Novel nano-drug Res (at) LDH as well as preparation method and application thereof

Abstract

The invention discloses a novel nano-drug Res (at) LDH and a preparation method and application thereof.Germanium-doped LDH powder and resveratrol are combined to obtain a nanoscale particle mixed material Res (at) LDH, electron paramagnetic resonance (EPR) tests show that both the germanium-doped LDH and the traditional drug resveratrol show excellent reactive oxygen species (ROS) removal characteristics, and the Res (at) LDH can be used for preparing a novel nano-drug Res (at) LDH. The nano-drug Res (at) LDH formed by combining the two has remarkable synergistic ROS removal efficiency which can reach 90%. Meanwhile, in-vivo biological distribution experiments show that the novel nano-drug Res (at) LDH has excellent blood-brain barrier permeability, enhances accumulation in brain tissues, improves neurological dysfunction, reduces infarct volume, shows strong antioxidant activity, and inhibits release of pro-inflammatory cytokines.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a novel nano drug Res@LDH, a preparation method thereof, and an application thereof in ischemic stroke. Background Art

[0002] Ischemic stroke refers to a series of clinical symptoms caused by local brain tissue hypoxia and ischemia due to occlusion of cerebral blood vessels. Ischemic stroke accounts for about 80% of all stroke cases worldwide and is one of the leading causes of death and disability worldwide, with more than 10 million new cases each year. Effective treatment strategies such as thrombolysis and thrombectomy, aimed at achieving early vascular recanalization, have been validated. Intravenous recombinant tissue plasminogen activator (rt-PA), used alone or in combination with various mechanical thrombectomy devices, has been approved by the US Food and Drug Administration (FDA) and has become the cornerstone of ischemic stroke treatment. However, the time window for effective thrombolytic therapy is only 4.5-6 hours, which limits its accessibility to a small number of patients. Therefore, there is an urgent need to explore innovative therapies for ischemic stroke. Many studies have emphasized that oxidative stress is the main factor in ischemic stroke injury. Whether or not patients receive thrombolytic therapy to restore cerebral blood flow, they are still susceptible to ischemic stroke and reperfusion injury. This susceptibility stems from the excessive production of reactive oxygen species (ROS) in the ischemic penumbra, including hydroxyl radicals (•OH), superoxide anions (O2−), hydrogen peroxide (H2O2), and nitric oxide (NO). In addition, excessive ROS generation triggers a series of cell signaling pathways, exacerbating blood-brain barrier permeability damage, brain edema, inflammation, and ultimately leading to neuronal cell death and brain tissue damage. Therefore, clearing ROS has become a key treatment for ischemic stroke damage.

[0003] Nanotechnology has shown great promise in the treatment of ischemic stroke due to its ability to combat ROS and tiny size. Nanomaterials such as cerium oxide (CeO2), manganese, carbon, and copper oxide (CuO) nanoparticles have shown potential in this regard. In particular, layered double hydroxides (LDH), also known as hydrotalcites, are a class of special layered inorganic crystalline materials composed of two or more metal elements. LDH not only has a small size but also has excellent properties, including coordinated structure, biocompatibility, scalable production, and economical preparation. LDH has shown antioxidant activity by incorporating appropriate metal ions into its structure, such as copper, manganese, ruthenium, gadolinium, and iron. Previous studies have reported that copper-incorporated LDH can alleviate oxidative stress, while atorvastatin-ferritin gadolinium LDH has been shown to play an important role in ischemic stroke by inhibiting oxidative stress. Despite significant progress in this field, challenges remain for LDH-based nanomaterials in terms of structural and functional stability.

[0004] Resveratrol (Res) is a natural polyphenol compound widely found in grapes and red wine, which plays an important neuroprotective role in ischemic stroke by inhibiting oxidative stress and inflammation. However, its application is greatly limited due to its low water solubility, poor bioavailability and the challenge of crossing the blood-brain barrier (BBB) ​​to reach ischemic brain tissue. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a novel nanomedicine Res@LDH and a preparation method thereof and its application in ischemic stroke, so as to solve the technical problems that resveratrol is limited in clinical application due to low water solubility, poor bioavailability and the existence of blood-brain barrier.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses for the first time a novel nano drug Res@LDH, which is prepared by dispersing germanium-doped LDH powder and resveratrol in water, fully stirring them in a light-proof condition, and collecting the product by centrifugation.

