CN115006345B - Preparation method and application of oral zero-valent molybdenum nano dots - Google Patents

Preparation method and application of oral zero-valent molybdenum nano dots Download PDF

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CN115006345B
CN115006345B CN202210748438.2A CN202210748438A CN115006345B CN 115006345 B CN115006345 B CN 115006345B CN 202210748438 A CN202210748438 A CN 202210748438A CN 115006345 B CN115006345 B CN 115006345B
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molybdenum
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zvmns
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CN115006345A (en
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王正廷
倪大龙
王韩
张晨
钟捷
唐忠敏
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Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

The invention provides a preparation method and application of an oral zero-valent molybdenum nano dot. The preparation method of the oral zero-valent molybdenum nano dot comprises the following steps: (1) Mixing molybdenum powder and isopropanol, and synthesizing zero-valent molybdenum nanodots by mechanical stripping in an ice bath; (2) Standing to obtain supernatant, removing isopropanol by a rotary evaporator, dissolving the obtained zero-valent molybdenum nanodots in deionized water, removing residual small amount of nano particles with larger particle size by high-speed centrifugation, and preserving the obtained solution in nitrogen atmosphere to obtain the oral zero-valent molybdenum nanodots. The zero-valent molybdenum nano dot provided by the invention can be used for treating inflammatory bowel disease by neutralizing excessive active oxygen and relieving intestinal inflammation after being orally taken, and is a safe, multi-effect enteritis treatment medicament with clinical transformation significance.

Description

Preparation method and application of oral zero-valent molybdenum nano dots
Technical Field
The invention belongs to the technical field of preparation of molybdenum nanodots, and particularly relates to a preparation method and application of an oral zero-valent molybdenum nanodot.
Background
Inflammatory Bowel Disease (IBD) is a non-specific chronic inflammatory disease of the gastrointestinal tract, often leading to extra-intestinal complications. The global IBD morbidity and prevalence are now on the rise, and about 680 thousands of IBD cases are reported worldwide by 2017 literature, which presents a great challenge to the health and medical system. However, at present, the clinical treatment means aiming at IBD are not satisfactory, and 5-aminosalicylic acid and glucocorticoid belong to first-line anti-inflammatory therapeutic drugs, but the nonspecific anti-inflammatory property of the drugs can cause systemic side effects; the novel biologicals such as the monoclonal antibody Infiniximab to TNF-alpha are not only expensive, but also present a 30% non-response rate and a 20% yearly drug resistance rate. Thus, there is a strong need to find more effective and more suitable methods of treating IBD.
Although the pathogenesis of IBD is very complex and currently not well defined, many studies indicate that Reactive Oxygen Species (ROS) play an important role in the development and progression of IBD. Upregulation of ROS was observed in both colitis tissue and mouse colitis models in IBD patients, ultimately leading to intestinal mucosal lesions. In one aspect, the pro-inflammatory cytokines can activate immune cells, release cytokines and produce ROS; on the other hand, excessive ROS production may further activate inflammatory/immune responses through NF- κb signaling pathways, resulting in increased expression and secretion of pro-inflammatory cytokines. At the same time, excessive ROS exposure causes oxidative damage to mitochondria, thereby inducing apoptosis of intestinal epithelial cells. Therefore, scavenging excessive ROS in the gut is important to suppress the development of IBD.
However, the anti-ROS formulations currently studied are not efficient, many of which may cause adverse immune responses. Studies have shown that natural antioxidants such as vitamin E, vitamin C and coenzyme Q have insignificant efficacy in IBD, and that the remaining ROS scavengers such as N-acetylcysteine (NAC) enhance Th17 cell (T helper cell) production in vivo in MINK 1-dependent manner, with the risk of eliciting autoimmune disease. Therefore, the development of novel antioxidants is of great significance.
In recent years, biological materials having an inflammation-regulating effect are gradually coming into the field of vision of people. MoS (MoS) 2 Molybdenum-based nano materials such as phosphomolybdate and the like are paid attention to in the field of antioxidation by good biocompatibility and effective antioxidation performance. Wherein the molybdenum nanodots are expected to become a novel antioxidant and anti-inflammatory material. Duan et al (Healing Diabetic Ulcers with MoO) 3-X Nanodots Possessing Intrinsic ROS-Scavenging and Bacteria-Killing Capacities, small 2022, 18, 2107137) to prepare a MoO 3-X Nanodots, research MoO 3-X The scavenging ability of nanodots to ROS indicates MoO 3-X The nano-dots can effectively reduce inflammatory reaction, promote epithelial cell regeneration, accelerate angiogenesis and promote recovery of DUs (diabetic ulcers).
