Preparation of nano-drug with NO catalytic performance and application thereof in treatment of infective endocarditis

Abstract

The application discloses a kind of nano-medicines with NO catalytic performance and its application in infective endocarditis treatment.The application dissolves molybdate and hydrogen phosphate in water respectively, then the two solutions are stirred and mixed to form Keggin type heteropoly acid of oxidized molybdenum base by self-assembly, i.e., the nano-medicines with NO catalytic performance (OX).The nano-medicines prepared by the application have GSNO catalytic properties and multiple antibacterial properties, and also have antithrombotic and vascular regulation functions, can efficiently catalyze endogenous GSNO to generate NO, through the synergistic effect of "oxidation antibacterial-NO regulation" dual functions, to realize efficient treatment of infective endocarditis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a preparation of a nano-drug with NO catalytic performance and application thereof in the treatment of infective endocarditis. BACKGROUND

[0002] Infective endocarditis (IE) is an intravascular infectious disease, which is usually caused by inflammation of the endocardium of the valve or ventricular wall after direct infection by bacteria, fungi or other pathogenic microorganisms. When the surface of the heart valve is damaged, Staphylococcus aureus in the blood flow is easy to adhere and form obvious vegetations. Although there has been significant progress in the diagnosis and treatment of IE in recent years, it is still a disease with poor prognosis and has a high mortality rate in clinic. Staphylococcus aureus has become the main pathogenic organism of IE, and the infection is the most serious. Since the combination of the vegetations is relatively loose, part of the vegetations will cause downstream organ embolism, visceral abscess and other complications after falling off, which is difficult to treat. At present, the clinical treatment relies on the combination of antibiotics and surgery, but the drug resistance is enhanced and the biofilm barrier effect is significantly reduced, so new treatment strategies are urgently needed.

[0003] Nitric oxide (NO) has become a research hotspot for the treatment of IE due to its multiple effects of broad-spectrum antibacterial, inhibition of platelet activation and regulation of vascular function. However, exogenous NO donors (such as NONOates, sodium nitroprusside, etc.) have defects such as poor stability, short half-life and insufficient targeting. Although nano-delivery systems (such as metal-organic frameworks, polymer nanoparticles) can partially improve the drug loading efficiency, the uncontrolled decomposition and leakage in circulation still cannot achieve the sustained release of NO at the lesion site.

[0004] Studies have shown that inflammation caused by harmful stimuli and infection, tissue damage, etc. can recruit immune cells (macrophages, neutrophils, etc.), cause an oxidative (or respiratory) burst, involve the rapid generation and release of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and help eliminate microorganisms. Excessive RNS further leads to the accumulation of S-nitrosyl thiol (SNO). The accumulation of reactive nitrogen species (RNS) can induce the formation of S-nitrosyl glutathione (GSNO) and other S-nitrosyl thiols (RSNO). GSNO, as a major source of endogenous NO, is chemically stable and can release NO through transition metal ion catalysis, and has both antibacterial and antithrombotic functions. Therefore, it is of important clinical significance to develop a new treatment system based on the generation of endogenous NO. SUMMARY

[0005] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a preparation method of a nano-drug with NO catalytic performance.

[0006] Another object of the present application is to provide the nano-drug with NO catalytic performance prepared by the method.

[0007] Still another object of the present application is to provide the application of the nano-drug with NO catalytic performance.

[0008] The object of the present application is achieved by the following technical solutions.

[0009] A preparation method of a nano-drug with NO catalytic performance, comprising the following steps:

[0010] (1) dissolving molybdate in water to obtain a molybdate solution; dissolving hydrogen phosphate in water to obtain a hydrogen phosphate solution;

[0011] (2) mixing the hydrogen phosphate solution with the molybdate solution, stirring at 400-2000 rpm until colorless, obtaining a mixed solution, continuing to stir until the reaction is completed, adding an alcohol solvent to precipitate, centrifuging to collect, washing, drying, and obtaining the nano-drug with NO catalytic performance.

[0012] The molybdate in step (1) is at least one of ammonium molybdate and ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 ·4H2O); preferably ammonium molybdate tetrahydrate.

[0013] The concentration of the molybdate solution in step (1) is 0.1-1 mol / L; further preferably 0.3-0.8 mol / L; still further preferably 0.4-0.6 mol / L; more preferably 0.5 mol / L.

[0014] The hydrogen phosphate in step (1) is at least one of disodium hydrogen phosphate and sodium dihydrogen phosphate dodecahydrate (NaH2PO4·12H2O); preferably sodium dihydrogen phosphate dodecahydrate.

[0015] The concentration of the hydrogen phosphate solution in step (1) is 0.1-0.5 mol / L; further preferably 0.2-0.4 mol / L; still further preferably 0.25-0.3 mol / L; more preferably 0.292 mol / L.

[0016] The molar ratio of the molybdate to the hydrogen phosphate in step (1) is (40-60):1; preferably (45-55):1; more preferably 50:1.

