CN114949376A - Anti-abdominal adhesion metal nanoenzyme Ru-PEG NDs and preparation method and application thereof - Google Patents
Anti-abdominal adhesion metal nanoenzyme Ru-PEG NDs and preparation method and application thereof Download PDFInfo
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/022—Metals or alloys
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- A—HUMAN NECESSITIES
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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Abstract
The invention discloses an anti-abdominal adhesion metal nano enzyme Ru-PEG NDs and a preparation method and application thereof, and relates to the technical field of metal nano materials. The preparation method comprises the following steps: s1 reaction: mixing ruthenium acetate [ Ru (OAc) 3 .xH2O]Dissolving polyethylene glycol monomethyl ether (PEG-SH) in deionized water, and then dropwise adding a reducing agent under continuous stirring for reaction until the reaction is completely finished to obtain a reaction mixture; s2 purification: and (4) transferring the reaction mixture prepared in the step S1 into a dialysis bag, dialyzing and purifying in deionized water, and finally performing vacuum freeze drying to obtain Ru-PEGNDs powder. The nanoparticles do not require a harsh reactionUnder the condition that active oxygen (H) can be catalyzed 2 O 2 Etc.), reduces cell damage, reduces the expression of inflammatory factors, achieves the effect of reducing the inflammation level, can effectively prevent the formation of postoperative abdominal adhesion, provides a brand new means for preventing the postoperative abdominal adhesion, and simultaneously has the advantages of simple preparation process, mild reaction conditions, easy synthesis and suitability for popularization and use.
Description
Technical Field
The invention relates to the technical field of metal nano materials, in particular to anti-abdominal adhesion metal nano enzyme Ru-PEG NDs and a preparation method and application thereof.
Background
The enzyme exists in each process of life activity of organisms, has strong catalytic activity and has high specificity to reaction substrates. The major components of most natural enzymes are proteins or RNAs, which are involved in human tissue metabolism, growth, repair, and the like. These natural enzymes have strict requirements on reaction environment, poor stability and are easy to lose catalytic activity. With the progress of technology, the nano enzyme is widely applied to the fields of medicine, biology and chemistry due to the advantages of high stability, good catalytic activity and the like.
Nanoenzymes are a class of nanoparticles with catalytic activity. Compared with natural enzymes, the artificially synthesized metal nano enzyme has the advantages of simple structure, convenience in synthesis, various designs, stable activity and no denaturation under extreme conditions. Common metal nano-enzymes such as titanium nano-enzyme, platinum nano-enzyme, manganese nano-enzyme and double-metal component gold/platinum nano-enzyme have large specific surface area, have higher catalytic activity and are widely applied in the field of biomedicine.
The abdominal adhesion is one of serious complications after surgical operation, the incidence rate of the abdominal adhesion after the surgical operation reaches 93 percent, and heavy disease burden and economic burden are brought to patients and society. In recent years, research and development of materials for preventing and treating postoperative abdominal adhesion mainly aim at isolating damaged parts, and an injured area and peripheral tissues are generally isolated in an early stage of adhesion formation by a physical barrier mode to prevent the formation of abdominal adhesion. The liquid anti-adhesion material is easy to dilute by body fluid and easy to leak; the gel anti-adhesion material is easy to smear unevenly and is easy to bond by self; the diaphragm anti-adhesion material is inconvenient to use and difficult to degrade. It is reported that oxidative stress pathway activation and macrophage polarization play an important role in the early stage of abdominal cavity adhesion, the generation of a large amount of active oxygen induces damage, and inflammatory factors such as IL-6, TNF-alpha and the like recruit macrophages to generate polarization, further aggravate inflammation and promote the generation of early adhesion. At present, no material for preventing abdominal cavity adhesion for eliminating Reactive Oxygen Species (ROS) exists, part of metal nanoenzyme has the effects of eliminating the ROS, protecting cells from being damaged by the ROS, inhibiting macrophage polarization and the like, and the material is widely researched in the fields of anti-inflammation, tissue repair and the like. Based on the situation, the inventor researches and develops the Ru-containing metal nanoenzyme Ru-PEG NDs, and at present, no relevant report on the preparation and application of the Ru-PEG NDs exists.
