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

Abstract

The invention discloses preparation of a nano-drug with NO catalytic performance and application of the nano-drug in treatment of infectious endocarditis. The preparation method comprises the following steps: respectively dissolving molybdate and hydrophosphate in water, then stirring and mixing the two solutions, and carrying out self-assembly to form oxidized molybdenum-based Keggin type heteropolyacid, namely the nano-drug (OX) with NO catalytic performance. The nano-drug prepared by the invention has GSNO catalytic characteristics and multiple antibacterial characteristics, also has antithrombotic and vascular regulation functions, can efficiently catalyze endogenous GSNO to generate NO, and realizes efficient treatment of infectious endocarditis through the synergistic effect of double functions of oxidation antibacterial-NO regulation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the preparation of a nano drug with NO catalytic performance and its application in the treatment of infective endocarditis. Background Art

[0002] Infective endocarditis (IE) is an intravascular infectious disease, typically inflammation of the endocardium of the heart valve or ventricular wall, following direct infection by bacteria, fungi, or other pathogens. Clinically, damaged heart valve surfaces allow Staphylococcus aureus in the bloodstream to attach and form prominent vegetations. Despite significant advances in diagnosis and treatment in recent years, IE remains a disease with a poor prognosis and a high mortality rate. Staphylococcus aureus has become the primary pathogen of IE, and its infectious forms are the most severe. Because the vegetations are loosely attached, detachment of some can lead to complications such as downstream organ embolism and visceral abscesses, making treatment challenging. Current clinical treatment relies on a combination of antibiotics and surgery, but increasing drug resistance and biofilm barriers significantly reduce efficacy, necessitating the urgent need for novel therapeutic strategies.

[0003] Nitric oxide (NO) has become a research hotspot for the treatment of IE due to its broad-spectrum antimicrobial activity, inhibition of platelet activation, and regulation of vascular function. However, exogenous NO donors (such as NONOates and sodium nitroprusside) suffer from limitations such as poor stability, short half-life, and limited targeting. Although nanodelivery systems (such as metal-organic frameworks and polymer nanoparticles) can partially improve drug loading efficiency, their uncontrolled decomposition and leakage in the circulation still prevent sustained NO release at the lesion site.

[0004] Research has shown that inflammation caused by noxious stimuli, infection, tissue damage, and other conditions recruits immune cells (macrophages, neutrophils, etc.), triggering an oxidative (or respiratory) burst. This involves the rapid generation and release of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which contribute to the elimination of microorganisms. Excessive RNS further leads to the accumulation of S-nitrosothiols (SNO). The accumulation of RNS can induce the formation of S-nitrosothiols (RSNO) such as S-nitrosoglutathione (GSNO). GSNO, as the primary source of endogenous NO, is chemically stable and can be decomposed and released by transition metal ions, exhibiting both antimicrobial and antithrombotic properties. Therefore, the development of novel therapeutic systems based on endogenous NO generation is of great clinical significance. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a nanomedicine with NO catalytic performance.

[0006] Another object of the present invention is to provide a nanomedicine with NO catalytic performance prepared by the method.

[0007] Another object of the present invention is to provide applications of the nanomedicine having NO catalytic performance.

[0008] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a nanomedicine having NO catalytic performance 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) The hydrogen phosphate solution and the molybdate solution are mixed and stirred at 400-2000 rpm until the mixture becomes colorless to obtain a mixed solution. The mixture is stirred and reacted until the reaction is completed. An alcohol solvent is added to precipitate the mixture. The mixture is collected by centrifugation, washed, and dried to obtain the nanomedicine having NO catalytic properties.

[0009] The molybdate described in step (1) is ammonium molybdate and ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 ·4H2O); preferably ammonium molybdate tetrahydrate.

[0010] The concentration of the molybdate solution in step (1) is 0.1 to 1 mol / L; more preferably 0.3 to 0.8 mol / L; further preferably 0.4 to 0.6 mol / L; and more preferably 0.5 mol / L.

[0011] The hydrogen phosphate described in step (1) is at least one of disodium hydrogen phosphate and sodium dihydrogen phosphate dodecahydrate (NaH2PO4·12H2O); preferably sodium dihydrogen phosphate dodecahydrate.

[0012] The concentration of the hydrogen phosphate solution in step (1) is 0.1 to 0.5 mol / L; more preferably 0.2 to 0.4 mol / L; further preferably 0.25 to 0.3 mol / L; and more preferably 0.292 mol / L.

[0013] The molar ratio of molybdate to hydrogen phosphate in step (1) is (40-60):1; preferably (45-55):1; more preferably 50:1.

[0014] The water in step (1) is ionized water or double deionized water (ddH2O).