[0017] The water in step (1) is ionized water or double deionized water (ddH2O).

[0018] In step (2), the hydrogen phosphate solution is slowly dripped into the ammonium molybdate solution for mixing.

[0019] The stirring speed in step (2) is preferably 1000-2000 rpm; further preferably 1200-2000 rpm; still further preferably 1400-1800 rpm; yet further preferably 1500-1700 rpm; most preferably 1600 rpm.

[0020] The reaction time in step (2) is 30-60 min.

[0021] The centrifugation condition in step (2) is 12000 rpm for 5-10 min; preferably 12000 rpm for 5 min.

[0022] The alcohol solvent in step (2) is preferably ethanol.

[0023] The volume ratio of the alcohol solvent to the mixed solution in step (2) is (3-10):1; preferably (4-8):1.

[0024] The washing in step (2) is washing with an alcohol solvent and water; preferably washing with ethanol and double deionized water (ddH2O).

[0025] The drying in step (2) is freeze-drying.

[0026] The vacuum degree of the freeze-drying is 10-20 Pa; preferably 10-18 Pa; further preferably 12-16 Pa; more preferably 14 Pa.

[0027] The temperature of the freeze-drying is -20℃ to -40℃; preferably -25℃ to -35℃; more preferably -30℃.

[0028] A nano-drug with NO catalytic performance is prepared by the method of any one of the above.

[0029] The particle size of the nano-drug is less than 10 nm.

[0030] The nano-drug with NO catalytic performance is used in the preparation of an antibacterial, antithrombotic (inhibiting platelet activation and promoting vascular smooth muscle relaxation) and / or anti-infective endocarditis (IE) drug.

[0031] The bacteria include gram-positive bacteria and gram-negative bacteria; preferably Staphylococcus aureus and / or Escherichia coli.

[0032] The nano-drug with NO catalytic performance is used in the preparation of an S-nitrosoglutathione (GSNO) catalyst.

[0033] The prepared nanodrug OX can break through the drug resistance limit, evade bacterial drug resistance through a non-antibiotic mechanism (oxidative damage + NO antibiosis), is effective on drug-resistant Staphylococcus aureus, realizes multiple synergistic treatment, simultaneously realizes antibiosis, antithrombus, vasodilation and inflammation regulation, improves the complex pathological links of IE, has an endogenous catalytic advantage, can generate NO in situ by using high-concentration GSNO (micromolar level) at the infection site, and avoids the pharmacokinetic defects of exogenous donors. This will effectively solve the limitations in the treatment of infective endocarditis, significantly improve the treatment effect, and has extremely important significance for realizing precise treatment of infective endocarditis and improving the prognosis of patients.

[0034] In the present application, the "having NO catalytic performance" means having the ability to catalyze GSNO to generate NO.

[0035] The present application has the following advantages and effects relative to the prior art:

[0036] 1. The prepared nanodrug OX of the present application is an oxidized molybdenum-based heteropoly acid, which is prepared by self-assembly of ammonium molybdate and sodium dihydrogen phosphate through coordination chemistry, and the molybdenum element exists in a +6 valence state (verified by XPS), has a Keggin-type heteropoly acid structure (chemical formula: [PMo 12 O 40 ] 3- ), and a particle size of about 2 nm, and has good dispersibility and excellent stability under physiological conditions (verified by DLS, with a particle size fluctuation of less than 10% for 1-7 days).

[0037] 2. The prepared nanodrug OX has catalytic properties and can efficiently catalyze the decomposition of GSNO, realizing the sustained and controllable release of NO with a half-life of ≥40 minutes, which is significantly better than traditional exogenous NO donors.

[0038] 3. The prepared nanodrug OX has multiple antibacterial mechanisms: ① oxidative damage: the high-valence molybdenum center of OX destroys the integrity of the bacterial cell membrane through oxidation, reducing the ATP synthesis of Staphylococcus aureus by 82.96%; ② biofilm inhibition: inhibits biofilm formation (inhibition rate 92.14%), breaking through the defect that traditional antibiotics are difficult to penetrate the biofilm; ③ NO synergistic antibiosis: the NO generated by catalyzing GSNO further enhances the bactericidal effect by degrading bacterial iron-sulfur clusters and inducing oxidative stress, with a minimum inhibitory concentration (MIC) of 250 μg / mL.

[0039] 4, The nano drug OX prepared by the application has the functions of anti-thrombosis and blood vessel regulation: ① platelet activation inhibition: NO activates the guanylate cyclase (GC) in the platelet, increases the cGMP level, inhibits the expression of CD62p on the surface of the platelet (inhibition rate 91.73%), and reduces thrombosis; ② blood vessel smooth muscle relaxation: NO diffuses to the blood vessel smooth muscle cells, activates the soluble guanylate cyclase (sGC), induces cGMP release, reduces the intracellular Ca 2+ concentration, realizes blood vessel relaxation (relaxation rate 77.89%), and reduces the risk of embolism; ③ inflammation microenvironment regulation: by regulating the levels of inflammatory factors (IL-8 is increased by 76.29%, IL-6 is increased by 72.98%, and IFN-γ is increased by 68.14%), the microenvironment of the heart tissue is improved, and the immune clearance capacity is enhanced.

