Bio-enzyme modified graded hydrogel film for spinal cord injury repair and preparation method of bio-enzyme modified graded hydrogel film

Abstract

The invention discloses a preparation method of a biological enzyme modified graded hydrogel film for spinal cord injury repair, which comprises the following steps: S1, dispersing silicon dioxide nanoparticles in absolute ethyl alcohol to obtain a dispersion liquid, and uniformly coating a glass slide with the obtained dispersion liquid for deposition to obtain a photonic crystal film template; s2, placing a glass slide above the obtained photonic crystal film template and separating the photonic crystal film template by using a gasket, placing a striped mask plate above the glass slide, then pouring a hydrogel precursor solution containing methacrylated gelatin and a photoinitiator into a space between the photonic crystal film template and the glass slide, and curing under the action of ultraviolet irradiation to obtain a hydrogel hydrogel; and finally, etching with hydrofluoric acid to obtain the graded hydrogel film. S3, the obtained graded hydrogel film is placed in an activating agent to be activated and then placed in a superoxide dismutase and catalase solution to be grafted, and a final product is obtained. The product provided by the invention can effectively improve the local microenvironment and promote the repair of spinal cord injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a bioenzyme-modified graded hydrogel film for repairing spinal cord injury and a preparation method thereof. Background Art

[0002] Spinal cord injury is one of the most common types of nerve injury. After the injury occurs, it will cause a series of complex pathological changes, which seriously affect axon regeneration and functional recovery. Among them, the injury activates multiple oxidative stress pathways, which in turn produces a large number of reactive oxygen molecules. These reactive oxygen molecules cause peroxidative damage to biological molecules such as intracellular lipids, proteins and DNA, thereby having obvious toxic effects on cells and tissues. In addition, due to the lack of a directional growth scaffold, newborn neurons cannot grow quickly and directional to connect to the injured site to promote spinal cord repair.

[0003] Superoxide dismutase (SOD) and catalase (CAT) are a group of biological enzymes that can remove reactive oxygen species. In the presence of SOD, superoxide anions are decomposed into oxygen and hydrogen peroxide, and then hydrogen peroxide is further decomposed into oxygen and water in the presence of CAT. In the body, SOD and CAT are important pathways for removing reactive oxygen species. When the spinal cord is injured, supplementing exogenous SOD and CAT is expected to achieve the removal of reactive oxygen species, thereby reducing oxidative stress damage.

[0004] Micro-nano structures are believed to have a significant regulatory effect on cell growth. When the surface of a material has an oriented micro-nano structure, the cells growing on the surface of the material can extend in a directional manner under the regulation of the micro-nano structure. Therefore, oriented micro-nanostructure materials are expected to be used to induce the directional growth of new cells at the site of spinal cord injury. Inverse opal colloidal crystal hydrogel is an ordered porous structure material that can be prepared using colloidal crystal films as templates. Thanks to its ordered through-macroporous structure, inverse opal colloidal crystal hydrogel has a huge specific surface area and can provide a large number of sites for cell growth and adhesion. Combining the photomask technology with the inverse opal hydrogel preparation process, a micro-nano hierarchical structure hydrogel with micron stripes and nanopores will be prepared to promote the repair of spinal cord injuries. Summary of the invention

[0005] The primary purpose of the present invention is to provide a bio-enzyme modified hierarchical hydrogel film for spinal cord injury repair and a preparation method thereof, which can effectively improve the local microenvironment to promote the repair of spinal cord injury.

[0006] To this end, the present invention provides the following technical solutions.

