CN111939270A - Double-nano enzyme antibacterial agent with continuous antibacterial effect and preparation method thereof - Google Patents
Double-nano enzyme antibacterial agent with continuous antibacterial effect and preparation method thereof Download PDFInfo
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- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/05—Metallic powder characterised by the size or surface area of the particles
- B22F1/054—Nanosized particles
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- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/02—Oxides; Hydroxides
- C01G49/08—Ferroso-ferric oxide (Fe3O4)
Abstract
The inventionProvides a double nano enzyme antibacterial agent (Au-Fe)3O4@ DMSNs), hydroxyl radical OH is generated through a cascade reaction between two supported nano enzymes, namely gold nano and ferroferric oxide, for sterilization, the problem that the natural enzyme is inactivated by the influence of the environment is solved because the nano enzyme is used instead of the natural enzyme, the two nano enzymes are simultaneously dispersed and supported in the mesoporous silica, the cascade reaction of the connection between the nano enzymes is ensured, and the common inactivation phenomenon of the nano enzymes caused by agglomeration is avoided because of the obstruction of the mesoporous silica. The preparation method is simple and suitable for industrial production.
Description
Technical Field
The invention relates to a nano enzyme antibacterial agent, in particular to a double nano enzyme antibacterial agent with a continuous antibacterial effect and a preparation method thereof, belonging to the technical field of preparation of antibacterial materials.
Background
Wound healing (wound healing) refers to the healing process after the body is subjected to external force, the tissues such as skin are separated or damaged, and the degree of damage and the regeneration capacity of the tissues determine the repairing mode, healing time and the size of scars. The healing of the wound is long-term and easily affected by the outside through the self-healing ability of the human body. For example, when a wound is not treated in time and is exposed to air after a wound is produced on a body surface, not only can the wound be further damaged when the wound is contacted with the outside, but also bacteria or external bacteria and viruses remained on the surface of the wound are more likely to enter the wound, so that the wound infection is worsened. Various pathogenic microorganisms and their variations make wound healing more challenging, causing wound infections that delay the time to wound healing, which has affected millions of people and caused billions of economic losses. The antibacterial material for treating the infected wound needs to clear external infection bacteria and viruses in time, does not harm the wound and promotes the healing of the wound. Therefore, high-quality antibacterial materials for promoting wound healing are always the focus of attention of scholars at home and abroad.
For drugs for promoting wound healing, antibiotics are widely used due to their highly effective antibacterial properties, save lives of countless patients in clinical medical applications for decades, have revolutionary effects, and have been used to date in a wide range. However, over decades of use and even abuse, the resulting spectrum of drug-resistant bacteria, superbacteria, has created even greater problems. Silver ions or silver nanoparticles possessing broad-spectrum antibacterial activity have thus been studied to replace antibiotics as new antibacterial agents, but their high toxicity limits their application in actual wounds. Even if it has been successful in numerous studies to try to inhibit toxicity while retaining some of the antimicrobial activity, it is still harmful to the wound itself. In addition, the common wound dressings on the market have the problem of adhering to the wound, and the wound is torn when the dressings are removed, so that secondary damage is caused to the wound.
It has been found that oxygen is involved in a series of reactions during cellular metabolism and is ultimately converted into Reactive Oxygen Species (ROS), such as hydroxyl radicals (. OH), hydrogen peroxide radicals (. OOH), H2O2And the like. These ROS are generated by cells in situ around the wound, and due to their high oxidative capacity, have antibacterial activity and can promote wound healing. Of these, OH is an effective antimicrobial agent that has been used in normal wound healing. Although the content of OH naturally produced by cells in vivo is relatively low, under the action of specific natural enzymes such as glucose oxidase and horseradish peroxidase, exogenous substrates such as glucose or H can be used2O2Generation of compensation. This process of production, which acts synergistically with the two enzymes mentioned above, is called a cascade reaction, the glucose molecule being first converted into H2O2Then converted to OH. The OH-like structure makes the cell membrane of the bacterium easily react with other reductive molecules of cells to be destroyed, thereby increasing oxidative stress and inhibiting bacterial infection.