[0007] LDH powder is a type of anionic clay mineral with a layered structure. Its layers are composed of two or more metal hydroxides, and there are exchangeable anions and water molecules between the layers.

[0008] Since the germanium-doped LDH powder combines the layered structure of LDH and the biological activity of germanium, the germanium-doped LDH powder may show potential application value in the biomedical field, such as drug carriers, biosensors, etc.

[0009] Preferably, the novel nanodrug Res@LDH has a layered structure, and Res is distributed in the double-layer LDH; Preferably, the particle size of the novel nanodrug Res@LDH is 15-30 nm.

[0010] Further preferably, the particle size of the novel nanodrug Res@LDH is about 20 nm.

[0011] The present invention also discloses a method for preparing the novel nano drug Res@LDH, wherein the particle size of the novel nano drug Res@LDH is: The germanium-doped LDH powder and resveratrol were dispersed in water at a molar ratio of 1:(100-150), stirred thoroughly in the dark, and then centrifuged at 8000-10000 rpm for 8-15 min. The product was collected to obtain the new nanodrug Res@LDH.

[0012] Preferably, the stirring treatment time is at least 24 hours.

[0013] Preferably, the method for preparing germanium-doped LDH powder comprises the following steps: 1) Add a mixed aqueous solution containing Mg(NO3)2·6H2O, Al(NO3)3·9H2O and Ge(NO3)3·6H2O to an aqueous solution containing NaNO3, and stir evenly at room temperature to prepare a metal salt solution; 2) At 80° C., the pH value of the metal salt solution is adjusted to 10-12, and the reaction is continued with stirring for 4-8 hours. The product is then collected by centrifugation and washing to obtain germanium-doped LDH powder.

[0014] Further preferably, in step 1), Mg(NO3)2·6H2O, Al(NO3)3·9H2O and Ge(NO3)3·6H2O are mixed in a molar ratio of 3:1:0.05.

[0015] Further preferably, in step 2), the solution used for adjusting the pH value is a NaOH solution, and during the treatment, 35 mL of a 0.2-0.3 mol / L NaOH solution is slowly dripped into the metal salt solution while continuously stirring.

[0016] More preferably, in step 2), the centrifugation is performed at a speed of 8000-10000 rpm for 8-12 min.

[0017] Further preferably, in step 2), washing is performed by washing with water three times, and the germanium-doped LDH powder product is collected with ethanol.

[0018] The present invention also discloses for the first time the application of the novel nano drug Res@LDH in the preparation of drugs for treating ischemic stroke.

[0019] Preferably, the drug is a drug that reduces oxidative stress and inflammation caused by ischemic stroke damage by reducing the activation of microglia and astrocytes.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a novel nano drug Res@LDH, which combines germanium-doped LDH powder and resveratrol to obtain a mixed material Res@LDH of nano-scale particles. Electron paramagnetic resonance (EPR) tests show that both germanium-doped LDH and traditional drug resveratrol exhibit excellent reactive oxygen species (ROS) scavenging properties, and the nano drug Res@LDH formed by the combination of the two has a significant synergistic ROS scavenging efficiency of up to 90%. At the same time, in vivo biodistribution experiments show that the novel nano drug Res@LDH has excellent blood-brain barrier permeability, enhances accumulation in brain tissue, improves neurological deficits, reduces infarct volume, exhibits strong antioxidant activity, and inhibits the release of pro-inflammatory cytokines.

