CN114053473A - Preparation method and application of ferroferric oxide composite nano-enzyme antibacterial agent - Google Patents
Preparation method and application of ferroferric oxide composite nano-enzyme antibacterial agent Download PDFInfo
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- CN114053473A CN114053473A CN202111324140.0A CN202111324140A CN114053473A CN 114053473 A CN114053473 A CN 114053473A CN 202111324140 A CN202111324140 A CN 202111324140A CN 114053473 A CN114053473 A CN 114053473A
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- ferroferric oxide
- nanoenzyme
- preparation
- antibacterial agent
- oxide composite
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims abstract description 35
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- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
Abstract
The invention discloses a preparation method and application of a ferroferric oxide composite nano enzyme antibacterial agentChemoenzymatic activity, using adenosine triphosphate and heme on Fe3O4Modulation of CNT peroxidase Activity to Fe3O4-CNT-Hemin/ATP nanoenzyme activity is enhanced while adenosine triphosphate can modulate catalytic activity under neutral conditions, generating more Reactive Oxygen Species (ROS); fe in the presence of potassium persulfate3O4CNT-Hemin/ATP has strong bacteriostatic and bactericidal effects on pathogenic bacteria; the nano enzyme has good biocompatibility, can be used as a medicine of a band-aid, can quickly promote wound healing and prevent wound infection, and has no stimulation, safety and toxic or side effect on the wound; compared with a positive control drug, the antibacterial effect on drug-resistant pathogenic bacteria is more excellent.
Description
Technical Field
The invention relates to the technical field of nano material antibiosis, in particular to a preparation method and application of a ferroferric oxide composite nano enzyme antibacterial agent.
Background
The prevalence of microbial contamination and drug-resistant bacteria is considered as a worldwide public health problem, and people pay high attention to problems such as related diseases caused by bacteria and environmental pollution, and meanwhile, with the use of antibiotics, the drug resistance of bacteria is gradually enhanced, and people are urgently required to develop novel antibacterial agents. The nano enzyme is a new generation of artificial mimic enzyme, has the unique physicochemical property and enzyme-like catalytic activity of nano materials, and is widely concerned due to the advantages of stable structure, low production cost and the like. The nano enzyme respectively catalyzes and generates strong oxidative active oxygen free radicals to destroy components of the bacterial biomembrane, generate hypohalous acid to interfere a quorum sensing system important for the survival of bacteria and degrade exogenous DNA in the bacterial biomembrane through the natural enzyme activity of the nano enzyme, thereby achieving the aim of antibiosis. Compared with natural enzymes and traditional antibacterial agents, the nano-enzyme has the advantages of good stability, low cost, easy functionalization and the like. The research of the nano enzyme as a novel antibacterial agent has been reported, but the antibacterial performance needs to be enhanced, especially the antibacterial performance and the biological safety performance of the nano enzyme to drug-resistant bacteria are improved. The antibacterial enzyme activity generated by the currently reported nano enzyme is mostly concentrated on the activity of the pseudoperoxidase, namely, H is required2O2Despite the participation of H2O2Is very low in concentration but is free of H2O2The antibacterial effect is greatly reducedLow but due to H2O2Is a liquid, which is inconvenient for subsequent application on a solid support.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a ferroferric oxide composite nanoenzyme antibacterial agent, which is characterized in that carbon nano tube modified ferroferric oxide, heme and adenosine triphosphate form composite nanoenzyme (Fe)3O4CNT-Hemin/ATP), the nano enzyme has the characteristic of mimic peroxidase, and generates more Reactive Oxygen Species (ROS) in the presence of potassium persulfate, so that a good antibacterial effect is achieved.
Composite nanoenzyme (Fe) of the invention3O4-CNT-Hemin/ATP), having a pseudoperoxidase activity, the antibacterial activity drops sharply under near-neutral pH conditions, since the pH-dependent activity of general peroxidases is limited to acidic environments only; the invention utilizes the adenosine triphosphate to adjust the catalytic activity under neutral pH (7.4), and simultaneously the adenosine triphosphate and the heme have Fe pairs3O4Modulation of CNT peroxidase Activity to Fe3O4-CNT-Hemin/ATP nanoenzyme activity enhancement with potassium persulfate instead of H2O2And exerting the activity of the pseudo-peroxidase. Fe3O4The CNT-Hemin/ATP has strong bacteriostatic and bactericidal effects on pathogenic bacteria including staphylococcus aureus, drug-resistant staphylococcus aureus, escherichia coli, drug-resistant escherichia coli, pseudomonas aeruginosa and the like. The nano enzyme has good biocompatibility, can be used for the application of the woundplast, can quickly promote the wound healing and prevent the wound infection, and has no stimulation, safety and toxic or side effect on the wound. Compared with a positive control drug, the antibacterial effect on drug-resistant pathogenic bacteria is more excellent.