After determining the use of OH as an effective factor for antibacterial, how to use OH which is easily inactivated and not easily preserved becomes an important problem. The generation of OH can be obtained by decomposing a substrate by the catalytic activity of enzymes, and hydrogen peroxide has the characteristic of being volatile and difficult to store, so that glucose can be used as a primary substrate of hydrogen peroxide, and the generated hydrogen peroxide is decomposed again to generate OH for antibiosis. In the process, the enzyme cascade reaction can be easily completed, namely glucose oxidase is used for catalyzing and decomposing glucose to generate hydrogen peroxide, and then peroxidase is used for catalyzing and decomposing hydrogen peroxide to generate OH for antibiosis. However, the glucolase and the peroxidase can be extracted from the nature, but the natural enzyme has the activity range of the natural enzyme, is easily inactivated by the influence of the external environment, and meanwhile, the problems of complicated purification, high cost and difficult preservation of the natural enzyme enable the nano-enzyme to be used as a substitute of the natural enzyme to simulate the activity of the enzyme for production.
The synergy between two nanoenzymes, i.e. the product of one enzyme is the substrate of the other, is called a cascade, but there is not only promotion and synergy between the two enzymes, but there is also an interaction between the two enzymes. And although the nano enzyme has better stability to external conditions compared with natural enzyme, the nano enzyme can lose activity due to agglomeration among particles with time. Therefore, coordination between nanoenzymes and stabilization of activity are also difficult problems that must be solved.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a double nano-enzyme antibacterial agent, which is Au-Fe3O4The @ DMSNs contains two nanoenzymes, namely gold nanoenzyme and ferroferric oxide, wherein the gold nanoenzyme can simulate glucose to be catalyzed by glucose oxidase to generate gluconic acid and hydrogen peroxide, the ferroferric oxide nanoenzyme can be synergistically reacted with the gold nanoenzyme to simulate peroxidase to catalyze the hydrogen peroxide to generate hydroxyl free radicals with strong oxidizing property, and the hydroxyl free radicals oxidize and damage cell membranes of bacteria to cause the rupture and death of the bacteria.
Except for special description, the percentages are mass percentages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a dual nanoenzyme antimicrobial agent, characterized by: the double nano enzyme antibacterial agent (Au-Fe)3O4@ DMSNs) is prepared by carrying gold nanoenzyme and ferroferric oxide nanoenzyme in pore channels of Dendritic Mesoporous Silica (DMSNs) through twice in-situ reduction.
Research shows that the Dendritic Mesoporous Silica (DMS)Ns) is prepared by carrying gold nanoenzyme and ferroferric oxide nanoenzyme through two-time in-situ reduction in pore canals, if the gold nanoenzyme (Au) and the ferroferric oxide (Fe) are simultaneously carried3O4) The nano enzyme, the interaction between two nano enzymes can cause agglomeration and lose activity; loading gold nanoenzyme firstly and then loading ferroferric oxide nanoenzyme can cause Au particles to be wrapped and cannot play a role; the ferroferric oxide nanoenzyme is loaded firstly, and then the gold nanoenzyme is loaded, so that the cascade reaction is not affected. Preferably, the double-nanoenzyme antibacterial agent is prepared by carrying gold nanoenzyme and ferroferric oxide nanoenzyme in a dendritic mesoporous silica pore channel through twice in-situ reduction; a cascade reaction is generated between the loaded gold nano enzyme and the ferroferric oxide nano enzyme to generate hydroxyl radical OH for sterilization; the method comprises the steps of loading ferroferric oxide nanoenzyme firstly and then loading gold nanoenzyme.
In the double-nanoenzyme antibacterial agent, the step of loading ferroferric oxide nanoenzyme is as follows: firstly, FeCl containing Dendritic Mesoporous Silica (DMSNs)2And FeCl3The mixed solution is added to NaOH solution and then added to N2Stirring and reacting for 20-40min under the atmosphere to obtain the ferroferric oxide nanoenzyme (Fe) loaded in the dendritic mesoporous silica3O4@ DMSNs). Further, the step of loading the ferroferric oxide nanoenzyme is to firstly load FeCl containing 0.1M2And 0.2M FeCl3Mixing 5mL of the solution with 0.1g of dendritic mesoporous silica, adding the mixture into 50mL of NaOH solution, and adding the mixture into N2Stirring and reacting for 30min under the atmosphere to obtain the ferroferric oxide nanoenzyme loaded in the dendritic mesoporous silica.
