Composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses composite hydrogel as well as a preparation method and application thereof. The composite hydrogel comprises the following components: gelatin, hyaluronic acid, a neurotrophic factor and a magnetic substance, and the composite hydrogel can generate double response to hyaluronidase and a magnetic field. The preparation method of the composite hydrogel comprises the following steps: dissolving hyaluronic acid in a hydrochloric acid solution; adding EDC into the hyaluronic acid solution, stirring, and adjusting the pH value of the solution to obtain an HA mixed solution; uniformly mixing the gelatin aqueous solution, the HA mixed solution, the neurotrophic factor and the magnetic substance; and standing overnight to prepare the hyaluronidase-magnetic field dual-response drug-loading composite hydrogel. The composite hydrogel provided by the invention can achieve the effect of generating double responses to hyaluronidase and a magnetic field at the same time.

Description

Technical Field

[0001] The invention relates to a biomedical material and a preparation method thereof, in particular to a composite hydrogel and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is a neurological disease that causes significant loss of sensory, motor and autonomic functions below the level of injury, and even paralysis in severe cases. In addition, SCI can cause a variety of complications, including bedsore ulcers, urinary tract infections, cardiovascular irregularities and sensory impairment, which can lead to respiratory failure in extreme cases. Current treatments for SCI mainly include surgical decompression and methylprednisolone shock therapy. However, these methods often fail to achieve the desired therapeutic effect and are accompanied by a large number of adverse reactions, highlighting the urgent need to develop more effective clinical treatments [YF Wang, Neurosci. Lett., 2014; WHO, Spinal cord injury: as many as 500000 people suffer each year, 2013]. Tissue engineering is an interdisciplinary technology that combines cells, drugs, molecules and advanced biomaterials, providing an innovative approach to repair SCI, namely using biomaterials as efficient delivery vehicles to precisely transport cells or drugs to the desired site of action, or promote their local release in situ. This approach alleviates the limitations of using cells or drugs alone, namely their slow proliferation or insufficient local concentration. Biomaterials used in tissue engineering fill defective tissues, regulate the local microenvironment, and create a favorable environment for repair and regeneration [H. Li, Adv. Healthc. Mater., 2016].

[0003] Li Ya et al. [Li Ya et al. Preparation and performance study of pH and hyaluronidase responsive antibacterial hydrogel [C] / / Antibacterial Industry Branch of the National Health Industry Enterprise Management Association, Zhongguancun Huizhi Antibacterial New Materials Industry Technology Innovation Alliance. 2020 (4th) Antibacterial Science and Technology Forum Paper Abstracts] reported the preparation of a pH and hyaluronidase responsive antibacterial hydrogel. The hydrogel has good mechanical properties and self-healing properties, and is expected to be able to provide an effective closed environment for wound healing through self-repair after being damaged by external forces. However, the hydrogel cannot respond to the magnetic field effect, cannot promote nerve growth, and cannot perform its corresponding functions again. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a composite hydrogel that can produce dual responses to hyaluronidase and magnetic field at the same time.

[0005] Another object of the present invention is to provide a method for preparing a composite hydrogel that can produce dual responses to hyaluronidase and magnetic field at the same time.

[0006] Another object of the present invention is to provide a composite hydrogel capable of generating dual responses to hyaluronidase and magnetic field at the same time for use in the preparation of a drug for treating spinal cord injury.

[0007] Technical solution: The composite hydrogel described in the present invention comprises the following components: gelatin, hyaluronic acid, neurotrophic factors and magnetic substances. The composite hydrogel can produce dual responses to hyaluronidase and magnetic field.

[0008] Preferably, the magnetic material is Fe3O4.

[0009] Preferably, the neurotrophic factor is one of nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophic factor 3 (NT-3), and neurotrophic factor 4 (NT-4).

[0010] The method for preparing the composite hydrogel of the present invention comprises the following steps:

[0011] S1. dissolving hyaluronic acid in hydrochloric acid solution;

[0012] S2, adding EDC to the hyaluronic acid solution obtained in S1, stirring, and adjusting the pH of the solution to obtain a HA mixed solution;

[0013] S3, uniformly mix the gelatin aqueous solution, the HA mixed solution prepared in S2, the neurotrophic factor and the magnetic substance; after standing overnight, a hyaluronidase-magnetic field dual-responsive drug-loaded composite hydrogel is prepared.

[0014] Preferably, the concentration of the hyaluronic acid in the hydrochloric acid solution in S1 is 0.5-1.5% (w / v), more preferably 1% (w / v); the pH of the hydrochloric acid solution is 4.0-5.5, more preferably 4.7.

[0015] Preferably, the mass ratio of the added EDC to the added hyaluronic acid in S2 is 1:0.5-1.5, more preferably 1:1; and the pH of the solution is adjusted to 4.0-5.5, more preferably 4.7.