[0021] In particular, in the middle cerebral artery occlusion (MCAO) mice and H2O2-induced HT22 cell toxicity models, the Res@LDH prepared by the present invention alleviated the oxidative stress and inflammation caused by ischemic stroke injury by reducing the activation of microglia and astrocytes during treatment. Therefore, the novel nanodrug Res@LDH prepared by the present invention using resveratrol and germanium doped LDH highlights its potential as a therapeutic intervention for ischemic stroke, provides a promising neuroprotective strategy for the treatment of ischemic reperfusion injury, and provides a new approach for the treatment of ischemic stroke, a disabling disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation of Res@LDH; Figure 2 The XRD patterns of Ge-LDH and Res@LDH powders measured by XRD according to the embodiment of the present invention; Figure 3 This is an electron microscope photo of the Res@LDH nanomaterial product prepared by the present invention; Figure 4 A test diagram of the ROS scavenging ability of the Res@LDH nanomaterial provided in an embodiment of the present invention; Figure 5 The ROS scavenging effect of Res@LDH in vitro; Figure 6 The neuroprotective effect of Res@LDH treatment on ischemic stroke; Figure 7 Res@LDH treatment alleviated oxidative stress in the ischemic penumbra; Figure 8 Res@LDH treatment alleviated inflammation mediated by astrocyte and microglia activation; Fig. 9 Diagram of the mechanism by which Res@LDH alleviates ischemic stroke. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings: 1. Preparation of the novel nanodrug Res@LDH See also Figure 1 , which is a preparation method of the novel nano drug Res@LDH provided in this embodiment, comprising the following steps: Step 1: Preparation of Ge-LDH nanomaterials A mixed aqueous solution containing Mg(NO3)2·6H2O (0.03 M), Al(NO3)3·9H2O (0.01 M) and Ge(NO3)3·6H2O (0.2 g) was added dropwise to an aqueous solution containing NaNO3 (0.4 g) while stirring continuously at room temperature. Subsequently, 35 mL of 0.25 M NaOH was slowly added dropwise to the metal salt solution at 80°C while stirring continuously. After 6 hours of reaction, the LDH product was collected by centrifugation at 10,000 rpm, washed three times with water, and then with ethanol. Figure 1 It can be seen that the sharp diffraction peaks verify that the Ge-LDH powder is of high purity.

[0026] Step 2: Prepare new nanodrug Res@LDH.

[0027] The prepared Ge-LDH powder and commercial resveratrol Res were dispersed in deionized water at a molar ratio of 1:(100-150). After stirring in the dark for 24 h, the Res@LDH product was collected by centrifugation at 10000 rpm for 10 min.

[0028] 2. Animal Experiments Adult male C57BL / 6 mice aged 8-10 weeks were obtained from Vital River (Beijing, China). The mice were randomly assigned to one of the following four groups: (A) sham operation group (Sham), (B) MCAO-R injury plus saline group (MCAO), (C) MCAO-R injury plus LDH treatment group, (D) MCAO-R injury plus Res@LDH treatment group. The mice were killed 24 hours after surgery.

[0029] 3. Cell Culture HT22 cells were cultured in DMEM containing 10% fetal bovine serum, streptomycin, and penicillin at 37°C in a 5% CO2 environment. HT22 cells were treated with H2O2-induced cytotoxicity for 4 h. In addition, HT22 cells were exposed to different doses of LDH and Res@LDH for 24 h and then collected for subsequent analysis.

[0030] 4. Middle Cerebral Artery Occlusion and Reperfusion (MCAO-R) Mice were subjected to MCAO-R injury by inserting silk thread. Briefly, mice were anesthetized with 2% pentobarbital and the body temperature was maintained at 37±0.5°C. Sutures were inserted from the left common carotid artery to the origin of the middle cerebral artery. After 1 hour, reperfusion was started by removing the silk thread. Subsequently, 20 mg / kg of LDH or Res@LDH was injected through the tail vein.

[0031] 5. Determination of blood-brain barrier (BBB) ​​permeability After reperfusion, 4 ml / kg of 2% Evans blue was injected into the tail vein. Mice were sacrificed 24 h after reperfusion. In the Evans blue extravasation experiment, coronal sections were cut into 1 mm slices and photographed. For quantitative measurements, the ischemic hemispheres were homogenized in N,N-dimethylformamide, followed by incubation and centrifugation. The supernatant was analyzed spectrophotometrically at 620 nm to calculate changes in BBB permeability.