[0040] 5, The nano drug OX prepared by the application can efficiently catalyze the endogenous S-nitrosoglutathione (GSNO) to generate NO, realize efficient treatment of infective endocarditis through the synergistic effect of the dual functions of “oxidation antibiosis-NO regulation”, and overcome the drug resistance of traditional treatment and the limitations of exogenous NO donors. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 It is the X-ray photoelectron spectroscopy (XPS) diagram of the nano drug OX in Example 1.

[0042] Figure 2 It is the dynamic light scattering (DLS) diagram of the nano drug OX in Example 1 in PBS or FBS solution for 1-7 days.

[0043] Figure 3 It is the release diagram of the nano drug OX in Example 1 catalyzing GSNO to generate nitric oxide (NO) over time.

[0044] Figure 4 It is the diagram of the influence of different stirring speeds (400, 800, 1600 rpm) on the hydration particle size of the nano drug OX in Example 1.

[0045] Figure 5 It is the diagram of the influence of different stirring speeds (400, 800, 1600 rpm) on the Zeta potential of the nano drug OX in Example 1.

[0046] Figure 6 It is the SEM image of Staphylococcus aureus after being treated by the nano drug OX in Example 2.

[0047] Figure 7 It is the statistical diagram of the ATP synthesis of Staphylococcus aureus after being treated by the nano drug OX in Example 2.

[0048] Figure 8PI fluorescence staining image of S. aureus after treatment with nanodrug OX in Example 2.

[0049] Figure 9 Three-dimensional confocal laser scanning microscope (CLSM) image of S. aureus biofilm formation after treatment with nanodrug OX in Example 2.

[0050] Figure 10 Graph of the results of the inhibition experiment of S. aureus and E. coli by nanodrug OX in Example 2.

[0051] Figure 11 Relaxation image of vascular smooth muscle cells after treatment with nanodrug OX in Example 3.

[0052] Figure 12 Schematic diagram of the construction steps of the mouse model of infective endocarditis in Example 4; wherein a is to stick the limbs of the mouse to the experimental plate with adhesive tape; b is to cut the skin at the right side of the mouse about 1 cm from the median line of the front neck; c is to separate the adipose tissue and free the right common carotid artery about 1 cm; d is to ligate the common carotid artery at the head end; e is to make a V-shaped incision in the middle part between the two ligations; f is to insert the catheter; g is to seal with nanogel; h is to suture the surgical incision and fix the end of the catheter subcutaneously.

[0053] Figure 13 Graph of the changes in the levels of IL-6, IL-8, and IFN-γ immune factors in mice after treatment with nanodrug OX in Example 4; wherein a is the expression level of IL-6; b is the expression level of IL-8; c is the expression level of IFN-γ.

[0054] Figure 14 LB plate image and statistical analysis graph of blood samples of mice in different treatment groups in Example 4; wherein a is the LB plate image; b is the statistical analysis result.

[0055] Figure 15 Graph of the results of the anti-neoplasm development experiment of OX in mice in Example 4; wherein a is the electronic image of the neoplasm samples of the hearts of mice in different treatment groups; b is the statistical analysis of the heart volumes of mice in different treatment groups; c is the H&E staining image of the neoplasm of the hearts of mice in different treatment groups.

[0056] Figure 16 Flow cytometry result graph and statistical analysis graph of platelet activation of mice in different treatment groups in Example 4; wherein a is the flow cytometry result graph; b is the statistical analysis result. DETAILED DESCRIPTION

[0057] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purposes, features and effects of the present application. The described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, the person skilled in the art can obtain his embodiments without creative labor, which all belong to the protection scope of the present application.

[0058] Unless otherwise specified, the reagents, methods and devices used in the present application are the conventional reagents, methods and devices in the technical field. The test methods in the following examples without specific experimental conditions are usually carried out according to the conventional experimental conditions or according to the experimental conditions suggested by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present application can be obtained by market.

[0059] In the embodiments of the present application, the preparation method of the nanodrug OX comprises the following steps:

[0060] S1, dissolving ammonium molybdate in water to prepare an ammonium molybdate solution; dissolving disodium hydrogen phosphate in water to prepare a disodium hydrogen phosphate solution.

[0061] S2, mixing the disodium hydrogen phosphate solution and the ammonium molybdate solution, stirring uniformly until colorless to obtain a mixed solution.

[0062] S3, after stirring reaction, adding an alcohol solvent for precipitation, centrifuging to collect, washing with an alcohol solvent and water for multiple times, freeze-drying to obtain the nanodrug OX.

