阅读说明：本技术 一种氧空位三氧化钼纳米颗粒的制备方法及应用 (Preparation method and application of oxygen vacancy molybdenum trioxide nanoparticles ) 是由 文玲 段广新 魏竹馨 曾剑峰 高明远 于 2021-11-08 设计创作，主要内容包括：本发明公开了一种氧空位三氧化钼纳米颗粒的制备方法及应用,包括S1)将包含功能团-COOH和/或-SH的聚合物溶于纯水中,制得溶液一；S2)将钼源溶于乙醇中,制得溶液二；S3)将溶液二加入溶液一种,在氮气保护下加热至沸腾,溶液颜色变为深蓝色；S4)将S3反应后产物冷却,去除未反应的原料,即制得纯化的氧化钼纳米颗粒分散液；S5)将氧化钼纳米颗粒分散液冷冻干燥,得到固体,即为氧空位三氧化钼纳米颗粒。本发明氧空位三氧化钼纳米颗粒一方面具有良好的自由基清除性能,另一方面具有强效的抗菌性能与生物安全性,通过使用氧化钼纳米材料,可以有效降低糖尿病创面的过氧化环境,减少创面区域细菌数量,进而促进血管生成,有效的加速糖尿病创面的愈合。(The invention discloses a preparation method and application of oxygen vacancy molybdenum trioxide nano particles, comprising S1) dissolving a polymer containing functional groups-COOH and/or-SH in pure water to prepare a solution I; s2) dissolving a molybdenum source in ethanol to prepare a solution II; s3) adding the solution II into the solution I, heating to boil under the protection of nitrogen, and changing the color of the solution into dark blue; s4) cooling the product obtained after the reaction of S3, and removing unreacted raw materials to obtain purified molybdenum oxide nanoparticle dispersion liquid; s5) carrying out freeze drying on the molybdenum oxide nano-particle dispersion liquid to obtain a solid, namely the oxygen vacancy molybdenum trioxide nano-particles. The oxygen vacancy molybdenum trioxide nano-particles have good free radical scavenging performance on one hand, and have strong antibacterial performance and biological safety on the other hand, and by using the molybdenum oxide nano-material, the peroxidation environment of a diabetic wound can be effectively reduced, the bacterial number in the wound area is reduced, so that angiogenesis is promoted, and the healing of the diabetic wound is effectively accelerated.)

1. A preparation method of oxygen vacancy molybdenum trioxide nano particles is characterized by comprising the following steps,

s1) dissolving polyvinyl acid or pentaerythritol-tetra (3-mercaptopropionate) -polymethacrylic acid polymer containing functional groups of-COOH and/or-SH in pure water to prepare 0.1-1% of aqueous solution I;

s2) dissolving a molybdenum source in ethanol to prepare a solution II with the Mo content of 0.1-0.5M;

s3) adding 1-2 mL of the solution II into 45-55 mL of the solution I, heating to boil under the protection of nitrogen, and changing the color of the solution into dark blue;

s4) cooling the product obtained after the reaction of S3, and removing unreacted raw materials to obtain purified molybdenum oxide nanoparticle dispersion liquid;

s5) carrying out freeze drying on the molybdenum oxide nano-particle dispersion liquid to obtain a solid, namely the oxygen vacancy molybdenum trioxide nano-particles.

2. The method for preparing oxygen-vacancy molybdenum trioxide nanoparticles as claimed in claim 1, wherein the dissolving condition in S1 is that the solution is heated to condensation reflux under the protection of nitrogen.

3. The method for preparing oxygen-vacancy molybdenum trioxide nanoparticles as claimed in claim 1, wherein the heating time in S3 is 1-3 h.

4. The method of claim 1, wherein the molybdenum source is MoCl₅。

5. The method for preparing oxygen-vacancy molybdenum trioxide nanoparticles as claimed in claim 1, wherein the product after the reaction in S4 is cooled to 20-25 ℃, and unreacted raw materials are removed by ultrafiltration using an ultrafiltration tube.

6. The method of claim 1, wherein the particle size of the molybdenum oxide nanoparticles in the molybdenum oxide nanoparticle dispersion of S4 is 3.1 ± 0.5 nm.

7. An oxygen-vacancy molybdenum trioxide nanoparticle prepared by the preparation method according to any one of claims 1 to 6, which is used for promoting recovery of diabetic drug-resistant bacterial infection wounds.

