Nano-drug with eNOS-like enzyme activity as well as preparation method and application of nano-drug

Abstract

The invention relates to a nano-drug with eNOS-like enzyme activity and a preparation method and application thereof, and belongs to the technical field of nano-drugs. The technical problems that in the prior art, medicine cannot penetrate through the blood brain barrier, neuron calcium overload cannot be inhibited at the same time, mitochondrial damage and endoplasmic reticulum stress cannot be relieved, and inflammatory storm cannot be inhibited are solved. The nano-drug provided by the invention is a NO-embedded molybdenum sulfide nano-material obtained by introducing an S-nitroso group on the surface of small-particle molybdenum sulfide. The nano-drug prepared by the invention has super-strong antioxidant activity and stable NO release ability, can effectively target CIRI to damage brain tissues and stably release pure NO, can effectively treat cerebral ischemia reperfusion injury, improves CIRI-induced intracellular Ca < 2 + > overload, oxidative stress and inflammatory storm, can effectively protect neuron mitochondria from being damaged by overload Ca < 2 + > and mtROS, and can be used for treating cerebral ischemia reperfusion injury. The mitochondrial function in the CIRI is obviously recovered, and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a nanomedicine with eNOS-like enzyme activity and a preparation method and application thereof. Background Art

[0002] Cerebral infarction (CI) is the leading cause of long-term disability worldwide, with more than 13.7 million new patients and more than 5.8 million related deaths each year. Early recanalization of occluded cerebral vessels using tissue plasminogen activator (tPA) or endovascular thrombectomy is the only intervention approved by the US Food and Drug Administration (FDA) for the treatment of CI. Unfortunately, less than 5% of patients benefit from these treatments, mainly because recanalization beyond the treatment time window can further induce cerebral ischemia-reperfusion injury (CIRI) and aggravate the pathological process of CI. Therefore, new strategies are urgently needed to prevent and treat CIRI.

[0003] The pathological mechanism of CIRI is complex, mainly involving neuronal apoptosis caused by calcium overload, oxidative stress, inflammatory storm, etc. Specifically, the large amount of glutamate released during CIRI induces intracellular Ca2+ in neurons through the postsynaptic N-methyl-D-aspartate receptor (NMDAR). 2+ Influx leads to mitochondrial dysfunction and mitochondrial reactive oxygen species (mtROS) burst, which subsequently triggers the release of pro-apoptotic factor cytochrome C (Cyt-C) and endoplasmic reticulum stress (ERS), ultimately leading to neuronal damage and death. In addition, mitochondrial deoxyribonucleic acid (mtDNA) released by damaged neurons can activate the microglial cyclic guanosine monophosphate-adenylate synthase-stimulator of interferon genes (cGAS-STING) signaling pathway, inducing microglia to polarize to a pro-inflammatory phenotype (M1), triggering a strong inflammatory storm and exacerbating neuronal death. Ultimately, calcium overload, oxidative stress and inflammatory storm promote each other, forming a vicious cycle of neuronal damage and death. Therefore, the therapeutic effect of a single target is limited and short-lived, and the development of a drug platform that can simultaneously reverse calcium overload, oxidative stress and inflammatory storm is crucial for the treatment of CIRI. However, due to the complex pathological mechanism of CIRI, there is currently no research that can simultaneously reverse these malignant progressions.

[0004] NO is a supramolecule with multiple beneficial biological effects, especially in cerebrovascular diseases. Loss of NO homeostasis plays a fundamental role in CIRI. NO mainly comes from three enzymes: eNOS from vascular endothelial cells, nNOS from neurons, and iNOS from glial cells or macrophages. Physiologically, under low reactive oxygen species (ROS) levels, appropriate concentrations of NO mainly come from eNOS and nNOS, and the NO they produce can inhibit thrombosis, improve ischemic collateral circulation, and inhibit inflammatory responses. More importantly, NO can inhibit Ca2+ by nitrosating the redox sites on NMDARs. 2+ Influx of intracellular Ca2+ can alleviate neurotoxicity mediated by inhibiting related protein kinases and calcium-derived nNOS. 2+ However, under the pathological condition of CIRI, eNOS and nNOS no longer generate NO, but instead generate superoxide (O 2 .- ) and other ROS because the electrons donated by NADPH are “decoupled” from nitric oxide synthase (NOS) activity and the availability of L-arginine / BH4 is reduced. At the same time, M1-polarized microglia produce a large amount of NO through iNOS, which can easily react with high concentrations of ROS (such as O 2 .- ) to form highly toxic peroxynitrite (ONOO - ), further exacerbating neuronal damage and death. In recent years, emerging NO-based treatments have also been developing, including NO donor S-nitrosoglutathione (GSNO), direct inhalation of NO gas, NO nanocarriers, etc. However, NO-based treatments have not yet achieved ideal therapeutic effects in the treatment of CIRI. On the one hand, the release rate of NO is difficult to control, and too high or too low NO concentrations may have harmful effects on CIRI; on the other hand, NO cannot remove ROS in situ, but will promote destructive ONOO - Therefore, the key problem that needs to be solved in the treatment of CIRI with NO-related therapies is to ensure the sustained, stable and “pure” release of NO. Summary of the invention

[0005] The present invention aims to solve the technical problems in the prior art that drugs cannot cross the blood-brain barrier and cannot simultaneously inhibit neuronal calcium overload, reduce mitochondrial damage and endoplasmic reticulum stress, inhibit inflammatory storm and other multiple effects, and provides a nano drug with eNOS enzyme activity and its preparation method and application.

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

[0007] A nanomedicine with eNOS-like enzyme activity is a NO-embedded molybdenum sulfide nanomaterial (MSNO) obtained by introducing S-nitroso (-SNO) groups on the surface of small-particle molybdenum sulfide (MS); the S-nitroso groups are doubly connected to the molybdenum sulfide surface via S-NO bonds and Mo-N coordination bonds.

[0008] In the above technical solution, preferably, the particle size of the small-particle molybdenum sulfide is in the range of 20-200 nm.

[0009] In the above technical solution, preferably, the S-nitroso (-SNO) group is introduced on the surface of small-particle molybdenum sulfide (MS) by reacting the small-particle molybdenum sulfide (MS) with a chemical drug containing the S-nitroso (-SNO) group.

[0010] In the above technical solution, it is further preferred that the chemical drug containing the S-nitroso (-SNO) group is sodium nitrite or sodium nitroprusside.

[0011] A method for preparing a nano drug having eNOS-like enzyme activity comprises the following steps:

[0012] Ammonium molybdate tetrahydrate (NH 4 ) 6 Mo 7 O 24 ·4H 2 O) and thiourea (CN 2 H 4 S) preparing MS by hydrothermal method; screening small particles of MS by differential centrifugation;

[0013] The small particle MS is washed with hydrochloric acid and reacted with a chemical containing an S-nitroso (-SNO) group to introduce an S-nitroso (-SNO) group on the MS surface to obtain NO-embedded MSNO.

[0014] In the above technical scheme, preferably, the reaction system prepared by the MS is an alkaline buffer solution with a pH of 8 to 10, the reaction temperature is 180°C, and the reaction time is 7 to 24 hours; the reaction system for introducing the S-nitroso (-SNO) group is 0°C, and the reaction time is 12 to 24 hours.

[0015] In the above technical solution, preferably, the mass ratio of the ammonium molybdate tetrahydrate to thiourea is between 5:1 and 1:5.

[0016] In the above technical scheme, preferably, the rotation speed for screening small particle MS by differential centrifugation is between 1000rpm and 5000rpm, and the centrifugation time is between 1 and 10min; when washing small particle MS, the concentration range of hydrochloric acid is 6mol / L to 12mol / L, and the washing time is 12 to 24h.