[0063] In a specific embodiment provided by the present application, the ammonium molybdate in step S1 can be specifically ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 ·4H2O); the water can be specifically double deionized water (ddH2O); the concentration of the ammonium molybdate solution can be specifically 0.1-1 mol / L, further preferably 0.3-0.8 mol / L, still further preferably 0.4-0.6 mol / L, and most preferably 0.5 mol / L.

[0064] In a specific embodiment provided by the present application, the disodium hydrogen phosphate in step S1 can be specifically disodium hydrogen phosphate dodecahydrate (NaH2PO4·12H2O); the water can be specifically double deionized water (ddH2O); the concentration of the disodium hydrogen phosphate solution can be specifically 0.1-0.5 mol / L, further preferably 0.2-0.4 mol / L, still further preferably 0.25-0.3 mol / L, and most preferably 0.292 mol / L.

[0065] In one specific embodiment provided by the present application, the sodium dihydrogen phosphate solution in step S2 is preferably slowly dripped into the ammonium molybdate solution for mixing; the molar ratio of ammonium molybdate to sodium dihydrogen phosphate is preferably (40-60): 1, further preferably (45-55): 1, more preferably 50: 1.

[0066] In one specific embodiment provided by the present application, the rotation speed of the mixing in step S2 is 400-2000 rpm, preferably 1000-2000 rpm, further preferably 1200-2000 rpm, again further preferably 1400-1800 rpm, more further preferably 1500-1700 rpm, most preferably 1600 rpm; the mixing is preferably carried out at room temperature. The ammonium molybdate and sodium dihydrogen phosphate compounds form Keggin-type heteropoly acids of oxidized molybdenum through self-assembly.

[0067] In one specific embodiment provided by the present application, the volume ratio of the added alcohol solvent to the mixed solution in step S3 is preferably (3-10): 1, further preferably (4-8): 1; the alcohol solvent is preferably ethanol.

[0068] In one specific embodiment provided by the present application, the washing in step S3 preferably uses ethanol and water, more preferably ethanol and double deionized water (ddH2O).

[0069] In one specific embodiment provided by the present application, the vacuum degree of the freeze-drying in step S3 is preferably 10-20 Pa, further preferably 10-18 Pa, again further preferably 12-16 Pa, most preferably 14 Pa; the temperature of the freeze-drying is preferably -20℃ to -40℃, further preferably -25℃ to -35℃, again further preferably -30℃.

[0070] Example 1, Preparation of OX and verification of catalytic activity

[0071] 1. Preparation of nanodrug OX

[0072] Step 1: Dissolve (NH4)6Mo7O 24 ·4H2O (ammonium molybdate tetrahydrate) in 10 mL ddH2O to prepare a (NH4)6Mo7O 24 ·4H2O solution (concentration 0.5 mol / L). Dissolve NaH2PO4·12H2O (sodium dihydrogen phosphate dodecahydrate) in 10 mL H2O to prepare a NaH2PO4·12H2O solution (concentration 0.292 mol / L).

[0073] Step 2: NaH2PO4·12H2O solution was slowly added into 10 mL of (NH4)6Mo7O24H2O solution at a molar ratio of 50:1 of ammonium molybdate to sodium dihydrogen phosphate, and stirred at room temperature on a magnetic stirrer at 1600 rpm until colorless. 24

[0074] Step 3: After stirring for 30-60 min, 80 mL of ethanol (C2H5OH) was added to precipitate MoNs, which were collected by centrifugation at 12000 rpm for 5 min, washed with ethanol and ddH2O, and freeze-dried (vacuum degree 14 Pa, temperature -30°C) to obtain nanoparticles, denoted as OX dry powder.

[0075] 2. Characterization of nanomedicine OX

[0076] The elemental composition and valence state of Mo-based POMs were analyzed by X-ray photoelectron spectroscopy (XPS). The Mo 3d peak is split into Mo 3d5 / 2and Mo 3d3 / 2sub-peaks due to spin-orbit coupling. The results are shown in Table 1. Figure 1 In the XPS spectrum of Mo3d, there are two main peaks of Mo3d at 233.35 eV and 236.4 eV, which correspond to Mo 3d5 / 2and Mo 3d3 / 2, respectively. The Mo3d5 / 2binding energy of Mo6 + is about 232.6-232.9 eV, and the Mo 3d3 / 2is about 235.7-236.0 eV, indicating that the valence state of molybdenum in OX is +6, with a high oxidation state.

[0077] To further evaluate the stability of OX, the change trend of the hydrated particle size (DLS) of OX was measured for 1-7 days. Specifically, the OX dry powder prepared above was dissolved in PBS buffer (pH=7.4) and 10% (v / v) fetal bovine serum (FBS), respectively, and placed at room temperature for 7 days. The hydrated particle size of OX was measured every day, with three replicates. The results are shown in Table 2. Figure 2 As shown in Table 2, the hydrated particle size of OX increased slightly in PBS or FBS, but still maintained at 1-3 nm, with a particle size fluctuation of <10%, indicating that OX has good dispersibility and excellent stability under physiological conditions.