[0007] One aspect of the present invention provides a method for preparing a bio-enzyme modified hierarchical hydrogel film for spinal cord injury repair, the method comprising the following steps: S1: dispersing silica nanoparticles in anhydrous ethanol to obtain a dispersion, and evenly applying the dispersion on a glass slide for deposition, and after the ethanol evaporates, a photonic crystal film template is obtained; dissolving methacrylated gelatin and a photoinitiator in water to obtain a hydrogel precursor solution; S2: placing a glass slide on the obtained photonic crystal film template and separating them with a spacer, and placing a stripe mask on the glass slide, and then pouring the obtained hydrogel precursor solution between the photonic crystal film template and the glass slide, and then curing it under ultraviolet light, and finally etching with hydrofluoric acid to remove the silica nanoparticles, so as to obtain a graded hydrogel film; S3: placing the obtained graded hydrogel film in an activator for activation, and then placing the activated graded hydrogel film in a superoxide dismutase and catalase solution for grafting to obtain a graded hydrogel film grafted with superoxide dismutase and catalase.

[0008] In a preferred embodiment of the present invention, in step S1, the mass fraction of the silicon dioxide nanoparticles in the dispersion is 5-30%.

[0009] In a preferred embodiment of the present invention, in step S1, the mass fraction of methacrylated gelatin in the hydrogel precursor solution is 5-30%, and the volume fraction of the photoinitiator is 0.25-2%.

[0010] In a preferred embodiment of the present invention, in step S1, the photoinitiator is selected from any one of 2-hydroxy-2-methyl-1-phenyl-1-propanone or 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

[0011] In a preferred embodiment of the present invention, in step S2, the height of the gasket is 50-400 µm.

[0012] In a preferred embodiment of the present invention, in step S2, the stripe width of the stripe mask is 10-60 μm.

[0013] In a preferred embodiment of the present invention, in step S2, the ultraviolet illumination conditions are: wavelength 365 nm, time 10-60 s.

[0014] In a preferred embodiment of the present invention, in step S2, the mass fraction of hydrofluoric acid is 1-8%, and the etching time is 1-3 h.

[0015] In a preferred embodiment of the present invention, in step S3, the activator is obtained by dissolving 40-300 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40-750 mM N-hydroxysuccinimide in 0.1 M pH 6.0 2-morpholineethanesulfonic acid buffer, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1-1:5.

[0016] In a preferred embodiment of the present invention, in step S3, the activation conditions are: room temperature, time 10-40 min.

[0017] In a preferred embodiment of the present invention, in step S3, the concentrations of superoxide dismutase and catalase are 1-5 kU / mL respectively.

[0018] In a preferred embodiment of the present invention, in step S3, the grafting conditions are: temperature 37° C., time 0.5-12 h.

[0019] Another aspect of the present invention also provides a use of a graded hydrogel film grafted with superoxide dismutase and catalase prepared according to the preparation method as described above in the preparation of a drug for promoting the repair of spinal cord injury.

[0020] By means of the above technical scheme, the present invention has at least the following advantages: the hydrogel stripe film with hierarchical micro-nano structure on the surface prepared by the method of the present invention has micron-scale stripes that are conducive to the directional induced growth of cells, and the nanopore structure increases its specific surface area, which can not only provide sufficient adhesion sites for cells, but also be used for functionalization. The hydrogel stripe film of the present invention can be grafted with superoxide dismutase and catalase to enable the stripe film to have the function of removing active oxygen, thereby improving the local microenvironment, and can be used in spinal cord injury to promote the repair of spinal cord injury. The hydrogel film has the advantages of easy preparation, safety and simplicity, and can be customized through mask patterns.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation process of the hierarchical hydrogel film of the present invention, wherein (1) is a photonic crystal template, (2) is the photonic crystal template being filled with glue and then cured under ultraviolet light, and (3) is a hierarchical hydrogel film; Figure 2are scanning electron microscope images of different products, among which a is a scanning electron microscope image of a photonic crystal template, b is a scanning electron microscope image of a photonic crystal glue-filled, and c is a scanning electron microscope image of a graded hydrogel film; Figure 3 is a scanning electron microscope cross-sectional view of the hierarchical hydrogel film of the present invention; Figure 4 is a scanning electron microscope image of cells growing directional on the surface of graded hydrogel film; Figure 5 It is the result of the active oxygen scavenging ability of the hydrogel films with different grades; Figure 6 It is the application effect of different graded hydrogel films in repairing spinal cord injury. DETAILED DESCRIPTION

