Strong-oxidation-resistance nano-drug with mitochondrial sequential targeting capacity as well as preparation method and application of strong-oxidation-resistance nano-drug

Abstract

The invention relates to a high-oxidation-resistance nano-drug with mitochondrial sequential targeting capacity as well as a preparation method and application thereof, and relates to the technical field of nano-drugs. The nano-drug with strong oxidation resistance disclosed by the invention is heteropoly acid jointly modified by dopamine and a polyphenol compound, or heteropoly acid modified by the polyphenol compound. The nano-drug with strong oxidation resistance is a nano-drug with strong oxidation resistance, which is prepared by a simple and green synthesis method and has mitochondrial sequential targeting capability, the adopted raw materials are low in price and easy to obtain, and the synthesis method is simple. The nano-drug with strong oxidation resistance disclosed by the invention can be used for effectively reducing the release of neuron mitochondria mtDNA to an external space and inhibiting the activation of a cGAS-STING signal of microglial cells, so that neuroinflammation induced by CIRI is reversed. The strong-oxidation-resistance nano-drug with the mitochondrial sequential targeting capability can be used for effectively treating CIRI by relieving oxidative stress.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a strong antioxidant nanomedicine with mitochondrial sequential targeting capability, and a preparation method and application thereof. Background Art

[0002] Cerebral infarction (CI) is the leading cause of disability and the second leading cause of death in adults worldwide. Tissue plasminogen activator (tPA) is the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of CI, but the tPA treatment time window is narrow (≤4.5h), and less than 5% of patients can benefit from treatment. In addition, tPA thrombolysis can induce cerebral ischemia-reperfusion injury (CIRI) and accelerate neuronal death. Therefore, new strategies are urgently needed to prevent and treat CIRI.

[0003] Neuronal apoptosis and immune inflammation caused by mitochondrial damage are the core mechanisms of CIRI. Neurons consume a lot of oxygen, are rich in mitochondria and are highly sensitive to mitochondrial damage. During CIRI, ischemia / reperfusion (I / R) induces electron leakage in the respiratory chain of neuronal mitochondria, which in turn leads to the massive production of mitochondrial reactive oxygen species (mtROS). The massive production of mtROS is the main source of ROS in CIRI, which directly damages mitochondria, impairs neuronal energy supply, and causes oxidative stress damage. In addition, mitochondria, as a relic of bacterial evolution, release their contents when damaged, inducing both neuronal apoptosis (such as cytochrome c, Cyt c) and strong inflammatory responses (such as mitochondrial DNA, mtDNA). Therefore, if neuronal mitochondrial damage can be effectively inhibited and mtROS can be eliminated, CIRI can be improved from the source.

[0004] In order to alleviate CIRI, various neuroprotective agents have been widely developed, including antioxidants (edaravone, N-acetylcysteine) and anti-inflammatory drugs (colchicine). However, due to the existence of the blood-brain barrier (BBB), the therapeutic effect of these neuroprotective agents is not significant. About 98% of small molecule drugs and almost 100% of macromolecule drugs cannot enter the brain through the BBB, which has become the main obstacle to the current treatment of CIRI. Fortunately, the BBB also provides an opportunity for nanodrugs to enter the brain to treat CIRI. First, the loss of glycocalyx on the surface of CIRI tissue capillaries exposes matrix proteins, which provides a target for drugs to specifically target CIRI lesions. In addition, the BBB of brain tissue is damaged during CIRI, and the loss of tight junctions of capillaries provides an opportunity for small-sized nanodrugs to penetrate damaged blood vessels and enter the CIRI site. In addition, in recent years, many mitochondrial targeting groups such as triphenylphosphine and SS-31 have been modified to the surface of drugs to improve the targeting of drugs to mitochondria. However, these mitochondrial targeting groups all carry strong positive charges to facilitate their targeting to mitochondria. However, after entering the blood, positively charged nanomedicines are not only easy to escape from CIRI lesions and accumulate in the liver and lungs, but also easy to induce inflammatory responses. Therefore, scavenging mtROS to effectively inhibit neuronal mitochondrial damage is an efficient and promising CIRI treatment strategy, but the realization of this goal faces huge challenges. First, in order to cope with the mtROS burst of neurons during CIRI, nanomedicines must have a strong ROS scavenging ability. Secondly, nanomedicines must be able to achieve continuous targeting of CIRI tissues and neuronal mitochondria, and the surface cannot be positively charged to avoid off-target effects and toxic side effects. Unfortunately, there is currently no strategy to overcome these huge challenges. Summary of the invention

[0005] The present invention aims to solve the technical problem that drugs in the prior art cannot cross the blood-brain barrier and target neuronal mitochondria to efficiently remove mtROS, and provide a strong antioxidant nano drug with mitochondrial sequential targeting capability, and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] A strong antioxidant nano drug with mitochondrial sequential targeting capability, which is a heteropoly acid modified by dopamine and polyphenol compounds, or a heteropoly acid modified by polyphenol compounds.

[0008] In the above technical solution, preferably, the heteropoly acid is a tungsten-based heteropoly acid or a molybdenum-based heteropoly acid.

[0009] In the above technical solution, it is further preferred that the heteropoly acid is phosphotungstic acid or phosphomolybdic acid.

[0010] In the above technical solution, preferably, the polyphenol compound is tannic acid or gallic acid.

[0011] In the above technical solution, preferably, the particle size of the strong antioxidant nanomedicine is between 5-15 nm.

[0012] A method for preparing a strong antioxidant nanomedicine with mitochondrial sequential targeting capability comprises the following steps:

[0013] Dopamine, polyphenolic compounds and heteropolyacids are used to prepare strong antioxidant nanomedicines through redox reactions under alkaline conditions.

[0014] Alternatively, polyphenol compounds and heteropolyacids are subjected to redox reactions under alkaline conditions to prepare nanomedicines with strong antioxidant properties.

[0015] In the above technical solution, preferably, the reaction system is an alkaline buffer solution with a pH of 8 to 10, and the reaction stirring time is 12 to 24 hours.

[0016] In the above technical solution, preferably, the mass ratio of dopamine to polyphenol compounds is between 5:1 and 0:1.

[0017] In the above technical solution, preferably, the prepared strong antioxidant nanomedicine is purified using a dialysis bag with a molecular weight cutoff of 3.5kD, and then freeze-dried at a temperature of -50 to -40°C for 48 to 72 hours.

[0018] Application of a strong antioxidant nanomedicine with mitochondrial sequential targeting capability in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

[0019] The beneficial effects of the present invention are:

[0020] The strong antioxidant nano drug with mitochondrial sequential targeting capability of the present invention is prepared by co-modifying tungsten / molybdenum-based heteropoly acid with dopamine and polyphenolic compounds, or by modifying tungsten / molybdenum-based heteropoly acid with polyphenolic compounds, and has an ultra-small size ( Figure 1 ) and strong antioxidant activity ( Figure 2 ).

[0021] The strong antioxidant nano drug with mitochondrial sequential targeting capability of the present invention is prepared by a simple and green synthesis method. The raw materials used are cheap and easily available, and the synthesis method is simple.

[0022] The strong antioxidant nanomedicine with mitochondrial sequential targeting ability of the present invention can effectively target the mitochondria of brain tissue and neurons damaged by CIRI ( Figure 3 ).

