronic acid / functional short peptide composite solution in step S3 is 20 mg / mL.

[0053] Preferably, the buffer solution in step S3 is PBS buffer.

[0054] Application of an injectable modified hyaluronic acid / functional short peptide composite hydrogel in 3D cell culture.

[0055] Preferably, the application is to disperse the cells in a modified hyaluronic acid / functional short peptide composite aqueous solution, and then inject it into a cell culture medium to form a hydrogel for 3D cell culture.

[0056] Application of an injectable modified hyaluronic acid / functional short peptide composite hydrogel in the preparation of spinal cord injury repair materials.

[0057] Preferably, the application is to inject the modified hyaluronic acid / functional short peptide composite aqueous solution into the spinal cord injury site to form a hydrogel, which serves as an artificial extracellular matrix to support nerve regeneration.

[0058] The dopamine-grafted functionalized hyaluronic acid in the present application is modified hyaluronic acid.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] 1. The present invention uses hyaluronic acid as one of the main components, utilizes dopamine grafting reaction to functionalize hyaluronic acid, and achieves the effect of increasing tissue viscosity through self-polymerization of catechol groups on dopamine; at the same time, a class of functional short peptides are designed to form a dynamic reversible π-π conjugated non-covalent bond with dopamine grafted functionalized hyaluronic acid, and finally obtains an injectable hydrogel with stronger mechanical properties and mechanical stability, which can improve the local microenvironment and also load bioactive substances.

[0061] 2. The preparation process conditions of the composite hydrogel of the present invention are simple, convenient, low-cost, and do not require additional additives, thus avoiding the problem of residual toxic initiators and catalysts. The prepared composite hydrogel has higher biosafety, higher mechanical properties and mechanical stability, and can perfectly fill the damaged area and connect the host tissues at both ends of the damage without causing additional tissue damage. It can be used as an extracellular matrix for 3D cell culture and as a tissue engineering material, used alone or in combination with cell transplantation and drug / active factor injection to the spinal cord injury site, to promote the repair of spinal cord injury and improve the repair ability after spinal cord injury. It has very broad application prospects and clinical value in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The modified hyaluronic acid / functional short peptide composite gel of Example 1 is injectable and has a structural diagram; A is an injectable diagram, and B is a scanning electron microscope diagram.

[0063] Figure 2 This is the growth and differentiation of neural stem cells after 7 days of 3D culture in Example 6; red: neuronal axons (NF200), green: astrocytes (GFAP), blue: cell nuclei (DAPI).

[0064] Figure 3 This is the growth and differentiation of dorsal root ganglia after 7 days of 3D culture in Example 7; green represents nerve fibers (NF200) and red represents Schwann cells.

[0065] Figure 4 The results of immunofluorescence staining of sagittal sections of the spinal cord transection of rats with spinal cord injury (T8-T9 complete transection, 2 mm defect) 60 days after injection of the hydrogel of Example 1; NF200 represents nerve fibers (red), GFAP represents astrocytes (green), and DAPI represents cell nuclei (blue).

[0066] Figure 5 The results of immunofluorescence staining of sagittal sections of the spinal cord transection 60 days after injection of the hydrogel of Example 4 in rats with spinal cord injury (complete transection of T8-T9, 2 mm defect); NF200 represents nerve fibers (red), GFAP represents astrocytes (green), and DAPI represents cell nuclei (blue).

[0067] Figure 6 This is a diagram of the tissue status of rat spinal cord bonded with composite hydrogel.

[0068] Figure 7 This is a diagram of the state after the composite aqueous solution of Comparative Example 1 was injected into PBS and inverted. DETAILED DESCRIPTION

[0069] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0070] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0071] Culture medium: DMEM / F12, Gibco (USA); 1% double antibody, Gibco (USA); 1% B27, Gibco (USA); 1% FBS, Gibco (USA); NGF, Gibco (USA); antibody NF200, Sigma (USA); antibody GFAP, Sigma (USA).