[0032] 6. Cell Counting Kit-8 (CCK-8) Cell Viability Assay The viability of HT22 cells in different groups was evaluated using a CCK-8 kit. Briefly, 10 μl of CCK-8 solution was added to 100 μL of culture medium in each well of a 96-well plate and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell activity = (experimental treatment group - blank group) / (negative control group - blank group) × 100%.

[0033] 7. Dihydroethidium (DHE) staining DHE staining was used to detect ROS levels. Frozen coronal sections of mouse brains and HT22 cells on coverslips were incubated with the fluorescent dye DHE for 30 min in a humidified chamber at 37°C and washed three times with PBS. The sections were then observed under an optical microscope.

[0034] 8. Immunofluorescence (IF) staining The mice were anesthetized and the brains were fixed by cardiac perfusion with 0.9% cold heparin saline and 4% paraformaldehyde. Subsequently, the brains were cut into 5 μm thick sections. These sections were used for DHE, HE, Nissl, TUNEL and IF staining.

[0035] For HE, Nissl, and TUNEL staining, sections were processed using HE, Nissl staining kits, and in situ cell death detection kits, respectively, according to the manufacturer's instructions. For IF staining, the primary antibodies used included mouse anti-GFAP antibody (1:200, Cell Signaling Technology, USA) and rabbit anti-IBA1 antibody (1:500, Wako, Japan). After primary antibody incubation, sections were observed under a fluorescence microscope.

[0036] 9. Statistical Analysis Statistical calculations were performed using GraphPad Prism 8.0. One-way analysis of variance (ANOVA) and Tukey's post hoc test were performed for comparisons between multiple groups. Garcia test results are presented as median and interquartile range and analyzed using Kruskal-Wallis test, while other values ​​are presented as mean ± SD. p < 0.05 was considered statistically significant.

[0037] Experimental results: 1. Synthesis and characterization of Res@LDH Nanohybrid Res@LDH with a size up to 20 nm was successfully synthesized through an intercalation process using nanosized LDH as a precursor. Figure 1 The schematic diagram of the preparation of Res@LDH is shown. The structure of the drug shows layered characteristics. Figure 2The X-ray diffraction data showed that the reflections of the LDH precursor were completely replaced by a new set of reflections that were systematically shifted to lower angles, a change that indicated that new molecular layers were introduced into the LDH layered structure. Figure 3 Electron microscope images of Res@LDH nanomaterials are shown.

[0038] 2. ROS scavenging synergistic effect of Res@LDH Given the goal of developing a therapeutic agent to mitigate ischemia-reperfusion injury, the ROS scavenging ability of the synthesized samples must be thoroughly investigated. The present invention uses electron paramagnetic resonance (EPR) spectroscopy for quantitative analysis, employing a •OH scavenging assay. In this assay, Fe 2+ / H2O2 system to generate •OH radicals and use DMPO (55'-dimethylpyrroline 1-oxide) as a scavenger (e.g. Figure 4 (shown in A).

[0039] In the absence of free radical scavengers, the Fenton reaction produced a strong EPR signal of DMPO-OH adduct ( Figure 4 (B and C). After LDH was added to the system, the EPR signal intensity of the DMPO-OH adduct decreased by about 50%, indicating the ROS scavenging ability of LDH. It is worth noting that typical LDH materials lack this ability, indicating that the incorporation of Ge ions into the lattice may have conferred this ability.

[0040] Res showed a significant reduction in EPR signal, consistent with its known ROS scavenging ability. In particular, the EPR signal of Res@LDH was very weak, indicating that the assembled nanohybrid had excellent ROS (•OH) scavenging properties. These results suggest that LDH and Res have the ability to scavenge ROS, especially hydroxyl radicals (•OH), respectively, and the interaction between Res and Ge ions in LDH nanosheets may promote the enhanced ROS scavenging activity observed for Res@LDH, with a significant synergistic enhancement.