In the double-nanoenzyme antibacterial agent, the steps of loading the gold nanoenzyme are as follows: adding HAuCl4Ferroferric oxide nanoenzyme (Fe) loaded in solution and dendritic mesoporous silica3O4@ DMSNs), stirring at 400-800rpm for 10-15h, and mixing the above solution with polyvinylpyrrolidone (PVP) in N2Mixing under atmosphere to remove O in the equipment2Reacting NaBH4Adding into the solution, reacting for 10-30min to obtain double-nanometer enzyme antibacterial agent (Au-Fe)3O4@DMSNs). Further, the step of loading the gold nano enzyme is to mix 1.25x10-4HAuCl of M4Mixing with ferroferric oxide nanoenzyme loaded in 20mg of dendritic mesoporous silica, stirring at 500rpm for 12h, and mixing the solution with polyvinylpyrrolidone (PVP) in N2Mixing under atmosphere to remove O in the equipment2(ii) a Reacting NaBH4Adding into the solution, and reacting for 20min to obtain the double nano enzyme antibacterial agent. Further, the NaBH described above4The dosage is NaBH4Au 5:1 in mass ratio.
In the double nano-enzyme antibacterial agent, the preparation steps of the dendritic mesoporous silica are as follows: dissolving Triethanolamine (TEA) in water, adding Cetyl Trimethyl Ammonium Bromide (CTAB) and salicylic acid (NaSal) as templates, adding tetraethyl orthosilicate (TEOS) into the solution, reacting for 2h, collecting the product, washing with ethanol, and removing residual reactants; then using HCl and CH3And (4) OH extracting the washed product, and drying to obtain the dendritic mesoporous silicon Dioxide (DMSNs). Further, the preparation step of the dendritic mesoporous silica comprises the steps of dissolving 0.068g of Triethanolamine (TEA) in water, stirring for 0.5h at 80 ℃, adding 380mg of Cetyl Trimethyl Ammonium Bromide (CTAB) and 168mg of salicylic acid (NaSal) as templates, and continuing stirring for 1 h; then adding 4ml of tetraethyl orthosilicate (TEOS) into the solution, keeping 300rpm and stirring for 2h, collecting the product, washing with ethanol, and removing residual reactants; finally using HCl and CH3CH2And extracting the washed product for at least 3 times by using the OH mixed solution at 60 ℃, and drying to obtain the dendritic mesoporous silica.
Specifically, the preparation method of the double nano enzyme antibacterial agent comprises the following steps:
(1) preparation of dendritic mesoporous silica
Dissolving 0.068g Triethanolamine (TEA) in water, stirring at 80 deg.C for 0.5h, adding 380mg Cetyl Trimethyl Ammonium Bromide (CTAB) and 168mg salicylic acid (NaSal) as template, and stirring for 1 h; then adding 4ml of tetraethyl orthosilicate (TEOS) into the solution, keeping 300rpm and stirring for 2h, collecting the product, washing with ethanol, and removing residual reactants; finally using HCl and CH3CH2Extracting the washed product at 60 ℃ for at least 3 times by using the OH mixed solution, and drying to obtain Dendritic Mesoporous Silica (DMSNs);
(2) load ferroferric oxide nanoenzyme
Firstly, 0.1M FeCl is contained2And 0.2M FeCl3Was mixed with 0.1g DMSNs and added to 50mL NaOH solution, then added under N2Stirring and reacting for 30min under the atmosphere to obtain the ferroferric oxide nanoenzyme (Fe) loaded in the dendritic mesoporous silica3O4@DMSNs)；
(3) Loading gold nano enzyme
Mix 1.25x10-4HAuCl of M4With ferroferric oxide nanoenzyme (Fe) loaded in 20mg of dendritic mesoporous silica3O4@ DMSNs), stirring at 500rpm for 12h, and then mixing the above solution with polyvinylpyrrolidone (PVP) in N2Mixing under atmosphere to remove O in the equipment2(ii) a Reacting NaBH4Adding into the solution to react for 20min, wherein NaBH4The dosage is NaBH4Au is 5:1 in mass ratio; obtaining the double nano enzyme antibacterial agent (Au-Fe)3O4@DMSNs)。
Has the advantages that:
the invention provides a double nano enzyme antibacterial agent (Au-Fe)3O4@ DMSNs), and hydroxyl radical OH is generated through a cascade reaction between two kinds of nano-enzymes of loaded gold nano-enzyme and ferroferric oxide to sterilize. The invention relates to a double nano enzyme antibacterial agent Au-Fe3O4The @ DMSNs is antibacterial by catalyzing added glucose to generate hydroxyl radicals by utilizing a cascade reaction of two nano enzymes. Au-Fe of the invention3O4The @ DMSNs double-nanoenzyme antibacterial agent can continuously and stably generate hydroxyl radicals for antibiosis under the condition that sufficient glucose sources exist, wherein Au nanoenzyme can simulate glucose oxidase to catalyze glucose into hydrogen peroxide and gluconic acid, and Fe3O4The nano enzyme can simulate peroxidase to catalyze the intermediate product hydrogen peroxide to generate hydroxyl radicals. The invention relates to a double nano enzyme antibacterial agent Au-Fe3O4Since the @ DMSNs uses nano enzyme, the day is avoidedHowever, the enzyme is easily inactivated by environmental influences. The invention relates to a double nano enzyme antibacterial agent Au-Fe3O4The preparation method of @ DMSNs is to load Fe on DMSNs firstly3O4The nano enzyme is loaded, the Au nano enzyme is loaded, the two nano enzymes are dispersedly loaded in the mesoporous silica, the cascade reaction of the connection between the nano enzymes is ensured, and the common inactivation phenomenon of the nano enzymes caused by agglomeration is avoided due to the obstruction of the mesoporous silica. The preparation method is simple and suitable for industrial production.
Drawings
FIG. 1 is Au-Fe of the present invention3O4A preparation flow chart of @ DMSNs;
FIG. 2 is Au-Fe of the present invention3O4The schematic diagram of the use of @ DMSNs.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure. The raw materials and the reagents are all commercial products.
(I) the double nano enzyme antibacterial agent Au-Fe of the invention3O4Preparation of @ DMSNs
The preparation process of Au-Fe3O4@ DMSNs is shown in figure 1, Triethanolamine (TEA) is dissolved in water (step (I)), Cetyl Trimethyl Ammonium Bromide (CTAB) and salicylic acid (NaSal) are added as templates (step (II)), tetraethyl orthosilicate (TEOS) is added into the solution (step (III)), Dendritic Mesoporous Silica (DMSNs) is prepared after extraction and cleaning, then ferroferric oxide nanoenzyme is loaded firstly, and then gold nanoenzyme is loaded.
Example 1
The invention relates to a double nano enzyme antibacterial agent Au-Fe3O4The preparation steps of @ DMSNs are as follows: 0.068g Triethanolamine (TEA) was dissolved in water and gently stirred at 80 ℃ for 0.5h, then 380mg cetyltrimethylammonium bromide (CTAB) and 168mg salicylic acid (NaSal) were added as templates and stirred againStirring for 1 h. Then, 4ml of tetraethyl orthosilicate (TEOS) was added to the above solution and stirred at 300rpm for 2h, and the product was collected and washed with ethanol to remove the residual reactant. Finally, the mixed solution (HCl and CH) is used3CH2OH) extracting the washed product at 60 deg.C for at least 3 times, and drying in oven to obtain DMSNs. The nano enzyme is loaded by firstly adding FeCl containing 0.1M2And 0.2M FeCl3Was mixed with 0.1g DMSNs and added to 50mL NaOH solution. Then the solution is in N2Stirring at high speed for 30min under atmosphere to generate ferroferric oxide (Fe) on DMSNs3O4) Form Fe3O4@ DMSNs; then 1.25x10-4HAuCl of M4Mixed with 20mg DMSNs and stirred at 500rpm for 12 h. Mixing the above solution with excessive polyvinylpyrrolidone (PVP) in N2Mixing under atmosphere to remove O in the equipment2. Reacting NaBH4(NaBH4Au-Fe (5: 1) is added into the solution and reacts for 20min to generate nano gold particles (AuNPs) which are prepared into Au-Fe3O4@ DMSNs double nano-enzyme antibacterial agent.
Referring to example 1, the inventors examined the loading order of two enzymes versus the prepared Au-Fe3O4The effect of @ DMSNs double nanoenzyme antibacterial agents. As a result, it was found that gold nanoenzyme (Au) and ferroferric oxide (Fe) were simultaneously supported3O4) The nano enzyme, the interaction between two nano enzymes can cause agglomeration and lose activity; loading gold nanoenzyme firstly and then loading ferroferric oxide nanoenzyme can cause Au particles to be wrapped and cannot play a role; the ferroferric oxide nanoenzyme is loaded firstly, and then the gold nanoenzyme is loaded, so that the cascade reaction is not affected.
