Preparation method and application of pyrogallic acid-zinc multifunctional nanomaterial

Abstract

The present invention belongs to the technical field of nanomaterial preparation and biomedical application, and specifically relates to a preparation method and application of a pyrogallic acid-zinc (PA-Zn) multifunctional nanomaterial. The method comprises dissolving pyrogallic acid and zinc acetate in ultrapure water, and adjusting the pH value to 8.0; pouring the mixed solution into a reactor for a solvothermal reaction; and after the reaction, cooling the mixture to room temperature, centrifuging, washing, and drying the mixture to obtain the pyrogallic acid-zinc multifunctional nanomaterial. The material has excellent superoxide dismutase-like and catalase-like activities and the ability to scavenge other free radicals. The preparation method is simple, preparation equipment is readily available, the preparation process is simple, the treatment effect of spinal cord injury is good, the material is harmless to the human body, the entire process is green and pollution-free, the application environment is friendly, and the market prospects are broad.

Description

Technical field:

[0001] The present invention belongs to the technical field of nanomaterial preparation and biomedical applications, and relates to a method for preparing and using a multifunctional nanomaterial with antioxidant and anti-inflammatory properties. Specifically, it relates to a method for preparing and using a multifunctional pyrogallic acid-zinc (PA-Zn) nanomaterial. The PA-Zn nanomaterial is prepared via a one-pot hydrothermal method. The PA-Zn nanomaterial exhibits superoxide dismutase- and catalase-like activities, effectively scavenging a broad spectrum of free radicals, and can be used to treat spinal cord injuries. Background technology:

[0002] Spinal cord injury (SCI) is a devastating disease. When the central nervous system is severely damaged, most patients lose their ability to move and care for themselves, placing a significant burden on their families and society. Regulating reactive oxygen species (ROS) levels is a potential therapeutic strategy that can significantly reduce the adverse effects associated with SCI. However, currently used clinical treatments are associated with severe side effects, such as gastric bleeding and intestinal spasms. Drugs or methods for treating inflammation caused by SCI remain to be developed.

[0003] Nanozymes have recently gained significant interest in biomedical therapeutics due to their exemplary ROS-modulating catalytic mechanisms, as well as their unique advantages of low cost, high stability, tunable catalytic activity, and ease of large-scale production. Currently, a variety of nanozymes with catalase (CAT) and / or superoxide dismutase (SOD) moieties, such as gold, ceria, and redox polymers, have been developed, enabling novel approaches to scavenge ROS in chemical or biological systems. A series of studies on superoxide dismutase (SOD) have demonstrated that the activity of SOD is significantly enhanced by the coordination and conformational relationship between the metal and the ligand. SOD has two subunits, each containing a zinc ion in its active site. The zinc ion is not only the structural centerpiece of the enzyme but also a crucial component in catalyzing SOD's high catalytic activity. Analysis of the SOD structure revealed that the zinc ion is primarily bound to the side chain of the histidine residue in a tetracoordinate manner. Therefore, mimicking the coordination structure between the zinc ion and the SOD ligand could be a promising approach to developing nanozymes with ROS-scavenging capabilities.

[0004] Pyrogallol, also known as pyrogallol, is a polysubstituted aromatic compound that can act as a metal chelating agent. Currently, there is limited research on the reaction products of pyrogallol with metals, and methods for preparing pyrogallol-zinc (PA-Zn) nanomaterials with a structure similar to superoxide dismutase have not yet been reported. Summary of the invention:

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a preparation method and application of a pyrogallate-zinc (PA-Zn) multifunctional nanomaterial. The pyrogallate-zinc (PA-Zn) multifunctional nanomaterial prepared by a one-pot hydrothermal method has the ability to efficiently scavenge a broad spectrum of free radicals and can effectively and quickly treat spinal cord injury.