[0041] In conclusion, the novel Res@LDH drug prepared in this invention exhibited a significant synergistic effect between the drug molecule and the nanocarrier, promoting the quenching of hydroxyl radicals (•OH) and inhibiting the generation of reactive oxygen species (ROS). This synergistic effect is of great significance in alleviating ischemia-reperfusion injury, prompting us to further explore the in vitro model.

[0042] 3. Res@LDH reduces cell damage and inhibits oxidative stress To further illustrate the excellent ROS scavenging properties of the novel Res@LDH drug prepared by the present invention, we conducted in vitro experiments in the mouse hippocampal neuronal cell line HT22 cells. First, the viability of HT22 cells after 24 h of treatment with LDH and Res@LDH at increasing doses was evaluated using CCK-8 cell viability assay. Figure 5 Figure (a) shows that the HT22 cell viability remains above 95%, indicating that LDH and Res@LDH have significant biocompatibility even at high concentrations (100 μg / mL).

[0043] Subsequently, H2O2-induced cytotoxicity assay was performed in HT22 cells, a cell model used for ischemic stroke injury, to investigate the antioxidant capacity of Res@LDH. Figure 5 As shown in (b) of Figure A, H2O2 injury reduced HT22 cell viability to 51.2%; after 24 hours of Res@LDH treatment, cell viability recovered to 62.8% even at a low concentration (10 μg / mL) and increased in a dose-dependent manner (85% at 50 μg / mL). Therefore, 50 μg / mL of Res@LDH was selected for subsequent in vitro experiments. LDH alone (50 μg / mL) also increased HT22 cell viability after H2O2 injury, highlighting the potential synergistic effect of LDH and resveratrol ( Figure 5 (c) in Figure A). In addition, the morphology of HT22 cells after 24 h of Res@LDH treatment was observed using transmission electron microscopy (TEM), showing efficient uptake and accumulation in lysosomes ( Figure 5 DHE staining was used to detect the level of ROS in HT22 cells after H2O2 injury ( Figure 5 C(a)). As expected, H2O2 injury resulted in significantly higher ROS levels than the control (saline) group, while LDH treatment (H2O2+LDH group) showed slight ROS scavenging ability ( Figure 5 C(b)). Res@LDH treatment (H2O2+Res@LDH group) showed a strong free radical scavenging effect in HT22 cells. These results indicate that Res@LDH has a strong antioxidant property, highlighting its promise as a potential therapeutic agent for the treatment of oxidative stress-induced cell damage in ischemic stroke, prompting us to test it in an in vivo model.

[0044] 4. Res@LDH reduces cerebral ischemic injury and blood-brain barrier disruption In the in vivo experiment, the neuroprotective effect of Res@LDH was studied using the MCAO-R mouse model. In order to visually observe the ischemia-reperfusion process, laser speckle imaging was used to measure cerebral blood flow (CBF). During the occlusion period, the cortical CBF value dropped to 15% of the pre-ischemia value and recovered to 70% of the baseline after reperfusion, confirming the successful establishment of the MCAO-R mouse model ( Figure 6 Middle A).

[0045] To evaluate the ability of LDH and Res@LDH to reach the brain and cross the blood-brain barrier (BBB), we studied their in vivo distribution in normal mice and MCAO-R model mice. By slightly modifying LDH and Res@LDH, nanomedicines labeled with the photosensitizer indocyanine green (ICG) were synthesized to track the distribution of these materials in the brain. Figure 6 Middle B shows that strong fluorescence signals of LDH and Res@LDH were detected in the brain of normal mice 1 hour after intravenous injection. Similarly, strong fluorescence signals were detected in the brain of both LDH and Res@LDH treatment groups 1 hour after MCAO-R injury. In MCAO-R mice, no obvious fluorescence intensity was observed in the brain of the LDH treatment group 6 hours after injection, while strong fluorescence signals were still present in the Res@LDH treatment group. These findings indicate that Res@LDH can cross the BBB and accumulate in the brains of healthy and MCAO-R-injured mice.

[0046] The neuroprotective effect of Res@LDH in ischemic stroke-injured mice was further investigated by neurological scoring and TTC staining to determine brain damage ( Figure 6 C and D). The neurological scoring criteria are shown in Table 1 below.