[0015] In step (2), the hydrogen phosphate solution is slowly dripped into the ammonium molybdate solution for mixing.

[0016] The stirring speed in step (2) is preferably 1000-2000 rpm; more preferably 1200-2000 rpm; further preferably 1400-1800 rpm; further preferably 1500-1700 rpm; and most preferably 1600 rpm.

[0017] The reaction time in step (2) is 30 to 60 minutes.

[0018] The centrifugation conditions in step (2) are: 12,000 rpm for 5 to 10 min; preferably 12,000 rpm for 5 min.

[0019] The alcohol solvent described in step (2) is preferably ethanol.

[0020] The volume ratio of the alcohol solvent to the mixed solution in step (2) is (3-10):1; preferably (4-8):1.

[0021] The washing in step (2) is performed using an alcohol solvent and water; preferably, ethanol and double deionized water (ddH2O) are used for washing.

[0022] The drying in step (2) is freeze-drying.

[0023] The vacuum degree of the freeze-drying is 10-20 Pa, preferably 10-18 Pa, more preferably 12-16 Pa, and more preferably 14 Pa.

[0024] The freeze-drying temperature is -20°C to -40°C; preferably -25°C to -35°C; more preferably -30°C.

[0025] A nano drug with NO catalytic performance is prepared by any of the methods described above.

[0026] The particle size of the nano drug is less than 10 nm.

[0027] The nanomedicine with NO catalytic performance is used in the preparation of antibacterial, antithrombotic (inhibiting platelet activation and promoting vascular smooth muscle relaxation) and / or infective endocarditis (IE) treatment drugs.

[0028] The bacteria include Gram-positive bacteria and Gram-negative bacteria; preferably Staphylococcus aureus and / or Escherichia coli.

[0029] The invention relates to an application of the nano drug with NO catalytic performance in the preparation of S-nitrosoglutathione (GSNO) catalyst.

[0030] The nanodrug OX prepared by this invention can overcome drug resistance limitations and circumvent bacterial resistance through a non-antibiotic mechanism (oxidative destruction + NO antibacterial effect), effectively targeting drug-resistant Staphylococcus aureus. It also achieves synergistic multi-effect therapy, simultaneously delivering antibacterial, antithrombotic, vasodilation, and inflammatory modulation, improving the complex pathological aspects of IE. Its endogenous catalytic advantage allows it to utilize the high concentrations of GSNO (micromolar levels) at the infection site to generate NO in situ, avoiding the pharmacokinetic drawbacks of exogenous donors. This approach will effectively address the limitations of infective endocarditis treatment, significantly improving therapeutic efficacy and possessing crucial significance for achieving precise treatment of infective endocarditis and improving patient outcomes.

[0031] In the present invention, the term "having NO catalytic performance" means having the ability to catalyze GSNO to generate NO.

[0032] The present invention has the following advantages and effects compared to the prior art: 1. The nano drug OX prepared by the present invention is an oxidized molybdenum-based heteropoly acid, which is prepared by coordination chemical self-assembly of ammonium molybdate and sodium dihydrogen phosphate. The molybdenum element exists stably in the +6 valence state (XPS verification) and has a Keggin-type heteropoly acid structure (chemical formula: [PMo 12 O 40 ] 3- ), with a particle size of approximately 2 nm, good dispersibility and excellent stability under physiological conditions (DLS verification of particle size fluctuation of <10% from 1 to 7 days).

[0033] 2. The nanomedicine OX prepared by the present invention has catalytic properties and can efficiently catalyze the decomposition of GSNO, achieving sustained and controlled release of NO with a half-life of ≥40 minutes, which is significantly better than traditional exogenous NO donors.

[0034] 3. The nanodrug OX prepared by the present invention has multiple antibacterial mechanisms: ① Oxidative damage: The high-valent molybdenum center of OX destroys the integrity of bacterial cell membranes through oxidation, reducing ATP synthesis in Staphylococcus aureus by 82.96%; ② Biofilm inhibition: Inhibits biofilm formation (inhibition rate 92.14%), overcoming the defect that traditional antibiotics have difficulty penetrating biofilms; ③ NO synergistic antibacterial: NO generated by catalyzing GSNO degrades bacterial iron-sulfur clusters and induces oxidative stress response, further enhancing the bactericidal effect, with a minimum inhibitory concentration (MIC) of 250 μg / mL.