The prior preparation of molybdenum nanodots is mostly concentrated on reports of molybdenum disulfide nanodots and molybdenum trioxide nanodots, but the preparation method of zero-valent molybdenum nanodots is not mentioned. Chinese patent No. 105999267B reports a molybdenum disulfide nanodot/polyaniline nanohybrid and a preparation method thereof, wherein the obtained molybdenum disulfide nanodot has wide particle size distribution, the size distribution is between 2 and 8nm, and the average particle size is larger and reaches about 6 nm. The Chinese patent document CN112209445A discloses a preparation method of a molybdenum trioxide nano dot antibacterial material, wherein the particle size of the obtained molybdenum trioxide nano dot is smaller than 10nm, the average particle size is 3.07+/-0.45 nm, but the particle size distribution still shows wider distribution, and the size distribution is between 1 and 5 nm.
Although the nano-dots can be prepared by adopting a mechanical stripping method, the nano-dot size obtained by the stripping method has randomness and cannot be quantitatively produced. Whether the zero-valent molybdenum nano-dots can be prepared, so that the zero-valent molybdenum nano-dots have the characteristics of narrow size distribution, high yield and small average particle size, become the technical problems to be solved urgently, and whether the zero-valent molybdenum nano-dots can be better used for treating IBD (infectious bursal disease) is still to be studied further.
Disclosure of Invention
The invention aims to solve the technical problems, and provides a preparation method and application of an oral zero-valent molybdenum nano dot. The technical purpose of the present invention is to provide a method for preparing zero-valent molybdenum nanodots with narrow size distribution, high yield and small average particle size, and to provide an oral zero-valent molybdenum nanodot which can maintain high stability in an acidic environment, and can accumulate and neutralize excessive ROS at colon parts after oral administration, thereby being used for treating IBD.
The first object of the invention is to provide a preparation method of oral zero-valent molybdenum nanodots, which comprises the following steps:
(1) Fully mixing molybdenum powder with isopropanol, and then mechanically stripping under ice bath, wherein the ultrasonic power of the mechanical stripping is 450W, the ultrasonic frequency is 1 second every 3 seconds, and the total ultrasonic stripping time is 20 hours, so that zero-valent molybdenum nanodots are obtained;
(2) And (3) standing the product obtained in the step (1), taking a supernatant, removing isopropanol by rotary evaporation, dissolving the obtained zero-valent molybdenum nano dots in deionized water, removing residual small amount of nano particles with larger particle size by high-speed centrifugation, obtaining a solution which is the oral zero-valent molybdenum nano dots, and storing the solution in a nitrogen atmosphere.
The novel oral zero-valent molybdenum nano dot is synthesized by mechanical stripping, has good biocompatibility and high-efficiency oxidation resistance, can be well used for treating IBD, and does not generate adverse immune reaction. Due to the high stability in an acidic environment, the oral zero-valent molybdenum nanodots can be orally administered and accumulated in the intestinal tract. Due to the strong reduction effect of the zero-valent molybdenum atom, the zero-valent molybdenum nano-dot can accurately neutralize excessive ROS in colon parts after being orally taken, thereby treating IBD, and the medicament is a safe, multi-effect and enteritis treatment medicament with clinical transformation significance.
According to the preparation method provided by the invention, the selection of the stripping solvent in the step (1) is very important. The present inventors have made extensive studies in order to select an appropriate stripping solution and to obtain zero-valent molybdenum nanodots with a narrow size distribution and high yield. The inventors initially selected common ethanol and N-methylpyrrolidone (NMP) as stripping solutions, and as a result found that when ethanol was used as a solvent, the yield of zero-valent molybdenum nanodots was extremely low, and quantitative production could not be achieved, thus excluding this scheme. When the common NMP is replaced as a solvent, although the yield of the zero-valent molybdenum nano dots is improved, the NMP has deliquescence property, the size distribution and the stability of the prepared zero-valent molybdenum nano dots are affected to a certain extent, and meanwhile, the NMP has a higher price, more importantly, the boiling point of the NMP is 202 ℃, the NMP is difficult to remove by simple rotary evaporation, and the separation cost of the solvent is greatly improved. Therefore, the inventors have adopted both solvents as stripping solutions, and have failed the solution for preparing zero-valent molybdenum nanodots.