[0078] 3. Verification of the catalytic activity of nanomedicine OX

[0079] ​To evaluate the rate of OX catalyzing S-nitrosoglutathione (GSNO) to generate NO, different concentrations of OX (31.25, 62.5, 125, 250, 500, 1000 pg / ml) were mixed with GSNO (100 pM) and the NO content was quantitatively determined at different time points using the kit described above. The experiment was set up in triplicate. The results are shown in Figure 3 OX can continuously catalyze GSNO to generate NO, and the duration can be up to 40 min or more.

[0080] 4. Effect of different stirring speeds on nanodrug OX

[0081] According to the above OX preparation method, the stirring speed in step 2 was adjusted to 800 rpm and 400 rpm, and the rest of the preparation steps were the same. The hydration particle size of OX was tested by dynamic light scattering, and the surface charge distribution-Zeta potential size was tested. The experiment was set up in triplicate.

[0082] The results are shown in Figure 4 and Figure 5 It can be seen that the stirring speed is related to the nucleation of nanomaterials. The faster the stirring speed, the faster the twelve water sodium hydrogen phosphate can fully react with ammonium molybdate tetrahydrate, and the faster the nucleation in a short period of time, forming a large number of nanomaterials with uniform and small particle size. Therefore, OX prepared under the condition of 1600 rpm was selected for the following experiments.

[0083] Example 2, Multiple antibacterial mechanism of OX

[0084] 1. Oxidation and NO synergistic antibacterial

[0085] To prove that the bacteria were damaged after OX and GSNO synergistic antibacterial, we used scanning electron microscopy (SEM) to observe the morphological changes of Staphylococcus aureus. The specific steps are as follows:

[0086] Use a loop to pick a colony of purchased Staphylococcus aureus (S. aureus) CMCC26003 (purchased from Yolee (Shanghai) Life Science Co., Ltd.) from the slope and inoculate it into 50 mL of sterilized LB liquid medium (0.5 g of NaCl, 0.5 g of tryptone and 0.25 g of yeast extract were dissolved in 50 mL of deionized water). The centrifuge tube containing the inoculated culture medium was placed in a constant temperature incubator and incubated at 37°C overnight. The bacterial suspension obtained by the above culture was diluted to 1x10 7CFU / mL. Then, 1 mL of the antibacterial experiment system (sterile PBS, vancomycin, OX, GSNO, GSNO + vancomycin, GSNO + OX) was prepared, respectively, and incubated at 37 ℃ for 6 h. After the incubation of the above-mentioned culture obtained antibacterial experiment system, silicon wafer sample preparation was used to characterize it by field emission scanning electron microscope, and ATP content of each group was tested using ATP synthesis kit. The experiment was set up three times.

[0087] The results are shown in Figure 6 and Figure 7 : Staphylococcus aureus in the control group and the GSNO group showed smooth spherical shape and intact surface; when treated with vancomycin (VIO) or only OX in the presence or absence of GSNO, the bacterial membrane was deformed and shrunk to a certain extent; when treated with OX + GSNO synergistic antibacterial, the bacterial membrane was severely damaged and ruptured Figure 6 . And according to Figure 7 , the relative ATP content of the GSNO group was significantly lower than that of the control group under OX treatment, indicating that the combination of GSNO and OX greatly affected the ATP synthesis process of bacteria, with an inhibition rate of 82.96%.

[0088] Further, dead and live staining imaging method was used, and 4 µM propidium iodide (PI) diluent was incubated for 5 minutes in a dark room at room temperature to observe the fluorescence release. The results are shown in Figure 8 : The control group and the GSNO group had almost no red fluorescence, indicating that the bacteria were less dead; when treated with VIO or only OX in the presence or absence of GSNO, the red fluorescence increased slightly; when treated with OX + GSNO synergistic antibacterial, the red fluorescence was the most, indicating that the bacteria were the most dead.

[0089] 2. Inhibition of biofilm

[0090] In order to visually observe the bacterial biofilm, laser confocal microscope was used to study the average thickness of the bacterial biofilm after different treatments. The results are shown in Figure 9 : When treated with OX and GSNO synergistic antibacterial, a very thin biofilm was formed; on the contrary, thick biofilm was observed on the surface of the antibiotic (VIO) or the control group, which was consistent with the previous results. The reason for this phenomenon can be attributed to the fact that the NO generated by OX catalyzing GSNO and the oxidizing property of OX can be superimposed to effectively eliminate the bacterial biofilm and inhibit the formation of biofilm (inhibition rate 92.14%), breaking through the defect that traditional antibiotics are difficult to penetrate the biofilm and enhancing the antibacterial effect.