Disclosure of Invention
The invention provides anti-abdominal adhesion metal nano enzyme Ru-PEG NDs and a preparation method and application thereof, aiming at solving the problems in the background technology.
In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:
one of the purposes of the invention is to provide a preparation method of anti-abdominal adhesion metal nano enzyme Ru-PEG NDs, which comprises the following steps:
s1 reaction: mixing ruthenium acetate hydrate [ Ru (OAc) 3 .xH 2 O]Dissolving methoxypolyethylene glycol mercapto in deionized water, and then dropwise adding a reducing agent under continuous stirring for reaction until the reaction is completely finished to obtain a reaction mixture;
s2 purification: and (4) transferring the reaction mixture prepared in the step S1 into a dialysis bag, dialyzing and purifying in deionized water, and finally performing vacuum freeze drying to obtain Ru-PEG NDs powder.
As one embodiment of the present invention, in step S1, the ruthenium acetate hydrate [ Ru (OAc) ] 3 .xH 2 O]The mass of (3) is 30-90 mg, and the mass of Ru in the ruthenium acetate hydrateThe mass fraction is 40-45%; the relative molecular mass of the polyethylene glycol monomethyl ether (PEG-SH) is 2000-10000 Da, and the mass is 20-100 mg.
As one of embodiments of the present invention, in step S1, the reducing agent is an aqueous solution of sodium borohydride.
In the invention, preferably, the concentration of the sodium borohydride aqueous solution is 2-20 mg/mL, and the reaction time is 12-48 h.
As one embodiment of the present invention, in step S2, the cut-off molecular mass of the dialysis bag is 3500-.
The second purpose of the invention is to provide the anti-celiac adhesion metal nano enzyme Ru-PEG NDs which is mainly prepared by the preparation method of the anti-celiac adhesion metal nano enzyme Ru-PEG NDs.
Preferably, the diameter of the metal nanoenzyme Ru-PEG NDs is 2-5 nm.
The third purpose of the invention is to provide the metal nano enzyme Ru-PEG NDs prepared by the preparation method of the anti-abdominal adhesion metal nano enzyme Ru-PEG NDs or the application of the anti-abdominal adhesion metal nano enzyme Ru-PEG NDs in preparing materials for preventing abdominal adhesion.
Specifically, the abdominal adhesion is postoperative early abdominal adhesion.
Compared with the prior art, the invention mainly has the following beneficial effects:
(1) the invention adopts a new liquid phase reduction method to synthesize the Ru-PEG NDs anti-adhesion metal nano enzyme, the diameter is 2-5 nm, the dispersibility is good, the biocompatibility is high, and the catalase activity and the anti-inflammatory activity are good. The nanoparticles can catalyze active oxygen (H) without harsh reaction conditions 2 O 2 And the like), reducing cell damage, reducing the expression of inflammatory factors and achieving the effect of reducing the inflammation level.
(2) The preparation process disclosed by the invention is simple, mild in reaction condition, easy to synthesize and suitable for popularization and use.
Drawings
FIG. 1 is a transmission electron microscope image of Ru-PEG NDs nanoenzyme;
FIG. 2 is a diagram of a Ru-PEG NDs nanoenzyme hydrated particle size distribution;
FIG. 3 shows the degradation of Ru-PEG NDs nanoenzyme H 2 O 2 A rate;
FIG. 4 shows the degradation of Ru-PEG NDs nanoenzyme H 2 O 2 Production of O 2 A rate;
FIG. 5 is a fluorescence diagram of the Ru-PEG NDs nanoenzyme scavenging Reactive Oxygen Species (ROS) in cells;
FIG. 6 is a diagram showing the results of the in vitro cytotoxicity experiments of Ru-PEG NDs nanobodies;
FIG. 7 is a diagram of Ru-PEG NDs nanoenzyme in the prevention of celiac adhesion based on rat caecum-celiac adhesion model;
FIG. 8 is a statistical chart of the adhesion area of Ru-PEG NDs nanoenzyme for preventing abdominal cavity adhesion formation;
FIG. 9 is a graph of staining of H & E and masson in two groups of rat cecum-abdominal adhesion tissues;
FIG. 10 is a graph of H & E staining of sections of major internal organs of two groups of rats.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to these embodiments.