[0047] Table 1 Neurological scoring criteria

[0048] As expected, MCAO-R injury significantly worsened neurological scores, but LDH and Res@LDH treatment improved these scores. In addition, the neurological deficit scores of MCAO-R mice treated with Res@LDH were higher than those of LDH alone ( Figure 6 In addition, the infarct volume in the MCAO-saline group was 37.5%±3.0%, which was reduced to 28.7%±3.4% and 12.3%±1.6% after LDH and Res@LDH treatment, respectively ( Figure 6 D(a) and D(b) in Figure 5). The significantly better neuroprotective effect of Res@LDH compared with LDH may be attributed to resveratrol as the active ingredient of LDH.

[0049] Next, BBB permeability among the four groups was measured by Evans blue staining ( Figure 6 E(a) and E(b)). Evans blue staining was significantly increased in the MCAO injury group, but was reduced by LDH and Res@LDH treatment, respectively. Res@LDH treatment showed the lowest Evans blue staining after MCAO-R injury.

[0050] In addition, HE, Nissl, and TUNEL staining were performed to evaluate neuronal damage and survival in the ischemic penumbra after MCAO-R injury. HE-stained cells (HE+) had intact nucleoli, compact structures, and clear outlines ( Figure 6 Figure 5 (a) and (b) of Figure F). The proportion of HE+ cells decreased significantly after MCAO-R injury (from 98.3%±2.6% in the sham group to 47.2%±4.0%). LDH and Res@LDH treatment increased the proportion of HE+ cells to 57.5%±3.4% and 74.1%±2.2%, respectively. In addition, the proportion of Nissl+ cells decreased significantly to 52.3%±5.1% in the MCAO-saline group and recovered to 58.8%±4.5% and 68.7%±4.6% after LDH and Res@LDH treatment, respectively. Among the four groups, Res@LDH showed the highest proportion of HE+ and Nissl+ cells after MCAO-R injury. TUNEL-stained cells (TUNEL+) represent apoptotic cells. Compared with the sham group, TUNEL+ cells increased significantly to 45.8%±4.4% after MCAO-R injury. However, LDH and Res@LDH treatment reduced neuronal apoptosis to 32.2%±2.5% and 18.8%±3.6%, respectively. Res@LDH showed the lowest neuronal apoptosis among the four groups, reflecting its excellent neuroprotective efficacy. These results indicate that Res@LDH significantly alleviated neuronal damage in the ischemic penumbra.

[0051] 5. Res@LDH inhibits oxidative stress in the ischemic penumbra Due to its high oxygen metabolism and blood perfusion, ischemic stroke injury is highly susceptible to high levels of ROS-induced oxidative stress.To evaluate the ROS scavenging properties of the nanocomposites in vivo, ROS levels in the ischemic penumbra were examined after reperfusion using dihydroethidium (DHE) staining.

[0052] Compared with the sham group, the proportion of ROS-positive cells in the MCAO-saline group increased significantly ( Figure 7 However, LDH or Res@LDH treatment resulted in a decrease in the level of ROS-positive cells. In particular, the level of ROS-positive cells in the MCAO-Res@LDH group was significantly reduced compared with that in the MCAO-saline group.

[0053] To further verify the antioxidant effect of Res@LDH, we detected the activity of superoxide dismutase (SOD), an important antioxidant enzyme that protects neural tissue from oxidative stress, and the level of malondialdehyde (MDA), a byproduct of lipid peroxidation. The SOD level in the sham group was 31.0±2.7 U / mL, while it was significantly reduced to 13.5±1.5 U / mL in the MCAO model. LDH or Res@LDH treatment increased the SOD level to 17.9±4.7 U / mL and 24.6±3.6 U / mL, respectively ( Figure 7 Similarly, the MDA level in the sham group was 14.0±1.4 U / mL, while it increased significantly to 26.8±1.2 U / mL after MCAO injury. LDH treatment reduced the MDA level to 22.6±1.1 U / mL, while Res@LDH treatment significantly reduced it to 20.4±1.5 U / mL, showing the highest SOD level and the lowest MDA level among all groups ( Figure 7 (as shown in C).