[0017] In the above technical solution, preferably, the mass ratio of the MS to the chemical drug containing the S-nitroso (-SNO) group is between 10:1 and 1:10.

[0018] Application of a nano drug with eNOS-like enzyme activity in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

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

[0020] The nano drug with eNOS-like enzyme activity of the present invention is prepared by further introducing S-nitroso (-SNO) on the surface of small particles of MS, and has super strong antioxidant activity and stable NO release ability ( Figure 1 ).

[0021] The nano drug with eNOS-like enzyme activity of the present invention is prepared by adopting a simple and green synthesis method. The raw materials adopted are cheap and easily available, and the synthesis method is simple.

[0022] The nanomedicine of the present invention having eNOS-like enzyme activity can effectively target brain tissue damaged by CIRI and stably release pure NO ( Figure 2 ).

[0023] The nano drug with eNOS-like enzyme activity of the present invention can effectively treat cerebral ischemia-reperfusion injury ( Figure 3 ).

[0024] The nanomedicine with eNOS-like enzyme activity of the present invention can simultaneously improve the intracellular Ca2+-induced CIRI. 2+ Overload, oxidative stress and inflammatory storm ( Figure 4 ).

[0025] The nanomedicine with eNOS-like enzyme activity of the present invention can effectively protect neuronal mitochondria from overloaded Ca 2+ and mtROS damage, and significantly restored mitochondrial function in CIRI ( Figure 5 ).

[0026] The nanomedicine of the present invention having eNOS-like enzyme activity 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 ).

[0027] The nanomedicine with eNOS-like enzyme activity of the present invention can effectively alleviate neuronal endoplasmic reticulum stress associated with mitochondrial damage and significantly reduce neuronal apoptosis ( Figure 7 ).

[0028] The nanomedicine with eNOS-like enzyme activity of the present invention has good biocompatibility, is non-toxic to hippocampal neuron cells (SH-5Y5Y cells) and microglia (BV2 cells) at the administration concentration of the present invention, and has no effect on the heart, liver, spleen, lung, kidney and brain of normal rats after long-term administration ( Figure 8-10 ). 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 The results of the synthesis and characterization of the nanodrug 1 (MSNO) synthesized in Example 1 are shown in Figure 1. Among them, a is a schematic diagram of the synthesis of MSNO; b is a transmission electron microscope (TEM) visualization of MSNO; c is a hydrodynamic diameter detection result of MSNO; d is a FT-IR spectrum of MSNO and MS; e is an XPS spectrum of Mo 3p of MS and Mo 3p+N 1s of MSNO; f is an XPS spectrum of S2p of MS and MSNO; g is a NO release behavior diagram of MSNO and GSNO at the same dose (1 mg / mL); h is the reaction of MSNO to O 2 .- The scavenging capacity diagram; i is GSNO, GSNO+O 2 .- 、MS+O 2 .- 、MSNO+O 2 .- ONOO - Generate an ability diagram; j is the scavenging ability diagram of MSNO for ·OH.

[0031] Figure 2 The results of the detection of the targeting of the nano drug 1 (MSNO) synthesized in Example 1 to the ischemic brain tissue of CIRI and the NO level in the brain tissue are shown in the figure. Among them, a is a schematic diagram of BBB damage during CIRI; b is a diagram of the MCAO model; c is a TEM image of brain tissue capillaries; d is a statistical diagram of the endothelial gap of the capillaries in I / R brain tissue; e is a representative image of the bright field and fluorescence imaging of the brain, heart, lung, liver, spleen, and kidney of the rats in the Sham group and I / R group 1 hour after the injection of FITC-MSNO; f is a diagram of the Mo content in the brain tissue of the rats in the Sham group and I / R group detected by ICP-MS at different time points after intravenous injection of MSNO; gh is a representative image (g) and quantitative statistical diagram (h) of the NO level in the cerebral infarction area detected by small animal imaging technology at different time points; ij is the brain tissue ONOO -Horizontal representative images (j) and quantitative statistical graphs (i); kl are graphs showing the changes in systolic blood pressure (k) and diastolic blood pressure (l) over time after administration of equal doses of GSNO and MSNO to SD rats.

[0032] Figure 3 The results of the efficacy of the nanodrug 1 (MSNO) synthesized in Example 1 in improving CIRI are shown in Figure 1. Among them, a is a flow chart of the efficacy study of MSNO in CIRI; b is a quantitative statistical graph of TTC staining of CIRI rats at different doses of MSNO; cd are representative images (c) and quantitative statistical graphs (d) of TTC staining of CIRI rat brain tissue treated with NAC, GSNO, MS, and MSNO 0.5 mg / kg; e is a neurological score graph of CIRI rat brain tissue treated with NAC, GSNO, MS, and MSNO 0.5 mg / kg; f is a representative image of Nissl staining of brain tissue of rats in different treatment groups (red arrows mark atrophic neurons, and black arrows mark tissue cavitation); g is a representative image of HE staining of brain tissue of different treatment groups.

[0033] Figure 4 The result diagram of the investigation of the mechanism of improving CIRI by the nanodrug 1 (MSNO) synthesized in Example 1. Among them, a is the correlation heat map between the samples in each group; b is the result diagram of the VENN / UpSetR graphical analysis showing the differentially expressed genes between the Sham group, the I / R group and the MSNO treatment group; c is the result diagram of the volcano map depicting the differentially expressed genes between the brain tissues of the MSNO group and the I / R group; d is the GO enrichment analysis diagram of the biological processes involved in the differentially expressed genes between the I / R group and the MSNO treatment group; ej is the visualization heat map of the differentially expressed genes in the biological processes involved in the GO enrichment analysis, including the calcium signaling pathway diagram (e), the response to inflammation diagram (f), the mitochondrial function diagram (g), the response and regulation of ROS diagram (h), the ERS diagram (i), and the apoptosis process diagram (j); k is the KEGG pathway enrichment analysis diagram of the DEGs of the MSNO treatment group and the I / R group.

[0034] Figure 5 The nano drug 1 (MSNO) synthesized in Example 1 effectively protects mitochondria from overloaded Ca 2+ The results of the experiment showed that the damage of mitochondria and mtROS in CIRI was significantly restored. Among them, a and b are the Ca2+ and Ca2+ in SH-SY5Y cells in different treatment groups. 2+Representative graph (a) and quantitative statistical graph (b) of content; c is the percentage of DCFH-DA (ROS fluorescent probe) positive cells in each treatment group analyzed by flow cytometry; d is the representative image and co-localization analysis graph of FITC-MSNO and mitochondria; e is the representative image of mtROS in SH-SY5Y cells in each group; fg are the representative images (f) and quantitative analysis graph (g) of JC-1 staining to evaluate the integrity of MMPs in cells in each group; h is the ATP generation determination graph in SH-SY5Y cells; i is the representative fluorescence image of ROS level in brain tissue of rats in each group; jl is the TEM image of mitochondria in brain tissue neurons of Sham group (j), I / R group (k), and MSNO treatment group (l).

