Preparation method and application of RuMo nanoflower

Abstract

The application discloses a preparation method of RuMo nanoflower and application thereof, and comprises the following steps: (1) dissolving ruthenium trichloride trihydrate in an organic solvent; (2) dispersing polyvinylpyrrolidone and molybdenum hexacarbonyl in the organic solvent; (3) mixing materials obtained in steps (1) and (2), and ultrasonic dispersing at room temperature, then carrying out programmed temperature rising, rising to 180 DEG C within 30 min, and cooling to room temperature after heat preservation reaction for 1-3 h; (4) centrifugally cleaning the material obtained in step (3) with a mixed solution of acetone and ethanol, and the RuMo nanoflower is obtained; the organic solvent is benzyl alcohol or ethylene glycol. The application has simple synthesis, high biosafety of the prepared RuMo nanoflower, good enzyme catalytic activity and good photo-thermal performance.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and particularly relates to a preparation method of RuMo nanoflowers and applications thereof. Background Art

[0002] Enzymes are natural catalysts possessed by organisms. They achieve maximum catalytic efficiency in the mild environment of the human body and participate in numerous metabolic processes throughout life, such as decomposition, synthesis, and transport. Within the human body, there are numerous enzymes, including amylase, lipase, and protease, each responsible for catalyzing corresponding metabolic processes and maintaining normal bodily function. However, the role of enzymes is not static. When the internal environment changes, or when certain enzymes are missing or malfunction due to illness or other causes, related diseases may occur.

[0003] With the advancement of science and technology, researchers have mastered the extraction of natural enzymes and applied them to the treatment of related diseases. For example, proteolytic enzymes (trypsin or serrapeptase) are used to treat inflammation in the body, and superoxide dismutase (SOD) is used to alleviate organ damage caused by reactive oxygen species (ROS) during hemorrhagic shock. Although synthetic proteases have shown some efficacy in disease treatment, the complex synthesis and extraction steps of natural enzymes, high production costs, harsh reaction conditions, and poor stability have greatly limited their application in biodiagnosis and treatment.

[0004] Nanozymes are a class of nanomaterials that possess the catalytic activity of natural enzymes and follow the Michaelis–Menten kinetics of enzyme catalysis. Nanozymes offer unique advantages, including flexibility in composition and structural design, ease of preparation, tunable catalytic activity, high catalytic stability in harsh environments, and excellent biocompatibility. These advantages can fully offset the shortcomings of natural enzymes in practical applications, giving them broad application prospects in fields such as biosensing, imaging, therapeutics, antimicrobial treatments, and environmental protection. Summary of the Invention

[0005] The present invention aims to provide a method for preparing RuMo nanoflowers.

[0006] Another object of the present invention is to provide applications of the RuMo nanoflowers prepared by the above preparation method.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing RuMo nanoflowers comprises the following steps:

[0009] (1) dissolving ruthenium trichloride trihydrate in an organic solvent;

[0010] (2) dispersing polyvinyl pyrrolidone and hexacarbonyl molybdenum in an organic solvent;

[0011] (3) The materials obtained in step (1) and step (2) are mixed and ultrasonically dispersed at room temperature, followed by programmed temperature increase to 180° C. within 30 min, kept at this temperature for 1-3 h, and then cooled to room temperature;

[0012] (4) The material obtained in step (3) is centrifuged and washed with a mixture of acetone and ethanol to obtain the RuMo nanoflowers.

[0013] In a preferred embodiment of the present invention, the mass ratio of ruthenium trichloride trihydrate, polyvinyl pyrrolidone and molybdenum hexacarbonyl is 2:4:1; in the step (1), each 1 mL of organic solvent corresponds to 12 mg of ruthenium trichloride trihydrate; in the step (2), each 1 mL of organic solvent corresponds to 24 mg of polyvinyl pyrrolidone.

[0014] The RuMo nanoflowers prepared by the above preparation method are used in the preparation of oxidase.