Technical Field

The invention relates to the technical field of molybdenum trioxide processing, in particular to a preparation method and application of oxygen vacancy molybdenum trioxide nano particles.

Background

Diabetes is the most common metabolic disease at present, diabetic ulcer is one of the most common complications of diabetes, mainly exists in feet or legs of patients, presents as a chronic wound surface which is not easy to heal, has the characteristics of high morbidity, long course of disease and the like, and causes great physical and psychological pain and economic burden to the patients. Even more feared, diabetic ulcers, if not effectively treated, are at risk of causing amputation, and the data suggest that diabetic ulcers are the leading cause of non-invasive amputation.

The poor peroxidation microenvironment of the wound surface in the diabetic ulcer area is the main reason for the difficult healing of the diabetic ulcer area. Studies have reported that the ulcerated wound area has a large number of neutrophil-dominated immune cells that produce excess Reactive Oxygen Species (ROS). Excessive ROS can damage epithelial cells on one hand and hinder the healing of the wound surface; on the other hand, angiogenesis is inhibited, oxygen and nutrition in the wound surface area are affected, and further deterioration of the ulcer area is caused. In addition, excessive ROS can also induce severe inflammatory reaction of macrophages, induce release of a large amount of inflammatory factors, enable a healing area to be easily broken, and further influence recovery of diabetic ulcers. Meanwhile, the diabetic ulcer wound area is open for a long time, has higher sugar content, and creates good conditions for the colonization of pathogenic microorganisms, so that the diabetic ulcer area is easy to have bacterial infection. Infection not only can strengthen the inflammatory reaction in ulcer area, disturb angiogenesis and epithelial cell proliferation, but also can further increase the generation of wound surface area ROS, and then form: excess ROS production-angiogenesis and epithelial cell proliferation inhibited-long term wound opening-bacterial infection-a vicious cycle of more ROS production.

At present, the operation debridement is mainly adopted clinically, and the antibiotic is combined to resist infection so as to treat the diabetic wound. Although the treatment can delay the deterioration of the disease to a certain extent, the treatment cannot effectively promote the recovery of the diabetic ulcer wound. In addition, the chronic wound surface is treated by antibiotics for a long time, so that the drug resistance of pathogenic bacteria is increased, and the difficulty of treating the diabetic ulcer is further increased. In recent years, researches prove that effective ROS removal is performed on diabetic ulcer wounds, and the wound healing can be remarkably promoted by combining strong sterilization. However, in these exploratory studies, complex compounds are used which are formed by simply combining various raw materials having single functions. Besides the complex components, the preparation process is relatively complicated, and the bottleneck problem of large-scale production and clinical popularization is caused. Therefore, the diabetes wound treatment drug with good oxidation resistance and bactericidal activity has the advantages of simple components and convenient preparation, can effectively break through the bottleneck of mass production and popularization of the drug, and has great market potential. However, no drug having the above properties has been reported at present.

With the rapid development of nanotechnology in recent years, a large number of nanometer materials with multiple functions emerge, and new references and ideas are provided for solving major scientific problems of energy storage, accurate disease diagnosis and treatment and the like. Based on the above, the invention aims to design a synthesis method of oxygen vacancy molybdenum oxide nanoparticles which have good treatment effect on diabetic wounds, simple components and convenient preparation.

Disclosure of Invention

The invention aims to provide a preparation method and application of oxygen vacancy molybdenum trioxide nano particles, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for preparing oxygen-vacancy molybdenum trioxide nanoparticles, comprising the step of S1) reacting a polyethylene acid or pentaerythritol-tetrakis (3-mercaptopropionate) -polymethacrylene containing functional groups of-COOH and/or-SHDissolving the acid polymer in pure water to prepare 0.1-1% of a first aqueous solution; s2) dissolving molybdenum source in ethanol to obtain solution II with Mo content of 0.1-0.5M, wherein the molybdenum source can be MoCl₅(ii) a S3) adding 1-2 mL of the solution II into 45-55 mL of the solution I, heating to boil under the protection of nitrogen, and changing the color of the solution into dark blue; s4) cooling the product obtained after the reaction of S3, and removing unreacted raw materials to obtain purified molybdenum oxide nanoparticle dispersion liquid; s5) carrying out freeze drying on the molybdenum oxide nano-particle dispersion liquid to obtain a solid, namely the oxygen vacancy molybdenum trioxide nano-particles.