[0091] 3. OX has broad-spectrum antibacterial effect

[0092] According to the above method for constructing the antibacterial experiment system of Staphylococcus aureus, an antibacterial system of Escherichia coli (CMCC44103, purchased from Oligo Shanghai Life Science Co., Ltd.) was constructed, and the drug used was OX with a concentration of 500 μg / ml, 1 mg / ml and 2 mg / ml. After the antibacterial system was constructed, 100 μL of the mixed solution (Staphylococcus aureus, Escherichia coli) was evenly coated on the solid LB culture medium, and then placed in a constant temperature incubator at 37°C for 14-16 hours. The number of bacterial colonies was counted by the CFU method, and the antibacterial effect was evaluated. The experiment was set in triplicate.

[0093] The results are shown in Table 1. Figure 10 As shown in Table 1, the OX synthesized in the application has a broad-spectrum antibacterial effect by combining the antibacterial properties of its own oxidation with the NO generated by its catalysis. It has good antibacterial effect on both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). OX can produce antibacterial effect on Staphylococcus aureus, and therefore can be used for clinical treatment of infective endocarditis.

[0094] Example 3, Antithrombus and Vessel Modulation of OX

[0095] In order to evaluate the relaxation of vascular smooth muscle cells, a Ca 2+ The content kit was used to evaluate the relaxation of vascular smooth muscle cells in each group. The specific method is as follows:

[0096] (1) Bacterial culture-liquid culture: Staphylococcus aureus (S. aureus) CMCC26003 (purchased from Oligo (Shanghai) Life Science Co., Ltd.) was inoculated into 50 mL of sterilized LB liquid medium (0.5 g of NaCl, 0.5 g of tryptone and 0.25 g of yeast extract were dissolved in 50 mL of deionized water) using an inoculation loop. The centrifuge tube containing the inoculated culture medium was placed in a constant temperature incubator at 37°C and 220 rev / min. -1 Cultured overnight.

[0097] (2) Vascular smooth muscle cell culture: immortalized mouse aortic vascular smooth muscle cells (MOVAS) were purchased from Xiamen Yimeng Biotechnology Co., Ltd. The MOVAS cells were cultured in DMEM medium (Thermo Fisher Scientific, USA) containing 10% (v / v) fetal bovine serum (FBS) (Thermo Fisher Scientific, USA) and 1% (v / v) penicillin-streptomycin (China Yixing Biological Technology Co., Ltd.). All cells were cultured in a humidified incubator at 37°C and 5% carbon dioxide.

[0098] (3) The MOVAS cells (5×103 Inoculated in each well of a 96-well plate. Then different groups of materials were co-incubated with cells for 6 h, and then the medium was updated. After standing for 4 hours, Ca 2+ The content kit was used to evaluate the relaxation of vascular smooth muscle cells in each group. The groups were sterile PBS, vancomycin, OX, GSNO, GSNO+vancomycin, and GSNO+OX, wherein the final concentration of OX was 500 μg / ml, the final concentration of GSNO was 100 μM, and the final concentration of vancomycin was 1 μg / ml.

[0099] The results are shown in Figure 11 , the fluorescence intensity of the GSNO+OX group was significantly lower than that of the other groups, indicating that OX catalyzed the generation of NO from GSNO, causing the Ca 2+ of vascular smooth muscle cells to flow out, relax, achieve vasodilation (relaxation rate 77.89%), and reduce the risk of embolism.

[0100] Example 4, in vivo experiment of OX

[0101] 1. Construction of a mouse model of infective endocarditis

[0102] 1.1 The steps for constructing the mouse model of infective endocarditis are as follows: the experimental animals used were ICR male mice (7 weeks old, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.). The ICR mice were caught, and 1% sodium pentobarbital solution (50 mg / kg) was injected intraperitoneally. After anesthesia, the limbs of the mice were taped to the experimental plate (a in Figure 12 ). The neck area of the mouse below the sternum was shaved, and after disinfection with 75% alcohol, the skin was cut with surgical scissors 1 cm to the right of the median line in the front of the neck (b in Figure 12 ). The layers were bluntly separated to prevent muscle tearing, and the adipose tissue was separated and the right common carotid artery was freed for about 1 cm (c in Figure 12 ). Two threads were passed under the right common carotid artery, and a loose knot was made on the proximal arterial trunk for fixing the arterial cannula, and the common carotid artery was ligated at the head end (d in Figure 12 ). The blood vessels were replaced with fine ophthalmic surgical scissors and fine forceps, and under a body microscope, the left hand held the fine forceps to pick up the blood vessels, and the right hand held the ophthalmic surgical scissors to make a V-shaped incision in the middle part between the two ligations (e in Figure 12 ). The left hand held the forceps to lift the blood vessel opening, and the right hand forceps clamped a catheter connected to a syringe and filled with normal saline, and the catheter was inserted into the blood vessel opening. The arterial clamp was removed, and the catheter was slowly pushed in until resistance was encountered. It was slightly withdrawn, and the catheter was inserted about 1.2-1.5 cm. After successful insertion of the catheter (f in Figure 12 ), the proximal end of the prepared knot was ligated, and the hemostat was clamped near the syringe end, cut off, and sealed with nano glue.Figure 12 (g) After clearing the blood from the surgical procedure, a strong pulsation of the catheter following the heartbeat indicates successful insertion. The surgical incision is then sutured, and the catheter tip is secured subcutaneously. Figure 12 (h in the original text). 24 h after successful cardiac cannulation, the mice were observed for their condition. Mice in good condition were injected via tail vein with 200 µl of overnight activated Staphylococcus aureus culture (7 × 10⁻⁶). 4 (CFU / ml) to induce infection.