Example 1
Synthesis of Ru-PEG NDs nano enzyme
30-90 mg of ruthenium acetate [ Ru (OAc) ] 3 .xH 2 O](wherein, the mass fraction of Ru in the ruthenium acetate hydrate is 40-45%) and 20-100 mg of methoxypolyethylene glycol thiol (mPEG-SH, 2000Da) are dissolved in 50-150 mL of deionized water. 5mL of aqueous sodium borohydride solution (NaBH) was added continuously dropwise with continuous stirring 4 2-20 mg/mL), the reaction is finished after 12-48 h, the color of the liquid is changed from dark green to black, and the mixture is transferred into a dialysis bag (Mw: 3500-14000), dialyzing in deionized water for 2-3 days, and changing one for every 6hAnd (3) dehydrating, and finally performing vacuum freeze-drying to obtain Ru-PEG NDs powder, namely the metal nano enzyme disclosed by the invention.
Example 2
Characterization of Ru-PEG NDs nanoenzyme prepared by the invention
1) Transmission electron microscope experiment of Ru-PEG NDs nanoenzyme
20 mu L of Ru-PEG NDs nano enzyme solution is respectively put into a 1.5mL centrifuge tube, 980 mu L of deionized water is added for dilution, 10 mu L of diluted sample is absorbed and dropped on a carbon-supported film copper net, the copper net is put in an electronic drying oven for overnight drying, and a Transmission Electron Microscope (TEM) sample is prepared.
The result observed by a transmission electron microscope is shown in figure 1, and the Ru-PEG NDs nanoenzyme is a tiny nanodot with the real diameter of about 2-5 nm.
2) Determination of hydrated particle size of Ru-PEG NDs nanoenzyme
And (3) taking the synthesized Ru-PEG NDs nano enzyme, and diluting the Ru-PEG NDs nano enzyme to 2mL by using deionized water. 1mL of the solution was used to determine the hydrated particle size. The water bath ultrasound was carried out for 15min, each set of samples was assayed in triplicate and the whole process was monitored for changes. Wherein the distribution diagram of the hydration particle size of the Ru-PEG NDs nano enzyme is shown in figure 2, the prepared Ru-PEG NDs nano enzyme has good dispersion, and the average hydration particle size is 50 nm.
Example 3
Ru-PEG NDs nano enzyme degradation H prepared by the invention 2 O 2 Situation(s)
1) Degradation of H by Ru-PEG NDs nano enzyme 2 O 2 Rate of speed
Taking the synthesized Ru-PEG NDs nano enzyme and using 1mM H 2 O 2 Dilute to 20. mu.g/mL. Control group is equal volume of H 2 O 2 . The remaining H was determined using a hydrogen peroxide detection kit (colorimetry) after reactions 0, 10, 20, 30, 40, 50, 60, 75min, respectively 2 O 2 The concentration and the result are shown in figure 3, and the prepared Ru-PEG NDs nano enzyme has rapid H 2 O 2 Catalytic ability.
2) Degradation of H by Ru-PEG NDs nano enzyme 2 O 2 Production of O 2 Rate of speed
Get the synthesisGood Ru-PEG NDs nanoenzyme with 1mM H 2 O 2 Dilute to 20 μ g/mL. Control group is equal volume of H 2 O 2 . Respectively monitoring H within 20min by using dissolved oxygen instrument 2 O 2 Decomposition of the liquid to produce O 2 The content of the modified Ru-PEG NDs is shown in figure 4, and the prepared trace Ru-PEG NDs nano enzyme can catalyze H within 5min 2 O 2 Fast decomposition of liquid to produce O 2 。
Example 4
Determination of active oxygen in cells eliminated by Ru-PEG NDs nano enzyme prepared by the invention
The DCF method is adopted to determine the intracellular ROS, and DCFH-DA has no fluorescence and can freely pass through cell membranes and be hydrolyzed by esterase in cells to generate DCFH. DCFH, however, does not permeate the cell membrane, thus allowing the probe to be easily loaded into the cell. Reactive oxygen species ROS in cells can oxidize non-fluorescent DCFH to generate fluorescent DCF. Therefore, the level of active oxygen in the cell can be known by detecting the fluorescence of DCF.