[0035] 4. The nano-drug OX prepared by the present invention has anti-thrombotic and vascular regulatory functions: ① Platelet activation inhibition: NO activates guanylate cyclase (GC) in platelets, increases cGMP levels, inhibits the expression of platelet surface activation marker CD62p (inhibition rate 91.73%), and reduces thrombosis; ② Vascular smooth muscle relaxation: NO diffuses into vascular smooth muscle cells, activates soluble guanylate cyclase (sGC), induces cGMP release, and reduces intracellular Ca 2+ concentration, achieving vasodilation (dilation rate 77.89%) and reducing the risk of embolism; ③ Regulation of inflammatory microenvironment: by regulating the levels of inflammatory factors (IL-8 increased by 76.29%, IL-6 increased by 72.98%, IFN-γ increased by 68.14%), the cardiac tissue microenvironment was improved and the immune clearance ability was enhanced.

[0036] 5. The nanomedicine OX prepared by the present invention can efficiently catalyze endogenous S-nitrosoglutathione (GSNO) to generate NO. Through the dual functional synergy of "oxidative antibacterial-NO regulation", it can achieve efficient treatment of infective endocarditis, overcoming the drug resistance of traditional treatments and the limitations of exogenous NO donors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the X-ray photoelectron spectroscopy (XPS) diagram of the nanodrug OX in Example 1.

[0038] Figure 2 The dynamic light scattering (DLS) graph of the nanodrug OX in Example 1 in PBS or FBS solution for 1-7 days.

[0039] Figure 3 This is a graph showing the release of nitric oxide (NO) generated by GSNO catalyzed by the nanomedicine OX in Example 1 over time.

[0040] Figure 4 Graph showing the effect of different stirring speeds (400, 800, 1600 rpm) on the hydrated particle size of nano drug OX in Example 1.

[0041] Figure 5 The figure shows the effect of different stirring speeds (400, 800, 1600 rpm) on the Zeta potential of nanomedicine OX in Example 1.

[0042] Figure 6 This is the SEM image of Staphylococcus aureus after treatment with nanodrug OX in Example 2.

[0043] Figure 7 This is a statistical chart of ATP synthesis by Staphylococcus aureus after treatment with nanodrug OX in Example 2.

[0044] Figure 8This is the PI fluorescence staining image of Staphylococcus aureus after treatment with the nanodrug OX in Example 2.

[0045] Figure 9 This is a three-dimensional confocal laser scanning microscopy (CLSM) image of the Staphylococcus aureus biofilm formed after treatment with the nanodrug OX in Example 2.

[0046] Figure 10 This is a graph showing the results of the inhibition experiment of the nanodrug OX on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in Example 2.

[0047] Figure 11 This is the relaxation image of vascular smooth muscle cells after treatment with the nanodrug OX in Example 3.

[0048] Figure 12 Schematic diagram of the steps for constructing the mouse model of infective endocarditis in Example 4; wherein, a is taping the mouse's limbs to the experimental plate with tape; b is cutting the skin 1 cm to the right of the mouse's anterior midline; c is separating the adipose tissue and freeing the right common carotid artery by about 1 cm; d is ligating the common carotid artery at the head end; e is making a V-shaped incision between the two ligatures; f is inserting a catheter; g is sealing with nano glue; and h is suturing the surgical incision and fixing the end of the catheter subcutaneously.

[0049] Figure 13 This is a graph showing changes in the levels of IL-6, IL-8, and IFN-γ immune factors in mice after treatment with the nanodrug OX in Example 4; wherein, a is the expression level of IL-6; b is the expression level of IL-8; and c is the expression level of IFN-γ.

[0050] Figure 14 LB plate images and statistical analysis graphs of blood samples from mice in different treatment groups in Example 4; wherein, a is the LB plate image; b is the statistical analysis result.

[0051] Figure 15 Graphs showing the results of the anti-vegetation development experiment of OX in mice in Example 4; wherein, a is an electronic image of cardiac vegetation samples from mice in different treatment groups; b is a statistical analysis of the heart volumes of mice in different treatment groups; and c is an H&E-stained image of cardiac vegetation from different treatment groups.

[0052] Figure 16 These are the flow cytometry results and statistical analysis diagrams of platelet activation in mice in different treatment groups in Example 4; wherein, a is the flow cytometry result diagram; b is the statistical analysis result. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making any creative efforts are all within the scope of protection of the present invention.

[0054] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional in the art. Experimental methods in the following examples, where specific experimental conditions are not specified, are generally performed under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0055] In an embodiment of the present invention, the method for preparing the nano drug OX comprises the following steps: S1. Dissolve ammonium molybdate in water to prepare an ammonium molybdate solution; dissolve disodium hydrogen phosphate in water to prepare a disodium hydrogen phosphate solution.

[0056] S2. Mix the sodium dihydrogen phosphate solution and the ammonium molybdate solution, and stir until they are colorless to obtain a mixed solution.

[0057] S3. After stirring the reaction, add an alcohol solvent to precipitate, collect by centrifugation, wash with an alcohol solvent and water for multiple times, and freeze-dry to obtain the nano drug OX.