The preparation method of the ferroferric oxide composite nano enzyme antibacterial agent comprises the following steps:
(1) preparation of carbon nano tube-ferroferric oxide-heme nano enzyme
Mixing 80-100 parts by weight of carbon nano tube, 30 parts by weight of ethylene glycol and 10 parts by weight of polyethylene glycol, and adding 1.5-2.0 parts by weight of FeCl3∙6H2O, 3.5-4.0 weight portions of NaAc and 0.02-0.05 weight portion of blood chloridePerforming ultrasonic treatment on red pigment for 1h, transferring into a muffle furnace, firing at 200 ℃ for 10-12h, cooling to room temperature, alternately washing with water and absolute ethyl alcohol for three times, and performing vacuum drying to obtain carbon nanotube-ferroferric oxide-heme nanoenzyme Fe3O4-CNT-Hemin；
(2) Preparation of carbon nano tube-ferroferric oxide-heme-adenosine triphosphate nanoenzyme
The nano enzyme Fe in the step (1)3O4Mixing the CNT-Hemin water solution and the adenosine triphosphate solution in equal volume, stirring for 5-6h, separating with an external magnet, washing the solid with ethanol for 2-4 times, and vacuum drying to obtain the ferroferric oxide composite nano enzyme antibacterial agent; wherein the nanoenzyme Fe3O4The concentration of the CNT-Hemin water solution is 20-50 mug/mL, and the concentration of the adenosine triphosphate solution is 2-4 mmol/L.
The vacuum drying condition is drying for 24-48h at 40-60 ℃.
The carbon nanotube includes single-walled carbon nanotube and multi-walled carbon nanotube.
The polyethylene glycol comprises polyethylene glycol 400 and polyethylene glycol 600.
The invention also aims to apply the ferroferric oxide composite nanoenzyme antibacterial agent prepared by the method in preparation of a band-aid, and specifically, the ferroferric oxide composite nanoenzyme antibacterial agent and potassium persulfate are added into a traditional Chinese medicine layer of the band-aid, wherein the weight ratio of the ferroferric oxide composite nanoenzyme antibacterial agent to the potassium persulfate is 1: 0.1-0.5.
The invention has the advantages that:
1. the invention adopts carbon nano-tube modified ferroferric oxide, heme and adenosine triphosphate to form composite nanoenzyme, the enzyme has the activity of pseudoperoxidase, and the adenosine triphosphate and the heme are used for treating Fe3O4Modulation of CNT peroxidase Activity to Fe3O4-CNT-Hemin/ATP nanoenzyme activity enhancement, while adenosine triphosphate can regulate catalytic activity at neutral pH (7.4), generate more Reactive Oxygen Species (ROS), and replace H with potassium persulfate2O2More active oxygen (ROS) is generated, so that the ferroferric oxide composite nano enzyme antibacterial agent can achieve a good antibacterial effect;
2. the nano enzyme antibacterial agent has good antibacterial effects on drug-resistant staphylococcus aureus, drug-resistant escherichia coli and the like, and compared with a positive control drug, the sterilization rate is improved by 60-70%;
3. the nano enzyme antibacterial agent is applied to the woundplast, so that the wound healing can be rapidly promoted, the wound infection can be prevented, and the nano enzyme antibacterial agent has no stimulation, safety and toxic or side effect on the wound;
the product of the invention is suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is a spectrum diagram of a nanoenzyme oxidation substrate TMB;
FIG. 2 is a graph showing the results of monitoring Reactive Oxygen Species (ROS) production;
FIG. 3 is a graph showing the antibiotic effect of nanoenzymes and antibiotics on Staphylococcus aureus resistance;
FIG. 4 is a graph showing the antibacterial effect of the nanoenzyme and the antibiotic on the drug-resistant bacteria of Pseudomonas aeruginosa.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1: fe3O4-CNT-Hemin/ATP+K2S2O8Preparation and antibacterial effect of antibacterial agent
1. Multi-walled carbon nanotube-ferroferric oxide-heme nanoenzyme (Fe)3O4Preparation of MWCNT-Hemin)