Referring to example 1, the inventors examined the effect of methanol/hydrochloric acid (1/1 to 10/1 (vol.) (38% concentrated hydrochloric acid) as an extract and ethanol/hydrochloric acid (1/1 to 10/1 (vol.) (38% concentrated hydrochloric acid)) as an extract on the reaction results in the preparation of DMSNs, and as a result, found that: ethanol/hydrochloric acid (10/1 by volume) can ensure the effect and reduce the cost and the danger as much as possible.
Referring to example 1, the inventors examined the influence of the reduction conditions on the formation of gold nanoparticles during the preparation of Au nanoenzymes. The inventor researches that sodium borohydride is rapidly added with reduced chloroauric acid within 2s during stirring in a nitrogen environment, and acetic acid is used as a protective agent; secondly, under the nitrogen environment, sodium borohydride is quickly added with reduced chloroauric acid within 2s while stirring, and PVP is taken as a protective agent; thirdly, under the nitrogen environment, sodium borohydride is added into reduced chloroauric acid drop by drop slowly within 2min while stirring, and acetic acid is taken as a protective agent; fourthly, under the nitrogen environment, sodium borohydride is added into the reduced chloroauric acid drop by drop slowly within 2min while stirring, and PVP is taken as a protective agent; under the nitrogen environment, rapidly adding reduced chloroauric acid into sodium borohydride within 2s while stirring, using PVP as a protective agent, and totally five reduction conditions are found out: under the nitrogen environment, sodium borohydride is rapidly added with reduced chloroauric acid within 2s while stirring, PVP is taken as a protective agent, and the gold nanoparticles with small particle size and glucose oxidase activity can be prepared.
(II) the double nano enzyme antibacterial agent Au-Fe of the invention3O4Antibacterial experiments of @ DMSNs
The principle of the double nano enzyme antibacterial agent is shown in the right figure 2, and Au-Fe3O4The @ DMSNs contains two nanoenzymes, namely gold nanoenzyme and ferroferric oxide, wherein the gold nanoenzyme can simulate glucose oxidase to catalyze glucose to generate gluconic acid and hydrogen peroxide, the ferroferric oxide nanoenzyme can synergistically react with the gold nanoenzyme to simulate peroxidase to catalyze the hydrogen peroxide to generate hydroxyl free radicals with strong oxidizing property, and the product hydroxyl free radicals generated by the gold nanoenzyme and the ferroferric oxide through a cascade reaction can oxidize and damage cell membranes of bacteria to cause the bacteria to break and die, so that the wound is prevented from being further infected and the wound is promoted to heal.
The inventor carries out an enzyme activity detection experiment, and the experimental method comprises the following steps: 0.2g of glucose is prepared into 1ml of solution for standby, 750 mul of 200 mul/mg of sample is added into the glucose solution, the mixed solution is cultured for 4 hours, the sample is removed by centrifugation, and the supernatant is taken for detection. 3.85mg of TMB was prepared into 1ml of solution, 50. mu.l of the solution was added to 750. mu.l of the supernatant, the mixed solution was incubated for 10min and color change was observed. The OPD and ABTS color reaction experimental method is the same as that of TMB. In the final solution, the double-nano enzyme antibacterial agent can generate OH under the catalysis of the existence of glucose, TMB is oxidized into blue, OPD is oxidized into orange red, ABTS is oxidized into green, and obvious peaks are respectively detected at 655nm, 450nm and 420nm by an ultraviolet spectrophotometer.