[0016] For adaptability, the concentration of the gelatin aqueous solution in S3 is 10-20% (w / v), and more preferably 15% (w / v); the dissolution temperature of gelatin in water is 45-60°C.

[0017] Preferably, the volume ratio of the gelatin aqueous solution to the HA mixed solution in S3 is 1:0.5-1.5, more preferably 1:1; the amount of neurotrophic factor added is More preferably The amount of magnetic material added is 0-5% (w / v), and more preferably 3‰ (w / v).

[0018] Preferably, the magnetic substance described in S3 is Fe3O4; the neurotrophic factor is one of nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophic factor 3 (NT-3), and neurotrophic factor 4 (NT-4).

[0019] Application of the composite hydrogel of the present invention in preparing medicine for treating spinal cord injury.

[0020] Principle of the invention: Stimuli-responsive materials have the ability to coordinate responses with external signals and play a key role in drug delivery and release systems, disease diagnosis and treatment. Studies have shown that after spinal cord injury, hyaluronidase is naturally produced at the site of injury and lasts for at least six weeks. It exhibits a significant degradation ability for hyaluronic acid. Low-frequency weak magnetic fields are often used in clinical practice to promote stem cell proliferation and differentiation, reduce inflammation and edema, relieve pain, reduce apoptosis, and enhance tissue regeneration; low-frequency weak magnetic fields can also promote the expression of growth factors such as FGF and VEGF, which helps reduce inflammation and promote vascular regeneration. Based on these results, the present invention designs an enzyme-magnetic dual-responsive drug-loaded hydrogel delivery platform with high biocompatibility and good mechanical properties. The platform uses the reaction characteristics of hyaluronic acid to hyaluronidase to target and deliver drugs such as neurotrophic growth factor (NGF) to the injury site and continuously release them. It also promotes the differentiation of neural stem cells, increases the expression of calcium ion channels, promotes the generation of neurons, regulates neurotransmitter levels, promotes the reconstruction of neural networks, accelerates the Wallerian degeneration process after nerve injury, promotes Schwann cell proliferation, and accelerates the regeneration of axons and myelin sheaths. In addition, the magnetic field increases nerve growth factor, accelerates axon growth and repair, helps the regeneration of nerve fibers, and strengthens the connection between nerves and muscles.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The hyaluronidase-magnetic field dual-responsive drug-loaded composite hydrogel prepared by the present invention has good biocompatibility and mechanical properties; (2) Through the reaction characteristics of hyaluronic acid to hyaluronidase, drugs such as NGF are targeted and delivered to the injured site and continuously released; the loaded magnetic Fe3O4 has a weak magnetic field response and can improve the local environment of the injured site and promote neurogenesis through external magnetic field stimulation; (3) The present invention introduces magnetic substances to make the hydrogel produce a dual effect on hyaluronidase and magnetic field, providing a new way for targeted drug delivery and local stimulation of nerve growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The infrared spectrum result of the composite hydrogel prepared in Example 1;

[0023] Figure 2 This is the SEM result of the composite hydrogel prepared in Example 1;

[0024] Figure 3 This is the mechanical curve of the composite hydrogel compression test prepared in Example 1;

[0025] Figure 4 This is the live and dead cell staining image of Example 1;

[0026] Figure 5 The immunohistochemical staining diagrams of Example 1, Comparative Example 1 and blank test are shown. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with embodiments.

[0028] The composite hydrogel of the present invention comprises the following components: gelatin, hyaluronic acid, neurotrophic factor and magnetic substance Fe3O4. The composite hydrogel can produce dual effects on hyaluronidase and magnetic field.

[0029] Example 1

[0030] The method for preparing the composite hydrogel of the present invention comprises the following steps:

[0031] S1. Dissolve hyaluronic acid at a concentration of 1% (w / v) in a hydrochloric acid solution at pH 4.7 at 25°C using mechanical stirring or magnetic stirring.

[0032] S2. Add EDC in an amount equal to that of hyaluronic acid to the hyaluronic acid solution obtained in S1, stir for 1 hour, and adjust the pH of the solution to 4.7 using 0.1 M HCl to obtain a HA mixed solution.

[0033] S3. Prepare a 15% (w / v) gelatin aqueous solution and dissolve it by mechanical stirring or magnetic stirring at 45°C; mix 0.5 mL of the gelatin aqueous solution, 0.5 mL of the HA mixed solution prepared in S2, 10 μg of NGF and 3 mg of Fe3O4 evenly by mechanical stirring or oscillating stirring; after standing overnight, a hyaluronidase-magnetic field dual-responsive drug-loaded composite hydrogel is prepared.