Firstly, inoculating human HMrSV on a 12-hole plate 5 Peritoneal mesothelial cells, the number of seeded cells was 10 ten thousand per well. Is divided into a control group,
200μM H 2 O 2 Group, group Ru-PEG NDs and group Ru-PEG NDs + 200. mu. M H 2 O 2 Group, Ru-PEG NDs concentration of 100. mu.g/mL. The cells were inoculated for 24 hours and then treated according to the above groups, after 24 hours, they were washed twice with sterile PBS, incubated in an incubator with a final concentration of 10. mu.M DCFH-DA (Sigma, Mw. gtoreq. 487) for 30min, stained and observed with a fluorescence microscope, and the results are shown in FIG. 5, where Ru-PEG NDs nanoenzymes can decompose H 2 O 2 Active oxygen is removed, and cells are protected from being damaged by the active oxygen.
Example 5
In vivo test for preventing abdominal cavity adhesion by Ru-PEG NDs nano enzyme prepared by the invention
1) Establishment of rat postoperative cecum-abdominal cavity adhesion model
In order to research the prevention effect of Ru-PEG NDs nano enzyme on the postoperative abdominal adhesion formation of rats, firstly, an international general abdominal adhesion model SD rat postoperative cecum-abdominal adhesion model is established. SD rats were anesthetized with 10% diluted sodium pentobarbital solution (20 mg/ml). The rats were placed in a supine position and the abdomen was prepared for skin loss by 4X 5 cm. Sterilized with 75% alcohol and iodophor, and then incised at the anterior midline of the peritoneum and abdominal wall to a length of 4 cm. The cecum was separated, exposing a 2 x 1cm area. The cecum serosa was rubbed 40 times with gauze until punctate bleeding appeared on the serosa surface but no perforation was observed, and then the peritoneum and the abdominal transverse muscle were scraped from the left abdominal wall to form a 2cm × 1cm defect. Then, 4-0 Aicherukang silk thread is used for hanging 1 needle on the worn cecum and the side of the defective abdominal wall, and finally, the abdominal cavity is closed by suturing layer by layer.
2) Ru-PEG NDs nano enzyme for preventing postoperative caecum-abdominal cavity adhesion of rats
In the molding process, the postoperative rats were randomly divided into 3 groups (Control group, commercial Chitosan Chitosan group, Ru-PEG NDs group) of 6 rats each. The Control group did not treat, and the commercial Chitosan Chitosan group injected 0.6mL of commercial Chitosan per one (15mg/mL, CHIOGELTM, Med. No.: 20143642114, Shanghai its peptide Biotechnology Co., Ltd.) into the abdominal wall defect and the injured cecum. The Ru-PEG NDs groups are sprayed with 0.6mL of 3mg/mL Ru-PEG NDs nano enzyme solution on the abdominal wall defect and the injured cecum. Specimens were collected 7 days later and photographs of the abdominal adhesions were taken, as shown in FIG. 7. And finally, quantifying the adhesion area by using Image J software, wherein the statistical result of the attached figure 8 shows that the adhesion area of the abdominal cavity of the Ru-PEG NDs group has statistical significance with the difference between the Control group and the commercialized chitosan. FIG. 9 shows the H & E and masson staining patterns for tissues. H & E is mainly used for observing histopathological morphology, and masson dyeing is used for observing the distribution of collagen fibers at tissue adhesion positions. AW (abdominal wall), AD (adhesion), CE (cecum) are shown on the graph. The results reflect that the Ru-PEG NDs nanoenzyme can effectively prevent the caecum-abdominal cavity adhesion formation of rats after the operation, and the effect is superior to that of the commercialized chitosan commonly used in the market.