[0023] The strong antioxidant nanomedicine with mitochondrial sequential targeting ability of the present invention can effectively treat CIRI by alleviating oxidative stress. Figure 4 ).

[0024] The strong antioxidant nanomedicine with mitochondrial sequential targeting ability of the present invention can effectively remove neuronal mitochondrial ROS and protect mitochondrial morphology and function ( Figure 5 ).

[0025] The strong antioxidant nanomedicine with mitochondrial sequential targeting ability of the present invention can effectively reduce the release of neuronal mitochondrial mtDNA into the external space, inhibit the activation of microglial cGAS-STING signal, and thus reverse the neuroinflammation induced by CIRI ( Figure 6 ).

[0026] The strong antioxidant nanomedicine with mitochondrial sequential targeting ability of the present invention can effectively alleviate neuronal endoplasmic reticulum stress associated with mitochondrial damage ( Figure 7 ).

[0027] The strong antioxidant nanomedicine with mitochondrial sequential targeting ability of the present invention can effectively reduce neuronal death through dual effects: effectively reducing oxidative stress and eliminating inflammatory cytokine storm ( Figure 8 ).

[0028] The strong antioxidant nanomedicine with mitochondrial sequential targeting capability of the present invention has good biocompatibility, is non-toxic to hippocampal neuron cells (HT-22 cells) and microglia (BV2 cells) at the administration concentration involved in the present invention, and has no effect on the heart, liver, spleen, lung, kidney and brain of normal rats after long-term administration ( Figure 9-10 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0030] Figure 1 This is a transmission electron microscope image of the nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 in water.

[0031] Figure 2 The nano drug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 has an activity against ROS (O 2. - 、OH、H2O2、ONOO - )’s clearance ability test results.

[0032] Figure 3The results of the targeting of the nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid to CIRI ischemic brain tissue and neuronal mitochondria in Example 1 are shown; wherein, a is a protein-coated plate experiment proving that MHT can bind to the matrix (collagen III) and mitochondrial membrane protein (Tom20); b is a schematic diagram of BBB damage induced by I / R; c is a schematic diagram of the MCAO model; d is a histogram of the size of the gaps between the vascular endothelial cells of the infarcted brain tissue; e is a TEM image of the blood vessels of the normal and I / R rat brain tissues; f is a diagram of the brain and heart of the I / R rat , lung, liver, spleen, and kidney MHT-FITC fluorescence imaging and bright field representative images; g is the tungsten content in brain tissue determined by ICP-MS at 1h, 3h, 6h, 12h, and 24h after intravenous injection of MHT; h is TEM and HAADF-TEM images of MHT in the infarcted area (the red dotted circle marks MHT); i is the TEM image of mitochondria in neurons in the I / R and MHT treatment groups, yellow indicates neuronal mitochondria, and the green arrow indicates MHT; j is a representative image and co-localization analysis of MHT-FITC and mitochondria.

[0033] Figure 4 The results of the study on the efficacy and mechanism of nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 in improving CIRI are shown in the figure; wherein, a is a schematic diagram of MHT treatment of CIRI in rats; b is the result of nervous system scoring; c is Nissl staining for studying brain tissue structure and neuronal function; de are TTC staining of brain tissue (d) and infarct area statistics (e), respectively; f is VENN / UpSetR graphical analysis showing differentially expressed genes (DEGs) between different comparison groups; g is a volcano plot depicting the DEGs between MHT-treated and I / R brain tissues; h is the GO enrichment analysis of the biological processes involved in the DEGs between the I / R and MHT-treated groups; i is the KEGG pathway enrichment analysis of the DEGs of the MHT-treated group and the I / R group.

[0034] Figure 5 The figure is a graph showing the detection results of the nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 to remove mtROS and protect neuronal mitochondrial function; wherein, a is a schematic diagram of MHT inhibiting mtROS and improving mitochondrial function; bc are the quantitative statistics (b) and representative images (c) of DHE staining of brain tissues in each group; d is a representative image of ROS in HT22 cells labeled with DCFH-DA; e is the MDA content, an indicator for detecting oxidative damage in brain tissue; fg are immunofluorescence staining (f) and quantitative analysis (g) of mitochondrial superoxide in HT22 cells; hi are immunofluorescence staining (h) and quantitative analysis (i) of JC-1 in HT22 cells; j is the ATP generation of HT22 cells under different treatments.

[0035] Figure 6 The nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 inhibits the activation of the cGAS-STING pathway and reverses neuroinflammation; wherein, a is dsDNA immunofluorescence staining of the cerebral infarction area in each treatment group; b is STING immunohistochemical staining of the cerebral infarction area in each treatment group; c is iNOS and CD206 immunofluorescence staining of the cerebral infarction area in each treatment group; d is the mechanism of action of MHT inhibiting the activation of the microglia cGAS-STING pathway; e is a schematic diagram of the experimental process of co-incubation of HT22 and BV2 cells; f is dsDNA immunofluorescence staining of BV2 cells in different treatment groups; g is WB of inflammation-related proteins cGAS, STING, IRF3, P-IRF3, P65, and P-P65 in the cerebral infarction area; hl are the contents of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-4, IL-10) in the cerebral infarction area of ​​each treatment group, respectively.

[0036] Figure 7 The figure is a graph showing the results of improving endoplasmic reticulum stress in CIRI by preparing nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1; wherein, a is a schematic diagram of endoplasmic reticulum stress in the I / R model and the protective effect of MHT; bk are respectively the expression levels of endoplasmic reticulum stress-related factors (Bip, PERK, eIf-2α, ATF-4, Chop, ATF-6, IRE1-α, XBP-1s, JNK, Caspase 12) in brain tissues after different treatments detected by real-time fluorescence PCR; l is a TEM image of the endoplasmic reticulum (yellow part) in neurons of brain tissues in the Sham, I / R, and MHT treatment groups.

[0037] Figure 8 The figure is a result diagram of the improvement of neuronal apoptosis caused by mitochondrial damage and neuroinflammation during CIRI by the nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1; wherein, a is a schematic diagram of cell apoptosis induced by endogenous and exogenous factors; bc are TUNEL staining (b) and quantitative analysis (c) of brain tissue sections in each treatment group; di are WB of apoptosis-related proteins in the infarcted area, including representative images (d) and analysis of Bax (e), Bcl-2 (f), Cleaved caspase3 (g), Complex I (h), and Cytosolic-Cyt c (i); jk are the Annexin V-FITC / PI flow cytometry results of HT22 cells under different treatment conditions (k) and the quantitative statistical results of cell apoptosis rate (j).

[0038] Fig. 9This is the HE staining result of the main organs of rats after the nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 was intravenously injected for 28 consecutive days.