[0006] To achieve the above objectives, the present invention provides a method for preparing a multifunctional nanomaterial of pyrogallic acid-zinc (PA-Zn) capable of reducing inflammation, the specific steps comprising:

[0007] Pyrogallic acid and zinc salt are dissolved in ultrapure water and stirred at room temperature until completely dissolved to obtain a mixed solution; the pH value of the mixed solution is adjusted to 8.0 with a KOH aqueous solution; the pH-adjusted mixed solution is poured into a reactor and then subjected to a solvothermal reaction; after heating, the reactor is naturally cooled to room temperature; the reaction solution is centrifuged to obtain a precipitate, which is washed several times with ultrapure water and anhydrous ethanol, and finally dried in a vacuum oven at 60°C to obtain a pyrogallic acid-zinc (PA-Zn) multifunctional nanomaterial.

[0008] The solvent thermal reaction temperature is 60-240° C. and the time is 8-48 hours.

[0009] The molar concentration ratio of pyrogallic acid to zinc salt in the mixed solution is 1:1 to 3:1.

[0010] The zinc salt is zinc acetate, zinc nitrate or zinc sulfate; the zinc acetate is zinc acetate dihydrate.

[0011] The structure of the pyrogallic acid-zinc (PA-Zn) multifunctional nanomaterial is similar to that of superoxide dismutase. Zinc, as a metal center, is coupled with pyrogallic acid through a Zn-OC bridge to form a four-coordinate structure centered on zinc ions.

[0012] The present invention also provides the use of the pyrogallic acid-zinc (PA-Zn) multifunctional nanomaterial in the preparation of a drug for treating aseptic inflammatory diseases. The pyrogallic acid-zinc nanomaterial can resist inflammation in aseptic inflammatory diseases and promote nerve function recovery; the aseptic inflammatory disease is spinal cord injury.

[0013] The multifunctional nanomaterial of zinc pyrogallate (PA-Zn) of the present invention has a strong anti-inflammatory ability both in vivo and in vitro, and can also promote the polarization of M1 cells to M2 cells.

[0014] The multifunctional nanometer material of zinc pyrogallate (PA-Zn) involved in the present invention is synthesized by simulating the structure of natural SOD and has the same high-efficiency broad-spectrum free radical scavenging ability as natural SOD enzyme.

[0015] The zinc pyrogallate (PA-Zn) nanomaterial has excellent superoxide dismutase- and catalase-like activities and is highly effective in scavenging a broad spectrum of free radicals. It can provide anti-inflammatory effects in sterile inflammation and effectively and rapidly treat spinal cord injuries. Therefore, when sterile inflammation occurs, the use of the PA-Zn nanomaterial can quickly and efficiently provide anti-inflammatory treatment, achieving the goal of effective treatment. Nanomedicines for disease treatment offer advantages such as low cost, low energy consumption, environmental friendliness, and sustainable development. Therefore, utilizing the anti-inflammatory properties of zinc pyrogallate (PA-Zn) nanomaterials is a highly significant and promising approach.

[0016] The present invention has developed a pyrogallate-zinc (PA-Zn) nanozyme with a structure similar to superoxide dismutase. Zinc, acting as a metal center, is coupled to pyrogallate via a Zn-OC bridge, forming a tetracoordinate structure centered on the zinc ion. The pyrogallate-zinc (PA-Zn) nanomaterial possesses enzyme-like activities similar to superoxide dismutase (SOD) and catalase (CAT), enabling anti-inflammatory treatment of aseptic inflammation of the injured spinal cord. This research has developed a nanomaterial with anti-inflammatory properties in aseptic inflammatory traumatic diseases that can be effectively used to treat spinal cord injuries, providing momentum for the development of nanomedicines.