[0023] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0025] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0026] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Embodiment 1: like Figure 1 As shown, the preparation process of the hierarchical hydrogel film of the present invention is shown, including the preparation of the photonic crystal template and the hierarchical hydrogel film, and the specific process is as follows: 1. Preparation of photonic crystal template Silica nanoparticles with a diameter of about 300 nm are dispersed in anhydrous ethanol to obtain a 20% silica nanoparticle dispersion. The obtained silica nanoparticle dispersion is evenly applied on a glass slide, and the ethanol is allowed to evaporate at room temperature, and the silica nanoparticles are deposited on the glass slide. This step can be repeated 1-5 times, and preferably repeated 3 times in this embodiment. Finally, after the ethanol evaporates completely, a photonic crystal film template is obtained, and its micro-nano structure is as follows: Figure 2 As shown in a.

[0030] 2. Preparation of graded hydrogel films Methacrylated gelatin (GelMA) and photoinitiator (HMPP) were dissolved in water to prepare a hydrogel precursor solution with a mass fraction of 10% GelMA and a volume fraction of 1% HMPP. The photonic crystal film template prepared above was placed between two glass slides, separated by a gasket with a thickness of 200 µm, and a mask with a stripe width of 30 µm was placed on top of the glass slides. The obtained hydrogel precursor solution was then poured into the two glass slides. After the pouring was completed, it was placed under 365 nm ultraviolet light for curing for 20 s to obtain a silica / hydrogel casting structure. Finally, the obtained silica / hydrogel casting structure was etched with 4% hydrofluoric acid for 3 h to remove the silica nanoparticles to obtain a graded hydrogel film.

[0031] The scanning electron microscopy images of the obtained silica / hydrogel casting structure and hierarchical hydrogel film are shown in Figure 2 b and 2c. The electron microscopy images of the cross-section of the graded hydrogel film are shown in Figure 3 As shown. Figure 2 It can be seen from ac that the pore size of the hierarchical hydrogel film structure is basically consistent with the diameter of the silica nanoparticles.

[0032] 3. Preparation of enzyme-modified hierarchical hydrogel films 35 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 55 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in 1 mL of 2-morpholineethanesulfonic acid (MES) buffer (0.1 M, pH 6.0) to prepare an activator. The obtained hierarchical hydrogel film was placed in the activator for activation for 30 min to obtain an activated hierarchical hydrogel film. Then, the activated hierarchical hydrogel film was placed in 5 kU / mL SOD and 5 kU / mL CAT solution and incubated at 37°C for 12 h to obtain a hierarchical hydrogel film grafted with SOD and CAT. Then, the obtained hierarchical hydrogel film grafted with SOD and CAT was incubated with 5% BSA for 2 h. After the incubation, it was washed with PBS for more than 3 times to obtain an inverse opal hydrogel stripe film with SOD and CAT activity.

[0033] Embodiment 2:

[0034] The preparation method of Example 2 is substantially the same as that of Example 1, with the only difference being that the mass fraction of GelMA in the hydrogel precursor solution used is 15%.

[0035] Embodiment 3: The preparation method of this embodiment 3 is substantially the same as that of embodiment 1, with the only difference being that the stripe width of the mask plate used is 40 μm.

[0036] Application Example 1: Effect of enzyme-active inverse opal hydrogel stripe film on cell growth ability Rat adrenal pheochromocytoma cells were cultured at a rate of 2×10 4 The number of cells / piece was inoculated on the inverse opal hydrogel stripe membrane with SOD and CAT activity obtained in Example 1 and cultured at 37°C and 5% CO2 for 7 days, with the culture medium replaced every day. The cell growth was then observed under a scanning electron microscope. The results are shown in Figure 4 .like Figure 4 As shown, it can be found that rat adrenal pheochromocytoma cells show an obvious directional growth trend on the anti-opal hydrogel stripe film.