[0015] The RuMo nanoflowers prepared by the above preparation method are used in the preparation of peroxidase.

[0016] The RuMo nanoflowers prepared by the above preparation method are used in the preparation of glutathione oxidase.

[0017] The RuMo nanoflowers prepared by the above preparation method are used in the preparation of photothermal therapy compositions.

[0018] An oxidase, whose effective components include the RuMo nanoflowers prepared by the above preparation method.

[0019] A peroxidase, the effective component of which includes the RuMo nanoflowers prepared by the above preparation method.

[0020] A glutathione oxidase, the effective component of which includes the RuMo nanoflowers prepared by the above preparation method.

[0021] A photothermal therapy composition, the raw materials of which include RuMo nanoflowers prepared by the above preparation method.

[0022] The beneficial effects of the present invention are: the synthesis of the present invention is simple, the prepared RuMo nanoflowers have high biological safety, good enzyme catalytic activity and good photothermal performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Figure 1 shows the growth process of the RuMo nanoflowers in Example 1 of the present invention. (a) UV-visible-near-infrared absorption spectra of the RuMo nanoflowers; (bh) Transmission electron microscopy images of the RuMo nanoflowers; (i) Schematic diagram of the growth process of the RuMo nanoflowers.

[0024] Figure 2 This is a transmission electron microscopy image of the RuMo nanoflower in Example 1 of the present invention, and the solvent is ethylene glycol.

[0025] Figure 3 The transmission electron micrographs of Example 1 of the present invention are shown below: (a) transmission electron micrograph of Ru nanoparticles; (b) transmission electron micrograph of Mo nanoparticles; (c) high-resolution transmission electron micrograph of RuMo nanoflowers; and (d) HAADF-STEM-EDX image of RuMo nanoflowers.

[0026] Figure 4 This is the X-ray diffraction pattern of the RuMo nanoflower in Example 1 of the present invention.

[0027] Figure 5 These are the atomic force microscope images, three-dimensional atomic force microscope images, and line scan thickness images of (a) single-layer RuMo and (b) RuMo nanoflowers in Example 1 of the present invention.

[0028] Figure 6 This is the XPS spectrum of the RuMo nanoflower in Example 1 of the present invention, where: (a) Ru 3p; (b) Mo 3d orbital narrow spectrum.

[0029] Figure 7 This is the hydrated particle size and surface potential diagram of RuMo nanoflowers in Example 1 of the present invention.

[0030] Figure 8 Figure 2 shows the near-infrared photothermal performance of the RuMo nanoflowers in Example 2 of the present invention. (a) Heating-cooling curve of the RuMo nanoflower dispersion under four consecutive laser on-off cycles; (b) Heating-cooling curve of the RuMo nanoflower dispersion; (c) Linear relationship between the cooling time of the RuMo nanoflowers and the negative logarithm (-lnθ).

[0031] Figure 9 Graphs showing the oxidase activity of the RuMo nanoflowers in Example 3 of the present invention. These graphs include: (a) the effect of RuMo nanoflower dosage on oxidase activity; (b) the change in oxidase activity of the RuMo nanoflowers over reaction time; the dependence of the oxidase activity of the RuMo nanoflowers on pH (c) and temperature (d); (e) a graph showing the reaction rate of the oxidase activity of the RuMo nanoflowers versus TMB concentration; and (f) a double reciprocal graph showing the reaction rate of the oxidase activity of the RuMo nanoflowers versus TMB concentration.

[0032] Figure 10 Figure 4 shows the peroxidase activity of the RuMo nanoflowers in Example 4 of the present invention. The figures include: (a) peroxidase activity of different combinations; (b) effect of RuMo nanoflower dosage on peroxidase activity; dependence of RuMo nanoflower peroxidase activity on pH (c) and temperature (d); (e) plot of the peroxidase activity reaction rate of the RuMo nanoflowers versus TMB concentration; (f) a double reciprocal plot of the peroxidase activity reaction rate of the RuMo nanoflowers versus TMB concentration; (g) a plot of the peroxidase activity reaction rate of the RuMo nanoflowers versus H2O2 concentration; and (h) a double reciprocal plot of the peroxidase activity reaction rate of the RuMo nanoflowers versus H2O2 concentration.