[0072] Example 1 Preparation of modified hyaluronic acid / functional short peptide composite gel

[0073] 1. Preparation of Modified Hyaluronic Acid

[0074] Dissolve hyaluronic acid in deionized water, adjust pH to 6.0 with 1M HCl to prepare a solution with a final concentration of 10 mg / mL; add sodium N-hydroxysuccinimide (NHS) to the above hyaluronic acid solution, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) after 15 minutes to obtain a mixed solution; dissolve dopamine in deionized water, adjust pH to 6.0 with 1M HCl, and prepare a dopamine (DA) solution, add the DA solution to the above mixed solution, and react at 25°C for 24 hours. During the whole process, the reaction system is maintained in an inert gas atmosphere. The mass ratio of hyaluronic acid to NHS and EDC is 1:1:1, and the mass ratio of hyaluronic acid to dopamine is 1:2. The reactants were placed in a dialysis bag with a molecular weight cutoff of 3000 to 4000, and dialyzed for 3 days with deionized water as the dialysate. The retentate was freeze-dried to obtain dopamine-hyaluronic acid composite biogel, i.e., dopamine grafted functionalized hyaluronic acid, i.e., modified hyaluronic acid. The freeze-drying conditions were a pressure of 50 Pa, a temperature of -50°C, and a time of 72 hours.

[0075] 2. Preparation of functional short peptide HGF-(RADA)4-RIKVAV

[0076] A peptide synthesizer was used to connect histidine-glycine-phenylalanine (HGF) at one end of the (arginine-alanine-aspartic acid-alanine)4 sequence, and the other end was connected to the functional amino acid sequence RIKVAV to obtain a functional short peptide HGF-(RADA)4-RIKVAV.

[0077] 1.3 Preparation of modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite hydrogel

[0078] The prepared dopamine grafted functionalized hyaluronic acid and functional short peptide HGF-(RADA)4-RIKVAV were dissolved in deionized water, wherein the concentration of the dopamine grafted functionalized hyaluronic acid solution was 20 mg / mL, the concentration of the functional short peptide HGF-(RADA)4-RIKVAV was 20 mg / mL, the pH value was adjusted to 7.0, the volume ratio of the dopamine grafted functionalized hyaluronic acid solution to the functional short peptide solution was 1:1, and the final composite solution concentration was 20 mg / mL; the dopamine grafted functionalized hyaluronic acid solution and the functional short peptide HGF-(RADA)4-RIKVAV solution were mixed, and the mixed composite aqueous solution was injected into PBS to form a hydrogel.

[0079] Example 2 Preparation of modified hyaluronic acid / functional short peptide composite gel

[0080] 1.1 Preparation of modified hyaluronic acid

[0081] Hyaluronic acid was dissolved in deionized water, and the pH was adjusted to 5.0 with 1M HCl to prepare a solution with a final concentration of 1 mg / mL; sodium N-hydroxysuccinimide (NHS) was added to the above hyaluronic acid solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added after 15 minutes to obtain a mixed solution; dopamine was dissolved in deionized water, and the pH was adjusted to 5.0 with 1M HCl to obtain a dopamine (DA) solution, and the DA solution was added to the above mixed solution, and the reaction was carried out at 20°C for 24 hours. During the whole process, the reaction system was maintained in an inert gas helium atmosphere. The mass ratio of hyaluronic acid to NHS and EDC was 1:0.6:0.6, and the mass ratio of hyaluronic acid to dopamine was 1:1. The reactants were placed in a dialysis bag with a molecular weight cutoff of 4000, and dialyzed for 1 day with deionized water as the dialysate. The retentate was freeze-dried to obtain dopamine-hyaluronic acid composite biogel, i.e., dopamine grafted functionalized hyaluronic acid, i.e., modified hyaluronic acid. The freeze-drying conditions were a pressure of 0 Pa, a temperature of -100°C, and a time of 24 hours.

[0082] 1.2 Preparation of functional short peptides

[0083] The functional short peptide HGF-(RADA)4-RYIGSR was synthesized by a peptide synthesizer.

[0084] 1.3 Preparation of modified hyaluronic acid / functional short peptide composite hydrogel

[0085] The prepared dopamine grafted functionalized hyaluronic acid and functional short peptide HGF-(RADA)4-RYIGSR were dissolved in deionized water to prepare solutions and then mixed to obtain a composite aqueous solution, wherein the concentration of the dopamine grafted functionalized hyaluronic acid solution was 5 mg / mL, and the concentration of the functional short peptide HGF-(RADA)4-RYIGSR was 2.5 mg / mL, the pH value was adjusted to 6.5, the volume ratio of the dopamine grafted functionalized hyaluronic acid solution to the functional short peptide solution was 1:2, and the final composite solution concentration was 5 mg / mL. The mixed composite aqueous solution was injected into PBS to form a hydrogel.