[0058] In a specific embodiment provided by the present invention, the ammonium molybdate in step S1 can be ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 ·4H2O); the water can be specifically double deionized water (ddH2O); the concentration of the ammonium molybdate solution is specifically 0.1-1 mol / L, more preferably 0.3-0.8 mol / L, further preferably 0.4-0.6 mol / L, and most preferably 0.5 mol / L.

[0059] In a specific embodiment provided by the present invention, the sodium dihydrogen phosphate in step S1 can be sodium dihydrogen phosphate dodecahydrate (NaH2PO4·12H2O); the water can be doubly deionized water (ddH2O); the concentration of the sodium dihydrogen phosphate solution is 0.1 to 0.5 mol / L, more preferably 0.2 to 0.4 mol / L, further preferably 0.25 to 0.3 mol / L, and most preferably 0.292 mol / L.

[0060] In a specific embodiment provided by the present invention, the sodium dihydrogen phosphate solution described in step S2 is preferably slowly dripped into the ammonium molybdate solution for mixing; the molar ratio of the ammonium molybdate to the sodium dihydrogen phosphate is preferably (40-60):1, more preferably (45-55):1, and more preferably 50:1.

[0061] In a specific embodiment provided by the present invention, the mixing speed in step S2 is 400 to 2000 rpm, preferably 1000 to 2000 rpm, more preferably 1200 to 2000 rpm, even more preferably 1400 to 1800 rpm, even more preferably 1500 to 1700 rpm, and most preferably 1600 rpm; the mixing is preferably performed at room temperature. The ammonium molybdate and sodium dihydrogen phosphate compounds self-assemble to form an oxidized molybdenum-based Keggin-type heteropolyacid.

[0062] In a specific embodiment provided by the present invention, the volume ratio of the added volume of the alcohol solvent described in step S3 to the mixed solution is preferably (3-10):1, more preferably (4-8):1; the alcohol solvent is preferably ethanol.

[0063] In a specific embodiment provided by the present invention, the washing in step S3 is preferably performed by washing with ethanol and water, more preferably by washing with ethanol and double deionized water (ddH2O).

[0064] In a specific embodiment provided by the present invention, the vacuum degree of the freeze-drying in step S3 is preferably 10-20 Pa, more preferably 10-18 Pa, further preferably 12-16 Pa, and most preferably 14 Pa; the temperature of the freeze-drying is preferably -20°C to -40°C, further preferably -25°C to -35°C, and further preferably -30°C.

[0065] Example 1. Preparation of OX and verification of its catalytic activity 1. Preparation of Nanodrug OX Step 1: (NH4)6Mo7O 24 4H2O (ammonium molybdate tetrahydrate) was dissolved in 10 mL ddH2O to make (NH4)6Mo7O 24 ·4H2O solution (concentration 0.5 mol / L). Dissolve NaH2PO4·12H2O (sodium dihydrogen phosphate dodecahydrate) in 10 mL of H2O to prepare a NaH2PO4·12H2O solution (concentration 0.292 mol / L).

[0066] Step 2: Slowly add NaH2PO4·12H2O solution dropwise into 10 mL of (NH4)6Mo7O according to the molar ratio of ammonium molybdate to sodium dihydrogen phosphate of 50:1. 24 ·4H2O solution, stir on a magnetic stirrer at 1600 rpm at room temperature until colorless.

[0067] Step 3: After stirring the reaction for 30 to 60 minutes, 80 mL of ethanol (C2H5OH) was added to precipitate MoNs. The MoNs were washed with ethanol and ddH2O and collected by centrifugation (speed: 12000 rpm, centrifugation time: 5 minutes). Nanoparticles were obtained after freeze-drying (vacuum degree: 14 Pa, temperature: -30°C), which was recorded as OX dry powder.

[0068] 2. Characterization of Nanodrug OX 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 / 2 and Mo 3d3 / 2 sub-peaks due to spin-orbit coupling. The results are as follows Figure 1 As shown in the XPS spectrum of Mo3d, Mo3d has two main peaks at 233.35 eV and 236.4 eV, which correspond to Mo 3d5 / 2 and Mo 3d3 / 2, respectively. + The binding energy of Mo3d5 / 2 is about 232.6-232.9 eV, and that of Mo 3d3 / 2 is about 235.7-236.0 eV, which indicates that the valence state of molybdenum in OX is +6, which means it has a high oxidation state.

[0069] To further evaluate the stability of OX, the hydrated particle size (DLS) trend of OX was measured from 1 to 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 left at room temperature for 7 days. Samples were taken daily to measure the hydrated particle size of OX. The experiment was repeated three times. Figure 2 As shown in the figure, although the hydrated OX particle size increased slightly in PBS or FBS, it still remained between 1-3 nm, with a particle size fluctuation of <10%, indicating that OX has good dispersibility and excellent stability under physiological conditions.