10g of multi-walled carbon nanotube, 3g of ethylene glycol and 1g of polyethylene glycol 400 are mixed, and then 0.15g of FeCl is added3∙6H2O, 0.35g NaAc and 0.002g hemin, carrying out ultrasonic treatment for 1h, transferring into a muffle furnace, firing for 10h at 200 ℃, cooling to room temperature, washing with water and absolute ethyl alcohol alternately for three times, and carrying out vacuum drying for 48h at 40 ℃ to obtain Fe3O4-MWCNT-Hemin nanoenzyme;
2. multi-walled carbon nanotube-ferroferric oxide-heme-adenosine triphosphate nanoenzyme (Fe)3O4Preparation of MWCNT-Hemin/ATP)
The nano enzyme Fe with the concentration of 50 mug/mL3O4Adding 5mL of CNT-Hemin aqueous solution into 5mL of 4mmol/L Adenosine Triphosphate (ATP) aqueous solution, stirring for 5 hr, separating with magnet, washing with ethanol for 3 times, removing excess ATP, and vacuum drying at 40 deg.C to obtain Fe3O4-MWCNT-Hemin/ATP nanoenzyme;
3. determination of Fe by TMB color reaction3O4Peroxidase Activity of MWCNT-Hemin/ATP Nanolase
100 mug/mLFe3O4100 muL of MWCNT-Hemin/ATP nanoenzyme, 100mmol/L of TMB50 muL, 12.5 mumol/L K2S2O8Adding 50 mu L of the supernatant into 2mL of phosphate buffer solution with the pH value of 7.4, fully and uniformly mixing, incubating at room temperature for 10min, separating by adding a magnet, taking supernatant, measuring absorbance at 655nm by using an ultraviolet-visible spectrophotometer, measuring each sample for 2-3 times, and taking an average value, wherein the result is shown in figure 1; as can be seen from FIG. 1, under neutral conditions, the nanoenzyme Fe3O4MWCNT-Hemin/ATP exhibits a considerably high peroxidase activity.
4. Monitoring of Reactive Oxygen Species (ROS) production: ascorbic Acid (AA) is used as a probe for monitoring, and the AA absorbs at 266nm, but after oxidation by ROS to generate dehydroascorbic acid, the absorption peak disappears. After 1h of co-incubation in Phosphate Buffered Saline (PBS), the nanoenzyme Fe3O4-MWCNT-Hemin/ATP+K2S2O8The absorbance of ascorbic acid at 266nm was greatly reduced, and the degree of reduction is shown in FIG. 2, Fe3O4-MWCNT-Hemin/ATP+K2S2O8＞Fe3O4-MWCNT-Hemin/ATP＞K2S2O8。
5. Killing experiment of nano enzyme on bacterial flat plate
(1) The experimental method comprises the following steps: with staphylococcus aureus (S. aureusATCC 25923 and Bacillus subtilis (Bacillus subtilis)B. subtilisATCC 6051) represents a gram-positive strain, Escherichia coli: (E.coli)E. coli，ATCC 25922 and Pseudomonas aeruginosaP.aeruginosa，ATCC 27853) is representative of gram-negative strains. In addition, methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA-1720) is used as an antibioticRepresentative of drug resistant strains. For each strain, 3-5 single colonies were inoculated into fresh Tryptone Soy Broth (TSB) and cultured at 37 ℃ for 16-18h to stationary phase. mu.L of the culture was diluted 100-fold with fresh TSB and cultured to mid-log phase at 37 deg.C (OD600= 0.5-0.7). After bacterial cell harvest, the cells were washed 1 time with sterile PBS by centrifugation and adjusted to 1.5X 10 with sterile PBS6CFU/mL; serial dilution of 2 times the nano enzyme dispersion with sterile PBS buffer solution, adding each nano enzyme dilution (100. mu.L) into each zero dilution hole of 96-hole microporous plate, inoculating 50. mu.L of the adjusted bacterial suspension into each zero dilution hole of preset microporous plate to make each hole reach 5X 105 CFU/mL (150. mu.L); the plate was then incubated at 37 ℃ for 3 h. Then, 10-fold serial dilution was performed with sterile PBS buffer, and the diluted bacterial solution (20. mu.L) was spread on TSB agar plates and incubated overnight at 37 ℃ to form colonies visible to the naked eye. Inoculum size was indicated by a control sample containing the same treated bacteria but without nanoenzymes. Each experiment was performed in 3 replicates and the results of the experiments are the average of two independent experiments. The Minimum Bactericidal Concentration (MBC) value is defined as the minimum concentration of the antibacterial drug or the nanoenzyme for inhibiting 99.9% of the bacterial growth; the Minimum Inhibitory Concentration (MIC) value is defined as the lowest concentration of antibiotic or nanoenzyme that inhibits 90% of bacterial growth.