[0103] 1.2 Grouping and Dosing

[0104] Following the experimental method described above, cardiac catheterization was performed on mice. Twenty-four hours later, each mouse was injected via tail vein with 0.5 ml of pre-cultured and diluted bacterial solution (10⁻⁶ ml / mL). 7 CFU / ml). Mice were randomly divided into three groups of five each. Twenty-four hours after infection, mice were treated daily via tail vein with the following drugs: blank control group (PBS), vancomycin (VIO, 80 mg / kg), and OX (20 mg / kg). Blood samples were collected from the maxillofacial region on days 1, 3, and 5 of treatment. Mice were euthanized by overdose anesthesia after the last treatment on day 7, and tissues and organs were collected.

[0105] 2. Regulation of the inflammatory microenvironment

[0106] To further investigate the biological effects of OX treatment in the microenvironment of infective endocarditis, circulating cytokine levels were measured to assess the systemic immune response.

[0107] The results are as follows Figure 13 As shown: After OX treatment, the circulating immune factor IL-6 ( Figure 13 a) IL-8 Figure 13 b) IFN-γ ( Figure 13 c) was significantly elevated, indicating that OX improves the cardiac tissue microenvironment and enhances immune clearance by regulating the levels of inflammatory factors (IL-8 increased by 76.29%, IL-6 increased by 72.98%, and IFN-γ increased by 68.14%).

[0108] 3. OX's in vivo anti-infective effect

[0109] To evaluate the anti-infective effect of OX in the treatment of infective endocarditis, equal amounts of venous blood samples were collected from mice on days 3, 5, and 7 of treatment and inoculated into LB medium overnight at 37°C. The number of colonies was then counted.

[0110] The results are as follows Figure 14The blood colony statistics results show that the number of colonies is relatively more in the control group (PBS treatment) at 3 days after infection treatment, the number of colonies in the VIO treatment group is lower than that in the control group, showing a certain bacteriostatic effect, and the number of colonies in the OX treatment group is very small, and the bacteriostatic effect is significant; at 5 days, the number of colonies in the control group increases significantly, the number of colonies in the VIO treatment group also increases, but is still lower than that in the control group, and the number of colonies in the OX treatment group still maintains a low level, and its ability to continuously inhibit bacterial growth is stronger; at 7 days, the number of colonies in the control group further increases, the number of colonies in the VIO treatment group decreases compared with that at 5 days due to the influence of the late bacteriostatic effect, and the number of colonies in the OX treatment group is always at a low level, indicating that the inhibitory effect of OX treatment on bacterial growth is stable and persistent during the entire observation period. Overall, the inhibitory effect of OX treatment on bacterial growth at each time point is better than that of VIO treatment, and OX treatment can more effectively control the number of bacteria in the blood sample.

[0111] 4. OX in vivo anti-tumor development

[0112] 4.1 In order to evaluate the inhibitory effect of the NO bio-catalytic ability of OX on tumor development, the mice in different groups were euthanized at 7 d after treatment, and the heart was carefully separated according to the above dissection method. The tumor was completely separated from the organ using ophthalmic surgical scissors and forceps. The tumor was photographed and recorded, and the volume of the tumor was measured using Image J.

[0113] As shown in the photograph of the heart tumor of different treatment groups (a) in FIG. 7, Figure 15 , the tumor in the PBS control group is relatively large, the tumor volume in the VIO treatment group is smaller than that in the control group, and the tumor in the OX treatment group is the smallest, and even the size of the tumor visible to the naked eye in some samples is very small; and as can be seen from the statistical data (b) in FIG. 7, Figure 15 , the thrombus volume in the PBS control group is the largest, about 30 mm³, the thrombus volume in the VIO treatment group is reduced, about 10 mm³, and the thrombus volume in the OX treatment group is the smallest, close to 0 mm³. It shows that the effect of OX treatment on inhibiting tumor growth is the most significant, VIO treatment also has a certain inhibitory effect, and the difference between the three groups is statistically significant.

[0114] 4.2 In order to further evaluate the internal structure and cell composition of the tumor, part of the tumor samples were subjected to histological sectioning, and the cell morphology and tissue structure were observed under a microscope by HE staining.