Finally, when isopropanol is selected as a solvent, the yield of the zero-valent molybdenum nano dots is higher, meanwhile, no deliquescence phenomenon exists, the boiling point of the isopropanol is 82.5 ℃, the isopropanol can be removed by adopting simple rotary evaporation, and the isopropanol collected by rotary evaporation can still be recycled for the production of the zero-valent molybdenum nano dots, so that the cost is further reduced. More importantly, isopropanol is adopted as a solvent for mechanical stripping, and the obtained zero-valent molybdenum nano dots have narrow size distribution, high yield and small average particle size and can be well used as an oral preparation.
After determining that the stripping solvent was isopropanol, the inventors have further explored the parameters of ultrasonic stripping. Since a large amount of energy acts on the solution in the ultrasonic stripping process, the temperature of the solution is rapidly increased, oxidation of zero-valent molybdenum nanodots is promoted, the yield and stability of the zero-valent molybdenum nanodots are affected, and meanwhile, in consideration of the service life of an ultrasonic probe, it is very important to select proper ultrasonic stripping parameters. The inventor adopts 500 watts, 450 watts and 300 watts to search ultrasonic stripping parameters respectively, and found that when the power of 500 watts is adopted, the stripping solution is difficult to cool by adopting a simple ice water bath, the instrument is often in overtemperature alarm, and meanwhile, an ultrasonic probe can give out abnormal sound after stripping for a period of time, so that the power is excluded from being used. While with 300 watts of power, although the solution temperature can be maintained at a low level, the yield of zero-valent molybdenum nanodots is low, and quantitative production cannot be performed, and rejection is also performed. When 450 watts is adopted for ultrasonic treatment, the stripping solution can be effectively cooled by an ice-water bath, and the yield of zero-valent molybdenum nanodots can still be maintained at a higher level, so that 450 watts is adopted as the power of ultrasonic stripping. Meanwhile, in order to obtain zero-valent molybdenum nano dots with narrow size distribution, high yield and small average particle size, the inventor creatively adopts a scheme that the ultrasonic frequency is 1 second every 3 seconds, and the total accumulated ultrasonic stripping time is 20 hours, so as to solve the defects of wider size distribution and larger average particle size of the molybdenum nano dots obtained by the traditional mechanical stripping method.
The method provided by the invention can prepare zero-valent molybdenum nano dots with uniform size, can stabilize quantitative production and is beneficial to clinical transformation. The zero-valent molybdenum nano-dot is highly stable in gastric acid after being orally taken, can safely reach colonic mucosa, plays a role in neutralizing ROS, and is used as a safe, multi-effect enteritis treatment drug with clinical transformation significance.
Further, the dosage ratio of the molybdenum powder to the isopropanol in the step (1) is 3g:50mL.
Further, the average diameter of the zero-valent molybdenum nano dots in the step (1) is 3nm.
Further, the step of high-speed centrifugation in the step (2) is as follows: centrifugation was performed at 13000rpm for 5 minutes.
A second object of the present invention is to provide an oral zero-valent molybdenum nanodot prepared by the above method. The oral zero-valent molybdenum nano-dots have the characteristics of narrow size distribution, high yield and small average particle size, and meanwhile, the oral zero-valent molybdenum nano-dots can maintain high stability in an acidic environment, and can accumulate and neutralize excessive ROS at colon parts after oral administration, so that IBD can be treated.
The third object of the invention is to provide the application of the oral zero-valent molybdenum nano-dots, which is to use the oral zero-valent molybdenum nano-dots for preparing medicines for treating inflammatory bowel diseases. Specifically, the medicine prepared from the oral zero-valent molybdenum nanodots can act on intestinal mucosa to play the roles of anti-inflammatory and flora double regulation. The oral zero-valent molybdenum nano-dot can be used as a safe, multi-effect and enteritis therapeutic drug with clinical transformation significance, and is an ideal candidate in IBD treatment.
Compared with the prior art, the invention has the following beneficial effects:
the invention prepares the oral zero-valent molybdenum nano-dots, which have good biocompatibility and active oxygen scavenging capability, have the characteristics of narrow size distribution, high yield and small average particle size, can keep high stability under an acidic environment, can realize energy production, can well play an anti-inflammatory role, and is used for treating enteritis. In vitro researches prove that the oral zero-valent molybdenum nanodot has the effects of neutralizing ROS and reducing secretion of inflammatory factors TNF-alpha, IL-1 beta and the like. The mouse model verifies that the oral zero-valent molybdenum nanodots can significantly relieve DSS-induced colitis; the nano material can be used for relieving inflammation through NF- κB pathway by sequencing RNA-seq of colon tissue of the mice. The zero-valent molybdenum nano dot provided by the invention is a safe, multi-effect and enteritis treatment drug with clinical transformation significance.