Then, an X-ray electron energy spectrometer is used for analyzing the nano-particle solid sample, and the obtained spectrogram is analyzed, so that the result shows that pentavalent molybdenum and hexavalent molybdenum which are oxygen vacancy MoO exist in the molybdenum oxide at the same time₃-the composition of X.

As a further optimization, the dissolving condition in S1 is that the solution is heated to condensation reflux under nitrogen protection.

As a further optimization, the heating time in S3 is 1-3 h.

As a further optimization, the product after the reaction in S4 is cooled to 20-25 ℃, and the unreacted raw materials are removed by ultrafiltration using an ultrafiltration tube.

As a further optimization, the particle size of the molybdenum oxide nanoparticles in the molybdenum oxide nanoparticle dispersion liquid in S4 is 3.1 + -0.5 nm.

The oxygen vacancy molybdenum trioxide nano particles are used for promoting recovery of diabetic bacterial infection wound surfaces, and the molybdenum oxide nano materials are used, so that the peroxidation environment of the diabetic wound surfaces can be effectively reduced, the bacterial number of the wound surface area is reduced, angiogenesis is promoted, and healing of the diabetic wound surfaces is effectively accelerated.

Compared with the prior art, the invention has the beneficial effects that: the oxygen vacancy molybdenum trioxide nano-particles have good free radical scavenging performance on one hand, strong antibacterial performance and biological safety on the other hand, and are single in component and simple to prepare.

According to the invention, the oxygen vacancy molybdenum trioxide nano particles are sprayed to treat the diabetes wound surface infected by bacteria, and the material improves the microenvironment of the wound surface through sterilization and antioxidation, so that the recovery of the diabetes wound surface is effectively accelerated.

Drawings

FIG. 1a is a transmission electron microscope image of oxygen-vacancy molybdenum trioxide nanoparticles of the present invention.

FIG. 1b is a picture of the particle size distribution of the oxygen-vacancy molybdenum trioxide nanoparticles of the present invention.

FIG. 2 is an X-ray electron energy spectrum of the inventive oxygen-vacancy molybdenum trioxide nanoparticles.

FIG. 3a is a test chart of hydroxyl radical (. OH) scavenging by oxygen vacancy molybdenum trioxide nanoparticles of the present invention.

FIG. 3b shows that the oxygen vacancy molybdenum trioxide nanoparticles eliminate superoxide anions (. O)₂ ^－) And (6) testing the graph.

FIG. 3c is a schematic representation of the oxygen vacancy molybdenum trioxide nanoparticles of the present invention for scavenging hydrogen peroxide (H)₂O₂) And (6) testing the graph.

FIG. 4a shows the detection of H scavenging by molybdenum trioxide of different concentrations using a hypersensitivity DCFH-DA probe₂O₂ROS assay profile induced in HUVEC cells.

FIG. 4b shows the detection of different concentrations of molybdenum trioxide vs H by CCK-8₂O₂Data comparison of protective effects on oxidative damage induced in HUVEC cells.

Fig. 5a is a graph testing antibacterial performance of molybdenum trioxide nanoparticles at different concentrations against escherichia coli (e. coli), staphylococcus aureus (s. aureus), and methicillin-resistant staphylococcus aureus (MRSA) by Colony Forming (CFU) experiments.

FIG. 5b is a statistical chart of the CFU experiment.

Fig. 6a is a picture of the healing process of a diabetic wound with MRSA infection after different treatments.

Figure 6b is a statistical plot of the healing of diabetic wounds with MRSA infection.

FIG. 6c is a hematoxylin-eosin staining chart of MRSA infected diabetic wound tissue sections.

FIG. 6d is a Masson staining chart of MRSA infected diabetic wound tissue sections.

FIG. 6e is a graph of Giemsa staining of MRSA infected diabetic wound tissue sections.

Detailed Description

The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described with reference to the drawings, but the present invention is not limited to these embodiments.