And (3) culturing drug-resistant bacteria: drug resistance is researched through a cycle inhibition experiment, pseudomonas aeruginosa is taken as a representative strain of gram-negative bacteria, staphylococcus aureus is taken as a representative strain of gram-positive bacteria, cefadroxil, gentamicin and nano enzyme are respectively used for treatment, and drug resistance is observed; the specific method comprises the following steps: circulating inhibition assays were performed using the same as the final bacterial inoculum for the next growth inhibition assay, using the same as the culture treated with the As MIC of the previous inhibition assay (300. mu.L, 100. mu.L per well) diluted to OD600=0.001, in 3 trials each, with the results being the average of two independent assays.
(2) And (3) antibacterial results: the Minimal Bactericidal Concentration (MBC) values of the nanoenzymes are shown in Table 1;
TABLE 1 Sterilization Effect of Nano-enzyme
The drug resistance test result shows that after 8 cycles, the inhibition effect of cefadroxil on staphylococcus aureus is obviously reduced, the survival rate of bacteria is improved to 78.6% from the initial 5.9%, and the survival rate of nano-enzyme on bacteria is improved to 58.5% from 3.6%; similarly, the inhibition of pseudomonas aeruginosa by gentamicin also decreased significantly after the circulation, and the survival rate of bacteria increased from 9.1% to 74.4% at the beginning, while the survival rate of bacteria increased from 7.9% to 47.1% only by nanoenzyme, as shown in fig. 3 and 4.
The results show that the nano enzyme not only has excellent performance in inhibiting drug-resistant bacteria, but also has excellent performance in slowing down the development of the drug resistance of bacteria. The ROS-producing nanoenzymes circumvent the drug resistance mechanisms of bacteria, because ROS destroy many small spherical substances (e.g., nucleic acids, proteins, lipids, etc.) that are critical to cell function simultaneously, rather than targeting specific intracellular metabolic pathways as antibiotics do.
Example 2: fe3O4-SWCNT-Hemin/ATP+K2S2O8Preparation of antibacterial agent and application of antibacterial agent in band-aid
1. Single-walled carbon nanotube-ferroferric oxide-heme nanoenzyme (Fe)3O4-SWCNT-Hemin) preparation
Mixing 8g of single-walled carbon nanotube, 3g of ethylene glycol and 1g of polyethylene glycol 400, and adding 0.2g of FeCl3∙6H2O, 0.4g NaAc and 0.005g hemin, carrying out ultrasonic treatment for 1h, transferring the mixture into a muffle furnace for firing at 200 ℃ for 12h, cooling the mixture to room temperature, washing the mixture with water and absolute ethyl alcohol alternately for three times, and carrying out vacuum drying at 60 ℃ for 24h to obtain Fe3O4-SWCNT-Hemin nanoenzyme;
2. single-walled carbon nanotube-ferroferric oxide-heme-adenosine triphosphate nanoenzyme (Fe)3O4-SWCNT-Hemin/ATP) preparation
Carrying out 20 mug/mL of nanoenzyme Fe in the step (1)3O4Adding 5mL of-SWCNT-Hemin/ATP aqueous solution to 5mL of 2mmol/L Adenosine Triphosphate (ATP) aqueous solution, stirring for 6 hr, separating with an additional magnet, and separating withWashing with ethanol for 3 times, removing excessive ATP, and vacuum drying at 50 deg.C to obtain Fe3O4-SWCNT-Hemin/ATP nanoenzyme;
3. preparing the nano enzyme antibacterial band-aid: mixing the Fe prepared in the step 23O4-SWCNT-Hemin/ATP nanoenzyme and K2S2O8Mixing the raw materials according to the weight ratio of 1:0.1 to form an antibacterial agent for the use of the band-aid, preparing the nano-enzyme antibacterial band-aid with the band-aid by the weight ratio of 0.05mg/g, and preparing the nano-enzyme antibacterial band-aid by a conventional band-aid preparation method;