[0035] Figure 6 The result graphs show that the nano drug 1 (MSNO) synthesized in Example 1 inhibits the activation of microglial cGAS-STING signals, thereby reversing the neuroinflammation induced by CIRI. Among them, a is the dsDNA / Tom20 / DAPI immunofluorescence staining of the cerebral infarction area of ​​each treatment group, and the white arrow indicates mtDNA; b is the result graph of immunofluorescence staining iNOS (M1) and CD206 (M2) to detect the phenotype of microglia in the infarction area; c is the expression graph of cGAS in the cerebral infarction tissue of each treatment group by immunohistochemistry analysis; d is the expression graph of STING in the cerebral infarction tissue of each treatment group by immunohistochemistry analysis; ei is the WB method to detect inflammation-related proteins in brain tissue homogenate (c The expression levels of GAS (f), STING (g), P-IRF3 / IRF3 (h), and P-P65 / P65 (i)) were measured and a representative graph was drawn (e); jo is the detection graph of brain tissue inflammatory factors, including pro-inflammatory factors IL-1β (j), IL-6 (k), iNOS (l), and TNF-α (m) and anti-inflammatory factors IL-4 (n) and IL-10 (o); p is the mechanism diagram of MSNO improving neuroinflammation by inhibiting the microglial cGAS-STING pathway.

[0036] Figure 7 This is a graph showing the results of the nanodrug 1 (MSNO) synthesized in Example 1 inhibiting neuronal endoplasmic reticulum stress and apoptosis.

[0037] Among them, ac are transmission electron microscopy photos of ER in rat brain neurons in the sham group (a), I / R group (b), and MSNO treatment group (c), and the yellow arrow indicates ER; d is a schematic diagram of the cascade of ERS and mitochondrial damage; eh is a diagram of the expression level of ERS-related factors in brain tissue detected by qPCR, including Bip (e), PERK (f), ATF-6 (g), and IRE-1α (h); ij is a diagram of TUNEL staining of brain tissue (j) and quantitative analysis (i); ko is a WB method to detect the expression level of apoptosis-related proteins (Bax (k), bcl-2 (l), C-Cas3 / Cas3 (m), C-Cyt C (n)) in brain tissue homogenate and draw a representative figure (o). pq is the flow cytometry analysis (q) and the quantitative analysis results of apoptosis of SH-SY5Y cells in different treatment groups (p).

[0038] Figure 8 The results of the in vivo biosafety assessment of the nano drug 1 (MSNO) synthesized in Example 1 are shown in Figure 1. Wherein, a is a HE staining of the main organs (heart, liver, spleen, lung, kidney and brain) of normal rats and rats 24 hours after injection of MSNO; be is a graph showing the results of liver function (alanine aminotransferase (b) and γ-glutamyl transferase (c)) and renal function (creatinine clearance (d) and blood urea nitrogen (e)) detection in rats 24 hours after sublingual intravenous injection of MSNO; fs is a graph showing the results of a routine blood test (24 hours after sublingual intravenous injection of MSNO), including white blood cell count (f), neutrophil count (g), lymphocyte count (h), monocyte count (i), neutrophil percentage (j), monocyte percentage (k), red blood cell count (l), hemoglobin count (m), hematocrit (n), mean corpuscular hemoglobin content (o), coefficient of variation of red blood cell distribution width (p), platelet count (q), mean platelet volume (r), and platelet distribution width (s).

[0039] Figure 9-10 The result diagram of the in vitro biosafety assessment of the nano drug 1 (MSNO) synthesized in Example 1, wherein: Fig. 9 This is a graph showing the effect of different concentrations of MSNO on cell viability after 24h and 48h of treatment of SH-SY5Y cells using CCK8 method; Fig.10 This is a graph showing the effect of different concentrations of MSNO on cell viability after 24h and 48h of treatment with BV2 cells using the CCK8 method. DETAILED DESCRIPTION

[0040] The inventive concept of the present invention is that NO has been identified as one of the most functional and unique molecules in the human body, and its protective effect in cerebrovascular diseases has also been widely verified. However, the beneficial effects of NO depend on its concentration and ROS levels, which makes the current NO donors face great difficulties in treating CIRI. Based on this, the present invention aims to develop a customized MoS-based 2 The NO donor (MSNO) double-links the nitrosyl group to MoS via S-NO bond and Mo-N coordination bond. 2 The two-dimensional plane enables it to stably release NO similar to the eNOS enzyme and avoid the production of highly toxic ONOO - After intravenous injection, MSNO with appropriate nanosize can penetrate the blood-brain barrier and effectively treat CIRI through multiple effects: inhibiting calcium overload, alleviating mitochondrial damage and endoplasmic reticulum stress, and inhibiting the inflammatory storm. This invention opens up a new way to treat CIRI and also provides a promising method for other ischemia-reperfusion injury diseases.

[0041] The following examples are used to clearly and completely describe the technical solutions of the present invention, but it should be understood that the following examples do not limit the protection scope of the present invention. The reagents used in the examples are all commercially available products.

[0042] Example 1

[0043] (1) Synthesis of molybdenum sulfide (MS)

[0044] (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (18.5375 g) and CN 2 H 4 S (7.9925 g) was fully dissolved in 500 mL of deionized water, and the pH value of the reaction system was detected to be about 9. It was then transferred to a high-pressure reactor and reacted at 180° C. for 24 h. The reaction product was repeatedly washed three times with ultrapure water to remove other impurities, and MS was obtained.

[0045] (2) Screening of small particle MS

[0046] The sample obtained in step (1) was centrifuged at 2000 rpm for 4 min to obtain small particles of MS. The particle size of the small particles of molybdenum sulfide was in the range of 20-200 nm.

[0047] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0048] Take the sample MS (0.5 g) obtained in step (2) and 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stir for 24 h. Transfer the system to ice, precool for 10 min, and then add 1 mL of NaNO 2 (0.1 g / mL), react in an ice bath in the dark, and wash the reaction product three times with ultrapure water to obtain the final product, nanodrug, which is called nanodrug 1 and denoted by MSNO.

[0049] Figure 1 Figure b is a TEM visualization of the eNOS enzyme-like nanomedicine prepared based on MS as a carrier. From the TEM image, it can be seen that the particle size of the nanomedicine is between 60-100nm.

[0050] Example 2

[0051] (1) Synthesis of molybdenum sulfide (MS)

[0052] (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (22.5 g) and CN 2 H 4 S (4.5 g) was fully dissolved in 500 mL of deionized water, and the pH value of the reaction system was detected to be about 10, and then transferred to a high-pressure reactor for reaction at 180°C for 24 h. The reaction product was repeatedly washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0053] (2) The screening of small particle MS is the same as in Example 1.

[0054] (3) The synthesis of NO-embedded molybdenum sulfide (MSNO) was the same as in Example 1, and the obtained nanomedicine was called nanomedicine 2.

[0055] Example 3

[0056] (1) Synthesis of molybdenum sulfide (MS)

[0057] (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (20.25 g) and CN 2 H 4 S (6.75 g) was fully dissolved in 500 mL of deionized water, and the pH value of the reaction system was detected to be about 9.5, and then transferred to a high-pressure reactor for reaction at 180°C for 24 h. The reaction product was repeatedly washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0058] (2) The screening of small particle MS is the same as in Example 1.

[0059] (3) The synthesis of NO-embedded molybdenum sulfide (MSNO) was the same as in Example 1, and the obtained nanomedicine was called nanomedicine 3.

[0060] Example 4

[0061] (1) Synthesis of molybdenum sulfide (MS)

[0062] (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (13.5 g) and CN 2 H 4 S (13.5 g) was fully dissolved in 500 mL of deionized water, and the pH value of the reaction system was detected to be about 8.5, and then transferred to a high-pressure reactor for reaction at 180°C for 12 h. The reaction product was repeatedly washed three times with ultrapure water to remove other impurities, and MS was obtained.

[0063] (2) The screening of small particle MS is the same as in Example 1.