[0086] Example 3 Preparation of modified hyaluronic acid / functional short peptide composite gel

[0087] 1.1 Preparation of modified hyaluronic acid

[0088] Hyaluronic acid was dissolved in deionized water, and the pH was adjusted to 6.5 with 1M HCl to prepare a solution with a final concentration of 20 mg / mL; sodium N-hydroxysuccinimide (NHS) was added to the above hyaluronic acid solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added after 15 minutes to obtain a mixed solution; dopamine was dissolved in deionized water, and the pH was adjusted to 6.5 with 1M HCl to obtain a dopamine (DA) solution, and the DA solution was added to the above mixed solution, and the reaction was carried out at 30°C for 24 hours. During the whole process, the reaction system was maintained in an inert gas nitrogen atmosphere. The mass ratio of hyaluronic acid to NHS and EDC was 1:2:2, and the mass ratio of hyaluronic acid to dopamine was 1:6. The reactants were placed in a dialysis bag with a molecular weight cutoff of 3000, and dialyzed for 3 days with deionized water as the dialysate. The retentate was freeze-dried to obtain dopamine-hyaluronic acid composite biogel, i.e., dopamine grafted functionalized hyaluronic acid, i.e., modified hyaluronic acid. The freeze-drying conditions were a pressure of -50 Pa, a temperature of -50°C, and a time of 72 hours.

[0089] 1.2 Preparation of functional short peptides

[0090] The functional short peptide HGF-(RADA)4-RRGD was synthesized by a peptide synthesizer.

[0091] 1.3 Preparation of modified hyaluronic acid / functional short peptide composite hydrogel

[0092] The prepared dopamine grafted functionalized hyaluronic acid and functional short peptide HGF-(RADA)4-RRGD were dissolved in deionized water to prepare solutions and then mixed to obtain a composite aqueous solution, wherein the concentration of the dopamine grafted functionalized hyaluronic acid solution was 50 mg / mL and the concentration of the functional short peptide HGF-(RADA)4-RRGD was 100 mg / mL, the pH value was adjusted to 7.5, the volume ratio of the dopamine grafted functionalized hyaluronic acid solution to the functional short peptide solution was 2:1, and the final composite solution concentration was 100 mg / mL. The mixed composite aqueous solution was injected into PBS to form a hydrogel.

[0093] Example 4 Preparation of modified hyaluronic acid / functional short peptide composite gel

[0094] 1.1 Preparation of modified hyaluronic acid

[0095] Dissolve hyaluronic acid in deionized water, adjust pH to 6.0 with 1M HCl to prepare a solution with a final concentration of 10 mg / mL; add sodium N-hydroxysuccinimide (NHS) to the above hyaluronic acid solution, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) after 15 minutes to obtain a mixed solution; dissolve dopamine in deionized water, adjust pH to 6.0 with 1M HCl, and prepare a dopamine (DA) solution, add the DA solution to the above mixed solution, and react at 25°C for 24 hours. During the whole process, the reaction system is maintained in an inert gas atmosphere. The mass ratio of hyaluronic acid to NHS and EDC is 1:1:1, and the mass ratio of hyaluronic acid to dopamine is 1:2. The reactants were placed in a dialysis bag with a molecular weight cutoff of 3000 to 4000, and dialyzed for 3 days with deionized water as the dialysate. The retentate was freeze-dried to obtain dopamine-hyaluronic acid composite biogel, i.e., dopamine grafted functionalized hyaluronic acid, i.e., modified hyaluronic acid. The freeze-drying conditions were a pressure of 50 Pa, a temperature of -50°C, and a time of 72 hours.

[0096] 1.2 Preparation of functional short peptide Fmoc-FDGDRGDS

[0097] The Fmoc was connected to the functional amino acid sequence FDGDRGDS using a peptide synthesizer to prepare the Fmoc-FDGDRGDS functional short peptide.

[0098] 1.3 Preparation of modified hyaluronic acid / functional short peptide Fmoc-FDGDRGDS composite hydrogel

[0099] The prepared dopamine grafted functionalized hyaluronic acid and functional short peptide Fmoc-FDGDRGDS were dissolved in deionized water, wherein the concentration of the dopamine grafted functionalized hyaluronic acid solution was 20 mg / mL, the concentration of the functional short peptide Fmoc-FDGDRGDS was 20 mg / mL, the pH value was adjusted to 7.0, the volume ratio of the dopamine grafted functionalized hyaluronic acid solution to the functional short peptide Fmoc-FDGDRGDS solution was 1:1, and the final composite solution concentration was 20 mg / mL; the dopamine grafted functionalized hyaluronic acid solution and the functional short peptide Fmoc-FDGDRGDS solution were mixed, and the mixed composite aqueous solution was injected into PBS to form a hydrogel.