[0017] Compared with the prior art, the pyrogallic acid-zinc (PA-Zn) nanomaterial synthesized by using pyrogallic acid and zinc acetate as precursors has the advantages of high catalytic activity, long-term storage, high tolerance to harsh environments, high stability, and adjustable catalytic activity; it has excellent superoxide dismutase-like and catalase-like activities and the ability to scavenge other free radicals; the preparation method is simple, the preparation equipment is readily available, the preparation process is simple, the treatment effect of spinal cord injury is good and it is harmless to the human body; the entire process is green and pollution-free, the application environment is friendly, and the market prospects are broad. Description of the drawings:

[0018] Figure 1 Schematic diagram of the synthesis (a) and treatment mechanism (b) of the pyrogallic acid-zinc (PA-Zn) nanomaterial involved in the present invention.

[0019] Figure 2 The morphology and elemental characterization diagrams of the pyrogallate-zinc (PA-Zn) nanomaterial involved in the present invention are shown, wherein a is a scanning electron microscope image (SEM), b is a transmission electron microscope image (TEM), c is an elemental distribution diagram (EDS), d is a nuclear magnetic resonance spectrum (NMR), e is a Fourier transform infrared spectrum (FTIR), and fg are X-ray photoelectron spectroscopy (XPS).

[0020] Figure 3Characterization diagrams of the free radical scavenging ability (a), catalase-like (b), and superoxide dismutase-like activity (c) of the zinc pyrogallate (PA-Zn) nanomaterial involved in the present invention.

[0021] Figure 4 Characterization diagrams of the in vitro free radical scavenging (a) and ROS quantification (b) cell polarization (c) abilities of the pyrogallate zinc (PA-Zn) nanomaterials of the present invention, as well as the quantification (d) of the representative marker of M2 cells (Arg-1).

[0022] Figure 5 Schematic diagram of the results of characterizing the in vivo anti-inflammatory ability of the zinc pyrogallate (PA-Zn) nanomaterials involved in the present invention, i.e., PA-Zn / gel inhibited the inflammatory response 14 days after spinal cord injury treatment: immunofluorescence images of CD11b and IL-1β staining in the injured spinal cords of mice in the Sham, SCI, Gel and PA-Zn / Gel groups (a), representative analysis of CD11b (b), IL-1β (c) and CD11b / IL-1β (d) positive cells in mice in the Sham, SCI, Gel and PA-Zn / Gel groups.

[0023] Figure 6 Schematic diagram showing the therapeutic effects of zinc pyrogallate (PA-Zn) nanomaterials on a mouse model of spinal cord injury. PA-Zn / Gel promotes functional recovery after SCI. Images (a) and semi-quantitative analysis (b) of surviving neurons in the sham, SCI, Gel, and PA-Zn / Gel groups of mice. Semi-quantitative analysis (c) of white matter areas in the sham, SCI, Gel, and PA-Zn / Gel groups of mice. Footprint analysis (d): step length (e) and step width (f) for the SCI, Gel, and PA-Zn / Gel groups.

[0024] Figure 7 This invention relates to in vivo biocompatibility experiments of zinc pyrogallate (PA-Zn) nanomaterials. Images of major organs (heart, liver, spleen, and lungs) of mice treated with PA-Zn / Gel stained by H&E staining 2 weeks after spinal cord injury (a). Analysis of AST, ALT (b), BUN, and CRE (c) in normal mice and mice treated with PA-Zn / Gel. Analysis of blood parameters in normal mice and mice treated with PA-Zn / Gel 2 weeks after spinal cord injury (dg). Specific implementation method:

[0025] The present invention will be further described below through examples with reference to the accompanying drawings.

[0026] Example 1:

[0027] This embodiment relates to a method for preparing a multifunctional pyrogallic acid-zinc (PA-Zn) nanomaterial, and the specific preparation steps are as follows:

[0028] (1) Accurately weigh 1.01 g of pyrogallic acid and 0.878 g of zinc acetate dihydrate using an electronic balance, place them in a 50 ml beaker, add 40 ml of ultrapure water, and stir thoroughly to disperse them evenly;

[0029] (2) The stirred clarified mixed solution was adjusted to pH 8.0 using a 5 mol / L KOH aqueous solution and then transferred to a 100 ml reactor;

[0030] (3) Place the reactor in an oven, heat it at 120°C for 12 hours, and then cool it to room temperature;

[0031] (4) The sample was taken out and centrifuged at 8000 rpm for 10 minutes. The precipitate was collected and washed with ethanol and distilled water several times, and dried in a vacuum oven at 60°C to obtain pyrogallic acid-zinc (PA-Zn) nanomaterials.