[0034] Infrared spectroscopy test of the composite hydrogel: The composite hydrogel was characterized by a 170SX Fourier transform infrared spectrometer. The composite hydrogel was freeze-dried and ground into powder, which was mixed with potassium bromide to prepare a pressed sample. The infrared spectrum scan range was 4000-400 cm -1 , resolution 4cm -1The results are as follows Figure 1 As shown, the surface gelatin and hyaluronic acid are successfully chemically cross-linked, and the addition of magnetic substances does not destroy the chemical cross-linking between gelatin and hyaluronic acid.

[0035] like Figure 2 As shown, the composite hydrogel prepared in this embodiment has a porous structure and a large specific surface area, which is beneficial to increase the drug loading amount so as to facilitate the growth of cells therein.

[0036] The mechanical properties were evaluated by compression testing with a strain rate of 1 mm / min, and the compression modulus was determined by taking the slope of the linear portion of the stress-strain curve in the 5%-10% strain region. Figure 3 As shown, the compression modulus of the composite hydrogel prepared in this example is 0.182 MPa.

[0037] Application of the composite hydrogel of the present invention in medicine for treating spinal cord injury,

[0038] Example 2

[0039] The same points as those in Example 1 are not described in detail here, except that the amount of Fe3O4 added in step S3 is 5 mg. The compression modulus of the composite hydrogel prepared in this example is 0.182 MPa.

[0040] Example 3

[0041] The same points as those in Example 1 are not described in detail here, except that the amount of Fe3O4 added in step S3 is 7 mg. The compression modulus of the composite hydrogel prepared in this example is 0.182 MPa.

[0042] Comparative Example 1

[0043] The same points as those in Example 1 are not described in detail here, except that Fe3O4 is not added in step S3. The compression modulus of the composite hydrogel prepared in this comparative example is 0.182 MPa.

[0044] like Figure 4 As shown, the green dye in the figure is the cell nucleus. It can be seen from the figure that the composite hydrogel prepared by the present invention is non-toxic to cells, has good cell compatibility, and has no obvious mortality rate.

[0045] like Figure 5 As shown, for the composite hydrogel prepared in Example 1, obvious CSPG positive staining can be seen in the lesion area, which indicates that not only astrocytes but also a large number of infiltrating cells secreting CSPG are present, which are gathered around the lesion; while for the hydrogel prepared in Comparative Example 1, there is no obvious CSPG positive staining in the lesion area. Figure 5This shows that the composite hydrogel prepared by this scheme can effectively restore spinal cord injury.

Claims

1. A composite hydrogel, characterized in that: Contains the following ingredients: 10-20% (w / v) gelatin aqueous solution, HA mixed solution, Neurotrophic factor and 0-5% (w / v) magnetic substance, the The solvent of neurotrophic factor and 0-5% (w / v) magnetic substance is a mixture of gelatin aqueous solution and HA mixed solution, wherein the HA mixed solution includes 0.5-1.5% (w / v) hyaluronic acid-hydrochloric acid solution and EDC with a mass ratio of 1:0.5-1.5 to hyaluronic acid.

2. The composite hydrogel according to claim 1, characterized in that The magnetic material is Fe3O4.

3. The composite hydrogel according to claim 1, characterized in that The neurotrophic factor is one of nerve growth factor, brain-derived growth factor, neurotrophic factor 3 and neurotrophic factor 4.

4. A method for preparing the composite hydrogel according to claim 1, characterized in that: The following steps are involved: S1. dissolving hyaluronic acid in hydrochloric acid solution; S2, adding EDC to the hyaluronic acid solution obtained in S1, stirring, and adjusting the pH of the solution to obtain a HA mixed solution; S3, uniformly mixing the gelatin aqueous solution, the HA mixed solution prepared in S2, the neurotrophic factor and the magnetic substance; After standing overnight, a hyaluronidase-magnetic field dual-responsive drug-loaded composite hydrogel was prepared.

5. The preparation method according to claim 4, characterized in that: The concentration of the hyaluronic acid in the hydrochloric acid solution described in S1 is 0.5-1.5% (w / v); the pH of the hydrochloric acid solution is 4.0-5.

5.

6. The preparation method according to claim 4, characterized in that: The mass ratio of the added EDC to the added hyaluronic acid described in S2 is 1:0.5-1.5; and the pH of the solution is adjusted to 4.0-5.

5.

7. The preparation method according to claim 4, characterized in that: The concentration of the gelatin aqueous solution described in S3 is 10-20% (w / v); the dissolution temperature of gelatin in water is 45-60°C.

8. The preparation method according to claim 4, characterized in that: The volume ratio of the gelatin aqueous solution to the HA mixed solution described in S3 is 1:0.5-1.5; the amount of neurotrophic factor added is The amount of magnetic material added is 0-5% (w / v).

9. The preparation method according to claim 4, characterized in that: The magnetic substance described in S3 is Fe3O4; the neurotrophic factor is one of nerve growth factor, brain-derived growth factor, neurotrophic factor 3, and neurotrophic factor 4.

10. Use of the composite hydrogel according to claim 1 in preparing a drug for treating spinal cord injury.

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