[0039] Fig.10 This is a graph showing the biocompatibility test results of the nanodrug 1 (MHT) prepared by co-modifying phosphotungstic acid with dopamine and tannic acid in Example 1 after acting on HT22 cells and BV2 cells for 24 / 48 hours. DETAILED DESCRIPTION

[0040] The inventive concept of the present invention is that as an emerging anion cluster material, tungsten / molybdenum-based heteropolyacids have attracted much attention due to their efficient ROS scavenging ability. 5+ / Mo 4+ The ratio of W in tungsten / molybdenum-based heteropolyacids is the key to scavenging ROS, and the modification of dopamine can increase the W in tungsten / molybdenum-based heteropolyacids. 5+ / Mo 4+ The content of polyphenols is high. Polyphenols are rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. They have strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which helps to achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria. Based on this, the present invention aims to develop a strong antioxidant tungsten / molybdenum-based nanomedicine with mitochondrial sequential targeting ability obtained based on heteropolyacids modified with dopamine and polyphenols or heteropolyacids modified with polyphenols for the treatment of CIRI.

[0041] The technical scheme of the present invention is clearly and completely described below through examples, but it should be understood that the following examples do not limit the protection scope of the present invention.

[0042] Example 1

[0043] (1) Synthesis of Nanomedicine

[0044] Dopamine hydrochloride (0.1 g) and tannic acid (0.2 g) were dissolved in 30 mL of deionized water, phosphotungstic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 100 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0045] (2) Purification

[0046] The sample obtained in step (1) was placed in a dialysis bag with a molecular weight cutoff of 3.5 kD. The dialysis bag was placed in 2 L of ultrapure water and dialyzed under magnetic stirring. The ultrapure water was changed every 4 hours for a total of 48 hours to remove unreacted impurities.

[0047] (3) Drying

[0048] The sample obtained in step (2) was divided into 50 mL centrifuge tubes (20 mL sample per tube), and then placed in a -20°C refrigerator for prefreezing for 2 hours. After 2 hours, the sample was placed in a freeze dryer and freeze-dried at -50°C under vacuum for 72 hours to obtain a nano drug powder.

[0049] Figure 1 The prepared dopamine and tannic acid co-modified tungsten heteropoly acid nanodrug (MHT) is called nanodrug 1. From the transmission electron microscope (TEM) image, it can be seen that the particle size of the nanodrug is between 8-10nm.

[0050] Example 2

[0051] (1) Synthesis of Nanomedicine

[0052] Dissolve tannic acid (0.3 g) in 30 mL of deionized water, add phosphotungstic acid (0.18 g) and fully dissolve and mix, then add 10 mL of 7.5% anhydrous sodium carbonate to form an alkaline environment (pH 8), pour the solution into a 150 mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at room temperature for 12 hours.

[0053] (2) Purification was the same as in Example 1.

[0054] (3) Drying is the same as in Example 1, and the obtained nanomedicine is referred to as nanomedicine 2.

[0055] The nano drug 2 prepared in this embodiment uses heteropoly acid phosphotungstic acid with efficient ROS scavenging ability, and tannic acid is rich in phenolic hydroxyl groups, which can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions, and has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, so that drugs can be targeted to CIRI damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0056] Example 3

[0057] (1) Synthesis of Nanomedicine

[0058] Dopamine hydrochloride (0.15 g) and tannic acid (0.15 g) were dissolved in 30 mL of deionized water, phosphotungstic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0059] (2) Purification was the same as in Example 1.

[0060] (3) Drying is the same as in Example 1, and the obtained nanomedicine is called nanomedicine 3.

[0061] Example 4

[0062] (1) Synthesis of Nanomedicine

[0063] Dopamine hydrochloride (0.2 g) and tannic acid (0.1 g) were dissolved in 30 mL of deionized water, phosphotungstic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0064] (2) Purification was the same as in Example 1.

[0065] (3) Drying is the same as in Example 1, and the obtained nanomedicine is referred to as nanomedicine 4.

[0066] The nano drugs 3 and 4 prepared in Examples 3 and 4, the heteropoly acid phosphotungstic acid used has a high ROS scavenging ability. 5+ The ratio of W in tungsten-based heteropolyacid is the key to scavenging ROS, and the modification of dopamine can increase the 5+ Tannic acid is rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. It has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which can achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0067] Example 5

[0068] (1) Synthesis of Nanomedicine

[0069] Dopamine hydrochloride (0.1 g) and gallic acid (0.2 g) were dissolved in 30 mL of deionized water, phosphotungstic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0070] (2) Purification was the same as in Example 1.

[0071] (3) Drying is the same as in Example 1. The obtained nanomedicine is referred to as nanomedicine 5.

[0072] The nano drug 5 prepared in this embodiment uses heteropoly acid phosphotungstic acid which has a high ROS scavenging ability. 5+ The ratio of W in tungsten-based heteropolyacid is the key to scavenging ROS, and the modification of dopamine can increase the 5+ Gallic acid is rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. It has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which can achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0073] Example 6

[0074] (1) Synthesis of Nanomedicine

[0075] Dissolve gallic acid (0.3 g) in 30 mL of deionized water, add phosphotungstic acid (0.18 g) and fully dissolve and mix, then add 10 mL of 7.5% by mass anhydrous sodium carbonate to form an alkaline environment (pH 8), pour the solution into a 150 mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at room temperature for 12 hours.

[0076] (2) Purification was the same as in Example 1.

[0077] (3) Drying is the same as in Example 1, and the obtained nanomedicine is referred to as nanomedicine 6.

[0078] The nano drug 6 prepared in this embodiment uses heteropoly acid phosphotungstic acid with efficient ROS scavenging ability, and gallic acid is rich in phenolic hydroxyl groups, which can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions, and has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, so that drugs can be targeted to CIRI damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0079] Example 7

[0080] (1) Synthesis of Nanomedicine

[0081] Dopamine hydrochloride (0.15 g) and gallic acid (0.15 g) were dissolved in 30 mL of deionized water, phosphotungstic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0082] (2) Purification was the same as in Example 1.

[0083] (3) Drying is the same as in Example 1. The obtained nanodrug is referred to as nanodrug 7.

[0084] Example 8

[0085] (1) Synthesis of Nanomedicine

[0086] Dopamine hydrochloride (0.2 g) and gallic acid (0.1 g) were dissolved in 30 mL of deionized water, phosphotungstic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0087] (2) Purification was the same as in Example 1.

[0088] (3) Drying is the same as in Example 1. The obtained nanomedicine is referred to as nanomedicine 8.

[0089] The nano drugs 7 and 8 prepared in Examples 7 and 8, the heteropoly acid phosphotungstic acid used has a high ROS scavenging ability. 5+ The ratio of W in tungsten-based heteropolyacid is the key to scavenging ROS, and the modification of dopamine can increase the 5+ Gallic acid is rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. It has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which can achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0090] Example 9

[0091] (1) Synthesis of Nanomedicine

[0092] Dopamine hydrochloride (0.1 g) and tannic acid (0.2 g) were dissolved in 30 mL of deionized water, phosphomolybdic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0093] (2) Purification was the same as in Example 1.

[0094] (3) Drying is the same as in Example 1. The obtained nanodrug is referred to as nanodrug 9.

[0095] The nano drug 9 prepared in this embodiment uses heteropoly acid phosphomolybdic acid which has a high ROS scavenging ability. 4+ The ratio of Mo in tungsten-based heteropolyacid is the key to scavenging ROS, and the modification of dopamine can increase the 4+ Tannic acid is rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. It has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which can achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0096] Example 10

[0097] (1) Synthesis of Nanomedicine

[0098] Dissolve tannic acid (0.3 g) in 30 mL of deionized water, add phosphomolybdic acid (0.18 g) and fully dissolve and mix, then add 10 mL of 7.5% by mass anhydrous sodium carbonate to form an alkaline environment (pH 8), pour the solution into a 150 mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at room temperature for 12 hours.