[0032] In this example, scanning electron microscopy (SEM), transmission electron microscopy (TEM), elemental distribution map (EDS), nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to characterize the morphology and elemental characteristics of pyrogallic acid-zinc (PA-Zn). Figure 2-Figure 4 shown.

[0033] from Figure 2 The SEM image shows that the pyrogallate zinc (PA-Zn) nanomaterial is in the form of flakes with a thickness of about 10 nm. Figure 2 The TEM image of b shows that some small protrusions and cracks can be seen on the surface of the zinc pyrogallate (PA-Zn) nanomaterial. These protrusions and cracks can make the atoms with catalytic activity more thoroughly exposed, and can also promote the binding of the zinc pyrogallate (PA-Zn) nanomaterial with the substrate, thereby making the zinc pyrogallate (PA-Zn) nanomaterial have higher catalytic activity. EDS can be used to show the uniform distribution of C, O and Zn in the zinc pyrogallate (PA-Zn) nanomaterial nanozyme ( Figure 2 c), which preliminarily confirmed the successful preparation of pyrogallate zinc (PA-Zn) nanomaterials.

[0034] from Figure 2 The NMR, FTIR and XPS patterns of dg indicate that the pyrogallate zinc (PA-Zn) nanomaterials were synthesized according to the structure of the simulated SOD enzyme.

[0035] This example uses the classic ABTS free radical assay to evaluate the free radical scavenging ability of PA-Zn. Figure 3 As shown in a, 0.5 μg / mL of zinc pyrogallate (PA-Zn) nanomaterials can scavenge about 60% of free radicals. When the concentration of zinc pyrogallate (PA-Zn) nanomaterials is 2 μg / mL, almost all free radicals have been scavenged, which shows that zinc pyrogallate (PA-Zn) nanomaterials have super strong scavenging ability for ABTS free radicals. In order to explore the scavenging activity of zinc pyrogallate (PA-Zn) nanomaterials on H2O2, EuTc was used as a probe to react with H2O2 to generate EuTc-HP. It is known that the fluorescence emission peak of EuTc-HP is around 620 nm (λex = 405 nm). The decomposition of H2O2 will terminate the formation of EuTc-HP, resulting in a decrease in fluorescence intensity. Therefore, the H2O2 concentration can be intuitively observed by the change in the EuTc fluorescence emission peak. As shown in Figure 3 As shown in b, 50 μg / mL PA-Zn can decompose about half of H2O2. In addition, the effect of PA-Zn on O2 ·- In simple terms, riboflavin, methionine and NBT reacted with each other under ultraviolet irradiation, and a strong absorption peak was generated at 600nm, which represented the generation of high concentration of superoxide. After PA-Zn nanozyme was added to the system for reaction, the absorption peak was significantly weakened. Figure 3 c shows that 50 μg / mL PA-Zn nanozyme can reduce the absorption peak by more than 60%, which shows that zinc pyrogallate (PA-Zn) nanomaterials can effectively remove O2 ·- , namely, pyrogallate zinc (PA-Zn) nanomaterials have ultra-high superoxide dismutase-like activity. In summary, this nanomaterial has the ability to efficiently scavenge a broad spectrum of free radicals and can effectively scavenge a variety of free radicals.