Drawings
Fig. 1 is: (a) electron microscopy of zero-valent molybdenum nanodots; (b) X-ray diffraction patterns of ZVMNs and molybdenum powder; (c) an X-ray diffraction pattern of hexavalent molybdenum nanoparticles; (d) X-ray photoelectron spectroscopy of zero-valent molybdenum nano-dots and hexavalent molybdenum nano-particles; (e) Mo K-edge XAFS R space of ZVMNs; (f) the R space of Mo K-edgeEXAFS of hexavalent molybdenum nanoparticles; (g) Wavelet transform of extended X-ray absorbing fine structures of ZVMNs; (h) Wavelet transformation of the extended X-ray absorbing fine structure of hexavalent molybdenum nanoparticles; (i) ZVMNs of different concentrations scavenge H 2 O 2 Is not limited in terms of the ability to perform; (j) Zeta potential of ZVMNs in water and HCl solution; (k) the hydration radius of ZVMNs in water and HCl solution; (l) photographs of ZVMNs in water and HCl solution.
Fig. 2 is a photograph of ZVMNs and hexavalent molybdenum nanoparticles.
Fig. 3 is an electron micrograph of hexavalent molybdenum nanoparticles.
Fig. 4 is: (a) Molybdenum powder and (b) MoO 3 R space of Mo K-edge EXAFS.
FIG. 5 shows EXAFS fitting parameters for various samples at Mo K-edge.
FIG. 6 is a graph reflecting ZVMNs biosafety by detecting cellular activity with CCK8 kit: cell activity curve after co-culture of ZVMNs with HCT 116.
FIG. 7 is a chart showing the staining of tissue HE of major organs such as heart, liver, lung, kidney, etc. of ZVMNs intragastric mice.
Fig. 8 is: (a) Representative ROS immunofluorescent staining (green fluorescence) of iBMDMs cell lines under different treatment conditions; (b) Flow-analyzing the ability of ZVMNs to scavenge ROS at cellular level LPS stimulation; (c) Quantitative analysis of the ability of ZVMNs to scavenge ROS at cellular level LPS stimulation (n=3, mean ± SD); qPCR to detect mRNA levels of pro-inflammatory cytokines IL-1β (d), IL-10 (e) and TNF- α (f), the experiment was repeated 3 times; (g) Extracellular acidification rate (ECAR) reflects the effect of ZVMNs on cellular glycolysis; (h) Oxygen Consumption Rate (OCR) represents the effect of ZVMNs on mitochondrial respiration capacity (n=3, mean ± SD); for analysis using Mann-Whitney U-test, ns was not significant, P <0.05, P <0.01, P <0.001, P <0.0001.
Fig. 9 is: (a) experimental design of DSS-induced IBD mouse model: mice were provided with sterile water or water containing 2.5% DSS for 7 days, and all mice were sacrificed after one day of cessation of DSS consumption starting from the second day; (b) representative colon pictures of each group, n=6; (c) measurement and analysis of colon length for each group, n=6; (d) representative spleen pictures of each group, n=6; (e) measurement and analysis of spleen weights for each group, n=6; (f) daily body weight change and analysis, n=6; (g) disease activity index analysis, n=6; (h) Representative colon tissue Hematoxylin Eosin (HE) staining images; for analysis using Mann-Whitney U-test, ns was not significant, P <0.05, P <0.01, P <0.001, P <0.0001.
Fig. 10 shows the diversity of different groups (n=3) for the primary coordinate analysis in RNA-seq.
FIG. 11 is a Venn diagram of signal pathway in RNA-seq analysis.
FIG. 12 is the results of analysis of RNA-seq sequencing of three groups of mice (healthy control group, DSS group, DSS+ZVMNs group): (a) Volcanic plot shows differential gene expression (fold change >1.5; p-adjust <0.05; up-regulated gene: red; down-regulated gene: blue) in DSS versus control (left) and DSS versus ZVMNs treated (right); (b) The venn plot of the RNA-seq analysis shows that DSS groups significantly up-regulated (bottom) or down-regulated (top) genes compared to control and DSS groups compared to ZVMNs treated groups; (c) A heat map of the response of the control group, DSS group and ZVMNs treated group to reactive oxygen species-related genes; (d) Enrichment Analysis (GSEA) of ROS-related gene sets under different conditions; (e) The KEGG pathway enriched in up-regulated genes in DSS compared to control (left) and DSS compared to ZVMNs treated (right).