[0054] These results suggest that Res@LDH treatment alleviates oxidative stress through the potential synergistic therapeutic effects of LDH and resveratrol, providing a promising new strategy for ischemic stroke treatment.

[0055] 6. Res@LDH inhibits inflammation mediated by astrocyte and microglia activation Neuroinflammation is a key mechanism in ischemic stroke injury. Therefore, we assessed the expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) 24 h after reperfusion. Figure 8 Middle A shows that the IL-1β level in the sham group was 123.8±5.3 pg / mL, while it increased significantly to 176.5±4.6 pg / mL in the MCAO-saline group. After LDH and Res@LDH treatment, the IL-1β level decreased to 143.4±10.9 pg / mL and 135.9±7.0 pg / mL, respectively ( Figure 8 Similarly, the IL-6 level in the sham group was 112.7±4.9 pg / mL, while it increased to 179.4±6.9 pg / mL in the MCAO-saline group. LDH treatment decreased its level to 154.6±15.2 pg / mL, while Res@LDH treatment significantly reduced it to 143.4±9.8 pg / mL ( Figure 8Regarding TNF-α, the level in the sham group was 690.0±13.4 pg / mL, while it increased significantly to 905.5±16.4 pg / mL after ischemic stroke injury. LDH treatment reduced its level to 838.0±17.4 pg / mL, while Res@LDH treatment further reduced it to 807.0±18.5 pg / mL ( Figure 8 (as shown in (c) in Figure A).

[0056] Our results showed that the levels of IL-1β, IL-6, and TNF-α were significantly increased after MCAO-R injury compared with the sham group. The levels of these proinflammatory cytokines decreased to varying degrees after LDH and Res@LDH treatment, and Res@LDH significantly reduced the levels of proinflammatory cytokines compared with the MCAO-saline group.

[0057] After ischemic stroke injury, microglia and astrocytes become overactivated, with large cell bodies and hypertrophic processes ( Figure 8 (a) and (b) in Figure B). Subsequently, these activated microglia and astrocytes release high levels of inflammatory factors, exacerbating neuroinflammation and ischemic stroke damage. Therefore, inhibiting the activation of microglia and astrocytes can significantly reduce ischemic stroke damage. Several studies have shown that resveratrol exerts anti-inflammatory effects in microglia and astrocytes by inhibiting proinflammatory cytokines. Therefore, the present invention also studied the role of Res@LDH in the activation of astrocytes and microglia, and measured their activation in the ischemic penumbra in the four groups. Compared with the sham operation group, the astrocytes (GFAP positive) and microglia (IBA1 positive) in the MCAO group showed high activation, manifested as short and thick cytoplasmic processes, enlarged cell bodies and hypertrophic processes ( Figure 8 However, LDH and Res@LDH treatment alleviated the activation of astrocytes and microglia caused by MCAO-R injury. In particular, Res@LDH treatment significantly alleviated the activation of astrocytes and microglia compared with the MCAO group.

[0058] These results suggest that Res@LDH effectively inhibited the activation of astrocytes and microglia during ischemic stroke injury, thereby alleviating inflammatory damage.

[0059] Conclusion In summary, the hybrid material Res@LDH with nanoscale particles (about 20 nm) was successfully synthesized through traditional chemical processes. Electron paramagnetic resonance (EPR) tests showed that both germanium-doped LDH and the traditional drug resveratrol exhibited excellent reactive oxygen species (ROS) scavenging properties. The nanohybrid therapeutic agent Res@LDH formed by their combination has a significant synergistic ROS scavenging efficiency (about 90%). In addition, in vivo biodistribution experiments showed that Res@LDH has excellent blood-brain barrier permeability and enhanced accumulation in brain tissue. In mice with middle cerebral artery occlusion (MCAO) and H2O2-induced HT22 cell toxicity models, Res@LDH treatment alleviated oxidative stress and inflammation caused by ischemic stroke injury by reducing the activation of microglia and astrocytes. Schematic diagram of Res@LDH treatment regulating ischemic stroke injury is shown in Fig. 9 As shown in the figure, Res@LDH is a new type of nanomaterial, which is composed of germanium-containing layered double hydroxide nanosheets (Ge-LDH) as a carrier and resveratrol as a neuroprotectant, showing enhanced blood-brain barrier penetration and high biocompatibility. Our experimental results show that Res@LDH can improve neurological deficits, reduce cerebral ischemic infarct volume, alleviate BBB damage, and has strong antioxidant and anti-inflammatory effects, while significantly inhibiting the activation of microglia and astrocytes. Through our innovative synthesis method, Res@LDH provides a promising therapeutic strategy for alleviating cerebral ischemia-reperfusion injury.