[0036] The present invention uses a series of free radical scavenging experiments and other methods to characterize the in vitro anti-inflammatory ability of the prepared zinc pyrogallate (PA-Zn) nanomaterials. The specific experimental method is a ROS kit fluorescence staining experiment. The free radical is ROS. The cells used are RAW264.7 cells. They are divided into a control group, an H2O2 group, and a PA-Zn group. The control group is not treated; the H2O2 group treats the cells with 250μM H2O2; the PA-Zn group treats the cells with 250μM H2O2 and then PA-Zn. The results are as follows: Figure 4 In the figure, DAPI is a live cell fluorescent dye, ROS is a reactive oxygen species dye, and Merge is a combination of the two fluorescent staining images.

[0037] from Figure 4As can be seen from the in vitro free radical scavenging and cell polarization ability characterization diagram, further ROS fluorescence staining experiments found that the ROS staining ratio increased significantly in the 250μM H2O2 group, while it decreased significantly in the PA-Zn group. Analysis of the ROS staining data showed that cells treated with H2O2 showed a significant upregulation of ROS (a 5.4-fold change compared to the untreated control), while the addition of PA-Zn effectively reversed this effect, indicating that zinc pyrogallate (PA-Zn) still has a strong free radical scavenging ability in vitro ( Figure 4 ab). To further investigate the effect of PA-Zn on macrophage polarization in vitro, immunofluorescence staining was used to detect IL-1β (M1 marker) and Arg-1 (M2 marker). Figure 4 As shown in Figures c-d, compared with RAW264.7 cells treated with H2O2, PA-Zn-treated cells showed significantly lower IL-1β and higher Arg-1 expression. These results indicate that PA-Zn treatment significantly promotes the polarization of M1 to M2 cells in vitro, demonstrating that PA-Zn nanomaterials have anti-inflammatory properties in vitro.

[0038] Example 2:

[0039] This embodiment relates to a method for preparing a multifunctional pyrogallic acid-zinc (PA-Zn) nanomaterial. Unlike Example 1, the molar concentration ratio of pyrogallic acid to zinc acetate dihydrate in the mixed solution is 1:1; the solvent thermal reaction temperature is 60°C, and the reaction time is 48 hours.

[0040] Example 3:

[0041] This embodiment relates to a method for preparing a multifunctional pyrogallic acid-zinc (PA-Zn) nanomaterial. Unlike Example 1, the molar concentration ratio of pyrogallic acid to zinc acetate dihydrate in the mixed solution is 3:1; the solvent thermal reaction temperature is 240°C, and the reaction time is 8 hours.

[0042] Example 4:

[0043] This example involves an experiment investigating the efficacy of zinc pyrogallate (PA-Zn) nanomaterials in a mouse model of traumatic spinal cord injury. The PA-Zn nanomaterial solution prepared in Example 1 was applied to mice with spinal cord injury. Sixteen C57 / BL6 mice underwent complete spinal cord transection (5 mm) at the T10 level using a rotary cutting needle modified from a syringe needle. The mice were divided into four groups: a sham group, a spinal cord injury (SCI) group, a gel (Gel) group, and a PA-Zn / Gel group. The sham group underwent laminectomy alone; the SCI group underwent complete spinal cord transection. The gel group received hydrogel treatment at the injury site, while the PA-Zn / Gel group received PA-Zn-loaded hydrogel (100 μL, 20 μg / mL). Bladder massage was performed twice daily until normal bladder function was achieved. On the 14th day after spinal cord injury, the CD11b-positive cells, IL-1β-positive cells and CD11b / IL-1β-double-positive cells of mice were detected. Figure 5 Quantification of CD11b-positive cells, IL-1β-positive cells, and CD11b / IL-1β double-positive cells showed that PA-Zn / Gel treatment significantly reduced cell apoptosis. These results demonstrate the anti-inflammatory effect of PA-Zn / Gel in inhibiting M1 macrophage activation and reducing inflammatory responses after spinal cord injury.