Fig. 13 is: (a) DHE stains (green fluorescence) representative ROS for each group of colon tissue; (b) Western blot analysis of phospho-NF- κbp65, total NF- κbp65, phospho-ikb- α and total ikb- α expression in DSS-induced colonic tissue of colitis mice; colon mRNA levels of IFN- γ (c), IL-6 (d), IL-1β (e), TNF- α (f) and IL-10 (g) (n=6, mean ± SD); the significance between each two groups, ns, was calculated using the Mann-Whitney U test, not significant, P <0.1, P <0.01, P <0.001, P <0.0001.
Figure 14 is qPCR evaluation of relative mitochondrial DNA copies in DSS-induced mice (n=6, mean ± SD); the significance between each two groups, ns, was calculated using the Mann-Whitney U test, with no significance.
Figure 15 is a schematic representation of ZVMNs for treating IBD.
FIG. 16 shows the results of 16s rRNA sequencing on the abundance of colonies from fecal flora from three groups of mice (healthy control group, DSS group, DSS+ZVMNs group): shannon and Simpson indices representing alpha-diversity of intestinal microflora.
Fig. 17 shows the β diversity of the intestinal microbiome (n=5) by principal coordinate analysis.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be specifically described with reference to the following examples, which are provided for explaining and illustrating the present invention only and are not intended to limit the present invention. Some non-essential modifications and adaptations of the invention according to the foregoing summary will still fall within the scope of the invention.
Example 1
The embodiment provides a preparation method of an oral zero-valent molybdenum nano dot, which comprises the following steps:
(1) 3g of molybdenum powder is mixed with 50mL of isopropanol, zero-valent molybdenum nanodots are synthesized by mechanical stripping under ice bath, the parameters of the mechanical stripping are 450 watts, 1 second is separated every 3 seconds of ultrasonic waves, and the total treatment time is 20 hours;
(2) And (3) standing the product obtained in the step (1), taking a supernatant, removing isopropanol by using a rotary evaporator, dissolving the rest product in deionized water, and removing residual particles with larger particle size by high-speed centrifugation (13000 rpm,5 min) to obtain zero-valent-molybdenum nanodots (ZVMNs for short).
Further experiments have shown that ZVMNs, after oral administration, can accumulate in the inflamed colonic mucosa, improving intestinal inflammation by scavenging excess ROS in the intestinal tract. The following experimental examples will explain the mechanism in detail.
Comparative example 1
Selection of stripping solution
The experimental investigation was conducted by selecting ethanol and N-methylpyrrolidone (NMP) which are commonly used for preparing nano-dots as stripping solutions, comparing the stripping solutions with isopropanol, and the preparation method of the zero-valent molybdenum nano-dots is all performed by referring to example 1, and the statistics of the yield of ZVMNs and the characteristics of the preparation process are performed, and the results are shown in Table 1.
TABLE 1
From the experimental results in table 1, it can be seen that although both ethanol and isopropanol have low boiling points, are easily removed by rotary evaporation, and are not deliquescent, ZVMNs produced using ethanol have extremely low yields and are therefore excluded. The NMP is used as stripping solution, the yield of ZVMNs is higher, but the NMP has high boiling point and is difficult to remove by simple rotary evaporation, so that the separation cost is greatly increased, and simultaneously, the NMP has high price and is easy to deliquesce, so that the stability and quantitative production of the ZVMNs are influenced, and the effect of preparing the ZVMNs by using isopropanol as the stripping solvent is optimal.
Comparative example 2
(II) selection of mechanical stripping conditions
The experimental search was conducted on ultrasonic stripping conditions, ultrasonic stripping powers of 500W, 450W and 300W were selected for the experimental search, the preparation method of zero-valent molybdenum nanodots was referred to example 1, and statistics were conducted on the yield and preparation process of ZVMNs, and the results are shown in table 2.
TABLE 2
Since a large amount of energy acts on the solution during ultrasonic stripping, the temperature of the solution is increased, thereby promoting the oxidation of ZVMNs and affecting the stability and yield of the ZVMNs. The research result of the inventor on ultrasonic peeling power parameters shows that: when using 500 watts of power, although the yield of ZVMNs is higher, simple ice water baths are difficult to cool the stripping solution, the instrument often gives out an overtemperature alarm, and the ultrasonic probe gives out abnormal sound after stripping for a period of time, so that the use of the power is eliminated. While with 300 watts of power, the ZVMNs yield is lower and therefore precluded, although the solution temperature can be maintained at a lower level. When using 450 watts of ultrasound, the ice water bath still effectively cools the stripping solution, and the ZVMNs yield remains high, thus using 450 watts as the stripping power.