[0070] 3. Verification of the catalytic activity of nanomedicine OX To evaluate the rate of NO production from S-nitrosoglutathione (GSNO) catalyzed by OX, different concentrations of OX (31.25, 62.5, 125, 250, 500, and 1000 µg / ml) and GSNO (100 µM) were added and the NO content was quantitatively determined using the above-mentioned kit at different time points. The experiment was repeated three times. The results are shown in Figure 2. Figure 3As shown in the figure, the results show that OX can continuously catalyze GSNO to produce NO, and the duration can reach more than 40 min.

[0071] 4. Effect of different stirring speeds on nanomedicine OX Following the aforementioned OX preparation method, adjust the rotation speed from 1600 rpm in step 2 to 800 rpm and then 400 rpm. The remaining preparation steps remain the same. Dynamic light scattering was used to determine the hydrated particle size of OX and the surface charge distribution (zeta potential) of OX. Three replicates were performed.

[0072] The results are as follows Figure 4 and Figure 5 As shown in the figure, stirring speed is correlated with nanomaterial nucleation. The faster the stirring speed, the more quickly the added disodium hydrogen phosphate dodecahydrate reacts with ammonium molybdate tetrahydrate, rapidly nucleating and forming a large number of uniformly sized nanomaterials. Therefore, OX prepared at 1600 rpm was selected for the following experiments.

[0073] Example 2: Multiple antibacterial mechanisms of OX 1. Oxidative antibacterial effect and NO synergistic antibacterial effect To demonstrate that the synergistic antibacterial effect of OX and GSNO was responsible for bacterial damage, we used scanning electron microscopy (SEM) to observe the morphological changes of Staphylococcus aureus. The specific steps are as follows: Use an inoculating loop to pick a colony of Staphylococcus aureus (S. aureus) CMCC26003 (purchased from Unicore (Shanghai) Life Sciences Co., Ltd.) from the slant and inoculate it into 50 mL of sterilized LB liquid medium (0.5 g NaCl, 0.5 g tryptone, and 0.25 g yeast extract dissolved in 50 mL deionized water). Place the centrifuge tube containing the inoculated medium in a constant temperature shaker at 37°C and incubate overnight. Dilute the bacterial suspension obtained above to 1 × 10 7 CFU / mL. 1 mL of each antibacterial experimental system (sterile PBS, vancomycin, OX, GSNO, GSNO + vancomycin, GSNO + OX; final concentrations of OX: 500 µg / mL, GSNO: 100 µM, and vancomycin: 1 µg / mL) was then prepared and incubated at 37°C for 6 h. After incubation, the resulting antibacterial experimental systems were characterized using silicon wafers prepared with field-emission scanning electron microscopy, and ATP content in each group was measured using an ATP synthesis kit. The experiment was repeated three times.

[0074] The results are as follows Figure 6 and Figure 7As shown: Staphylococcus aureus in the control group and GSNO group showed a smooth spherical shape with an intact surface; when vancomycin (VIO) was used in the presence or absence of GSNO or when OX was used alone for treatment, the bacterial membranes of both groups were deformed and shrunk to a certain extent; when OX+GSNO was used for synergistic antibacterial treatment, the bacterial membranes were severely damaged and ruptured ( Figure 6 ). And according to Figure 7 As shown in the figure, under OX treatment, the relative ATP content of the GSNO group was significantly lower than that of the control group, indicating that the combined action of GSNO and OX greatly affected the bacterial ATP synthesis process, with an inhibition rate of 82.96%.

[0075] The live-death staining imaging method was further used to observe the fluorescence release by incubating with 4 μM propidium iodide (PI) dilution for 5 minutes at room temperature in the dark. Figure 8 As shown, there was almost no red fluorescence in the control group and GSNO group, indicating that the bacterial death was less; when VIO or OX alone was used in the presence or absence of GSNO, the red fluorescence increased slightly; when OX+GSNO was used for synergistic antibacterial treatment, the red fluorescence was the highest, indicating that the bacterial death was the highest.

[0076] 2. Biofilm inhibition In order to visualize the bacterial biofilm, laser confocal microscopy was used to study the average thickness of the bacterial biofilm after different treatments. Figure 9 As shown, when OX and GSNO were used synergistically for antibacterial purposes, a very thin biofilm formed. In contrast, much thicker biofilms were observed on surfaces treated with antibiotics (VIO) or the control group, consistent with the aforementioned results. This phenomenon can be attributed to the fact that the NO generated by OX-catalyzed GSNO and the oxidative properties of OX can effectively eliminate bacterial biofilms and inhibit biofilm formation (inhibition rate of 92.14%), overcoming the difficulty of traditional antibiotics in penetrating biofilms and enhancing their antibacterial efficacy.