[0033] Figure 11 The glutathione oxidase activity of the RuMo nanoflowers in Example 5 of the present invention. (a) The scavenging capacity of RuMo nanoflowers on reduced glutathione at different reaction times; (b) The scavenging capacity of RuMo nanoflowers on reduced glutathione at different doses.

[0034] Figure 12 This is a graph showing the results of evaluating the toxicity of RuMo nanoflowers to PC12 cells using the MTT method in Example 6 of the present invention.

[0035] Figure 13 This is a comparison chart of the hemolysis rates of RuMo nanoflowers with different concentrations in Example 7 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0037] Example 1 Preparation of RuMo Nanoflowers

[0038] In this example, ruthenium trichloride trihydrate was purchased from Beijing Inokai Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; molybdenum hexacarbonyl was purchased from Beijing Bailingwei Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; polyvinyl pyrrolidone was purchased from Wokai, and benzyl alcohol and ethylene glycol were purchased from Sinopharm and stored in the dark at room temperature.

[0039] In this example, RuMo nanoflowers were prepared by wet chemical synthesis:

[0040] (1) Dissolve 60 mg of ruthenium trichloride trihydrate in 5 mL of benzyl alcohol / ethylene glycol;

[0041] (2) Disperse 120 mg of polyvinylpyrrolidone and 30 mg of hexacarbonyl molybdenum in 5 mL of benzyl alcohol / ethylene glycol;

[0042] (3) The materials obtained in step (1) and step (2) were mixed and ultrasonicated at room temperature for 20 minutes. After the ultrasonication, the temperature was programmed to rise to 180°C within 30 minutes, kept at this temperature for 3 hours, and then cooled to room temperature;

[0043] (4) The material obtained in step (3) was centrifugally washed three times with a mixture of acetone and ethanol, and the product was dispersed in water to obtain RuMo nanoflowers.

[0044] The RuMo nanoflowers prepared above were subjected to physical and chemical characterization (UV-visible absorption spectroscopy, transmission electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy (XPS), dynamic light scattering, and X-ray diffraction (XRD)).

[0045] like Figure 1 As shown in Figure a, during the synthesis process, the reaction solution will undergo color changes from orange to yellow to green to brown as the reaction temperature and time change. This color change actually reflects the growth and change of the RuMo nanoflowers during the synthesis process. At the same time, this example also analyzed the UV-visible absorption spectrum of the reaction solution and found that as the reaction proceeds, the multiple absorption peaks of its UV-visible absorption spectrum gradually disappear, indicating that the morphology and structure of the RuMo nanoflowers are changing. Figure 1 Transmission electron microscopy images clearly show that the RuMo nanoflowers initially grow from small particles, gradually increasing in size as the reaction proceeds. After incubation at 180°C for one hour, the nanoflowers gradually develop into a "snowflake"-like sheet structure. As the incubation time increases, the sheet structures gradually accumulate, eventually forming multiple layers of RuMo.

[0046] like Figure 2 As shown in Figure 3, after the solvent was replaced with ethylene glycol, the morphology of RuMo changed to bean-shaped particles, indicating that benzyl alcohol promoted the assembly of nanoparticles to a certain extent.

[0047] like Figure 3 As shown, Ru and Mo alone cannot form a sheet structure, indicating that the assembly of RuMo is affected by both the solvent and the effect of molybdenum hexacarbonyl. High-resolution transmission electron microscopy clearly reveals the lattice distribution on the material surface, as well as the elemental distribution of ruthenium and molybdenum.