Experimental example 1
Characterization of the ZVMNs synthesized in example 1, transmission Electron Microscope (TEM) images showed that the size of the prepared ZVMNs was about 3nm (part a in fig. 1). The X-ray diffraction pattern (XRD) of the molybdenum powder is the same as the standard powder diffraction pattern (part b in fig. 1). However, after mechanical exfoliation, there is no distinct peak in the ZVMNs, indicating that the ZVMNs have ultra-small particle size (part b in fig. 1).
Next, through ZVMNs and H 2 O 2 Reflecting the ability of ZVMNs to scavenge ROS. The aqueous solution of ZVMNs was blue-violet, while the oxidized product (HVMNs) showed no color (fig. 2). XRD patterns showed the product to be between MoO 3 And MoO 3 ·H 2 Between O (part c in FIG. 1). The peak expansion of XRD and TEM images showed that the size of the oxidized product was quite small (fig. 3). The increase in molybdenum 3d orbital binding energy of the oxidation product in X-ray photoelectron spectroscopy (XPS) demonstrates the increase in the valence state of molybdenum (part d in fig. 1). Thus, the oxidized product is called hexavalentMolybdenum nanoparticles (hexavalent-molybdenum nanoparticles, abbreviated as HVMNs).
The structures of ZVMNs and HVMNs were further analyzed using MoK-edge extended X-ray absorption fine structure (EXAFS). As can be seen by comparison with the standard samples, ZVMNs contain a large number of Mo-O bonds, and contain a small number of Mo-Mo bonds (FIG. 1, parts e, FIG. 4, parts a and b); whereas HVMNs are mainly Mo-O bonds (part f in fig. 1 and part b in fig. 4), and the coordination number fitted (fig. 5) is similar to ZVMNs. The wavelet transform of EXAFS provides more information about the difference between ZVMNs and HVMNs, as shown in parts g and h in fig. 1, most of the coordinate atoms in ZVMNs are farther away than the atomic positions in HVMNs, indicating that ZVMNs have a more relaxed structure. Based on the above data we hypothesize that ZVMNs are composed of loosely assembled monoatomic Mo and O-containing ligands (e.g. hydroxyl and H 2 O), HVMNs consist of closely packed MoO 3 Clusters and bound water. Then ZVMNs clear H was detected 2 O 2 As shown in part i of FIG. 1, ZVMNs can effectively reduce H in solution 2 O 2 In terms of quantity, about 0.27H can be removed per molybdenum atom 2 O 2 A molecule.
Experimental example 2
Zero-valent molybdenum nanodots have good ROS scavenging ability, while a large body of evidence suggests that excessive ROS play an important role in the development of IBD. Thus, this experimental example demonstrates the ROS scavenging ability of ZVMNs in vitro.
Molybdenum is known to be a trace element required by the human body, so ZVMNs have better biocompatibility. The experimental results showed that HCT116 cells (human colon cancer cells) exposed to different concentrations of ZVMNs for 24 hours showed normal cell morphology, and the CCK-8 assay showed that the tested concentrations of ZVMNs did not show significant cytotoxicity (fig. 6). In addition, histopathological analysis of major organs (including heart, liver, kidney and lung) after oral ZVMNs showed: the ZVMNs did not cause any observable lesions in the body (fig. 7), indicating that the ZVMNs have good biocompatibility.
Since ZVMNs have very strong antioxidant properties, their potential ability to scavenge ROS was measured in vitro. Cellular inflammation models were established by treatment of immortalized bone marrow-derived macrophages (iBMDMs) with Lipopolysaccharide (LPS). After LPS stimulation, cells were stained with ROS fluorescent dye 2',7' -dichlorofluorescein diacetate (DCFH-DA). As shown in part a of fig. 8, the intracellular green fluorescent signal of iBMDMs increased significantly after LPS treatment; in contrast, intracellular ROS levels were significantly reduced when iBMDMs cells were treated with ZVMNs. Further quantitative analysis of intracellular ROS levels by flow cytometry showed (parts b and c in fig. 8) that ZVMNs cleared approximately 66.7% of ROS in iBMDMs cells, demonstrating good ROS clearance of ZVMNs.
To further confirm the anti-inflammatory activity of ZVMNs, mRNA expression levels of LPS-stimulated inflammatory cytokines in iBMDMs cell lines were examined. As shown in parts d and f of fig. 8, ZVMNs treatment group showed a significant decrease in interleukin 1 beta (IL-1 beta), interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha) levels, indicating that ZVMNs can significantly alleviate inflammatory responses in vitro.