[0077] 3.OX has a broad-spectrum antibacterial effect Following the method for constructing the antibacterial system for Staphylococcus aureus, an antibacterial system for Escherichia coli (CMCC44103, purchased from Unico Shanghai Life Sciences Co., Ltd.) was constructed. OX was used at concentrations of 500 µg / ml, 1 mg / ml, and 2 mg / ml. After the antibacterial system was constructed, 100 µL of each mixed solution (S. aureus and E. coli) was evenly spread onto solid LB medium and incubated in a 37°C incubator for 14–16 hours. The number of bacterial colonies was counted using the CFU method, and the antibacterial efficacy was evaluated. The experiment was repeated three times.

[0078] The results are as follows Figure 10As shown, the synthesized OX, through its inherent oxidative antibacterial properties and the catalytic production of NO, produces a broad-spectrum antibacterial effect, exhibiting significant antibacterial activity against both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). OX's antibacterial activity against S. aureus suggests its potential clinical application in the treatment of infective endocarditis.

[0079] Example 3: Antithrombotic and Vascular Regulation of OX To assess vascular smooth muscle cell relaxation, Ca 2+ The assay kit is used to evaluate the relaxation of vascular smooth muscle cells in each group. The specific method is as follows: (1) Bacterial culture - liquid culture: Use an inoculation loop to pick a colony of Staphylococcus aureus (S. aureus) CMCC26003 (purchased from Unico (Shanghai) Life Sciences Co., Ltd.) from the slant and inoculate it into 50 mL of sterilized LB liquid culture medium (0.5 g NaCl, 0.5 g tryptone and 0.25 g yeast extract dissolved in 50 mL deionized water). Place the centrifuge tube containing the inoculated culture medium in a constant temperature shaker at 37 °C and 220 rev min -1 Incubate overnight.

[0080] (2) Vascular smooth muscle cell culture: Immortalized mouse aortic vascular smooth muscle cells (MOVAS) were purchased from Xiamen Yimo Biotechnology Co., Ltd. MOVAS cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific, USA) supplemented with 10% (v / v) fetal bovine serum (FBS) (Thermo Fisher Scientific, USA) and 1% (v / v) penicillin-streptomycin (Yisheng Biotechnology Co., Ltd., China). All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0081] (3) MOVAS cells (5×10 3 The different grouped materials were then incubated with the cells for 6 h, and then the culture medium was renewed. After standing for 4 hours, Ca 2+ The assay kit was used to assess vascular smooth muscle cell relaxation in each group. The groups included sterile PBS, vancomycin, OX, GSNO, GSNO + vancomycin, and GSNO + OX. The final concentrations of OX, GSNO, and vancomycin were 500 µg / ml, 100 µM, and 1 µg / ml, respectively.

[0082] The results are as follows Figure 11 As shown in the figure, the fluorescence intensity of cells in the GSNO+OX group was significantly lower than that in other groups, which indicates that OX catalyzes GSNO to generate NO, which causes the Ca2+ production of vascular smooth muscle cells to increase. 2+Outflow and dilation occur, achieving vasodilation (dilation rate 77.89%) and reducing the risk of embolism.

[0083] Example 4: In vivo experiments on OX 1. Establishment of a Mouse Model of Infective Endocarditis 1.1 The steps for constructing a mouse model of infective endocarditis are as follows: The experimental animals used were ICR male mice (7 weeks old, purchased from Spefox (Beijing) Biotechnology Co., Ltd.). ICR mice were captured and intraperitoneally injected with 1% sodium pentobarbital solution (50 mg / kg). After anesthesia, the mouse's limbs were taped to a test plate ( Figure 12 The mouse's neck area from the chin to slightly below the sternum was depilated, and the skin was cut open 1 cm to the right of the anterior midline of the neck using surgical scissors after being disinfected with 75% alcohol ( Figure 12 b), perform blunt dissection layer by layer to prevent muscle tearing, separate the fat tissue, and free the right common carotid artery about 1 cm ( Figure 12 c). Pass two strands of thread under the free right common carotid artery and tie a loose knot on the proximal arterial trunk to fix the arterial cannula. Ligate the common carotid artery at the head end ( Figure 12 d in the figure). Use fine ophthalmic surgical scissors and fine forceps to operate on the blood vessels. Under a stereo microscope, use the fine forceps in your left hand to lift the blood vessels, and use the ophthalmic surgical scissors in your right hand to make a V-shaped incision between the two ligatures. Figure 12 e). Hold the tweezers in your left hand to lift the blood vessel opening. Use the tweezers in your right hand to clamp a catheter with a syringe at one end filled with saline. Insert the catheter through the blood vessel opening. Remove the arterial clamp and slowly push the catheter in until resistance is encountered. Slightly withdraw the catheter and insert approximately 1.2-1.5 cm. After the catheter is successfully inserted ( Figure 12 f), tie the proximal end with a knot, clamp it with hemostatic forceps near the syringe end, cut it off, and seal it with nano glue ( Figure 12 After cleaning the blood from the operation, the catheter was observed to have a strong beating sensation along with the heart, indicating that the insertion was successful. The surgical incision was sutured and the end of the catheter was fixed under the skin ( Figure 12 24 h after successful cardiac cannulation, the mice were observed and the tail veins of normal mice were injected with 200 μl of overnight activated Staphylococcus aureus culture medium (7×10 4 CFU / ml) for infection.