[0099] (2) Purification was the same as in Example 1.

[0100] (3) Drying is the same as in Example 1. The obtained nanodrug is referred to as nanodrug 10.

[0101] The nano drug 10 prepared in this embodiment uses heteropoly acid phosphomolybdic acid with high ROS scavenging ability, and tannic acid is rich in phenolic hydroxyl groups, which can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions, and has strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, so as to achieve drug targeting of CIRI damaged brain tissue and enter the damaged brain tissue through the gaps of damaged capillaries and further sequentially target neuronal mitochondria.

[0102] Embodiment 11

[0103] (1) Synthesis of Nanomedicine

[0104] Dopamine hydrochloride (0.15 g) and tannic acid (0.15 g) were dissolved in 30 mL of deionized water, phosphomolybdic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0105] (2) Purification was the same as in Example 1.

[0106] (3) Drying is the same as in Example 1, and the obtained nanodrug is called nanodrug 11.

[0107] Example 12

[0108] (1) Synthesis of Nanomedicine

[0109] Dopamine hydrochloride (0.2 g) and tannic acid (0.1 g) were dissolved in 30 mL of deionized water, phosphomolybdic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0110] (2) Purification was the same as in Example 1.

[0111] (3) Drying is the same as in Example 1, and the obtained nanodrug is referred to as nanodrug 12.

[0112] The nano drugs 11 and 12 prepared in Examples 11 and 12, the heteropoly acid phosphomolybdic acid used has a high ROS scavenging ability, Mo 4+ The ratio of Mo in tungsten-based heteropolyacid is the key to scavenging ROS, and the modification of dopamine can increase the 4+ Tannic acid is rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. It has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which can achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0113] Example 13

[0114] (1) Synthesis of Nanomedicine

[0115] Dopamine hydrochloride (0.1 g) and gallic acid (0.2 g) were dissolved in 30 mL of deionized water, phosphomolybdic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0116] (2) Purification was the same as in Example 1.

[0117] (3) Drying is the same as in Example 1, and the obtained nanodrug is called nanodrug 13.

[0118] Embodiment 14

[0119] (1) Synthesis of Nanomedicine

[0120] Dissolve gallic acid (0.3 g) in 30 mL of deionized water, add phosphomolybdic acid (0.18 g) and fully dissolve and mix, then add 10 mL of 7.5% by mass anhydrous sodium carbonate to form an alkaline environment (pH 8), pour the solution into a 150 mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at room temperature for 12 hours.

[0121] (2) Purification was the same as in Example 1.

[0122] (3) Drying is the same as in Example 1, and the obtained nanodrug is referred to as nanodrug 14.

[0123] The nano drug 14 prepared in this embodiment uses heteropoly acid phosphomolybdic acid with high ROS scavenging ability, and gallic acid is rich in phenolic hydroxyl groups, which can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions, and has strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, so as to achieve drug targeting CIRI damaged brain tissue and enter the damaged brain tissue through the gaps of damaged capillaries and further sequentially target neuronal mitochondria.

[0124] Embodiment 15

[0125] (1) Synthesis of Nanomedicine

[0126] Dopamine hydrochloride (0.15 g) and gallic acid (0.15 g) were dissolved in 30 mL of deionized water, phosphomolybdic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by weight anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0127] (2) Purification was the same as in Example 1.

[0128] (3) Drying is the same as in Example 1. The obtained nanodrug is referred to as nanodrug 15.

[0129] Example 16

[0130] (1) Synthesis of Nanomedicine

[0131] Dopamine hydrochloride (0.2 g) and gallic acid (0.1 g) were dissolved in 30 mL of deionized water, phosphomolybdic acid (0.18 g) was added and fully dissolved and mixed, and then 10 mL of 7.5% by mass anhydrous sodium carbonate was added to form an alkaline environment (pH 8), and the solution was poured into a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at room temperature for 12 hours.

[0132] (2) Purification was the same as in Example 1.

[0133] (3) Drying is the same as in Example 1, and the obtained nanodrug is referred to as nanodrug 16.

[0134] The nanomedicines 13, 15 and 16 prepared in Examples 13, 15 and 16 respectively, the heteropoly acid phosphomolybdic acid used has a high ROS scavenging ability, Mo 4+ The ratio of Mo in tungsten-based heteropolyacid is the key to scavenging ROS, and the modification of dopamine can increase the 4+ Gallic acid is rich in phenolic hydroxyl groups and can bind to proline-rich proteins through multiple hydrogen bonds and hydrophobic interactions. It has a strong binding ability to vascular matrix proteins and mitochondrial outer membrane proteins, which can achieve drug targeting of CIRI-damaged brain tissue and enter the damaged brain tissue through the gaps in damaged capillaries and further sequentially target neuronal mitochondria.

[0135] Embodiment 17

[0136] Taking the tungsten-based heteropoly acid nanodrug 1 (MHT) co-modified with dopamine and tannic acid synthesized in Example 1 as an example, the antioxidant properties of the nanodrug were evaluated. The specific steps are as follows:

[0137] Superoxide anion (O 2. - ) Clearance ability test: The nitro blue tetrazolium (NBT) method was used to detect the effect of nano drug 1 on O 2. - In the presence of methionine, riboflavin can be reduced by light to further generate O 2. - , O 2. -It can reduce NBT to blue methylhydrazone, with a maximum absorption at 560nm. Brief description: 390μL methionine (0.1M), 6μL riboflavin (20μM), 23μL NBT (0.01M), 1.5mL PBS (pH7.4, 0.1M), 1.1mL deionized water and 15μL MHT solution of different concentrations (0μg / mL, 200μg / mL, 400μg / mL, 800μg / mL, 1600μg / mL) are fully mixed, added to a quartz cuvette, placed on a UV / visible spectrophotometer to measure the baseline, then taken out and irradiated under UV light for 5 minutes, after irradiation, the UV absorption peak of the solution at 560nm is detected, and the pseudo-SOD activity of the sample to be tested is calculated. When the inhibition rate is 50%, the SOD enzyme activity in the test sample is recorded as 1 unit.

[0138] Hydroxyl radical (·OH) scavenging ability test: The terephthalic acid fluorescence spectrophotometry was used to detect the scavenging ability of MHT to ·OH. Ferrous sulfate and hydrogen peroxide catalyze the generation of ·OH through the Fenton reaction, converting non-fluorescent terephthalic acid into fluorescent 2-hydroxyterephthalic acid. Brief description: A PBS solution containing FeSO4 (0.5mM), terephthalic acid (0.5mM), H2O2 (1mM) and different concentrations of MHT (0ng / mL, 125ng / mL, 250ng / mL, 500ng / mL, 1μg / mL) was reacted at room temperature for 6min, mixed thoroughly and added to a quartz cuvette. The characteristic absorption peak of fluorescent 2-hydroxyterephthalic acid was detected by fluorescence spectrophotometer at 430nm.