Furthermore, since ROS in cells are mainly produced by mitochondria, the Seahorse technique was used to analyze cellular mitochondrial function. Extracellular acidification rate (ECAR) showed a significant change between LPS-stimulated cells and ZVMNs-treated cells, indicating that ZVMNs affected glycolytic function (part g in fig. 8), basal Oxygen Consumption Rate (OCR) showed no change in basal mitochondrial respiratory function (part h in fig. 8).
Experimental example 3
In this experimental example, the therapeutic effect of ZVMNs on colitis in vivo was examined by constructing a DSS (bissuccinimidyl suberate) -induced colitis mouse model. DSS induced colitis is one of the most commonly used animal models of IBD, and when dissolved in drinking water, DSS can damage epithelial cells and destroy intestinal barrier, causing invasion of intestinal microbiota, generating excessive ROS, which in turn causes inflammation.
The experimental method is as follows:
mice were randomly assigned to control, DSS, DSS+ZVMNs, DSS+5-ASA. The control group was given normal drinking water and the remaining groups were given drinking water with 2.5% w/v dss, two of the other groups were ZVMNs and 5-ASA (5-aminosalicylic acid) treated by gavage once daily (part a in fig. 9). As a result, it was found that the administration of ZVMNs gastric lavage treatment significantly maintained the body weight level of DSS-induced colitis mice (P <0.0001, part f in fig. 9), and the disease activity index (P <0.0001, part g in fig. 9) reflected the general health status of mice as well as better than the control group. Upon dissection of the mice, dss+zvmns groups were found to have significantly longer colon length than DSS groups (P <0.0001, parts b and c in fig. 9), and spleen weights were also significantly lighter than DSS groups (P <0.001, parts d and e in fig. 9). From the HE staining of part h in fig. 9, ZVMNs were found to have a role in maintaining colonic epithelial integrity and reducing infiltration of mucosal inflammatory cells during treatment of DSS-induced colitis. The evidence indicates that ZVMNs can significantly alleviate intestinal inflammation in mice by oral administration.
Furthermore, although 5-ASA is a first-line drug for the treatment of IBD, there is a risk of relapse and multiple adverse effects, and thus this experimental example compares the therapeutic effects of ZVMNs and 5-ASA on DSS-induced enteritis. From the results of fig. 9, it can be seen that the therapeutic effect of ZVMNs is more remarkable than that of 5-ASA in various aspects of body weight, colon length, spleen weight, colon injury score, etc.
Experimental example 4
To reveal the specific mechanism by which ZVMNs protect mice from DSS-induced colitis, RNA sequencing (RNA-seq) was performed on colon tissue of mice in this experimental example. PCA results revealed a significant difference in transcriptome spectra between the control, DSS, and dss+zvmns groups (fig. 10). Volcanic images show 3229 genes up-regulated and 2818 genes down-regulated in DSS-induced colitis mice compared to control. After ZVMNs treatment, 2550 genes were up-regulated and 1863 genes were down-regulated (part a in fig. 12). Of the genes up and down regulated, the two sets of comparisons overlap 1731 genes and 1234 genes, respectively, which can be attributed to the presence of inflammation (part b in fig. 12).
Likewise, in the top-ranked upregulated signaling pathway, 15 overlapping signaling pathways were found in the ZVMNs treated colitis mice (fig. 11). Gene Set Enrichment Analysis (GSEA) showed that up-regulated genes in response to ROS were significantly enriched in dss+zvmns groups and control groups (part d in fig. 12). Up-regulated genes for ROS response were reduced to normal levels under ZVMNs treatment. Further analysis showed that gene expression associated with ROS response was significantly reduced in IBD mice after ZVMNs treatment (part c in fig. 12). Functional pathway enrichment analysis showed that TNF signaling pathway, IL-17 signaling pathway, chemokine signaling pathway and NF- κb signaling pathway are highly correlated with the therapeutic mechanisms of ZVMNs (part e of fig. 12). It can be concluded that ZVMNs can alleviate mouse colitis by reducing ROS levels to inhibit NF- κb signaling pathways.
To further demonstrate the therapeutic mechanism of ZVMNs in vivo, ROS levels in mouse colonic mucosa in DSS models were first detected by using DHE staining. As shown in part a of fig. 13, ROS levels in ZVMNs-treated IBD mice were similar to healthy mice, indicating that ZVMNs can scavenge ROS in vivo as an antioxidant. Furthermore, it is well known that mitochondria are the centers of cellular energy metabolism and are closely related to the production of active oxygen. Excessive ROS can lead to reduced ATP production, inhibition of intracellular electron transfer chains, and DNA damage in mitochondria. However, we found no significant differences in mitochondrial DNA (mtDNA) between the three groups (control, DSS, and dss+zvmns) (fig. 14), probably due to the short modeling time, the cell mitochondria were not significantly altered.