A method for preparing oxygen vacancy molybdenum trioxide nano-particles comprises the following steps of S1) dissolving a polyvinyl acid polymer containing a functional group-COOH in pure water to prepare a 0.3% aqueous solution, and preparing a first solution; s2) adding MoCl₅Dissolving a molybdenum source in ethanol to obtain a solution II, wherein the concentration of the molybdenum source in the ethanol is 0.34M; s3) adding 1 mL of the solution II into 45 mL of the solution I, heating to boil under the protection of nitrogen, and changing the color of the solution into dark blue; s4) cooling the product obtained after the reaction of S3, and removing unreacted raw materials to obtain purified molybdenum oxide nanoparticle dispersion liquid; s5) carrying out freeze drying on the molybdenum oxide nano-particle dispersion liquid to obtain a solid, namely the oxygen vacancy molybdenum trioxide nano-particles. Data for oxygen-vacancy molybdenum trioxide nanoparticles made according to the present invention are shown in fig. 1a, 1b, and 2.

As shown in fig. 3a to 3c, the performance of scavenging free radicals of oxygen vacancy molybdenum trioxide nanoparticles was tested: (1) and (3) detecting the OH removal performance of the oxygen vacancy molybdenum trioxide nanoparticles: the production of. OH was induced using the Fenton reaction, and the. OH signal was detected by Electron Spin Resonance (ESR) using DMPO as the. OH trapping agent. Adding water and Fe into a 1.5 mL centrifuge tube in sequence²⁺ (final concentration 50. mu.M), DMPO (final concentration 25. mu.M), molybdenum trioxide nanoparticles and H₂O₂ (final concentration: 50. mu.M) the reaction solution was prepared in a total volume of 50. mu.L. 5 minutes after the start of the reaction (from H)₂O₂At the start of addition), the reaction solution was sucked into a capillary having an inner diameter of 0.9 mm, and the ESR spectrum was measured using a microwave power of 20 mW and a parameter modulated by a 1G field.

(2) Oxygen vacancy molybdenum trioxide nanoparticle pair O₂ ^－Cleaning performance detection of (1): production of O by reaction of xanthine with xanthine oxidase₂ ^－Detection of. O by ESR with the aid of DMPO trapping agent₂ ^－A signal. Water, xanthine (final concentration: 1. mu.M), PBS, and trioxane were added to a 1.5 mL centrifuge tubeMolybdenum oxide nanoparticles, DMPO (final concentration 25. mu.M) and xanthine oxidase (final concentration 1U) in a total volume of 50. mu.L, after reacting for 3min, the reaction liquid was drawn into a capillary, and then the ESR spectrum was detected.

(3) Oxygen vacancy molybdenum trioxide nanoparticle pair H₂O₂Cleaning capability detection of (1): using H₂O₂Evaluation of molybdenum oxide nanometer scavenging H by detection kit₂O₂The ability of the cell to perform. Mixing different concentrations of molybdenum oxide nanoparticles with H₂O₂Incubating for 3H (100 μ M), adding 50 μ L mixed solution into 100 μ L prepared working solution, incubating at 37 deg.C for 30 min, collecting absorbance of sample 560 nm with microplate reader, analyzing molybdenum oxide nanoparticles to remove H₂O₂Activity of (2).

From the above examples, it was found that OH and O were detected as the concentration of molybdenum trioxide nanoparticles increased₂ ^－The signal gradually decreases, and as the addition of the molybdenum trioxide nanoparticles increases, H₂O₂The concentration is gradually reduced, which shows that the molybdenum trioxide nano particles prepared by the invention are opposite to OH and O₂ ^－And H₂O₂Several common ROS have concentration-dependent scavenging effects.

As shown in fig. 4a and 4b, the performance of oxygen vacancy molybdenum trioxide scavenging cells for free radical protection in cells was tested: human Umbilical Vein Endothelial Cells (HUVEC) were cultured at 5X 10⁴Inoculating the cell/well concentration into a 24-well plate, and culturing in an incubator for 24 h; diluting the hypersensitive DCFH-DA probe for ROS detection to a working concentration, adding into a 24-well plate, and incubating the cells for 30 min; then, discarding the DCFH-DA probe, and washing the stained cells twice with a PBS solution; using a catalyst containing H₂O₂Treating the cells for 1 h; then discarding the fraction containing H₂O₂And adding a culture medium containing 0, 50, 100, 200 mug/mL molybdenum trioxide nanoparticles, and treating the cells for 1 h; the medium containing the molybdenum trioxide material was discarded, the cells were washed twice with PBS, then fresh medium was added to each 24-well plate, and finally cell ROS production was observed using a fluorescence microscope (485 nm excitation, 525 nm emission). HUVEC cells were cultured at 5X 10³The concentration of each cell/well was inoculated in a 96-well plate and cultured overnight in an incubator; after the cells are attached to the wall, the cells are respectively subjected to H₂O₂、H₂O₂+ 50. mu.g/mL molybdenum trioxide nanoparticles, H₂O₂+ 100. mu.g/mL molybdenum trioxide nanoparticles and H₂O₂+ 200. mu.g/mL molybdenum trioxide nanoparticles for 24 h; then, removing the culture medium, washing the cells twice by PBS, and adding a fresh culture medium containing 10% CCK-8 into each hole of the cells respectively; and (3) placing the cells in an incubator for continuous culture for 2 h, finally detecting the absorbance of the cells in each hole by using an enzyme-labeling instrument, and analyzing the performance of removing free radicals in the cells and protecting the cells by using molybdenum trioxide to calculate the survival rate of the cells.