[0037] According to the method described above, the cell growth ability of the inverse opal hydrogel stripe films with SOD and CAT activities obtained in Examples 2 and 3 was tested respectively, and the results obtained were consistent with the test results of the inverse opal hydrogel stripe films with SOD and CAT activities obtained in Example 1: rat adrenal pheochromocytoma cells showed an obvious directional growth trend on the inverse opal hydrogel stripe films of Examples 2 and 3. Therefore, the inverse opal hydrogel stripe films with SOD and CAT activities of Example 1 were used as representatives for various tests.

[0038] Application Example 2: Study on the active oxygen scavenging performance of inverse opal hydrogel stripe films with different enzyme activities (1) The superoxide anion scavenging ability was determined using iodonitronitrile tetrazolium blue chloride (INT). First, prepare 1 mL of a mixed solution of 75 mM xanthine and 1 U / mL xanthine oxidase and react at 37°C for 15 min. Divide the resulting mixture into three equal groups: GelMA group refers to adding one piece of graded hydrogel film without SOD and CAT grafted into the mixture and incubating for 15 min; The GelMA@SC group refers to adding one piece of the inverse opal hydrogel stripe film with SOD and CAT activity obtained in Example 1 to the mixed solution and incubating for 15 minutes; The SC group refers to the mixed solution of 5 kU / mL SOD and 5 kU / mL CAT added directly to the mixed solution and incubated for 15 min.

[0039] Each group was treated according to the above method. Then, a 5 mM INT probe solution was prepared, and 100 μL of the INT probe solution was added to the solution obtained after the incubation reaction of each group and reacted at room temperature for 15 min to obtain a reaction solution. Finally, the absorbance of the obtained reaction solution was measured at 505 nm.

[0040] The calculation formula for the elimination rate of superoxide anions is: Elimination rate (%) = [(A1 - A2) / (A1 - A0)] × 100%, where A1 and A2 are the absorbances of the inverse opal hydrogel stripe film with and without enzyme activity, respectively, and A0 is the absorbance of INT.

[0041] (2) The catalase activity was determined by adding one hydrogel stripe membrane into a 3% H2O2 solution and recording the dissolved oxygen value every minute using a dissolved oxygen meter.

[0042] Results Figure 5 .like Figure 5 As shown in a, the results show that the superoxide anion scavenging ability of the GelMA group of the graded hydrogel film without SOD and CAT grafted is low (2.78%) and can be ignored, while the GelMA@SC group of the inverse opal hydrogel stripe film grafted with SOD and CAT has the corresponding superoxide anion scavenging ability (74.24%), and the scavenging ability is close to that of the SC group mixed with SOD and CAT (98.99%). Figure 5 As shown in b, compared with the GelMA group of the graded hydrogel film without SOD and CAT grafted, the GelMA@SC group of the inverse opal hydrogel stripe film grafted with SOD and CAT has a more obvious ability to decompose hydrogen peroxide.

[0043] Application Example 3: Repair of spinal cord injury in rats by reverse opal hydrogel stripe films with different enzyme activities After the rats were anesthetized by isoflurane inhalation, the T9 spinal cord was exposed to obtain model rats. The obtained model rats were divided into the following groups (1) to (4): (1) SCI group refers to the group of model rats with 2 mm spinal cord segment transection and no other treatment; (2) GelMA group: The rats in this group had their spinal cord segments transected at 2 mm, and then the inverse opal hydrogel stripe membrane without SOD and CAT activity was implanted in the injured area; (3) GelMA@SC group refers to the group of model rats in which the spinal cord segment was transected by 2 mm, and then the inverse opal hydrogel stripe membrane with SOD and CAT activity obtained in Example 1 was implanted in the injured area; (4) Sham group refers to blank control, that is, the spinal cord of the model rats in this group was only exposed by surgery without spinal cord injury.

[0044] The model rats in each group were treated according to the above grouping. When there was no obvious bleeding or cerebrospinal fluid leakage, the muscles, deep fascia, and skin were sutured in turn. Penicillin (50 mg / kg) was injected intramuscularly 3 days after surgery to prevent infection, and the bladder was massaged (8 h / d) until reflex urination was restored. The spinal cord injury repair was observed 8 weeks later. The results are shown in Figure 6 .