[0139] Hydrogen peroxide (H2O2) scavenging ability test: A hydrogen peroxide detection kit was used to determine the hydrogen peroxide scavenging ability, specifically: a hydrogen peroxide solution containing different concentrations of MHT was prepared, and after reacting for 30 minutes, the hydrogen peroxide concentration was detected using a kit.

[0140] Peroxynitrite anion (ONOO - ) Scavenging ability test: The pyrogallol red method was used to detect the effect of MHT on ONOO - Pyrogallol red has a specific absorption peak at 540nm, ONOO - It can quench pyrogallol red and reduce its specific absorption peak. Method: 10 μL pyrogallol red (5 mM), 9 μL ONOO - The mixture of MHT (1.73 mM) and different concentrations (0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL) was used to prepare the pyrogallol red solution and ONOO - After 15 min of reaction, the absorbance of pyrogallol red at 540 nm was measured by UV-visible spectrophotometry to calculate ONOO- cleaning ability.

[0141] The results are as follows Figure 2 As shown, nanodrug 1 (MHT) can efficiently remove ROS, including superoxide anions, hydroxyl radicals, hydrogen peroxide, and peroxynitrite anions, at low doses, indicating that it has strong antioxidant properties.

[0142] Embodiment 18

[0143] Taking the synthesized dopamine and tannic acid co-modified tungsten heteropoly acid nanodrug 1 (MHT) in Example 1 as an example, the targeting of the nanodrug to the ischemic brain tissue and neuronal mitochondria of CIRI rats was evaluated. The specific steps are as follows:

[0144] Animal level: Rats in the sham operation group (Sham group) and CIRI group (I / R group) were given fluorescently labeled nanodrug 1 (MHT-FITC) through the sublingual vein, and 1 hour later, the brain, heart, liver, spleen, lung, and kidney of the rats were taken and placed under a stereo fluorescence microscope (Leica, M205FCA) to observe the distribution of the drug in the main organs of the rats and collect images; Rats in the Sham group and I / R group were given nanodrug 1 (MHT) through the sublingual vein, and then the brain, heart, liver, spleen, lung, and kidney of the rats were taken 1h, 3h, 6h, 12h, and 24h after injection, and the tungsten content in the main organs of the rats was determined by ICP-MS; brain tissues (less than 1mm) of rats in the Sham group, I / R group, and MHT group were taken 3 ), fixed with fresh TEM fixative overnight, then washed the tissue three times with PBS (15 minutes each time), and then replaced with 1% osmium acid mixture to soak for 2 hours. After gradient dehydration, the tissue was embedded and solidified. The sample was cut into 60-80nm thick slices with a slicer, double-stained with 3% uranyl acetate-lead citrate, and the neuronal mitochondrial morphology and the distribution of nanodrug 1 (MHT) in mitochondria were observed using a Jeol 1200EX transmission electron microscope. Using high-angle annular dark field (HAADF) STEM, it was confirmed that MHT in cerebral infarction tissue was rich in W.

[0145] Cell level: HT22 cells were cultured at 1×10 4 / well were inoculated in a 24-well plate. After 24 hours, HT22 cells were treated with medium containing MHT-FITC + 400 μM cobalt chloride for 16 hours, incubated with Mitotracker fluorescent probe for 20 minutes, washed twice with buffer, incubated cell nuclei with Hoechst stain for 10 minutes, washed twice with buffer, and the co-localization of MHT and mitochondria was observed under a fluorescence microscope.

[0146] The results are as follows Figure 3 As shown by Figure 3a It can be seen that MHT has a high affinity for proline-rich matrix proteins (type III collagen) and mitochondrial membrane proteins (Tom20), but has a repulsive force on negatively charged serum albumin and glycocalyx; Figure 3 b It can be seen that in CIRI, the brain-blood barrier is damaged by inflammatory cytokines, matrix metalloproteinases (MMPs), and ROS, resulting in the exposure of vascular matrix proteins and the loss of endothelial cell tight junctions, which is the key to MHT targeting CIRI tissues; Figure 3 c As can be seen, the CIRI rat model was constructed by simulating cerebral ischemia in male SD rats by middle cerebral artery occlusion (MCAO) and removing the suture 2 hours after ischemia to simulate the reperfusion period; Figure 3 d shows that the average gap between the capillaries in the brain tissue of CIRI rats is 97.4 nm (much larger than the average size of MHT, 8-10 nm), so MHT can enter the CIRI tissue through these gaps; Figure 3 e It can be seen intuitively that the blood-brain barrier of the brain tissue in the Sham group was intact, and the endothelial cell gap was 0; the vascular endothelial cells of the CIRI brain tissue were obviously swollen, and the tight junctions were lost; Figure 3 f shows that only 1 hour after sublingual intravenous injection of fluorescently labeled MHT (MHT-FITC), MHT can specifically target the I / R area of ​​brain tissue; Figure 3 g shows that the MHT content in brain tissue began to decrease gradually 3 hours after intravenous injection, indicating that MHT in brain tissue can be effectively cleared; Figure 3 hWET enrichment in cerebral infarction tissue can be directly detected, further confirming the specific targeting of MHT to damaged brain tissue. Figure 3 It can be seen that MHT can target neuronal mitochondria and significantly improve the morphology and function of I / R-induced neuronal mitochondria in brain tissue; Figure 3 As far as we know, the Pearson coefficient of co-localization between MHT and mouse hippocampal neurons (HT22 cells) mitochondria is as high as 0.93, indicating that MHT has a strong targeting effect on neuronal mitochondria. Figure 3 The results showed that nanodrug 1 (MHT) could specifically target cerebral infarction tissue and accumulate in the mitochondria of neurons. The sequence targeting characteristics of nanodrug 1 (MHT) provided a basis for the elimination of neuronal mtROS in CIRI.

[0147] Embodiment 19

[0148] Taking the tungsten-based heteropoly acid nanodrug 1 (MHT) co-modified with dopamine and tannic acid synthesized in Example 1 as an example, the therapeutic effect of the nanodrug on rat CIRI was evaluated. The specific steps are as follows:

[0149] TTC and neurological function score: Male SD rats (8 weeks old, 260-280g) were subjected to middle cerebral artery occlusion (MCAO) to simulate the cerebral ischemia stage. After 2 hours of ischemia, the occluded wire was removed and blood supply was restored for 24 hours to simulate the reperfusion stage to establish the rat CIRI model. In order to evaluate the effect of drug intervention, MHT was given before reperfusion. After 24 hours of reperfusion, the neurological dysfunction of the rats was evaluated by neurological function scoring criteria. Subsequently, the rat brain tissue was obtained and the cerebral infarction area of ​​the rats was evaluated by triphenyltetrazolium (TTC) staining. To evaluate the efficacy of the drug, the clinically approved antioxidant drug NAC and the nanodrug (HT) without dopamine modification at the same dose were used as controls. (The neurological function score was based on the ZeaLonga 5-point standard: 0 points represent no symptoms of neurological deficit; 1 point represents the inability to fully extend the contralateral forelimb; 2 points represent turning to the hemiplegic side when walking; 3 points represent the body leaning to the hemiplegic side when walking; 4 points represent the inability to walk independently and impaired consciousness; 5 points represent death.)