[0048] like Figure 4 , XRD data also showed the Ru and Mo crystal plane information. And through atomic force microscopy ( Figure 5 )You can also see the surface structure and atomic thickness of RuMo nanoflowers.

[0049] By X-ray photoelectron spectroscopy analysis ( Figure 6), the composition and valence information of RuMo nanoflowers can be obtained.

[0050] The hydrated particle size and surface charge of RuMo nanoflowers can be obtained by Malvern analyzer test ( Figure 7 ) Example 2 Photothermal conversion efficiency of RuMo nanoflowers

[0051] This example uses an 808nm laser to study the near-infrared photothermal conversion properties of the RuMo nanoflowers prepared in Example 1 (using benzyl alcohol as the organic solvent). Figure 8 As shown in the figure, after four cycles of 808nm laser "on-off" irradiation, the RuMo nanoflower dispersion still maintained a stable temperature increase trend with no significant temperature decay, indicating that the RuMo nanoflowers have good photothermal conversion performance. Furthermore, by fitting the linear relationship between the cooling time and the negative logarithm (-lnθ) of the RuMo nanoflower dispersion after laser irradiation, the photothermal conversion efficiency of the RuMo nanoflowers was calculated to be 23.67%.

[0052] Example 3 Oxidase (OXD) activity of RuMo nanoflowers

[0053] In this example, ruthenium trichloride trihydrate was purchased from Beijing Inokai Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; molybdenum hexacarbonyl was purchased from Beijing Bailingwei Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from Sigma-Aldrich and stored in a -4°C refrigerator in the dark; dimethyl sulfoxide (DMSO) and benzyl alcohol were purchased from Sinopharm and stored in the dark at room temperature.

[0054] In this example, 3,3',5,5'-tetramethylbenzidine (TMB) was used as the reaction substrate, and the OXD activity of the RuMo nanoflowers (using benzyl alcohol as the organic solvent) prepared in Example 1 was evaluated by recording the absorbance change of its oxidation product at 652nm. In a HAc-NaAc buffer solution (pH = 3.51), TMB (0.5mM, dissolved in DMSO) and an aqueous solution of RuMo nanoflowers were thoroughly mixed. After reacting for 5 minutes, the absorption spectrum of the mixed solution was collected on a UVmini-1280. Subsequently, the optimal conditions for the OXD activity of RuMo nanoflowers were confirmed by changing the pH and temperature of the reaction. Then, based on the kinetic reaction mechanism of the enzyme activity, the Michaelis-Menten double reciprocal equation Lineweaver-Burk was used to calculate the Michaelis constant of the RuMo nanoflower OXD enzyme-catalyzed reaction.

[0055] The results are as follows Figure 9As shown in the figure, RuMo nanoflowers exhibit optimal OXD activity at a pH of 4.5. When the pH value gradually increases above this optimal value, the OXD activity of RuMo nanoflowers gradually decreases, and after the pH value exceeds 7, almost no OXD activity can be detected. At the same time, this example also observed that RuMo nanoflowers have a wide temperature adaptability range, and the optimal temperature for their OXD activity can reach 60°C. And within the temperature range of 20-70°C, their OXD activity remains basically unchanged. By calculating the Michaelis constant, its V max (2.67×10 -3 mM·min -1 ) and K m (2.99×10 -4 mM).

[0056] Example 4 Peroxidase (POD) activity of RuMo nanoflowers

[0057] In this example, hydrogen peroxide, dimethyl sulfoxide, benzyl alcohol, and ethylene glycol were purchased from Chinese medicine and stored at room temperature in the dark.

[0058] In a HAc-NaAc buffer solution (pH = 3.51), TMB (0.5mM, dissolved in DMSO), H2O2 (0.5mM), and an aqueous solution of the RuMo nanoflowers prepared in Example 1 (using benzyl alcohol as the organic solvent) were thoroughly mixed. After reacting for 5 minutes, the absorption spectrum of the mixed solution was collected using a UVmini-1280. Subsequently, the optimal conditions for the POD activity of the RuMo nanoflowers were determined by varying the pH and temperature of the reaction. The Michaelis-Menten double reciprocal equation (Lineweaver-Burk) was then used to calculate the Michaelis-Menten constant for the POD enzyme-catalyzed reaction of the RuMo nanoflowers based on the kinetic reaction mechanism of the enzyme activity.