The inventor carries out an in vitro hemolysis test, and the experimental method comprises the following steps: fresh blood (5mL) of a New Zealand white rabbit was added to anticoagulated 3.8% sodium citrate, added to PBS (10mL), and then centrifuged at 2500rpm for 5min to remove serum, thereby obtaining erythrocyte RBCs. Samples were pre-warmed at 37 ℃ with different concentrations and 3ml PBS, and 60. mu.L RBCs were added. The mixture was incubated at 37 ℃ for 1 h. PBS (3ml) and 60 μ L RBCs were set as a negative control group (-), which was in a clear state, and 3ml deionized water and 60 μ L RBBCs were set as a positive control group (+), which was shown to be turbid. After incubation, the fabric samples were removed from the mixture and centrifuged at 2500rpm for 5 minutes. The absorbance of the supernatant of the mixture was measured at a wavelength of 545nm with an ultraviolet-visible spectrophotometer. The hemolysis rate is calculated as follows: hemolysis ratio (%) - (Abs)(s)-Abs(-))/(Abs(+)-Abs(-)) X 100%, wherein, Abs(s)Is the absorbance of the sample; abs(+)Absorbance as positive control; abs(-)Absorbance of the negative control. The results show that the hemolysis rate of different samples is kept less than or equal to 1 percent, and the hemocyte has good biocompatibility with the hemocyte.
The inventor carries out a biocompatibility experiment, and the experimental method comprises the following steps: to assess cytotoxicity, L929 mouse fibroblast cell line (ATCC, cc-1) was cultured with 10% fetal bovine serum and 1% antibiotic/antifungal in Dulbecco's modified Eagle's medium. The samples were incubated in serum-free medium (2ml) at 37 ℃ for 24h, and then the sample-free extract (100. mu.L) was distributed to 96-well plates. Then, 1X 104L 929 cells were cultured in 37 ℃ leachate for 24h and 48h, with pure cells as control. A solution of tetramethylazoazolium salt (MTT) (10 μ Ι _) was added to each well; after 4h, further solutions were added to dimethyl sulfoxide (100 μ L) and incubated for 4h to dissolve formazan crystals. Finally, the absorbance of the sample at 490nm wavelength was recorded. For visual observation of biocompatibility, live and dead cells were simultaneously stained with Calcein-AM/PI double staining kit (40747ES76, Yeasen Biological Technology co., ltd., Shanghai, China) and observed with a fluorescence microscope (Nikon TS 100, Tokyo, Japan). The experimental results show that: the cells cultured with the sample grow well, the cell survival rate is over 80 percent, and the cells are not dead too much in cell staining.
The inventor carries out animal experiments, and the experimental method comprises the following steps: new Zealand white rabbits were used as animal experimental models. All rabbits were injected with xylazine hydrochloride, and after shaving, 4 full-thickness round wounds with a diameter of 2cm were made on the back. Infection of the wound with Staphylococcus aureus (about 10)7CFU/mL), covered with sterile gauze, and cultured for 24 h. In which 2 wounds were treated with Au-Fe3O4The application method comprises the following steps of @ DMSNs coating, adding hydrogen peroxide and glucose respectively, setting one wound as a blank, and coating 1 wound with a commercialized antibacterial agent dressing. Subsequently, the rabbits were kept individually in cages in a rabbit house, the temperature being kept around 25 ℃ (daily check to ensure the health of the animals). Euthanasia was performed after measuring the wound area on days 5, 10, and 15 post-infection, respectively. Wound tissue specimens were excised, soaked in 10% formaldehyde, stored at 4 ℃ and sectioned for analysis. The experimental results show that: the double nano enzyme antibacterial agent shows more excellent antibacterial effect and wound healing effect in the same group of experiments, the number of wound bacteria is reduced most obviously in different days, and the excellent continuous antibacterial effect is shown3O4The wounds of the @ DMSNs sample all cleared infection and healed faster than the blank.
Claims (9)
1. A dual nanoenzyme antimicrobial agent, characterized by: the double-nanoenzyme antibacterial agent is prepared by carrying gold nanoenzyme and ferroferric oxide nanoenzyme in a pore channel of dendritic mesoporous silica through twice in-situ reduction; a cascade reaction is generated between the loaded gold nano enzyme and the ferroferric oxide nano enzyme to generate hydroxyl radical OH for sterilization; the method comprises the steps of loading ferroferric oxide nanoenzyme firstly and then loading gold nanoenzyme.
2. As claimed in claim 1The double-nanoenzyme antibacterial agent is characterized in that the step of loading the ferroferric oxide nanoenzyme is as follows: firstly FeCl2And FeCl3Adding the solution into NaOH solution containing the dendritic mesoporous silica, and then adding the solution into N2Stirring and reacting for 20-40min under the atmosphere to obtain the ferroferric oxide nanoenzyme loaded in the dendritic mesoporous silica.