[0150] Nissl staining: 24 hours after reperfusion, the rat brain tissue was obtained, fixed with 4% PFA for 24 hours, dehydrated and embedded in paraffin blocks, cut into 4 μm sections, and Nissl staining was used to observe the pathological changes of each tissue.

[0151] RNA sequencing analysis: Total RNA was extracted from brain tissues of the Sham, I / R, and MHT groups using TRIzol according to the manufacturer's protocol. Subsequent analysis and data mining were performed on the Dr.Tom Multi-omics Data Mining System (https: / / biosys.bgi.com), and clean reads were mapped to the reference genome using HISAT2, and clean reads were aligned to the gene set using Bowtie2.

[0152] The results are as follows Figure 4 As shown, Figure 4 a is the treatment regimen of MHT for the treatment of CIRI; Figure 4 b shows that the neurological function of CIRI rats is severely impaired, and MHT can significantly improve the neurological function of CIRI rats, and its therapeutic effect is significantly better than HT. Figure 4 c shows that the number and morphology of Nissl bodies in brain tissue of CIRI rats were severely damaged, and MHT treatment could significantly reverse the number and morphology of neuronal Nissl bodies, and the therapeutic effect was significantly better than HT. Figure 4 It can be seen that the cerebral ischemic area of ​​CIRI rats has severe cellular infarction, and MHT treatment can almost reverse the infarction, reducing the infarction area of ​​CIRI rats from 42.75% to 1.95%, and the therapeutic effect is significantly better than HT. Figure 4f shows that there are 2114 differentially expressed genes (DEGs) between the Shan group and the I / R group, while there are only 63 differentially expressed genes between the MHT group and the Sham group, indicating that MHT treatment can significantly improve the expression of differentially expressed genes in I / R-induced ischemic brain tissue; Figure 4 g It can be seen that compared with the I / R group, 340 genes were upregulated and 1344 genes were downregulated in the MHT group; Figure 4 hIt can be seen that MHT treatment of CIRI mainly involves five biological processes: oxidative stress response, inflammatory response, cell apoptosis, mitochondrial function, and endoplasmic reticulum stress; Figure 4 It can be seen that MHT treatment of CIRI mainly acts through mitochondrial damage signaling pathways and inflammation-related signaling pathways, including apoptosis pathways, cytoplasmic DNA sensing pathways, cytokine-cytokine receptor interactions, tumor necrosis factor (TNF) signaling pathways, and NF-kappa B signaling pathways. Figure 4 The results showed that nanodrug 1 (MHT) had a good therapeutic effect on CIRI, and the modification of dopamine could significantly improve the efficacy of nanodrug on CIRI.

[0153] Embodiment 20

[0154] Taking the synthesized dopamine and tannic acid co-modified tungsten heteropoly acid nanodrug 1 (MHT) in Example 1 as an example, the nanodrug's ability to scavenge mtROS in the cerebral infarction area of ​​CIRI rats and its protective effect on mitochondrial function were evaluated. The specific steps are as follows:

[0155] Animal level: 24 hours after reperfusion, brain tissues of CIRI rats were taken for frozen sections, and the overall level of ROS in brain tissues was evaluated by immunofluorescence staining with a ROS probe (DHE); 24 hours after reperfusion, brain tissues of CIRI rats were taken, brain tissue homogenates were prepared, and the supernatants were centrifuged to measure the MDA content in the brain tissues to evaluate mitochondrial damage in the brain tissues.

[0156] Cell level: HT22 cells were cultured at 1×10 4 / well inoculated in a 24-well plate, HT22 cells were treated with medium containing different doses of MHT and 400 μM cobalt chloride for 16 h after 24 h, and then replaced with medium containing only different doses of MHT for another 8 h. The cells were incubated with ROS fluorescent probe (DCFH-DA), mitochondrial ROS fluorescent probe (MitoSox), and mitochondrial membrane potential detection fluorescent probe (JC-1) for 25 min, washed twice with buffer, and observed and collected images under a fluorescence microscope; HT22 cells were plated at 1×10 4 / well were inoculated in a 24-well plate. After 24 hours, HT22 cells were treated with medium containing different doses of MHT and 400 μM cobalt chloride for 16 hours, and then replaced with medium containing only different doses of MHT for another 8 hours. The ATP production content of HT22 cells was evaluated using an ATP detection kit.

[0157] The results are as follows Figure 5 As shown by Figure 5 aIt can be seen that CIRI is mainly mediated by ROS burst, and the antioxidant effect of MHT has been preliminarily studied in CIRI; Figure 5 It can be seen that MHT can effectively clear the ROS level in the brain tissue induced by I / R. 2mg / kg MHT treatment can reduce the ROS level in the ischemic brain tissue to 1.16 times that of the Sham group, and the therapeutic effect is significantly better than HT. Figure 5 d It can be seen that MHT can also significantly reduce the level of ROS in H / R-induced HT22 cells, and its therapeutic effect is significantly better than HT; in CIRI, mtROS destroys the lipids in the mitochondrial membrane to release malondialdehyde (MDA), which reflects the degree of mitochondrial damage in the CIRI process to a certain extent; Figure 5 As shown in e, MHT can significantly reduce the MDA level in CIRI, and its therapeutic effect is significantly better than HT; MitoSOX probe indicates the mtROS level in HT22 cells. Figure 5 fg shows that MHT can effectively reduce the level of mtROS in H / R-induced HT22 cells, and its therapeutic effect is significantly better than HT. Figure 5 It can be seen that MHT can effectively reverse the decrease in mitochondrial membrane potential of HT22 cells induced by H / R and restore the integrity of MMP (the percentage of MMP in the MHT treatment group increased from 8.23% in the model group to 81.1%), and the therapeutic effect is significantly better than HT. Figure 5 It can be seen that MHT can effectively reverse the decrease in ATP production in HT22 cells induced by H / R, and its therapeutic effect is significantly better than HT. Figure 5 The results showed that nanodrug 1 (MHT) can effectively protect mitochondria from mtROS damage and significantly restore mitochondrial function through mitochondrial targeted therapy. Modification of dopamine can significantly improve the efficacy of nanodrugs.

[0158] Embodiment 21

[0159] Taking the synthesized dopamine and tannic acid co-modified tungsten-based heteropoly acid nanodrug 1 (MHT) in Example 1 as an example, the effect of the nanodrug on inhibiting the activation of microglial cGAS-STING signal and reversing CIRI neuroinflammation was evaluated. The specific steps are as follows:

[0160] Animal level: After 24h of reperfusion, brain tissues of CIRI rats were collected for paraffin sections. The sections were incubated with primary antibodies against dsDNA / Tom 20, iNOS, and CD206 at 4°C overnight, and with the corresponding secondary antibodies at room temperature for 2 hours. The sections were sealed with anti-fluorescence quenching solution containing DAPI, and then the enlarged fluorescence images were acquired using LSM 900 equipped with Airyscan2 (Carl Zeiss). For quantification, at least three random imaging fields within the lesion area were selected for each brain tissue section and analyzed using Image J; the expression levels of inflammation-related proteins in rat brain tissue were detected. Specifically: After 24h of reperfusion, brain tissues of CIRI rats were collected by tissue homogenizer, and proteins were separated using RIPA (protease and phosphatase inhibitors). Protein concentration was analyzed using a BCA kit, and 30μg of total protein was separated using 8%-12% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% milk in TBST for 1 hour at room temperature, and the specific primary antibodies were incubated overnight at 4°C. After that, the membrane was washed three times with TBST and then incubated with the secondary antibody at room temperature for 1 hour. The bands were visualized using a gel imaging system (Bio-Rad, USA) and quantified using ImageJ (V 1.53a) software; the expression levels of inflammatory factors in rat brain tissue were detected. Specifically: 24 hours after reperfusion, the brain tissue of CIRI rats was collected by a tissue homogenizer and the supernatant was taken. The expression levels of inflammatory factors in brain tissue were detected by ELISA detection kits, including pro-inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and anti-inflammatory factors interleukin-4 (IL-4) and interleukin-10 (IL-10).