[0059] The results are as follows Figure 10 As shown in the figure, RuMo nanoflowers have good POD activity when pH < 7, and their POD activity is relatively stable in the pH range of 3.5-6. At the same time, RuMo nanoflowers also have strong high temperature resistance, and their POD optimum temperature can reach 60°C, which undoubtedly provides a good guarantee for their stability in practical applications. Next, this example uses the Michaelis-Menten equation to calculate the V of the two reaction substrates TMB and H2O2. max (TMB: 8.38×10 -3 mM·min -1 ;H2O2:0.20×10 - 3 mM·min -1 ) and K m (TMB: 4.27 × 10-5 mM; H2O2:8.85×10 -4 mM).

[0060] Example 5 Glutathione oxidase activity of RuMo nanoflowers

[0061] In this embodiment, ruthenium trichloride trihydrate was purchased from Beijing Inokai Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; molybdenum hexacarbonyl was purchased from Beijing Bailingwei Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; glutathione (reduced form) was purchased from Shanghai Boer Chemical Reagent Co., Ltd. and stored in a -4°C refrigerator in the dark; 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from TCI and stored in a -4°C refrigerator in the dark; polyvinyl pyrrolidone was purchased from Wokai, and benzyl alcohol was purchased from Sinopharm and stored in the dark at room temperature.

[0062] This example uses the properties of the yellow product (i.e., 5-thio-2-nitrobenzoic acid) generated by the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with reduced glutathione (GSH) at a wavelength of 412 nm to evaluate the GSH scavenging ability of the RuMo nanoflowers prepared in Example 1 (using benzyl alcohol as the organic solvent), i.e., the glutathione oxidase activity. The specific steps are as follows: GSH is mixed with RuMo nanoflowers at different concentrations (5.6–90 μg / mL), protected from light, and reacted at 37°C at a speed of 120 rpm for a period of time (1, 2, 4, and 6 hours). After the reaction is completed, the reaction solution is mixed with DTNB and Tris-HCl buffer, waited for 5-10 minutes, and the absorbance value of the mixture at 410 nm is measured.

[0063] The results are as follows Figure 11 As shown in the figure, as the dosage of RuMo nanoflowers increases, their GSH clearance effect becomes better. When the dosage of RuMo nanoflowers reaches 90μg / mL, its GSH clearance rate can reach nearly 80% within 6 hours. And the GSH clearance rate will continue to increase over time.

[0064] Example 6 Cytotoxicity of RuMo Nanoflowers

[0065] In this embodiment, ruthenium trichloride trihydrate was purchased from Beijing Inokai Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; molybdenum hexacarbonyl was purchased from Beijing Bailingwei Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; thiazolyl blue tetrazolium bromide (MTT) was purchased from Abcam and stored in a -4°C refrigerator in the dark; polyvinyl pyrrolidone was purchased from Wokai, and dimethyl sulfoxide and benzyl alcohol were purchased from Sinopharm and stored in the dark at room temperature.

[0066] In this embodiment, the number is 10 5The cells were seeded in a 96-well plate and incubated for 24 hours. They were then incubated with DMEM medium containing different concentrations of RuMo nanoflowers prepared in Example 1 for 24 hours. The old medium was then removed and 200 μL of MTT (0.5 mg / mL) was added and placed in an incubator for incubation for 4 hours. The MTT residual solution was discarded, and 150 μL of DMSO was added and placed in an incubator for a further 30 minutes until the formazan crystals were completely dissolved. Finally, the optical density at 570 nm was measured on a microplate reader to calculate the cell survival rate (reference wavelength was 630 nm).