[0084] 1.2 Grouping and Dosing According to the above experimental method, the mice were catheterized in the heart, and 24 hours later, each mouse was injected with 0.5 ml of pre-cultured and diluted bacterial solution (10 7CFU / ml). Mice were randomly divided into three groups, 5 in each group. Twenty-four hours after infection, drugs were administered daily via the tail vein: blank control (PBS), vancomycin (VIO, 80 mg / kg), or OX (20 mg / kg). Blood samples were collected from the maxillofacial area on days 1, 3, and 5 of treatment. After the final treatment on day 7, mice were sacrificed by overdose of anesthesia, and tissues and organs were harvested.

[0085] 2. Regulation of the inflammatory microenvironment To further investigate the biological effects of OX therapy in the microenvironment of infective endocarditis, circulating cytokine levels were measured to assess systemic immune responses.

[0086] The results are as follows Figure 13 As shown: After OX treatment, circulating immune factor IL-6 ( Figure 13 a), IL-8 ( Figure 13 b), IFN-γ ( Figure 13 This indicates that OX improves the cardiac tissue microenvironment and enhances immune clearance ability 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%).

[0087] 3. OX's anti-infection effect in vivo To evaluate the anti-infective effect of OX in the treatment of infective endocarditis, equal amounts of venous blood samples from mice were collected on days 3, 5, and 7 of treatment and inoculated into LB medium for overnight culture at 37°C. The number of colonies was counted.

[0088] The results are as follows Figure 14 As shown in the figure, blood colony counts showed that on day 3 after infection treatment, the control group (PBS-treated) had a relatively high colony count, while the VIO-treated group had a lower colony count than the control group, indicating a moderate antibacterial effect. The OX-treated group had very few colonies, demonstrating a significant antibacterial effect. On day 5, the control group had a significant increase in colony counts, while the VIO-treated group also saw a slight increase, though still lower than the control group. The OX-treated group maintained a low colony count, demonstrating its continued ability to inhibit bacterial growth. On day 7, the control group had a further increase in colony counts, while the VIO-treated group had a decreased colony count compared to day 5 due to changes in the antibacterial effect at later stages. The OX-treated group maintained a low colony count, indicating that OX treatment had a stable and sustained inhibitory effect on bacterial growth throughout the observation period. Overall, OX treatment was superior to VIO treatment in inhibiting bacterial growth at all time points, demonstrating a more effective control of bacterial numbers in blood samples.

[0089] 4. Development of OX in vivo anti-neoplastic effects 4.1 To evaluate the inhibitory effect of OX's NO biocatalytic activity on the development of neoplasia, mice in the different groups were euthanized on the seventh day of treatment. The hearts were carefully dissected using the aforementioned dissection technique, and the neoplasia was completely separated from the organ using ophthalmic surgical scissors and forceps. The neoplasia was photographed and its volume was statistically measured using Image J.

[0090] As shown in the pictures of cardiac vegetation in different treatment groups ( Figure 15 As shown in a), the vegetation in the PBS control group was relatively large, the vegetation in the VIO treatment group was smaller than that in the control group, and the vegetation in the OX treatment group was the smallest. In some samples, the vegetation was even extremely small in size visible to the naked eye. And through statistical data ( Figure 15 As shown in Figure b), the thrombus volume was the largest in the PBS control group, approximately 30 mm³. The thrombus volume decreased to approximately 10 mm³ in the VIO treatment group, and the thrombus volume was the smallest in the OX treatment group, approaching 0 mm³. This indicates that OX treatment was most effective in inhibiting vegetation growth, while VIO treatment also had a certain inhibitory effect, with statistically significant differences among the three groups.

[0091] 4.2 In order to further evaluate the internal structure and cell composition of the vegetation, some vegetation samples were sliced ​​and processed, and their cell morphology and tissue structure were observed under a microscope using HE staining.