[0064] (3) The synthesis of NO-embedded molybdenum sulfide (MSNO) was the same as in Example 1, and the obtained nanodrug was called nanodrug 4.

[0065] Example 5

[0066] (1) Synthesis of molybdenum sulfide (MS)

[0067] (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (6.75 g) and CN 2 H 4 S (20.25 g) was fully dissolved in 500 mL of deionized water, and the pH value of the reaction system was detected to be about 8.3, and then transferred to a high-pressure reactor for reaction at 180°C for 24 h. The reaction product was repeatedly washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0068] (2) The screening of small particle MS is the same as in Example 1.

[0069] (3) The synthesis of NO-embedded molybdenum sulfide (MSNO) was the same as in Example 1, and the obtained nanodrug was called nanodrug 5.

[0070] Example 6

[0071] (1) Synthesis of molybdenum sulfide (MS)

[0072] (NH 4 ) 6 Mo 7O 24 ·4H 2 O (4.5 g) and CN 2 H 4 S (22.5 g) was fully dissolved in 500 mL of deionized water. The pH value of the reaction system was detected to be about 8, and then it was transferred to a high-pressure reactor for reaction at 180 °C for 24 h. The reaction product was washed repeatedly with ultrapure water three times to remove other impurities, and MS was obtained.

[0073] (2) The screening of small-particle MS was the same as that in Example 1.

[0074] (3) The synthesis of molybdenum sulfide embedded with NO (MSNO) was the same as that in Example 1, and the obtained nanomedicine was called nanomedicine 6.

[0075] Example 7

[0076] (1) The synthesis of molybdenum sulfide (MS) was the same as that in Example 1.

[0077] (2) The screening of small-particle MS

[0078] The sample obtained in step (1) was taken and centrifuged and screened at 1000 rpm for 4 min, and the precipitate was discarded to obtain small-particle MS.

[0079] (3) The synthesis of molybdenum sulfide embedded with NO (MSNO) was the same as that in Example 1, and the obtained nanomedicine was called nanomedicine 7.

[0080] Example 8

[0081] (1) The synthesis of molybdenum sulfide (MS) was the same as that in Example 1.

[0082] (2) The screening of small-particle MS

[0083] The sample obtained in step (1) was taken and centrifuged and screened at 3000 rpm for 4 min, and the precipitate was discarded to obtain small-particle MS.

[0084] (3) The synthesis of molybdenum sulfide embedded with NO (MSNO) was the same as that in Example 1, and the obtained nanomedicine was called nanomedicine 8.

[0085] Example 9

[0086] (1) The synthesis of molybdenum sulfide (MS) was the same as that in Example 1.

[0087] (2) The screening of small-particle MS

[0088] The sample obtained in step (1) was taken and centrifuged and screened at 4000 rpm for 10 min, and the precipitate was discarded to obtain small-particle MS.

[0089] (3) The synthesis of molybdenum sulfide embedded with NO (MSNO) was the same as that in Example 1, and the obtained nanomedicine was called nanomedicine 9.

[0090] Example 10

[0091] (1) The synthesis of molybdenum sulfide (MS) is the same as in Example 1.

[0092] (2) Screening of small particle MS

[0093] The sample obtained in step (1) was centrifuged at 5000 rpm for 4 min, and the precipitate was discarded to obtain small particles of MS.

[0094] (3) The synthesis of NO-embedded molybdenum sulfide (MSNO) was the same as in Example 1, and the obtained nanodrug was called nanodrug 10.

[0095] Embodiment 11

[0096] (1) The synthesis of molybdenum sulfide (MS) is the same as in Example 1.

[0097] (2) The screening of small particle MS is the same as in Example 1.

[0098] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0099] Take the sample MS (0.54 g) obtained in step (2) and 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stir for 24 h. Transfer the system to ice, precool for 10 min, and then add 1 mL of NaNO 2 (0.06 g / mL), reacted in an ice bath in the dark for 7 h, and the reaction product was repeatedly washed three times with ultrapure water to obtain the final product called nanodrug 11.

[0100] Example 12

[0101] (1) The synthesis of molybdenum sulfide (MS) is the same as in Example 1.

[0102] (2) The screening of small particle MS is the same as in Example 1.

[0103] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0104] Take the sample MS (0.3 g) obtained in step (2) and 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stir for 24 h. Transfer the system to ice, precool for 10 min, and then add 1 mL of NaNO 2 (0.3 g / mL), reacted in an ice bath in the dark for 7 h, and the reaction product was repeatedly washed three times with ultrapure water to obtain the final product called nanodrug 12.

[0105] Example 13

[0106] (1) The synthesis of molybdenum sulfide (MS) is the same as in Example 1.

[0107] (2) The screening of small particle MS is the same as in Example 1.

[0108] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0109] Take the sample MS (0.1 g) obtained in step (2) and 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stir for 24 h. Transfer the system to ice, precool for 10 min, and then add 1 mL of NaNO 2 (0.5 g / mL), reacted in an ice bath in the dark for 24 h, and the reaction product was repeatedly washed three times with ultrapure water to obtain the final product called nanodrug 13.

[0110] Embodiment 14

[0111] (1) The synthesis of molybdenum sulfide (MS) is the same as in Example 1.

[0112] (2) The screening of small particle MS is the same as in Example 1.

[0113] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0114] Take the sample MS (0.06 g) obtained in step (2) and 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stir for 24 h. Transfer the system to ice, precool for 10 min, and then add 1 mL of NaNO 2 (0.54 g / mL), reacted in an ice bath in the dark for 12 h, and the reaction product was repeatedly washed three times with ultrapure water to obtain the final product called nanodrug 14.

[0115] Embodiment 15

[0116] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the structure of the nanodrug was characterized, and the NO release and antioxidant properties of the nanodrug were evaluated. The specific steps are as follows:

[0117] NO detection kit (Biyuntian) was used to measure NO release in vitro. NO released by MSNO and S-nitrosoglutathione (GSNO) will be rapidly oxidized to NO in aqueous solution. 2 - , react with diazonium salt sulfonamide to form a diazo reaction, and then couple with naphthyl-vinyl diamine to form a product with a maximum absorption peak at 540nm. Place a 1.0mg / mL MSNO solution (or GSNO solution) in a 37℃ water bath, take out 1mL of sample at 0min, 1h, 3h, 6h, 12h, 24h, 2d, 3d, 4d, and 5d, and take the supernatant through a water filter (0.22μm). Add GriessⅠ and GriessⅡ respectively, and measure the absorbance of the mixture at a wavelength of 540nm using an enzyme reader.

[0118] 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, which has 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 MSNO solution of different concentrations (0μg / mL, 0.2mg / mL, 0.4mg / mL, 0.8mg / mL, 1.6mg / mL, 3.2mg / mL) are fully mixed, added to a quartz cuvette, placed on a UV / visible spectrophotometer to measure the baseline, and 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.

[0119] Hydroxyl radical (·OH) scavenging ability test: The terephthalic acid fluorescence spectrophotometry method was used to test the scavenging ability of MSNO 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. 4 (0.5 mM), terephthalic acid (0.5 mM), H 2 O 2 PBS solution of 1 mM and different concentrations of MSNO (0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL) was reacted at room temperature for 6 min, mixed thoroughly and added to a quartz cuvette. The characteristic absorption peak of fluorescent 2-hydroxyterephthalic acid was detected at 430 nm by a fluorescence spectrophotometer.