This example found that molybdenum trioxide nanoparticles can significantly reduce H₂O₂Induced ROS production in cells and effective reduction of H₂O₂The oxidative damage of HUVEC is induced, and the survival rate of cells is improved to 79.6 percent by adding 200 mug/mL of molybdenum trioxide nano particles, which is obviously higher than that of the group without molybdenum trioxide by 47.6 percent.

As shown in fig. 5a and 5b, the oxygen vacancy molybdenum trioxide nanoparticles were tested for in vitro antibacterial performance: choose bigE. coli(ATCC 2922)、S. aureus(ATCC 25923) andMRSAas representative of gram-negative, gram-positive and drug-resistant bacterial models to evaluate the antibacterial activity of molybdenum trioxide nanoparticles. Adding the frozen bacterium liquid into a liquid LB culture medium, and performing shake culture at 37 ℃ and 250 rpm overnight; sucking 1 mL of overnight-cultured bacterial liquid, adding the overnight-cultured bacterial liquid into 9 mL of fresh liquid LB culture medium, carrying out shake culture at 37 ℃ for 3h, and enabling the bacteria to be in an exponential growth phase; centrifugally collecting exponential phase bacteria liquid, cleaning by using 0.9% of normal saline, and dispersing thalli; molybdenum trioxide nano particles are added to be 0, 25, 50, 100 and 200 mu g/mL, then the molybdenum trioxide nano particles are mixed with diluted bacteria liquid, and shaking culture is carried out at 37 ℃ and 250 rpm for 2 hours; subsequently, the mixed solution was diluted to an appropriate concentration, 100. mu.L of the mixed solution was uniformly spread on a solid LB plate, the plate was placed in a biochemical incubator at 37 ℃ for overnight culture, and the antibacterial activity of the molybdenum trioxide nanoparticles was analyzed by counting the number of bacterial colonies.

This example found that molybdenum trioxide nanoparticle pairsE. coli、S. aureusAndMRSAall have good antibacterial effect. When the concentration of the molybdenum trioxide reaches 100 mug/mL, the antibacterial effect can reach 100 percent.

As shown in fig. 6a to 6e, the oxygen-vacancy molybdenum trioxide nanoparticles are used for testing the curative effect of diabetic wound healing infected by drug-resistant bacteria: (1) selecting 8-week-old male BALB/c mice, injecting streptozotocin into the mice through the abdominal cavity according to the dose of 50 mg/kg, once a day, and continuously administering for five days; (2) detecting the blood sugar of the mice by using blood sugar test paper after stopping the drug for five days, wherein the mice with the blood sugar value more than or equal to 13.6 mmol/L are hyperglycemic mice; (3) after the back of a hyperglycemic mouse is treated by hair, anesthesia treatment is carried out by injecting anesthesia medicine into the abdominal cavity; (4) preparing a prototype wound surface with the diameter of 5 mm in the area of the skin hair of the diabetic mouse by using sterilizing scissors, and dripping MRSA bacteria to the wound surface area to prepare the MRSA-infected diabetic wound surface. (5) Mice with MRSA infected wounds were randomly divided into four groups: PBS group (control group), general antibiotic group (cefathiamidine, 10. mu.g/body/time), vancomycin group (10. mu.g/body/time) and molybdenum trioxide group (100. mu.g/body/time). The medicines in each group are administrated twice a day, and are continuously administrated for 3 days for treatment, and the wound recovery condition is analyzed by observing the wound size and pathological sections. This example demonstrates that molybdenum trioxide promotes the recovery of MRSA-infected diabetic wounds with superior efficacy to cefathiamidine and vancomycin.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.