[0092] The results are as follows Figure 15 As shown in Figure (c), numerous red areas were visible in the control group, primarily reflecting the aggregation of red blood cells. Furthermore, numerous areas of relatively dense tissue structure, likely composed of fibrous tissue, were observed, indicating that the vegetation contained a rich supply of blood components and extracellular matrix. In the VIO-treated group, the red areas were relatively reduced, indicating a decrease in the red blood cell content. The tissue structure appeared relatively loose, suggesting that VIO treatment had a certain effect on the composition and structure of the vegetation, possibly inhibiting the accumulation of certain components. In the OX-treated group, the red areas were further reduced, and the changes in tissue structure were more pronounced compared to the control group. The distribution of cellular components and extracellular matrix was also altered, indicating that OX treatment not only inhibited vegetation growth but also significantly affected its internal structure. These results indicate that OX treatment was the most effective in inhibiting the growth of cardiac vegetation and altering its internal structure, followed by VIO treatment.

[0093] 5. OX inhibits platelet activation in vivo To evaluate the inhibitory effect of OX on the NO biocatalytic activity of endogenous GSNO on platelet activation, venous whole blood was collected from mice in different groups, 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 No. 104305; CD62p, Catalog No. 148304; both purchased from Biolegend) at room temperature for 30 min. The fluorescence intensity was then measured by flow cytometry to evaluate the degree of platelet activation in each group.

[0094] The results are as follows Figure 16 As shown, flow cytometry scatter plot ( Figure 16 a) and platelet activation rate histogram ( Figure 16 Figure b) shows that compared to the control group, the proportion of activated platelets increased slightly after VIO treatment, while this proportion decreased significantly after OX treatment. This is consistent with the results of in vitro platelet activation. Furthermore, VIO treatment promotes platelet activation, while OX treatment effectively inhibits platelet activation by catalyzing the production of NO from endogenous GSNO, reducing platelet aggregation and thus controlling the development of neoplasia.

[0095] In summary, the present invention provides an antibacterial drug that utilizes GSNO in the microenvironment to generate NO to exert anti-infection and anti-vegetative effects. This nanomedicine utilizes an oxidizing material formed from the transition metal Mo as a NO synthase, catalyzing the stable generation of NO from endogenous GSNO. This nanomedicine combines the vasodilatory effects of NO with its broad-spectrum antibacterial properties, effectively treating infective endocarditis while preventing the development of antibiotic resistance. This drug has significant potential for clinical application in the treatment of infective endocarditis.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a nanomedicine having NO catalytic performance, characterized in that: The steps include: (1) dissolving molybdate in water to obtain a molybdate solution; dissolving hydrogen phosphate in water to obtain a hydrogen phosphate solution; (2) The hydrogen phosphate solution and the molybdate solution are mixed and stirred at 400-2000 rpm until the mixture becomes colorless to obtain a mixed solution. The mixture is stirred and reacted until the reaction is completed. An alcohol solvent is added to precipitate the mixture. The mixture is collected by centrifugation, washed, and dried to obtain the nanomedicine having NO catalytic properties.

2. The method according to claim 1, wherein: The molybdate described in step (1) is at least one of ammonium molybdate and ammonium molybdate tetrahydrate; The hydrogen phosphate described in step (1) is at least one of disodium hydrogen phosphate and sodium dihydrogen phosphate dodecahydrate; The alcohol solvent described in step (2) is ethanol.

3. The method according to claim 1, wherein: The molar ratio of molybdate to hydrogen phosphate in step (1) is 40 to 60:1; The stirring speed in step (2) is 1000-2000 rpm.

4. The method according to claim 3, wherein: The molar ratio of molybdate to hydrogen phosphate in step (1) is 45-55:1; The stirring speed in step (2) is 1200 to 2000 rpm.

5. The method according to claim 1, wherein: The concentration of the molybdate solution in step (1) is 0.1 to 1 mol / L; The concentration of the hydrogen phosphate solution in step (1) is 0.1 to 0.5 mol / L; The molar ratio of molybdate to hydrogen phosphate in step (1) is 50:1; The stirring speed in step (2) is 1400-1800 rpm.

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

7. The method according to claim 1, wherein: The reaction time in step (2) is 30 to 60 minutes; The centrifugation conditions described in step (2) are: 12000 rpm, centrifugation for 5-10 min; The washing in step (2) is carried out using an alcohol solvent and water.

8. A nanomedicine with NO catalytic performance, characterized by: It is prepared by the method according to any one of claims 1 to 7.

9. Use of the nanomedicine with NO catalytic performance according to claim 8 in the preparation of antibacterial, antithrombotic and / or infective endocarditis treatment drugs.

10. Use of the nanomedicine with NO catalytic performance according to claim 8 in the preparation of S-nitrosoglutathione catalyst.

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