[0120] Peroxynitrite anion (ONOO - ) Scavenging ability test: The pyrogallol red method was used to detect the effect of MSNO on ONOO - Pyrogallol red has a specific absorption peak at 540nm, ONOO -It can quench pyrogallol red and reduce its specific absorption peak. Methods: 0.1M methionine (390μL), 20μM riboflavin (6μL), 0.1M (pH7.4) PBS (1.5mL), and 1.1mL ultrapure water were added to the cuvette in sequence and mixed well, then exposed to ultraviolet light for 5min to generate O 2 ·- The solution was transferred to a centrifuge tube, and 24 μL (1 mg / mL) of ultrapure water / GSNO / MS / MSNO and an equal amount of pure GSNO solution were added, respectively. After being kept at 37°C for 12 hours, 10 μL of pyrogallol red solution was added, and the characteristic absorption peak of pyrogallol red at 540 nm was measured after reacting at room temperature for 20 minutes. The control sample was a pyrogallol red solution without UV irradiation and drug.

[0121] The results are as follows Figure 1 As shown. Figure 1 From a, we can see that MSNO is prepared by introducing nitroso groups on the surface of MS; Figure 1 In b, we can see that MSNO is a three-dimensional flower-like structure composed of many nanosheets, and high-resolution TEM can be used to observe that the MSNO nanosheets are derived from MoS 2 The (002) crystal plane has an interlayer spacing of Depend on Figure 1 From c, we can see that the hydrodynamic average particle size of MSNO is 84.49nm; Figure 1 From the above, we can know that both MSNO and MS are at 591cm -1 and 932cm -1 The stretching vibration of Mo-S is contained at 695 cm -1 and 1576cm -1 There are characteristic peaks at , which are attributed to the stretching vibration of SN and N=O respectively; Figure 1 From the above, we can see that the N1s in MSNO are mainly attributed to the NO bond at 401.7 eV and the Mo-N bond at 398.8 eV, indicating that the nitroso group in MSNO can form a coordination bond with the adjacent Mo, thereby achieving a slow release of NO. Figure 1 From the figure, we can see that the SH bond at 164.2 eV in MSNO is significantly reduced compared with that in MS, indicating that -SH is successfully converted to -SNO during the preparation of MSNO. Figure 1As can be seen from the figure, it only takes 10 minutes for the NO in GSNO to be completely released, indicating that GSNO exhibits a bursty NO release behavior. In sharp contrast to GSNO, MSNO slowly releases NO over 120 hours, indicating that the coordination bond between NO and Mo in MSNO greatly slows down the self-cleavage rate of -SNO in aqueous solution. In addition, the NO release curve of MSNO shows an approximately linear correlation between NO concentration and time within the first 24 hours, indicating that MSNO has a controllable NO release behavior and that the steady state of NO concentration can be easily maintained by optimizing the dose of MSNO; Figure 1 It can be seen from the above that MSNO inherits the powerful O 2 .- Its pseudo-SOD activity is as high as 105U / mg; Figure 1 As we can see, in O 2 .- In the system, NO released by GSNO produces a large amount of ONOO - , while ONOO produced by MSNO group - Significantly less than the GSNO group, only 1 / 22 of the GSNO group; Figure 1 It can be seen from Figure 5 that MSNO inherits the scavenging ability of MS to OH. Figure 1 The results showed that MSNO was successfully prepared and had controllable NO release and antioxidant capabilities.

[0122] Example 16

[0123] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the targeting of the nanodrug to the ischemic brain tissue of CIRI rats and the NO level in the brain tissue were evaluated. The specific steps are as follows:

[0124] In vivo fluorescence tracking of drug distribution: Rats in the sham operation group (Sham group) and CIRI group (I / R group) were given fluorescently labeled nanodrug 1 (MSNO-FITC) through the sublingual vein. One 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.

[0125] ICP-MS detection of drug content in tissues: Rats in the sham group and the MSNO group were injected with MSNO, and the brain, heart, liver, spleen, lung, and kidney were collected 1h, 3h, 6h, 12h, and 24h later. The Mo content of each organ was determined by ICP-MS.

[0126] Brain tissue TEM: Brain tissues (less than 1 mm) of rats in the Sham group, I / R group, and MSNO group were collected. 3), fixed with fresh TEM fixative overnight, then washed with PBS three times (15 minutes each time), and then replaced with 1% osmium acid mixture for 2 hours. After gradient dehydration, the tissue was embedded and solidified. The sample was cut into 60-80nm thick slices with a microtome, double-stained with 3% uranyl acetate-lead citrate, and the neuronal mitochondrial morphology and the distribution of nanodrug 1 (MSNO) in mitochondria were observed with a Jeol 1200EX transmission electron microscope.

[0127] Brain tissue NO: Fresh brain tissues were collected from rats in the Sham, I / R, GSNO, MS, and MSNO groups at 10 min, 30 min, 1 h, and 6 h after drug injection, respectively. The brain tissues were washed three times with PBS to remove the surface blood, and then the brain tissues were cut into 5 slices vertically on the coronal plane. The brain tissue slices were immersed in DAF-FM DA dye solution, incubated at 37 ° C in the dark for 15 min, and the residual dye was washed with PBS, and then the small animal in vivo optical imaging system was used to collect images.

[0128] The results are as follows Figure 2 As shown. Figure 2 As shown in Figure a, during CIRI, the BBB is affected by ROS burst, proinflammatory cytokines, matrix metalloproteinases (MMPs), etc., and the tight junctions of capillary endothelial cells and the integrity of the basement membrane are destroyed, providing an opportunity for the specific enrichment of MSNO in CIRI lesions. Figure 2 As shown in Figure b, the rat CIRI model was constructed by simulating cerebral ischemia through the middle cerebral artery occlusion (MCAO) method, and the occlusive wire blocking the middle cerebral artery was removed 2 hours after ischemia to restore blood supply for 24 hours; Figure 2 As shown in Figure c, compared with the Sham group, 2 hours after ischemia, the vascular endothelial cells in the brain tissue of the I / R group were significantly swollen, and the tight junctions and basement membrane integrity were lost; Figure 2 As shown in Figure d, the average gap between tight junctions of capillary endothelial cells in brain tissue of the I / R group was about 120 nm, which provided an opportunity for MSNO with a smaller particle size (about 85 nm) to penetrate the BBB gap and target CIRI lesions with high specificity. Figure 2 As shown in Figure e, FITC-MSNO in the sham group was mainly distributed in the liver and kidneys, and was almost not distributed in the brain tissue of the sham group and the healthy brain tissue of the I / R group. This is because the BBB of the healthy brain tissue prevented the entry of MSNO. In contrast, only 1 hour after intravenous injection of FITC-MSNO in rats in the I / R group, FITC-MSNO specifically targeted CIRI lesions, and the fluorescence intensity of FITC-MSNO in the CIRI lesions of the I / R group was 27 times that of the sham group; Figure 2It can be seen from the figure that MSNO can achieve targeted distribution in ischemic brain tissue 1 hour after intravenous injection, and reaches a peak at around 6 hours (25 times that of the Sham group), and then the content gradually decreases, indicating that MSNO can target ischemic brain tissue and be degraded. These evidences fully confirm that MSNO can specifically target CIRI tissue and is biodegradable; Figure 2 It can be seen that in the MSNO group, NO was only distributed in the I / R brain tissue (right side of the yellow line), and a relatively stable NO concentration was maintained after 1 hour, but it was not distributed in the non-I / R brain tissue (left side of the yellow line). Unlike the MSNO group, in the GSNO group, the NO concentration in the non-I / R brain tissue was higher than that in the I / R brain tissue at 15 minutes. This interesting and diametrically opposite phenomenon stems from the different physicochemical properties of MSNO and GSNO and the pathological characteristics of the BBB during CIRI. As a large-sized nanodrug, MSNO tends to be enriched in CIRI lesions through damaged BBB gaps. The distribution of GSNO is closely related to the amount of blood perfusion because GSNO is a small molecule. Since the blood supply of healthy brain tissue is better than that of CIRI lesions, the NO level in CIRI lesions in the GSNO group is lower than that in healthy brain tissue. In addition, due to the shorter half-life of GSNO in the body, NO disappears faster in the GSNO group; Figure 2 It can be seen that ONOO in I / R group and GSNO group - Both were significantly increased, which were 6.21 times and 6.67 times of the sham group, respectively. MS and MSNO can significantly reduce ONOO - level, this is because MS has a strong ROS scavenging ability; Figure 2 As far as we know, GSNO caused a "cliff-like" drop in blood pressure in the GSNO group within 15 minutes, with both systolic and diastolic blood pressure dropping to half of normal blood pressure. In contrast, MSNO has no significant effect on systemic blood pressure due to its strong targeting of CIRI lesions and controllable release of NO. Figure 2 It can be seen that MSNO can target CIRI lesions with high specificity and can treat CIRI by scavenging high concentrations of ROS and stably releasing "pure NO".