[0100] Example 5 Observation of composite hydrogel

[0101] The composite hydrogels prepared in Examples 1 to 4 were observed. The hydrogels prepared in Examples 1 to 4 were able to form gels after being injected with PBS. Scanning electron microscopy was used to observe that the hydrogels were all porous structures, with pores directly connected to each other.

[0102] The observation results of the composite hydrogel in Example 1 are as follows Figure 1 , Figure 1 A is a dopamine-grafted functionalized hyaluronic acid / functional short peptide composite hydrogel injectable. Figure 1 B is a scanning electron micrograph of dopamine grafted functionalized hyaluronic acid / functional short peptide composite hydrogel.

[0103] Example 6 Three-dimensional culture of neural stem cells

[0104] Neural stem cells were cultured in three dimensions using modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite hydrogel.

[0105] The neural stem cell spheres were dispersed in the modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite aqueous solution prepared in Example 1 to obtain a mixed solution, and the mixed solution was injected into a neural stem cell differentiation medium (differentiation medium was DMEM / F12, 1% double antibody, 1% B27 (containing VA), 1% FBS) to form a hydrogel encapsulating neural stem cells. The cells were cultured in an incubator at 37°C and 5% CO2 for 7 days, fixed and stained, and observed using a laser confocal microscope.

[0106] Neural stem cell growth and differentiation Figure 2 As shown, from Figure 2 It can be seen that neural stem cells differentiate into neuronal axons and astrocytes, indicating that the modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite gel can be used for the three-dimensional culture of neural stem cells.

[0107] Example 7 Three-dimensional culture of dorsal root ganglia

[0108] Dorsal root ganglia were cultured in three dimensions using modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite gel.

[0109] The dorsal root ganglion was dispersed in the modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite aqueous solution prepared in Example 1 to obtain a mixed solution, which was injected into the dorsal root ganglion culture medium (2w / v% B27, 0.3wt% L-glutamine, 100ngmL-1NGF were added to the Neurobasal-A basal culture medium and mixed evenly) to form a hydrogel encapsulating the dorsal root ganglion. The solution was fixed and stained in an incubator at 37°C and 5% CO2 for 7 days and observed using a laser confocal microscope.

[0110] Growth and differentiation of dorsal root ganglia Figure 3 As shown, from Figure 3 It can be seen that nerve fibers and Schwann cells appeared in the hydrogel, indicating that the modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite gel can be used for the three-dimensional culture of dorsal root ganglia.

[0111] Example 8 Spinal cord injury repair

[0112] 1. Use modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite gel to repair spinal cord injury.

[0113] A rat spinal cord T8 to T9 complete transection (2 mm defect) injury was constructed, and the modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite aqueous solution prepared in Example 1 was injected into the injury site to form a hydrogel. The dura mater, muscle and skin were sutured in sequence. After 2 months, the animals were perfused with 4% paraformaldehyde solution and the spinal cord tissue was taken and frozen into sections. NF200 and GFAP were used as primary antibodies, incubated overnight at 4°C, and then incubated with fluorescently labeled secondary antibodies at room temperature for 2 hours. Laser confocal microscopy was used to take pictures and detect the distribution of regenerated nerve fibers at the transection of the spinal cord.

[0114] Immunofluorescence staining results of sagittal sections of the spinal cord Figure 4 As shown, from Figure 4 It can be seen that the regenerated nerve fibers grow toward the injured area, indicating that the modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite gel can be used for spinal cord injury repair.

[0115] 2. Use modified hyaluronic acid / functional short peptide Fmoc-FDGDRGDS composite gel to repair spinal cord injury.

[0116] A rat spinal cord T8 to T9 full transection (2 mm defect) injury was constructed, and the hyaluronic acid / functional short peptide Fmoc-FDGDRGDS composite aqueous solution prepared in Example 4 was injected into the injury site to form a hydrogel. The dura mater, muscle and skin were sutured in sequence. After 2 weeks, the animals were perfused with 4% paraformaldehyde solution and the spinal cord tissue was taken and frozen. NF200 and GFAP were used as primary antibodies, incubated overnight at 4 ° C, and then incubated with fluorescently labeled secondary antibodies at room temperature for 2 hours. Laser confocal microscopy was used to take pictures and detect the distribution of regenerated nerve fibers at the transection of the spinal cord.