[0161] Cell level: Cell slides were placed in 24-well plates. Then, HT22 cells were cultured at 1×10 4 / well were inoculated in a 24-well plate. After 24 hours, HT22 cells were treated with medium containing MHT-FITC + 400μM cobalt chloride for 16 hours, and then replaced with medium containing only different doses of MHT for another 8 hours. The cell slides were taken out and incubated with the primary antibody of dsDNA / Tom20 at 4°C overnight and with the corresponding secondary antibody at room temperature for 2 hours. The slides were sealed with anti-fluorescence quenching solution containing DAPI, and then the enlarged fluorescence images were obtained using LSM 900 equipped with Airyscan2 (Carl Zeiss).

[0162] The results are as follows Figure 6 As shown, Figure 6As shown in a, the DAPI / dsDNA / Tom20 co-staining method was used to detect the distribution of mtDNA in ischemic brain tissues of different groups; in the I / R group, mitochondria were severely damaged, resulting in a large amount of mtDNA released from damaged mitochondria into the cytoplasm and outside neurons; MHT could protect mitochondria and significantly reduce mtDNA release; at the same dose, HT also reduced the release of mtDNA to a certain extent, but its effect was significantly lower than that of MHT; mtDNA released from mitochondria induced microglia to polarize to the M1 type and caused a strong inflammatory response through the cGAS / STING pathway; as shown in Figure 6 As shown in b, the number of STING positive spots in the brain tissue of the I / R group increased significantly, while MHT significantly inhibited the activation of STING, and the therapeutic effect was better than HT; the ischemic brain tissue was immunofluorescently stained with M1 microglia marker protein (iNOS) and M2 microglia marker protein (CD206) fluorescent probes; as shown in Figure 6 As shown in Figure c, after I / R, almost all microglia in ischemic brain tissue were activated to M1 type; in the MH and MHT treatment groups, the M1 phenotype of microglia decreased, and the M2 phenotype increased, indicating that MHT significantly promoted the transformation of microglia from M1 phenotype to M2 phenotype, and the effect was significantly higher than HT; Figure 6 e, HT22 cells were co-cultured with microglia (BV2) to construct an in vitro H / R cell model to simulate the I / R brain tissue microenvironment; Figure 6 As shown in (f), compared with the normoxic group, the abnormal distribution of mtDNA in the microglial cytoplasm of the H / R group co-stained with DAPI / dsDNA / Tom20 was more, indicating that neuronal mtDNA was delivered to the microglial cytoplasm; MHT significantly reduced the distribution of mtDNA in the microglial cytoplasm, and its therapeutic effect was better than HT; as shown in (f), Figure 6 As shown in g, WB analysis was used to detect the expression levels of key proteins of the cGAS-STING pathway in brain tissue in vivo, including cGAS, STING, and downstream P-IRF3 / IRF3 and P-P65 / P65; MHT significantly reduced the high expression of cGAS / STING and I / R-induced phosphorylation of IRF3 and P65, and the effect was significantly better than HT; Figure 6 As shown in hj, the ELISA method was used to detect the levels of inflammatory factors in the brain tissue homogenate of each group; after I / R, the expression of proinflammatory factors IL-1β, IL-6, and TNF-α in the I / R group was significantly increased, and the HT treatment group could inhibit the production of proinflammatory factors to a certain extent, but the inhibitory effect of MHT on the production of proinflammatory factors was significantly better than that of HT; Figure 6 As shown in k and l, HT and MHT treatment can significantly increase the expression of anti-inflammatory factors IL-4 and IL-10, and MHT treatment is always more effective than HT treatment. Figure 6The results showed that nanodrug 1 (MHT) can reverse neuroinflammation during CIRI by reducing the release of mtDNA, inhibiting the activation of the cGAS-STING pathway, and regulating the transformation of microglia M1 to M2. The modification of dopamine can significantly improve the efficacy of nanodrugs.

[0163] Embodiment 22

[0164] Taking the tungsten-based heteropoly acid nanodrug 1 (MHT) co-modified with dopamine and tannic acid synthesized in Example 1 as an example, the protective effect of the nanodrug on endoplasmic reticulum stress in the infarct area of ​​CIRI rats was evaluated. The specific steps are as follows:

[0165] Quantitative real-time PCR analysis: Total RNA was extracted from frozen brain tissue / cells using TRIzol and reverse transcribed to obtain cDNA. Quantitative real-time PCR was performed using an Applied Biosystems Step One Plus instrument and TB Green Premix Ex TaqTM (TliRNaseH Plus). Gene expression was evaluated using the comparative Ct value method, using β-Actin as a reference gene.

[0166] The results are as follows Figure 7 As shown. Figure 7 aIt can be seen that under I / R conditions, mtROS amplifies endoplasmic reticulum stress, leading to protein misfolding and inducing neuronal death. Endoplasmic reticulum stress promotes cell apoptosis by activating three functional transmembrane proteins: PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). qPCR was used to verify the regulatory mechanism of MHT on endoplasmic reticulum stress in vivo. Figure 7 As shown in bk, the expression of Bip in I / R brain tissue was significantly increased, and PERK, ATF6, IRE-1α and their mediated pathways were also significantly activated, including eIf-2α, ATF-4, Chop, XBP-1s, JNK and Caspase12, indicating that ER stress can further promote neuronal apoptosis, while MHT can effectively inhibit ER stress, and the effect is significantly higher than HT. In addition, as Figure 7 As shown in Figure 1, with the destruction of neuronal mitochondria, the endoplasmic reticulum of neurons in the I / R group swelled significantly, confirming that the level of neuronal endoplasmic reticulum stress increased after I / R. The endoplasmic reticulum edema of neurons in the MHT group was significantly improved, and the structure was similar to that in the Sham group, indicating that MHT can effectively improve I / R-induced endoplasmic reticulum stress. Figure 7 The results showed that nanodrug 1 (MHT) could significantly improve the endoplasmic reticulum stress in the brain tissue of CIRI rats. The modification of dopamine could significantly improve the efficacy of nanodrugs.

[0167] Embodiment 23

[0168] Taking the synthesized dopamine and tannic acid co-modified tungsten-based heteropoly acid nanodrug 1 (MHT) in Example 1 as an example, the nanodrug was evaluated for its dual effects (strongly reducing oxidative stress and eliminating inflammatory cytokine storm) to effectively reduce neuronal death. The specific steps are as follows:

[0169] Animal level: TdT-mediated dUTP end labeling (TUNEL) staining was used to analyze the apoptosis of cerebral infarction tissues in different groups. WB method was used to detect the expression of apoptosis-related proteins in brain tissues induced by ischemia / reperfusion (I / R). The detailed methods of tissue section fluorescence staining and WB protein level detection were the same as those in Example 21.