[0129] Embodiment 17

[0130] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the therapeutic effect of the nanodrug on CIRI in rats was evaluated.

[0131] The specific steps are as follows:

[0132] 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, MSNO was given before reperfusion. After 24 hours of reperfusion, the neurological dysfunction of the rats was evaluated by the neurological function score standard. 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 drugs NAC and GSNO and MS at the same dose were used as controls. (The neurological function score was based on the ZeaLonga 5-point standard: 0 points represent no neurological deficit symptoms; 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.)

[0133] HE staining and 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 HE staining and Nissl staining were used to observe the pathological changes of each tissue.

[0134] The results are as follows Figure 3 As shown. Among them, Figure 3 a in the table is the treatment regimen of MSNO for the treatment of CIRI; Figure 3 As shown in Figure b, even at ultra-low therapeutic doses, MSNO can significantly reduce the area of ​​cerebral infarction, and the efficacy increases in a dose-dependent manner. At the optimal dose (0.5 mg / kg), the area of ​​cerebral infarction treated with MSNO was reduced to 7.89%, which is much lower than the 44.58% of the I / R group; Figure 3 From the CD, we can see that the infarct area in the MSNO group was the smallest, much smaller than that in the MS group, NAC group, GSNO group and I / R group. MS also has a certain therapeutic effect on CIRI due to its strong ability to clear ROS, while the effects of NAC and GSNO are minimal; Figure 3 It can be seen from Figure 5 that the therapeutic effect of MSNO in improving the neurological function of I / R rats is far greater than that of MS, NAC and GSNO; Figure 3 As shown in Figure 5, the number of Nissl staining spots (Nissl bodies) in CIRI lesions in the I / R group was significantly reduced, and the morphology was reduced from regular tiger-striped circles to irregular dots or vacuoles. Both MS and MSNO treatments can improve the number and morphology of Nissl bodies, and the therapeutic effect of MSNO is significantly better than that of MS. Figure 3 It can be seen from the figure that MSNO treatment can significantly improve brain tissue structure. Figure 3It can be seen that MSNO has a strong therapeutic effect on CIRI, and the effect is much better than MS, GSNO and NAC, suggesting that the efficacy of MSNO comes from its high targeting to CIRI lesions, ROS scavenging ability and controllable and precise release of NO.

[0135] Embodiment 18

[0136] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the possible mechanism of MSNO in treating CIRI was evaluated.

[0137] RNA sequencing analysis: Total RNA was extracted from brain tissues of the Sham, I / R, and MSNO 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.

[0138] The results are as follows Figure 4 As shown. Figure 4 From a, we can see that the Pearson correlation coefficients between the three samples in each group are all greater than 0.94, indicating that the sample data are highly consistent. Figure 4 As shown in Figure b, VENN / UpSetR analysis found that there were 1937 differentially expressed genes (DEGs) between the Sham group and the I / R group, while there were only 99 DEGs between the MSNO group and the Sham group. Figure 4 As shown in Figure c, compared with the I / R group, 1647 genes were up-regulated and 790 genes were down-regulated in the MSNO group; Figure 4 As shown in Figure d, the DEGs between the Sham group and the I / R group were compared with the gene ontology (GO) database, and the biological processes involved mainly included calcium ion channels, inflammatory response, mitochondrial function, oxidative stress, ERS, and apoptosis; Figure 4 As can be seen from Figure 5, the gene expression levels of the above six biological processes in the I / R group changed significantly compared with the Sham group, while the gene expression levels in the MSNO treatment group were close to those in the Sham group, which fully confirmed the effective regulatory effect of MSNO on the genes related to the above six biological processes; Figure 4 As shown in Figure 5, compared with the I / R group, the DEGs in the MSNO group were enriched in Ca 2+Related signaling pathways, mitochondrial damage signaling pathways (including ER function, cytokine-cytokine receptor interaction and cell apoptosis) and inflammation-related signaling pathways (cytoplasmic DNA sensing pathway, tumor necrosis factor (TNF) signaling pathway and NF-κB signaling pathway) are strongly activated during CIRI, and MSNO can effectively inhibit the activation of these pathways. Figure 4 It can be seen that MSNO can simultaneously improve CIRI-induced intracellular Ca 2+ Overload, oxidative stress, and inflammatory storm.

[0139] Embodiment 19

[0140] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the nanodrug was evaluated to effectively protect mitochondria from overloaded Ca 2+ The results of this study showed that the damage of mtROS and the significant restoration of mitochondrial function in CIRI were induced. The specific steps are as follows:

[0141] 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).

[0142] Cell level: SH-SY5Y cells were cultured at 1×10 4 / well inoculated in a 24-well plate, SH-SY5Y cells were treated with a medium containing (2μg / mL) MSNO and 400μM cobalt chloride for 16h after 24h, and then replaced with a medium containing only 2μg / mL MSNO for 8h. 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 25min, washed twice with buffer, and observed and collected images under a fluorescence microscope; SH-SY5Y cells were plated at 1×10 4 / well were inoculated in a 24-well plate. After 24 hours, SH-SY5Y cells were treated with medium containing (2μg / mL) MSNO and 400μM cobalt chloride for 16 hours, and then replaced with medium containing only 2μg / mL MSNO for another 8 hours. The ATP production content of SH-SY5Y cells was evaluated using an ATP detection kit.