[0117] Immunofluorescence staining results of sagittal sections of the spinal cord Figure 5 As shown, from Figure 5 It can be seen that regenerated nerve fibers (arrows) grow toward the injured area.

[0118] 3. Take fresh rat spinal cord tissue, cut it in the middle, and insert the modified hyaluronic acid / functional short peptide Fmoc-FDGDRGDS composite hydrogel in the middle, let it stand for 5 minutes, and then observe.

[0119] The results showed that the modified hyaluronic acid / functional short peptide Fmoc-FDGDRGDS composite hydrogel can well adhere to rat spinal cord tissue. Figure 6 .

[0120] Comparative Example 1 Preparation of modified hyaluronic acid / functional short peptide HGF-(RADA)4-RIKVAV composite gel

[0121] The dopamine grafted functionalized hyaluronic acid and the functional short peptide HGF-(RADA)4-RIKVAV prepared in Example 1 were dissolved in deionized water respectively, the concentration of the dopamine grafted functionalized hyaluronic acid solution was 20 mg / mL, the concentration of the functional short peptide HGF-(RADA)4-RIKVAV was 20 mg / mL, and the pH value was adjusted to 5.0. The volume ratio of the dopamine grafted functionalized hyaluronic acid solution to the functional short peptide solution was 1:1, and the final composite solution concentration was 20 mg / mL. The dopamine grafted functionalized hyaluronic acid solution and the functional short peptide HGF-(RADA)4-RIKVAV solution were mixed, and the resulting composite aqueous solution could not form a hydrogel after being injected into PBS.

[0122] The composite aqueous solution was injected into PBS and then inverted. Figure 7 As shown, from Figure 7 It can be seen that the solution flows to the tube mouth after inversion, indicating that the composite aqueous solution cannot form a hydrogel after being injected into PBS.

[0123] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An injectable modified hyaluronic acid / functional short peptide composite hydrogel, It is characterized in that The invention is obtained by compounding modified hyaluronic acid and functional short peptide, wherein the modified hyaluronic acid is hyaluronic acid functionalized by dopamine grafting; and the functional short peptide is HGF-(RADA) 4 -RIKVAV, HGF-(RADA) 4 -RYIGSR, HGF-(RADA) 4 -RRGD or Fmoc-FDGDRGDS; the pH value of the composite hydrogel is 6.5-7.

5.

2. A method for preparing an injectable modified hyaluronic acid / functional short peptide composite hydrogel, It is characterized in that The following steps are involved: S1. Preparation of modified hyaluronic acid: After the hyaluronic acid is dissolved, the pH is adjusted to 5.0-6.5; sodium N-hydroxysuccinimide is added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added after 12-15 minutes to obtain a mixed solution; dopamine is dissolved, and the pH is adjusted to 5.0-6.5 to obtain a dopamine solution, and then the dopamine solution is mixed with the mixed solution, and the reaction is carried out at 20-30° C. for 24 hours to obtain a reactant, and the reaction system is maintained in an inert gas atmosphere during the whole process; the reactant is dialyzed for 1-3 days, and the retentate is freeze-dried to obtain a modified hyaluronic acid; S2. Preparation of functional short peptides: The functional short peptide is HGF-(RADA) 4 -RIKVAV, HGF-(RADA) 4 -RYIGSR, HGF-(RADA) 4 -RRGD or Fmoc-FDGDRGDS; S3. Preparation of modified hyaluronic acid / functional short peptide composite hydrogel: The modified hyaluronic acid described in step S1 and the functional short peptide described in step S2 are dissolved and then mixed, and the pH is adjusted to 6.5-7.5 to obtain a composite aqueous solution, which is then injected into a buffer solution or a cell culture medium to obtain a composite hydrogel.

3. The preparation method according to claim 2, It is characterized in that In step S1, the mass ratio of hyaluronic acid, NHS and EDC is 1:0.6-2:0.6-2; the mass ratio of hyaluronic acid to dopamine is 1:1-6.

4. The preparation method according to claim 2, It is characterized in that The volume ratio of the modified hyaluronic acid and the functional short peptide mixed in step S3 is 1-2:1-2.

5. Use of the composite hydrogel according to claim 1 in 3D cell culture for non-diagnostic and non-therapeutic purposes.

6. Use of the composite hydrogel according to claim 1 in preparing spinal cord injury repair materials.

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