[0170] Cell level: HT22 cells were cultured at 1×10 4 / well were inoculated in a 24-well plate. After 24 hours, HT22 cells were treated with medium containing MHT-FITC + 400μM cobalt chloride for 16 hours, and then replaced with medium containing only different doses of MHT for another 8 hours. The cells were washed three times with cold PBS and collected from the dish. HT22 cells were stained with Annexin V and PI according to the Annexin V-FITC / PI apoptosis detection protocol, and then detected by flow cytometry.

[0171] The results are as follows Figure 8 As shown. Figure 8 It can be seen from a that after I / R, excessive mtROS generated by neurons can cause mitochondrial damage, release pro-apoptotic factors (Cyt c) and activate the endoplasmic reticulum stress signaling pathway to jointly induce neuronal apoptosis; in addition, mtDNA released from damaged neuronal mitochondria will stimulate microglia to secrete pro-inflammatory factors, which can reversely act on neuronal mitochondria, inhibit the activity of neuronal mitochondrial complex I, and aggravate neuronal mitochondrial damage again; Figure 8 As shown in b and c, the apoptosis rate of cells in the injured brain tissue of rats in the I / R group was as high as 72.6%, and MHT could significantly reduce the apoptosis rate of cells in the injured brain tissue (12.5%); however, at the same dose, the apoptosis rate of HT cells was only reduced to 40.75%; Figure 8 As shown in dg and 8i, the expression levels of pro-apoptotic proteins Bax, Cleaved-caspase 3, and Cytosolic Cyt c in the brain tissue of the I / R group were significantly higher than those in the Sham group, while the expression level of anti-apoptotic protein Bcl-2 was significantly reduced; HT and MHT could reverse the expression of the above proteins, and the effect of MHT was significantly higher than that of HT, which indicated that both HT and MHT could effectively reduce I / R-induced neuronal apoptosis, and the therapeutic effect of MHT was better than that of HT; Figure 8As shown in h, the expression of mitochondrial complex I in the I / R group was significantly reduced; HT and MHT treatment reversed the expression of mitochondrial complex I, and the therapeutic effect of MHT was better than HT, indicating that MHT can more effectively reduce inflammation-induced neuronal apoptosis; Figure 8 As shown in j and k, MHT significantly reduced H / R-induced apoptosis in HT22 cells, and its therapeutic effect was significantly better than that of HT. Figure 8 The results showed that nanodrug 1 (MHT) could significantly reduce neuronal apoptosis through endogenous and exogenous pathways. The modification of dopamine could significantly improve the efficacy of nanodrugs.

[0172] Embodiment 24

[0173] Taking the synthesized dopamine and tannic acid co-modified tungsten heteropoly acid nanodrug 1 (MHT) in Example 1 as an example, the biocompatibility of the nanodrug was evaluated at the cell level and the animal level. The specific steps are as follows:

[0174] Animal level: After 20 mg / kg MHT nanodrug (dissolved in saline) was injected into the tail vein of healthy SD mice for 28 consecutive days, the mice were euthanized and the main organs (brain, heart, liver, spleen, lung, and kidney) were collected. Paraffin sections of individual tissues and organs were prepared and the tissue morphology was observed by hematoxylin-eosin staining. Specifically, after the paraffin sections were dewaxed, the hematoxylin stain was added to the tissue to ensure that the tissue was completely covered and stained for 10-15 minutes. Rinse with running water to wash away the excess stain. Differentiate with 1% hydrochloric acid alcohol solution, soak in water to turn blue for 2 minutes, add eosin stain to the tissue for about 10 seconds, and after the eosin staining is completed, immediately immerse in anhydrous ethanol for dehydration twice (2 minutes each time). The samples were immersed in xylene twice (2 minutes each time), dried naturally in a fume hood for 10-30 minutes, sealed with neutral resin, and observed and imaged under a microscope. Blood samples were taken to evaluate the liver function-related indicators (alanine aminotransferase (ALT) and γ-glutamyltransferase (γ-GT)) and renal function indicators (creatinine (CR) and urea nitrogen (BUN)) of rats.

[0175] Cell level: HT22 cells and BV2 cells were cultured at 1×10 4 / well was inoculated into a 96-well plate and incubated for 24 hours. MHT was dispersed in the culture medium to prepare cell culture fluids of different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μg / mL, respectively). The above cell culture fluid was added to HT22 cells and BV2 cells and incubated for another 24 / 48 hours, and then 10 μL of CCK-8 reagent was added to each well and incubated for half an hour, and cell viability was detected by measuring absorbance at 450 nm.

[0176] The results are as follows Figure 9-10As shown, nanodrug 1 (MHT) had no obvious toxicity to HT22 cells and BV2 cells. In addition, HE staining results showed that normal rats had no obvious damage to the brain, heart, liver, spleen, lung, and kidney after long-term intravenous injection of MHT nanodrugs, and liver and kidney function and blood routine indicators were all within the normal range.

[0177] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A strong antioxidant nanomedicine with mitochondrial sequential targeting capability, characterized in that: It is a heteropoly acid modified by dopamine and a polyphenol compound, or a heteropoly acid modified by a polyphenol compound.

2. The strong antioxidant nanomedicine with mitochondrial sequential targeting ability according to claim 1, characterized in that: The heteropoly acid is a tungsten-based heteropoly acid or a molybdenum-based heteropoly acid.

3. The strong antioxidant nanomedicine with mitochondrial sequential targeting ability according to claim 2, characterized in that: The heteropoly acid is phosphotungstic acid or phosphomolybdic acid.

4. The strong antioxidant nanomedicine with mitochondrial sequential targeting ability according to claim 1, characterized in that: The polyphenol compound is tannic acid or gallic acid.

5. The strong antioxidant nanomedicine with mitochondrial sequential targeting ability according to claim 1, characterized in that: The particle size of the strong antioxidant nano drug is between 5-15 nm.

6. A method for preparing a strong antioxidant nanomedicine with mitochondrial sequential targeting capability according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dopamine, polyphenolic compounds and heteropolyacids are used to prepare strong antioxidant nanomedicines through redox reactions under alkaline conditions. Alternatively, polyphenol compounds and heteropolyacids are subjected to redox reactions under alkaline conditions to prepare nanomedicines with strong antioxidant properties.

7. The preparation method according to claim 6, characterized in that: The reaction system is an alkaline buffer solution with a pH of 8 to 10, and the reaction stirring time is 12 to 24 hours.

8. The preparation method according to claim 6, characterized in that: The mass ratio of dopamine to polyphenol compounds is between 5:1 and 0:

1.

9. The preparation method according to claim 6, characterized in that: The prepared strong antioxidant nanomedicine was purified using a dialysis bag with a molecular weight cutoff of 3.5kD, and then freeze-dried at a temperature of -50 to -40°C for 48 to 72 hours.

10. Use of the strong antioxidant nanomedicine with mitochondrial sequential targeting capability as claimed in any one of claims 1 to 5 in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

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