[0045] like Figure 6 As shown in the figure, compared with the simple spinal cord injury group (SCI group), the GelMA@SC film group grafted with SOD and CAT can effectively reduce the cavity area by 59%. This result shows that the implant can effectively bridge the injury gap and promote nerve regeneration, thereby reducing the cavity area (upper left figure). In addition, Nissl bodies are widely distributed in mature nerve cells and can be used as a marker for neuronal regeneration. In the same area, the number of Nissl bodies in the GelMA@SC group (41) was the largest, and closer to the number of Nissl bodies in the sham group (59) (lower left). β-Tubulin (Tuj-1) and glial fibrillary acid protein (GFAP) are specific markers of neurons and astrocytes. Neurons in the spinal cord injury area bridge the injured spinal cord, while astrocyte enrichment leads to glial scars that hinder axon regeneration. The results showed that there was a strong Tuj-1 signal distribution in the GelMA@SC group, which showed abundant newborn neurons in the treatment area of ​​the rat (upper right). At the same time, the GFAP signal in the SCI group was the strongest, while the GFAP signal in the GelMA@SC group was weaker (lower right). The above results show that the GelMA@SC film group grafted with SOD and CAT can promote nerve regeneration and avoid the formation of glial scars in the spinal cord injury area of ​​rats.

[0046] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a bio-enzyme modified hierarchical hydrogel film for spinal cord injury repair, characterized in that: The method comprises the following steps: S1: dispersing silica nanoparticles in anhydrous ethanol to obtain a dispersion, and evenly applying the dispersion on a glass slide for deposition, and after the ethanol evaporates, a photonic crystal film template is obtained; dissolving methacrylated gelatin and a photoinitiator in water to obtain a hydrogel precursor solution; S2: placing a glass slide on the obtained photonic crystal film template and separating them with a spacer, and placing a stripe mask on the glass slide, and then pouring the obtained hydrogel precursor solution between the photonic crystal film template and the glass slide, and then curing it under ultraviolet light, and finally etching with hydrofluoric acid to remove the silica nanoparticles, so as to obtain a graded hydrogel film; S3: placing the obtained graded hydrogel film in an activator for activation, and then placing the activated graded hydrogel film in a superoxide dismutase and catalase solution for grafting to obtain a graded hydrogel film grafted with superoxide dismutase and catalase.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass fraction of the silicon dioxide nanoparticles in the dispersion is 5-30%.

3. The preparation method according to claim 1, characterized in that: In step S1, the mass fraction of methacrylated gelatin in the hydrogel precursor solution is 5-30%, and the volume fraction of the photoinitiator is 0.25-2%.

4. The preparation method according to claim 1 or 3, characterized in that: In step S1, the photoinitiator is selected from any one of 2-hydroxy-2-methyl-1-phenyl-1-propanone or 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

5. The preparation method according to claim 1, characterized in that: In step S2, the height of the gasket is 50-400 μm; The stripe width of the stripe mask is 10-60 μm; The ultraviolet irradiation conditions are: wavelength 365 nm, time 10-60 s.

6. The preparation method according to claim 1, characterized in that: In step S2, the mass fraction of hydrofluoric acid is 1-8%, and the etching time is 1-3 h.

7. The preparation method according to claim 1, characterized in that: In step S3, the activator is prepared by dissolving 40-300 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40-750 mM N-hydroxysuccinimide in 0.1 M 2-morpholineethanesulfonic acid buffer at pH 6.0, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1-1:5; The activation conditions are: room temperature, time 10-40 min.

8. The preparation method according to claim 1, characterized in that: In step S3, the concentrations of superoxide dismutase and catalase are 1-5 kU / mL respectively.

9. The preparation method according to claim 1, characterized in that: In step S3, the grafting conditions are: temperature 37° C., time 0.5-12 h.

10. Use of the graded hydrogel film grafted with superoxide dismutase and catalase prepared by the preparation method according to any one of claims 1 to 9 in the preparation of a drug for promoting the repair of spinal cord injury.

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