[0060] In summary, the present invention considers the role of germanium (Ge)-doped LDH in ischemic stroke, and the study found that germanium-doped LDH nanoparticles showed potential as ROS scavengers in alleviating ischemic stroke damage. The use of germanium-doped LDH nanomaterials may be a solution to improve the bioavailability and efficacy of resveratrol, while potentially reducing the cost of ischemic stroke treatment. Therefore, the present invention provides a new nano drug synthesized using resveratrol and Ge-LDH, providing a promising neuroprotective strategy for the treatment of ischemia-reperfusion injury. The present invention demonstrates the synergistic effect of drug molecules and nanocarriers in the new Res@LDH nano drug, highlighting its potential as a therapeutic intervention for ischemic stroke, providing a new approach for the treatment of this disabling disease.

[0061] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A novel nano drug Res@LDH, characterized in that: The method is prepared by dispersing germanium-doped LDH powder and resveratrol in water, fully stirring in a light-proof condition, and collecting the product by centrifugation.

2. The novel nano drug Res@LDH according to claim 1, characterized in that: The new nanodrug Res@LDH has a layered structure, and Res is distributed in the double-layer LDH; the particle size of the new nanodrug Res@LDH is 15~30 nm.

3. The method for preparing the novel nano drug Res@LDH according to claim 1 or 2, characterized in that: The method comprises: dispersing germanium-doped LDH powder and resveratrol in water at a molar ratio of 1:(100-150), stirring the mixture evenly in a light-proof condition, and then centrifuging the mixture at a speed of 8000-10000 rpm for 8-15 minutes, and collecting the product to obtain a novel nano drug Res@LDH.

4. The method for preparing the novel nano drug Res@LDH according to claim 3, characterized in that: The stirring treatment time is at least 24 hours.

5. The method for preparing the novel nano drug Res@LDH according to claim 3, characterized in that: The preparation method of germanium-doped LDH powder comprises the following steps: 1) Add a mixed aqueous solution containing Mg(NO3)2·6H2O, Al(NO3)3·9H2O and Ge(NO3)3·6H2O to an aqueous solution containing NaNO3, and stir evenly at room temperature to prepare a metal salt solution; 2) At 80° C., the pH value of the metal salt solution is adjusted to 10-12, and the reaction is continued with stirring for 4-8 hours. The product is then collected by centrifugation and washing to obtain germanium-doped LDH powder.

6. The method for preparing the novel nano drug Res@LDH according to claim 5, characterized in that: In step 1), Mg(NO3)2·6H2O, Al(NO3)3·9H2O and Ge(NO3)3·6H2O are mixed in a molar ratio of 3:1:0.

05.

7. The method for preparing the novel nano drug Res@LDH according to claim 5, characterized in that: In step 2), the pH value is adjusted using a NaOH solution with a concentration of 0.2-0.3 mol / L.

8. The method for preparing the novel nano drug Res@LDH according to claim 5, characterized in that: In step 2), the centrifugation is performed at a speed of 8000-10000 rpm for 8-12 min.

9. Use of the novel nano drug Res@LDH according to claim 1 or 2 in the preparation of a drug for treating ischemic stroke.

10. The use according to claim 9, characterized in that The drug is a drug that reduces oxidative stress and inflammation caused by ischemic stroke damage by reducing the activation of microglia and astrocytes.

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