[0044] After the spinal cord injured mice were treated with PA-Zn / Gel, the histological changes were observed by Nissl staining. Figure 6 As shown. Nissl staining analysis showed that the number of ventral horn neurons increased significantly after PA-Zn / Gel treatment compared with that in spinal cord injured mice. In addition, after PA-Zn / Gel treatment, the area of ​​the cavity near the injured area in spinal cord injured mice was significantly reduced on the 14th day after spinal cord injury. These data indicate that PA-Zn / Gel administration effectively improved the white matter area of ​​the brain after spinal cord injury. In addition, footprint analysis was also used in this experiment to evaluate the rehabilitation effect of PA-Zn / Gel treatment on spinal cord injured mice. Figure 6 In Figure d, it can be clearly observed that the SCI mice had difficulty standing, with noticeable dislocation of their hind limbs. In the Gel-treated group, mice occasionally stood on their hind limbs. However, the hind limbs of mice treated with PA-Zn / Gel were able to contact the ground, and gait analysis showed a more stable gait than those in the SCI and Gel groups. Further analysis revealed that the stride length and stride width of the SCI mice were significantly reduced, which was attributed to the improvement of these results by PA-Zn / Gel treatment. Based on these results, we found that PA-Zn / Gel treatment effectively improved the motor dysfunction caused by SCI.

[0045] Example 5:

[0046] This example is an application experiment of zinc pyrogallate (PA-Zn) nanomaterials in vivo biocompatibility. To evaluate the possible adverse reactions of the PA-Zn / Gel group treatment in Example 2, mice in the healthy normal group (normal C57 / BL6 mice) and the PA-Zn / Gel group in Example 4 were sacrificed after 2 weeks of treatment. Blood samples and major organ tissues were collected from both groups of mice and blood tests and H&E staining were performed. The results are shown in Figure 2. Figure 7 As shown. Figure 7 As can be seen, after two weeks of PA-Zn / Gel treatment, no necrosis, congestion, or hemorrhage was observed in major organs (heart, spleen, liver, kidneys, and lungs). Furthermore, these blood chemistry data showed no difference in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) concentrations between the PA-Zn / Gel group and the healthy control group, indicating that PA-Zn / Gel has negligible toxicity to liver and kidney function in vivo. Furthermore, blood test results in the PA-Zn / Gel-treated group were similar to those in the healthy control group. These results demonstrate that PA-Zn / Gel is non-toxic. These results confirm the good biocompatibility of PA-Zn / Gel in vivo.

Claims

1. A method for preparing a pyrogallic acid-zinc multifunctional nanomaterial, characterized in that: The specific steps are as follows: dissolving pyrogallic acid and zinc salt in ultrapure water, adjusting the pH value to 8.0, pouring into a reactor for solvothermal reaction at a temperature of 60-240°C for 8-48 hours; after the reaction is completed, cooling to room temperature, centrifuging, washing, and drying to obtain pyrogallic acid-zinc multifunctional nanomaterials; The pyrogallic acid-zinc multifunctional nanomaterial is in the form of a sheet and has a structure in which zinc, as a metal center, is coupled to pyrogallic acid via a Zn-OC bridge to form a four-coordinate structure centered on the zinc ion. The pyrogallic acid-zinc multifunctional nanomaterial has superoxide dismutase-like and catalase-like activities. The molar concentration ratio of the pyrogallic acid to the zinc salt is 1:1 to 3:1; The zinc salt is zinc acetate dihydrate.

2. A pyrogallic acid-zinc multifunctional nanomaterial prepared by the preparation method according to claim 1, characterized in that: The structure of the nanomaterial is as follows: zinc as the metal center is coupled with pyrogallic acid through a Zn-OC bridge to form a four-coordinate structure centered on the zinc ion.

3. The use of the pyrogallic acid-zinc multifunctional nanomaterial as claimed in claim 2 in the preparation of a drug for treating spinal cord injury, characterized in that: Pyrogallol-zinc nanomaterials can fight inflammation and promote neurological function recovery in spinal cord injury.

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