Next, the effect of ZVMNs on key proteins in NF-. Kappa.B signaling pathways, including NF-. Kappa.Bp 65, phosphorylated NF-. Kappa.Bp 65, I.kappa.B, and phosphorylated I.kappa.B, was demonstrated. As shown in part B of FIG. 13, phosphorylation of NF- κBp65 and IκB was significantly enhanced in DSS-induced colitis mice, indicating activation of NF- κB signaling pathways in IBD. According to the RNA-seq analysis result, phosphorylation of NF- κBp65 and IκB in ZVMNs treatment group was significantly reduced, indicating that ZVMNs could inhibit NF- κB signaling pathway. In addition, the analysis results of the downstream inflammatory factor mRNA expression of NF- κB signaling pathway and the cytokine level in serum were consistent with those found in vitro and in vivo (parts c to g in FIG. 13). Cytokines, including IFN-gamma, IL-6, IL-1 beta, TNF-alpha, were highly increased in the IBD group but significantly decreased in the ZVMNs treated group compared to the control group. According to previous studies, IL-1β hypersecretion is due to increased activity of NLRP3 inflammatory bodies in the development of IBD. Meanwhile, secreted IL-1 beta can also stimulate neutrophils to release ROS, and further increase the synthesis of TNF-alpha. IL-6 and IFN-gamma are also the primary pro-inflammatory cytokines that activate immune cells. In IBD patients, IL-6 and IFN-gamma levels are elevated and mediate T cell activation, ultimately exacerbating intestinal inflammation. In summary, IL-1 beta, IL-6, IFN-gamma and TNF-alpha produced by immune cells and Intestinal Epithelial Cells (IEC) are key pro-inflammatory factors in the initiation of IBD, and the decrease in expression of these inflammatory factors also demonstrates the therapeutic efficacy of ZVMNs. Figure 15 illustrates a schematic of ZVMNs for treating IBD that can protect colon tissue from oxidative stress by inhibiting NF- κb signaling and reducing excessive cytokine production.
Furthermore, it is well known that disorders of intestinal microorganisms are closely related to the occurrence of IBD, and intestinal microorganisms are also considered as an important source of ROS. Overproduction of ROS may also lead to intestinal barrier dysfunction and the expansion of potentially harmful bacteria. Thus, to explore whether ZVMNs affected the composition or abundance of intestinal flora, 16srRNA sequencing techniques were further sampled to conduct in-depth analysis of fecal flora in three groups of mice (healthy control group, DSS group, dss+zvmns group), and the results showed no significant differences in alpha diversity between the three groups (control group, DSS group, and dss+zvmns group) (fig. 16). Principal Coordinate Analysis (PCA) showed that the therapeutic fraction of ZVMNs altered the microbiota composition of the colitis mice (fig. 17).

Claims (6)

1. The preparation method of the oral zero-valent molybdenum nano dot is characterized by comprising the following steps of:
(1) Thoroughly mixing molybdenum powder with isopropanol, the dosage ratio of the molybdenum powder to the isopropanol being 3g:50mL, then carrying out mechanical stripping under ice bath, wherein the ultrasonic power of the mechanical stripping is 450W, the ultrasonic frequency is 1 second every 3 seconds, and the total accumulated ultrasonic stripping time is 20 hours, so as to obtain zero-valent molybdenum nanodots;
(2) And (3) standing the product obtained in the step (1), taking a supernatant, removing isopropanol by rotary evaporation, dissolving the obtained zero-valent molybdenum nano dots in deionized water, removing residual small amount of nano particles with larger particle size by high-speed centrifugation, obtaining a solution which is the oral zero-valent molybdenum nano dots, and storing the solution in a nitrogen atmosphere.
2. The method of claim 1, wherein the average diameter of the zero-valent molybdenum nanodots in step (1) is 3nm.
3. The method according to claim 1, wherein the step of high-speed centrifugation in step (2) is: centrifugation was performed at 13000rpm for 5 minutes.
4. An oral zero-valent molybdenum nanodot produced by the method of any one of claims 1-3.
5. The use of the oral zero-valent molybdenum nanodots of claim 4 in the manufacture of a medicament for the treatment of inflammatory bowel disease.
6. The use according to claim 5, wherein the oral zero-valent molybdenum nanodots are prepared to act on intestinal mucosa as a medicament, exerting anti-inflammatory and flora dual-modulation effects.
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