[0143] The results are as follows Figure 5 As shown. Figure 5 As shown in Figure 1, the intracellular Ca 2+ The level was 3.45 times that of SH-SY5Y cells in the Normoxia group, indicating that H / R caused severe calcium overload in SH-SY5Y cells. 2+The level dropped to close to that of the Normoxia group, and the effect was significantly better than that of MS. 2+ In addition to overload, ROS burst is a key factor leading to mitochondrial damage in CIRI neurons; Figure 5 As shown in Figure c, by flow cytometry, after H / R, the percentage of DCFH-DA-positive cells in SH-SY5Y cells increased from 4.08% to 64.07%, while the percentage of DCFH-DA-positive cells in cells treated with MS and MSNO decreased to 7.53% and 6.39%, respectively, indicating that MSNO and MS can effectively eliminate the ROS burst caused by H / R. The main source of ROS in neuronal cells is mitochondria, and MSNO targeting mitochondria is the key to ROS elimination; Figure 5 As shown in Figure d, co-labeling with Mito-tracker and FITC-MSNO confirmed that MSNO efficiently targeted mitochondria of SH-SY5Y cells under H / R conditions, with a Pearson coefficient as high as 0.84. Figure 5 As shown in Figure e, H / R increased mtROS in SH-SY5Y cells by 3.36 times. As expected, MS and MSNO significantly reduced the level of mtROS in SH-SY5Y cells induced by H / R, and the mtROS level was close to that of the normal group; Figure 5 As shown in Figure fg, after H / R treatment, the proportion of normal MMP in SH-SY5Y cells decreased from 100% to 4.39%, while the proportion of normal MMP in the MSNO treatment group was significantly restored (80.26%). Figure 5 It can be seen from the figure that MSNO effectively reversed the decrease in ATP production caused by H / R, and the effect was better than that of MS, indicating that MSNO can effectively restore the mitochondrial function damaged by H / R in SH-SY5Y cells; Figure 5 As shown in Figure 1, the ROS level in the brain tissue of the post-I / R group increased to about 3.4 times that of the Sham group, while the ROS level in the CIRI lesions of the MS group and the MSNO group decreased to a level close to that of the Sham group; Figure 5 It can be seen from the results that the mitochondria of brain tissue neurons in the I / R group were severely damaged, with swelling, dissolution, unclear ridges and even vacuolization, while the mitochondrial structure was significantly restored after MSNO treatment, providing direct evidence for the protective effect of MSNO on mitochondria. Figure 5 It can be seen that MSNO can effectively protect mitochondria from Ca 2+ and mtROS overload-induced damage, and significantly restored mitochondrial function in CIRI.

[0144] Embodiment 20

[0145] Taking the nanodrug 1 (MSNO) synthesized 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:

[0146] Animal level: 24 hours after reperfusion, brain tissues of CIRI rats were obtained for paraffin sections. The sections were incubated with primary antibodies against dsDNA / Tom 20, iNOS, and CD206 (Anti-dsDNA antibody, Abcam, AB27156; Tom20 Polyclonal antibody, Proteintech, 11802-1-AP; iNOS antibody, Affinity, AF0199; CD206 Recombinant antibody, Proteintech, 81525-1-RR) at 4°C overnight, and incubated with corresponding secondary antibodies (Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488, Invitrogen, A11029; Goat anti-Rabbit IgG (H+L) Cross-AdsorbedSecondary Antibody, Alexa Fluor TM555, Invitrogen, A21428) was incubated at room temperature for 2 hours. The slides were sealed with an anti-fluorescence quenching solution containing DAPI, and then the enlarged fluorescence images were acquired using an LSM 900 equipped with Airyscan2 (Carl Zeiss). For quantification, for each brain tissue section, at least three random imaging fields within the lesion area were selected and analyzed using Image J; the expression levels of inflammation-related proteins in rat brain tissue were detected. Specifically: After 24h of reperfusion in CIRI rats, rat brain tissue was collected by tissue homogenizer, and proteins were separated using RIPA (protease and phosphatase inhibitors). The 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 specific primary antibodies (cGAS, STING, P65, IRF3, P-IRF3, P-P65) were incubated overnight at 4°C. After that, the membrane was washed three times with TBST and then incubated with secondary antibodies (Goat antimice IgG H&L (HRP); Goat anti Rabbit IgG H&L (HRP)) 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 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).

[0147] Cell level: Cell slides were placed in 24-well plates. Then, SH-SY5Y cells were cultured at 1×10 4 / well were inoculated in a 24-well plate. After 24 hours, SH-SY5Y cells were treated with a medium containing 2μg / mL MSNO-FITC + 400μM cobalt chloride for 16 hours, and then replaced with a medium containing only 2μg / mL MSNO-FITC for 8 hours. The cell slides were taken out and incubated with the primary antibody of dsDNA / Tom20 (Anti-dsDNA antibody, Abcam, AB27156; Tom20 Polyclonal antibody, Proteintech, 11802-1-AP;) at 4°C overnight, and with the corresponding secondary antibody (Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488, Invitrogen, A11029; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 555, Invitrogen, A21428) were incubated at room temperature for 2 hours. The slides were mounted with anti-fluorescence quenching solution containing DAPI, and then magnified fluorescence images were acquired using LSM 900 equipped with Airyscan2 (Carl Zeiss).

[0148] The results are as follows Figure 6 As shown. Figure 6 As shown in Figure a, a large amount of mtDNA was released from the damaged mitochondria in the CIRI lesions of the I / R group, and MSNO could significantly reduce the release of mtDNA, and its therapeutic effect was significantly better than that of MS. Figure 6 As shown in Figure b, most microglia in the CIRI lesions of the I / R group were activated to the M1 type, with an iNOS / CD206 / ratio of 4.02. Surprisingly, the M1 phenotype of microglia in the MSNO group decreased, while the M2 phenotype increased, suggesting that MSNO can promote the transformation of microglia from the M1 phenotype to the M2 phenotype; Figure 6 As can be seen from the CD, the number of cGAS and STING positive cells in the CIRI lesions of the I / R group increased significantly, while MSNO significantly inhibited the activation of cGAS-STING, indicating that MSNO regulates the phenotypic transformation of microglia by inhibiting the activation of the cGAS-STING pathway; Figure 6It can be seen from the data that the expression levels of cGAS, STING and downstream molecules P-IRF3 / IRF3, P-P65 / P65 in the brain tissue of the I / R model group were more than twice that of the Sham group, confirming that the cGAS-STING signaling pathway was significantly activated in CIRI. MSNO treatment can significantly reduce the I / R-induced high expression of cGAS and STING and the phosphorylation levels of IRF3 and P65, and can significantly reduce the release of I / R-induced proinflammatory factors (IL-1β, IL-6, iNOS and TNF-α), and increase the secretion of anti-inflammatory factors (IL-4 and IL-10). In summary, Figure 6 It can be seen from the p that mtDNA released by neurons can be quickly recognized by the dsDNA receptor cGAS on the microglia membrane, activating the downstream STING signaling pathway and causing a strong inflammatory response. MSNO inhibits the release of mtDNA by protecting neuronal mitochondria, significantly reduces the activation of the cGAS-STING pathway in microglia, eliminates neuroinflammation, and ultimately reduces the external apoptosis of neurons in CIRI.

[0149] Embodiment 21

[0150] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the protective effect of the nanodrug on endoplasmic reticulum stress (ERS) and apoptosis in the infarct area of ​​CIRI rats was evaluated. The specific steps are as follows:

[0151] Animal level: TdT-mediated dUTP end labeling (TUNEL) staining was used to analyze the apoptosis of cerebral infarction tissues in different groups. WB 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 in Example 20. The detailed methods of tissue TEM were the same as in Example 16.

[0152] 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.

[0153] Cell level: SH-SY5Y cells were collected at 1×10 5The cells were seeded at a density of 100 μg / mL in a 6-well plate. After the cells adhered to the wall, the cells in the normoxic group were incubated normally. The cells in the H / R model group were incubated with a culture medium containing 400 μM cobalt chloride for 16 h and then replaced with a normal culture medium for another 8 h. The cells in the drug administration group were incubated with a culture medium containing 400 μM cobalt chloride and MS / MSNO for 16 h, and then incubated with a culture medium containing only MS / MSNO for another 8 h. Subsequently, the cells in each group were collected, treated according to the instructions of the Annexin V-FITC / PI reagent, and then detected by flow cytometry.