Example 6
Safety evaluation of Ru-PEG NDs nano-enzyme prepared by the invention
1) Cytotoxicity test
The CCK8 method is adopted in the experiment to explore the toxicity of the nano-enzyme on the peritoneal mesothelial cells of the human HMrSV 5. Firstly, 8 elements are added into each hole of 96 holes10 3 Culturing the cells for 24h to allow the cells to grow adherent to the cells, diluting the Ru-PEG NDs nanoenzyme with a culture medium to obtain final concentrations of 0, 12.5, 25, 50, 100 and 200 mu g/mL, adding 200 mu L of the Ru-PEG NDs nanoenzyme into each hole for co-culture for 24h and 48h, discarding the upper culture medium, adding 100ul of fresh serum-free culture medium into each hole, adding 10 mu L of CCK8 detection solution into each hole, culturing for 2h in a dark place, and measuring the absorbance value at 450nm by using an enzyme labeling instrument. As shown in figure 6, at the highest concentration of 200 mug/mL, the cell viability of the peritoneal mesothelial cells of the human HMrSV5 is still more than 95% when the peritoneal mesothelial cells are incubated for 24h and 48 h. This indicates that the Ru-PEG NDs nanoenzyme has lower cytotoxicity.
2) Evaluation of visceral safety
After preventing postoperative abdominal cavity adhesion for 7 days by using the Ru-PEG NDs nanoenzyme, rats were euthanized, and the main viscera (heart, liver, spleen, lung and kidney) of the rats of the control group and the Ru-PEG NDs nanoenzyme group were subjected to H & E staining to analyze whether the Ru-PEG NDs nanoenzyme has toxicity to the internal organs of the mice. Comparing the H & E staining of the major organs of the Ru-PEG NDs nanoenzyme group as shown in fig. 10, no significant histomorphological differences were observed between all major organs of the control group and the Ru-PEG NDs nanoenzyme group, which further confirmed the low in vivo toxicity and good biocompatibility of the Ru-PEG NDs nanoenzyme.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A preparation method of anti-abdominal adhesion metal nano enzyme Ru-PEG NDs is characterized by comprising the following steps:
s1 reaction: mixing ruthenium acetate hydrate [ Ru (OAc) 3 .xH 2 Dissolving O and methoxypolyethylene glycol thiol (PEG-SH) in deionized water, and then dropwise adding a reducing agent under continuous stirring for reaction until the reaction is completely finished to obtain a reaction mixture;
s2 purification: and (4) transferring the reaction mixture prepared in the step S1 into a dialysis bag, dialyzing and purifying in deionized water, and finally performing vacuum freeze drying to obtain Ru-PEG NDs powder.
2. The method for preparing the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs according to claim 1, which is characterized in that: in step S1, the ruthenium acetate hydrate [ Ru (OAc) 3 .xH 2 O]The mass of the ruthenium acetate is 30-90 mg, and the mass fraction of Ru in the ruthenium acetate hydrate is 40-45%; the relative molecular mass of the polyethylene glycol monomethyl ether (PEG-SH) is 2000-10000 Da, and the mass is 20-100 mg.
3. The method for preparing the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs according to claim 1, which is characterized in that: in step S1, the reducing agent is an aqueous solution of sodium borohydride.
4. The method for preparing the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs according to claim 1, which is characterized in that: the concentration of the sodium borohydride aqueous solution is 2-20 mg/mL, and the reaction time is 12-48 h.
5. The preparation method of the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs, according to claim 1, is characterized in that: in step S2, the cut-off molecular mass of the dialysis bag is 3500-.
6. An anti-abdominal adhesion metal nano enzyme Ru-PEG NDs is characterized in that: the preparation method of the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs as defined in any one of claims 1-5.
7. The anti-celiac adhesion metal nanoenzyme Ru-PEG NDs as claimed in claim 6, wherein: the diameter of the metal nano enzyme Ru-PEG NDs is 2-5 nm.
8. The use of the metal nanoenzyme Ru-PEG NDs prepared by the method for preparing the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs according to any one of claims 1 to 5 or the anti-celiac adhesion metal nanoenzyme Ru-PEG NDs according to any one of claims 6 to 7 in the preparation of a material for preventing celiac adhesion.
9. Use according to claim 8, characterized in that: the abdominal adhesion is postoperative early abdominal adhesion.
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