3. The dual nanoenzyme antimicrobial of claim 2, wherein: the step of loading the ferroferric oxide nanoenzyme is to firstly load FeCl containing 0.1M2And 0.2M FeCl3Is mixed with a solution containing 0.1g of dendritic mesoporous silica, added to 50mL of NaOH solution, and added to the solution in N2Stirring and reacting for 30min under the atmosphere to obtain the ferroferric oxide nanoenzyme loaded in the dendritic mesoporous silica.
4. The dual nanoenzyme antimicrobial of any one of claims 1 to 3, wherein the step of loading the gold nanoenzyme is: adding HAuCl4Mixing the solution with ferroferric oxide nanoenzyme loaded in dendritic mesoporous silica, stirring for 10-15h at 400-800rpm, and then mixing the solution with polyvinylpyrrolidone in N2Mixing under atmosphere to remove O in the equipment2Reacting NaBH4Adding into the solution, and reacting for 10-30min to obtain the double-nano enzyme antibacterial agent.
5. The dual nanoenzyme antimicrobial of claim 4, wherein: the step of loading the gold nano enzyme is to mix 1.25x10-4HAuCl of M4Mixing with ferroferric oxide nanoenzyme loaded in 20mg of dendritic mesoporous silica, stirring at 500rpm for 12h, and mixing the solution with polyvinylpyrrolidone in N2Mixing under atmosphere to remove O in the equipment2(ii) a Reacting NaBH4Adding into the solution, and reacting for 20min to obtain the double nano enzyme antibacterial agent.
6. The dual nanoenzyme antimicrobial of claim 5, wherein the NaBH is4Dosage ofNaBH4: Au is 5:1 in mass ratio.
7. The double nanoenzyme antimicrobial agent of claim 1 or 2, wherein the dendritic mesoporous silica is prepared by the steps of: dissolving triethanolamine in water, adding cetyl trimethyl ammonium bromide and salicylic acid as templates, adding tetraethyl orthosilicate into the solution, reacting for 10-152h, collecting a product, washing with ethanol, and removing residual reactants; then extracting the washed product by HCl and CH3CH2OHCH3OH, and drying to obtain the dendritic mesoporous silicon dioxide.
8. The double nanoenzyme antimicrobial agent of claim 7, wherein the dendritic mesoporous silica is prepared by the steps of: dissolving 0.068g triethanolamine in water, stirring at 80 deg.C for 0.5h, adding 380mg cetyl trimethyl ammonium bromide and 168mg salicylic acid as template, and stirring for 1 h; adding 24ml tetraethyl orthosilicate into the solution, keeping the stirring at 300rmp for reaction for 10-152h, collecting the product, washing the product with ethanol, and removing residual reactants; then extracting the washed product by HCl and CH3CH2OHCH3OH, and drying to obtain the dendritic mesoporous silicon dioxide.
9. A preparation method of a double nano-enzyme antibacterial agent comprises the following steps:
(1) preparation of dendritic mesoporous silica
Dissolving 0.068g triethanolamine in water, stirring at 80 deg.C for 0.5h, adding 380mg cetyl trimethyl ammonium bromide and 168mg salicylic acid as template, and stirring for 1 h; then adding 4ml of tetraethyl orthosilicate into the solution, keeping 300rpm and stirring for 2 hours, collecting a product, washing the product with ethanol, and removing residual reactants; finally using HCl and CH3CH2Extracting the washed product for at least 3 times at 60 ℃ by using the OH mixed solution, and drying to obtain the dendritic mesoporous silica;
(2) load ferroferric oxide nanoenzyme
Firstly, 0.1M FeCl is contained2And 0.2M FeCl3After mixing with 0.1g DMSNsAdded to 50mL NaOH solution, then in N2Stirring and reacting for 30min under the atmosphere to obtain ferroferric oxide nanoenzyme loaded in the dendritic mesoporous silica;
(3) loading gold nano enzyme
Mix 1.25x10-4HAuCl of M4Mixing with 20mg of ferroferric oxide nanoenzyme loaded in the dendritic mesoporous silica prepared in the step (2), stirring at 500rpm for 12h, and then mixing the solution with polyvinylpyrrolidone in N2Mixing under atmosphere to remove O in the equipment2(ii) a Reacting NaBH4Adding into the solution to react for 20min, wherein NaBH4The dosage is NaBH4Au is 5:1 in mass ratio; the double nano enzyme antibacterial agent is obtained.
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