[0115] The results are shown in FIG. 8, Figure 15As shown in Figure c, a large number of red areas are visible in the field of view of the control group, mainly indicating the aggregation of red blood cells. At the same time, many areas with relatively dense tissue structure are also visible, possibly containing fibrous tissue and other components, suggesting that the vegetation contains abundant blood components and extracellular matrix. In the VIO-treated group, the red areas are relatively reduced, indicating a decrease in red blood cell content, and the tissue structure appears relatively looser, suggesting that VIO treatment has a certain impact on the composition and structure of the vegetation, possibly inhibiting the accumulation of certain components. In the OX-treated group, the red areas are further reduced, and the changes in tissue structure are more pronounced compared to the control group. The distribution of cellular components and extracellular matrix also changes, indicating that OX treatment not only inhibits the growth of vegetation but also significantly affects its internal tissue structure. These results indicate that OX treatment is most effective in inhibiting the growth of cardiac vegetation and altering its internal structure, followed by VIO treatment.

[0116] 5. OX in vivo antiplatelet activation

[0117] To evaluate the inhibitory effect of OX on platelet activation by the NO biocatalytic activity of endogenous GSNO, whole blood was collected from different groups of mice, and platelets were separated using a platelet separation kit. The platelet suspension was then incubated with fluorescently labeled anti-platelet activation marker antibodies CD61 and CD62p (CD61, catalog number 104305; CD62p, catalog number 148304; both purchased from Biolegend) at room temperature for 30 min. The fluorescence intensity was then detected by flow cytometry to assess the degree of platelet activation in each group.

[0118] The results are as follows Figure 16 As shown, the flow cytometry scatter plot ( Figure 16 a) and platelet activation rate bar chart ( Figure 16 Figure b) shows that, compared to the control group, VIO treatment increased the proportion of activated platelets, while OX treatment significantly decreased this proportion. This is consistent with the in vitro platelet activation results. Furthermore, VIO treatment promotes platelet activation, while the OX treatment group effectively inhibits platelet activation and reduces platelet aggregation by catalyzing the generation of NO from endogenous GSNO, thereby controlling the development of vegetations.

[0119] In summary, this invention provides an antibacterial drug that can utilize GSNO in the microenvironment to generate NO, exerting anti-infective and anti-vegetation effects. This nanomedicine utilizes a transition metal Mo to form an oxidizing material as a NO synthase, catalyzing the stable generation of NO from endogenous GSNO, thereby combining the vasodilatory and broad-spectrum antibacterial effects of NO to achieve effective treatment of infective endocarditis, avoiding the development of antibiotic resistance, and has great application potential in the clinical application of infective endocarditis.

[0120] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for preparing a nano-drug having NO catalytic performance, characterized in that, The method comprises the following steps: (1) dissolving molybdate in water to obtain a molybdate solution; dissolving hydrogen phosphate in water to obtain a hydrogen phosphate solution; (2) mixing the hydrogen phosphate solution with the molybdate solution, stirring at 400-2000 rpm until colorless, obtaining a mixed solution, continuing to stir until the reaction is completed, adding an alcohol solvent to precipitate, centrifuging to collect, washing, drying, and obtaining the nano drug with NO catalytic performance; The molybdate in step (1) is at least one of ammonium molybdate and ammonium molybdate tetrahydrate; The hydrogen phosphate in step (1) is at least one of disodium hydrogen phosphate and sodium dihydrogen phosphate dodecahydrate; The concentration of the molybdate solution in step (1) is 0.1-1 mol / L; The concentration of the hydrogen phosphate solution in step (1) is 0.1-0.5 mol / L; The molar ratio of the molybdate to the hydrogen phosphate in step (1) is 50:1; The alcohol solvent in step (2) is ethanol.

2. The method of claim 1, wherein: The stirring speed in step (2) is 1000-2000 rpm.

3. The method of claim 2, wherein: The stirring speed in step (2) is 1200-2000 rpm.

4. The method of claim 3, wherein: The stirring speed in step (2) is 1400-1800 rpm.

5. The method of claim 1, wherein: The volume ratio of the alcohol solvent to the mixed solution in step (2) is 3-10:1; The drying in step (2) is freeze-drying; The vacuum degree of the freeze-drying is 10-20 Pa; The temperature of the freeze-drying is -20℃ to -40℃.

6. The method of claim 1, wherein: The reaction time in step (2) is 30-60 min; The centrifugation condition in step (2) is 12000 rpm for 5-10 min; The washing in step (2) is washing with an alcohol solvent and water.

7. A nano-drug with NO catalytic performance, characterized in that: Prepared by the method of any one of claims 1-6.

8. The nano drug with NO catalytic performance of claim 7 for use in the preparation of an antibacterial, antithrombotic and / or treatment of infective endocarditis drug.

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