[0154] The results are as follows Figure 7 As shown. Figure 7 As shown in the ac, accompanied by the destruction of neuronal mitochondria, the endoplasmic reticulum of neurons in the I / R group was significantly swollen under transmission electron microscopy, confirming that severe ERS occurred in CIRI neurons. As a control, the ER edema of neurons in the MSNO group was significantly improved, and its structure was similar to that of the Sham group, indicating that MSNO can effectively improve the ERS of neurons in CIRI. Figure 7 As shown in Figure d, neurons are extremely sensitive to the rate of protein synthesis. mtROS causes misfolding of neuronal proteins in CIRI. The misfolded proteins bind to Bip on the endoplasmic reticulum membrane and activate three ERS transmembrane molecules: inositol requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF-6), thereby inducing ERS and further promoting cell apoptosis. Specifically, PERK can phosphorylate eukaryotic initiation factor-2α (eIf-2α) and increase the expression of transcription factors such as activating transcription factor 4 (ATF-4). ATF-6 is transported to the Golgi apparatus, where it is cleaved by endopeptidases S1P and S2P to release cytoplasmic ATF6 fragments. ATF-4 and ATF-6 initiate the transcription of Chop and promote neuronal apoptosis. IRE1 activates the transcription factor X-box binding protein (XBP1), catalyzing the apoptosis signaling pathway mediated by JNK and Caspase-12. By Figure 7 As shown in Figure e, the expression of Bip was significantly increased in I / R brain tissue and H / R group SH-SY5Y cells, indicating that ER stress was triggered both in vivo and in vitro. Figure 7 As shown in Figure 5, PERK, ATF6, and IRE-1α were significantly activated, and further stimulated the expression of downstream factors, including eIf-2α, ATF-4, Chop, XBP-1s, JNK, and Caspase 12. Since MSNO can effectively eliminate mtROS, MSNO can inhibit ERS and almost completely reverse the activation of these pathways. Figure 7 It can be seen that the apoptosis rate of brain tissue cells in the I / R group was as high as 74.75%, and the apoptosis rate of cells in the MSNO treatment group (9.05%) was significantly reduced. The MS group had a certain effect (30.94%), but the effect was not as good as that in the MSNO group. Figure 7 The ko results showed that the expression levels of pro-apoptotic proteins Bax, cleaved-caspase3 / caspase3 (C-Cas / Cas), and cytoplasmic-cytochrome C (C-Cyt C) in the I / R group were significantly increased, which were 3.75, 3.57, and 2.3 times that of the Sham group, respectively. The expression of anti-apoptotic factor Bcl-2 was significantly reduced, which was 0.58 times that of the Sham group. Both MS and MSNO treatments can significantly reverse the expression of the above proteins, and the therapeutic effect of MSNO is significantly better than that of MS. Flow cytometry was further used to detect the improvement effect of MSNO on H / R-induced SH-SY5Y cell apoptosis. Figure 7 From the pq, we can see that the apoptosis rate of SH-SY5Y cells after H / R induction was as high as 39.81%, the apoptosis rate of cells in the MS treatment group dropped to 24.25%, and the apoptosis rate of cells in the MSNO treatment group dropped to 12.30%, indicating that MSNO can also effectively inhibit H / R-induced neuronal apoptosis in vitro. Figure 6 It can be seen that MSNO can effectively alleviate ERS and significantly reduce neuronal apoptosis, and its effect is far better than MS which can only clear ROS.

[0155] Embodiment 22

[0156] Taking the nanodrug 1 (MSNO) synthesized 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:

[0157] Animal level: 24 hours after the tail vein injection of healthy SD mice with 5mg / kg MSNO nanodrug (dissolved in saline), the mice were euthanized and the main organs (brain, heart, liver, spleen, lung, kidney) were collected. Paraffin sections of various 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. Differentiation with 1% hydrochloric acid alcohol solution, soak in water to turn blue for 2 minutes, eosin stain was added to the tissue for about 10s, and eosin staining was completed, and it was immediately immersed in anhydrous ethanol for dehydration twice (2 minutes each time). Soak in xylene twice (2 minutes each time), dry naturally in a fume hood for 10-30 minutes, seal with neutral resin, observe and collect images under a microscope; take blood samples to evaluate rat liver function-related indicators (alanine aminotransferase (ALT) and γ-glutamyl transferase (γ-GT)), renal function indicators (creatinine (CR) and urea nitrogen (BUN))

[0158] Cell level: SH-SY5Y cells and BV2 cells were cultured at 1×10 4 / well was inoculated into a 96-well plate and incubated for 24 hours. MSNO was dispersed in the culture medium to prepare cell culture fluids of different concentrations (0, 0.25, 0.5, 1, 5, 2, 4, 8, 16, 32 and 64 μg / mL, respectively). The above cell culture fluid was added to SH-SY5Y 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.

[0159] The results are as follows Figure 8-10 Nanodrug 1 (MSNO) had no obvious toxicity to SH-SY5Y 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 MSNO nanodrugs, and liver and kidney function and blood routine indicators were within the normal range.

[0160] 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 nano drug having eNOS-like enzyme activity, characterized in that: It is a NO-embedded molybdenum sulfide nanomaterial (MSNO) obtained by introducing S-nitroso (-SNO) groups on the surface of small-particle molybdenum sulfide (MS), wherein the S-nitroso groups are doubly connected to the molybdenum sulfide surface through S-NO bonds and Mo-N coordination bonds.

2. The nano drug having eNOS-like enzyme activity according to claim 1, characterized in that: The particle size of the small particles of molybdenum sulfide is in the range of 20-200 nm.

3. The nano drug having eNOS-like enzyme activity according to claim 1, characterized in that: The S-nitroso (-SNO) group is introduced on the surface of small-particle molybdenum sulfide (MS) by reacting the small-particle molybdenum sulfide (MS) with a chemical drug containing the S-nitroso (-SNO) group.

4. The nano drug having eNOS-like enzyme activity according to claim 3, characterized in that: Chemical drugs containing the S-nitroso (-SNO) group are sodium nitrite or sodium nitroprusside.

5. A method for preparing a nano drug having eNOS-like enzyme activity according to any one of claims 1 to 4, characterized in that: The following steps are involved: Ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 ·MS was prepared by hydrothermal method using 4H2O) and thiourea (CN2H4S); Small particles of MS were screened out by differential centrifugation; The small particle MS is washed with hydrochloric acid and reacted with a chemical containing an S-nitroso (-SNO) group to introduce an S-nitroso (-SNO) group on the MS surface to obtain NO-embedded MSNO.

6. The method for preparing the nano drug having eNOS-like enzyme activity according to claim 5, characterized in that: The reaction system for the MS preparation is an alkaline buffer solution with a pH of 8 to 10, a reaction temperature of 180° C., and a reaction time of 7 to 24 hours; the reaction system for the introduction of the S-nitroso (-SNO) group is 0° C. and a reaction time of 12 to 24 hours.

7. The method for preparing the nano drug having eNOS-like enzyme activity according to claim 5, characterized in that: The mass ratio of the ammonium molybdate tetrahydrate to thiourea is between 5:1 and 1:

5.

8. The method for preparing the nano drug having eNOS-like enzyme activity according to claim 5, characterized in that: The rotation speed of the differential centrifugation method for screening small particle MS is between 1000rpm and 5000rpm, and the centrifugation time is between 1 and 10 minutes; the concentration range of hydrochloric acid when washing small particle MS is 6mol / L to 12mol / L, and the washing time is 12 to 24h.

9. The method for preparing the nano drug having eNOS-like enzyme activity according to claim 5, characterized in that: The mass ratio of the MS to the chemical drug containing an S-nitroso (-SNO) group is between 10:1 and 1:

10.

10. Use of the nano drug having eNOS-like enzyme activity according to any one of claims 1 to 4 in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

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