4. wound healing test of nano enzyme antibacterial band-aid
The wound healing test adopts a 6-8 week healthy ICR female mouse, a round wound with the diameter of 1cm is made on the back of the mouse, after the mouse is infected with pseudomonas aeruginosa and staphylococcus aureus for 24 hours, a nano-enzyme antibacterial band-aid group, a white drug band-aid group and an untreated group are respectively arranged, the band-aids are replaced once every 24 hours, and the wound healing condition is observed; the results show that after 24h, the wounds treated by the nano-enzyme antibacterial band-aid and the white drug band-aid begin to heal, while the untreated mice have the signs of wound healing after 72h, the wounds of the mice treated by the nano-enzyme antibacterial band-aid substantially heal on day 7, the wounds of the mice treated by the white drug band-aid substantially heal on day 9, and the wounds of the mice treated by the untreated mice substantially heal on day 16.
Claims (5)
1. A preparation method of a ferroferric oxide composite nano enzyme antibacterial agent is characterized by comprising the following steps:
(1) preparation of carbon nano tube-ferroferric oxide-heme nano enzyme
Mixing 80-100 parts by weight of carbon nano tube, 30 parts by weight of ethylene glycol and 10 parts by weight of polyethylene glycol, and adding 1.5-2.0 parts by weight of FeCl3∙6H2O, 3.5-4.0 parts of NaAc and 0.02-0.05 part of hemin by weight, ultrasonically treating for 1h, transferring into a muffle furnace, firing for 10-12h at 200 ℃, cooling to room temperature, alternately washing with water and absolute ethyl alcohol for three times, and vacuum drying to obtain the carbon nano tube-ferroferric oxide-heme nanoenzyme Fe3O4-CNT-Hemin；
(2) Preparation of carbon nano tube-ferroferric oxide-heme-adenosine triphosphate nanoenzyme
The nano enzyme Fe in the step (1)3O4Mixing the CNT-Hemin water solution and the adenosine triphosphate solution in equal volume, stirring for 5-6h, separating with an external magnet, washing the solid with ethanol for 2-4 times, and vacuum drying to obtain the ferroferric oxide composite nano enzyme antibacterial agent; wherein the nanoenzyme Fe3O4The concentration of the CNT-Hemin water solution is 20-50 mug/mL, and the concentration of the adenosine triphosphate solution is 2-4 mmol/L.
2. The preparation method of the ferroferric oxide composite nanoenzyme antibacterial agent according to claim 1, characterized by comprising the following steps: the carbon nanotube is one of a single-walled carbon nanotube and a multi-walled carbon nanotube.
3. The preparation method of the ferroferric oxide composite nanoenzyme antibacterial agent according to claim 1, characterized by comprising the following steps: the polyethylene glycol is one of polyethylene glycol 400 and polyethylene glycol 600.
4. The preparation method of the ferroferric oxide composite nanoenzyme antibacterial agent according to claim 1, characterized by comprising the following steps: the vacuum drying condition is drying at 40-60 deg.C for 24-48 h.
5. The application of the ferroferric oxide composite nanoenzyme antibacterial agent prepared by the preparation method of the ferroferric oxide composite nanoenzyme antibacterial agent according to any one of claims 1 to 4 in preparation of a band-aid is characterized in that: mixing the ferroferric oxide composite nano enzyme antibacterial agent and potassium persulfate, and adding the mixture into the traditional Chinese medicine layer of the band aid, wherein the weight ratio of the ferroferric oxide composite nano enzyme antibacterial agent to the potassium persulfate is 1: 0.1-0.5.
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