[0067] The results are as follows Figure 12 As shown, the experimental results show that under the action of different concentrations of RuMo nanoflowers, the survival rate of PC12 cells can basically be maintained above 90%, which shows that RuMo nanoflowers have very low toxicity to PC12 cells.

[0068] Example 7 Hemolysis rate of RuMo nanoflowers

[0069] In this example, ruthenium trichloride trihydrate was purchased from Beijing Inokai Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; molybdenum hexacarbonyl was purchased from Beijing Bailingwei Technology Co., Ltd. and stored in a -4°C refrigerator in the dark; benzyl alcohol was purchased from Sinopharm and stored in a room temperature in the dark.

[0070] In this example, fresh mouse blood was centrifuged at 1600 rpm for 15 min, and the obtained precipitate was washed four times with a phosphate buffer solution (PBS) at pH 7.4. Then, 1.5 mL of the precipitate was mixed with 5.5 mL of PBS to prepare a stock solution. 200 μL of the stock solution was added to 1 mL of a PBS solution of RuMo nanoflowers (using benzyl alcohol as the organic solvent) prepared in Example 1 (the solvent was PBS at pH 7.4, and the specific concentration of RuMo nanoflowers in it is shown in FIG. 1 ). Figure 13 ), the mixture was centrifuged at 10,000 rpm for 20 min, and the supernatant was measured for absorbance at 540 nm using a microplate reader.

[0071] Hemolysis rate (%) = (A s -A n ) / (A p -A n )×100%

[0072] A s :Red blood cell stock solution is mixed with materials; A n :Red blood cell stock solution mixed with PBS; A p : Red blood cell stock solution is mixed with H2O.

[0073] Through the above hemolysis test ( Figure 13), it can be observed that the hemolysis rates of PBS solutions of RuMo nanoflowers with different concentrations are all lower than 5%, which indicates that the RuMo nanoflowers prepared in Example 1 (using benzyl alcohol as the organic solvent) have good biosafety.

[0074] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing RuMo nanoflowers, characterized in that: The steps include: (1) Dissolve ruthenium trichloride trihydrate in an organic solvent, with 12 mg of ruthenium trichloride trihydrate per 1 mL of organic solvent; (2) Dispersing polyvinyl pyrrolidone and molybdenum hexacarbonyl in an organic solvent, with 24 mg of polyvinyl pyrrolidone per 1 mL of organic solvent; (3) The materials obtained in step (1) and step (2) were mixed and ultrasonically dispersed at room temperature, followed by programmed temperature increase to 180°C within 30 minutes, kept at this temperature for 1-3 hours, and then cooled to room temperature; (4) washing the material obtained in step (3) by centrifugation with a mixture of acetone and ethanol to obtain the RuMo nanoflowers; The above-mentioned organic solvent is benzyl alcohol or ethylene glycol; The mass ratio of ruthenium trichloride trihydrate, polyvinylpyrrolidone and molybdenum hexacarbonyl is 2:4:

1.

2. Use of the RuMo nanoflowers prepared by the preparation method according to claim 1 in the preparation of oxidase.

3. Use of the RuMo nanoflowers prepared by the preparation method according to claim 1 in the preparation of peroxidase.

4. Use of the RuMo nanoflowers prepared by the preparation method according to claim 1 in the preparation of glutathione oxidase.

5. Use of the RuMo nanoflowers prepared by the preparation method according to claim 1 in preparing a photothermal therapy composition.

6. An oxidase, characterized in that: The effective ingredients include RuMo nanoflowers prepared by the preparation method according to claim 1.

7. A peroxidase, characterized in that: The effective ingredients include RuMo nanoflowers prepared by the preparation method according to claim 1.

8. A glutathione oxidase, characterized in that: The effective ingredients include RuMo nanoflowers prepared by the preparation method according to claim 1.

9. A photothermal therapy composition, characterized in that: The raw materials include RuMo nanoflowers prepared by the preparation method according to claim 1.

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